The GUT and GABA

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So I recently started drinking Kefir and it was relaxation in a glass so I look into it and those lil microbes help us out my friends..

 

Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve

 

    Javier A. Bravoa,1,

    Paul Forsytheb,c,1,

    Marianne V. Chewb,

    Emily Escaravageb,

    Hélène M. Savignaca,d,

    Timothy G. Dinana,e,

    John Bienenstockb,f,2, and

    John F. Cryana,d,g,2

Abstract

 

There is increasing, but largely indirect, evidence pointing to an effect of commensal gut microbiota on the central nervous system (CNS). However, it is unknown whether lactic acid bacteria such as Lactobacillus rhamnosus could have a direct effect on neurotransmitter receptors in the CNS in normal, healthy animals. GABA is the main CNS inhibitory neurotransmitter and is significantly involved in regulating many physiological and psychological processes. Alterations in central GABA receptor expression are implicated in the pathogenesis of anxiety and depression, which are highly comorbid with functional bowel disorders. In this work, we show that chronic treatment with L. rhamnosus (JB-1) induced region-dependent alterations in GABAB1b mRNA in the brain with increases in cortical regions (cingulate and prelimbic) and concomitant reductions in expression in the hippocampus, amygdala, and locus coeruleus, in comparison with control-fed mice. In addition, L. rhamnosus (JB-1) reduced GABAAα2 mRNA expression in the prefrontal cortex and amygdala, but increased GABAAα2 in the hippocampus. Importantly, L. rhamnosus (JB-1) reduced stress-induced corticosterone and anxiety- and depression-related behavior. Moreover, the neurochemical and behavioral effects were not found in vagotomized mice, identifying the vagus as a major modulatory constitutive communication pathway between the bacteria exposed to the gut and the brain. Together, these findings highlight the important role of bacteria in the bidirectional communication of the gut–brain axis and suggest that certain organisms may prove to be useful therapeutic adjuncts in stress-related disorders such as anxiety and depression.

 

    brain–gut axis

    irritable bowel syndrome

    probiotic

    fear conditioning

    cognition

 

There is increasing evidence suggesting an interaction between the intestinal microbiota, the gut, and the central nervous system (CNS) in what is recognized as the microbiome–gut–brain axis (1–4). Studies in rodents have implicated dysregulation of this axis in functional bowel disorders, including irritable bowel syndrome. Indeed, visceral perception in rodents can be affected by alterations in gut microbiota (5). Moreover, it has been shown that the absence and/or modification of the gut microflora in mice affects the hypothalamic–pituitary–adrenal (HPA) axis response to stress (6, 7) and anxiety behavior (8, 9), which is important given the high comorbidity between functional gastrointestinal disorders and stress-related psychiatric disorders, such as anxiety and depression (10). In addition, pathogenic bacteria in rodents can induce anxiety-like behaviors, which are mediated via vagal afferents (9, 11).

 

GABA is the main inhibitory neurotransmitter of the CNS, the effects of which are mediated through two major classes of receptors—the ionotropic GABAA receptors, which exist as a number of subtypes formed by the coassembly of different subunits (α, β, and γ subunits; ref. 12), and the GABAB receptors, which are G protein coupled and consist of a heterodimer made up of two subunits (GABAB1 and GABAB2), both of which are necessary for GABAB receptor functionality (13). These receptors are important pharmacological targets for clinically relevant antianxiety agents (e.g., benzodiazepines acting on GABAA receptors), and alterations in the GABAergic system have important roles in the development of stress-related psychiatric conditions.

 

Probiotic bacteria are living organisms that can inhabit the gut and contribute to the health of the host (14). Accumulating clinical evidence suggests that probiotics can modulate the stress response and improve mood and anxiety symptoms in patients with chronic fatigue and irritable bowel syndrome (15, 16). One such organism is Lactobacillus rhamnosus (JB-1), which has been demonstrated to modulate the immune system because it prevents the induction of IL-8 by TNF-α in human colon epithelial cell lines (T84 and HT-29) (17) and modulates inflammation through the generation of regulatory T cells (18). Moreover, it inhibits the cardio–autonomic response to colorectal distension (CRD) in rats (19), reduces CRD-induced dorsal root ganglia excitability (20), and affects small intestine motility (21).

 

It is currently unclear whether potential probiotics such as L. rhamnosus (JB-1) could affect brain function, especially in normal, healthy animals. To this end, we sought to assess whether this bacteria could mediate direct effects on the GABAergic system. In parallel, behaviors relevant to GABAergic neurotransmission and the stress response were assessed subsequent to L. rhamnosus (JB-1) administration. Finally, the role of the vagus nerve in mediating such effects was also investigated by examining these parameters in subdiaphragmatically vagotomized mice.

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Results

Behavioral Effects of L. rhamnosus (JB-1) Administration.

 

A battery of behavioral tests relevant to anxiety and depression was carried out. The stress-induced hyperthermia (SIH) and elevated plus maze (EPM) tests are widely used for assessing functional consequences of alterations in GABA neurotransmission (22, 23). Chronic administration of L. rhamnosus (JB-1) produced a nonsignificant reduction in SIH (t = 1.567, df = 34; P = 0.1263; Fig. 1A). On the EPM, animals treated with L. rhamnosus (JB-1) had a larger number of entries to the open arms than broth-fed animals, suggesting anxiolytic effects (open arm entry defined as all four paws entering the arms of the EPM apparatus) (t = 4.662, df = 34; P < 0.001; Fig. 1A). This effect is also reflected in the percentage of time spent in the open arms, although this observation did not reach statistical significance [broth v. L. rhamnosus (JB-1): 25.28 ± 6.67% vs. 38.36 ± 7.99%; t = 1.267, df = 34; P = 0.2146].

 

Effects of L. rhamnosus (JB-1) on GABA Receptor Expression.

GABAB1b mRNA.

 

There was a differential expression of this transcript in the different studied areas. Higher levels of GABAB1b mRNA were found in cingulate cortex 1 (CG1) (Fig. 2A) and prelimbic (PrL) (Fig. 2B) cortical areas of L. rhamnosus (JB-1)-fed mice in comparison with broth-fed mice (t = 3.485, df = 10, P < 0.01; and t = 2.965, df = 10, P < 0.05, respectively), but no differences were observed in the infralimbic (IL) cortex (t = 0.4558, df = 10, P = 0.658; Fig. 2C). Conversely, L. rhamnosus (JB-1)-fed mice had lower levels of GABAB1b mRNA in the basolateral amygdala (BLA) (t = 8.778, df = 10, P < 0.001; Fig. 2D) and central amygdala (CeA) (t = 3.372, df = 10, P < 0.01; Fig. 2E), locus coeruleus (LC) (t = 5.339, df = 10, P < 0.001; Fig. 2F), hippocampal sub areas of the dentate gyrus (DG) (t = 5.555, df = 10, P < 0.001; Fig. 2G), cornus ammonis area 3 (CA3) (t = 3.207, df = 10, P < 0.01; Fig. 2H), and cornus ammonis area 1 (CA1) (t = 3.826, df = 10, P < 0.01; Fig. 2I) compared with broth-fed control mice.

 

 

GABAAα2 mRNA.

 

A differential expression of GABAAα2 mRNA within the studied areas was also found (Fig. 3). In L. rhamnosus (JB-1)-fed animals, there were low levels of GABAAα2 mRNA in CG1 (t = 2.611, df = 10, P < 0.05; Fig. 3A), PrL (t = 2.267, df = 10, P < 0.05; Fig. 3B), and IL (t = 2.803, df = 10, P < 0.05; Fig. 3C) cortical areas, as well as in the BLA (t = 7.541, df = 10, P < 0.001; Fig. 3D) and CeA (t = 7.150, df = 10, P < 0.001; Fig. 3E), in comparison with broth-fed mice. In addition, no differences in GABAAα2 mRNA were found in the LC between the two groups of mice (t = 1.190, df = 10, P = 0.2616; Fig. 3F); however, higher levels of GABAAα2 mRNA were found in the DG of L. rhamnosus (JB-1)-fed mice in comparison with broth-fed control animals (t = 5.967, df = 10, P < 0.001; Fig. 3G). No differences in GABAAα2 mRNA were found in CA3 (t = 0.403, df = 10, P = 0.6955; Fig. 3H) and CA1 (t = 2.161, df = 10, P = 0.0560; Fig. 3I) neuronal layer of the hippocampus of L. rhamnosus (JB-1) compared with broth-fed mice.

Effects of L. rhamnosus (JB-1) Administration on the Behavior of Vagotomized Mice.

 

To further understand the role of the vagus nerve in communicating sensory information to the brain, subdiaphragmatic vagotomy (Vx) was carried out, and behavioral parameters were determined. As shown in Fig. 4A, two-way ANOVA revealed that there was an overall effect of Vx [F(1, 36) = 8.91; P < 0.01], an overall effect of L. rhamnosus (JB-1) treatment [F(1, 36) = 5.80; P < 0.05], and an interaction between Vx and L. rhamnosus (JB-1) [F(1, 36) = 5.690; P < 0.05]. In terms of time in the center of the open field arena, Vx prevented the anxiolytic effects of L. rhamnosus (JB-1) in mice, which is reflected in a reduction of the time spent in the center of the open field compared with sham surgery animals fed with L. rhamnosus (JB-1) (P < 0.05). That Vx prevented the anxiolytic effect of L. rhamnosus (JB-1) is further verified because the analysis of the number entries to the central area of the open field reflects a similar profile as in the percentage of time spent in the central part of the arena [Fig. 6A; effect of Vx: F(1, 36) = 5.56, P < 0.05; effect of L. rhamnosus (JB-1): F(1, 36) = 4.64, P < 0.05; interaction between Vx and L. rhamnosus (JB-1): F(1, 36) = 7.66, P < 0.01]. This exploratory behavior seems to be related to an anxiolytic effect, because the total distance traveled by the mice in each experimental condition did not differ between them [F(1, 36) = 0.44, P = 0.51; Fig. 4A].

 

In addition, FST revealed that there was an overall effect of Vx [F(1, 36) = 5.14, P < 0.05], an overall effect of L. rhamnosus (JB-1) treatment [F(1, 36) = 10.47, P = 0.01], and an interaction between Vx and L. rhamnosus (JB-1) [F(1, 36) = 6.22, P < 0.05] in terms of immobility time. Post hoc analysis showed that sham animals fed with L. rhamnosus (JB-1) had significantly lower mobility time (P < 0.05) compared with sham animals fed with broth (Fig. 4A). This effect was prevented by Vx, because immobility time of Vx animals fed with L. rhamnosus (JB-1) was similar to the immobility time of control mice (P > 0.05).

Effects of L. rhamnosus (JB-1) Administration on GABA Receptor mRNA Expression: Role of Vagus Nerve.

 

In our first series of studies, we showed that administration of L. rhamnosus (JB-1) for 28 d had marked and distinct effects on the expression of transcripts for GABAB1b and GABAAα2 receptors subunits in prefrontal cortex, amygdala, hippocampus, and LC compared with broth-fed animals. These findings suggest that the behavioral changes observed could be due to the effects of L. rhamnosus (JB-1) on brain mRNA expression. Thus, to elucidate a mechanistic means as to how L. rhamnosus (JB-1) can affect GABA receptor mRNA expression, in situ hybridization of the two major GABAA receptor subunits was performed in the brains of Vx mice.

GABAAα2 mRNA.

 

Statistical analysis revealed that there is a significant interaction between L. rhamnosus (JB-1) treatment and Vx on GABAAα2 mRNA levels [F(1, 20) = 5.674, P = 0.0273] in the BLA and also in the CeA [F(1, 20) = 4.756, P = 0.0413]. There is also an effect of Vx in both areas [bLA: F(1, 20) = 8.532, P = 0.0084; CeA: F(1, 20) = 4.84, P = 0.0397] and an effect of treatment only in the BLA [F(1, 20) = 12.75, P = 0.0019], but not in the CeA [F(1, 20) = 3.586, P = 0.0728; Fig. S1]. Post hoc analysis found that in sham animals, L. rhamnosus (JB-1) significantly reduced the levels of GABAAα2 mRNA in the BLA (P < 0.001) and CeA (P < 0.05) areas of the amygdala in comparison with sham animals fed with broth (Fig. S1 A and B), which is consistent with our initial findings (Fig. 3 D and E). This effect on the GABAAα2 transcript was completely prevented by Vx (Fig. S1 C and D).

 

In the hippocampus, ANOVA revealed that there was no interaction between L. rhamnosus (JB-1) and Vx on the levels of GABAAα2 mRNA in any of the studied areas [DG: F(1, 20) = 3.47, P = 0.0772; CA3: F(1, 20) = 1.84, P = 0.1900; CA1: F(1, 20) = 1.51, P = 0.2327]. However, it did show an effect of L. rhamnosus (JB-1) in the DG [F(1, 20) = 6.36, P = 0.02038] and also in CA3 [F(1, 20) = 6.66, P = 0.0179], but not in CA1 [F(1, 20) = 4.13, P = 0.0557]. Additionally, an effect of Vx was only observed in the DG [F(1, 20) = 5.86, P = 0.0248], but not in CA3 [F(1, 20) = 3.09, P = 0.0941] or CA1 [F(1, 20) = 1.47, P = 0.2393]. Post hoc analysis showed that sham animals fed with L. rhamnosus (JB-1) had significantly higher levels of GABAAα2 mRNA in the DG (P < 0.05) and CA3 (P < 0.05; Fig. 4B), in comparison with sham animals fed with broth. Vx in broth-fed animals increased the levels of GABAAα2 mRNA in the different hippocampal areas, while L. rhamnosus (JB-1) did not affect the action of Vx on the hippocampus (Fig. 4B; representative images in Fig. S2).

GABAAα1 mRNA.

 

Densitometric analysis of GABAAα1 mRNA showed an interaction between Vx and L. rhamnosus (JB-1) treatment in both studied areas of the amygdala [bLA: F(1, 20) = 33.43, P < 0.0001; CeA: F(1, 20) = 15.19, P = 0.0009; Fig. S3]. This analysis revealed an effect of Vx on GABAAα1 mRNA [bLA: F(1, 20) = 49.80, P < 0.0001; CeA: F(1, 20) = 73.91, P < 0.0001) and an effect of L. rhamnosus (JB-1) administration on this same transcript [bLA: F(1, 20) = 44.53, P < 0.0001; CeA: F(1, 20) = 12.77, P = 0.0019). Post hoc analysis showed that animals that had sham Vx surgery and were fed with L. rhamnosus (JB-1) showed significant reduction in GABAAα1 mRNA in the BLA (P < 0.0001; Fig. S3A) and CeA (P < 0.0001; Fig. S3B), in comparison with sham animals fed with broth. In addition, no differences in GABAAα1 mRNA were found in L. rhamnosus (JB-1) or broth-fed Vx animals compared with sham control mice.

 

In the hippocampus, analysis of the levels of GABAAα1 mRNA revealed an interaction between Vx and L. rhamnosus (JB-1) treatment in all studied areas [DG: F(1, 20) = 21.80, P = 0.0001; CA3: F(1, 20) = 19.133, P = 0.0003; CA1: F(1, 20) = 22.87, P = 0.0001; Fig. 4C]. In addition, an effect of L. rhamnosus (JB-1) was observed in the DG [F(1, 20) = 12.49, P = 0.0021], CA3 [F(1, 20) = 13.49, P = 0.0015], and in CA1 [F(1, 20) = 25.66, P < 0.0001]. However, an effect of Vx was only observed in the DG [F(1, 20) = 9.751, P = 0.0054], but not in the CA3 [F(1, 20) = 2.357, P = 0.1404] or CA1 [F(1, 20) = 1.28, P = 0.2713]. Post hoc analysis found significant reductions in GABAAα1 mRNA in the DG (P < 0.0001), CA3 (P < 0.0001), and CA1 (P < 0.0001; Fig. 4C) in comparison with sham control animals only fed with broth. Vx did not affect the expression of GABAAα1 mRNA in broth-fed animals, and it prevented the effects of L. rhamnosus (JB-1) on GABAAα1 mRNA expression in the analyzed areas (Fig. 4C; representative images in Fig. S4).

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Discussion

 

These data demonstrate specific, previously undescribed neurochemical changes induced by modulation of intestinal microbiota using a potential probiotic [L. rhamnosus (JB-1)] in normal, healthy animals (Table S1). Moreover, we show that L. rhamnosus (JB-1) can have a direct effect upon associated behavioral and physiological responses in a manner that is dependent on the vagus nerve. L. rhamnosus (JB-1) consistently modulated GABAAα2, GABAAα1, and GABAB1b receptor mRNA expression—receptors implicated in anxiety behavior—in a regional-dependent manner.

 

Furthermore, in this study we observed that L. rhamnosus (JB-1) administration reduces the stress-induced elevation in corticosterone, suggesting that the impact of the Lactobacillus on the CNS has an important effect at a physiological level. Alterations in the HPA axis have been linked to the development of mood disorders and have been shown to affect the composition of the microbiota in rodents (26). Our data are in line with previous studies showing that subchronic or chronic treatment with antidepressants can prevent forced swim stress-induced increases in plasma corticosterone in both mice and rats (27). Moreover, it has been shown that alterations in HPA axis modulation can be reversed by treatment with Lactobacillus and Bifidobacterium (28, 29). However, caution is needed when extrapolating from single timepoint neuroendocrine studies (30). Nonetheless, these data clearly indicate that in the bidirectional communication between the brain and the gut, the HPA axis is a key component that can be affected by changes in the enteric microbiota.

 

Accumulating evidence suggests that metabotropic GABA receptors are crucial for the maintenance of normal behavior. Indeed, genetic and pharmacological studies have implicated that GABAB receptors play a key role in mood and anxiety disorders (13). In the present study, the mRNA of the GABAB1b subunit, the main isoform of the GABAB1 receptor in the adult brain (13), was increased in the prefrontal cortex of L. rhamnosus (JB-1)-fed animals. Studies have shown that animal models of depression have reductions in GABAB receptor expression in frontal cortices (13). Thus, it is tempting to speculate that the changes induced by the Lactobacillus might provide an advantage toward stressful situations in comparison with broth-fed control animals. This difference is consistent with behavioral and neuroendocrine responses seen. In the other analyzed areas (amygdala, hippocampus, and LC), L. rhamnosus (JB-1) administration reduced the expression of GABAB1b mRNA, which is consistent with the antidepressant-like effect of GABAB receptor antagonists (31). L. rhamnosus (JB-1)-fed animals showed an enhanced memory to an aversive cue and context in comparison with broth-fed mice—an observation that implies changes at the level of the amygdala and hippocampus (24). These findings are consistent with data generated from GABAB1b-deficient animals, highlighting an important role for this subunit in the development of cognitive processes, including those relevant to fear (32, 33). In line with these results, it has recently been shown that treatment with certain bacteria improves memory function in infected mice (34) as well as cognitive abilities in humans (35). However, unlike GABAB1b knockout mice (36), L. rhamnosus (JB-1)-fed mice are able to extinguish learned fear, behaviors dependent on the PrL cortex (37), which may reflect the actual up-regulation of this receptor subunit in this brain region.

 

The amygdala is crucial for manifestation of fear and anxiety responses and for modulation of the affective components of visceral perception. Given increased levels of GABAAα2 mRNA in the amygdala are found in stressed animals (38), the reductions in GABA receptor subunits induced by the Lactobacillus suggest that this bacteria could have promoted an adaptive advantage over broth-fed animals in terms of interaction with stressful situations. The amygdala is also necessary for conditioning of a relatively simple stimulus or cue (conditioned stimulus) and the context in which the unconditioned stimulus is delivered (24, 25). Component analysis revealed that animals fed with L. rhamnosus (JB-1) had significantly higher freezing behaviors during the last cues and context in the second day (recall phase) of testing than broth-fed animals—an observation that is in line with previous reports on BALB/c mice (24). Interestingly, it has been shown that alterations in the expression of GABAA receptor subunits affect fear-related behaviors, as genetic ablation of the GABAAα1 subunit in mice enhances freezing behavior (39). It is worth noting that this increased emotional learning may also be interpreted as increased anxiety behavior; this interpretation suggests that L. rhamnosus (JB-1) has differential effects on conditioned compared with unconditioned aspects of anxiety.

 

GABAergic neurotransmission in the hippocampus has been related to the modulation of behavior and memory processes (40). Additionally, this structure is required for contextual conditioning, and evidence suggest that inactivation of hippocampal GABAB receptors improves spatial working memory (41). In the present study, hippocampal GABAB1b mRNA is reduced in L. rhamnosus (JB-1)-fed mice, which is consistent with an enhanced memory consolidation in the fear conditioning test and further suggests that the changes in hippocampal gene expression induced by the Lactobacillus could in part account for these differences in behavior. L. rhamnosus (JB-1) administration also affected the transcripts of GABAA receptor subunits in the hippocampus. Although differences in the expression of the transcript for GABAAα2 and GABAAα1 have been found in the hippocampus of rats subjected to different learning tasks, these changes are not consistent (38, 42). Nevertheless, it has been shown that GABAA receptors bearing the GABAAα2 subunit mediate the anxiolytic effects of benzodiazepines, whereas GABAA receptors that have the α1 subunit mediate the sedative and amnesic effects of benzodiazepines (12). In the present study, the difference in hippocampal expression of GABAAα2 and GABAAα1 mRNAs support the behavioral findings because L. rhamnosus (JB-1)-fed mice were less anxious and displayed antidepressant-like behaviors in comparison with broth-fed controls. Furthermore, it can be suggested that the effects of L. rhamnosus (JB-1) on fear-related behavior could be due to its effects on stress-induced corticosterone levels. Administration of corticosterone to BALB/c mice after the acquisition phase (day 1) destabilizes fear memory consolidation and allows faster extinction (24), suggesting a mechanism by which corticosterone itself could directly affect fear-related behavior. In the present work, L. rhamnosus (JB-1) reduced the stress-induced levels of corticosterone, which suggest that these “lower” levels could underlie the behavioral alterations observed.

 

The vagus nerve plays a major role in communicating changes in the gastrointestinal tract to the CNS (3). In the present study, Vx prevented the anxiolytic and antidepressant effects of L. rhamnosus (JB-1) and also the changes in GABAAα2 and GABAAα1 mRNAs in the amygdala (SI Materials and Methods), as well as GABAAα1 mRNA in the hippocampus. Nevertheless, Vx on its own was able to increase the levels of GABAAα2 mRNA in the hippocampus, although it prevented any further effect produced by L. rhamnosus (JB-1) supporting the observation that the changes in the hippocampus could reflect an indirect consequence of the Lactobacillus-induced changes in structures receiving direct visceral sensory inputs that can project afferents toward the hippocampus. Indeed, it has been shown that vagus nerve stimulation in rats can affect hippocampal functions (43), and therefore the changes in hippocampal GABAAα2 mRNA expression could occur as a result of Vx. Moreover, vagus nerve stimulation has been described as a successful approach to treat some (44), but not all (45), patients with treatment-resistant depression, which further suggests the importance of the vagus nerve in the modulation of behavior. We cannot exclude the possibility that there are physiological changes in the gut associated with Vx that may indirectly alter functional aspects of the Lactobacillus. However, feeding and weight gain were similar in vagotomized and sham-treated animals, and we have previously demonstrated that the ability of this organism to protect against colitis in a murine model is not influenced by subdiaphragmatic Vx (46)—a strong indication that local intestinal anti-inflammatory actions are not altered. The molecular mechanisms underlying how the bacteria affects vagal afferents needs to be resolved in future studies.

 

One of the important aspects of these studies is that the behavioral changes observed were consistent across two different laboratories using slightly different protocols, which is important, given the perceived problems in replication of behavioral data between laboratories (47). It is important to note that the present neurochemical observations only represent changes at the mRNA level, and not protein, and they could only represent a more complex situation involving other neurotransmitter systems (48) and a variety of intracellular cascades that can affect the expression of these transcripts in the different studied areas. Moreover, probiotic effects are strain dependent; for example, in contrast to L. rhamnosus (JB-1), Lactobacillus salivarius had no neurally dependent effects on murine gut smooth muscle contractions indicating the unlikelihood of L. salivarius having an effect on the enteric nervous system, which must occur before the signals are communicated via the vagus nerve to the brain (21). Considerable further investigation needs to be conducted to the molecular mechanisms at a microbiome level underlying the effects observed. Moreover, future studies using dead bacteria, killed in such a way as to exclude structural alteration, are needed to further insight into the mechanism of action of these bacteria (19, 21). Nonetheless, our data conclusively demonstrate that a potential probiotic can robustly alter brain neurochemistry and behavior relevant to anxiety- and depression-related behavior in mice.

 

In summary, our data with L. rhamnosus (JB-1) suggest that nonpathogenic bacteria can modulate the GABAergic system in mice and therefore may have beneficial effects in the treatment of depression and anxiety. Moreover, it is worth noting that, in the present study, the effects were observed in healthy animals, whereas most studies examining the effects of potential probiotics on microbiome–gut–brain axis function rely on using infected, germ-free, or antibiotic-treated animals (2, 14); thus, the ramifications of these findings is manifold for the therapeutic potential of bacteria in modulating brain and behavior. Changes in transcripts for GABA receptor subunits emphasize a possible mechanistic insight into the potential effect of L. rhamnosus (JB-1) on anxiety-like behavior (12, 13). However, the participation of other neurotransmitter and neuropeptide systems that are of relevance to stress-related psychiatric disorders—such as 5-hydroxytryptamine, norepinephrine, glutamate, and corticotrophin-releasing factor—cannot be ruled out. Thus, future studies should investigate whether chronic treatment with L. rhamnosus (JB-1) can modulate such systems and, if so, how long such changes may last. Furthermore, the effects of L. rhamnosus (JB-1) on neurotransmitter levels are probably downstream of the effects on the HPA axis. In addition, the vagus nerve is responsible for some of the behavioral and molecular changes induced by L. rhamnosus (JB-1), demonstrating a clear pathway for the functional communication between bacteria, the gut, and the brain that modulates the behavioral responses toward different stressful situations. It is worth noting that the majority of studies on the microbiome–gut–brain axis are rodent-based, and future validation of the role of this axis in modulation in behavior is now warranted. Nonetheless, our current studies offer the intriguing opportunity of developing unique microbial-based strategies for the adjunctive treatment of stress-related psychiatric disorders.

 

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Title: Modulating the human gut microbiome as an emerging therapeutic paradigm

Author(s): Deepak K. Rajpal and James R. Brown

Source: Science Progress. 96.3 (Fall 2013): p224.

Document Type: Report

DOI: http://dx.doi.org.ezproxy.flinders.edu.au/10.3184/003685013X13691404141587

Copyright: COPYRIGHT 2013 Science Reviews 2000 Ltd.

http://www.sciencereviews2000.co.uk/view/journal/science-progress

Full Text:

 

ABSTRACT

 

The human body is actually a vast and changing ecosystem comprised of billions of microbial organisms, known collectively as the microbiome. Within the last few years, the study of the microbiome and its impact on human health has been a rapidly growing area of biomedical science. The gut intestinal tract microbiome has been a particular focus of research given its potential role in many inflammatory and metabolic diseases as well as drug metabolism. Although a nascent field, the potential for modulating the gut microbiome or human host interactions associated with these microbes offers new therapeutic strategies for many chronic diseases, in particular obesity, diabetes and inflammatory bowel diseases. Here we provide an overview of present knowledge about the gut microbiome, its putative role in metabolic diseases and the potential for microbiome focused treatments from the drug development perspective.

 

Keywords: microbiome, metabolic disease, obesity, diabetes, drug discovery, human genome

 

1. Introduction

 

Studies of the human genome have led to the advancement of biomedical science and drug discovery. However, it is increasingly apparent that determinants of our health are not solely controlled by our own genomes. Rather, many disease pathologies involve the interplay between the human body, the external environment and the complex communities of microorganisms residing on respiratory (1), vaginal/urogenital (2) and gastrointestinal (GIT) tract (3) and skin (4) surfaces. The complement of microbial cells co-inhabiting an individual, the microbiota, exceeds at least 10-fold the number of cells in the human body (5,6). Furthermore, the gene collection of this residing microbial community, the microbiome, exceeds by at least 100 times the complement of genes present in the human nuclear genome (7). While our knowledge of the detrimental disease impact of many bacterial, viral and eukaryotic pathogens is well-established, the roles of complex nonpathogenic microbiota communities in sustaining health or promoting disease are only recently studied.

 

Over the last five years, the microbiome has been one of the fast growing areas of biomedical research. The advent of sensitive, high volume DNA sequencing and metabolomics technologies has led to a rapid expansion of datasets from microorganism populations associated with various chronic disease phenotypes. Major public funding initiatives such as the US National Institutes of Health (NIH) Human Microbiome Project, initiated in 2007 (, and the EU MetaHIT Consortium, started in 2008, are driving the characterization of microbiomes from hundreds, soon thousands, of individuals of different ages, geographical, dietary and disease backgrounds. New tools and methodologies are also being developed for modulating the microbiota in model organisms as well as sampling not only genomes but also metabolites. Concurrently, more powerful computational biology approaches are necessary to make sense of the vast volumes of genomic, metabolic and phenotypic data being generated from both pre-clinical and clinical microbiome related studies.  The status of gut microbiome is increasingly being studied in a wide variety of other diseases. One central aspect of the human-microbiota symbiosis is the dialogue between the microbiota and the immune system (58,59). The microbiota contributes to the development of both the mucosal and systemic immune systems. It is now appreciated that loss of homeostasis or dysbiosis in the GIT immune system plays a central role in numerous disease conditions. Clinically, antibiotic usage has also been associated with increased incidence of Crohn's disease and ulcerative colitis in both adults and children (60,61). Homeostasis of the mucosal immune system requires the development of both tolerance to the residential microbiota and, at the same time, regulation to avoid overgrowth and invasion of internal tissues. Both the microbiota and the innate and adaptive immune systems contribute to the establishment of an optimal equilibrium whereas disturbances can lead to dysbiosis (62) and disease states through the development of intestinal inflammation (63,64). This is reflected on the host side by genetic predispositions to inflammatory bowel diseases (IBD), which point to the importance of the immune system and microbial sensing. For example, mutations in human genes NOD2, ATG16L1 and those encoding defensins are known predispositions to IBD. Recent meta-analysis of 15 GWAS studies of Crohn's disease and/or ulcerative colitis with a combined total of more than 75,000 cases and controls revealed a significant overlap between susceptibility loci for IBD and mycobacterial infection which reinforces the role of host-pathogen responses in IBD pathology (65).

 

Specific microbiome changes have also been observed in patients with colon cancer (66,67). Moreover, mechanisms for the microbiome to promote colon cancer progression have been suggested. These include microbial stimulation of the c-Jun/JNK and STAT3 signaling pathways (68) and the production of genotoxin colibactin, a polyketide synthase encoded by the gene pks found in enteric E. coli strains (69). Other areas of interest are the potential roles of the GIT microbiota in diseases of distal organs such as asthma (70).

 

Early stage but intriguing research suggests that some behavioral disorders might be manifestations of a CNS-gut microbiota axis mediated by immune, neural and endocrine pathways (71). Many bioactive neural activating metabolites, including GABA, norepinephrine, serotonin, dopamine and acetylcholine, are produced by microbial species (72). Chronic administration of Lactobacillus rhamnosus to mice was shown to modulate GABA receptor expression via the vagus nerve and reduce anxiety and depression behaviors (73). Children with autism spectrum disorders have been found to have abnormal amino acid metabolism, increased oxidative stress, and altered gut microbiomes (74).

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And I thought this was a little interesting plus to get in some exercise

 

Title: Exercise attenuates PCB-induced changes in the mouse gut microbiome

Author(s): Jeong June Choi, Sung Yong Eum, Evadnie Rampersaud, Sylvia Daunert, Maria T. Abreu and Michal Toborek

Source: Environmental Health Perspectives. 121.6 (June 2013): p725.

Document Type: Report

DOI: http://dx.doi.org.ezproxy.flinders.edu.au/10.1289/ehp.1306534

Copyright: COPYRIGHT 2013 National Institute of Environmental Health Sciences

http://www.ehponline.org/

Abstract:

 

BACKGROUND: The gut microbiome, a dynamic bacterial community that interacts with the host, is integral to human health because it regulates energy metabolism and immune functions. The gut microbiome may also play a role in risks from environmental toxicants.

 

OBJECTIVES: We investigated the effects of polychlorinated biphenyls (PCBs) and exercise on the composition and structure of the gut microbiome in mice.

 

METHODS: After mice exercised voluntarily for 5 weeks, they were treated by oral gavage with a mixture of environmentally relevant PCB congeners (PCB153, PCB138, and PCB180; total PCB dose, 150 [micro]mol/kg) for 2 days. We then assessed the microbiome by determination of 16S rRNA using microarray analysis.

 

RESULTS: Oral exposure to PCBs significantly altered the abundance of the gut microbiome in mice primarily by decreasing the levels of Proteobacteria. The activity level of the mice correlated with a substantial shift in abundance, biodiversity, and composition of the microbiome. Importantly, exercise attenuated PCB-induced changes in the gut microbiome.

 

CONCLUSIONS: Our results show that oral exposure to PCBs can induce substantial changes in the gut microbiome, which may then influence their systemic toxicity. These changes can be attenuated by behavioral factors, such as voluntary exercise.

 

The gut microbiome is a collection of bacteria that resides in the host gastrointestinal tract. As many as [10.sup.14] microbes are in the human gut (Ley et al. 2006), accounting for 15,000-36,000 species of bacteria (Frank et al. 2007). The main bacterial phyla comprising the human gut microbiome are the gram-negative Bacteroidetes and Proteobacteria and the gram-positive Actinobacteria and Firmicutes (Frank et al. 2007). Each host has a unique composition of gut microbiota, which implies highly individual responses to environmental stressors and suggests a role for gut microbiota in future personalized health strategies (Kinross et al. 2011).

 

Recent evidence has implicated the gut microbiome in the development of a wide range of disorders, including obesity, diabetes, metabolic dysfunctions, vascular disease, and inflammatory bowel disease (Kinross et al. 2011). Strong evidence also indicates the critical role of the gut microbiota in drug metabolism and toxicity, energy metabolism, immune functions, and postsurgical recovery (Holmes et al. 2011; Kinross et al. 2011; Tilg and Kaser 2011). Moreover, the gut microbiome has been reported to regulate psychiatric health and influence etiopathology of autism. For example, Bravo et al. (2011) reported that chronic administration of Lactobacillus rhamnosus induced anxiolytic and antidepressant effects by modulating the expression of GABA receptors in the brain, and Lyte et al. (2006) observed that infection with Citrobacter rodentium induced anxiety-like behaviors via vagal sensory regulation.

 

Despite these diverse effects on human health, the influence of the microbiome on the toxicity of environmental pollutants and its role in disease risk are largely unknown (Betts 2011). It was recently suggested that preabsorptive metabolism can modify toxicity of environmental pollutants, influencing their health effects (Lapanje et al. 2007; Pinyayev et al. 2011). The most compelling evidence illustrating this phenomenon was obtained in studies on biotransformation of heavy metals by the gut microbiome. For example, Pinyayev et al. (2011) reported that anaerobic microbiota of mouse cecum converts arsenate into oxyarsenicals and thioarsenicals; Lapanje et al. (2007) observed that exposure to mercury altered the bacterial community in the gut of a terrestrial isopod (Porcellio scaber); and Liebert et al. (1997) found that gram-negative fecal bacteria were involved in the biotransformation of mercury. Experiments on germ-free mice also provided evidence that the gut microbiome can regulate the expression of cytochrome P450 enzymes, which are involved in the metabolism of a variety of xenobiotics, including environmental chemicals (Claus et al. 2011). Indeed, human colon microbiota can transform polycyclic aromatic hydrocarbons (PAHs) to estrogenic metabolites (Van de Wiele et al. 2005). These findings are significant because PAH toxicity has been linked to estrogenicity of the compounds, thus suggesting that PAH bioactivation in the colon should be taken into consideration when assessing risk (Van de Wiele et al. 2005). In addition, the role of gut microbiota, as well as its variability in relation to the disposition of environmental chemicals in the human body and its contribution to the development of obesity and diabetes, have recently been recognized (Snedeker and Hay 2012).

 

The present study was designed on the basis of recent evidence implicating the role of the gut microbiome in risks associated with exposure to environmental chemicals (Betts 2011). We examined whether exposure to polychlorinated biphenyls (PCBs) could affect the abundance and composition of the gut microbiome. There is growing interest in the role of behavioral factors in modulating toxicity of environmental pollutants; although the role of nutrition has been explored (e.g., Majkova et al. 2008), the impact of exercise on the health effects of toxicants is unknown. Exercise can influence the outcomes of disorders associated with alterations in the gut microbiome (Martin 2011); therefore, we hypothesized that physical activity may affect the composition of the gut microbiota and thus influence the impact of environmental toxicants. Our results indicate that PCBs can induce profound changes in the microbial composition of the gut and that exercise can attenuate these PCB-induced effects on the intestinal microbiome.

 

Results

 

PCB exposure decreases the abundance of the gut microbiota. We first analyzed the effects of PCB exposure on the gut microbiome in sedentary mice. The Welch's test revealed that exposure to the PCB mixture significantly (p < 0.05 by Student's t-test) altered the abundance of 1,223 bacterial taxa in these mice (1,133 taxa had decreased abundance and 90 taxa had increased abundance). As a result of these changes, the overall abundance of bacteria significantly diminished (by 2.2%) in PCB-exposed mice. Table 1 lists bacterial taxa with the greatest decrease in abundance (4.0- to 5.6-fold) after PCB treatment. These taxa belong primarily to phylum Proteobacteria, but the classes and families are diverse. The group of bacterial taxa with increased abundance after PCB treatment was relatively modest, and the changes did not exceed 2-fold; the bacterial taxa of these with the highest increases in abundance are listed in Table 2. They belong to several difference phyla, including Bacteriodetes, Actinobacteria, Verrucomicrobia, and Firmicutes.

 

Exposure to the PCB mixture did not alter biodiversity of the gut microbiome; all 11,229 taxa were detected in at least one sample from sedentary mice. After PCB exposure, the number of taxa dropped to 10,798, but the change was not statistically significant.

 

Exercise alters the composition of the gut microbiome. As analyzed by weighted Unifrac distance, the structure of the gut microbiome of the exercised mice was significantly different that of the sedentary mice (Adonis test, p < 0.05). PCoA of unweighted Unifrac distance with given presence/absence metrics also showed prominent categorization of the composition of the gut microbiome between the exercised and sedentary mice (Figure 2A).

 

Among detected bacterial taxa, 93 were present exclusively in either exercised or sedentary mice. Specifically, 67 taxa were detected only in the exercised mice [see Supplemental Material, Table S1 (http://dx.doi.org.ezproxy.flinders.edu.au/10.1289/ ehp.1306534)], and 26 taxa were unique to sedentary mice (see Supplemental Material, Table S2).

 

A group of 2,510 taxa showed differences in abundance between the exercised and sedentary mice. These taxa were then analyzed by PCoA with weighted Unifrac distance, which indicated significant differences in the composition of the microbial communities between the exercised and sedentary mice (Figure 2B). HC-AN analysis based on weighted Unifrac distance confirmed a shift of the composition of the gut microbiome related to physical activity (exercised vs. sedentary mice) (Figure 2C).

 

[FIGURE 2 OMITTED]

 

Further examination using the PAM identified 10 taxa with substantially different abundance between the exercised and sedentary mice (Table 3). The taxa that were more abundant in the exercised group were in phylum Firmicutes, class Bacilli, and most of these were in order Lactobacillales. The taxa that were decreased in the exercised group belonged to phyla Tenericutes, Bacteroidetes, and Firmicutes. The most striking change in exercised mice was a decrease in Erysipelotrichaceae bacterium C11_K211 from phylum Tenericutes, which decreased dramatically in exercised compared with sedentary mice.

 

Exercise attenuates PCB-induced alterations of gut microbiome composition. Comparison of the gut microbiome among all experimental groups (sedentary and exercised mice with or without PCB exposure) identified 1,568 bacterial taxa that were differentially abundant in at least one of these groups. Analysis of these taxa for dissimilarity between the groups using PCoA with weighted Unifrac indicated significant differences between the composition of the gut microbiome before and after PCB exposure in sedentary mice. Importantly, exercise altered PCB-mediated effects on the gut microbiome, as indicated by a loss of bacterial clustering (Figure 3A). This phenomenon was subsequently confirmed by HC-AN analysis (Figure 3B).

 

We detected 10,799 taxa in sedentary mice exposed to the PCB mixture, compared with 13,383 taxa in the exercised mice treated with PCBs. Although there appeared to be a tendency toward increased biodiversity in the exercise-plus-PCB group, these changes were not significant. In contrast, the abundance of bacterial species was elevated by 2.9% (Student's mest, p < 0.05) in PCB-treated exercised mice as compared PCB-treated sedentary mice, providing additional evidence that exercise can protect against PCB-mediated alterations in the gut microbiota. As shown in Figure 3C, exercise appeared to prevent a PCB-induced decrease in abundance of Proteobacteria in sedentary mice (Figure 3C).

 

Discussion

 

In view of recent evidence indicating that gut bacteria can be involved in the preabsorptive metabolism of heavy metals and organic chemicals (Lapanje et al. 2007; Liebert et al. 1997; Pinyayev et al. 2011), the gut microbiome has been proposed to play a role in the assessment of health risks associated with environmental chemicals (Betts 2011; Snedeker and Hay 2012). Therefore, our demonstration that short-term exposure to an environmentally relevant PCB mixture resulted in profound changes in the gut microbiome in mice is highly significant. The most striking change in the intestinal microbial profiles was a decrease in the overall abundance of bacterial species. Although these results are the first to show effects of PCBs on the gut microbiome, the decrease in bacterial abundance we observed corresponds with the finding that PCB-contaminated soil is characterized by a shift in structure and abundance of bacterial community (Petric et al. 2011). Incubation of soil slurries with higher-chlorinated PCB congeners (e.g., PCB28, PCB77, Aroclor 1242) has been reported to result in lower bacterial numbers (Correa et al. 2010). Even though the gut and soil provide completely different bacterial environments, PCB exposure appears to elicit environmental stress on the structure and composition of bacterial communities that results in diminished bacterial abundance. In the present study, these changes selectively affected bacterial phylotypes in phylum Proteobacteria. Such changes may be most relevant to immune functions of the host, because the gut microbiome has been shown to play important roles in mucosal immunity and interactions with intestinal and colonic epithelial cells, dendritic cells, and T and B immune cells. Microbiota composition has functional effects on T-effector- and T-regulatory-cell balance, immune responsiveness, and homeostasis (Kelly and Mulder 2012). Thus, it is likely that alterations of the gut microbiota compromise a novel mechanism leading to immunological alterations, which develop in response to exposure to PCBs (Maule et al. 2005; Weisglas-Kuperus et al. 2004). In addition, alterations of the gut microbiome can affect PCB-induced disruption of the intestinal barrier and translocation of lipopolysaccharides into the blood stream (Choi JJ et al. 2012; Choi YJ et al. 2010). In the present study, we observed no statistical differences in bacterial community structure in the exercised mice before and after PCB treatment. Thus, exercise provided protection against PCB-induced changes in the gut microbiome. In particular, exercise prevented a PCB-induced decrease in abundance of Proteobacteria, which was observed in sedentary mice.

 

[FIGURE 3 OMITTED]

 

Although the data we reported here are novel, effects of physical activity on several other aspects of gut physiology (e.g., peristalsis; Song et al. 2012) and pathology have been reported. For example, exercise was demonstrated to decrease the risk of developing several intestinal diseases, including colon cancer (Friedenreich et al. 2006), inflammatory bowel disease and irritable bowel syndrome (Lustyk et al. 2001), and other disorders that are accompanied by changes in the gut microbiome (De Hertogh et al. 2012; Nelson et al. 2011; Walker et al. 2011).

 

The mechanisms of exercise-mediated changes in gut ecology are not known; however, they are likely to be mediated by altering the host factors that influence the intestinal microenvironment. For example, physical activity has been reported to increase excretion of primary bile acids to the gastrointestinal tract (Meissner et al. 2011) and to suppress the formation of secondary bile acids (Hagio et al. 2010). The primary bile acids, such as cholic, deoxycholic, or chenodeoxycholic acids, have established antimicrobial activity, which is mediated by the reduction in internal pH levels of bacteria, dissipation of their transmembrane electrical potential, and disturbances of membrane integrity, leading to leakage of ions and cell death (Kurdi et al. 2006). In support of this hypothesis, Islam et al. (2011) demonstrated that cholic acid induced substantial changes in the cecal microbiome composition by stimulating the growth of Firmicutes at the expense of Bacteroidetes and outgrowth of several bacteria in the classes Clostridia and Erysipelotrichi. Thus, the antimicrobial activity of the bile acids may elicit selective pressure on the bacterial communities in exercised mice, leading to a shift of the gut microbiome structure as observed in the present study.

 

Short-chain fatty acids (SCFAs) may be another factor that regulates the gut microbiome in response to exercise. Indeed, Matsumoto et al. (2008) showed that rats that participated in voluntary running exercise had increased butyrate concentration in the cecum compared with sedentary rats. These authors directly linked this effect to the beneficial effect of exercise on the gut microbiota and the development of gastrointestinal disorders. SCFAs (e.g. butyrate and acetate) increase colonic epithelial cell proliferation and decrease the risk of colorectal cancer. Their influence on the composition of microbial environment has been linked to a decreased pH in the gut (Wong et al. 2006). Nevertheless, compared with the results of the present study, the effects of butyrate infusion on the rumen microbiome in cows was relatively minor because only 19 genera and 43 bacterial taxa were significantly affected in response to butyrate (Li et al. 2012); these data suggest that this SCFA may be only one of several factors involved in exercise-mediated changes in the gut microbiome. Treatment with SCFAs may also affect host-related intestinal factors because butyrate promotes cell differentiation and cell-cycle arrest, inhibits the enzyme histone deacetylase, and decreases the transformation of primary to secondary bile acids as a result of colonic acidification (Wong et al. 2006).

 

Finally, exercise may influence the composition of the gut microbiome by altering the intestinal immune system. Viloria et al. (2011) observed that physical activity increased expression of IgA and cytokines such as interleukin-6 and tumor necrosis factor-[alpha]. These changes in the intestinal immune system may lead to secondary alternations of the host-bacterial interaction and induce selective pressure on bacterial selection.

 

The development of chronic diseases related to the exposure to environmental toxicants are associated with age, and the benefits of exercise are also being emphasized in older individuals; therefore, we used aged mice (11-13 months of age) in the present study. Older mice tend to have higher body mass than younger animals. In fact, the average body weight of sedentary mice in the present study was 46.8 [+ or -] 1.4 g, and body weight of exercised mice was approximately 30% lower. Recent evidence from Ley et al. (2005) indicated a strong association of the intestinal microbiome with the development of obesity. Genetically obese ob/ob mice were characterized by a major decrease in the abundance of Bacteroidetes and an increase in Firmicutes compared with lean ob/+ wild-type littermates and lean ob/+ mothers fed the same diets. Similar changes were observed in wild-type mice fed a high fat/high polysaccharide diet (Turnbaugh et al. 2008) and in obese humans (Ley et al. 2005). An increase in Firmicutes (such as Lactobacilli) and a decrease in Bacteroidetes have been confirmed in obese humans (Armougom et al. 2009). In another study, overweight pregnant patients had reduced abundance of Bifidobacteria and Bacteroidetes and increased abundance of selected Firmicutes (e.g., Staphylococcus) and Proteobacteria (e.g., Enterobacteriaceae) (Santacruz et al. 2010). In line with these reports, it is relevant that we detected an increased abundance of several Firmicutes, primarily Enterococcaceae (e.g., Enterococcus faecium), in the exercised mice.

 

Although Enterococci are commensal bacteria, they are also important nosocomial pathogens that cause bacteremia, endocarditis, and other infections in humans. Some strains are resistant to multiple antibiotics and possess virulence factors, such as adhesins, invasins, pili, and haemolysin (Willems et al. 2011). Nevertheless, E. faecium isolates from clinical outbreaks are different strains than E. faecium from animals, food, and humans in the community (Franz et al. 2011). In fact, several enterococci, including E. faecium strains, are used as probiotics in the form of pharmaceutical preparations. They are administered to treat diarrhea, antibiotic-associated diarrhea, and irritable bowel syndrome; to lower cholesterol levels; or to improve host immunity (Franz et al. 2011).

 

The most striking change we found in the gut microbiome of exercised mice was a > 300-fold decrease in the abundance of Erysipelotrichaceae (Table 3). This family plays an important role in metabolic disorders and energy metabol ism (Chen et al. 2012). Chen et al. (2012) reported that Erysipelotrichaceae were enriched in obese humans and mice, as well as in mice fed a high-fat diet, and noted that the abundance of Erysipelotrichaceae was also increased in patients with colorectal cancer. Thus, our data are consistent because we observed a decrease in the number of Erysipelotrichaceae in exercised mice that had lost a substantial amount of body weight; this is associated with the role of Erysipelotrichaceae in energy production and adiposity (Claus et al. 2011; Goodman et al. 2011; Zhang et al. 2009).

 

Conclusions

 

Here we demonstrate that oral exposure to a mixture of environmentally relevant PCB congeners significantly altered the abundance of the gut microbiome by decreasing the levels of Proteobacteria. These results suggest that the gut microbiome may be one of the primary targets of PCB-induced toxicity in subjects exposed orally to these environmental toxicants. We observed that PCB-induced alterations of the gut microbiome were attenuated by voluntary exercise.

 

Caption: Figure 1. Experimental design indicating treatment and sampling times.

 

Caption: Figure 2. Exercise alters the structure and composition of the gut microbiome. (A) PCoA based on unweighted Unifrac distance between exercised and sedentary mice (PCoA1, 33% of variation; PCoA2, 15% of variation). (6) PCoA and © HC-AN analysis based on weighted Unifrac distance between exercised and sedentary mice of the 2,510 taxa with significant abundance differences across at least one of the categories (PCoA1, 84% of variation; PCoA2, 6% of variation).

 

Caption: Figure 3. Exercise prevents PCB-induced alterations of the gut microbiome. (A) PCoA and (6) HC-AN analysis based on weighted Unifrac distance of the 1,568 taxa with significant differences in abundance in at least one of the categories (PCoA1, 71% of variation; PCoA2, 11% of variation). © Proteobacteria content in vehicle-treated control and PCB-treated sedentary and exercised mice; values are mean [+ or -] SE of pooled taxa in phylum Proteobacteria.

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Front Neurol. 2014; 5: 43.

Published online Apr 4, 2014. doi:  10.3389/fneur.2014.00043

PMCID: PMC3983497

The Gastrointestinal Tract Microbiome and Potential Link to Alzheimer’s Disease

James M. Hill,1,2,3 Surjyadipta Bhattacharjee,1 Aileen I. Pogue,4 and Walter J. Lukiw1,3,4,5,*

Author information ► Article notes ► Copyright and License information ►

This article has been cited by other articles in PMC.

 

Accumulating clinical- and scientific research-based evidence is driving our increased awareness of the significance of the human microbiome (HM) to the healthy and homeostatic operation of the human central nervous system (CNS). HM communities occupy several different but distinct microbial ecosystems on and within the human body, including nasal, oral, and otic cavities, the surface of the skin and the urogenital and the gastrointestinal (GI) tracts. The complex symbiotic inter-relationship between the GI-tract microbiome and its host is strongly influenced by diet and nutrition, and when optimized can be highly beneficial to food digestion, nutrient intake, and immune health (1–6). For example, dietary composition ultimately affects the structure, organization, function, and speciation of the HM occupying the GI tract, in part by supplying multiple substrates for microbial metabolism. Typical Western diets containing high fat–cholesterol, low amounts of soluble and insoluble fiber, and sugar- and salt-enrichment not only impart deleterious nutrition but also dietary constraints on the HM. This in turn impacts the supply of microbiome-generated molecules absorbed into the systemic circulation for transport into the extensive neurovasculature of the CNS. This short communication will focus on emerging ideas concerning the contribution of the GI-tract microbiome to human neurological disease with emphasis on Alzheimer’s disease (AD) wherever possible.

 

It is the HM of the GI tract that contains the largest reservoir of microbes in humans, containing about 1014 microorganisms from at least 1000 distinct microbial species, and outnumbering human somatic cells by about 100 to 1 (1, 7). The total HM has been estimated to encode about 4 × 106 genes versus the ~26,600 genes of the human host, so again the quantity of HM genes outnumbers host genes in the order of about 150 to 1 (4). Of the 55 bacterial divisions currently identified, only two are prominent in mammalian GI-tract microbiota, including the anaerobic Bacteroidetes (~48%) and Firmicutes (~51%), with the remaining 1% of phylotypes distributed amongst the Proteobacteria, Verrucomicrobia, Fusobacteria, Cyanobacteria, Actinobacteria, and Spirochetes, with various species of fungi, protozoa, viruses, and other microorganisms making up the remainder (http://www.genome.gov/pages/research/sequencing/seqproposals/hgmiseq.pdf). Interestingly, microorganisms making up the smallest proportion of the HM seem to have a disproportionately large effect on host health and disease (see below). Of all GI-tract microbiota, bacterial densities of 1011–1012/ml are the highest recorded density in any known microbial ecosystem of any living organism (1, 4, 7–10). There is currently expanding interest in the ability of these high density GI-tract bacteria to influence host innate-immune, neuromodulatory-, and neurotransmission-functions (3, 4, 11–14). Established pathways of GI–CNS communication and mutualism currently include the autonomic nervous system (ANS), the enteric nervous system (ENS), the immune system, and the neuroendocrine system (15–21). Remarkably, neuronal signaling pathways along this bidirectional GI–CNS axis remain incompletely understood despite their important roles: (1) in coordinating metabolic-, nutritive-, and homeostatic-functions, and (2) in their functional disruption in chronic diseases such as anxiety, autoimmune-disease, diabetes, metabolic-syndrome, obesity, and stress-induced and progressive neuropsychiatric diseases including AD (3, 11, 12, 20, 22–24).

 

Here we list six specific, highly illustrative examples and recent insights into the interactive nature of the HM with a healthy, homeostatic CNS, and examples of a dysfunctional or altered HM contribution to the development of age-associated neurological disease:

 

    (1)

    studies of the ENS in germ-free “gnotobiotic” mice, i.e., those missing their microbiome, indicate that commensal GI-tract microbiota are critically essential for membrane electrical characteristics, including ion fluxes, action potentials, and GI-tract sensory neuron excitability, thus providing a potential mechanistic link for the initial exchange of signaling information between the GI-tract microbiome and the ANS, ENS, CNS neuroimmune–neuroendocrine systems (4, 5, 20, 23, 25);

    (2)

    GI-tract-abundant Gram-positive facultative anaerobic or microaerophilic Lactobacillus, and other Bifidobacterium (Actinobacteria) species such as Lactobacillus brevis and Bifidobacterium dentium are capable of metabolizing glutamate to produce gamma-amino butyric acid (GABA), the major inhibitory neurotransmitter in the human CNS (26). Increased GI-tract GABA appears to correlate with increased CNS GABA levels, but the systemic pathways that contribute to this gut–brain linkage require additional study (3, 26). In CNS dysfunctions in GABA-mediated neuromodulatory and neurotransmission functions have been linked to the development of anxiety, behavioral deficits, epilepsy, defects in synaptogenesis, depression, and cognitive impairment including AD (16, 17, 23, 27–29). Interestingly, epileptic activities including complex partial-seizures and non-convulsive seizures are commonly associated with AD, especially in its early stages, but the contribution of GI-tract microbiome to epileptiform events via GABA modulation is not well understood (30);

    (3)

    the secreted, dimeric, 238 amino acid brain-derived neurotrophic factor (BDNF) essential in the maintenance and survival of neurons, has pleiotropic effects on neuronal development, differentiation, synaptogenesis, and the synaptic plasticity that underlies neuronal circuit formation and cognition, and has been found to be decreased in brains and serum from patients with anxiety, behavioral defects, schizophrenia, and AD (27, 31, 32). Interestingly, mice deficient in BDNF have altered development of GI-tract innervations including the vagus nerve, which normally serves as a major constitutive, modulatory communication pathway across the GI–CNS axis (33, 34). In experimental infection models known to lead to significant alterations in the microbiota profiles, BDNF expression was found to be reduced in the hippocampus and cortex of germ-free “gnotobiotic” mice, and the reduction in the expression of BDNF was found to specifically associate with increased anxiety and progressive cognitive dysfunction (20, 31, 32);

    (4)

    glutamate is the most abundant excitatory neurotransmitter in the human CNS; the N-methyl-d-aspartate (NMDA) glutamate receptor, a CNS-enriched transmembrane sensor that regulates synaptic plasticity and cognition has some intriguing and potentially direct interactions with the HM; for example, the NMDA-, glutamate-targeting, glutathione-depleting, and oxidative-stress-inducing neurotoxin β-N-methylamino-l-alanine (BMAA), found elevated in the brains of patients with amyotrophic-lateral sclerosis (ALS), the Parkinson-dementia complex of Guam, and AD, has been hypothesized to be generated by Cyanobacteria of the GI-tract microbiome, and anxiety, stress, chronic intestinal inflammatory disease, or malnutrition may further induce BMAA generation to ultimately contribute to neurological dysfunction (13, 35). Interestingly, BMAA, a neurotoxic amino acid not normally incorporated into the polypeptide chains that constitute brain proteins, has been linked with intra-neuronal protein misfolding, a hallmark feature of the amyloid peptide-enriched senile plaque lesions, and resultant inflammatory neurodegeneration, that characterize AD, ALS, PD, and prion disease (21, 23, 36). These and other HM-resident Cyanobacteria-generated neurotoxins including saxitoxin and anatoxin-α may further contribute to neurological disease, especially over the course of aging when the intestinal epithelial barrier of the GI tract becomes significantly more permeable (13, 37);

    (5)

    the HM not only secretes nutritive molecules, including essential vitamins of the B and K group, but also release molecular factors that may potentially modulate or alter systemic- and CNS-amyloidosis, CNS neurochemistry, and neurotransmission. For example, HM organisms widely utilize their own naturally secreted peptides and amyloids as structural materials, adhesion molecules, and neurotoxins that ultimately function in host auto-immunity and immune-protection. The specific contribution of the HM and bacterial amyloid to protein misfolding and amyloidogenic diseases such as AD are however not well understood, although bacterial components such as endotoxins are often found within the senile plaque lesions that characterize the AD brain (5, 21, 38). The HM further appears to condition host immunity to foreign microbes, including viral infection and xenobiotics, while regulating autoimmune responses that can impact homeostatic metabolic- and neural-signaling functions within the CNS (4, 14, 23, 39). Progressive neurological disorders such as AD have been increasingly linked to altered autoimmune and faulty innate-immune responses (12, 40, 41). An increased incidence of auto-immunity, exposure to pathogens both pre- and postnatally, and findings of antibodies to brain-specific antigens are common in disorders as diverse as anxiety, autism, depression, obsessive–compulsive disorder, schizophrenia, Parkinson’s disease (PD), and AD, together suggesting that differences in exposure and genetic vulnerability toward HM-mediated auto-immunity may be significant determinants of age-related neurological disease course and outcome as humans age (14, 17, 23, 39, 42–46);

    (6)

    secretory products of the GI-tract microbiome and translocation of these signaling molecules via the lymphatic and systemic circulation throughout the CNS are just beginning to be identified. Recent advances in metagenomics, RNA sequencing, metatranscriptomics, metaproteomics, and metabolomics continue to clarify our perceptions of the GI-tract HM and its contribution to health and disease. Just as each individual has a unique “stoichiometrically proportioned” composition of microorganisms in their microbiome, individuals appear to be variably sensitive to age-related neurological disorders such as AD through the concept of “human biochemical individuality” (11, 16, 47). Importantly, dietary and GI-tract HM manipulation and the emergence of personalized medicine may be poised to revise and modernize our remedial efforts in the clinical management of brain disorders including AD, and the progressive transformation to more favorable clinical outcomes (30, 48, 49).

 

In summary, the human GI tract is a natural habitat for large, diverse, and host-specific microbial communities including multiple species from the kingdoms of Archaea, Bacteria, the Viruses, and other symbiotic microbiota. How humans co-evolved with these complex microbial ecosystems, and how certain microbial species were specifically selected for mutual symbiotic benefit is of extreme interest when assessing critical HM–host interactions involving food digestion, nutrition supply and uptake, metabolic interactions, protection against pathogens and immune system development, maintenance, and dyshomeostasis in both health and disease. To cite another relevant example, abundant evidence suggests that human mitochondria originated from bacteria via endosymbiotic relationships from very early in the evolutionary history of eukaryotes, so cross-reactivity of mitochondria and host immunological responses to selective bacterial GI constituents may have deleterious effects on human mitochondrial function through molecular mimicry (4, 12, 42). This is evidenced by multiple findings in common autoimmune, inflammation-linked systemic, and neurological disorders including ALS, anxiety, diabetes, epilepsy, metabolic disease, obesity, rheumatic fever, schizophrenia, Sydenham’s chorea, PD, AD, and other age-related pathologies, including transgenic animal models for these diseases (2, 4, 12, 23, 44–46, 50–54).

 

Lastly, since the early investigations of Koch, Metchnikoff, Pasteur, Von Leeuwenhoek, and others on the microbial basis of pathogenicity and disease transmission, Westernized societies have very successfully reduced the incidence of microbial-borne infectious disease, while an environment of autoimmune, cardiovascular, metabolic, and neuroinflammatory diseases continues to flourish. We have only recently begun to truly appreciate the potential for complex and beneficial contributions of the GI-tract HM to host genetics, phenotype, and the development and course of CNS disease. With advancement in next-generation, high throughput sequencing and metagenomic technologies our further investigations into the complex microbial ecosystems within us should yield novel HM manipulative strategies for both the optimization of our health and the more effective clinical management of human metabolic, neuropsychiatric, and neurological disorders.

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Gut, bugs, and brain: Role of commensal bacteria in the control of central nervous system disease

 

    Javier Ochoa-Repáraz PhD1,*,

    Daniel W. Mielcarz PhD2,

    Sakhina Begum- Haque PhD1 and

    Lloyd H. Kasper MD1

 

Article first published online: 8 MAR 2011

 

DOI: 10.1002/ana.22344

 

Copyright © 2011 American Neurological Association

 

Issue

Annals of Neurology

Annals of Neurology

 

Volume 69, Issue 2, pages 240–247, February 2011

 

Abstract

 

The mammalian gastrointestinal track harbors a highly heterogeneous population of microbial organisms that are essential for the complete development of the immune system. The gut microbes or “microbiota,” coupled with host genetics, determine the development of both local microbial populations and the immune system to create a complex balance recently termed the “microbiome.” Alterations of the gut microbiome may lead to dysregulation of immune responses both in the gut and in distal effector immune sites such as the central nervous system (CNS). Recent findings in experimental autoimmune encephalomyelitis, an animal model of human multiple sclerosis, suggest that altering certain bacterial populations present in the gut can lead to a proinflammatory condition that may result in the development of autoimmune diseases, in particular human multiple sclerosis. In contrast, other commensal bacteria and their antigenic products, when presented in the correct context, can protect against inflammation within the CNS. Ann Neurol 2011

 

Microbiome

 

The largest epithelial surface of the human body is the gastrointestinal (GI) tract. As such, it could be considered the largest surface area of exposure and interaction with both exogenous pathogens and intrinsic commensal microorganisms. Although mammals are born sterile, microorganisms soon colonize the gut and other mucosal surfaces after birth. This colonization evolves into a highly diverse endogenous microbial population comprising over 1 × 1013–14 resident bacteria, creating a relationship that confers benefits to both microorganisms and host.1 However, this environment can be shared by multiple pathogens that utilize the mucosa as invasion and infection sites. It is the role of the immune system to concurrently control the responses to commensal and pathogenic organisms.2 The microbiota survive, proliferate, and interact with the large mucosal surface of the GI tract, which is defined by the host genetic background. The genetics of the host both determines the physical barrier where the gut microbes are found and influences the immunological responses by which the host controls the heterogeneous population of microorganisms.3 At the same time, the microbiota modulate the normal function and development of the GI tract.4 The interactions of microbes with the host have evolved into a complex balance of host genes, environment, and microorganisms defined as the microbiome.

 

It has been estimated that at least 1,000 different bacteria species cohabit the human gut, although recent studies suggest this number may approach 35,000.5 Recent molecular approaches provide new methods for the identification of those species unable to be cultured in laboratories6; specifically, the detection of 16S ribosomal RNA from microbes significantly increased the number of species described.7 Recently developed metagenomic technology to examine 16S recombinant DNA (rDNA) has provided further functional and metabolic analysis of the microbial genes present in the gut.8 Metaproteomics (whole community proteome analysis) can identify and compare levels of proteins present in the environment that may provide a more relevant biological readout than a functional gene analysis.9, 10 This proteomic approach allows for the acquisition of additional information regarding protein interactions between the host and microorganism. The importance of the microbiome and human disease has been brought to the forefront by a recent National Institutes of Health (NIH) initiative to establish the “Human Microbiome Project.” This $140 million project is to focus and compare the human microbiome between individuals, and to assess how changes in the microbiome correlate with human disease,6 using metagenomic and genomic DNA sequencing techniques.

 

As studies related to the human microbiome are difficult at the experimental level, animal models are utilized to study interactions between the populations that comprise the microbiota and the interactions of these populations within the host. Germ-free (gnotobiotic) mice are born and raised in sterile conditions. These mice lack any prior exposure to either gut microbial populations or antigens. The model has significant intrinsic limitations, because the absence of any commensal organisms could provoke the development of an altered immune system, and the lack of any metabolic benefit conferred by the commensal flora. Although interpreting results obtained from studies in germ-free mice to the etiology or disruption of immune homeostasis in human disease is difficult, what it has allowed for is the capacity of specific commensal organisms to modify the peripheral immune system. For example, monocolonization of germ-free mice with Bacteroides fragilis has been found to provide a sufficient stimulus in the early development of the gut-associated lymphoid tissue (GALT) to allow for normal organogenesis in the spleen and thymus, and development of a balanced immune system.4 Germ-free animals have been successfully colonized with a single commensal population or with distinct combinations of bacterial species. An alternative to germ-free animals is the use of selective combinations of antibiotics to obtain the depletion of certain commensal organisms.11 This approach allows for the comparison of animals subjected to antibiotic treatments when the immune system is fully developed and competent with untreated intact animals.

 

In this review, we discuss the effect that the gut microbiome has in the development and control of experimental neurological disorders and speculate about potential implications in human disease. Moreover, we explore how selective alterations of the microbiome may lead to a new therapeutic paradigm to treat central nervous system (CNS) diseases, in particular human multiple sclerosis (MS).

 

Gut Microbiome Involvement in Nervous System Diseases

 

Although the association between CNS infection and neurological diseases has been extensively studied,41 much less is known about the impact of gut-derived microbes and CNS pathologies. There is no epidemiological evidence to connect the microbiome with the control of CNS disease. However, the metagenomic approaches supported by the NIH Human Microbiome Project should assist in the resolution of this discrepancy. The regulatory cells induced and harbored within the gut as well as the mature APC could traffic from the GALT to other peripheral lymphoid sites including the CNS. Inhibition of migration and trafficking of inflammatory cells from the periphery into the CNS is the presumptive mechanism of action for several current U.S. Food and Drug Administration (FDA)-approved treatments for MS. Some of the FDA-approved and yet-to-be approved oral treatment for MS include natalizumab, fingolimod, IFNβ, and liquinimod. The effect of these trafficking and migration inhibiting therapies on the induction of Tregs within the GALT has yet to be determined. The potential role of gut commensal bacteria in the initiation and maintenance of this regulatory population is of interest to both understanding disease mechanisms and perhaps as a novel therapeutic paradigm. We have recently shown that CD103+ DCs accumulate in the mesenteric and cervical LN of EAE mice after oral treatment with PSA purified from B. fragilis.40 This accumulation was not observed in mice treated with PSA but not subjected to EAE induction. In the context of CNS inflammation, these cells could migrate from the mesenteric LN to the CNS or the associated lymphoid tissues, such as the cervical LN. These DC may possibly enter the brain parenchyma.42, 43 The accumulation of DCs during CNS inflammation has been documented and suggests that these accumulating DCs may be derived from elsewhere, including the bone marrow, rather than differentiated from CNS-resident cell precursors.44, 45 Regulatory cell populations induced within the gut could cross the blood-brain barrier and within the CNS be reactivated by the appropriate resident or peripheral APC, including B cells.

Autism and Behavioral Disorders

 

As recently reviewed by Sekirov and colleagues,6 changes in the microbiome have been associated with autism. The use of antibiotics could alter the gut composition, promoting the survival and growth of specific populations, such as Clostridium tetani.46 It has been shown that a significantly higher number of Clostridium spp. are present in the fecal samples of autistic children when compared to healthy individuals. Disease onset, relapses, and exacerbation of the symptoms have been linked to antibiotic treatments.47, 48 It was hypothesized that the mechanisms by which commensal flora might be triggering autism could be associated with the toxin production of overgrown Clostridium populations with neurological effect. Changes in the gut microbiome have been also associated to major depressive disorder (MDD). MDD patients show enhanced blood levels of proinflammatory TNF-α and IL-6 cytokines49 and metabolic disorders associated to the gut. Moreover, MDD-related stress could be related to the reduced relative percentages of specific commensal populations such as Lactobacilli and Bifidobacterium.

Guillain-Barré Syndrome

 

Campylobacter jejuni is a Gram-negative bacterium that naturally inhabits the intestinal tract of poultry. In humans, consumption of food contaminated with C. jejuni is the major cause of enteritis, characterized by diarrhea, abdominal pain, fever, and malaise. Preinfection with C. jejuni has been associated with different chronic conditions such as reactive arthritis,50 carditis,51 appendicitis,52 as well as Guillain-Barré syndrome (GBS).50, 53 Campylobacter-induced GBS is an autoimmune disorder of the peripheral nervous system triggered by the antibodies produced to combat the infection. GBS patients develop loss of reflexes, and weakness of the limbs and respiratory muscles. As reviewed by Tam and colleagues,54 preceding infection by other gut-associated pathogens such as Haemophilus pneumoniae, Mycoplasma pneumoniae, influenza, and Epstein-Barr virus has been associated with GBS. Pathogen-induced antibodies cross-react with neural surface antigens in a “molecular mimicry” process, causing neuronal damage that leads to acute flaccid paralysis. The mechanism by which preinfection by C. jejuni or other gut-associated pathogen could induce GBS may differ from the proposed effect of the microbiome in the regulation of the inflammatory CNS diseases. Despite the immunological differences, gut-pathogen–associated GBS illustrates how gut-residing microorganisms can lead to autoimmune diseases, not only associated with GALT, as in Crohn's diseases, but also in the nervous system.

CNS Demyelinating Diseases

 

As discussed previously, there is no epidemiological evidence to date suggesting that the microbiome could be related to the control of CNS demyelinating diseases. As clinicians it is well appreciated that there are a number of purported treatments for MS including diet (eg, Swank diet and others such as probiotics) that have never been scientifically scrutinized but could be related to shifts in bacterial strain balance in the microbiome.55 Different experimental approaches have clearly demonstrated that modifications of the gut commensal populations alter the outcome of demyelinating diseases in murine EAE, the generally accepted experimental model for human MS. This experimental MS model has allowed for the generation of various immunomodulatory therapeutics currently utilized in relapsing MS. However, EAE is not a spontaneous disease and the immunological and pathological manifestations may differ significantly from human disease.

 

Oral immunization with an attenuated Salmonella Typhimurium expressing the colonization factor antigen I (CFA/I) fimbriae of enterotoxigenic E. coli conferred prophylactic56 and therapeutic57, 58 protection against EAE. IL-10 and/or TGF-β–producing Tregs elicited after oral immunization with foreign antigens suggest a potentially critical association between the GALT and the CNS. The role of neuropeptides in the induction of Treg-inducer DCs was proposed when vasoactive intestinal peptide (VIP) was reported to induce human IL-10–producing DCs that enhanced the conversion of effector CD4+ T cells and CD8+ T cells into Tr1 and CD8+CD28−CTLA4+ Tregs.59 VIP was able to induce FoxP3+ Tregs protective against collagen-induced rheumatoid arthritis in mice.60 Moreover, therapeutic treatment with VIP reduced CNS inflammation and diminished EAE severity.61

 

The modification of the bacterial populations of the gut has been demonstrated to alter the clinical outcome of EAE in mice.62 Oral treatment of mice with antibiotics reduced EAE severity by diminishing proinflammatory responses and enhancing FoxP3+ Tregs that accumulated in mesenteric and cervical LN. FoxP3+ Tregs obtained from the cervical LN of mice treated with antibiotics produced elevated IL-10 levels and conferred protection against EAE after adoptive transfer. The implication of natural killer T (NKT) cells in the protective role provided by oral antibiotics in the EAE model has been studied.63 In the report by Yokote et al.,63 oral treatment of mice with a combination of kanamycin, colistin, and vancomycin protected against EAE. Reduced IL-17 responses were observed in mesenteric LN and LPs that appeared to be mediated by the regulatory effect of invariant NKT (iNKT) cells. EAE protection by oral treatment with antibiotics was abrogated in iNKT cell–deficient mice. We have shown that oral antibiotic treatment can render EAE susceptible strains of mice (C57BL/6 and SJL) resistant to disease induction and persistence. Resistance to disease was associated with a significant accumulation of protective FoxP3+ Tregs in the cervical LN.62 When antibiotic treated mice were depleted of CD25+ T cells, a significant increase in the disease severity was observed. However, the disease severity was significantly less when compared to CD25+-depleted EAE mice not treated with antibiotics, suggesting that other regulatory cell populations such as iNKT cells may be important in protection against disease.63 We further demonstrated that alteration of the gut microbiota enhances a population of IL-10–producing CD19+ B cells that express high levels of CD1d,64 a regulatory cell population currently under extensive evaluation in the context of EAE.65–67 Adoptive transfer of IL-10–producing CD19+ B cells of mice treated with oral antibiotics into naive recipient mice significantly reduced the severity of EAE. This protection was associated with a shift of the cytokine patterns favoring Th2-type polarization vs proinflammatory Th1/Th17.

 

Different Lactobacillus and Bifidobacterium strains have been used to study the effect of orally-administered probiotics in the control of EAE. In a recent study by Lavasani and colleagues,55 EAE-induced C57BL/6 mice were treated orally with several stains of Lactobacillus. Prophylactic treatment with L. paracasei and L. plantarum strains abrogated EAE clinical symptoms and proinflammatory responses.55 Of more important clinical interest, the combination of 3 different strains of L. plantarum reduced the severity of established EAE. The EAE protection by the probiotics was associated to the induction of FoxP3+ Tregs, and was found to be an IL-10–dependent mechanism. Other commensal bacteria have been associated with EAE clinical severity in rodents.68 Interestingly, the same authors reported that specific probiotic bacteria could in fact reverse and enhance EAE severity.69 Other commensals or their products have been shown to contribute to disease severity; it was recently described that administration of phosphorylated dihydroceramides, lipids derived from the oral commensal Porphyromonas gingivalis enhanced EAE in a TLR2-dependent mechanism promoted by IL-6–producing DCs that reduced FoxP3+ Treg frequencies.70 Moreover, as noted earlier, when EAE disease-resistant germ-free mice are monocolonized with SFB, disease susceptibility is restored (Fig   B).24 In accordance with these results, it appears that specific commensal bacteria can be involved in either the induction or enhancement of autoimmune conditions and conversely protect against disease, thus leading us to a major new paradigm in our understanding of autoimmune disease pathogenesis. These results add further credence to the role of commensal bacteria in maintaining the fine balance of immune homeostasis and the regulation of autoimmune processes in the periphery.

thumbnail image

 

Figure  . CD4+ T cells induced by gut commensal bacteria can exacerbate or reduce the severity of experimental CNS demyelinating disease. (A) Gut microbes are sampled by professional antigen-presenting cells (1) from the intestinal lumen or in the Peyer's Patches. After the recognition and the process, APCs present the antigen to naive T cells in the lamina propria (2). Activated T cells migrate to the mesenteric lymph nodes (LNs), where activation can also occur (3). Activated T cells become traffic through the blood stream and disseminate through distal lymph nodes and can migrate back to the lamina propria (4). (B) Gut commensal Bacteroides fragilis and segmented filamentous bacteria (SFB) mediate the T cell differentiation into proinflammatory Th17 or anti-inflammatory Tregs. PSA produced by B. fragilis is recognized by tolerogenic APCs in a TLR2-dependent manner, inducing the differentiation of naive T cells into IL-10–producing FoxP3+ Tregs. In contrast, absence of PSA in B. fragilis and the murine commensal SFB promote the differentiation of T cells into proinflammatory Th17 cells through the activation of inflammatory APCs. Gut colonization with SFB enhances the severity of EAE in germ-free mice24 through increased Th17 cell responses. Reconstitution of mice previously treated with antibiotics with PSA-deficient (5PSA) B. fragilis restores EAE severity that was reduced upon gut microbiota reduction.67 In contrast, reconstitution of mice treated with antibiotics with PSA-producing B. fragilis maintains protection to the disease after treatment with antibiotics.67 Moreover, oral treatment with a highly purified preparation of PSA reduces EAE severity in mice.40 Treatment with other commensal bacteria (Lactobacillus spp.) can also protect against disease.50

Inducing Protection Against CNS Demyelination by Human Gut Commensal Bacteria

 

The studies of Mazmanian and Kasper28 and Dasgupta and Kasper71 have provided extensive evidence regarding the induction of regulatory responses induced by PSA. We compared reconstitution of mice with wild-type (WT) PSA-producing vs PSA-deficient B. fragilis in EAE mice that were treated with disease-protecting oral antibiotics (see Fig  B).72 Recolonization of antibiotic-treated mice with PSA-deficient B. fragilis restored the susceptibility to disease development, whereas mice recolonized with the intact strain of B. fragilis remained protected against EAE. Recolonization of antibiotic-treated EAE mice with PSA-deficient B. fragilis induced the production of IL-17 and IL-6. In contrast, IL-10 production was enhanced following recolonization with the intact B. fragilis but not PSA-deficient B. fragilis. The presence or absence of this single polysaccharide expressed on the surface of B. fragilis could determine protective or pathogenic outcomes in EAE. Reconstitution with either WT or PSA-deficient B. fragilis enhanced the frequency of FoxP3+ Tregs isolated from the cervical LN as soon as 3 days after bacterial recolonization. Susceptibility to EAE was observed following CD25+ cell depletion in antibiotic-treated mice as well as in mice recolonized with WT B. fragilis. Interestingly, FoxP3+ Treg conversion by CD103+ DCs purified from mice recolonized with PSA-deficient B. fragilis was significantly diminished when compared to DCs from PSA-producing B. fragilis. Moreover, FoxP3−CD4+ T cells obtained from the cervical LN of PSA-producing B. fragilis recolonized mice were more efficiently converted into FoxP3+ Tregs than a phenotypically similar population isolated from PSA-deficient B. fragilis recolonized mice. In vitro converted FoxP3+ Tregs derived from mice recolonized with PSA-producing B. fragilis produced enhanced levels of IL-10 and induced EAE protection following adoptive transfer. In vitro FoxP3+ cells converted from cells of PSA-deficient B. fragilis recolonized mice did not produce IL-10 and failed to protect against the disease. These results suggest that despite phenotypic similarities, deficiency of PSA expression by B. fragilis can influence the functional role of immunomodulatory dendritic cells and FoxP3+ Tregs induced by B. fragilis.

 

The capacity of this single polysaccharide antigen to influence the outcome of CNS demyelination was confirmed in studies using a highly purified preparation of PSA. Oral administration of PSA conferred both prophylactic and therapeutic protection against EAE in 2 genetically distinct strains of mice (SJL/J and C57BL/6) that were not subjected to previous treatment with antibiotics.40 Protection against disease was associated with a significant accumulation of CD103+ DCs and FoxP3+ Tregs in the cervical LN. CD103+ DCs induced the conversion of naive CD4+ T cells into IL-10–producing FoxP3+ Tregs. Disease resistance was completely abrogated in IL-10–deficient mice. The findings obtained in these studies provide direct evidence that effective immunization by a single polysaccharide purified from a human commensal bacterium is plausible, and establishes a potentially important novel therapeutic approach to treating CNS demyelinating disease.

Conclusions: Altering the Gut Microbiome as a New Paradigm to Treat CNS Disease

 

The clinical implications of recently published experimental approaches suggest an important and novel role for commensal bacteria in regulating peripheral immune homeostasis. The capacity of specific bacterial antigens such as PSA derived from the commensal B. fragilis to mediate trafficking and migration of a population of gut-derived APCs to CNS-associated lymphoid tissue suggests an important biologic interaction between the gut mucosal tissue and the brain and spinal cord. Moreover, these observations would suggest that the reservoir for the effector and regulatory populations of immune cells involved in the pathogenesis of human MS may lie within the gut-associated lymphoid tissue. Although epidemiological studies in man connecting the gut commensal bacteria with CNS demyelinating disease have not yet been reported, perhaps due to the complexity of the human microbiome, repopulation of gut microbiota or even oral immunization with commensal bacterial antigens may be a reasonable pathway by which to control disease pathogenesis. Selective alteration of the gut microbiome or treatment with immune-modifying probiotic therapeutics would provide a safe and important alternative with nominal side effects for the treatment of patients with MS or perhaps other neurological diseases. This approach would reduce the complications associated with current FDA-approved platform therapies as well as the immune ablative treatments that are now in the development pipeline for treating MS.

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So this isn't just about the Gut but it is still really interesting and touches on some of my other pet interests like inflammation

 

Trends in Neurosciences

 

Volume 36, Issue 11, November 2013, Pages 674–684

Cover image

Review

Towards a ‘systems’-level understanding of the nervous system and its disorders

 

It is becoming clear that nervous system development and adult functioning are highly coupled with other physiological systems. Accordingly, neurological and psychiatric disorders are increasingly being associated with a range of systemic comorbidities including, most prominently, impairments in immunological and bioenergetic parameters as well as in the gut microbiome. Here, we discuss various aspects of the dynamic crosstalk between these systems that underlies nervous system development, homeostasis, and plasticity. We believe a better definition of this underappreciated systems physiology will yield important insights into how nervous system diseases with systemic comorbidities arise and potentially identify novel diagnostic and therapeutic strategies.

 

Why focus on systems physiology?

 

Long-standing clinical observations and recent epidemiological and scientific studies suggest that many diseases classically thought to be nervous system-specific disorders actually have more complex phenotypes, including manifestations in other physiological systems and at brain–systemic interfaces (Box 1), profound in some cases and more subtle in others. Most, if not all, major neurological and psychiatric disorders display immunological abnormalities such as high levels of inflammation and aberrant profiles of innate and adaptive immune system activity 1 and 2. Many nervous system disorders also exhibit failure to maintain energy homeostasis, occurring not only at cellular and subcellular levels (i.e., mitochondrial dysfunction) but also in select brain regions and at an organismal level with overt signs of metabolic deregulation (i.e., alterations in body weight and composition and in glucose, amino acid, and lipid homeostasis) 3, 4 and 5. A spectrum of other, less well-characterized impairments in additional organ systems are also emerging as features of disorders classically considered nervous system specific. These include, as one exceptionally interesting example, manifestations vis-à-vis the gut microbiome (see Glossary) that are likely to be as pervasive and important as – and intimately linked to – immunological and bioenergetic abnormalities 6, 7 and 8. Considering the significance of these observations calls for taking a whole-organism or ‘systems’-level view.

 

This contemporary, constructionist approach – centered on understanding the dynamics of the whole organism in a more integrated manner – will not only provide novel insights into how neurological and psychiatric disease states and their comorbidities arise; it will also help to predict and explain the range of effects associated with modulating molecular targets that are shared by the nervous system and these other systems and will serve as the basis for developing innovative diagnostic and treatment modalities that complement and enhance existing approaches.

Neuroimmune interactions

 

The central nervous system (CNS) is subject to active immune surveillance throughout life by the innate and adaptive immune systems. There is increasing evidence that this form of immune surveillance is linked not only to pathology but is also important for promoting normal brain development and adult activity.

CNS development, homeostasis, and plasticity

 

One key mechanism responsible for this crosstalk is that the nervous system and immune system express and secrete common sets of molecules that are implicated in a diverse range of system-specific and interrelated functions (Table 1). These include factors with roles traditionally ascribed to the immune system or to the nervous system as well as novel mediators with emerging and conjoint immunological and neural roles. Indeed, many so-called immune molecules are found in specific regional, cellular, and subcellular distributions in the CNS and their expression levels are modulated by neural activity. These factors can have roles in regulating neural development and synaptic function and morphology 9 and 10. For example, components of the complement system, immunoglobulin superfamily proteins (e.g., major histocompatibility complex proteins), and cytokines and chemokines have well-characterized immunological roles, including the mediation of cell migration, antigen presentation, cell–cell interactions, and signaling. It is now clear that many of these molecules also modulate CNS development through effects on cellular migration, axonal and dendritic targeting, and synapse formation and its adult activity by regulating synaptic plasticity and de novo neurogenesis. These factors are also increasingly being linked to susceptibility to and the clinical phenotypes of neurological disorders

 

Likewise, neurotransmitters, neuropeptides, and their receptors, canonically thought to subserve neural signaling and associated functions, have roles in the immune system. For example, T cells express neurotransmitter receptors and can be activated or suppressed in a context-dependent manner by various neurotransmitters. These factors do not simply mediate neural-to-immune signaling, because T cells produce many neurotransmitters and can be found in immune organs such as the thymus.

 

In addition, the nervous system affects the composition, mobilization, and activity of the immune system. For example, the sympathetic division of the autonomic nervous system (ANS) mediates the activity and numbers of distinct subsets of T regulatory (Treg) cells that are involved in orchestrating central and peripheral tolerance, via a transforming growth factor-β-dependent mechanism [16]. Further, the ANS modulates hematopoietic stem and progenitor cell (HSPC) proliferation, mobilization, peripheral migration, and differentiation into lymphoid and myeloid cellular elements in a circadian fashion through the actions of adrenergic signaling 17, 18, 19 and 20. Disease states that perturb the ANS, such as diabetes mellitus, which leads to abnormalities in sympathetic nerve termini, impair HSPC mobilization [21]. In addition, neural circuits regulate cytokine production in health and disease. For example, the ANS controls innate immunity through innervation of the spleen, regulation of T cell mediated production of acetylcholine, and modulation of the ‘inflammatory reflex’ associated with proinflammatory cytokine production [22]. Correspondingly, post-stroke systemic immunosuppression is, at least in part, mediated by noradrenergic signaling acting on hepatic invariant natural killer T cells [23].

CNS disease and clinical implications

 

While immune surveillance plays a role in maintaining neural cell identity, homeostasis, connectivity, and plasticity, diverse CNS pathologies are associated with abnormalities in immune surveillance [24]. Specifically, the onset and progression of CNS disease states is often characterized by deregulation of systemic and CNS-specific T and B cells and microglia, CNS-resident mononuclear phagocytes, and associated inflammatory cascades 25, 26, 27, 28, 29 and 30. One particularly intriguing study recently highlighted the importance of proper microglial functioning in the brain. It reported that, in a mouse model of Rett syndrome, engraftment of brain parenchyma from wild type bone marrow-derived microglia or targeted expression of wild type methyl-CpG binding protein 2 (Mecp2) in myeloid cells ameliorates disease symptoms and pathology [31]. These effects are dependent on microglial phagocytic activity. Similarly, missense mutations in the triggering receptor expressed on myeloid cells 2 gene, which encodes an anti-inflammatory signaling protein expressed on dendritic cells, macrophages, and microglia, impart significant risk for developing Alzheimer's disease (AD) 32 and 33. In some instances, these immune responses can be protective [34]. For example, after injury, monocyte-derived macrophages exhibit neuroprotective effects in the retina and spinal cord 35 and 36, and T cells secrete factors that promote neuronal survival by modulating astrocyte functions 37 and 38. Alternatively, these impairments can be mechanistically linked to known pathogenic factors. For example, in Huntington's disease (HD), the mutant huntingtin protein is known to impair the migration of immune cells [39]. Moreover, there are observations that are notable but whose significance is yet to be determined. For example, Down syndrome (DS) is associated with deregulation of AIRE, which mediates central and peripheral tolerance, and thymic dysplasia 40 and 41.

 

Overall, these observations indicate that, although our understanding of neuroimmune interactions is advancing, it remains incomplete. It is clear that immune surveillance plays a central role in promoting nervous system health. One interesting hypothesis is that, through strategically placed molecules that serve as substrates for neuroimmune crosstalk, the immune system monitors the functional integrity of neural pathways and responds actively to changes in their fidelity. Subtle impairments in these homeostatic processes may even represent sentinel events in preclinical stages of disease, suggesting novel therapeutic windows. Further investigations are necessary to more precisely define these cellular mechanisms (e.g., the roles of microglia) and intracellular communications (e.g., at the stem cell niche) and the corresponding effects of brain aging and disease states on these processes.

The nervous system and energy homeostasis

 

Multiple organs, from the gut microbiome and immune system to the brain, are involved in a highly integrated manner in maintaining energy homeostasis by modulating energy intake, storage, and expenditure. In turn, energy balance and metabolic signals serve as key regulators of the development, programming, and function of these different organ systems. For example, changes in the gut microbiome, such as those associated – perhaps even causally – with obesity, increase the efficiency of harvesting energy from the diet [42]. Conversely, altering dietary fat and sugar content rapidly shifts the composition of the gut microbiome [43]. Likewise, immune system functioning and energy homeostasis are interdependent, as shown by long-standing observations regarding the immunosuppressive effects of malnutrition and the recent emergence of the field of immunometabolism, which is focused on studying crosstalk between these systems [44]. These links include, for example: (i) the confluence of immune and bioenergetic signaling pathways in quiescent and activated immune cells; (ii) the role of metabolic stress responses, such as autophagy, in innate and adaptive immune system activity; (iii) immune surveillance in traditional metabolic tissues; and (iv) immune system activation and inflammation in metabolic diseases. Bidirectional relationships between nervous system processes and energy homeostasis are similarly complex and are now being investigated.

CNS development, homeostasis, and plasticity

 

The brain senses, integrates, and responds to fluxes in energy states throughout life via a range of complementary mechanisms. The central regulation of energy balance is complex and mediated by distributed neural networks such as those in the limbic system and cerebral cortex underpinning reward and motivation, food anticipatory circadian rhythms, and other feeding behaviors 45 and 46. The hypothalamus and brainstem are essential centers for controlling these processes, with various nuclei and subpopulations of neurons having specific roles in regulating feeding behavior and satiety, lipid and glucose levels, body weight, and related metabolic parameters 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60. The best characterized is the arcuate nucleus (ARC) of the hypothalamus, which contains subpopulations of anorexigenic pro-opiomelanocortin (POMC)-expressing neurons and orexigenic agouti-related peptide (AgRP)- and neuropeptide Y (NPY)-expressing neurons.

 

Correspondingly, nutrient levels, gut- and adipose-derived peptides and hormones, and sundry metabolic signaling pathways influence CNS development, homeostasis, and plasticity [61]. Most notably, hypothalamic development and functioning are regulated by factors that mediate feeding behavior and satiety, energy balance, and metabolism. During developmentally critical periods, for example, the adipocyte-derived anorexigenic hormone leptin promotes the programming of metabolism and establishment of feeding circuitry through activation of POMC and AgRP/NPY cell type-specific developmental signaling pathways 62, 63, 64 and 65. Leptin and the gut-derived orexigenic hormone, ghrelin also promote synaptic plasticity in the ARC of adult mice 66 and 67. Interestingly, one of the potential mechanisms by which hypothalamic energy balance circuits undergo remodeling during adult life is through ongoing neurogenesis [68] and it has been suggested that leptin and ghrelin can modulate neurogenesis in these and other contexts 69, 70 and 71. In addition, these and other factors involved in mediating feeding behavior and satiety, energy homeostasis, and metabolism are implicated in regulating learning and memory, reward and motivation, anxiety, and depression via extrahypothalamic actions, underscoring the highly integrated but widely distributed effects of energy balance and nutrition on the brain 72 and 73.

 

Furthermore, like neuroimmune interactions, a common set of signaling pathways affects energy homeostasis within the nervous system and in other organ systems (Table 2). These common molecules play diverse roles in nutrient sensing, lipid and glucose homeostasis, and mitochondrial biogenesis and activity and evidence suggests that they simultaneously regulate aspects of neural development, synaptic plasticity, and stress responses 4, 74, 75, 76 and 77.

 

CNS disease and clinical implications

 

Unsurprisingly, hypothalamic abnormalities such as inflammation, autophagy, neuronal injury, and aberrant circuitry are associated with disorders of energy homeostasis such as obesity. For example, a recent study reported that, within 1–3 days of consuming a high-fat diet (HFD), rodents exhibit increased expression levels of inflammation-related genes, reactive gliosis, neuronal injury, and autophagy in the ARC [78]. These very early changes do not occur peripherally in liver and adipose tissues and are transient. With chronic consumption of a HFD and the development of obesity, however, these abnormalities recur both in the ARC and peripherally. This time course raises the possibility that the early hypothalamic changes lead to impairments in the regulation of energy balance and ultimately to obesity. A related study found that obese mice have defects in the normal profiles of dynamic cellular remodeling within the ARC [71]. The authors observed that hypothalamic neuronal turnover is suppressed in a HFD-induced obesity model, with a decrease in proliferating NPCs and neurogenesis and an increase in apoptosis of newborn neurons. Similarly, they found that levels of hypothalamic neurogenesis are decreased in a leptin-deficiency obesity model (ob/ob mice) as a result of a depleted pool of hypothalamic neural stem cells (NSCs). Another report suggests that hypothalamic neurogenesis acts as a compensatory mechanism for maintaining energy balance in response to environmental and physiologic insults (and in the context of neurodegeneration) [79]. In a related study with implications for developing treatments, it was found that transplanting dissociated developmentally appropriate leptin-responsive hypothalamic cells into analogous regions of a leptin receptor-deficient obesity model (db/db mice) leads to their functional integration into the hypothalamic circuitry and mitigates disease processes [80]. Specifically, the transplanted cells survive and differentiate into multiple hypothalamic neuronal subtypes with appropriate electrophysiological and ultrastructural features and responsiveness to leptin, glucose, and insulin, leading to a decrease in adiposity. Leptin also modulates T cell subsets and promotes a proinflammatory milieu, and ob/ob mice are resistant to the induction of experimental autoimmune encephalomyelitis (EAE) 81 and 82. These findings link energy homeostasis signals with immune system activity and CNS disease.

 

Deregulation of bioenergetic signaling pathways is also implicated in the pathogenesis of CNS disorders, particularly those associated with age-related neurodegeneration, such as AD, HD, Parkinson's disease, and amyotrophic lateral sclerosis, all of which exhibit distinctive metabolic phenotypes 83 and 84. These same pathways are often deregulated in metabolic disorders. Thus, an important question to be answered is whether there is crosstalk when metabolic disorders are comorbid with these neurological diseases and, if so, what is its precise nature. Is it protective, detrimental, or somehow more complex over the courses of the different diseases?

 

Importantly, many bioenergetic pathways can be targeted with existing and emerging therapeutic modalities for treating metabolic disorders [85] and it has been suggested that they might also be beneficial in nervous system diseases. In fact, studies suggest that peroxisome proliferation-activated receptor (PPAR) agonists such as bezafibrate and thiazolidinediones (e.g., ciglitazone, pioglitazone, and troglitazone) have neuroprotective effects in neurodegenerative disease models. Similarly, analogs of GLP-1 (exendin-4) and GLP-1 receptor agonists (liraglutide) have neurotrophic and neuroprotective effects. The molecular and cellular mechanisms for the apparent benefits of these agents are currently a matter of debate and include putative effects on microglia, inflammation, mitochondria, and oxidative stress. Nevertheless, these preliminary observations imply that both dietary interventions and FDA approved and emerging drugs for metabolic disorders, such as insulin sensitizers and secretagogues and related agents, can modify CNS disease processes, including potentially during preclinical stages of disease. Clinical trials evaluating these treatments are underway (NCT01280123, NCT00811681, NCT01174810, and NCT01255163).

The brain–gut microbiome axis

 

Microbiota (bacteria, viruses, and fungi) are important mediators of health and disease. Commensal microbial populations are associated with various tissues (gut, skin, and vagina). These communities engage in quorum sensing – intercellular communication among bacteria – and in complex interactions with the host.

 

The dynamic equilibrium that exists between gut microbiota and their associated genomes, host-related factors (age, gender, and pregnancy), and environmental influences (diet) is termed the gut microbiome. Although it is thought to play a primary role in digestion and energy metabolism in the gut lumen, the gut microbiome also modulates development and maturation of the immune system, including effects on both the innate and adaptive immune responses [86]. For example, the gut is colonized after birth with a skin- or vagina-like composition that evolves into a relatively stable community through the induction of tolerance to particular bacteria, mediated by recognition of symbiotic bacterial molecules such as those affecting Toll-like receptor (TLR) signaling, and the generation of bacterial antigen-specific populations of Treg cells [87]. Emerging evidence suggests that the gut microbiome plays a similar instructive role in the CNS, either directly or indirectly through immune regulation, neuroendocrine signaling, and other processes. Indeed, there are efforts under way aimed at elucidating functional interconnections between the gut microbiome and the nervous system, mediated by gut intrinsic and extrinsic mechanisms including the enteric and autonomic nervous systems, the hypothalamic–pituitary–adrenal (HPA) and sympathoadrenal axes, gut-associated lymphoid tissue, immune cells, enteroendocrine cells, neurotransmitters, and gut peptides and hormones 6 and 8.

CNS development, homeostasis, stress, and behavioral responses

 

It is becoming clear that the gut microbiome can modulate CNS development and homeostasis, stress and behavioral responses, and disease processes. Specifically, observations suggest that, during a developmentally critical period, the gut microbiome plays a role in the programming of regional neural gene expression levels, signaling pathways, and behavioral repertoires present later in life 6 and 8. One seminal study demonstrated that adult mice raised in germ-free (GF) conditions exhibit higher levels of motor activity and lower levels of anxiety-like behavior than specific pathogen-free (SPF) mice, which have a normal gut microbiota [88]. This phenotype is associated with increased rates of striatal neurotransmitter turnover and differential expression in various brain regions of genes involved in synaptic plasticity, cAMP signaling, and other pathways. Furthermore, exposing GF mice early in life, but not in adulthood, to microbiota obtained from SPF mice results in a phenotype similar to that of SPF mice. The mechanisms by which these processes are mediated are emerging. A recent study found that male GF animals have increased 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in the hippocampus and higher concentrations of their precursor, tryptophan, in plasma, suggesting that the microbiome impacts hippocampal serotonergic neurotransmission via a humoral mechanism [89]. Another study showed that chronic ingestion of a particular Lactobacillus strain modulates regional expression profiles of GABA receptor subtypes in the brain and associated behavioral phenotypes in adult mice and that these effects are abrogated by vagotomy [90]. These observations imply that the vagus nerve serves as a key mediator of gut-to-brain signaling and, further, that the effects of the gut microbiome are not simply restricted to the developing brain but are also involved in adult brain functions.

CNS disease and clinical implications

 

It is intriguing to speculate that the gut microbiome influences susceptibility to and pathogenesis of CNS diseases, as it does for other organ systems. One study found that the composition of the fecal microbiota is more diverse in autistic children with gastrointestinal symptoms compared with controls [91]. Although these findings are correlative, an important study utilizing a mouse model for multiple sclerosis [92] supports a more causal relationship [93]. It reported that commensal microbiota are necessary for the development of spontaneous relapsing–remitting EAE. Although 80% of mice raised under SPF conditions develop this form of EAE within 3–8 months of age, those raised under GF conditions exhibit impaired differentiation of proinflammatory T helper 17 cells and do not develop EAE. However, exposing GF mice to conventional commensal microbiota leads to the rapid development of EAE. Corresponding studies have demonstrated that strategies aimed at modifying the composition of the gut microbiota can influence the course of EAE (and other disorders), probably mediated by microbiota-induced changes in the milieu of cytokines with pro- and anti-inflammatory effects and the balance between different T cell subsets 7, 94 and 95.

 

The gut microbiome may have an impact on the pathogenesis of a broader range of CNS disorders directly or through indirect effects: (i) on the immune system, given how neuroimmune interactions are responsible for mediating CNS health and disease; (ii) on energy metabolism, given the complex interrelationships that exist between the brain and energy balance; and (iii) on other physiological processes. Conversely, the CNS may exert effects on the gut microbiome through these same interconnections. For example, in the example of autism above, it is a reasonable conjecture that the CNS pathology is responsible for giving rise to the abnormal profile of fecal microbiota by inducing impairments in the activity of the immune system or the gut. Indeed, stress during development and adulthood can alter the composition of gut microbial populations 96 and 97.

 

These findings imply that defining personal enterotypes, interrogating host–gut microbiome interactions, and identifying dysbiosis might yield insights into CNS disease states and have important therapeutic implications [98]. Approaches used to modify gut microbiota, including dietary interventions, pre- and probiotic agents, antibiotics, fecal transplants, and other modalities, might impact neurobiological programming during developmentally critical periods and brain function throughout life. Further, the effects of drugs for neurological and psychiatric diseases, including specific therapeutic responses and side effects, can potentially be mediated by the microbiome. For example, chronic treatment of rats with olanzapine induces changes in the gut microbiome that might influence the weight gain and metabolic dysfunction associated with this atypical antipsychotic agent [99].

Concluding remarks

 

The traditional view is that the brain acts as a central regulator of homeostatic processes. However, this brain–body connection is not unidirectional. Brain development and functioning, along with disease onset and progression, occur within the context of the whole organism. Seminal neural processes are highly responsive to environmental and interoceptive cues, including those derived from circadian pacemakers. Recent studies have started elucidating how these are also mediated, at a mechanistic level, by dynamic crosstalk that occurs between the nervous system and other organ systems. Here, we have highlighted emerging roles for immune surveillance, bioenergetic factors, and the gut microbiome. Further interrelationships between the brain and various other organ systems are also now being recognized. These encompass complex signals propagated across a broad range of local, widely distributed, and specialized organ-specific brain–systemic interfaces through both existing and novel mechanisms (intercellular trafficking of exosomes), representing intriguing areas for future study.

 

Collectively, these evolving insights raise many interesting questions (Box 2). For example, it is known that predispositions to a subset of neurological and psychiatric diseases – as well as metabolic phenotypes, cancers, and other systemic disorders – can be programmed during developmentally critical periods, but these states are difficult to assess functionally because they are subtle or relatively inaccessible or their biological substrates are unknown. Might it be possible to better characterize these preclinical vulnerabilities or frank disease states by interrogating elements of brain–systemic crosstalk, especially because these complex disorders often have manifestations in multiple organ systems? If so, can diagnostic and therapeutic modalities targeting these signals be developed, perhaps as extensions of the ‘systems’ approaches for biomarker discovery and pharmacology that are currently in vogue

[[co...]]

Whew!  I'll take the Reader's Digest version, thanks...something about bugs are good, and the poor mice!

[Guest [jc...]]

I've been drinking kefir with good results, how much do you drink per day and do  you drink Lifeway brand?  Thanks.

[[pr...]]

Whew!  I'll take the Reader's Digest version, thanks...something about bugs are good, and the poor mice!

 

Hahahaha ... :laugh: 

 

Cookie, that was simply hilarious.

[[...]]

Right over my head but I've been drinking kefir all thru taper.  i do lifeway and others.  about 1/2 to 1 cup in my smoothie.  have no idea if it helps cuz don't know what it would be like without it.  my stomach is pretty good right now.  finally at lower dose of benzo gettin appetite back.

[[Sm...]]

So yea sorry about all the papers with zero run down

I'm busy at the moment but when I can I'll try to cut them down and highlight in bold what matters

 

You are right though: upshot is lots of good bacteria and healthy gut = better functioning nervous system and brain

[[kl...]]

Doesn't Kefir have alcohol in it? I thought I read it can have up to 3% abv.

[Guest [jc...]]

No alcohol in it.

http://en.wikipedia.org/wiki/Kefir

[Guest [jc...]]

The slow-acting yeasts, late in the fermentation process, break lactose down into ethanol and carbon dioxide: depending on the process, ethanol concentration can be as high as 1-2% (achieved by small-scale dairies early in the 20th century), with the kefir having a bubbly appearance and carbonated taste: most modern processes, which use shorter fermentation times, result in much lower ethanol concentrations of 0.2-0.3%.

[[On...]]

Smiff, these studies are truly awesome. Thank you so much for posting them. It has taken some time for me to carefully 'digest' them, lol.

 

For the record, a year ago I was down to 77 pounds with horrendous GI pain and dysfunction. I felt like I had scar tissue throughout the gut, loads of adhesions, and swelling to the point of fearing I would explode.

 

I credit lactobaccilus rhamnosis and a probiotic mix called "lactate free" made by Custom Probiotics for my healing. Also, avoiding msg and foods very high in natural glutamates helped greatly. 

 

One of your posted studies stated this: "lactobaccilus rhamnosus and bifida dentium are capable of metabolizing glutamate to produce gamma-amino butyric acid (GABA), the major inhibitory neurotransmitter in the human CNS (26). Increased GI-tract GABA appears to correlate with increased CNS GABA levels, but the systemic pathways that contribute to this gut–brain linkage require additional study."

 

What a great find!!!  So this must be how it all came together for me?  Thanks for the info and taking the time to post it.  Invaluable!

 

~OneLove 

 

 

 

 

[[Sm...]]

I'm glad it was of some help One!

 

And thanks for the tips re the probiotics that worked for you. I'm currently working out what probiotics sit with me.

 

It is clear that some strains cause issues particularly to sensitive people like ourselves.

 

L.rhamnosis comes in a single strain formula at Custom probiotics so I'm definitely getting that. I was wondering about the lactate free. It all gets rather complicated - some cause lactic acid, others cause histamine reactions, some lower histamine reactions.

It is a mine field. I think if one has had reactions to probiotics before but, like me, still believe there is something valuable here the safest thing is probably to try to attempt single strains and see what you react to.

[[Sm...]]

I left it too late to modify previous posts. I might see if mods can help me out since via my being slack I was breaking copyright

Anyhow, when I get moments I'll repost some of what I posted with un-necessary bits cut out and important bits bolded

 

So I recently started drinking Kefir and it was relaxation in a glass so I look into it and those lil microbes help us out my friends..

 

Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve

 

    Javier A. Bravoa,1,

    Paul Forsytheb,c,1,

    Marianne V. Chewb,

    Emily Escaravageb,

    Hélène M. Savignaca,d,

    Timothy G. Dinana,e,

    John Bienenstockb,f,2, and

    John F. Cryana,d,g,2

Abstract

 

There is increasing, but largely indirect, evidence pointing to an effect of commensal gut microbiota on the central nervous system (CNS). However, it is unknown whether lactic acid bacteria such as Lactobacillus rhamnosus could have a direct effect on neurotransmitter receptors in the CNS in normal, healthy animals. GABA is the main CNS inhibitory neurotransmitter and is significantly involved in regulating many physiological and psychological processes. Alterations in central GABA receptor expression are implicated in the pathogenesis of anxiety and depression, which are highly comorbid with functional bowel disorders. In this work, we show that chronic treatment with L. rhamnosus (JB-1) induced region-dependent alterations in GABAB1b mRNA in the brain with increases in cortical regions (cingulate and prelimbic) and concomitant reductions in expression in the hippocampus, amygdala, and locus coeruleus, in comparison with control-fed mice. In addition, L. rhamnosus (JB-1) reduced GABAAα2 mRNA expression in the prefrontal cortex and amygdala, but increased GABAAα2 in the hippocampus. Importantly, L. rhamnosus (JB-1) reduced stress-induced corticosterone and anxiety- and depression-related behavior. Moreover, the neurochemical and behavioral effects were not found in vagotomized mice, identifying the vagus as a major modulatory constitutive communication pathway between the bacteria exposed to the gut and the brain. Together, these findings highlight the important role of bacteria in the bidirectional communication of the gut–brain axis and suggest that certain organisms may prove to be useful therapeutic adjuncts in stress-related disorders such as anxiety and depression.

 

    brain–gut axis

    irritable bowel syndrome

    probiotic

    fear conditioning

    cognition

 

There is increasing evidence suggesting an interaction between the intestinal microbiota, the gut, and the central nervous system (CNS) in what is recognized as the microbiome–gut–brain axis (1–4). Studies in rodents have implicated dysregulation of this axis in functional bowel disorders, including irritable bowel syndrome. Indeed, visceral perception in rodents can be affected by alterations in gut microbiota (5). Moreover, it has been shown that the absence and/or modification of the gut microflora in mice affects the hypothalamic–pituitary–adrenal (HPA) axis response to stress (6, 7) and anxiety behavior (8, 9), which is important given the high comorbidity between functional gastrointestinal disorders and stress-related psychiatric disorders, such as anxiety and depression (10). In addition, pathogenic bacteria in rodents can induce anxiety-like behaviors, which are mediated via vagal afferents (9, 11).

 

GABA is the main inhibitory neurotransmitter of the CNS, the effects of which are mediated through two major classes of receptors—the ionotropic GABAA receptors, which exist as a number of subtypes formed by the coassembly of different subunits (α, β, and γ subunits; ref. 12), and the GABAB receptors, which are G protein coupled and consist of a heterodimer made up of two subunits (GABAB1 and GABAB2), both of which are necessary for GABAB receptor functionality (13). These receptors are important pharmacological targets for clinically relevant antianxiety agents (e.g., benzodiazepines acting on GABAA receptors), and alterations in the GABAergic system have important roles in the development of stress-related psychiatric conditions.

 

Probiotic bacteria are living organisms that can inhabit the gut and contribute to the health of the host (14).

 

 

Results

Behavioral Effects of L. rhamnosus (JB-1) Administration.

 

A battery of behavioral tests relevant to anxiety and depression was carried out. The stress-induced hyperthermia (SIH) and elevated plus maze (EPM) tests are widely used for assessing functional consequences of alterations in GABA neurotransmission (22, 23). Chronic administration of L. rhamnosus (JB-1) produced a nonsignificant reduction in SIH (t = 1.567, df = 34; P = 0.1263; Fig. 1A). On the EPM, animals treated with L. rhamnosus (JB-1) had a larger number of entries to the open arms than broth-fed animals, suggesting anxiolytic effects (open arm entry defined as all four paws entering the arms of the EPM apparatus) (t = 4.662, df = 34; P < 0.001; Fig. 1A). This effect is also reflected in the percentage of time spent in the open arms, although this observation did not reach statistical significance [broth v. L. rhamnosus (JB-1): 25.28 ± 6.67% vs. 38.36 ± 7.99%; t = 1.267, df = 34; P = 0.2146].

 

 

Discussion

 

These data demonstrate specific, previously undescribed neurochemical changes induced by modulation of intestinal microbiota using a potential probiotic [L. rhamnosus (JB-1)] in normal, healthy animals (Table S1). Moreover, we show that L. rhamnosus (JB-1) can have a direct effect upon associated behavioral and physiological responses in a manner that is dependent on the vagus nerve. L. rhamnosus (JB-1) consistently modulated GABAAα2, GABAAα1, and GABAB1b receptor mRNA expression—receptors implicated in anxiety behavior—in a regional-dependent manner.

 

Furthermore, in this study we observed that L. rhamnosus (JB-1) administration reduces the stress-induced elevation in corticosterone, suggesting that the impact of the Lactobacillus on the CNS has an important effect at a physiological level. Alterations in the HPA axis have been linked to the development of mood disorders and have been shown to affect the composition of the microbiota in rodents (26). Moreover, it has been shown that alterations in HPA axis modulation can be reversed by treatment with Lactobacillus and Bifidobacterium (28, 29). However, caution is needed when extrapolating from single timepoint neuroendocrine studies (30). ... these data clearly indicate that in the bidirectional communication between the brain and the gut, the HPA axis is a key component that can be affected by changes in the  microbiota.

 

Accumulating evidence suggests that metabotropic GABA receptors are crucial for the maintenance of normal behavior. Indeed, genetic and pharmacological studies have implicated that GABAB receptors play a key role in mood and anxiety disorders (13). In the present study, the mRNA of the GABAB1b subunit, the main isoform of the GABAB1 receptor in the adult brain (13), was increased in the prefrontal cortex of L. rhamnosus (JB-1)-fed animals. Studies have shown that animal models of depression have reductions in GABAB receptor expression in frontal cortices (13). Thus, it is tempting to speculate that the changes induced by the Lactobacillus might provide an advantage toward stressful situations in comparison with broth-fed control animals. This difference is consistent with behavioral and neuroendocrine responses seen. In the other analyzed areas (amygdala, hippocampus, and LC), L. rhamnosus (JB-1) administration reduced the expression of GABAB1b mRNA, which is consistent with the antidepressant-like effect of GABAB receptor antagonists (31). L. rhamnosus (JB-1)-fed animals showed an enhanced memory to an aversive cue and context in comparison with broth-fed mice—an observation that implies changes at the level of the amygdala and hippocampus (24). These findings are consistent with data generated from GABAB1b-deficient animals, highlighting an important role for this subunit in the development of cognitive processes, including those relevant to fear (32, 33). In line with these results, it has recently been shown that treatment with certain bacteria improves memory function in infected mice (34) as well as cognitive abilities in humans (35). However, unlike GABAB1b knockout mice (36), L. rhamnosus (JB-1)-fed mice are able to extinguish learned fear, behaviors dependent on the PrL cortex (37), which may reflect the actual up-regulation of this receptor subunit in this brain region.

 

The amygdala is crucial for manifestation of fear and anxiety responses and for modulation of the affective components of visceral perception. Given increased levels of GABAAα2 mRNA in the amygdala are found in stressed animals (38), the reductions in GABA receptor subunits induced by the Lactobacillus suggest that this bacteria could have promoted an adaptive advantage over broth-fed animals in terms of interaction with stressful situations. The amygdala is also necessary for conditioning of a relatively simple stimulus or cue (conditioned stimulus) and the context in which the unconditioned stimulus is delivered (24, 25). Component analysis revealed that animals fed with L. rhamnosus (JB-1) had significantly higher freezing behaviors during the last cues and context in the second day (recall phase) of testing than broth-fed animals—an observation that is in line with previous reports on BALB/c mice (24). Interestingly, it has been shown that alterations in the expression of GABAA receptor subunits affect fear-related behaviors, as genetic ablation of the GABAAα1 subunit in mice enhances freezing behavior (39). It is worth noting that this increased emotional learning may also be interpreted as increased anxiety behavior; this interpretation suggests that L. rhamnosus (JB-1) has differential effects on conditioned compared with unconditioned aspects of anxiety.

 

GABAergic neurotransmission in the hippocampus has been related to the modulation of behavior and memory processes (40). Additionally, this structure is required for contextual conditioning, and evidence suggest that inactivation of hippocampal GABAB receptors improves spatial working memory (41). In the present study, hippocampal GABAB1b mRNA is reduced in L. rhamnosus (JB-1)-fed mice, which is consistent with an enhanced memory consolidation in the fear conditioning test and further suggests that the changes in hippocampal gene expression induced by the Lactobacillus could in part account for these differences in behavior. L. rhamnosus (JB-1) This may not be so good particularly in the context of Perserverence's LTP thoughts on benzodiazepine withdrawal administration also affected the transcripts of GABAA receptor subunits in the hippocampus. Although differences in the expression of the transcript for GABAAα2 and GABAAα1 have been found in the hippocampus of rats subjected to different learning tasks, these changes are not consistent (38, 42). Nevertheless, it has been shown that GABAA receptors bearing the GABAAα2 subunit mediate the anxiolytic effects of benzodiazepines, whereas GABAA receptors that have the α1 subunit mediate the sedative and amnesic effects of benzodiazepines (12). In the present study, the difference in hippocampal expression of GABAAα2 and GABAAα1 mRNAs support the behavioral findings because L. rhamnosus (JB-1)-fed mice were less anxious and displayed antidepressant-like behaviors in comparison with broth-fed controls. Furthermore, it can be suggested that the effects of L. rhamnosus (JB-1) on fear-related behavior could be due to its effects on stress-induced corticosterone levels. Administration of corticosterone to BALB/c mice after the acquisition phase (day 1) destabilizes fear memory consolidation and allows faster extinction (24), suggesting a mechanism by which corticosterone itself could directly affect fear-related behavior. In the present work, L. rhamnosus (JB-1) reduced the stress-induced levels of corticosterone, which suggest that these “lower” levels could underlie the behavioral alterations observed.

 

The vagus nerve plays a major role in communicating changes in the gastrointestinal tract to the CNS (3). In the present study, Vx (vagus nerve removal) prevented the anxiolytic and antidepressant effects of L. rhamnosus (JB-1) and also the changes in GABAAα2 and GABAAα1 mRNAs in the amygdala (SI Materials and Methods), as well as GABAAα1 mRNA in the hippocampus. Nevertheless, Vx on its own was able to increase the levels of GABAAα2 mRNA in the hippocampus, although it prevented any further effect produced by L. rhamnosus (JB-1) supporting the observation that the changes in the hippocampus could reflect an indirect consequence of the Lactobacillus-induced changes in structures receiving direct visceral sensory inputs that can project afferents toward the hippocampus. Indeed, it has been shown that vagus nerve stimulation in rats can affect hippocampal functions (43), and therefore the changes in hippocampal GABAAα2 mRNA expression could occur as a result of Vx. Moreover, vagus nerve stimulation has been described as a successful approach to treat some (44), but not all (45), patients with treatment-resistant depression, which further suggests the importance of the vagus nerve in the modulation of behavior.

 

In summary, our data with L. rhamnosus (JB-1) suggest that nonpathogenic bacteria can modulate the GABAergic system in mice and therefore may have beneficial effects in the treatment of depression and anxiety. Moreover, it is worth noting that, in the present study, the effects were observed in healthy animals, whereas most studies examining the effects of potential probiotics on microbiome–gut–brain axis function rely on using infected, germ-free, or antibiotic-treated animals (2, 14); thus, the ramifications of these findings is manifold for the therapeutic potential of bacteria in modulating brain and behavior. Changes in transcripts for GABA receptor subunits emphasize a possible mechanistic insight into the potential effect of L. rhamnosus (JB-1) on anxiety-like behavior (12, 13). However, the participation of other neurotransmitter and neuropeptide systems that are of relevance to stress-related psychiatric disorders—such as 5-hydroxytryptamine, norepinephrine, glutamate, and corticotrophin-releasing factor—cannot be ruled out. Thus, future studies should investigate whether chronic treatment with L. rhamnosus (JB-1) can modulate such systems and, if so, how long such changes may last. Furthermore, the effects of L. rhamnosus (JB-1) on neurotransmitter levels are probably downstream of the effects on the HPA axis. In addition, the vagus nerve is responsible for some of the behavioral and molecular changes induced by L. rhamnosus (JB-1), demonstrating a clear pathway for the functional communication between bacteria, the gut, and the brain that modulates the behavioral responses toward different stressful situations. It is worth noting that the majority of studies on the microbiome–gut–brain axis are rodent-based, and future validation of the role of this axis in modulation in behavior is now warranted. Nonetheless, our current studies offer the intriguing opportunity of developing unique microbial-based strategies for the adjunctive treatment of stress-related psychiatric disorders.

[[On...]]

Thanks again for highlighting the most important points. Since my early career working with teenage girls in crisis, most diagnosed Borderline Personality Disordered, I have been very I there's ted in the amygdala and hippocampus.  Who would guess that benzos would bring me back to the subject? 

 

I'm glad your previous post mentioned that some specific probiotics can affect histamine and some create lactates which are highly irritating to the brain. Back when I was being a good little patient and taking my lorazepam only once a day as prescribed  , I tried several different very powerful mixes available over the counter and had horrible reactions to them. I wondered if I was just losing my mind. Later, after etown introduced me to the scd diet, I made their home made yogurts and STILL had that reaction. I now know why. It took a good while for me to be brave enough to try the rhamnosus. But, hey, since the mice sacrificed themselves so that we could benefit, I felt duty bound to give it a go.  .  What a great delight that it worked so dadgum well!

    Smiff, I think in times to come, the info you dug up will be used widely to help the benzo challenged population AND so many others suffering from a number of different gut-brain illnesses. You are up front there on the leading edge, woman. 

[[Lo...]]

I've been taking a probiotic for the last month and i have been feeling so much better. i should try the kefir too. thanks for the reminder on this. glad to know it helps/

[[sc...]]

So this is good for tummy troubled or does it help with any other aspect of w/d

[[On...]]

So this is good for tummy troubled or does it help with any other aspect of w/d

 

It helps with many aspects of benzo withdrawal because it is calming to the nervous system and also helps repopulate gaba receptors.

[[je...]]

I drank kefir months ago and it seemed to not help as much so I stopped drinking it. I just started drinking it again and my sleep has been amazing- like 9 hrs. Do you think it could be related?

[[Sm...]]

It could have helped Jenny!

It helps lots of people

 

My particular bout with kefir has ended because I started getting a histamine reaction to it.

 

Like all things, this stuff is very complicated and what is good for less sensitive people may not work as well for us. For me, I haven't given up on probiotics but I'm finding out what strains I tolerate using single strain compounds.

 

The two biggest things that can go wrong with probiotics

 

- acidosis from excess lactic acid: to avoid that you can get a 'D-Lactate' free probiotic

 

- histamine reaction. To avoid this you can go for the ones that reduce histamine or are neutral

 

-  Histamine producing bacteria: Lactobacillus casei, Lactobacillus reuteri, and Lactobacillus bulgaricus (Found in most yogurts and fermented foods).

   

- Neutral bacteria: Streptococcus thermophiles (also in yogurt) and Lactobacillus rhamnosus (shown to down regulate histamine receptors and up-regulate anti-inflammatory agents)

 

-  Histamine degrading bacteria: Bifidobacterium infantis (found in breast milk), Bifidobacterium longum, Lactobacillus plantarum, and some soil-based organisms.

 

from this site which is partly an advertisement for a particular low histamine diet but I've read the same information on these probiotics https://www.bulletproofexec.com/why-yogurt-and-probiotics-make-you-fat-and-foggy/

 

To be continued as always 

 

[[je...]]

Thanks for all the info smiff! What do you mean by histamine reaction?

[[Sm...]]

Thanks for all the info smiff! What do you mean by histamine reaction?

 

That sight I gave a link to runs down histamines and when they can become problematic. They are the things that cause an allergic reaction. The things that get blocked with anti-histamines. They are part of our immune system but can get a bit over excited.

 

Also you can google 'histamine intolerance'. Beyond meds has a few articles on people trying to eat low histamine to heal themselves from med damage. http://beyondmeds.com/2013/02/25/a-mini-histamine-intolerance/