Using a validated model of chronic stress and depression [18, 19], we demonstrate, for the first time, the influence of a single orally administered bacteria strain, Lactobacillus rhamnosus JB-1, on behavioural deficits and systemic immune alterations caused by chronic exposure to a psychosocial stressor. While we observed no effects on baseline behaviour, JB-1 attenuated stress-induced behavioural deficits, including changes in sociability and anxiety-like behaviour, and prevented immunoregulatory alterations associated with the stress phenotype. Notably, the tempering of stress-induced changes occurred in the absence of any effects of treatment on stress-related disruptions in the microbiota, suggesting that JB-1 directly modulates gut-brain signalling pathways independently of the microbial community.

Following CSD, JB-1-treated stressed mice, as opposed to vehicle-treated, did not show avoidance of novel social stimuli, exhibited more frequent rearing behaviour in the OFT, and showed reduced aversion towards the light chamber (LDT). These data support the emerging literature suggesting that administration of specific bacterial strains decreases anxiety- and depressive-like behaviours [9, 10]. Indeed, we have previously demonstrated that chronic administration of JB-1 in Balb/c mice altered baseline levels of anxiety-like behaviour [10]. That the effects of JB-1 here were limited to deficits produced by chronic stress and not baseline behaviour (Fig. 2), may be indicative of intrinsic differences between Balb/c and C57BL/6 mice, the latter of which exhibit reduced apprehension, neophobia, and anxiety-like behaviour on baseline behavioural assays [47]. These mouse strain-specific effects may have implications for translational studies in humans, suggesting that, in keeping with a recent report [48], JB-1 would not be expected to have an anxiolytic effect in non-anxious individuals. Similarly, anti-depressants have very limited effects on healthy subjects [49].

In models of psychiatric conditions, repeated aggression and defeat lead to persistent conditioned submissive behaviour and aversion towards social stimuli [18, 50]. These behavioural manifestations bear similarity to symptoms of social withdrawal in depression and phobic avoidance of trauma-related stimuli in PTSD [51]. It is notable that the ameliorating effects of JB-1 on deficits in social behaviour were limited to interactions involving a non-threatening conspecific, while avoidance of the novel trauma-related stimulus was maintained. Previous research has suggested dissociation of social and non-social forms of anxiety-like behaviour [52]. For instance, treatment with a human commensal organism, Bacteroides fragilis, in a model of autism spectrum disorder attenuated deficits in anxiety-like behaviour, but did not affect sociability [31]. Our findings suggest that social anxiety may be further dissociated into discrete, differentially modulated behaviours expressed towards non-threatening versus threatening stimuli, the latter of which is experience-dependent [18, 19]. Thus, the disparate effects of JB-1 on behaviours expressed by defeated mice may be due to independent underlying neural circuitry. Such dissociable circuitry has been indicated by work on the stimulation of nucleus accumbens afferents, which alters behaviour towards a novel aggressor, but not anxiety-like behaviour [27]. This concept is further supported and emphasized in the current study given the recovery of anxiety-like behaviour but not of aggressor avoidance behaviour 3 weeks post-defeat (Fig. 2 h, i). In addressing neural mechanisms underlying the effect of microbial treatment on the expression of stress-related behaviours, we examined a limited number of genes related to the stress circuitry in the frontal cortex and hippocampus. While stress exposure reduced GABA receptor expression in the prefrontal cortex and glucocorticoid receptor expression in the hippocampus, there was no effect of microbe treatment on these measures. This contrasts the previously demonstrated effects of JB-1 administration on baseline expression of central GABA receptors in Balb/c mice [10]. While these results further emphasize the mouse strain-dependent effects of microbe exposure on gut-brain signalling, a more extensive assessment of additional neural pathways in multiple brain regions will be required to identify potential circuitry involved in JB-1-induced attenuation of stress-related behaviour.

Consistent with the immunomodulatory role of gut bacteria [15] and previous studies with JB-1 [24], microbial treatment influenced systemic changes in the CSD-induced immune phenotype. Social defeat increased the population of activated splenic DCs—a shift completely prevented by JB-1. Furthermore, treatment with the bacteria induced systemic expansion of Treg: a population that produces high levels of the anti-inflammatory cytokine, IL-10 [53]. Coordination between multiple host systems—and dysregulation thereof—likely contributes to the phenotypic changes in stress and related psychiatric conditions, during which systemic disruptions and allostatic load accumulate over extended periods of time. For instance, a pro-inflammatory milieu and a decrease in Tregs are commonly observed in severe stress and PTSD [17, 54] and form the central premise of the inflammation theory of depression [55]. Indeed, stress-induced trafficking of peripheral monocytes to the brain appears to play a crucial role in anxiety-like behaviour [56]. Disruption of the host-microbiota relationship during chronic stress may contribute to exaggerated inflammation and immune dysregulation and is associated with colitis and inflammatory bowel disease [11, 57]. The observed acute increase in the Treg population (Fig. 3a) [13] following stress may be a counteractive response to the pro-inflammatory shifts described in the literature upon stress induction [13, 56, 58]; such responses are a well-documented reaction to host inflammation in an attempt to restore homeostasis [59]. Although this natural allostatic mechanism does not prevent an inflammatory environment during maladaptive stress, JB-1-induced modulation of host-initiated immunoregulatory responses may be one mechanism contributing to the behavioural effects of the bacteria. Similar mechanisms were posited to explain the stress-mitigating effects of Mycobacterium vaccae immunization, which were demonstrated to depend on Tregs [11]. These data suggest that recruitment of immune pathways in bottom-up (gut-to-brain) signalling is important. The current study was limited to two immune cell lineages: dendritic cells and T cells. Clearly, additional immune cell types may make important contributions to gut-brain signalling. Future studies should include a broader assessment of the immune system and more detailed examination of microbiota-immune-neural coordination and dysregulation of these systems in stress.

It has been proposed widely that modifying the resident intestinal bacteria in disease can reverse microbial dysbiosis and restore homeostatic function [60, 61]. Such an approach is especially relevant given evidence of microbiota disruption in severe stress and psychiatric conditions and its association with adverse gastrointestinal outcomes [37]. Thus, we investigated whether the improved neurobehavioural phenotype due to microbial treatment was associated with alterations to the existing microbiota community. Prior to stress exposure, administration of JB-1 did not alter the profile of the microbiota—data that parallel observations in humans who were administered a different strain of L. rhamnosus [62]. Furthermore, in our study, microbial treatment did not prevent any of the shifts in the microbiota community due to stress exposure. JB-1 treatment also completely failed to restore the diversity and richness of the microbiota or correct the relative abundances of specific OTUs altered by stress. Thus, the neuroactive properties of the beneficial microbe may be mediated independently of restoring microbial community balance, and might be dependent on its functional activity and direct modulation of host signalling pathways.

Not unexpectedly, the stress-induced dybiosis was accompanied by a significant change in levels of various faecal metabolites, while, perhaps more surprisingly, JB-1 treatment alone significantly modulated the levels of 75 metabolites, many of which have immunomodulatory and neuroactive properties. While the source of these metabolites, host or microbe, cannot be identified, these observations suggest that JB-1 could alter the function of the existing gut microbiota without influencing composition. Most notably, the reduction in tyramine levels induced by CSD was the only metabolite change significantly inhibited by JB-1 treatment. Tyramine is a monoaminergic neuromodulator, acting as an agonist for trace amine-associated receptor 1 (TAAR1) [63]. Tyramine also causes the release of norepinephrine from sympathetic nerves, reversing re-uptake through the norepinephrine transporter and has been demonstrated to induce serotonin (5-HT) production by enterochromaffin cells [64]. Given that intestinal 5-HT [65] and catecholamines [66] have been proposed as mediators of microbe-gut-brain signalling via modulation of the enteric nervous system, the impact of luminal tyramine levels on the gut-brain axis may warrant further investigation. The current study focused on faecal metabolites, with the understanding that gut lumen metabolites acting at the level of the gut epithelium, enteroendocrine cells, and enteric nervous system may play a role in microbe-gut-brain signalling. However, future assessment of plasma metabolites may identify circulating factors, produced by gut microbes or induced in the host, which have more direct effects on the central nervous system.

One limitation of the current study is that we only assessed the faecal microbiota, and it is possible that JB-1 stabilized site-specific microbiota, for example, in the small intestine or specifically associated with the epithelium elsewhere, that are involved in gut-brain signalling. However, a direct action of JB-1 on gut-brain signalling is further supported by previous studies using in vivo and ex vivo models, demonstrating that it can directly or indirectly activate the vagus nerve and that an intact vagus is required to mediate the effects of this bacterium, at least on the baseline behaviour of Balb/c mice [22]. Collectively, these data suggest that JB-1, independently of changes in the microbiota, can recruit host signalling pathways, likely including vagal afferents that mediate the effects of the bacteria on severe CSD-induced neurobehavioural changes. Investigation of the role of the vagus in mediating microbe-induced modulation of behaviour in the CSD model is certainly warranted.

Although numerous studies have demonstrated the effect of environmental adversity on disruption of gut microbiota [11–13], there is very little evidence on the permanence of these changes in stress-related disorders or on whether microbial supplementation can facilitate the recovery of dysbiosis. A limited number of observations suggest a complex relationship between environmental factors and perturbations of the gut microbiota. Certain factors impart transient changes in the community, while others, for instance, antibiotic usage, leave behind a more persistent signature [67, 68]. Furthermore, factors such as birth delivery mode have marked effects on the microbiota community during early life that are no longer distinguishable in adulthood [69]. Our own observations suggest that stress-induced disruptions in the microbiota appear stable for a prolonged period following stress exposure. Examination of defeated mice 3 weeks following CSD revealed enduring structural changes in the faecal microbial community: defeated mice continued to show reduced diversity and richness in the variety of species represented while exhibiting broad-scale changes in overall composition and profile. The long-term stress-induced changes in the microbiome were not significantly altered with JB-1 treatment.