Oral treatment with Lactobacillus rhamnosus attenuates behavioural deficits and immune changes in chronic social stress

Abstract

Background: Stress-related disorders involve systemic alterations, including disruption of the intestinal microbial community. Given the putative connections between the microbiota, immunity, neural function, and behaviour, we investigated the potential for microbe-induced gut-to-brain signalling to modulate the impact of stress on host behaviour and immunoregulation.

Methods: Male C57BL/6 mice treated orally over 28 days with either Lactobacillus rhamnosus (JB-1) ™ or vehicle were subjected to chronic social defeat and assessed for alterations in behaviour and immune cell phenotype. 16S rRNA sequencing and mass spectrometry were employed to analyse the faecal microbial community and metabolite profile.

Results: Treatment with JB-1 decreased stress-induced anxiety-like behaviour and prevented deficits in social interaction with conspecifics. However, JB-1 did not alter development of aggressor avoidance following social defeat. Microbial treatment attenuated stress-related activation of dendritic cells while increasing IL-10+ regulatory T cells. Furthermore, JB-1 modulated the effect of stress on faecal metabolites with neuroactive and immunomodulatory properties. Exposure to social defeat altered faecal microbial community composition and reduced species richness and diversity, none of which was prevented by JB-1. Stress-related microbiota disruptions persisted in vehicle-treated mice for 3 weeks following stressor cessation.

Conclusions: These data demonstrate that despite the complexity of the gut microbiota, exposure to a single microbial strain can protect against certain stress-induced behaviours and systemic immune alterations without preventing dysbiosis. This work supports microbe-based interventions for stress-related disorders.

Keywords: Behaviour; Chronic social defeat; Gut-brain axis; Immune system; Microbiota; Psychiatric disease.

Fig. 1

Schematic diagram of experimental approach. Mice were treated with JB-1 on 20 instances over a period of 28 days, including 7 instances over the final 10 days, during which mice were exposed to chronic social defeat (CSD) stress every day. OFT open field test, LDT light-dark box test

Fig. 2

Chronic stress induces deficits in social and anxiety-like behaviours in mice that are partially corrected by microbial treatment. a Microbial treatment does not alter baseline sociability, measured by time spent in the social and non-social chambers, in vehicle-treated (n = 10) versus JB-1-treated (n = 8) unstressed mice. b Vehicle-treated defeated mice (n = 15) exhibit avoidance of the social chamber—deficits that are corrected in defeated mice treated with JB-1 (n = 10). c Data demonstrating the time spent in the social and non-social chambers as a log ratio across all four groups. d Aggressor approach-avoidance test paradigm. e Socially defeated mice exhibit avoidance of a novel aggressor, independent of treatment. f Chronic stress reduced rearing behaviour on the OFT, but was partially rescued by JB-1 treatment (n: CON/VEH = 29, CON/JB-1 = 13, DEF/VEH = 27, DEF/JB-1 = 16). g Chronic stress reduced the number of entries into the light zone on the LDT, but was partially rescued by JB-1 treatment (n: CON/VEH = 18, CON/JB-1 = 12, DEF/VEH = 15, DEF/JB-1 = 16). h Anxiety-like behaviour across time, as measured by the number of entries into the light zone, at 24 h and at 3 weeks following cessation of CSD treatment (n: CON/VEH = 11, DEF/VEH = 9, DEF/JB-1 = 9). i Avoidance behaviour on the aggressor approach-avoidance test, at 24 h and at 3 weeks following cessation of CSD treatment (n: DEF/SAL = 9, DEF/JB-1 = 10). *p < 0.05, **p <0.01, and ***p < 0.001. Data are represented as mean ± SEM

Fig. 3

Effect of chronic social defeat stress and JB-1 treatment on splenocyte phenotype (n = 5/group). a IL-10+ CD4+ CD25+ T cells in mice following exposure to chronic social defeat and JB-1 treatment. b JB-1 treatment prevents the stress-induced increase in CD80+ MHCII+ CD11c + splenocyte levels in defeated mice c CD86+ MHCII+ CD11c + splenocytes in mice following exposure to chronic social defeat and JB-1 treatment. d Fluorescence-activated cell sorting (FACS) gating strategy for IL-10+ CD4+ CD25+ T cells (CD3+). e FACS gating strategy for CD80+ and CD86+ on MHCII+ DCs (CD11c+). *p < 0.05. Data are represented as mean ± SEM

Fig. 4

JB-1 treatment does not affect stress-induced structural changes in the microbiota community. a Effect of chronic social defeat and JB-1 treatment on phylogenetic diversity and Chao1 richness estimates from the rarefied 16S rRNA data (n: CON/VEH = 18, CON/JB-1 = 13, DEF/VEH = 24, DEF/JB-1 = 17, 7923 reads/sample). b, c Principle coordinates analysis (PCoA) of Bray-Curtis distances from the average rarefied 16S rRNA data (n: CON/VEH = 18, CON/JB-1 = 13, DEF/VEH = 24, DEF/JB-1 = 17; n = 999 rarefactions, 6339 reads/sample) indicate a significant effect of social defeat on group clustering (p = 0.013), but no effect of JB-1 treatment (median ± min/max). d Effect of chronic social defeat and JB-1 treatment on the taxonomic distribution of OTUs at the phylum level (n = 17–24/group). e Bray-Curtis distances from the average rarefied 16S rRNA data (n = 999 rarefactions, 44,648 reads/sample) three weeks after stressor and treatment cessation indicate a persistent significant effect of social defeat on group clustering (p = 0.022), but no difference between the control group and defeated mice treated with JB-1 (median ± min/max). f Phylogenetic diversity and Chao1 richness estimates from the rarefied 16S rRNA data (55,810 reads/sample) 3 weeks after stressor and treatment cessation indicate a persistent significant effect of social defeat, but no difference between the control group and defeated mice treated with JB-1 (n = 9–11/group). *p < 0.05, **p <0.01, and ***p < 0.001. Data are represented as mean ± SEM unless otherwise indicated

Fig. 5

Effect of chronic stress and JB-1 treatment on the faecal metabolome. a–d Metabolites whose levels were altered by chronic stress but prevented in JB-1-treated mice (n: CON/VEH = 10, CON/JB-1 = 5, DEF/VEH = 10, DEF/JB-1 = 10). a 1-methylnicotinamide, b 4-hydroxybutryrate, c tyramine, d kynurenine. *p < 0.05, **p <0.01. Data are represented as median ± min/max