Psychological stress impairs IL22-driven protective gut mucosal immunity against colonising pathobionts

Crohn's disease (CD) is an inflammatory disorder of the gastrointestinal tract triggered by microbial and environmental insults. The global burden is rising, and current therapies have limitations, underscoring the need to understand microbial and environmental triggers to inform new preventions and treatments. The CD-associated microbiome typically shows decreased diversity, reductions in Clostridiales, and increased Proteobacteria, with bacteria often closely associated with the mucosa, especially Enterobacteriaceae enriched in virulence pathways. Adherent-invasive Escherichia coli (AIEC) are abundant pathobionts enriched in CD and can exist as planktonic, biofilm, and intracellular forms that activate host inflammation. Mucosal immunity elements disrupted in CD include barrier integrity, mucus production, antimicrobial peptides (AMPs), and metal ion sequestration (nutritional immunity). IL-22 regulates multiple aspects of mucosal defense, including AMP and nutritional immunity pathways; however, some enteric pathogens circumvent IL-22–dependent defenses. The role of IL-22 in host control of CD-associated pathobionts is not fully defined. CD disease course is relapsing-remitting, and psychological stress is a known disease modifier associated with flares and increased inflammatory markers. Animal models show stress can worsen inflammation, disrupt barriers, and increase infection susceptibility, but mechanisms—particularly in the ileum—remain unclear. Here, in a pre-clinical model, the authors test how psychological stress reshapes the ileal microbiome and host immunity, focusing on IL-22 pathways and AIEC colonization dynamics, and whether immunomodulation can counteract stress-induced dysbiosis.

Prior work has characterized CD microbiome features: reduced diversity, reduced Clostridiales, increased Proteobacteria, and enrichment of mucosa-associated Enterobacteriaceae in CD. AIEC are frequently enriched in CD and can induce inflammation through multiphasic lifestyles (planktonic, biofilm, intracellular) in epithelial cells and macrophages. Mucosal immunity and nutritional immunity (metal sequestration) are central in spatially restricting bacteria; IL-22 promotes AMPs and nutritional immunity, though some pathogens (Salmonella, Citrobacter) evade IL-22–dependent defenses via AMP resistance and alternative metal acquisition. IL-22 can induce iron-scavenging host proteins (hemopexin, haptoglobin) controlling enteric pathogens. Psychological stress is clinically associated with IBD flares and increased inflammatory markers; rodent stress models demonstrate increased inflammation, barrier dysfunction, reactivation of chemically induced colitis, and heightened infection susceptibility. Chronic stress can alter cecal/colonic microbiota and inflammation, but mechanisms, especially in the ileum and in relation to IL-22 and AIEC, were previously undefined.

Study design: Pre-clinical mouse model to assess the impact of acute psychological stress on gut microbiota, mucosal immunity (with a focus on IL-22), and colonization by AIEC, with interventional testing of glucocorticoid receptor blockade and exogenous IL-22. Animals: 6–8-week-old male C57BL/6N mice (SPF), with some confirmation in females; TNF knockout mice for selected experiments. Psychological stress: overnight (16 h) restraint stress in ventilated 50 mL conical tubes during the dark cycle; matched control mice underwent food and water deprivation for 16 h. AIEC colonization: streptomycin pretreatment (20 mg gavage, 1 day prior); infection with 2 × 10^6 CFU AIEC strain NRG857c by oral gavage. Competitive infections used a 1:1 mixture of WT and ΔiroB mutant. Sampling: feces at baseline, 6 h, 24 h post-stress; tissues (four sequential 8 cm small intestine segments, cecum, colon, stomach) harvested immediately after stress for CFU enumeration; liver for dissemination. Microbiome profiling: 16S rRNA v3–v4 sequencing (Illumina), taxonomic assignment with Silva v132 via DADA2; diversity (Shannon), PCA, and LefSe analyses. Barrier function: ex vivo Ussing chambers on ileal segments assessing paracellular flux using 51Cr-EDTA probes. Gene expression: RT-qPCR (2^-ΔΔCT) on distal ileum (segment 4), normalized to RPLP0; targets included cytokines (e.g., TNF, IFN-γ, IL-23, IL-17A), barrier genes (mucins, tight junctions), TLR-4, IL-6, and antimicrobial/nutritional immunity genes (Lcn2, S100a8/a9, HMOX1, NRAMP1, HPX, haptoglobin, Reg3 family, defensins, Pla2, Duox, Fut2, Nos2). Protein measurements: ELISAs for Lcn2/NGAL, S100a8 in tissue; corticosterone in serum; liver LPS (endotoxin units) via chromogenic endotoxin assay; serum cytokines by multiplex. Flow cytometry: isolation of epithelial and lamina propria cells; staining panels for CD45, CD90, lineage markers, TCRβ, CD4, CD8, RORγt, glucocorticoid receptor (GR), pSTAT-3, Annexin V; quantification of CD45+CD90+ populations (T cells and ILCs), CD11b+Gr1+ cells (neutrophil-like), epithelial pSTAT-3. Explant assays: 1-cm ileal explants cultured 24 h with/without rIL-23 (20 ng/mL), supernatant IL-22 by ELISA. RNA sequencing: bulk distal ileum RNA-seq (ribodepletion, NEBNext library, HiSeq 2 × 150 bp), quantification with Salmon, differential expression via DESeq2; heatmaps via pHeatmap; data deposited (GSE180342). Interventions: RU486 (glucocorticoid receptor antagonist; 50 mg/kg i.p. 1 h pre-stress); LPS i.p. (0.25 mg/kg) to induce Lcn2 without stress; neutralizing antibodies: anti-IL-22 (150 µg i.p. daily; or every other day for 11 days in long course), anti-CD90 (200 µg i.p. every other day starting day of infection), IL-10R blockade (200 µg i.p. 1 h pre-stress). Recombinant IL-22-Fc (150 µg i.p. prior to stress; 50 µg every other day post-stress). DSS colitis: 2% DSS in drinking water from day 3 post-infection; stress on day 5; water restored on day 8/9; body weight tracked; IL-22-Fc dosing as above. Statistics: Mann–Whitney for two-group comparisons; one- or two-way ANOVA for ≥3 groups (generally not corrected for multiple comparisons unless specified); Spearman correlations; significance p < 0.05.

• Acute psychological stress drives Enterobacteriaceae expansion and dysbiosis: 16S profiling showed Enterobacteriaceae became dominant post-stress across gut regions, reaching >80% of sequences in ileum vs <1% in controls; Escherichia-Shigella was most increased; Enterococcus, Proteus, and Mucispirillum were enriched; beneficial taxa (e.g., Lachnospiraceae, Bifidobacterium) decreased. Ileal Shannon diversity significantly reduced (p = 0.0002; p = 0.0004 in AIEC-colonized cohort); stressed groups clustered distinctly by PCA.
• Stress triggers rapid and recurrent AIEC outgrowth: In AIEC-colonized mice, fecal AIEC increased by 4–5 logs at 6 h post-stress vs pre-stress, returning toward baseline by 24 h; starvation control showed no change. Weekly episodic stress produced stereotyped AIEC expansion-contraction cycles, with sustained high burdens through week 4, whereas controls cleared AIEC by weeks 2–3. AIEC expanded along the entire intestine in stressed mice, including proximal small intestine segments typically low/negative in controls.
• Stress worsens colitis in presence of AIEC: In DSS colitis, stress alone caused ~10% transient weight loss with recovery; stress plus AIEC caused ~20% loss with poor recovery.
• Barrier dysfunction and inflammation are exacerbated by stress (especially with AIEC): Increased ileal cytokine mRNA (TNF, IFN-γ, IL-23, IL-17A) in AIEC-colonized stressed mice; downregulation of barrier genes (Muc2, Muc3, Muc4; claudin-2; occludin), and increased GATA-4 (epithelial repair marker). Ussing chambers showed increased paracellular flux after stress in both naive and AIEC-colonized mice. Enhanced bacterial translocation (higher liver LPS; greater frequency of positive liver CFU), elevated ileal TLR-4 (~8–16×) and IL-6 (~64×) mRNA, and neutrophil-like CD11b+Gr1+ recruitment.
• Nutritional immunity induced by stress correlates with AIEC expansion: Upregulation of HMOX1 and NRAMP1, and increased Lcn2 and S100a8/a9 mRNA and proteins across intestine and liver early (4 h) during stress. Lcn2 remained elevated during stress and after release; AIEC burdens tracked with Lcn2 kinetics. Strong positive correlations between AIEC burdens and Lcn2 (ileum r = 0.77, p < 0.0001; cecum r = 0.600, p < 0.0001) and S100a8 (ileum r = 0.701, p < 0.0001; cecum r = 0.701, p < 0.0001). AIEC ΔiroB (salmochelin-deficient) was outcompeted by WT in stressed mice (ileum p = 0.0148; cecum p = 0.0005; colon p = 0.0201), but not under non-stressed conditions, indicating advantage under Lcn2 pressure.
• Stress-induced glucocorticoids deplete IL-22–producing lymphocytes: Stress elevated serum corticosterone; most CD45+CD90+ cells expressed glucocorticoid receptor. Early apoptosis marker Annexin V increased in CD45+CD90+ cells at 8 h of stress; RU486 (GR antagonist) prevented Annexin V upregulation. Overnight stress reduced frequency and absolute number of CD45+CD90+ cells (rescued by RU486). Depletion was indiscriminate across CD90+ T cells and ILCs (including TH17 and ILC3), reducing absolute numbers without changing subset frequencies.
• IL-22 axis is functionally impaired by stress: Ileal explants from controls produced IL-22 in response to IL-23 ex vivo; stressed explants failed to do so; RU486 restored IL-23–induced IL-22. In vivo, in absence of stress, anti-IL-22 or anti-CD90 did not increase AIEC. Under stress with RU486 (preserving CD90+ cells), AIEC expansion was prevented; co-administration of anti-CD90 or anti-IL-22 restored AIEC outgrowth. Reconstituting both pathways without stress (LPS to induce Lcn2 + anti-CD90 depletion) reproduced AIEC expansion, indicating requirement for both nutritional immunity and immune cell depletion downstream of stress.
• Exogenous IL-22 corrects defects and prevents dysbiosis: IL-22-Fc treatment during stress significantly blunted AIEC expansion in ileum (p = 0.0030) and other regions; restored epithelial pSTAT-3 positivity; RNA-seq showed stress-induced transcriptomic shifts largely normalized by IL-22-Fc. IL-22-Fc increased IL-22-inducible AMPs (Reg3b, Reg3g), Fut2, Pla2 family, Nos2, and reactive oxygen/nitrogen pathways; normalized pro-inflammatory cytokines (IL-23, IL-17A, IFN-γ, IL-6). IL-22-Fc ameliorated DSS-associated weight loss during stress and prevented Enterobacteriaceae overgrowth in 16S profiles.

The study addresses how psychological stress modifies gut immunity and microbiota to favor expansion of CD-associated pathobionts. Acute stress induced a profound ileal dysbiosis characterized by Enterobacteriaceae (including AIEC) overgrowth, reduced microbial diversity, compromised barrier function, and heightened inflammation. Mechanistically, stress exerted a dual impact: (1) it activated nutritional immunity (e.g., Lcn2, S100a8/a9, HPX, haptoglobin, HMOX1, NRAMP1), selecting for Enterobacteriaceae with siderophore strategies (e.g., salmochelin) that evade host sequestration; and (2) it induced glucocorticoid receptor–dependent apoptosis and attrition of CD45+CD90+ IL-22–producing lymphocytes (TH17/ILC3), blunting IL-22-driven mucosal defenses. Either pathway alone was insufficient to reproduce AIEC outgrowth, but together they created a niche favoring pathobiont expansion. Blocking glucocorticoid signaling preserved CD90+ cells and prevented AIEC expansion despite ongoing nutritional immunity, while neutralizing IL-22 or depleting CD90+ cells reinstated susceptibility. Conversely, exogenous IL-22 restored epithelial STAT3 activation, enhanced AMPs and barrier-supportive programs, normalized proinflammatory cytokines, reduced AIEC expansion, and ameliorated colitis severity. These findings place IL-22 deficiency, secondary to stress-induced lymphocyte attrition, at the center of stress-related dysbiosis and suggest that augmenting IL-22 signaling can counteract stress-driven disease-relevant microbiome shifts in the ileum.

Psychological stress impairs IL-22–dependent mucosal immunity in the small intestine through glucocorticoid receptor–mediated apoptosis of CD45+CD90+ lymphocytes, leading to decreased IL-22 responses, compromised epithelial defenses, and a dysbiotic expansion of Enterobacteriaceae, including AIEC. Concurrent induction of nutritional immunity further selects for pathobionts with Lcn2-evading iron acquisition, jointly driving dysbiosis and exacerbating barrier dysfunction and inflammation. Interventions that block glucocorticoid signaling or deliver exogenous IL-22 restore mucosal immunity, limit AIEC expansion, normalize stress-induced transcriptomic changes, reduce Enterobacteriaceae overgrowth, and mitigate DSS colitis severity. These results highlight IL-22 modulation as a potential therapeutic avenue to counteract stress-associated dysbiosis and disease activity in CD. Future work should delineate translational relevance in humans, the impact of chronic vs acute stress, sex differences, and interactions with broader microbiome and host genetic contexts.

The study is a pre-clinical mouse model using acute overnight restraint stress and streptomycin-facilitated AIEC colonization, which may limit direct generalization to human CD and to chronic or diverse stressors. Most experiments used male mice (to avoid estrus cycle confounding), which may not capture sex-specific responses. Statistical analyses often did not correct for multiple comparisons, potentially inflating type I error in multi-analyte assays. Findings were centered on a single AIEC strain (NRG857c) and specific siderophore genetics (ΔiroB), which may not represent all AIEC or Enterobacteriaceae. The reliance on food/water deprivation as the control for restraint stress may not fully control for all physiological variables introduced by restraint.