Microbiota-Independent Ameliorative Effects of Antibiotics on Spontaneous Th2-Associated Pathology of the Small Intestine

Abstract

We have previously generated a mouse model of spontaneous Th2-associated disease of the small intestine called TRAF6ΔDC, in which dendritic cell (DC)-intrinsic expression of the signaling mediator TRAF6 is ablated. Interestingly, broad-spectrum antibiotic treatment ameliorates TRAF6ΔDC disease, implying a role for commensal microbiota in disease development. However, the relationship between the drug effects and commensal microbiota status remains to be formally demonstrated. To directly assess this relationship, we have now generated TRAF6ΔDC bone marrow chimera mice under germ-free (GF) conditions lacking commensal microbiota, and found, unexpectedly, that Th2-associated disease is actually exacerbated in GF TRAF6ΔDC mice compared to specific pathogen-free (SPF) TRAF6ΔDC mice. At the same time, broad-spectrum antibiotic treatment of GF TRAF6ΔDC mice has an ameliorative effect similar to that observed in antibiotics-treated SPF TRAF6ΔDC mice, implying a commensal microbiota-independent effect of broad-spectrum antibiotic treatment. We further found that treatment of GF TRAF6ΔDC mice with broad-spectrum antibiotics increases Foxp3+ Treg populations in lymphoid organs and the small intestine, pointing to a possible mechanism by which treatment may directly exert an immunomodulatory effect. To investigate links between the exacerbated phenotype of the small intestines of GF TRAF6ΔDC mice and local microbiota, we performed microbiotic profiling of the luminal contents specifically within the small intestines of diseased TRAF6ΔDC mice, and, when compared to co-housed control mice, found significantly increased total bacterial content characterized by specific increases in Firmicutes Lactobacillus species. These data suggest a protective effect of Firmicutes Lactobacillus against the spontaneous Th2-related inflammation of the small intestine of the TRAF6ΔDC model, and may represent a potential mechanism for related disease phenotypes.

Fig 1. Germ-free conditions exacerbate inflammation of TRAF6ΔDC small intestine.

(A) Representative gut images from control (WT) and TRAF6ΔDC (ΔDC) bone marrow chimera established in specific pathogen free (SPF) or germ-free (GF) condition at 8 weeks post-reconstitution. (B) Histological analyses were performed by H&E staining of duodenum from each mice. Scale bars represent 100 μm. (C) Lengths and masses of small intestines and colons were measured in control (WT) and TRAF6ΔDC (ΔDC) bone marrow chimera (n = 6). SPF, specific pathogen free; GF, germ-free. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig 2. Germ-free conditions exacerbate Th2-associated immune pathology in TRAF6ΔDC small intestine.

(A and B) Increases of IL-13⁺ cells in mesenteric lymph nodes of TRAF6ΔDC (ΔDC) bone marrow chimera in germ-free (GF) condition compared to specific pathogen free (SPF) condition. The representative FACS plots gated on CD4 T cells of mesenteric lymph nodes show intracellular staining for interferon-γ (IFN-γ) and IL-13. (C) Serum immunoglobulin levels in the chimera were measured by ELISA. (D) Fibrosis markers (Acta2 and Igf-1) and Th2 cell cytokines (IL-13, IL-5, and IL-4) mRNA expression levels in the ileum region of small intestines from SPF or GF control and TRAF6Δ bone marrow chimera. Histograms (mean ± SD) are representative of three independent experiments. *p < 0.05; **p < 0.01.

Fig 3. Broad-spectrum antibiotics ameliorate TRAF6ΔDC immune pathology exacerbated by GF conditions.

(A) FACS plots gated on CD4⁺ T cells show intracellular staining for interferon-γ (IFN-γ) and IL-13 from each indicated organ of TRAF6ΔDC bone marrow chimera (ΔDC-BMC) in germ-free (GF) condition at 8 weeks post-reconstitution. Some of TRAF6ΔDC bone marrow chimeras (ΔDC) were provided with broad-spectrum antibiotic water (Abx). The antibiotic water, containing 1 g/L Ampicillin, 1 g/L Neomycin, 0.5 g/L Vancomycin, and 1 g/L Metronidazole, was provided ad libitum in water to TRAF6ΔDC bone marrow chimera (ΔDC) for last 2 weeks. (B) Percentage of IL-13⁺ CD4 T cells were shown in each organs of TRAF6ΔDC bone marrow chimera (ΔDC-BMC) in germ-free (GF) or germ-free under antibiotics (GF-Abx). (C) Lengths and masses of small intestines were measured in GF and GF-Abx TRAF6ΔDC bone marrow chimera (ΔDC-BMC). (D) Fibrosis markers (Acta2 and Igf-1) and Th2 cell cytokines (IL-13, IL-5, and IL-4) mRNA expression levels in the ileum region of small intestines from GF and GF-Abx TRAF6ΔDC bone marrow chimera (ΔDC-BMC). Histograms (mean ± SD) are representative of four independent experiments. SP, spleen; MLN, mesenteric lymph node. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4. Increased Tregs in GF TRAF6ΔDC lymphoid organs following antibiotic treatment.

(A) FACS plots gated on CD4⁺ T cells show intracellular staining Foxp3 from each indicated organ of germ-free (GF) TRAF6ΔDC bone marrow chimera (ΔDC-BMC) at 8 weeks post-reconstitution. Broad-spectrum antibiotic water was provided to a group of TRAF6ΔDC bone marrow chimeras (ΔDC) for the last 2 weeks. (B) Percentage of Foxp3⁺ CD4 T cells were determined from the indicated organs of TRAF6ΔDC bone marrow chimera (ΔDC-BMC) in germ-free (GF) or germ-free under antibiotics (GF-Abx). (C) Representative FACS plots show Foxp3⁺ CD4 T cells from each indicated organ of germ-free (GF) B6 mice. Some of the mice were provided with broad-spectrum antibiotic water (GF-Abx) for last 2 weeks. Percentage of Foxp3⁺ CD4 T cells were shown in each organs of germ-free B6 mice with (GF-Abx) or without antibiotics (GF). SP, spleen; MLN, mesenteric lymph node; SI, small intestine; COL, colon. **p < 0.01.

Fig 5. TRAF6ΔDC mice exhibit disruption of microbiotic homeostasis localized to the small intestine.

(A) Pie charts represent bacterial composition categorized by phylum in the small bowl of control (WT) or TRAF6ΔDC (ΔDC) mice. (B) Heatmap analysis shows relative abundance of taxa as a percentage of total 16S rRNA, organized according to genus or most specific assigned taxon. Color scales reflect proportion contributed by each taxon. Total bacterial genome was isolated from small intestinal contents of control (WT) or TRAF6ΔDC (ΔDC) mice (n = 4, 20 week old littermate and co-housed group). (C) The copy number of total bacterial 16S rRNA bacteria from fecal or small intestine contents of control (WT) and TRAF6ΔDC (ΔDC) was measured by comparing to reference E. coli 16S rRNA plasmids. Some groups were fed by broad-spectrum antibiotic water (Abx) for last 2 weeks before collecting samples. **p < 0.001; n.s., not significant. Data were analyzed by Anova on Prism software (n = 3).