Parasite-induced TH1 cells and intestinal dysbiosis cooperate in IFN-γ-dependent elimination of Paneth cells

Abstract

Activation of Toll-like receptors (TLRs) by pathogens triggers cytokine production and T cell activation, immune defense mechanisms that are linked to immunopathology. Here we show that IFN-γ production by CD4(+) T(H)1 cells during mucosal responses to the protozoan parasite Toxoplasma gondii resulted in dysbiosis and the elimination of Paneth cells. Paneth cell death led to loss of antimicrobial peptides and occurred in conjunction with uncontrolled expansion of the Enterobacteriaceae family of Gram-negative bacteria. The expanded intestinal bacteria were required for the parasite-induced intestinal pathology. The investigation of cell type-specific factors regulating T(H)1 polarization during T. gondii infection identified the T cell-intrinsic TLR pathway as a major regulator of IFN-γ production in CD4(+) T cells responsible for Paneth cell death, dysbiosis and intestinal immunopathology.

Figure 1. T. gondii infection results in intestinal dysbiosis

(a) qRT-PCR analysis of Proteobacteria and Bacteroidetes loads in the lumens of small intestines of wild-type mice left untreated (d0) or infected orally with 20 cysts of the T. gondii ME49 strain per mouse at the indicated time post-infection. The results are representative of four independent experiments, each involving at least five mice per group. * P< 0.05; ** P< 0.01. (b) Genomic DNA analysis of microbial communities from the lumens of naïve or T. gondii-infected wild-type mice on day 7 post-infection (n= 5–7 mice/group). For each mouse, 192 colonies containing cloned 16rRNA amplicons were processed for sequencing. The relative frequencies of Proteobacteria (magenta), Bacteroidetes (turquoise), Firmicutes (Blue), and other phyla (purple) are shown. Bacteroidetes were not detected in samples prepared from T. gondii-infected mice. (c) Heatmap of the relative abundance of the 40 most dominant genera in the small intestines of naïve or T. gondii-infected wild-type mice as determined by genomic DNA analysis by 454-based DNA pyrosequencing. Samples are sorted based on hierarchical clustering of weighted unifrac distances. (d) Bacterial loads in the lumens of the small intestines of non-infected (d0) or T. gondii-infected mice (d7) analyzed by plating bacteria on blood agar or CHROMagar plates under aerobic conditions. * P< 0.05; ** P< 0.001 (e) In situ hybridization of Proteobacteria (green) in non-infected or (f) T. gondii-infected mice. The results are representative of four independent experiments, each involving at least five mice per group.

Figure 2. T. gondii infection results in loss of Paneth cells

(a) qRT-PCR analysis of Lyz1, Defcr1 (alpha-defensin-1), Defa-rs1, Defa21, and CR2 were measured in the small intestines of untreated mice (n=6, blue) or infected orally with 20 cysts per mouse of the ME49 on day 7 post-infection (n=6, purple). * P< 0.001 (b) qRT-PCR analysis of RegIII-γ in the small intestines of naïve (n=6) and infected (n=6) mice. (c) Histological visualization of Paneth cells in the small intestines of naïve and infected (d7) mice. The results are representative of four independent experiments, each involving at least six mice per group. The blue arrows indicate Paneth cells and the black arrows point toward bases of the crypts lacking these cells. (d) Electron microscopy analysis of the bases of the crypts in naïve and T. gondii-infected mice (3 mice per group). The blue arrows indicate Paneth cells. The results are representative of four independent experiments.

Figure 3. T. gondii-induced dysbiosis contributes to the intestinal pathology

(a) Germ-free (GF) mice were left untreated, colonized with the Enterobactericeae isolate (Supplemental Fig. 3a), or with Bacteroides fragiles (B. frag), and infected orally with 20 cysts of the T. gondii ME49 strain. Histological analysis of the small intestine was performed on day 7 post-infection. The results are representative of two independent experiments. (b) Germ-free (GF) mice were mock colonized, colonized with the Enterobactericeae isolate (Supplemental Fig. 3a), or with B. frag, and additionally infected orally with T. gondii. Histological visualization of Paneth cells in the small intestines were performed on day 7 post-infection. The blue arrows indicate Paneth cells and the black arrows point toward bases of the crypts lacking these cells. The results are representative of two independent experiments.

Figure 4. TLR11-mediated activation of MyD88 triggers Paneth cell death and intestinal dysbiosis

(a) WT (not shown), Tlr11^−/−, and Myd88^−/− mice (five mice per group) were left untreated or were infected orally with 20 cysts per mouse of the ME49 strain of T. gondii. Histological visualization of Paneth cells in the small intestines were performed on day 7 post-infection. The blue arrows indicate Paneth cells and the black arrows point toward bases of the crypts lacking these cells. (b) The relative expression levels of Defcr1 (alpha-defensin-1), Defa- rs1, Defa21, CR2, and RegIII-γ were measured by qRT-PCR in the small intestines of WT (n=3), Tlr11^−/− (n=6), and Myd88^−/− (n=4) mice on day 7 post-infection. * P< 0.05; ** P< 0.01. (c) qRT-PCR analysis of Proteobacteria and Bacteroidetes in the lumens of small intestines of naïve (blue) or T. gondii-infected (purple) mice. The data shown are the means ± SD. * P< 0.05; ** P< 0.01; *** P< 0.001. The results are representative of >10 independent experiments, each involving 3–7 mice per group.

Figure 5. IFN-γ mediates loss of Paneth cells

(a) WT mice were treated with isotype control (IC), α-IFN-γ, α-CD4, or α-CD8 antibodies and then infected orally with 20 cysts per mouse of the ME49 strain of T. gondii. Histological analyses of Paneth cells in small intestines were performed on day 7 post-infection. (b) qRT-PCR analysis of Lyz1, Defcr1 (alpha-defensin-1), Defa-rs1, Defa21, CR2, and RegIII-γ were measured in the small intestines of naïve (blue) or T. gondii-infected WT mice treated with IC, α-IFN-γ, α-CD4, or α-CD8 antibodies (purple). * P< 0.05; ** P< 0.01. The results are representative of three independent experiments, each involving 4–7 mice per group.

Figure 6. Intrinsic T-cell MyD88 signaling regulates T_H1 polarization

(a) WT, Myd88^−/−, and mice with cell type-specific inactivation of MyD88 in DCs, epithelial cells (E), macrophages (M), T cells (T), DCs and epithelial cells (DC&E), DCs and macrophages (DC&M), and DCs and T cells (DC&T) were infected orally with 20 cysts per mouse of the ME49 strain of T. gondii. Mesenteric lymph nodes (mLNs) were harvested on day 7 post-infection, and IFN-γ production by CD4⁺ T cells was analyzed by flow cytometry. The data shown are representative of five independent experiments each involving 4–6 mice per group. (b) Average frequency and (c) absolute quantification of T_H1 cells (CD4⁺IFN-γ⁺) in the mLNs of mice infected orally with the parasite. The data shown are representative of five independent experiments each involving 4–6 mice per group. * P< 0.01; ** P< 0.001.

Figure 7. Intrinsic T-cell MyD88 signaling mediates loss of Paneth cells intestinal dysbiosis

(a) qRT-PCR analysis of relative expression of Lyz1, Defcr1 (alpha-defensin-1), Defa-rs1, Defa21 in the small intestines of WT, Myd88^−/−, and cell type-specific MyD88-deficient mice on day 7 post-infection. The results are representative of five independent experiments each involving 4–5 mice per group. (b) Histological visualization of Paneth cells in small intestines of DC-Myd88^−/−, T-Myd88^−/−, and DC&T-Myd88^−/− mice compared to complete MyD88-deficient mice (Myd88^−/−). The results are representative of five independent experiments each involving 4–5 mice per group. (c) Bacterial loads in the lumens of small intestines of naïve (blue) or infected WT, Myd88^−/− and cell type-specific Myd88^−/− mice (purple) were analyzed by plating bacteria on blood agar plates on day 7 post-infection. * P< 0.05; ** P< 0.01. The results are representative of five independent experiments each involving 4–5 mice per group.