A TNF-IL-1 circuit controls Yersinia within intestinal granulomas

Abstract

Tumor necrosis factor (TNF) is a pleiotropic inflammatory cytokine that mediates antimicrobial defense and granuloma formation in response to infection by numerous pathogens. Yersinia pseudotuberculosis colonizes the intestinal mucosa and induces recruitment of neutrophils and inflammatory monocytes into organized immune structures termed pyogranulomas that control the bacterial infection. Inflammatory monocytes are essential for control and clearance of Yersinia within intestinal pyogranulomas, but how monocytes mediate Yersinia restriction is poorly understood. Here, we demonstrate that TNF signaling in monocytes is required for bacterial containment following enteric Yersinia infection. We further show that monocyte-intrinsic TNFR1 signaling drives production of monocyte-derived interleukin-1 (IL-1), which signals through IL-1 receptor on non-hematopoietic cells to enable pyogranuloma-mediated control of Yersinia infection. Altogether, our work reveals a monocyte-intrinsic TNF-IL-1 collaborative circuit as a crucial driver of intestinal granuloma function, and defines the cellular target of TNF signaling that restricts intestinal Yersinia infection.

Figure 1.. TNFR1 is required for organized pyogranuloma formation and restriction of Yersinia in intestine and periphery

(A) H&E-stained paraffin-embedded longitudinal small intestinal sections from Yp-infected mice at day 5 post-infection. Dashed line highlights pyogranuloma (left) or necrosuppurative lesion (right). Images representative of two independent experiments. Scale bars = 500 μm (top) and 200 μm (bottom). (B) Histopathological scores of small intestinal tissue from uninfected or Yp-infected mice at day 5 post-infection. Each mouse was scored between 0–4 (healthy-severe) for indicated sign of pathology. Each circle represents one mouse. Lines represent median. Pooled data from two independent experiments. (C) Bacterial burdens in small intestinal PG− and PG+ tissue isolated day 5 post-infection. Each circle represents the mean CFU of 3–5 pooled punch biopsies from one mouse. Lines represent geometric mean. Pooled data from three independent experiments. (D) Total numbers and frequencies of CD45⁺ cells, monocytes, macrophages, and neutrophils in uninfected, PG−, and PG+ small intestinal tissue isolated 5 days post-infection. Each circle represents the mean of 3–10 pooled punch biopsies from one mouse. Lines represent median. Pooled data from three independent experiments. (E) Mean fluorescence intensity (MFI) of CD11b expression on neutrophils in PG+ tissue at day 5 post-infection. Each circle represents the mean of 3–10 pooled punch biopsies from one mouse. Lines represent median. Data representative of three independent experiments. (F) Bacterial burdens in indicated organs at day 5 post-infection. Each circle represents one mouse. Lines represent geometric mean. Pooled data from four independent experiments. (G) Survival of infected WT (n=9) and Tnfr1^−/− (n=21) mice. Pooled data from two independent experiments. (H) Bacterial burdens in small intestinal PG− and PG+ tissue at day 5 post-infection of indicated chimeric mice. Each circle represents the mean Yp-CFU of 3–5 pooled punch biopsies from one mouse. Lines represent geometric mean. Pooled data from two independent experiments. (I) Bacterial burdens in indicated organs at day 5 post-infection of indicated chimeric mice. Each circle represents one mouse. Lines represent geometric mean. Pooled data from two independent experiments. Statistical analysis by Mann-Whitney U test (B, C, D, E, F), Mantel-Cox test (G), and Kruskal-Wallis test with Dunn’s multiple comparisons correction (H, I) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant.

Figure 2.. Autocrine TNF signaling in monocytes is required for control of Yersinia

(A) Schematic of mixed bone marrow chimeras. (B) H&E-stained paraffin-embedded transverse small-intestinal sections from chimeric WT mice reconstituted with Ccr2^gfp/gfp + WT (left), Ccr2^gfp/gfp + Tnfr1^−/− (middle), or Ccr2^gfp/gfp (right) bone marrow, at day 5 post-infection. Dotted lines highlight lesions. Scale bars = 100 μm. Images representative of two independent experiments. (C) Bacterial burdens in small intestinal PG− and PG+ tissue of chimeric WT mice reconstituted with either Ccr2^gfp/gfp + WT (white), Ccr2^gfp/gfp + Tnfr1^−/− (light gray), or Ccr2^gfp/gfp (dark gray) at day 5 post Yp-infection. Each symbol represents one mouse. Lines represent geometric mean. Pooled data from two independent experiments. (D) Bacterial burdens in indicated organs at day 5 post-infection. Each circle represents one mouse. Lines represent geometric mean. Pooled data from two independent experiments. (E) Bacterial burdens in small intestinal PG− and PG+ tissue of chimeric WT mice reconstituted with either Tnf^−/− + WT (white), Tnf^−/− + Ccr2^gfp/gfp (light gray), or Tnf^−/− (dark gray) at day 5 post Yp-infection. Each symbol represents one mouse. Lines represent geometric mean. Pooled data from three independent experiments. (F) Bacterial burdens in indicated organs at day 5 post-infection. Each circle represents one mouse. Lines represent geometric mean. Pooled data from three independent experiments. Statistical analysis by Kruskal-Wallis test with Dunn’s multiple comparisons correction. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant.

Figure 3.. TNFR1 signaling in monocytes controls Yp infection independently of RIPK1 kinase-induced cell death

(A) Bacterial burdens in small intestinal PG− and PG+ tissue of WT (white) and Ripk1^K45A (blue) mice at day 5 post Yp-infection. Each symbol represents one mouse. Lines represent geometric mean. Pooled data from two independent experiments. (B) Bacterial burdens in indicated organs at day 5 post-infection. Each circle represents one mouse. Lines represent geometric mean. Pooled data from two independent experiments. (C) H&E-stained paraffin-embedded longitudinal small intestinal sections from WT (left) and Ripk1^K45A (right) mice at day 5 post Yp-infection with dotted line highlighting lesion. Scale bars = 100 μm. Representative images of two independent experiments. (D) H&E-stained paraffin-embedded transverse small-intestinal sections from chimeric WT mice reconstituted with either Ccr2^gfp/gfp + WT (left), Ccr2^gfp/gfp + Ripk1^K45A (middle), or Ccr2^gfp/gfp (right) bone marrow, at day 5 post Yp-infection with dotted line highlighting lesion. Scale bars = 100 μm. Representative images of two independent experiments. (E) Bacterial burdens in small intestinal PG− and PG+ tissue of chimeric WT mice reconstituted with either Ccr2^gfp/gfp + WT (white), Ccr2^gfp/gfp + Ripk1^K45A (light gray), or Ccr2^gfp/gfp (dark gray) at day 5 post Yp-infection. Each symbol represents one mouse. Lines represent geometric mean. Pooled data from two independent experiments. (F) Bacterial burdens in indicated organs at day 5 post-infection. Each circle represents one mouse. Lines represent geometric mean. Pooled data from two independent experiments. (G) H&E-stained paraffin-embedded longitudinal small intestinal sections from WT and Tnfr1^−/− mice infected with either WT or ΔyopJ Yp at day 5 post-infection. Scale bars = 100 μm. Representative images of three independent experiments. (H) Bacterial burdens in indicated organs at day 5 post-infection. Each circle represents one mouse. Lines represent geometric mean. Pooled data from four independent experiments. (I) Survival of WT (white) and Tnfr1^−/− (gray) mice infected with WT (circles) or ΔyopJ (squares) Yp. n = 9–12 mice per group. Pooled data from two independent experiments. (J) Survival of WT (white) or Tnfr1^−/− (gray) mice infected with WT (circles) or yopEH (squares) Yp. n = 11–15 mice per group. Pooled data from two independent experiments. Statistical analysis by Mann-Whitney U test (A, B), Kruskal-Wallis test with Dunn’s multiple comparisons correction (E, F, H), or Mantel-Cox test (I, J). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant.

Figure 4.. Cell-intrinsic TNFR1 signaling is required for maximal IL-1 production within intestinal pyogranulomas during Yersinia infection

(A) Cytokine levels were measured by cytometric bead array in tissue punch biopsy homogenates isolated 5 days post-infection from chimeric WT mice reconstituted with indicated donor cells. Lines represent median. Pooled data from two independent experiments. (B) Intracellular cytokine levels in monocytes and neutrophils isolated from small intestinal PG+ tissue 5 days post-infection. Each circle represents the mean of 3–10 pooled punch biopsies from one mouse. Lines represent median. Pooled data from three independent experiments. (C) Flow cytometry plots of intracellular IL-1 in monocytes (CD64⁺ Ly-6C^hi) from small intestinal PG+ tissue at day 5 post-infection. Plots representative of two independent experiments. (D) Aggregate datasets from (C) for intracellular IL-1 staining in monocytes and neutrophils in small intestinal PG+ tissue at day 5 post-infection. Each circle represents the mean of 3–10 pooled punch biopsies from one mouse. Lines connect congenic cell populations within individual mice. Pooled data from two independent experiments. Statistical analysis by Kruskal-Wallis test with Dunn’s multiple comparisons correction (A), Mann-Whitney U test (B), congenic cells within mice: Wilcoxon test; across groups: Mann-Whitney U test (D). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant.

Figure 5.. IL-1 signaling is required for organized pyogranuloma formation and intestinal control of Yersinia

(A) Bacterial burdens in small intestinal Peyer’s patches (PP), PG−, and PG+ tissues isolated 5 days post-infection. For PP, each circle represents pooled tissue from one mouse. For PG− and PG+, each circle represents the mean of 3–5 pooled punch biopsies from one mouse. Lines represent geometric mean. Pooled data from three independent experiments. (B) H&E-stained paraffin-embedded longitudinal small intestinal sections from Yp-infected mice at day 5 post-infection. Representative images of one experiment. Scale bars = 250 µm. (C) Survival of infected WT (n=26) and Il1r1^−/− (n=20) mice. Pooled data from two independent experiments (D) Bacterial burdens in small intestinal PP, PG−, and PG+ tissues at day 5 post-infection of indicated genotypes. For PP, each circle represents pooled tissue from one mouse. For PG− and PG+, each circle represents the mean of 3–5 pooled punch biopsies from one mouse. Lines represent geometric mean. Pooled data from three independent experiments. (E) Survival of infected WT (n=27, n=19), Il1a^−/− (n=22), and Il1b^−/− (n=21) mice. Pooled data from three (WT vs Il1a^−/−) and two (WT vs Il1b^−/−) independent experiments. Statistical analysis by Mann-Whitney U test (A, D) or Mantel-Cox test (C, E) *p<0.05, **p<0.01, ****p<0.0001, ns = not significant.

Figure 6.. Monocyte-derived IL-1 signals to nonhematopoietic cells to restrict Yersinia infection in intestinal pyogranulomas

(A) Bacterial burdens in small intestinal Peyer’s patches (PP), PG−, and PG+ tissues at day 5 post-infection of indicated chimeric mice. For PP, each circle represents pooled tissue from one mouse. For PG− and PG+, each circle represents the mean of 3–5 pooled punch biopsies from one mouse. Lines represent geometric mean. Data pooled from two independent experiments. (C) Bacterial burdens in small intestinal Peyer’s patches (PP), PG−, and PG+ tissues isolated 5 days post-infection of indicated chimeric mice. For PP, each circle represents one mouse. For PG− and PG+, each circle represents the mean of 3–5 pooled punch biopsies from one mouse. Lines represent geometric mean. Pooled data from three independent experiments. (C) Bacterial burdens in indicated organs at day 5 post-infection of indicated chimeric mouse. Each circle represents one mouse. Lines represent geometric mean. Data pooled from three independent experiments. (D) Model of TNF-IL-1 circuit mediated by monocyte and stromal compartment to promote Yp restriction within intestinal pyogranulomas. All statistical analysis by Kruskal-Wallis test with Dunn’s multiple comparisons correction. *p<0.05, **p<0.01, ***p<0.001, ns = not significant.