Context-dependent role of group 3 innate lymphoid cells in mucosal protection

Abstract

How group 3 innate lymphoid cells (ILC3s) regulate mucosal protection in the presence of T cells remains poorly understood. Here, we examined ILC3 function in intestinal immunity using ILC3-deficient mice that maintain endogenous T cells, T helper 17 (T_H17) cells, and secondary lymphoid organs. ILC3s were dispensable for generation of T_H17 and T_H22 cell responses to commensal and pathogenic bacteria, and absence of ILC3s did not affect IL-22 production by CD4 T cells before or during infection. However, despite the presence of IL-22-producing T cells, ILC3s and ILC3-derived IL-22 were required for maintaining homeostatic functions of the intestinal epithelium. T cell-sufficient, ILC3-deficient mice were capable of pathogen clearance and survived infection with a low dose of Citrobacter rodentium. However, ILC3s promoted pathogen tolerance at early time points of infection by activating tissue-protective immune pathways. Consequently, ILC3s were indispensable for survival after high-dose infection. Our results demonstrate a context-dependent role for ILC3s in immune-sufficient animals and provide a blueprint for uncoupling of ILC3 and T_H17 cell functions.

Figure 1.. RORγ^STOP/CD4 mice specifically lack ILC3 in the presence of T cells, including Th17 cells.

(A) Schematic of generation of RORγ^STOP/CD4 (STOP/CD4) mice. (B) Total thymus cellularity in WT, STOP, and STOP/CD4 mice. (C) Thymoma-mediated mortality of WT, STOP, and STOP/CD4 mice. n = 27–33 mice per genotype. (D-F) Thymocyte development in WT, STOP, and STOP/CD4 mice, including (E) double-positive (DP) and (F) CD4 single-positive (CD4 SP) thymocytes. One out of two independent experiments with similar results, n = 4–5 mice per group. (G-I) Small intestinal RORγ⁺Foxp3^– Th17 cells (G, H) and Foxp3⁺RORγt^– Tregs (I) in WT, STOP, and STOP/CD4 mice. Plots gated on TCRβ⁺CD4⁺ cells (G, H) or total lymphocytes (I). Combined data from two out of four independent experiments, n = 5–8 mice per group. (J-L) Proportions (J, K) and number (L) of small intestinal ILC3 in WT, STOP and STOP/CD4 mice. Plots gated on Lin^– cells (J) or total lymphocytes (K). Combined data from several independent experiments, n= 7–12 mice per group.

Figure 2.. ILC3 control steady state intestinal epithelial cell function.

(A) Multidimensional scaling (MDS) ordination of transcriptomes of SI LP CD4 T cells (top) and terminal ileum intestinal epithelial cells (IEC) (bottom) from WT, STOP and ΔILC3 mice. n = 2–4 mice per group. (B) Differentially expressed genes decreased in STOP compared to WT mice are plotted and compared between WT and ΔILC3 mice for either LP CD4 T cells (top) or IEC (bottom). “Like WT”, similar expression between WT and ΔILC3 mice. “Low in ΔILC3”, decreased in ΔILC3 compared to WT. T-cell receptor (TCR) genes, Th17 signature genes or IL-22 controlled genes are highlighted below the heatmaps. (C) Th17 cell signature profile genes in RNA-Seq data from SI LP CD4 T cells from WT, STOP and ΔILC3 mice. (D) Pathways similarly enriched in LP CD4 T cells from WT and ΔILC3 mice compared to STOP mice; Top 5 enriched pathways of GO biological process and KEGG categories are shown. Pathways related to intestinal T cell functionality are shaded in red. (E) IL-22-controlled genes in RNA-Seq data from SI LP IEC from WT, STOP and ΔILC3 mice. (F) Gene set enrichment analysis (GSEA) of IL-22-regulated genes in transcriptomes of IEC from WT versus ΔILC3 mice. (G) Quantitative RT-PCR for Il22 mRNA from purified CD4 T cells from SI LP of WT, STOP and ΔILC3 mice. (H) IL-22 protein levels in supernatants of in vitro stimulated SI LP CD4 T cells. (I) Intracellular staining for IL-22 in SI LP TCRβ⁺CD4⁺ T cells from WT, STOP and ΔILC3 mice (see also Figure S5D). Data from one of two independent experiments with similar results, n = 3–4 mice per group.

Figure 3.. ILC3 and bystander Th17 cells participate in early Citrobacter protection.

(A, B) Survival (A) and pathogen CFUs in feces at day 4 post infection (B) of WT, STOP and ΔILC3 mice infected with high dose (10⁹ CFU/animal) Citrobacter rodentium (C. rod). One out of several independent experiments with similar results, n = 4–5 mice per group. (C) Survival of WT, STOP and ΔILC3 mice following infection with low dose C. rod (5×10⁷ CFU/animal). Data from two out of six independent experiments with similar results, n = 5–6 mice per group. p value refers to ΔILC3 vs STOP mice. (D) Lethally irradiated ILC3/Th17-deficient and LN/PP-deficient RORγ-KO mice were reconstituted with BM from WT, STOP or ΔILC3 mice. (E, F) Pathogen CFUs at day 5 (E) and survival (F) of RORγ-KO BM chimeras (D) following infection with low dose C. rod. Data from two independent experiments with similar results, n = 7–9 mice per group. (G) STOP (left) and ΔILC3 (right) mice were pre-treated with anti-CD4 antibody to deplete CD4 T cells, or isotype antibody as control, and infected with low dose C. rod. One out of two independent experiments with similar results, n = 4–6 mice per group. (H) STOP (left) and ΔILC3 (right) mice were treated with neutralizing anti-IL-22 antibody (or isotype control) and infected with low dose C. rod. One out of two independent experiments with similar results, n = 2–5 mice per group. (I) Low dose C. rod infection in SFB-negative or SFB-positive STOP and ΔILC3 mice. One experiment, n = 3–5 mice per group. (J) RORγ-KO BM chimeras (D) were infected with low dose C. rod and supplemented with exogenous IL-22-Fc or isotype control every other day starting at Day 0. One out of two independent experiments with similar results, n = 3–5 mice per group. Statistics survival curves, Mantel-Cox test.

Figure 4.. ILC3 control intestinal epithelial cell IL-22 program in the presence of lymph nodes and Peyer’s patches.

(A) Schematic of generation of LN/PP-sufficient, Th17 cell-sufficient, ΔILC3 mice (ΔILC3^LN/PP) and experimental controls. (B, C) RORγt⁺ Th17 cells in SI LP of ΔILC3^LN/PP mice and controls. Plots gated on CD4⁺TCRβ⁺ cells. (D) RORγt⁺Foxp3⁺ Treg cells in SI LP of ΔILC3^LN/PP mice and controls. Plots gated on CD4⁺TCRβ⁺ cells. (E-G) ILC3 in SI LP of ΔILC3^LN/PP mice and controls. Plots gated on Lin^– lymphocytes. Combined data from several independent experiments, n = 5–6 mice per group. (H-J) RORγt⁺ (H, I) and RORγt^neg (H, J) IL-22 producing Lin^– lymphocytes in SI LP of ΔILC3^LN/PP mice and controls. Combined data from two independent experiments, n = 5–6. (K) RNA-seq of terminal ileum from WT^LN/PP, STOP^LN/PP, and ΔILC3^LN/PP mice. Differentially expressed genes decreased in STOP^LN/PP compared to WT^LN/PP mice are plotted and compared between WT and ΔILC3^LN/PP mice. Only mice without residual ILC3. “Like WT^LN/PP”, similar expression between WT^LN/PP and ΔILC3^LN/PP mice. “Low in ΔILC3^LN/PP”, decreased in ΔILC3^LN/PP compared to WT^LN/PP. IL-22 induced genes marked in green. n = 4 mice per group. (L) Quantitative PCR for IL-22 controlled IEC genes in terminal ileum of WT^LN/PP, STOP^LN/PP, and ΔILC3^LN/PP mice. Only mice without residual ILC3. RE, relative expression. n = 5–7 mice per group. (M) Top GO biological process pathways enriched in terminal ileum of WT^LN/PP compared to ΔILC3^LN/PP mice. Only mice without residual ILC3. Pathways related to anti-microbial defense are indicated in blue.

Figure 5.. ILC3 are essential for survival of high dose Citrobacter rodentium infection.

(A) Survival of WT^LN/PP and ΔILC3^LN/PP mice infected with high dose C. rod. One out of two independent experiments with similar results, n = 3–5 mice per group (B) Quantitative PCR for Il22 and Reg3g transcripts in colon at day 4 post infection. RE, relative expression. (C, D) Number of IL-22⁺ ILC3 and CD4 T cells in colon LP of infected WT^LN/PP and ΔILC3^LN/PP mice at Day 4. Plots in C gated on Lin^– lymphocytes. (E-G) Pathogen burden in mLN (E), liver (F), and spleen (G) on day 4 post infection. (H) Multidimensional scaling (MDS) ordination of transcriptomes of colon tissue from infected WT^LN/PP and ΔILC3^LN/PP mice at Day 4. (I) Differentially expressed genes increased upon infection in WT^LN/PP mice at Day 4. Genes controlled by IL-22 are marked in green and intestinal epithelial cell (IEC) genes are marked in red. n = 2 mice per group. (J) Enrichment of top infection-activated pathways in infected WT^LN/PP mice versus infected ΔILC3^LN/PP mice. Infection-activated pathways were defined as pathways increased in infected WT^LN/PP mice compared to uninfected WT^LN/PP mice. GSEA, gene-set enrichment analysis. MPS, module perturbation score. (K) Differential gene expression in colon of infected ΔILC3^LN/PP versus WT^LN/PP mice at Day 4. Red, transcripts increased in ΔILC3^LN/PP mice compared to WT^LN/PP mice. Blue, transcripts decreased in ΔILC3^LN/PP mice compared to WT^LN/PP mice. Green, IL-22-induced genes.

Figure 6.. ILC3 decrease disease severity during low dose Citrobacter rodentium infection.

WT^LN/PP, STOP^LN/PP and ΔILC3^LN/PP mice were infected with low dose C. rod. (A-C) Pathogen levels in feces (A), spleen (B), and colonic tissue (C) at day 4 post infection. (D, E) Fecal C. rod levels (D) and survival (E). (F) Weight loss. Data from one of two independent experiments with similar results, n=3–5 mice per group. (G-J) Quantitative PCR for Ccl2 (G), Cxcl1 (H), Tnfa (I), and Lcn2 (J) transcripts in distal colon at day 8 post infection. Combined data from two independent experiments, n = 4–6 mice per group. (K, L) Intestinal pathology in colon of infected WT^LN/PP and ΔILC3^LN/PP mice in early (Day 8) and late (Day 14) timepoints. Images taken at 50x magnification. Scale bars, 500 μm. Combined data from two independent experiments, n = 5–6 mice per group.

Figure 7.. Effects of ILC3 on primary T cell responses following Citrobacter rodentium infection.

(A-D) ΔILC3^LN/PP mice and LN/PP-sufficient controls were infected with low dose C. rod and effector CD4 T cell responses in colonic LP examined at early (Day 8) and late (Day 14) stages of infection by flow cytometry. Plots gated on CD4⁺TCRβ⁺ cells. Data from two independent experiments, n = 3–7 mice per group.

Figure 8.. ILC3 are not required for induction of SFB-specific Th17 cells.

(A) Fecal SFB levels in ΔILC3 mice and littermate controls. (B, C) Endogenous Th17 cells in SI LP of SFB-colonized WT, STOP and ΔILC3 mice. Plots gated on TCRβ⁺CD4⁺ cells. (D) Schematic of adoptive transfer experiments. (E, F) Expansion of CD45.1⁺ SFB-specific 7B8 TCR Tg CD4 T cells in SI LP of SFB-colonized WT, STOP and ΔILC3 recipient mice. Plots gated on TCRβ⁺CD4⁺ cells. (G, H) IL-17A (GFP) expression in transferred CD45.1⁺Vβ14⁺ SFB-specific 7B8.IL-17A-GFP TCR Tg CD4 T cells in SI LP of SFB-colonized WT, STOP and ΔILC3 recipient mice. Plots gated on CD45.1⁺TCRβ⁺CD4⁺ cells. Data from two independent experiments. n = 3–5 mice per group.