Interleukin-10 production by innate lymphoid cells restricts intestinal inflammation in mice

Abstract

Interleukin-10 (IL-10) is an immunomodulatory cytokine critical for intestinal immune homeostasis. IL-10 is produced by various immune cells but IL-10 receptor signaling in intestinal CX₃CR1⁺ mononuclear phagocytes is necessary to prevent spontaneous colitis in mice. Here, we utilized fluorescent protein reporters and cell-specific targeting and found that Rorc-expressing innate lymphoid cells (ILCs) produce IL-10 in response to anti-CD40-mediated intestinal inflammation. Deletion of Il10 specifically in Rorc-expressing ILCs led to phenotypic changes in intestinal macrophages and exacerbated both innate and adaptive immune-mediated models of experimental colitis. The population of IL-10⁺ producing ILCs shared markers with both ILC2 and ILC3 with nearly all ILC3s being of the NCR⁺ subtype. Interestingly, Ccl26 was enriched in IL-10⁺ ILCs and was markedly reduced in IL-10-deficient ILC3s. Since CCL26 is a ligand for CX₃CR1, we employed RNA in situ hybridization and observed increased numbers of ILCs in close proximity to Cx3cr1-expressing cells under inflammatory conditions. Finally, we generated transgenic Rorc^tdTomato reporter mice that faithfully marked RORγt⁺ cells that could rescue disease pathology and aberrant macrophage phenotype following adoptive transfer into mice with selective Il10 deficiency in ILC3s. These results demonstrate that IL-10 production by a population of ILCs functions to promote immune homeostasis in the intestine possibly via direct effects on intestinal macrophages.

Keywords: CCL26; CX(3)CR1; IBD; IL-10; ILC3.

Figure 1.. IL-10 is induced in ILCs during intestinal inflammation.

A) Schematic of anti-CD40 colitis model in Rag1^−/−;Il10^GFP;Rosa26^lsl-tdTomato;Rorc^Cre mice. B) Time course of body weight change following anti-CD40 treatment. C) Hematoxylin and Eosin (H&E)-stained colonic sections (left) and inflammation score quantified by a pathologist blinded to genotype and treatment (right). (n ≥ 7), scale bar = 100μm. D) Representative flow cytometric analysis of GFP expressing cells gated on Live,CD45⁺,Thy1.2⁺ at day 0 and 7 days following anti-CD40 treatment (left) with longitudinal assessment of IL-10-GFP expressing cells (right) (n ≥ 5). E) Heatmap showing normalized expression of differentially expressed genes between GFP⁺ and GFP⁻ ILCs sorted from the colons of Rag1^−/−;Il10^GFP;Rosa26^lsl-tdTomato;Rorc^Cre mice 7 days post-anti-CD40 treatment, n=4 mice per group. F) Representative flow cytometric analysis of IL-10-GFP expression on colon ILCs 7 days post anti-CD40 (left) assessed for expression of ILC3 markers (CCR6, NKp46) and ILC2 markers (KLRG1, IL-25R) (right) and quantified in G. H) IL-10-GFP⁺ ILCs sorted from the colon 7 days post-anti-CD40 treatment were pooled from 5 mice and restimulated with PMA/ionomycin for 4 hours in the presence of a Golgi inhibitor. Cells were then stained for intracellular IL-22, IFNγ, and IL-17A. I) Flow cytometry plot depicting GFP⁺ and GFP⁻ ILCs that were sorted from pooled colons of 5 mice 7 days post-anti-CD40 treatment and stained for nuclear expression of RORγt, TBET, and GATA3. Data are pooled from 2 or more independent experiments. Each dot represents an individual mouse. Bars are the mean ± SEM. **P < 0.01, ***P < 0.001. one-way ANOVA: A; unpaired t-test: C,D,G.

Figure 2.. Selective deletion of Il10 in Rorc-expressing cells augments anti-CD40-mediated colitis.

A) Schematic of Rag1^−/−;Il10^flox/flox;Rosa26^lsl-tdTomato;Rorc^Cre (Il10^ΔILC3) mice. B) Confirmation of selective deletion of Il10 in ILC3s by PCR of sorted cells. C) Depiction of anti-CD40 model in Il10^ΔILC3 mice and IL-10-sufficient littermate controls (Rag1^−/−). D) Kaplan-Meier survival curve following PBS or anti-CD40 treatment of Rag1^−/− and Il10^ΔILC3 mice. E) Weight loss as a percentage of initial body weight over the course of 7 days following anti-CD40 administration. F) Representative H&E-stained colonic sections of Rag1^−/− and Il10^ΔILC3 mice following anti-CD40 (left) with quantified histological score (right). Each dot represents an individual mouse. Data are pooled from four independent experiments. (PBS: n =2, anti-CD40 groups: n ≥ 8), Bars are the mean ± SEM. *P < 0.05, **P < 0.01, ^###, *** P < 0.001. Gehan-Breslow-Wilcoxon test: E; multiple comparisons: F.

Figure 3.. IL-10 deficiency in Rorc-expressing ILCs drives intestinal macrophage dysfunction and permits T cell-mediated colitis.

A) Representative flow cytometry plots depicting Ly6C^hiMHCII^hi vs. Ly6C^loMHCII^hi monocyte/macrophage in colonic tissue of Rag1^−/− and Il10^ΔILC3 mice (left) and quantified (right) (n ≥ 4). B) Peritoneal macrophages were isolated from Rag1^−/− and Il10^ΔILC3 mice and stimulated with 100 ngml⁻¹ LPS for 6 hours. cDNA was generated from purified RNA and subjected to multiplex qPCR using select Taqman probes with Tbp set as an internal reference gene (n ≥ 6). C) Schematic of WT total CD4⁺ T cell transfer into Rag1^−/− and Il10^ΔILC3 mice. D) Representative flow cytometry plots depicting the frequency of colonic Treg cells in Rag1^−/− and Il10^ΔILC3 mice transferred with unfractionated WT CD4⁺ T cells (n ≥ 7). E) Representative H&E-stained colonic sections of Rag1^−/− and Il10^ΔILC3 mice 8-weeks post T cell transfer (left) and quantified by a pathologist blinded to genotype and treatment (right) (n ≥ 9). F) Representative flow cytometry plots of colonic macrophage and G) neutrophil populations recovered from Rag1^−/− and Il10^ΔILC3 mice 6 weeks post-transfer of unfractionated CD4⁺ T cells (n ≥ 4). H) Representative flow cytometry plot of CD4⁺ T cells recovered from transferred mice restimulated with PMA/ionomycin for 4 hours in the presence of a Golgi inhibitor and stained for intracellular TNF, IL-17A, and IFNγ. Each dot represents an individual mouse (n ≥ 3). Data are pooled from two or more independent experiments. Bars are the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired t-test: A,B,D-H.

Figure 4.. Il10^ΔILC3 mice exhibit enhanced susceptibility to inflammation-associated carcinogenesis.

A) Cartoon of AOM/DSS colitis-associated cancer protocol in Rag51^−/− and Il10^ΔILC3 mice that were reconstituted with total WT CD3⁺ T cells. B) Weight curve shown as % initial body weight over the course of the experiment with gray box depicting each DSS cycle. C) Frequency of splenic CD3⁺ T cells at experimental endpoint. D) Tumor number and E) tumor volume determined at experimental endpoint. F) Representative H&E-stained colonic sections of Rag1^−/− and Il10^ΔILC3 mice highlighting intestinal tumors (left) with dysplasia graded by a pathologist (right) (n=9/genotype). Each dot represents an individual mouse except for tumor volume where each data point is an individual tumor. Data are pooled from two independent experiments. Bars are the mean ± SEM. **P < 0.01, unpaired t-test: C-E.

Figure 5.. Ccr6 is enriched in IL-10 expressing ILCs.

A) Volcano plot comparing transcripts in colonic GFP⁺ and GFP⁻ ILCs sorted from Rag1^−/−;Il10^GFP;Rosa26^lsl-tdTomato;Rorc^Cre mice 7 days post-anti-CD40 treatment, (n=4 mice/group). B) Volcano plot depicting differentially expressed genes in Live,CD45⁺,Thy1.2⁺,CD127⁺,Tomato⁺ ILCs isolated from colonic lamina propria of unmanipulated Rag1^−/−;Il10^flox/flox;Rosa26^lsl-tdTomato;Rorc^Cre mice or IL-10-sufficient littermates. C) qPCR analysis for relative expression of Ccl26 in ILC3s sorted from the colonic lamina propria of Rag1^−/−;Il10^GFP;Rosa26^lsl-tdTomato;Rorc^Cre mice and stimulated for 0 or 24 hrs with 20 ngml⁻¹ recombinant IL-23. Each dot is an individual mouse (n ≥ 3). Bars are the mean ± SEM. *P < 0.05; paired t-test: C.

Figure 6.. Increased number of ILCs found in close proximity to CX₃CR1⁺ macrophages following anti-CD40 treatment in IL-10-sufficient but not Il10^DILC3 mice.

A) Schematic of analysis pipeline on colon sections 7 days post-PBS or anti-CD40 treatment in Rag1^−/− (WT) or Il10^ΔILC3 mice. B) 3-color fluorescence images captured on an Aperio VERSA 200 of RNA in situ hybridization for murine Cx3cr1 (green), Thy1 (red), DAPI (gray) with cell boundaries outlined in blue. C) Zoomed in view of dashed yellow box from (B) highlighting RNAscope signal in nuclei. D) Spatial profiling of computationally defined Thy1 and Cx3cr1 positive cells to calculate cell-cell distances located within the same crypt (left) with the mean Euclidian distances and number of nearest neighbors quantified (right). ***P < 0.001; unpaired t-test: D.

Figure 7.. Rorc^tdTomato transgenic mice faithfully mark endogenous RORγt expression.

A) Schematic of a targeting strategy of a bacterial artificial chromosome (BAC) covering the Rorc locus to introduce tdTomato 3’ of the ATG start site of the RORγt specific isoform. B) qPCR analysis for Rorc and Tbx21 transcripts in sorted tomato⁺ or tomato⁻ CD4⁺ splenic T cells from Rorc^tdTomato mice (n = 3). C) Naïve CD4⁺ T cells from Rorc^tdTomato mice cultured in vitro under Th17 polarizing conditions for 4 days, sorted based on tdTomato, and stained for RORγt protein for examination by flow cytometry. D) tdTomato⁻ naïve CD4⁺ T cells sorted from Rorc^tdTomato mice and cultured in vitro under Th1-, Th2-, or Th17- polarizing conditions respectively. Representative flow cytometry plots showing frequency of tdTomato⁺ IFNγ producing Th1 cells, IL-4 producing Th2 cells, and IL-17 producing Th17 cells (left) and quantified (right) (n ≥ 6). E) Representative flow cytometry data showing tdTomato expression of colonic CD4⁺ T cells in Rorc^tdTomato and Rorc^tdTomato;Il6^−/− mice (left) with quantification (right) (n ≥ 3). F) Representative flow cytometric analysis of splenic ILC3s from Rorc^tdTomato mice assessed for expression of CCR6 and NKp46 compared to ILCs gated on RORγt intracellular staining using cells from the same donor mouse (n ≥ 3). G) IL-22 expression in Tomato⁺ ILC3s following overnight IL-23 treatment of colonic leukocytes isolated from Rorc^tdTomato mice (Lin-1: B220, CD11b, CD11c; Lin-2: CD3, CD5, CD8, NK1.1) (n = 3). Each dot represents a biological replicate for in vitro data or an individual mouse for in vivo data. Data are pooled from three independent experiments. Bars are the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired t-test: B,E,F; multiple comparisons: D.

Figure 8.. Transfer of WT ILC3s reverses pro-inflammatory macrophage phenotype and improves survival and inflammation following anti-CD40 treatment.

A) Schematic depicting transfer of IL-10-sufficient CD45.1⁺Lin⁻Thy1.2⁺Tomato⁺ ILC3s into Il10^ΔILC3 mice then treated with anti-CD40. B) Kaplan-Meier survival curve for Il10^ΔILC3 mice treated with anti-CD40 previously injected with/without IL-10-sufficient ILC3s (n ≥ 7). C) Representative H&E-stained colonic sections of Il10^ΔILC3 mice +/− WT ILC3s treated with anti-CD40 (left) and scored (right) (n ≥ 4). D) Flow cytometry plots showing colonic macrophages defined by Ly6C and MHCII expression isolated from Il10^ΔILC3 mice and Il10^ΔILC3 mice transferred with IL-10-sufficient CD45.1⁺;tdTomato⁺ ILC3s (left) with Ly6C^hiMHCII^hi, Ly6C^loMHCII^hi monocyte/macrophage quantified (right) (n ≥ 3). E) Persistence of CD45.1⁺ ILC3s post-transfer into Il10^ΔILC3 mice versus non-transferred Il10^ΔILC3 mice. Each dot represents an individual mouse. Data are pooled from two independent experiments. *P < 0.05; Log-rank (Mantel-Cox) test: B; unpaired t-test: C,D.