Citrobacter rodentium infection activates colonic lamina propria group 2 innate lymphoid cells

Abstract

Group 3 innate lymphoid cells (ILC3s) play a major role in protecting against infection with the enteric mouse pathogen Citrobacter rodentium (CR) used to model infections with enteropathogenic and enterohaemorrhagic Escherichia coli. ILC3s-secreted IL-22 induces secretion of IL-18, antimicrobial peptides and nutritional immunity proteins as well as activation of tissue regeneration processes. While ILC2s have traditionally been associated with immune responses to helminth infection and allergic inflammation via the production of type 2 cytokines (e.g. IL-4, IL-5, IL-9 and IL-13), more recently they have been implicated in protection against Clostridium difficile and Helicobacter pylori infections. Here we show that colonic lamina propria ILC2s expand in response to CR infection and secrete IL-4, IL-5 and IL- 13, which are involved in maintenance of the intestinal barrier function, tissue repair and mucus secretion. When stimulated with IL-18, and IL-33 as a control, colonic ILC2s from uninfected mice secreted type 2 cytokines. Injection of IL-18 binding protein (IL18 BP), at 2- and 3-days post CR infection, blocked expansion of ILC2s in vivo. While ILC2s do not expand in CR-infected Il22-/- mice, injection of IL-18 into Il22-/- mice at 2- and 3-days post CR infection triggered ILC2s expansion. Importantly, injection of anti-IL-13, at 2- and 4-days post CR infection, diminished local secretion of IL-10 and IL-22. These data show that ILC2s are activated in response to infection with an enteric Gram-negative pathogen. Moreover, stimulation with IL-18 plays a role in ILC2s expansion and secretion of type 2 cytokines, which may participate in shaping the local immunological landscape.

Fig 1. Colonic lamina propria ILC2s expand in response to CR at 4 dpi.

A. Faecal CR (CR) at 4 dpi. Mock infected (PBS) were used as controls. B. Crypt lengths measured in H&E-stained colon sections. C. Representative H&E sections from the distal colons of uninfected (PBS) and infected mice at 4 dpi (scale bar 100 μm). D. Representative and E. quantitative colon length. F. Faecal MPO levels measured by ELISA. G. Flow cytometry gating strategy used to identify ILC2s from mice colonic lamina propria cells. Debris (SSC-A vs. FSC-A) and doublets (FSC-H vs. FSC-A) were excluded, and live/dead discrimination was determined using the LIVE/DEAD fixable blue dead cell stain kit (FSC-A vs. Live/Dead). Total ILCs were defined as CD45 + NKp46 + /-Lineage- [CD3/B220/CD19/TER119/Gr-1/CD5/CD11c/CD4/Nk1.1]. Expression of the CD127, KLRG1 and GATA3 markers were used to identify ILC2 (CD127 + KLRG1 + GATA3+). H. Numbers and frequency of ILC2s increased upon infection. dpi: days post infection. Data shown are pooled values from three independent experiments. P values were determined on data plotted as mean ± SEM using Student’s-t-test. ns: non-significant; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig 2. ILC2 cytokine profile during CR infection.

A. Total ILC2s numbers sorted from pooled colons of uninfected and infected mice. B. IL-4, IL-5 and IL-13 levels in the supernatant from the same sorted cells measured by ELISA. C. Representative flow cytometry plots of cytokine expression from fluorescent-minus-one (FMO), unstimulated and stimulated samples. D. Total number of IL-4⁺, IL-5⁺ and IL-13⁺ of sorted ILC2 cells from colons pooled from uninfected and infected mice. Cells were stimulated for 4 h in the presence of protein transport inhibitor cocktail to assess cytokine intracellularly by flow cytometry. Data shown are pooled values from three independent experiments. P values were determined on data plotted as mean ± SEM using Student’s-t-test. *p < 0.05; ***p < 0.001; ****p < 0.0001.

Fig 3. Colonic lamina propria ILC2s proliferate in response to IL-18.

A. RT-qPCR analysis of Il18, Il25 and Il33 transcript expression in colons of CR-infected and PBS-dosed mice at 4 dpi. Each data point represents one mouse. B. IL-18 levels secreted from explant colonic tissue and detected by ELISA. C. Representative flow cytometry plots and D. Summary data for Ki67 expression (represented as percentage of ILC2) by FACS sorted colonic lamina propria ILC2s. Cells were cultured for 3 days in vitro with recombinant IL-2 and IL-7 (control) in addition to IL-18, IL-22 or IL-33. E. Histogram showing positive and negative peaks of proliferative ILC2s in response to different cytokines. F. IL-5, IL-9 and IL-13 levels in the supernatant from cultured ILC2s measured by ELISA. Cells pooled from n = 10 mice per experiment. Data shown are pooled values from four independent experiments. P values were determined on data plotted as mean ± SEM using Student’s-t-test (A-B) and ANOVA with Bonferroni post-test for multiple comparisons (D, F). ns: non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig 4. Injection of IL-18 BP into CR-infected mice results in lower ILC2s expansion.

A. Experimental plan. B. Faecal CR shedding at 4 dpi. C. Colon length at 4 dpi. D. Frequency and absolute number of ILC2s is reduced in the colon of CR-infected mice treated with IL-18 BP. Data shown are pooled values from three independent experiments. P values were determined on data plotted as mean ± SEM using Student’s-t-test. ns: non-significant; *p < 0.05; **p < 0.01.

Fig 5. Il22^-/- mice display lower IL-18-mediated ILC2s expansion.

A. Frequency and absolute number of ILC2s in CR infected Il22^-/- mice at 4 dpi. B. Experimental plan of rIL18 treatment. C. Faecal CR shedding at 4 dpi. D. Frequency and absolute number of ILC2s post rIL-18 treatment. Data shown are pooled values from three independent experiments. P values were determined on data plotted as mean ± SEM using Student’s-t-test. ns: non-significant; **p < 0.01; ***p < 0.001.

Fig 6. Neutralisation of IL-13 post CR infection results in lower anti-inflammatory cytokine levels.

A. Experimental plan. B. Faecal CR shedding at 4 dpi. C. Representative and quantitative colon length. D. Weight-to-length ration of colon at 6 dpi. E. Faecal MPO levels measured by ELISA. F. Levels of IL-10 and IL-22 in colonic explant culture. Data shown are pooled values from two independent experiments with three mice in each group per experiment. P values were determined on data plotted as mean ± SEM using Student’s-t-test (B, F) and ANOVA with Bonferroni post-test for multiple comparisons (C-E). ns: non-significant; *p < 0.05; **p < 0.01; ***p < 0.001.