ILC2s activated by IL-25 promote antigen-specific Th2 and Th9 functions that contribute to the control of Trichinella spiralis infection

Abstract

IL-25, an IL-17 family cytokine, derived from epithelial cells was shown to regulate Th2- and Th9-type immune responses. We previously reported that IL-25 was important in promoting efficient protective immunity against T. spiralis infection; however, the cellular targets of IL-25 to elicit type-2 immunity during infection have not yet been addressed. Here, we investigated IL-25-responding cells and their involvement in mediating type-2 immune response during T. spiralis infection. ILC2 and CD4+ Th2 cells residing in the gastrointestinal tract of T. spiralis infected mice were found to express high levels of surface interleukin-17 receptor B (IL-17RB), a component of the IL-25 receptor. Following T. spiralis infection, activated ILC2s upregulated surface MHCII expression and enhanced capacity of effector T helper cell in producing antigen-specific Th2 and Th9 cytokines through MHCII-dependent interactions. Reciprocally, lack of CD4+ T helper cells impaired ILC2 function to produce type 2-associated cytokines in responding to IL-25 during T. spiralis infection. Furthermore, mice deficient in IL-17RB showed markedly reduced ILC2 numbers and antigen-specific Th2 and Th9 cytokine production during T. spiralis infection. The Il17rb-/- mice failed to mount effective antigen specific Th2 and Th9 functions resulting in diminished goblet cell and mast cell responses, leading to delayed worm expulsion in the intestines and muscles. Thus, our data indicated that ILC2s and CD4+ Th2 cells are the predominant cellular targets of IL-25 following T. spiralis infection and their collaborative interactions may play a key role in mounting effective antigen-specific Th2 and Th9 cytokine responses against T. spiralis infection.

Fig 1. Type 2 innate lymphoid cells (ILC2s) and CD4⁺Th2 cells are the major immune cells expressed IL-25 receptor (IL-17RB) in response to T. spiralis infection.

(A) BALB/c mice were infected with T. spiralis and their lamina propria cells were analyzed for the expression of IL-17RB at day 7 following infection. The gated IL-17RB⁺ population from infected mice was stained with lineage markers (CD11b, CD11c, B220, CD19, DX5, Gr.1, CD8), IL-7Rα, ST2, CD3 and CD4. The expression of IL-7Rα and ST2 on Lin^- cells from IL-17RB⁺ (open histogram) were compared with those from IL-17RB^- (shaded histogram) population. (B and C) Phenotypic analysis and frequencies of IL-17RB⁺GFP⁺ by CD3⁺CD4⁺ and Lin^- cells in the lamina propria of the small intestines (B) and mesenteric lymph nodes (C) of 4GET mice following T. spiralis infection at days 3, 7 and 14. (D) Cell surface marker expression (IL-7 receptor α, KLRG1, ST2, c-KIT and Thy1) in the IL-17RB⁺GFP⁺Lin^- cells in the lamina propria of the small intestines after T. spiralis infection for 7 days. Flow cytometric data shown are a representative histogram profile of one of three independent experiments. The solid gray line indicates the plot profile of isotype-matched control antibody and shaded histogram represents the profile of the indicated antibody. The data represent one of three independent experiments (n = 4 mice each group). Error bars denoted mean ± SD. Significance was determined using one-way ANOVA * p <0.05, ** p <0.01*** and p <0.001 (compared with data in naïve mice).

Fig 2. ILC2s induced by IL-25 facilitates antigen-specific Th2 and Th9 cytokine production in response to T. spiralis infection.

(A) Mesenteric lymph node cells of naïve mice or mice infected with T. spiralis stimulated with or without T. spiralis antigen in the presence or absence of IL-25 for 72 hours were measured for cytokines as indicated and compared. (B) Gated ILC2s (Lin^-CD4^-IL-17RB⁺CD127⁺T1/ST2⁺GFP⁺) in the lamina propria isolated from the small intestine of naïve and T. spiralis–infected mice were analyzed for the expression of MHCII and compared. (C) ILC2s sorted from IL-25-treated mice and pulsed with T. spiralis antigen or OVA antigen were co-cultured with or without purified effector/memory CD4⁺ T cells (CD44⁺CD62L^-CD4⁺CD3⁺) from mesenteric lymph nodes of T. spiralis–infected mice for 72 hours. An anti-MHCII antibody was added to effector/memory CD4⁺ T cells co-cultured with T. spiralis-pulsed ILC2 cells. The indicated cytokines in the supernatant were examined by ELISA. Data represented one of three independent experiments (n = 4 mice each group). Error bars denote mean ± SD. Significance was determined by one-way ANOVA * p <0.05, ** p <0.01 and *** p <0.001.

Fig 3. ILC2s failed to function in T. spiralis–mice ablated CD4+ T cells.

BALB/c mice were intraperitoneally given anti-CD4 or rat IgG antibody every other day after infection with T. spiralis. (A) Frequency and numbers of CD3⁺CD4⁺ cells at 7 and 14 days postinfection were evaluated in the mesenteric lymph node of infected mice treated with anti-CD4 or rat IgG antibody. (B) At 7 and 14 days postinfection, the small intestines were harvested and subjected for worm burden. (C) Detection and frequency of ILC2s (CD3^-CD4^-IL-17RB⁺CD127⁺T1/ST2⁺) in the mesenteric lymph nodes of infected mice treated with anti-CD4 or rat IgG antibody. (D) Mesenteric lymph node cells of T. spiralis-infected mice treated with anti-CD4 or rat IgG antibody were treated with IL-25 with or without T. spiralis antigen for 72 hours and the indicated cytokines in the supernatant were assessed using ELISA. (E) Lin^- cells from the mesenteric lymph node of T. spiralis-infected mice treated with anti-CD4 or rat IgG antibody were enriched using a MACS column. The enriched ILC2 cells were then restimulated with IL-25 or PMA/ionomycin for 72 hours, and the indicated cytokines in the supernatant were assessed using ELISA. Data represented one of three independent experiments (n = 4 mice each group). Error bars denote mean ± SD. Significance was determined by one-way ANOVA * p <0.05, ** p <0.01 and *** p <0.001.

Fig 4. Signaling through IL-17RB is essential to induce ILC2 and antigen specific Th2 and Th9 responses.

Il17rb^-/-/4GET or wild type 4GET mice were infected with T. spiralis. (A) Detection of IL-17RB in lineage negative (CD11b^-CD11c^-B220^-CD49b^-Gr.1^-CD335^-CD3^-CD4^-CD8^-) and CD3⁺CD4⁺ T cells. (B) Frequency and numbers of CD3⁺CD4⁺GFP⁺ cells at 7 and 14 days postinfection were evaluated in the mesenteric lymph node of infected Il17rb^-/-/4GET or wild type mice. (C) Detection and frequency of ILC2s (CD3^-CD4^-CD127⁺T1/ST2⁺KLRG1⁺GFP⁺) in the lamina propria of the small intestines of Il17rb^-/-/4GET or wild type 4GET mice infected with T. spiralis at 7 and 14 days postinfection. (D) Effector/memory (CD3⁺CD4⁺CD44⁺) T cells from mesenteric lymph nodes of T. spiralis-infected wild type and Il17rb^-/- mice were sorted and subjected to analyze the expression of indicated cytokine using real-time PCR. Data are expressed as fold induction over actin (Actb) expression, with the mRNA levels in the naïve group set as 1. (E) At day 7 and 14 postinfection, mesenteric lymph node cells were harvested and single cell suspensions were then cultured with or without T. spiralis extract antigen (10 μg/ml). After three days, the supernatant was collected and analyzed for T. spiralis-specific cytokine production by ELISA. Graphs depict mean±SD and are a representative of at least three independent experiments with three to four mice each group. Significance was determined by one-way ANOVA with Tukey’s post hoc analysis * p <0.05, ** p <0.01 and *** p <0.001.

Fig 5. Absence of IL-17RB resulted in reduced intestinal Th2 and Th9 effector responses and attenuated intestinal and muscle T. spiralis worm clearance.

Il17rb^-/- or BALB/c mice were infected with T. spiralis. (A) At days 7 and 14 postinfection, the small intestines (jejunum) were harvested and subjected to RNA extraction, followed by cDNA synthesis and cytokine gene expression using real-time PCR analysis. Data are expressed as fold induction over actin (Actb) expression, with the mRNA levels in the naïve group set as 1. (B) The small intestines (jejunum) were fixed with 10% formalin buffer and subjected to histological analysis of goblet cells by Periodic acid–Schiff (PAS) staining. Numbers of goblet cells were expressed per villus crypt unit (VCU). (C) At days 7 and 14 postinfection, the whole intestines of T. spiralis-infected Il17rb^-/- or BALB/c mice were harvested and analyzed for adult worms. (D) At day 30 postinfection, whole carcasses of infected mice from Il17rb^-/- or wild type groups were analyzed for muscle larvae burden. Graphs depict mean±SD and are a representative of at least three independent experiments with four mice each group. Significance was determined using one-way ANOVA with Tukey’s post hoc analysis * p <0.05, ** p <0.01 and *** p <0.001.