Macrophages as IL-25/IL-33-responsive cells play an important role in the induction of type 2 immunity

Abstract

Type 2 immunity is essential for host protection against nematode infection but is detrimental in allergic inflammation or asthma. There is a major research focus on the effector molecules and specific cell types involved in the initiation of type 2 immunity. Recent work has implicated an important role of epithelial-derived cytokines, IL-25 and IL-33, acting on innate immune cells that are believed to be the initial sources of type 2 cytokines IL-4/IL-5/IL-13. The identities of the cell types that mediate the effects of IL-25/IL-33, however, remain to be fully elucidated. In the present study, we demonstrate that macrophages as IL-25/IL-33-responsive cells play an important role in inducing type 2 immunity using both in vitro and in vivo approaches. Macrophages produced type 2 cytokines IL-5 and IL-13 in response to the stimulation of IL-25/IL-33 in vitro, or were the IL-13-producing cells in mice administrated with exogenous IL-33 or infected with Heligmosomoides bakeri. In addition, IL-33 induced alternative activation of macrophages primarily through autocrine IL-13 activating the IL-4Rα-STAT6 pathway. Moreover, depletion of macrophages attenuated the IL-25/IL-33-induced type 2 immunity in mice, while adoptive transfer of IL-33-activated macrophages into mice with a chronic Heligmosomoides bakeri infection induced worm expulsion accompanied by a potent type 2 protective immune response. Thus, macrophages represent a unique population of the innate immune cells pivotal to type 2 immunity and a potential therapeutic target in controlling type 2 immunity-mediated inflammatory pathologies.

Figure 1. IL-33 stimulated macrophages to produce IL-13 in vitro.

A, B, Time- and concentration-dependent upregulation of IL-13 mRNA expression (A, qPCR) or IL-13 secretion from cell culture supernatants (B, ELISA) in BMDM stimulated with IL-33. C, After IL-33 stimulation, BMDM were stained with antibodies, and analyzed by FACS for surface F4/80 and intracellular IL-13 expression. D, BMDM from WT, STAT6^−/−, IL-4Rα^−/−, or IL-4Rα^−/−Rag2^−/− mice were stimulated with IL-33, and analyzed for IL-13 mRNA expression (72 hours) by qPCR. Data shown in bar graphs are the mean ± s.e.m and representative of at least two independent experiments.

Figure 2. IL-33 induced alternative activation of macrophages in vitro.

BMDM from WT, STAT6^−/−, or IL-4Rα^−/− mice were stimulated with IL-33 (24 and 72 hours), and analyzed for the expression of arginase I (A) or YM-1 (B) by qPCR (*p<0.01 vs respective vehicle; ^φp<0.001 vs WT-IL-33, one-way ANOVA followed by Neuman-Keuls test). Data shown in bar graphs are the mean ± s.e.m and representative of two independent experiments with similar results.

Figure 3. Exogenously administrated IL-33 in mice promoted type 2 immunity.

Mice were i.p. injected with BSA or IL-33 (1 µg/mouse) daily for 3 days. Tissues were collected for functional or molecular analysis. A, IL-33 injection increased IL-13 expression in the small intestine and spleen independently of STAT6 or T/B cells. B, mRNA expression of IL-13Rα2 or mast cell protease I (mMCP-1) in the small intestine (n = 3–5 mice per group, *p<0.05 vs respective vehicle for A&B). C, IL-33 injection induced increase in the permeability of intestinal mucosa (decrease in transepithelial electrical resistance, TEER) (n = 5 per group, **p<0.01 vs BSA-treated, t test). D, IL-33 induced intestinal smooth muscle hypercontractility. Smooth muscle responses to acetylcholine and electric field stimulation (EFS), as well as amplitude of spontaneous contraction (SC) are shown (n = 5 per group, p<0.01 was generated from two-way ANOVA with repeated measures between the two curves). Data shown in bar and line graphs are the mean ± s.e.m and representative of two independent experiments with similar results.

Figure 4. Exogenously administrated IL-33 in mice induced IL-13-expressing macrophages.

C57BL/6 mice were i.p. injected with BSA or IL-33 (1 µg/mouse) daily for 3 days. A, Sections of small intestine were stained with anti-F4/80 (green) and anti-IL-13 (red). Representative images from 5 animals per group were shown. B, Peritoneal exudate cells (PECs) were collected and analyzed for IL-13 expression by qPCR or ELISA in triplicates (n = 3–5 per group, *p<0.05 vs respective BSA, t test). C, Representative plots from PECs stained with anti-F4/80 and anti-IL-13 for analyzing IL-13-expressing macrophages by FACS. D, Number of total PECs and F4/80⁺/IL-13⁺ PECs collected per mouse after injection of BSA or IL-33 (n = 3–5 per group, *p<0.05 vs respective BSA). Data shown in bar graphs are the mean ± s.e.m and representative of at least two independent experiments with similar results.

Figure 5. Macrophage depletion in mice impaired type 2 immunity induced by exogenous IL-33.

A–C, IL-33-induced upregulation of IL-13 and IL-5 was attenuated in the small intestine (A), spleen (B), and mesenteric lymph nodes (C), in mice treated with Cl₂MDP. D, E, IL-33-induced smooth muscle hypercontractility was attenuated in mice with decreased number of macrophage as shown by the response to electric field stimulation (D, EFS) and the amplitude of spontaneous contraction (E, SC) (n = 5 per group, *p<0.05 vs respective BSA; ^φp<0.05 vs PBS-IL-33, one-way ANOVA followed by Neuman-Keuls test; p = 0.002 was generated from two-way ANOVA with repeated measures between the two curves). Data shown in bar and line graphs are the means ± s.e.m, and are representative of two independent experiments.

Figure 6. Adoptive transfer of IL-33-activated macrophages promoted protective immunity against H. bakeri (Hb) infection.

A–C, Numbers of adult worms in the lumen of small intestine in mice receiving BMDM (A), PECs (B), or CD11b MicroBead-enriched PECs (C). D, E, Enhanced type 2 immunity in mice that received IL-33-activated CD11b MicroBead-enriched PECs (IL-33-Macs). Intestinal smooth muscle function (D) and in situ IL-13 production in the small intestine and spleen (E) were shown. Data shown in bar and line graphs are the mean ± s.e.m, and are representative of two independent experiments (n = 3–6 mice per group, *p<0.05 vs respective vehicle-Macs, t test; p = 0.02 was generated from two-way ANOVA with repeated measures between the two curves). ND, not detected.