白介素33

Contribution of IL-33–activated type II innate lymphoid cells to pulmonary eosinophilia in intestinal nematode-infected mice Koubun Yasudaa,1, Taichiro Mutoa,1, Tatsukata Kawagoeb,1, Makoto Matsumotoa, Yuki Sasakia, Kazufumi Matsushitac, Yuko Takia, Shizue Futatsugi-Yumikuraa,c, Hiroko Tsutsuid, Ken J. Ishiib,e, Tomohiro Yoshimotoc, Shizuo Akirab,2, and Kenji Nakanishia,2 aDepartment of Immunology and Medical Zoology, cLaboratory of Allergic Diseases, Institute for Advanced Medical Sciences, and dDepartment of Microbiology, Hyogo College of Medicine, Nishinomiya 663-8501, Japan; bLaboratory of Host defense, World Premier International Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan; and eLaboratory of Adjuvant Innovation, National Institute of Biomedical Innovation, Osaka 567-0085, Japan Contributed by Shizuo Akira, January 19, 2012 (sent for review December 9, 2011) When animals are infected with helminthic parasites, resistant hosts show type II helper T immune responses to expel worms. Recently, natural helper (NH) cells or nuocytes, newly identified type II innate lymphoid cells, are shown to express ST2 (IL-33 receptor) and produce IL-5 and IL-13 when stimulated with IL-33. Here we show the relevant roles of endogenous IL-33 for Strong- yloides venezuelensis infection-induced lung eosinophilic inflam- mation by using Il33−/− mice. Alveolar epithelial type II cells (ATII) express IL-33 in their nucleus. Infection with S. venezuelensis or intranasal administration of chitin increases in the number of ATII cells and the level of IL-33. S. venezuelensis infection induces pul- monary accumulation of NH cells, which, after being stimulated with IL-33, proliferate and produce IL-5 and IL-13. Furthermore, S. venezuelensis infected Rag2−/−mice increase the number of ATII cells, NH cells, and eosinophils and the expression of IL-33 in their lungs. Finally, IL-33–stimulated NH cells induce lung eosinophilic inflammation and might aid to expel infected worms in the lungs. helminth | Th2 cytokine | Loeffler syndrome Animals, infected with intestinal nematodes, develop type IIhelper T (Th2) immune responses, which induce high level of IgE production, systemic eosinophilia, and local eosinophilic infiltration, particularly in the lung (1–4). We still do not know why only lungs develop such severe eosinophilic inflammation (Löffler syndrome) under helminth infection. We recently reported IL-33 is important for acute eosinophilic inflammation by using allergic conjunctivitis model (5). IL-33, a member of IL-1 family cytokine, is a ligand of ST2 (IL- 1RL1) (6). IL-33 was originally reported as a nuclear factor protein in endothelial cells of high endothelial venules (7). IL-33 is synthesized as an active full-length form and the processing by caspases abrogates its function (8–10). It is well documented that IL-33 is important for innate-type mucosal immunity in the lungs and gut (11) and for airway inflammation and peripheral antigen- specific responses (12). Th2 cells and various types of innate cells including basophils, mast cells, eosinophils, natural helper (NH) cells, and nuocytes express ST2 and produce Th2 cytokines in response to IL-33 (5, 13–16). Thus, IL-33–stimulated Th2 cells and innate cells play a critical role in various allergic inflam- mation by production of IL-4, IL-5, IL-13, and chemokines (5, 6, 14–19). Among innate cells, as we previously reported, only basophils and eosinophils produce IL-4 in response to IL-33, and as Moro et al. (15) and Neill et al. (16) showed, only NH cells and nuocytes produce IL-5 in response to IL-33. Helminthic parasite infection induces Th2 immune response. However, it remains uncertain how parasite infection stimulates innate cells to produce Th2 cytokines. Chitin, a widespread environmental biopolymer, provides structural rigidity to fungi, crustaceans, helminths, and insects (20). Because intranasal administration of chitin induces pulmonary eosinophilia (21), we focused on the function of chitin as a stimulator for IL-33 production. In this article, we first demonstrated the alveolar epithelial type II cells (ATII) express IL-33 and Strongyloides venezuelensis in- fection or intranasal administration of chitin markedly increases the number of ATII cells. Second, we demonstrated that S. venezuelensis infection induces severe eosinophilic inflammation and goblet-cell hyperplasia in the lungs almost dependently on IL- 33. Third, this parasite infection induces pulmonary eosinophilia even in Rag2−/− mice, suggesting the contribution of IL-33–stimu- lated NH cells. Fourth, this infection strongly and IL-33–depen- dently increases the number of NH cells. Finally, IL-33–stimulated NH cells induce lung eosinophilic inflammation through their production of IL-5 and IL-13. Result IL-33 Is Induced in the Lungs after S. venezuelensis Infection. We examined histological differences of the lungs before and after S. venezuelensis infection. C57BL/6 (B6) WT mice, infected with third-stage larvae (L3) of S. venezuelensis, developed eosinophil- dominated leukocyte infiltration at days 5 and 7 (Fig. 1 A and B). Immunohistochemical analysis of lung tissues revealed that there were a small number of cells that expressed IL-33 in their nucleus even before infection (Fig. 1A). S. venezuelensis infection in- creased the number of these IL-33+ cells particularly at days 5 and 7 (Fig. 1A). These kinetics seemed to be proportional to that of induction of eosinophil infiltration in the bronchoalveolar lavage fluid (BALF) and of IL-33 protein production in the lung (Fig. 1 B and C). Next, we performed kinetic study of Il33 mRNA expres- sion in the lungs after infection. We found S. venezuelensis in- fection increased the expression of mRNA for Il33 at day 4 and elevated further this expression at day 7 (Fig. 1D). These kinetics parallel well with that for Il5 or Il13, and the appearance of lung inflammation after S. venezuelensis infection (Fig. 1A). Nippos- trongylus brasiliensis infection induced a similar kinetics of in- duction of Il33, Il5, and Il13 mRNA in BALB/c mice (Fig. S1A). Next we sought the IL-33–producing cells in the lung. DAPI staining data confirmed that IL-33 is present in the nucleus. These IL-33+ cells are ATII cells because they were also positively Author contributions: K.Y., H.T., K.J.I., T.Y., S.A., and K.N. designed research; K.Y., T.M., T.K., M.M., Y.S., K.M., Y.T., and S.F.-Y. performed research; T.K. and K.J.I. contributed new reagents/analytic tools; K.Y., T.M., M.M., T.Y., S.A., and K.N. analyzed data; and K.Y. and K.N. wrote the paper. The authors declare no conflict of interest. 1K.Y., T.M., and T.K. contributed equally to this work. 2Towhomcorrespondencemaybeaddressed. E-mail: sakira@biken.osaka-u.ac.jp or nakaken@ hyo-med.ac.jp. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1201042109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1201042109 PNAS Early Edition | 1 of 6 IM M U N O LO G Y stained for prosurfactant protein C, a specific marker for ATII cells (22) (Fig. 1E,Left). To determine whether other types of cells, such as macrophages, also express IL-33, we stained macrophages with anti–IL-33 antibody and anti-F4/80 antibody, and found that they do not express IL-33 (Fig. 1E, Right). As chitin is a component of the outer membrane of parasites (20), and is included in soluble extracts from Strongyloides stercoralis (23), we examined the ca- pacity of chitin to induce IL-33 production in the lungs. Immuno- histochemical analysis of lung tissues revealed that an intranasal administration of chitin promptly increased the number of IL-33+- ATII cells in the lungs (Fig. 1F). This treatment promptly increased the IL-33 level in the BALF (Fig. S2A). We examined whether administration of chitin indeed increased the number of ATII cells. We counted the number of cells negative for T1α, CD16/32, and CD45.2 and positive for MHC class II as ATII cells (22, 24) (Fig. S3) and concluded that chitin treatment increased the number of ATII cells at least 72 h after treatment (Fig. 1F). Taken together, these results indicated that S. venezuelensis infection induces IL-33 production in the lung possibly by the action of chitin. Generation and Immunological Investigation of Il33−/− Mice. We wished to determine whether endogenous IL-33 is critically re- quired for establishment of lung eosinophilic inflammation in S. venezuelensis infected mice. For this purpose, we generated Il33 gene-deficient mice (Fig. S4 A and B) and examined their immu- nological properties. RT-PCR and Western blot analysis showed that the expression of IL-33 was completely abrogated in their lung tissues (Fig. S4 C and D). Proportions of T cells, B cells, dendritic cells, neutrophils, and eosinophils in the spleens of Il33−/− mice were comparable to those of Il33+/+ and Il33+/− mice (Fig. S4E). Anti-CD3–induced cytokine production responses revealed no skewing of splenic CD4+ T cells into Th1 or Th2 phenotype (Fig. S4F). Next, we examined the susceptibility of Il33−/− mice to S. venezuelensis infection.We simultaneously measured their systemic Th2/IgE response andmucosal mast cell activation in vivo. CD4+ T cells prepared from mesenteric lymph nodes of Il33−/−mice exhibited normal differentiation into Th2 cells (Fig. S5A). How- ever, the measurement of serum levels of IgE andmouse mast cell protease 1, an activation marker of mucosal mast cells, indicated that the absence of IL-33 partly but significantly diminished these responses (Fig. S5 B and C). Furthermore, their capacity to expel S. venezuelensis was also modestly impaired (Fig. S5D). Thus, IL-33 is partly involved in the host defense against S. venezuelensis infection. Il33−/− Mice Show Reduced Accumulation of Eosinophils in the Lungs After S. venezuelensis Infection. After S. venezuelensis infection, Il33+/+ mice developed eosinophilic inflammation and goblet-cell 0 10 20 30 0 2 4 6 8 10 0 20 40 60 80 100 120 0 2 4 7 10 14 0 3 6 9 A Il33IL-33 protein D ** * 0 1 2 4 7 10 re la tiv e ex pr es sio n * ** ** ** * Days post infection x1 04 eosinophils * ** * Days post infection ** Il5 Il13 0 1 2 4 7 10 0 1 2 4 7 100 1 2 4 7 10 E IL-33, Pro-SPC, DAPI IL-33, F4/80, DAPI 0 150 300 450 pg /m g F Pre 6 h 72 h IL-33 Pro-SPC DAPI Chitin IL-33 0 5 10 15 20 25 ce ll nu m be r ( x1 05) ** pre 72 h ATII cells B C Days post infection HE Pre Day 2 Day 4 Day 5 Day 7 IL-33 IL-33, DAPI Fig. 1. S. venezuelensis infection increases IL-33 expression in the lungs. (A–E) WT mice were infected with S. venezuelensis at day 0. (A) Histological analysis of lungswas performed at indicated days. (Upper) HE, stainedwith H&E. (Scale bar, 100 μm.) (Lower) Confocalmicroscopic analysis of the IL-33 expression. Red, IL-33; blue, DAPI. (Scale bar, 50 μm.) (B) The number of eosinophils in BALFs at indicated days (n = 3∼4). (C andD) IL-33 concentration in the lung lysateswas examined by ELISA. The amounts of IL-33were normalized by the total protein concentration (C). Quantitative RT-PCR (qPCR) analysis of the expression levels ofmRNA for Il33, Il5, or Il13 in the lungs (D). Data are representative of two independent experiments and expressed as themeans± SD (n = 5) *P < 0.01, **P < 0.001 (B–D; one-way ANOVA with Dunnett’s post test). (E) IL-33 (red) in the lungs at day 7 postinfection was costained with pro-SPC (green, Left) or F4/80 (green, Right). Blue, DAPI. (Scale bars, 20 μm.) (F) Chitinwas intranasally administered into B6mice. (Left) Confocal microscopic analysis of the lungs at indicated time points. Red, IL-33; blue, DAPI; green, pro-SPC. (Scale bar, 50 μm.) (Right) Lung cells were prepared from nontreated mice or mice treated with chitin 72 h before (n = 3). The numbers of ATII cells were calculated as described in Fig. S3. 2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1201042109 Yasuda et al. hyperplasia in the lungs at day 7, but Il33−/− mice only modestly developed these changes (Fig. 2A), suggesting critical involvement of IL-33 in these responses. Consistent with this modest eosino- philic inflammation, expressions of mRNA for eosinophil markers (25) Epx (eosinophil peroxidase) and Prg2 (major basic protein) in the lungs were significantly lower in Il33−/− mice than those in Il33+/+ mice (Fig. 2B). Because the development and the re- cruitment of eosinophils are regulated by IL-5, IL-13, and che- mokines (e.g., CCL11) (26), respectively, we measured their mRNA expressions. We also measured the mRNA expression for the epithelial cells-derived cytokines, IL-18, IL-33, thymic stromal lymphopoietin and IL-25, all of which are shown to up-regulate allergic inflammation (27, 28). The expressions of Il5, Il13, and Ccl11 were strongly increased in the lungs of Il33+/+ mice, but significantly diminished in those of Il33−/− mice, suggesting that IL-33 is responsible for the productions of IL-5, IL-13, and CCL11, which in turn stimulate eosinophils to grow and infiltrate into the lung (Fig. 2B). Simultaneous measurement of epithelial cytokines revealed that S. venezuelensis infection selectively in- creased the expression of Il33 mRNA among these cytokines (Figs. 1D and 2B). These results strongly indicated the IL-33– dependent production of IL-5, IL-13, and CCL11 is essential for goblet-cell hyperplasia and eosinophilic inflammation in the lung after S. venezuelensis infection. Next we examined the proportion of eosinophils in the BALFs. There were very few eosinophils in the BALFs of uninfected mice. However, at day 7 after S. ven- ezuelensis infection, we observed high proportion of eosinophils (Fig. 2C and Fig. S5E) in the BALFs from Il33+/+ mice. In con- trast, this proportion in the BALFs from Il33−/− mice was rela- tively low (Fig. 2C and Fig. S5E). As chitin is shown to induce IL- 33 production in the lung (Fig. 1E and Fig. S2A), we examined whether Il33+/+mice developed eosinophilia after treatment with chitin. We found that mice treated with chitin, displayed in- filtration of inflammatory cells around the chitin particles and marked goblet-cell hyperplasia at 72 h (Fig. S2B). In contrast, chitin-treated Il33−/− mice failed to develop these changes (Fig. S2C). Furthermore, chitin treatment increased the number of eosinophils in the BALF and the expression of Il5 and Il13mRNA by BALF cells in an IL-33–dependent manner (Fig. S2 D and E). S. venezuelensis Infection Induces Pulmonary Eosinophilia Even in the Absence of Acquired Immune Cells. We wished to directly demon- strate that S. venezuelensis infection induces pulmonary eosino- philia without help from Th2 cells. We infected WT, Rag2−/−, or γc−/−Rag2−/− mice with S. venezuelensis. All of these mice in- creased the number of IL-33+ ATII cells in their lungs (Fig. 3A). Expectedly, like WT mice, Rag2−/− mice developed pulmonary eosinophilia (Fig. 3B). WT mice and Rag2−/− mice also increased the number of eosinophils in the BALFs and the expression of Il33 mRNA and Il5 mRNA in their lungs (Fig. 3 C and E). As innate cells, such as NH cells, were reported to produce IL-5 in response to IL-33 stimulation (29), we tried to show the presence of these innate cells in the BALF cells. Expectedly, these cells appeared as Sca-1+ST2+ cells in the FSClowSSClowLin− cells in the BALF cells from WT and Rag2−/− mice after infection (Fig. 3D). To further identify the phenotype of Lin−ST2+ cells, we examined the ex- pression of other surface markers; then, we found they expressed Sca-1, Thy1.2, IL-7Rα, CD25, c-Kit, and ICOS and had limited expression ofMHC class II, as described in NH cells (Fig. S6) (30). In contrast to WT and Rag2−/−, γc−/−Rag2−/−mice, which have no NH cells in mesenteric tissues (15), failed to develop these changes, suggesting the importance of the expression of the γc chain for the induction of pulmonary eosinophilia, NH cell pro- liferation, and IL-5 expression. NH cells emerged around day 7 after infection and expanded at least until day 14 inWTmice (Fig. 3F). Along with their expansion, degree of eosinophilia and ex- pression of IL-5 and IL-13 are simultaneously up-regulated (Fig.1 B and D). Similar increases in the number of NH cells in the lungs were also observed at day 7 after N. brasiliensis infection (Fig. S1B). NH Cells Are Induced in S. venezuelensis Infected Mice in an IL-33– Dependent Manner. We demonstrated that S. venezuelensis infec- ted Il33+/+ mice but not Il33−/− mice markedly increased the ex- pression of Il5 and Il13 mRNA in their lungs (Fig. 2B). Thus, we examined whether S. venezuelensis infection increased the number of NH cells by induction of IL-33 production in the lungs. Com- pared with Il33+/+ mice, Il33−/− mice exhibited significantly re- duced number of NH cells in the BALFs (Fig. 4A), suggesting the importance of endogenous IL-33 for the induction of NH cells. To determine whether IL-33 is directly responsible for increasing NH cells and IL-5 expression, we examined the effects of IL-33 on these responses by intranasal administration. Intranasal adminis- tration of IL-33 strongly increased the number of NH cells and eosinophils in the BALF of Il33−/− mice (Fig. 4B). At the same time, this treatment strongly increased the expression of Il5 mRNA in the lungs. This treatment also significantly accelerated the worm expul- sion in Il33−/− mice (Fig. 4B, Right). Finally, we tried to identify the cells that produce IL-5 in response to IL-33 in the lungs of S. venezuelensis infected mice. We prepared BALF cells from WT mice at day 7 after infection and divided them into two fractions: lineage marker- (CD3, CD4, CD8, CD19, NK1.1, Gr-1, siglec F, IgE) positive and negative fractions. We could not find IL-5– producing cells in Lin+ cells, thus excluding the presence of IL- 0 20 40 60 80 0 1 2 3 0 2 4 6 8 10 0 1 2 3 4 5 6 7 0 2 4 6 8 0 3 6 9 Il33+/+ Il33-/- HE PAS cont Sv day 7 cont Sv day 7 re la tiv e ex pr es si on Ccl11Epx Prg2 A * ** * **** cont B * Il33+/+ Il33-/- % C Eo Il13Il5 Sv cont Sv cont Sv cont Sv cont Sv Neu Lym Mono Il18 Il25Tslp 0 0.5 1 1.5 2 2.5 0 1 2 3 4 0 0.5 1 1.5 Il33+/+ Il33-/- cont Sv cont Sv cont Sv Fig. 2. Il33−/− mice showed reduced accumulations of eosinophils in the lungs after S. venezuelensis infection. (A) Histological analysis of lungs of Il33+/+ and Il33−/− was performed before (cont) and after S. venezuelensis infection (Sv). PAS, periodic acid-Schiff stain. (Scale bars, 100 μm.) (B) Total RNA was prepared from lungs and the levels of mRNA expressions for Epx, Prg2 and indicated cytokines were determined by qPCR. Data are repre- sentative of two independent experiments and expressed as the means ± SD (n = 3 ∼5) *P < 0.01, **P < 0.001, ***P < 0.05 (Student’s t test). (C) Pro- portions of eosinophils (Eo), neutrophils (Neu), lymphocytes (Lym), or mon- ocytes (Mono) in the BALF were calculated from flow cytometric analysis of CD45+ BALF cells from S. venezuelensis infected mice (Sv). Data are repre- sentative of two independent experiments and expressed as the means ± SD (n = 3 ∼5) *P < 0.05, (Student’s t test). Yasuda et al. PNAS Early Edition | 3 of 6 IM M U N O LO G Y 5–producing Th2 cells in the BALF (Fig. 4C). However, we found a substantial proportion of ST2+ cells in Lin− fraction produced IL-5 (Fig. 4C). Taking these data together, IL-33 contributes to the induction of NH cells, which in turn protect host against S. venezuelensis infection by inducing lung eosino- philia through their production of IL-5 and IL-13. Discussion We demonstrated that S. venezuelensis infection of mice induced severe eosinophilic inflammation, goblet-cell hyperplasia, and accumulation of NH cells, and increased the number of IL-33– producing ATII cells and the expressions of mRNA for Il5 and Il13 in the lungs, even without help from a