BASIC RESEARCH www.jasn.org IL-25 Elicits Innate Lymphoid Cells and Multipotent Progenitor Type 2 Cells That Reduce Renal Ischemic/ Reperfusion Injury † ‡ ‡ ‡ Qingsong Huang,* Zhiguo Niu,* Jing Tan, Jun Yang, Yun Liu, Haijun Ma, ‡ Vincent W.S. Lee,§ Shuming Sun,* Xiangfeng Song,* Minghao Guo, Yiping Wang,§ and Qi Cao*§ *Research Center for Immunology, Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine, Xinxiang Medical University, Xinxiang, China; †Department of Nephrology, Third Affiliated Hospital of Xinxiang Medical University, Xinxiang, China; ‡Department of Nephrology, First Affiliated Hospital of Xinxiang Medical University, Xinxiang, China; and §Centre for Transplant and Renal Research, Westmead Millennium Institute, University of Sydney, Sydney, New South Wales, Australia ABSTRACT IL-25 is an important immune regulator that can promote Th2 immune response-dependent immunity, in- flammation, and tissue repair in asthma, intestinal infection, and autoimmune diseases. In this study, we examined the effects of IL-25 in renal ischemic/reperfusion injury (IRI). Treating IRI mice with IL-25 significantly improved renal function and reduced renal injury. Furthermore, IL-25 treatment increased the levels of IL-4, IL-5, and IL-13 in serum and kidney and promoted induction of alternatively activated (M2) macrophages in kidney. Notably, IL-25 treatment also increased the frequency of type 2 innate lymphoid cells (ILC2s) and multipotent progenitor type 2(MPPtype2) cells in kidney. IL-25–responsive ILC2 and MPPtype2 cells produced greater amounts of Th2 cytokines that associated with the induction of M2 macrophages and suppression of classically activated (M1) macrophages in vitro. Finally, adoptive transfer of ILC2s or MPPtype2 cells not only reduced renal functional and histologic injury in IRI mice but also induced M2 macrophages in kidney. In conclusion, our data identify a mechanism whereby IL-25-elicited ILC2 and MPPtype2 cells regulate macrophage phenotype in kidney and prevent renal IRI. J Am Soc Nephrol 26: 2199–2211, 2015. doi: 10.1681/ASN.2014050479 IL-25 (also known as IL-17E) is a member of the IL-17 and administration of IL-25 attenuated renal injury cytokine gene family and is produced by several cell in mice with adriamycin nephropathy (AN) via in- types, including T lymphocytes, mast cells, eosinophils, ducing Th2 immune responses.11 basophils, and epithelial cells in the lung and intestine.1–3 Four independent research groups recently iden- Administration of IL-25 to mice has been shown to tified previously unrecognized innate immune cell induce a Th2 immune response characterized by the populations that were capable of contributing to overproduction of IL-4, IL-5, and IL-13.4 IL-25 facili- tates pathogenic Th2 cell responses, increases serum levels of IgE and blood eosinophilia, and enhances Received May 16, 2014. Accepted November 16, 2014. the recruitment of inflammatory cells in asthma and Q.H. and Z.N. contributed equally to this work. fl 1,5,6 allergic in ammation. However, IL-25 is an im- Published online ahead of print. Publication date available at portant regulator of host defense and promotes im- www.jasn.org. munity to helminth infections.7–9 Moreover, IL-25 is fl Correspondence: Dr. Qi Cao, Centre for Transplant and Renal also required to limit chronic intestinal in ammation Research, Westmead Millennium Institute, University of Sydney, and experimental autoimmune encephalomyelitis Darcy Road, Westmead, NSW 2145, Australia. Email: qi.cao@ through controlling Th1/Th17 cell responses.3,10 sydney.edu.au The role of IL-25 in CKD was recently investigated Copyright © 2015 by the American Society of Nephrology J Am Soc Nephrol 26: 2199–2211, 2015 ISSN : 1046-6673/2609-2199 2199 BASIC RESEARCH www.jasn.org Th2 cytokine responses in vivo. These cell populations were (Figure 1, C and D). Gr-1+ neutrophil infiltration in the outer named natural helper cells, nuocytes, innate type 2 helper medulla of postischemic kidney was significantly increased (Ih2)cells,ormultipotentprogenitortype2(MPPtype2) compared with that of sham kidney and significantly reduced cells.12–15 Based on developmental, phenotypic, and functional in IRI mice treated with IL-25 (Figure 1E). However, interstitial similarities, natural helper cells, nuocytes, and Ih2 cells have infiltration with F4/80+ macrophages was not reduced in the been collectively categorized as group 2 or type 2 innate outer medulla of IRI mice treated with IL-25 compared with lymphoid cells (ILC2s).16,17 In response to the epithelial cytokines that of control IRI mice (Figure 1F). Together, IL-25 attenuated IL-25 and IL-33, ILC2s expand and produce large amounts of postischemic renal failure and renal IRI. type 2 cytokines, particularly IL-13 and IL-5 through the expres- sion of the receptors of these cytokines, IL-17RB and ST2, re- IL-25 Induced Th2 Responses and Alternatively spectively. ILC2s play critical roles in promoting immunity to Activated Macrophages In Vivo helminth parasites, allergic airway inflammation, and lung To define the mechanisms underlying the protective effect of epithelial repair.12,14,18,19 In contrast with ILC2s, MPPtype2 IL-25 against renal injury, we examined Th2 responses in the cells express unique cell surface markers and exhibit the ability periphery and in kidney. In IRI mice treated with IL-25, serum to differentiate into cells of the monocyte and granulocyte levels of the Th2 cytokines IL-4, IL-5, and IL-13 were lineages, suggesting that MPPtype2 cells may be a distinct pop- significantly increased compared with those of control and ulation.15,20 Administration of IL-25 promotes the accumula- IRI mice (Figure 2A). In addition, the mRNA expression of tion of ILC2 and MPPtype2 cells at multiple tissue sites, whereas IL-4, IL-5, and IL-13 in kidney was significantly increased in whether IL-25 induces expansion of ILC2 and MPPtype2 cells in IRI mice given IL-25 compared with that of control and IRI kidney is unknown. mice (Figure 2B). To further investigate the mechanisms of Ischemic/reperfusion injury (IRI) is the primary cause of IL-25’s protective effects, we examined the activation status of AKI and is also relevant to a number of clinical situations, endogenous macrophages in the kidney. Interestingly, the including kidney transplantation. Macrophages contribute to kidney macrophages from IRI mice treated with IL-25 had the initiation of IRI through secretion of cytokines, recruit- elevated mRNA expression of M2 macrophage markers, in- ment of neutrophils, and induction of epithelial cell apoptosis cluding mannose receptor (MR), arginase, FIZZ1, YM1, and and also play an important role in recovery or regeneration IL-10 (Figure 2C). Similarly, FACS analysis demonstrated that MR processes from IRI by modulating immune responses against expression was significantly increased in the kidney macrophages inflammation.21–23 We previously reported that IL-25 induced from IRI mice treated with IL-25 compared with those from Th2 immune responses by increasing levels of IL-4, IL-5, and control IRI mice (Figure 2D). In addition, the expression of M2 IL-13 in serum, kidney, and kidney draining lymph nodes macrophage markers, such as MR and arginase, was signifi- (KLDNs), and thereby induced alternatively activated (M2) cantly increased in macrophages isolated from spleen, liver, macrophages and protected against renal injury in AN.11 In and lung of IRI mice treated with IL-25 (Supplemental Figure this study, we evaluated IL-25’s ability to protect against renal 1), indicating the influence of IL-25 in systemic immune re- injury in mice with IRI, and further examined possible mech- sponses rather than a specific effect on renal macrophage anisms underlying its effect on ILC2s, MPPtype2 cells, and function. By contrast, the expression of M1 macrophage macrophages in kidney. Here, we provide evidence that IL-25 markers, including inducible nitric oxide synthase (iNOS), is a critical cytokine in both promoting Th2 immune responses TNF-a,IL-1b, IL-6, and CCL2, was significantly lower in the and preventing renal injury in murine IRI. Interestingly, ILC2 kidney macrophages from IRI mice treated with IL-25 than that and MPPtype2 cells were expanded in kidney of mice treated with of control IRI mice (Figure 2, E and F). Thus, IL-25 induced Th2 IL-25, and adoptive transfer of ILC2 or MPPtype2 cells attenuated responses in the periphery and kidney, thereby inducing M2 renal injury in mice with IRI via the induction of M2 macrophages macrophages in kidney, which are known to trigger immuno- in kidney. regulation and tissue repair. Alternatively Activated Macrophages Promoted RESULTS Tubular Cell Survival In Vitro To determine the effects of M1 and M2 macrophages on renal IL-25 Protected against Renal Injury in IRI Mice tubular cells in vivo,anin vitro coculture model was established to BUN and serum creatinine were significantly increased in mimic the in vivo macrophage interaction with injured tubular bilateral IRI mice compared with those of control mice and cells. Simulated ischemic renal tubular epithelial cells (TECs) were were significantly improved in bilateral IRI mice treated with induced by immersing the cellular monolayer in mineral oil, and IL-25 (Figure 1, A and B). In renal IRI, renal injury was char- were then cocultured with M0, M1, or M2 macrophages for 1–3 acterized by tubular necrosis, tubular dilation, cast formation, days. The apoptosis of ischemic TECs was significantly in- and tubular cell vacuolization. Tubular injury of postischemic creased compared with control TECs, and was further enhanced kidney was significantly increased compared with that of sham by coculture with M1 macrophages, whereas coculture with M2 kidney and significantly reduced in IRI mice treated with IL-25 macrophages resulted in a reduction of apoptosis in ischemic 2200 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 2199–2211, 2015 www.jasn.org BASIC RESEARCH Figure 1.