IL-17A Produced by Innate Lymphoid Cells Is Essential for Intestinal Ischemia-Reperfusion Injury

This information is current as Mayya Geha, Maria G. Tsokos, Robin E. Bosse, Tatyana of September 28, 2021. Sannikova, Yoichiro Iwakura, Jurandir J. Dalle Lucca, Rene De Waal Malefyt and George C. Tsokos J Immunol published online 6 September 2017 http://www.jimmunol.org/content/early/2017/09/05/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 6, 2017, doi:10.4049/jimmunol.1700655 The Journal of Immunology

IL-17A Produced by Innate Lymphoid Cells Is Essential for Intestinal Ischemia-Reperfusion Injury

Mayya Geha,*,† Maria G. Tsokos,† Robin E. Bosse,† Tatyana Sannikova,‡ Yoichiro Iwakura,x Jurandir J. Dalle Lucca,{ Rene De Waal Malefyt,‖ and George C. Tsokos†

Ischemia-reperfusion (IR) injury to the small intestine following clamping of the superior mesenteric artery results in an intense local inflammatory response that is characterized by villous damage and neutrophil infiltration. IL-17A, a produced by a variety of cells in response to inflammatory released following tissue injury, has been implicated in IR injury. Using Il17a2/2, Il23r2/2,andRorc2/2 mice and administration of anti–IL-17A and anti–IL-23 neutralizing Abs to wild-type mice, we demonstrate that intestinal IR injury depends on IL-17A and that IL-17A is downstream of the binding of autoantibody to ischemia-conditioned tissues and subsequent complement activation. Using chimeras, we demonstrate that the IL-17A required for intestinal Downloaded from IR injury is derived from hematopoietic cells. Finally, by transferring autoantibody-rich sera into Rag2gc2/2 and Rag22/2 mice, we demonstrate that innate lymphoid cells are the main producers of IL-17A in intestinal IR injury. We propose that local production of IL-17A by innate lymphoid cells is crucial for the development of intestinal IR injury and may provide a therapeutic target for clinical exploitation. The Journal of Immunology, 2017, 199: 000–000.

ntestinal ischemia-reperfusion (IR) injury occurs when the IL-17A is a proinflammatory cytokine that causes epithelial cells http://www.jimmunol.org/ blood supply to the intestine is re-established following a period to secrete neutrophil chemoattractant , such as CXCL1, of transient disruption of blood flow. Intestinal IR injury occurs CXCL2, and IL-8 (14). Cells that produce IL-17A include TCRa/ I 2 in a variety of clinical conditions, including shock, trauma, sepsis, b T cells (15, 16), TCRg/d T cells (17), and CD45+CD4+TCR aortic surgery, and acute mesenteric artery occlusion (1, 2). Intestinal IL-7R+ type 3 innate lymphoid cells (ILC3s) (18, 19), which reside IR injury triggers a cascade of events that results in local and remote at mucosal surfaces. Other sources of IL-17A include dendritic cells organ injury. Intestinal damage after IR injury is characterized by (DCs), , neutrophils, and NK cells (20). The IL-17A severe villus destruction, with disruption and dysfunction of the in- receptor is a heterodimer of the IL-17RA and IL-17RC chains that testinal (3). Re-establishment of the blood supply to the bind IL-17A and its homolog, IL-17F, and it is expressed pre- by guest on September 28, 2021 intestine initiates an intense local inflammatory response with neu- dominantly on epithelial cells (21, 22). Because of its ability to trophil infiltration (4). IR injury depends on elements of the innate mobilize neutrophils, IL-17A is important in the pathogenesis of and adaptive immune responses (5). Contributors to the tissue dam- autoimmune diseases and in chronic steroid-resistant that is age include IgM (6), natural Abs (7), complement (8, 9), neutrophils characterized by neutrophil predominance (23, 24). It is also im- (4), platelets (10, 11), B (12), and T lymphocytes (13). portant for host defense against candida infection, as illustrated by the susceptibility of patients who carry mutations in IL17A or genes encoding IL-17A receptor chains to candida infection (25). Naive *Department of Pediatrics, Massachusetts General Hospital, Harvard Medical TCRa/b cells differentiate into Th17 cells following TCR ligation School, Boston, MA 02114; †Department of Medicine, Beth Israel Deaconess Med- ical Center, Harvard Medical School, Boston, MA 02115; ‡Department of Pediatrics, in the presence of the inflammatory cytokines IL-1, IL-6, and x Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115; Center for TGF-b (26). The differentiation of TCR CD4+ Th17 cells is pro- Animal Disease Models, Research Institute for Biomedical Sciences, Tokyo Univer- sity of Science, Chiba 278-0022, Japan; {Translational Medical Division, Department moted by the cytokine IL-23 (20), whereas the rapid production of of Chemical and Biological Technologies, Defense Threat Reduction Agency, Fort IL-17A by TCRg/d T cells and ILC3s is directly driven by IL-23 ‖ Belvoir, VA 22060; and Merck Sharp & Dohme Corp., Palo Alto, CA 94304 (27). IL-23 is a heterodimer of the 40-kDa chain (p40), which is ORCIDs: 0000-0001-8072-1358 (M.G.T.); 0000-0001-5463-9617 (R.E.B.); 0000- shared with IL-12, and the IL-23–specific 19-kDa chain (p19) (28) 0002-3749-576X (T.S.); 0000-0003-1424-0999 (J.J.D.L.); 0000-0001-9589-2360 and is produced by epithelial cells and DCs (29, 30). IL-23R is a (G.C.T.). heterodimer of the IL-12R b1-chain, which is shared with Received for publication May 5, 2017. Accepted for publication August 11, 2017. IL-12R, and an IL-23R–specific chain (30, 31). This work was supported by U.S. Department of the Army Medical Research and Material Command Grant W81XWH-12-1-0526 and National Institutes of Health Using immunofluorescence microscopy, we previously showed Grant T32 HD-007466 (to Stella Kourembanas, principal investigator). an increase in IL-17A expression in the intestine after IR injury Address correspondence and reprint requests to Dr. George C. Tsokos, Department of (13). Importantly, IL-17A, via its role in neutrophil recruitment, Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 has been shown to play a critical role in intestinal IR injury (32, Brookline Avenue, CLS-937, Boston, MA 02115. E-mail address: gtsokos@bidmc. harvard.edu 33), as well as in IR injury in other organ systems, including heart The online version of this article contains supplemental material. (34, 35), liver (36, 37), lung (38), kidney (39, 40), and brain (41, Abbreviations used in this article: B6, C57BL/6J; B6.lpr, B6.MRLTnfrsf6lpr; BM, 42). It has been claimed that Paneth cells store IL-17A and are the bone marrow; CycloA, cyclophilin A; DC, ; HPF, high-power field; ILC, major source of IL-17A in intestinal IR injury (33). However, innate lymphoid cell; ILC3, type 3 innate lymphoid cell; IR, ischemia-reperfusion; because published gene array analyses do not show that Paneth qRT-PCR, quantitative RT-PCR; SMA, superior mesenteric artery; WT, wild-type. cells express Il17a (43, 44), we set out to analyze the source of Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 IL-17A relevant to intestinal IR injury. We have confirmed the role

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700655 2 ILC-DERIVED IL-17A IS ESSENTIAL FOR INTESTINAL IR INJURY of IL-17A and demonstrated a role for IL-23 in intestinal IR in- IR, as described (47). Control animals were given 200 mg of rat IgG jury, and we provide evidence that ILCs are the relevant source of isotype control Ab (R&D Systems) also in three doses 96, 48, and 24 h IL-17A in intestinal IR injury. prior to IR. To neutralize IL-23, 10 mg of goat anti-mouse anti–IL-23 Ab (R&D Systems) was given in three doses by i.p. injection 96, 48, and 24 h prior to IR, as described (48). Control animals were given 20 mg goat IgG Materials and Methods isotype control Ab (R&D Systems) also in three doses 96, 48, and 24 h Mice prior to IR. 2/2 2/2 2/2 Il17a2/2 and Il23r2/2 mice on a C57BL/6J (B6) background were gen- Reconstitution of Rag2 and Rag2 gc mice with IgG erated as described (45, 46). Age-matched B6 wild-type (WT) CD45.2 from B6.lpr mice mice (The Jackson Laboratory, Bar Harbor, ME) were used as controls. Serum from B6.lpr mice was obtained by cardiac puncture. The Melon Gel CD45.1 B6 WT mice (The Jackson Laboratory) were used to create the 2 2 2 2 2 2 IgG Purification Kit (Thermo Fisher Scientific) was used to isolate and bone marrow (BM) chimeras. Rag2 / mice and Rag2 / gc / mice on purify IgG. A buffer exchange to PBS was performed on the eluted ma- the B6 background were obtained from Taconic (Hudson, NY). B6. 2/2 2/2 lpr terial. A total of 200 mg of IgG was injected i.v. into Rag2 or Rag2 MRLTnfrsf6 (B6.lpr) female mice (age 7–8 mo) were purchased from 2/2 2 2 gc mice 30 min prior to ischemia or sham operation, as previously The Jackson Laboratory and were used to isolate IgG from serum. Tcrd / 2 2 described (49). and Rorc / mice on the B6.129 background were backcrossed onto a B6 background for at least 12 and 6 generations, respectively. Mice underwent Generation of BM radiation chimeras $7 d of acclimatization before experimentation. All mice used in this 2 2 study were 8–12-wk-old males, with the exception of the BM chimeras Eight-week-old recipient CD45.2+ WT and Il17a / mice were lethally that were 18–22 wk old, and were maintained in pathogen-free conditions irradiated (1100 rad delivered in two doses of 550 rad each with a 3-h in the animal research facility at the Beth Israel Deaconess Medical Center. interval) and injected i.v. with 5 3 106 BM cells obtained from congenic All experiments were performed in accordance with the guidelines and CD45.1+ WT mice and vice versa. Chimerism was assessed by measuring Downloaded from approval of the Harvard University Institutional Animal Care and Use the percentages of CD45+ donor and recipient cells in the chimeric mice 8 Committee. wk after BM reconstitution using FACS analysis for the CD45.1 and CD45.2 markers on blood after RBC lysis. IR injury Quantitative RT-PCR analysis Mice were randomly assigned to sham or IR groups. Mice were anesthetized by i.p. injection of 72 mg/kg pentobarbital or 250 mg/kg 2,2,2-tri- Intestines were harvested and placed in RNAlater (Thermo Fisher Scien- bromoethanol, and anesthesia was maintained by s.c. injection of 36 mg/kg tific). Total RNA was isolated using the RNeasy Mini Kit (QIAGEN). http://www.jimmunol.org/ pentobarbital or 125 mg/kg 2,2,2-tribromoethanol. A midline laparotomy Reverse transcription was performed using the High-Capacity cDNA Re- incision was made, and the superior mesenteric artery (SMA) was iden- verse Transcription Kit (Applied Biosystems), according to the manufac- tified, isolated, and clamped for 30 min using a microvascular clip (Roboz turer’s protocol. Quantitative RT-PCR was performed (Light Cycler 480; Surgical Instrument, Rockville, MD) delivering ∼8.5 3 g of pressure. The Roche) for Il17a, Il23, Cxcl1, Cxcl2, GAPDH, and cyclophilin A (CycloA) clip was removed after 30 min of ischemia, and the intestines were with 40 cycles at 94˚C for 12 s and 60˚C for 60 s using appropriate murine reperfused for 2 h. The laparotomy incision was sutured closed using TaqMan assays for Il17a, Il23, Cxcl1, Cxcl2, and GAPDH (Applied 4.0 Prolene suture, and the mice were resuscitated with 1 ml of warm Biosystems) or using SYBR Green Brilliant I Master Mix (Stratagene, PBS injected s.c. The mice were monitored throughout the experiment. La Jolla, CA) with the appropriate primers for Il17a, Il23, and CycloA. Body temperature was maintained at 37˚C throughout the experiment on Ct values were determined using Mx3000P software. All PCR reactions a temperature-controlled heating pad. Sham-operated mice underwent were run in triplicates. The averaged cycle threshold values for each target by guest on September 28, 2021 identical abdominal manipulations (laparotomy, intestinal retraction, and gene were normalized for GAPDH or CycloA mRNA, and relative positioning) as mice subjected to SMA clamping. Intestines were collected expression of the target gene mRNA was calculated with the DDCt relative 2 h after sham operation or intestinal IR injury, unless otherwise noted. quantification method. Histological analysis of intestinal IR injury Statistical analysis Small intestine (jejunum and ) was washed in ice-cold PBS and fixed Numeric data are presented as mean 6 SD or mean 6 SEM. Statistical overnight in 10% formalin. After automated dehydration through a graded analyses were performed using GraphPad Prism 6.0 (GraphPad, San alcohol series, tissues were embedded in paraffin, sectioned at 5 mm, and Diego, CA). The ordinal values of the injury scores and polymorpho- stained with H&E. Intestinal H&E-stained sections were graded for in- nuclear cells per HPF were analyzed by the nonparametric Mann–Whitney testinal IR-induced mucosal injury. Villi were scored using a published U test. The two-tailed Student t test for unpaired samples was used to algorithm (3) to measure intestinal damage in a blinded manner by one of compare the means of two groups. A p value ,0.05 was considered sta- us (M.G.T.). tistically significant.

Immunohistochemistry Results Formalin-fixed paraffin sections of small intestine were subjected to re- IL-17A is important for mesenteric IR injury hydration, and endogenous peroxidase activity was quenched with 3% H2O2. Then Ag retrieval was performed using Retrievagen A (BD Phar- We confirmed the critical role of IL-17A in our model of mesenteric mingen, San Jose, CA), according to the manufacturer’s directions. The IR injury in which mice are subjected to 30 min of SMA occlusion, sections were blocked with 10% BSA/PBS containing the serum from host followed by 2 h of reperfusion. Il17a2/2 mice developed markedly species of secondary Ab. The primary Ab rat anti-mouse Ly-6B.2 (Gr1) less intestinal damage, with lower injury scores, compared with clone 7/4 (1/250 dilution; Bio-Rad, Hercules, CA) was used to stain for 2/2 neutrophils. Primary Ab or isotype-control Ab prepared in 10% BSA/PBS WT mice (Fig. 1A, 1B). Il17a mice subjected to mesenteric IR was applied overnight at 4˚C. The slides were incubated with HRP- injury had significantly less neutrophil infiltration in the intestine conjugated secondary Ab for 60 min at room temperature, developed than did WT controls, as determined by examination of intestinal with NovaRED (Vector Laboratories, Burlingame, CA), counterstained sections stained immunohistochemically for Gr1 (Fig. 1C, 1D). with hematoxylin, and dehydrated. The sections were mounted in mounting medium (Thermo Fisher Scientific, Waltham, MA) and evalu- Mesenteric IR injury in WT mice was associated with a sig- ated with a Nikon Eclipse 80i microscope (Nikon, Melville, NY). Images nificant increase in Il17a mRNA levels in the small intestine were analyzed using Nikon NIS-Elements software. For neutrophil infil- compared with sham-operated mice, as determined by quantitative tration, positive-staining cells in the area of injury were counted in 20 RT-PCR (qRT-PCR) (Fig. 1E). IL-17A is known to drive neutro- high-power fields (HPFs) at 2003, and the average was calculated and expressed as the number of neutrophils per HPF. phil infiltration into tissues by inducing resident cells to express neutrophil-attracting chemokines, including Cxcl1 and Cxcl2 (50). Neutralization of endogenous IL-17A and IL-23 Cxcl1 and Cxcl2 mRNA levels were significantly higher in the To neutralize IL-17A, 200 mg of rat anti-mouse anti–IL-17A Ab (R&D small intestine of WT mice subjected to mesenteric IR injury Systems) was given in three doses by i.p. injection 96, 48, and 24 h prior to compared with sham-operated mice. In contrast, there was no The Journal of Immunology 3

FIGURE 1. IR injury is attenuated in Il17a2/2 mice. Representative H&E- stained sections (A), injury scores (B), representative immunohistochemical stain- ing for Gr1 (C), quantitation of infiltrating + Gr1 cells per HPF (D), and qRT-PCR Downloaded from analysis of Il17a (E)andCxcl1 and Cxcl2 (F) expression in Il17a2/2 mice and WT controls subjected to intestinal IR injury or sham operation. Results are derived from three independent experiments, each with three or four mice per group. Data in bar graphs are mean + SEM. Scale bars, http://www.jimmunol.org/ 100 mM(A), 50 mM(C). *p , 0.05, **p , 0.01, ****p , 0.0001. by guest on September 28, 2021

significant increase in Cxcl1 and Cxcl2 mRNA levels in the small in intestinal IR injury (49, 52–55). To circumvent these limita- intestine of Il17a2/2 mice subjected to mesenteric IR injury tions, we examined the effect of neutralizing IL-17A on intestinal compared with WT controls (Fig. 1F). IR injury in WT mice. WT mice were treated with a neutralizing Natural Abs (7), which include self-reacting autoantibodies (49, IgG Ab to IL-17A or with IgG isotype-control Ab on days 4, 2, 51), are raised in response to the bacterial flora and play a critical and 1 prior to IR injury. WT mice treated with neutralizing anti– role in intestinal IR injury by cross-reacting with neoantigens IL-17A Ab developed significantly lower intestinal injury scores expressed on damaged intestinal cells and triggering complement after IR (Supplemental Fig. 2A) and significantly diminished activation. Given the interaction between IL-17A and the micro- neutrophil infiltration in the (Supplemental Fig. biome, we considered the possibility that the failure of Il17a2/2 2B) compared with mice treated with IgG isotype control. Col- mice to develop IR injury is due to defective production of natural lectively, these results confirm that IL-17A plays a critical role in Abs. Administration of serum IgG from B6.lpr mice, which con- intestinal IR injury. tains autoantibodies known to restore IR injury in Rag-deficient recipients (49, 52–55), failed to restore IR injury in Il17a2/2 The RORgt is essential for intestinal IR mice (Supplemental Fig. 1). These observations conclusively place injury IL-17A downstream from the recognition of damaged cells in is- The transcription factor RORgt encoded by Rorc plays an im- chemic intestinal tissue by autoantibodies and subsequent fixation portant role in driving Il17a gene expression (57). We used Rorc2/2 and activation of complement. mice to examine whether RORgt is essential for IR injury. Lack of IL-17A could avert IR injury by exerting effects during Rorc2/2 mice developed significantly less intestinal IR injury development. Furthermore, IL-17A plays an important role in the compared with WT controls, as assessed by injury scores and the maintenance and composition of the gut normal (56). numbers of infiltrating neutrophils in the small intestine (Fig. 2). Alteration of the intestinal microbiota, in the absence of IL-17A, These results indicate that RORgt plays an essential role in in- may affect the profile of natural Abs, which play an essential role testinal IR injury. 4 ILC-DERIVED IL-17A IS ESSENTIAL FOR INTESTINAL IR INJURY

IL-23 plays an important role in intestinal IR injury hematopoietic origin are the major source of IL-17A important for IL-23 plays an important role in IL-17A production and is primarily intestinal damage after intestinal IR. expressed by epithelial cells and DCs (50, 58). Intestinal IR injury TCRgd cells do not contribute significantly to intestinal IR did not cause a significant increase in Il23 mRNA levels in the injury small intestine of WT mice or Il17a2/2 mice (Supplemental Fig. The intestine is rich in TCRgd cells and ILC3s, both of which can 3). Nevertheless, intestinal damage, neutrophil infiltration, and rapidly release IL-17A in response to stimulation with IL-23 (59). expression of Cxcl1 and Cxcl2 mRNA after intestinal IR injury 2/2 2 2 We used Tcrd mice to examine the role of TCRgd cells in IR were significantly, although partially, attenuated in Il23r / mice injury. Intestinal damage and neutrophil infiltration were not sig- compared with WT controls (Fig. 3A–E). nificantly different in Tcrd2/2 mice compared with WT controls To confirm the importance of IL-23 in IR injury, WT mice were (Fig. 5). These results indicate that TCRgd cells do not contribute treated with neutralizing IgG Ab to IL-23 or with IgG isotype significantly to intestinal IR injury. control on days 4, 2, and 1 prior to IR. WT mice treated with neutralizing IL-23 Ab developed significantly less small intestinal ILCs play an important role in intestinal IR injury that depends damage after IR injury than did mice treated with IgG isotype control, on IL-17A as evidenced by significantly lower injury scores (Supplemental Fig. It has been previously demonstrated that intestinal IR injury depends 4A) and significantly lower numbers of infiltrating neutrophils on the presence of natural Abs or autoantibodies (49). Furthermore, it (Supplemental Fig. 4B). These results suggest that IL-23 constitu- 2 2 has been shown that Rag2 / mice, which lack mature T and B cells, tively expressed in the small intestine plays an important role in are resistant to intestinal IR injury and that administration of IgM Downloaded from intestinal IR injury. directed against intestinal neoantigensorofIgGfromautoimmune- IL-17A produced by hematopoietic cells is essential for prone B6.lpr mice restores intestinal IR injury in these mice (6, 7). intestinal IR injury To investigate the potential role of ILCs in intestinal IR injury, we assessed the ability of IgG from autoimmune-prone B6.lpr mice to It has been reported that Paneth cells are the major IL-17A– 2/2 2/2 containing intestinal cells in IR injury (33), but it is not clear restore intestinal IR injury in Rag2 gc mice, which lack ILCs whether they produce IL-17A or simply store and release IL-17A in addition to mature T and B cells. http://www.jimmunol.org/ We first verified that the batch of IgG that we used restores IR injury made by other cells. We used BM chimeras to ascertain whether 2/2 2/2 the IL-17A in IR injury is of hematopoietic or nonhematopoietic in Rag2 mice. As previously reported (13, 51, 55), Rag2 mice cell origin. To determine the contribution of IL-17A derived from had minimal intestinal IR injury compared with WT controls and + neutrophil infiltration (Fig. 6A, 6B). Administration of IgG from B6. hematopoietic cells, BM from CD45.1 WT donors was used to 2/2 reconstitute lethally irradiated CD45.2+ Il17a2/2 or WT recipi- lpr mice restored IR injury in Rag2 mice, albeit to a level lower than that in WT controls (Fig. 6A, 6B). Restoration of intestinal IR ents. To assess the contribution of IL-17A derived from non- 2/2 hematopoietic cells, BM from CD45.2+ WT Il17a2/2 donors was injury in Rag2 mice was associated with the induction of Il17a, used to reconstitute lethally irradiated CD45.1+ WT recipients or Cxcl1,andCxcl2 expression in the small intestine (Fig. 6C). Im- + 2/2 portantly, coadministration of anti–IL-17A neutralizing Ab prevented by guest on September 28, 2021 CD45.2 Il17a recipients. FACS analysis 8 wk after BM re- 2/2 constitution revealed that .91% of the blood cells in WT → the restoration of intestinal IR injury in Rag2 mice treated with Il17a2/2,WT→ WT, and Il17a2/2 → WT BM chimeras were IgG from B6.lpr mice (Fig. 6A, 6B), demonstrating that this resto- ration depended on IL-17A. donor derived (Fig. 4A–C). We could not assess the percentage of 2/2 2/2 2/2 2/2 → 2/2 Like Rag2 mice, Rag2 gc mice developed minimal donor cells in Il17a Il17a BM chimeras, because donors 2/2 and recipients were on the CD45.2 background; however, Il17a2/2 → intestinal IR injury (Fig. 7A). However, in contrast to Rag2 2/2 mice, administration of IgG from B6.lpr mice failed to restore Il17a BM chimeras had numbers of leukocytes in blood and 2/2 2/2 that were comparable to those in the other three chimeras intestinal IR injury in Rag2 gc mice (Fig. 7A, 7B). It also (data not shown). Intestinal IR injury was comparable between failed to induce Il17a, Cxcl1,orCxcl2 expression in the small WT → Il17a2/2 and WT → WT BM chimeras, as assessed by intestine (Fig. 7C). These results indicate that ILCs play an es- injury scores and the numbers of infiltrating neutrophils in the sential role in intestinal IR injury, likely by producing IL-17A. small intestine (Fig. 4D, 4E). In contrast, minimal intestinal injury after IR was observed in Il17a2/2 → WT and Il17a2/2 → Il17a2/2 Discussion BM chimeras (Fig. 4D, 4E). These results indicate that cells of We demonstrate a critical role for IL-17A in the development of intestinal epithelial damage and neutrophil-dominated inflamma- tion during intestinal reperfusion after mesenteric IR injury. We also show that IL-23 is important for intestinal IR injury. In ad- dition, we demonstrate for the first time, to our knowledge, that the major source of IL-17A in this model is ILCs, which are known to rapidly release IL-17A in response to IL-23. Intestinal IR injury was associated with a significant increase in Il17a expression in the small intestine and was markedly attenu- ated in Il17a2/2 mice, as evidenced by a significant decrease in injury scores and neutrophil infiltration in the lamina propria. Similar findings were observed in WT mice treated with anti–IL- 17A neutralizing Ab, ruling out a role for secondary effects of the lack of IL-17A on the microbiome and on the development of FIGURE 2. The transcription factor RORgt is essential for intestinal IR 2/2 injury. Quantitative injury scores (A) and quantitation of infiltrating Gr1+ natural Abs, which are essential for intestinal IR injury, in Il17a cells (B)inRorc2/2 mice and WT controls subjected to intestinal IR injury mice. These observations conclusively place IL-17A down- or sham operation (n = 4 mice per group). Data are mean + SD. *p , 0.05, stream from the recognition of damaged cells in ischemic tissue by Mann–Whitney U test. natural Abs and subsequent fixation and activation of complement. The Journal of Immunology 5

FIGURE 3. IR injury is attenuated in Il23r2/2 mice. Representative H&E-stained sections (A), quantitative injury scores (B), representative immunohistochemical staining for Gr1 (C), quantitation of in- + D filtrating GR1 cells ( ), and qRT-PCR Downloaded from analysis of Il17a (E)andCxcl1 and Cxcl2 (F) expression in Il23r2/2 mice and WT controls subjected to intestinal IR injury or sham operation. Results are derived from three independent experiments, each with three or four mice per group. Data in bar graphs are mean + SEM. Scale http://www.jimmunol.org/ bars, 100 mM(A), 50 mM(C). *p , 0.05, ***p , 0.001, ****p , 0.0001. by guest on September 28, 2021

The role of IL-17A in intestinal IR injury was further supported by Il23r2/2 mice. We did not detect a significant increase in intes- the novel observation that intestinal IR injury was significantly at- tinal Il23 mRNA levels in WT mice following IR injury, sug- tenuated in Rorc2/2 mice, which lack the transcription factor RORgt gesting that preformed IL-23 is the culprit. Notably, IL-23 is that is essential for Il17a gene expression. The mechanism of villous constitutively expressed in the intestines by endothelial cells and, damage in intestinal IR injury is largely dependent on neutrophils to a lesser extent, by epithelial cells (63). Upregulation of Il7a (60). Consistent with the role of IL-17A in driving expression of expression in the intestine following IR was abrogated in Il23r2/2 neutrophil chemoattractants, the expression of Cxcl1 and Cxcl2 was mice, indicating that this upregulation is strictly dependent on significantly upregulated following IR injury in WT mice but not IL-23. Upregulation of Cxcl1 and Cxcl2 expression was signifi- in Il17a2/2 mice. The residual neutrophil infiltration observed in cantly diminished, but not abolished, in Il23r2/2 mice. Because Il17a2/2 mice could be due to cytokines other than IL-17A that upregulation of Cxcl1 and Cxcl2 expression was abolished in cause neutrophil recruitment to injured tissues. These may include Il17a2/2 mice, these findings together suggest that the release of IL-1 and IL-6, which are known to be upregulated in intestines preformed IL-17A contributes to the intestinal upregulation of subjected to IR injury (61, 62). Cxcl1 and Cxcl2 expression caused by IR injury. This may explain We demonstrate for the first time, to our knowledge, that in- why intestinal IR injury was attenuated less in Il23r2/2 mice testinal IR injury was significantly attenuated in Il23r2/2 mice, as compared with Il17a2/2 mice and in WT mice treated with anti– evidenced by decreased injury scores and by decreased neutrophil IL-23 neutralizing Ab compared with WT mice treated with anti– infiltration in the lamina propria. This is consistent with our pre- IL-17A neutralizing Ab. In addition, pathways independent of the vious report that intestinal IR injury is attenuated in IL-23–defi- IL-23–IL-23R signaling pathway may also be involved in driving cient p192/2 mice (13). Furthermore, administration of anti–IL-23 intestinal IR injury. neutralizing Ab to WT mice attenuated intestinal IR injury, indi- We definitively demonstrate, using BM chimeras, that the source of cating that IL-23–IL-23R signaling is important for IR injury in- IL-17A important for intestinal IR injury is a cell of hematopoietic dependent of potential alterations in the microbiota in p192/2 and origin. WT → Il7a2/2 chimeras exhibited intestinal injury that was 6 ILC-DERIVED IL-17A IS ESSENTIAL FOR INTESTINAL IR INJURY Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. IL-17A derived from hematopoietic cells is essential for intestinal IR injury. Representative FACS analysis of CD45.1 and CD45.2 ex- pression (A) and quantitative analysis of the percentages of CD45.1+ and CD45.2+ cells (B and C) in blood leukocytes from WT → WT, WT → Il17a2/2, Il17a2/2 → Il17a2/2, and Il17a2/2 → WT BM radiation chimeras. Gating was on live cells following red cell lysis. Injury scores (D) and quantitation of infiltrating GR1+ cells (E) in the four BM radiation chimeras (n = 4–6 mice per group). Data in bar graphs are mean + SD. n.s., not significant. The Journal of Immunology 7

depletion of IL-17A from the Paneth cells of Il7a2/2 → WT chi- meras, explaining their minimal intestinal IR injury that was no different from that of Il7a2/2 → Il7a2/2 control chimeras. TCRgd T cells rapidly express Il17a and secrete IL-17A in response to IL-23 released by tissue injury (64). However, there was no reduction in the severity of intestinal IR injury in TCRgd- deficient Tcrd2/2 mice, suggesting that TCRgd T cells are not the important source of IL-17A in our model. In addition to releasing preformed IL-17A in response to IL-23 stimulation, ILCs, like TCRgd T cells, rapidly upregulate Il17a mRNA expression in FIGURE 5. TCRgd T cells do not contribute to intestinal IR injury. 2/2 Quantitative injury scores (A) and quantitation of infiltrating GR1+ cells response to IL-23 stimulation. In contrast to Rag2 mice, 2/2 2/2 (B)inTcrd2/2 mice and WT controls subjected to small intestinal injury or Rag2 gc mice reconstituted with IgG from B6.lpr mice sham operation (n = 4 mice per group). Data are mean + SD. n.s., not developed minimal intestinal IR injury. Furthermore, intestinal IR 2 2 2 2 2 2 significant. in Rag2 / mice, but not in Rag2 / gc / mice, reconstituted with B6.lpr IgG was associated with robust expression of Il17a, comparable to that observed in WT → WT chimeras. In contrast, Cxcl1, and Cxcl2 in the intestine. As previously mentioned, in- Il7a2/2 → WT chimeras developed minimal intestinal IR injury. testinal IR injury in the reconstituted mice depends on IL-17A, Paneth cells have been demonstrated to store IL-17A and to be because it was abrogated by administration of anti–IL-17A neu- important for IR injury (33); however, gene expression array anal- tralizing Ab. Taken together with the observation that intestinal IR Downloaded from yses do not reveal detectable expression of Il17a mRNA in Paneth injury depends on RORgt, these findings strongly suggest that cells. This suggests that Paneth cells store IL-17A that is normally RORgt-expressing IL-17A–producing ILC3s are essential for the produced by a cell of hematopoietic origin and, thereby, contribute to development of intestinal IR injury. intestinal IR injury. The 8–9-wk interval after irradiation of WT Based on our current and previous data (65, 66), we propose a mice and their reconstitution with Il7a2/2 BM likely resulted in the mechanism for intestinal IR injury in which ischemia causes the http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. Reconstitution of injury in Rag22/2 is IL-17A dependent. Quantitative injury scores (A) and quantitation of small intestinal infiltrating GR1+ cells (B)inRag22/2 mice, WT mice, and in Rag22/2 mice administered IgG from B6.lpr mice alone, rat anti–IL-17A IgG neutralizing Ab, or rat IgG isotype control and subjected to intestinal IR injury or sham operation (n = 3–5 mice per group). (C) Il17a, Cxcl1, and Cxcl2 expression in the small intestine in WT mice, Rag22/2 mice, and Rag22/2 mice administered IgG from B6.lpr mice (n = 3–5 mice per group) and subjected to small intestinal IR injury or sham operation. Data are mean + SD. ***p , 0.001, ****p , 0.0001. 8 ILC-DERIVED IL-17A IS ESSENTIAL FOR INTESTINAL IR INJURY Downloaded from http://www.jimmunol.org/

FIGURE 7. ILCs are important in intestinal IR injury by producing IL-17A. Injury scores (A), quantitation of small intestinal infiltrating GR1+ cells (B), and Il17a, Cxcl1, and Cxcl2 expression (C)inRag22/2gc2/2 mice administered IgG from B6.lpr mice subjected to small intestinal IR injury compared with Rag22/2gc2/2 and WT mice (n = 3–6 mice per group) subjected to small intestinal IR injury. Data are mean + SD. n.s., not significant. by guest on September 28, 2021 expression of damage-associated neoantigens on intestinal cells. 5. Diepenhorst, G. M., T. M. van Gulik, and C. E. Hack. 2009. Complement- Binding of natural Abs that cross-react with intestinal neoantigens mediated ischemia-reperfusion injury: lessons learned from animal and clini- cal studies. Ann. Surg. 249: 889–899. results in fixation and activation of complement (7). Complement 6. Zhang, M., W. G. Austen, Jr., I. Chiu, E. M. Alicot, R. Hung, M. Ma, N. Verna, binding to receptors, such as C3aR and C5aR, on intestinal cells M. Xu, H. B. Hechtman, F. D. Moore, Jr., and M. C. Carroll. 2004. Identification releases IL-23, which drives rapid IL-17A production and release by of a specific self-reactive IgM antibody that initiates intestinal ischemia/ reperfusion injury. Proc. Natl. Acad. Sci. USA 101: 3886–3891. ILC3s. The locally released IL-17A acts on its receptors on epithelial 7. Fleming, S. D., T. Shea-Donohue, J. M. Guthridge, L. Kulik, T. J. Waldschmidt, and other stromal cells to induce the expression and release of M. G. Gipson, G. C. Tsokos, and V. M. Holers. 2002. Mice deficient in com- chemokines that attract neutrophils. The release of granular contents plement receptors 1 and 2 lack a tissue injury-inducing subset of the natural antibody repertoire. J. Immunol. 169: 2126–2133. from neutrophils causes tissue damage, resulting in intestinal IR 8. Maroko, P. R., C. B. Carpenter, M. Chiariello, M. C. Fishbein, P. Radvany, injury. Accordingly, blockade of the IL-23/IL-17A axis may provide J. D. Knostman, and S. L. Hale. 1978. Reduction by cobra venom factor of a powerful therapeutic strategy to attenuate intestinal IR injury. myocardial necrosis after coronary artery occlusion. J. Clin. Invest. 61: 661–670. 9. Hill, J., T. F. Lindsay, F. Ortiz, C. G. Yeh, H. B. Hechtman, and F. D. Moore, Jr. 1992. Soluble complement receptor type 1 ameliorates the local and remote Acknowledgments organ injury after intestinal ischemia-reperfusion in the rat. J. Immunol. 149: 1723–1728. We thank Stella Kourembanas (Division of Newborn Medicine, Children’s 10. Lapchak, P. H., L. Kannan, A. Ioannou, P. Rani, P. Karian, J. J. Dalle Lucca, and Hospital) and Paul H. Lerou (Division of Newborn Medicine, Mas- G. C. Tsokos. 2012. Platelets orchestrate remote tissue damage after mesenteric sachusetts General Hospital) for support and Drs. Rene De Waal Malefyt ischemia-reperfusion. Am. J. Physiol. Gastrointest. Liver Physiol. 302: G888– 2 2 2 2 and J. Kolls for providing Il23r / and Il7a / mice. G897. 11. Pamuk, O. N., P. H. Lapchak, P. Rani, P. Pine, J. J. Dalle Lucca, and G. C. Tsokos. 2010. Spleen tyrosine kinase inhibition prevents tissue damage Disclosures after ischemia-reperfusion. Am. J. Physiol. Gastrointest. Liver Physiol. 299: The authors have no financial conflicts of interest. G391–G399. 12. Chen, J., J. C. Crispı´n, T. F. Tedder, J. Dalle Lucca, and G. C. Tsokos. 2009. B cells contribute to ischemia/reperfusion-mediated tissue injury. J. Autoimmun. 32: 195–200. References 13. Edgerton, C., J. C. Crispı´n, C. M. Moratz, E. Bettelli, M. Oukka, M. Simovic, 1. Kong, S. E., L. R. Blennerhassett, K. A. Heel, R. D. McCauley, and J. C. Hall. 1998. A. Zacharia, R. Egan, J. Chen, J. J. Dalle Lucca, et al. 2009. IL-17 producing CD4+ Ischaemia-reperfusion injury to the intestine. Aust.N.Z.J.Surg.68: 554–561. T cells mediate accelerated ischemia/reperfusion-induced injury in autoimmunity- 2. Stoney, R. J., and C. G. Cunningham. 1993. Acute mesenteric ischemia. Surgery prone mice. Clin. Immunol. 130: 313–321. 114: 489–490. 14. Yeremenko, N., J. E. Paramarta, and D. Baeten. 2014. The -23/ 3. Chiu, C. J., A. H. McArdle, R. Brown, H. J. Scott, and F. N. Gurd. 1970. In- interleukin-17 immune axis as a promising new target in the treatment of testinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, spondyloarthritis. Curr. Opin. Rheumatol. 26: 361–370. and metabolic reappraisal. Arch. Surg. 101: 478–483. 15. Ivanov, I. I., B. S. McKenzie, L. Zhou, C. E. Tadokoro, A. Lepelley, J. J. Lafaille, 4. Shandall, A. A., G. T. Williams, M. B. Hallett, and H. L. Young. 1986. Colonic D. J. Cua, and D. R. Littman. 2006. The orphan nuclear receptor RORgammat healing: a role for polymorphonuclear leucocytes and oxygen production. directs the differentiation program of proinflammatory IL-17+ T helper cells. Br.J.Surg.73: 225–228. Cell 126: 1121–1133. The Journal of Immunology 9

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Platelets, complement and T. Iwaki, Y. Okada, M. Iida, D. J. Cua, et al. 2009. Pivotal role of cerebral tissue inflammation. Autoimmunity 46: 1–5. 5 Sham IR e 4 r o

c 3 S

y r 2 u j n

I 1 0 - - +PBS +B6.lpr IgG Il17a-/-

Figure S1. Administration of autoantibody rich B6.lpr IgG fails to restore IR injury in

Il17a-/- mice. A. Injury scores in Il17a-/- mice administered PBS or B6.lpr IgG compared to sham operated control (n = 3-4 mice per group). Columns and bars represent the mean+SD. n.s.: not significant.

Supplementary Figure 1 A B ** ** 5 20

e 4 r 15 o F c 3 P S 10 H y / r 2 N u j

M 5 n I

1 P 0 0 Sham IR Sham IR Sham IR Sham IR WT WT + αIL17A Ab WT WT + α-L17A Ab

Figure S2. IL-17A neutralization attenuates small intestinal IR injury. A-B. Injury scores

(A) and quantitation of infiltrating GR1+ cells (B) in WT mice pretreated with rat anti-IL-17A

neutralizing IgG Ab. or IgG isotype control, then subjected to IR injury compared to WT

controls (n = 3-4 mice per group). Columns and bars represent the mean+SD. *p< 0.05, ** p<

0.01.

Supplementary Figure 2 A 10 Il-23 8

6

4 n.s. n.s.

mRNA/CycloA 2 expression over sham 0 Sham IR Sham IR WT Il17a-/-

Figure S3. Intestinal IR injury does not increase in Il23 mRNA expression in the small

intestine of WT mice or Il17a-/- mice. Il23 expression in WT and Il17a-/- mice subjected to

intestinal IR injury or sham operated.

Supplementary Figure 2 A B ** ** 5 20 e r 4 15 F o P c 3 S H

/ 10 y N r 2 u M j P

n 5 I 1 0 0 Sham IR Sham IR Sham IR Sham IR WT WT + αIL23p19 Ab WT WT + αIL23p19 Ab

Figure S4. IL-23 neutralization attenuates small intestinal IR injury. A-B. Injury scores

(A) and quantitation of infiltrating GR1+ cells (B) in WT mice pretreated with goat anti-IL-23

neutralizing monoclonal IgG Ab., or IgG isotype control mAb then subjected to IR injury

compared to WT controls (n = 3-4 mice per group). Columns and bars represent the mean+SD.

*p< 0.05, ** p< 0.01.

Supplementary Figure 3