IL-17A produced by innate lymphoid cells is essential for intestinal ischemia reperfusion injury

Abstract

Ischemia-reperfusion (IR) injury to the small intestine following clamping of the superior mesenteric artery results in an intense local inflammatory response characterized by villous damage and neutrophil infiltration. IL-17A, a cytokine produced by a variety of cells in response to inflammatory cytokines released following tissue injury, has been implicated in IR injury. Using Il17a^−/−, Il23r^−/− and Rorγt^−/− mice and administration of anti-IL17A and anti-IL-23 neutralizing antibodies to wild-type mice, we demonstrate that intestinal IR injury depends on IL17A and that IL-17A is downstream of the binding of autoantibody to ischemia-conditioned tissues and subsequent complement activation. Using bone marrow chimeras, we demonstrate that the IL17A required for intestinal IR injury is derived from hematopoietic cells. Finally, by transferring autoantibody-rich sera into Rag2γc^−/− and Rag2^−/− mice we demonstrate that innate lymphoid cells (ILCs) are the main producers of IL-17A in intestinal IR injury. We propose that local production of IL-17A by ILCs is crucial for the development of intestinal IR injury and may provide a therapeutic target for clinical exploitation.

INTRODUCTION

Intestinal ischemia-reperfusion (IR) injury occurs when the blood supply to the intestine is re-established following a period of transient disruption of blood flow. Intestinal IR injury occurs in a variety of clinical conditions including shock, trauma, sepsis, aortic surgery, and acute mesenteric artery occlusion (1, 2). Intestinal IR injury triggers a cascade of events that result in both local and remote organ injury. Intestinal damage after IR injury is characterized by severe villus destruction with disruption and dysfunction of the intestinal epithelium (3). Re-establishment of the blood supply to the intestine initiates an intense local inflammatory response with neutrophil infiltration (4). IR injury depends on elements of both the innate and the adaptive immune response (5). Contributors to the tissue damage include IgM (6), natural antibodies (7), complement (8, 9), neutrophils (4), platelets (10, 11), B lymphocytes (12), and T (13) lymphocytes.

IL-17A is a proinflammatory cytokine that causes epithelial cells to secrete neutrophil chemoattractant chemokines such as CXCL1, CXCL2 and IL-8 (14). Cells that produce IL-17A include TCRα/β T cells (15, 16), TCRγ/δT cells (17) and CD45⁺CD4⁺TCR⁻IL-7R⁺ type 3 innate lymphoid cells (ILC3s) (18, 19), which reside at mucosal surfaces. Other sources of IL-17A include dendritic cells (DCs), macrophages, neutrophils and natural killer cells (NK) (20). The IL17A receptor is a heterodimer of the IL-17RA and IL-17RC chains that bind IL-17A and its homologue IL-17F, and is expressed predominantly on epithelial cells (21, 22). Because of its ability to mobilize neutrophils, IL-17A is important in the pathogenesis of autoimmune diseases and in chronic steroid resistant asthma characterized by neutrophil predominance (23, 24). It is also important for host defense against candida infection, as illustrated by the susceptibility to candida infection of patients who carry mutations in IL17A or genes encoding IL-17A receptor chains (25). Naïve TCRα/β cells differentiate into T helper 17 (Th17) cells following TCR ligation in the presence of the inflammatory cytokines IL-1, IL-6 and TGFβ(26). The differentiation of TCR CD4⁺ Th17 cells is promoted by the cytokine IL-23 (20), whereas the rapid production of IL-17A by TCRγ/δT cells and ILC3s is directly driven by IL-23 (27). IL-23 is a heterodimer of the 40 kD chain (p40), shared with IL-12, and the IL-23 specific 19 kD chain p19 (28), and is produced by epithelial cells and DCs (29, 30). The IL-23R is a heterodimer of the IL12Rβ1 chain, shared with the IL-12R, and an IL-23R specific chain (30, 31).

We have previously shown, using immunofluorescence microscopy, an increase in IL-17A expression in the intestine after IR injury (13). Importantly, IL-17A, via its role in neutrophil recruitment, has been shown to play a critical role in intestinal IR injury (32, 33), as well as in IR injury in other organ systems including heart (34, 35), liver (36, 37), lung (38), kidney (39, 40), brain (41, 42). It has been claimed that Paneth cells store IL-17A and are the major source of IL-17A in intestinal IR injury (33). However, because published gene array analyses do not show that Paneth cells express Il17a (43, 44); we set out to analyze the source of IL-17A relevant to intestinal IR injury. We have confirmed the role of IL-17A and demonstrated a role for IL-23 in intestinal IR injury, and we provide evidence that ILCs are the relevant source of IL-17A in intestinal IR injury.

METHODS

Mice

Il17a^−/− and Il23r^−/− mice on a C57BL/6J (B6) background were generated as described (45) (46). Age-matched C57BL/6J WT (Jackson Laboratory, Bar Harbor, ME) wild type (WT) CD45.2 mice were used as controls. CD45.1 C57BL/6J WT mice (Jackson Laboratory, Bar Harbor, ME) were used in making the bone marrow chimeras. Rag2^−/− mice and Rag2^−/−γ_c^−/− mice on C57BL/6J background were obtained from Taconic, (Hudson, NY). B6.MRLTnfrsf6^lpr (B6.lpr) female mice age 7–8 months were purchased from The Jackson Laboratory (Bar Harbor, ME) and used to isolate IgG from serum. Tcrd^−/− and Rorc^−/− mice on B6.129 background were backcrossed on to a C57BL/6 background for at least 12 and 6 generations respectively. Mice underwent at least 7 days of acclimatization before experimentation. All mice used in this study were 8–12 week old males, except the bone marrow chimeras that were 18–22 weeks old, and were maintained in pathogen-free conditions in the animal research facility at the Beth Israel Deaconess Medical Center, Boston, MA. All experiments were performed in accordance with the guidelines and approval of the Harvard University Institutional Animal Care and Use Committee.

Ischemia-Reperfusion (IR) Injury

Mice were randomly assigned to sham or IR groups. Mice were anesthetized by intraperitoneal injection of 72 mg/kg pentobarbital or 250 mg/kg Avertin and anesthesia was maintained by subcutaneous 36 mg/kg pentobarbital injected or 125 mg/kg Avertin. A midline laparotomy incision was made and the superior mesenteric artery (SMA) was identified, isolated and clamped for 30 min using a microvascular clip (Roboz Surgical Instruments, Rockville MD) delivering ~8.5 g of pressure. The clip was removed after 30 minutes of ischemia and the intestines were re-perfused for 2 hours. The laparotomy incision was sutured closed using 4.0 prolene suture and the mice were resuscitated with 1 ml warm PBS injected subcutaneously. The mice were monitored throughout the experiment. Body temperature was maintained at 37°C throughout the experiment on a temperature controlled heating pad. Sham-operated mice underwent identical abdominal manipulations (laparotomy, intestinal retraction, and positioning) as mice subjected to SMA clamping. Intestines were collected 2 h after sham operation or intestinal IR injury unless otherwise noted.

Histological analysis of intestinal IR injury

Small intestine (jejunum and ileum) was washed in ice-cold PBS and fixed overnight in 10% formalin. After automated dehydration through a graded alcohol series, tissues were embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin-eosin (H&E). Intestinal H&E sections were graded for intestinal IR-induced mucosal injury. Villi were scored using a published algorithm (3) to measure intestinal damage in a blinded manner by one of us (MGT).

Immunohistochemistry

Formalin-fixed paraffin sections of small intestine were subjected to rehydration, and endogenous peroxidase activity was quenched with 3% H₂O₂. Then antigen retrieval was performed using Retrievagen A (BD Pharmingen, San Jose, CA) according to the manufacturer’s directions. The sections were blocked with 10% BSA/PBS containing the serum from host species of secondary antibody. The primary antibody rat-anti-mouse Ly-6B.2 (Gr1) clone 7/4 at 1/250 dilution (Bio-Rad, Herculese, CA) was used to stain for neutrophils. Primary antibody or isotype control antibody prepared in 10% BSA/PBS were applied overnight at 4°C. The slides were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 60 min at room temperature, developed with NovaRED (Vector Laboratories, Burlingame, CA), counterstained with hematoxylin, and dehydrated. The sections were mounted in mounting medium (Thermo Scientific, Waltham, MA) and evaluated with Nikon eclipse 80i microscope. Images were analyzed using Nikon NIS-Elements software (Nikon, Melville, NY). For neutrophil infiltration positive staining cells in the area of injury were counted in 20 high power fields (HPF) at 200× and the average was calculated and expressed as number of neutrophils per HPF.

Neutralization of endogenous IL-17A and IL-23

To neutralize IL-17A, 200 μg of rat-anti-mouse anti-IL-17A Ab (R&D Systems) were given in 3 doses by intraperitoneal (i.p.) injection 96, 48 and 24 hours prior to IR as described (47). Control animals were given 200 μg rat IgG isotype control antibody (R&D systems) also in 3 doses 96, 48 and 24 hours prior to IR. To neutralize IL-23, 10 μg of goat-anti-mouse anti-IL-23 Ab (R&D Systems) were given in 3 doses by i.p. injection 96, 48 and 24 hours prior to IR as described by (48). Control animals were given 20 μg goat IgG isotype control antibody (R&D systems) also in 3 doses 96, 48 and 24 hours prior to IR.

Reconstitution of Rag2^−/− and Rag2^−/−γ_c^−/− mice with IgG from B6.lpr mice

Serum from B6.lpr mice was obtained by cardiac puncture. The Melon^TM Gel IgG Purification Kit (Thermo Scientific) was used to Isolate and purify IgG. A buffer exchange to PBS was performed on the eluted material. 200 μg of IgG was injected intravenously in Rag2^−/− or Rag2^−/−γ_c^−/− mice 30 minutes prior to ischemia or sham operation as previously described (49).

Generation of Bone Marrow (BM) Radiation Chimeras

Eight-week-old recipient CD45.2⁺ WT and Il17a^−/− mice were lethally irradiated (1,100 rads delivered in 2 doses of 550 rads each at 3 hrs. intervals), and injected i.v. with 5×10⁶ BM cells obtained from congenic CD45.1⁺ WT mice and vice versa. Chimerism was assessed by measuring the percentages of CD45⁺ donor and recipient cells in the chimeric mice 8 weeks after BM reconstitution using FACS analysis for the CD45.1 and CD45.2 markers on blood after red blood cell lysis.

qRT-PCR analysis

Intestines were harvested and placed in RNAlater (Thermo Fisher Scientific). Total RNA was isolated using the RNeasy Mini kit (Qiagen). Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to manufacturer protocol. Quantitative real-time PCR were performed (Light Cycler 480; Roche) for Il17a, Il23, Cxcl1, Cxcl2, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Cyclophilin A (CycloA) with 40 cycles at 94 °C for 12 s and 60 °C for 60 s using appropriate murine TaqMan assays for Il17a, Il23, Cxcl1, Cxcl2 and GAPDH (Applied Biosystems) or using SYBR Green I Brilliant Mastermix (Stratagene, La Jolla, CA) with appropriate primers for Il17a, Il23 and CycloA Ct values were determined by using Mx3000P software. All PCR reactions were run in triplicates. The averaged cycle threshold values for each target gene were normalized for GAPDH or CycloA mRNA, and relative expression of the target gene mRNA was calculated with the ΔΔCt relative quantification method.

Statistical analysis

Numeric data are presented as mean ± SD or mean ± SEM. Statistical analyses were performed using GraphPad Prisim 6.0 (GraphPad Software, San Diego, CA). The ordinal values of the injury scores and PMN/HPF were analyzed by the non-parametric Mann-Whitney U. The two-tailed Student’s t test for unpaired samples was used in the comparison of means of two groups. A value of P < 0.05 was considered statistically significant.

RESULTS

IL-17A is important for mesenteric IR injury

We confirmed the critical role of IL-17A in our model of mesenteric IR injury in which mice are subjected to 30 minutes of SMA occlusion followed by 2 hours of reperfusion. Il17a^−/− mice developed markedly less intestinal damage, with lower injury scores, compared to WT mice (Fig. 1A & B). Il17a^−/− mice subjected to mesenteric IR injury had significantly less neutrophil infiltration in the intestine than WT controls as determined by examination of intestinal sections stained immunohistochemically for Gr1 (Fig. 1C & D).

Figure 1. IR injury is attenuated in Il17a^−/− mice.

A–E. Representative H&E sections (A) Injury scores (B), representative immunohistochemical staining for Gr1 (C) quantitation of infiltrating Gr1⁺ cells/HPF (D), and qRT-PCR analysis of Il17a (E), Cxcl1 and Cxl2 expression (F) in Il17a^−/− mice and WT controls subjected to intestinal IR injury or sham operated. Results are derived from three independent experiments each with 3–4 mice per group. Columns and bars represent the mean+SEM. *p< 0.05, ** p< 0.01, *** p<0.001. Photomicrographs in A 100× magnification, scale bars represent 100 μM. Photomicrographs in C 200× magnification, scale bars represent 50 μM.

Mesenteric IR injury in WT mice was associated with a significant increase in Il17a mRNA levels in the small intestine when compared to sham operated mice, as determined by qRT-PCR (Fig. 1E). IL-17A is known to drive neutrophil infiltration into tissues by inducing resident cells to express neutrophil attracting chemokines, including Cxcl1 and Cxcl2,(50). Cxcl1 and Cxcl2 mRNA levels were significantly higher in the small intestine of WT mice subjected mesenteric IR injury compared to sham operated mice. In contrast, there was no significant increase in Cxcl1 and Cxcl2 mRNA levels in the small intestine of Il17a^−/− mice subjected to mesenteric IR injury when compared to WT controls (Fig. 1F).

Natural antibodies (7) that include self-reacting autoantibodies (49, 51) are raised in response to the bacterial flora and play a critical role in intestinal IR injury by cross reacting with neoantigens expressed on damaged intestinal cells and triggering complement activation. Given the interaction between IL-17A and the microbiome, we considered the possibility that the failure of Il17a^−/− mice to develop IR injury may be due to defective production of natural antibodies. Administration of serum IgG from B6.lpr mice, which contains autoantibodies known to restore IR injury in Rag deficient recipients (49, 52–55), failed to restore IR injury in Il17a^−/− mice (Fig S1). These observations conclusively place IL-17A downstream from the recognition of damaged cells in ischemic intestinal tissue by autoantibodies and subsequent fixation and activation of complement.

Lack of IL17-A could avert IR injury by exerting effects during development. Furthermore, IL-17A plays an important role in the maintenance and composition of the gut normal microbiome (56). Alteration of the intestinal microbiota, in the absence of IL-17A, may affect the profile of natural antibodies, which play an essential role in intestinal IR injury (49, 52–55). To circumvent these limitations, we examined the effect of administration of neutralizing IL-17A on intestinal IR injury in WT mice. WT mice were treated with a neutralizing IgG antibody to IL-17A, or IgG isotype control antibody, on days -4, -2 and -1 prior to IR injury. WT mice treated with neutralizing anti-IL-17A antibody developed significantly lower intestinal injury scores after IR (Fig. S2 A) and significantly diminished neutrophil infiltration in the lamina propria (Fig. S2 B) compared to mice treated with IgG isotype control. Collectively, these results confirm that that IL-17A plays a critical role in intestinal IR injury.

The transcription factor RORγt is essential for intestinal IR injury

The transcription factor RORγt encoded by Rorc plays an important role in driving Il17a gene expression(57). We used Rorc^−/− mice to examine whether RORγt is essential for IR injury. Rorc^−/− mice developed significantly less intestinal IR injury compared to WT controls, as assessed by injury scores and numbers of infiltrating neutrophils in the small intestine (Fig. 2A & B). These results indicate that RORγt plays an essential role in intestinal IR injury.

Figure 2. The transcription factor RORγt is essential for intestinal IR injury.

A–B. Quantitative Injury scores (A) and quantitation of infiltrating Gr1⁺ cells (B), in Rorc^−/− mice and WT controls subjected to intestinal IR injury or sham operated (n = 4 mice per group). Columns and bars represent the mean+SD statistical analysis was performed by the Mann-Whitney U test. *p< 0.05

IL-23 plays an important role in intestinal IR injury

IL-23 plays an important role in IL-17A production and is primarily expressed by epithelial cells and dendritic cells (50, 58). Intestinal IR injury did not cause a significant increase in Il23 mRNA levels in the small intestine of WT mice or Il17a^−/− mice (Fig S3). Nevertheless, intestinal damage, neutrophil infiltration and expression of Cxcl1 and Cxcl2 mRNA after intestinal IR injury were significantly, albeit partially, attenuated in Il23r^−/− mice compared to WT controls (Fig. 3A–E).

Figure 3. IR injury is attenuated in Il23r^−/− mice.

A–E. Representative H&E sections (A) quantitative Injury scores (B), representative immunohistochemical staining for Gr1 (C) quantitation of infiltrating GR1⁺ cells (D), and qRT-PCR analysis of Il17a (E), Cxcl1 and Cxl2 expression (F) in Il23r^−/− mice and WT controls subjected to intestinal IR injury or sham operated. Results are derived from three independent experiments each with 3–4 mice per group. Columns and bars represent the mean+SEM. *p< 0.05, ** p< 0.01, *** p<0.001. Photomicrographs in A 100× magnification, scale bars represent 100 μM. Photomicrographs in C 200× magnification, scale bars represent 50 μM.

To confirm the importance of IL-23 in IR injury, WT mice were treated with neutralizing IgG antibody to IL-23, or IgG isotype control, on days -4, -2 and -1 prior to IR. WT mice treated with neutralizing IL-23 antibody developed significantly less small intestinal damage after IR injury than mice treated with IgG isotype control, as evidenced by significantly lower injury scores (Fig. S4 A), and significantly lower numbers of infiltrating neutrophils (Fig. S4 B). These results suggest that IL-23 constitutively expressed in the small intestine plays an important role in intestinal IR injury.

IL-17A produced by hematopoietic cells is essential for intestinal IR injury

It has been reported that Paneth cells are the major IL-17A-containing intestinal cells in IR injury (33) but it is not clear whether Paneth cells produce IL-17A or simply store and release IL-17A made by other cells. We used BM chimeras to ascertain whether the source of IL-17A in IR injury is of hematopoietic or non-hematopoietic cell origin. To determine the contribution of IL-17A derived from hematopoietic cells BM from CD45.1⁺ WT donors was used to reconstitute lethally irradiated CD45.2⁺ Il17a^−/− or WT recipients. To assess the contribution of IL-17A derived from non-hematopoietic cells BM from CD45.2⁺ WT Il17a^−/− donors were used to reconstitute lethally irradiated CD45.1⁺ WT recipients or CD45.2⁺ Il17a^−/− recipients. FACS analysis eight weeks after BM reconstitution revealed that >91% of the blood cells in the WT->Il17a^−/−, WT->WT and Il17a^−/− ->WT BM chimeras were donor-derived (Fig. 4A–C). We could not assess the percentage of donor cells in Il17a^−/− ->Il17a^−/− BM chimeras, as both donors and recipients were on the CD45.2 background; however Il17a^−/− ->Il17a^−/− BM chimeras had numbers of leukocytes in blood and spleen that were comparable to those in the other three chimeras (data not shown). Intestinal IR injury was comparable between WT->Il17a^−/− and WT->WT BM chimeras as assessed by injury scores and numbers of infiltrating neutrophils in the small intestine (Fig. 4D & E). In contrast, minimal intestinal injury after IR was observed in Il17a^−/− ->WT and Il17a^−/− ->Il17a^−/− BM chimeras (Fig. 4D & E). These results indicate that the major source of IL-17A important for intestinal damage after intestinal IR is a cell of hematopoietic origin.

Figure 4. IL-17A derived from hematopoietic cells is essential for intestinal IR injury.

A–C. Representative FACS analysis of CD45.1 and CD45.2 expression (A) and quantitative analysis of the percentages of CD45.1+ and CD45.2+ cells (B & C) in blood leukocytes from WT->WT, WT ->Il17a^−/−, Il17a^−/−->WT Il17a^−/−-> Il17a^−/− bone marrow radiation chimeras. Gating was on live cells following red cell lysis. D, E. Injury scores (D), and quantitation of infiltrating GR1⁺ cells (E) in the four bone marrow radiation chimeras (n = 4–6 mice per group). Columns and bars represent the mean+SD. n.s.: not significant.

TCRγδ cells do not contribute significantly to intestinal IR injury

The intestine is rich in TCRγδ cells and type 3 ILCs, both of which can rapidly release IL-17A in response to stimulation with IL-23 (59). We used Tcrd^−/− mice to examine the role of TCRγδ cells in IRI. Intestinal damage and neutrophil infiltration was not significantly different Tcrd^−/− mice compared to WT controls (Fig. 5A & B). These results indicate that TCRγδ cells do not contribute significantly to intestinal IR injury.

Figure 5. TCRγδ T cells do not contribute to intestinal IR injury.

A–B. Quantitative Injury scores (A) and quantitation of infiltrating GR1⁺ cells (B), in Tcrd^−/− mice and WT controls subjected to small intestinal injury or sham operated (n = 4 mice per group). Columns and bars represent the mean+SD. n.s.: not significant.

Innate lymphoid cells (ILCs) play an important role in intestinal IR injury that depends on IL-17A

It has been previously demonstrated that intestinal IR injury depends on the presence of natural antibodies or autoantibodies (49). Furthermore it has been shown that Rag2^−/− mice, which lack mature T and B cells, are resistant to intestinal IR injury and that administration of IgM directed against intestinal neoantigens, or of IgG from autoimmune-prone B6.lpr mice, restores intestinal IR injury in these mice (6, 7). To investigate the potential role of ILCs in intestinal IR injury, we assessed the ability of IgG from autoimmune-prone B6.lpr mice to restore intestinal IR injury in Rag2^−/−γc^−/− mice, which lack ILCs in addition to lacking mature T and B cells.

We first verified that the batch of IgG we used restores IR injury in Rag2^−/− mice. As previously reported (13, 51, 55), Rag2^−/− mice had minimal intestinal IR injury compared to WT controls and neutrophil infiltration (Fig. 6A & B). Administration of IgG from B6.lpr mice restored IR injury in Rag2^−/− mice, albeit to a level lower than that in WT controls (Fig. 6A & B). Restoration of intestinal IR injury in Rag2^−/− mice was associated with the induction of Il17a, Cxcl1 and Cxcl2 expression in the small intestine (Fig. 6C). Importantly, co-administration of anti-IL-17A neutralizing antibody prevented the restoration of intestinal IR injury in Rag2^−/− mice treated with IgG from B6.lpr mice (Fig. 6A & B), demonstrating that this restoration depended on IL-17A.

Figure 6. Reconstitution of injury in Rag2^−/− is IL-17A dependent.

A, B. Quantitative injury scores (A) and quantitation of small intestinal infiltrating GR1⁺ cells (B) in Rag2^−/−, WT and in Rag2^−/− mice administered IgG from B6.lpr mice alone, or with rat anti-IL-17A IgG neutralizing antibody or with rat IgG isotype control subjected to intestinal IR injury or sham operated (n=3–5 mice per group). C. Il17a, Cxcl1 and Cxcl2 expression in the small intestine in WT, Rag2^−/− and Rag2^−/− mice administered IgG from B6.lpr mice (n=3–5 mice per group) subject to small intestinal IR injury or sham operated. Columns and bars represent the mean+SD. *** p<0.001.

Like Rag2^−/− mice, Rag2^−/−γc^−/− mice developed minimal intestinal IR injury (Fig. 7A). However, in contrast to Rag2^−/− mice, administration of IgG from B6.lpr mice failed to restore intestinal IR injury in Rag2^−/−γc^−/− mice (Fig. 7A & B). It also failed to induce Il17a, Cxcl1 and Cxcl2 expression in the small intestine (Fig. 7C). These results indicate that ILCs play an essential role in intestinal IR injury, likely by producing IL-17A.

Figure 7. Innate lymphoid cells are important in intestinal IR injury by producing IL-17A.

A–C. Injury scores (A) quantitation of small intestinal infiltrating GR1⁺ cells (B) and Il17a, Cacl1 and Cxcl2 expression (C) in Rag2^−/−γc^−/− mice administered IgG from B6.lpr mice subject to small intestinal IR injury compared to Rag2^−/−γc^−/− and WT (n= 3–6 mice per group) subject to small intestinal IR injury. Columns and bars represent the mean+SD. n.s.: not significant.

DISCUSSION

We demonstrate a critical role for IL-17A in the development of intestinal epithelial damage and neutrophil-dominated inflammation during intestinal reperfusion after mesenteric IR injury. We also show that IL-23 is important for intestinal IR injury. In addition, we demonstrate for the first time that the major source of IL-17A in this model are ILCs, which are known to rapidly release IL-17A in response to IL23.

Intestinal IR injury was associated with a significant increase in Il17a expression in the small intestine, and was markedly attenuated in Il17a^−/− mice, 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 antibody, ruling out a role for secondary effects of lack of IL-17A on the microbiome and on the development of natural antibodies, essential for intestinal IR injury, in Il17a^−/− mice. These observations conclusively place IL-17A downstream from the recognition of damaged cells in ischemic tissue by natural antibodies and subsequent fixation and activation of complement. The role of IL-17A in intestinal IR injury was further supported by the novel observation that intestinal IR injury was significantly attenuated in Rorc^−/− mice, which lack the transcription factor RORγt essential for Il17a gene expression. The mechanism of villous damage in intestinal IR injury is largely dependent on neutrophils (60). Consistent with the role of IL-17A in driving expression of neutrophil chemoattractants, the expression of Cxcl1 and Cxcl2 was significantly upregulated following IR injury in WT mice, but not in Il17a^−/− mice. The residual neutrophil infiltration observed in Il17a^−/− mice could be due to cytokines other than IL-17A that cause neutrophil recruitment to injured tissues. These may include IL-1 and IL-6, which are known to be upregulated in intestines subjected to IR injury (61, 62)

We demonstrate for the first time that intestinal IR injury was significantly attenuated in Il23r^−/− mice, as evidenced by decreased injury scores as well as by decreased neutrophil infiltration in the lamina propria. This is consistent with our previous report that intestinal IR injury is attenuated in IL-23 deficient p19^−/− mice (13). Furthermore, administration of anti-IL-23 neutralizing antibody to WT mice attenuated intestinal IR injury, indicating that IL-23-IL-23R signaling is important for IR injury independent of potential alterations in the microbiota in p19^−/− and Il23r^−/− mice. We did not detect a significant increase in intestinal Il23 mRNA levels in WT mice following IR injury, suggesting that preformed IL-23 is the culprit. Notably IL-23 is constitutively expressed in the intestines by endothelial cells, and to a lesser extent by epithelial cells (63). Upregulation of Il7a expression in the intestine following IR was abrogated in Il23r^−/− mice, indicating that this upregulation is strictly dependent on IL-23. Upregulation of Cxcl1 and Cxcl2 expression was significantly diminished, but not abolished, in Il23r^−/− mice. Since upregulation of Cxcl1 and Cxcl2 expression was abolished in Il17a^−/− mice, these findings together suggest that the release of preformed IL-17A contributes to the intestinal upregulation of Cxcl1 and Cxcl2 expression caused by IR injury. This may explain why intestinal IR injury was attenuated less in Il23r^−/− mice compared to Il17a^−/− mice and in WT mice treated with anti-IL-23 neutralizing antibody compared to WT mice treated with anti-IL-17A neutralizing antibody. In addition, pathways independent of IL-23-IL-23R signaling pathway may also be involved in driving intestinal IR injury.

We definitively demonstrate, using BM chimeras, that the source of IL-17A important for intestinal IR injury is a cell of hematopoietic origin. WT->Il7a^−/− chimeras exhibited intestinal injury that was comparable to that observed in WT->WT chimeras. In contrast, Il7a^−/−->WT chimeras developed minimal intestinal IR injury. Paneth cells have been demonstrated to store IL-17A and to be important for IR injury (33). However, gene expression array analyses do not reveal detectable expression of Il17a mRNA in Paneth cells. This suggests that Paneth cells store IL-17A normally produced by a cell of hematopoietic origin, and thereby contribute to intestinal IR injury. The 8–9 week interval after irradiation of WT mice and their reconstitution with Il7a^−/− BM likely resulted in the depletion of IL-17A from the Paneth cells of Il7a^−/−->WT chimeras explaining their minimal intestinal IR injury, that was no different from that of Il7a^−/−-> Il7a^−/− control chimeras.

TCRγδT 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 TCRγδ deficient Tcrd^−/− mice suggesting that TCRγδ 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 TCRγδ T cells, rapidly upregulate Il17a mRNA expression in response to IL-23 stimulation. In contrast to Rag2^−/− mice, Rag2^−/−γc^−/− mice reconstituted with IgG from B6.lpr mice developed minimal intestinal IR injury. Furthermore, intestinal IR in Rag2^−/− mice, but not Rag2^−/−γc^−/− mice, reconstituted with B6.lpr IgG was associated with robust expression of Il17a, Cxcl1 and Cxcl2 in the intestine. As previously mentioned, intestinal IR injury in the reconstituted mice depends on IL-17A, because it was abrogated by administration of anti-IL-17A neutralizing antibody. Taken together with the observation that intestinal IR injury depends on RORγt, these findings strongly suggest that RORγt expressing, IL-17A producing ILC3s are essential for the development of intestinal IR injury

Based on our current and previous data (65, 66) we propose a mechanism for intestinal IR injury in which ischemia causes the expression of damage-associated neoantigens on intestinal cells. Binding of natural antibodies that crossreact with intestinal neoantigens results in fixation and activation of complement (7). Complement binding to receptors, such as C3aR and C5aR, on intestinal cells releases IL-23, which drives rapid IL-17A production and release by ILC3s. The locally released IL-17A acts on its receptors on epithelial and other stromal cells to induce the expression and release of chemokines that attract neutrophils. The release of granular contents from neutrophils causes tissue damage, resulting in intestinal IR injury. Accordingly, blockade of the IL-23/IL-17A axis may provide a powerful therapeutic strategy to attenuate intestinal IR injury.

Abbreviations used in this manuscript

IR

ischemia-reperfusion

ILC

innate lymphoid cells

B6

C57BL/6J

WT

wild type

B6.lpr

B6.MRLTnfrsf6^lpr

SMA

superior mesenteric artery

HPF

high power field

qRT-PCR

quantitative RT-PCR

CycloA

Cyclophilin A

BM

bone marrow