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

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 results in 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 the innate and adaptive immune responses (5). Contributors to the tissue damage include IgM (6), natural Abs (7), complement (8, 9), neutrophils (4), platelets (10, 11), B lymphocytes (12), and T lymphocytes (13).

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/δ T 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 NK cells (20). The IL-17A receptor is a heterodimer of the IL-17RA and IL-17RC chains that bind IL-17A and its homolog, IL-17F, and it 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 that is characterized by neutrophil predominance (23, 24). It is also important 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 TCRα/β cells differentiate into Th17 cells following TCR ligation in the presence of the inflammatory cytokines IL-1, IL-6, and TGF-β (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/δ T cells and ILC3s is directly driven by IL-23 (27). IL-23 is a heterodimer of the 40-kDa chain (p40), which is shared with IL-12, and the IL-23–specific 19-kDa chain (p19) (28) and is produced by epithelial cells and DCs (29, 30). IL-23R is a heterodimer of the IL-12R β1-chain, which is shared with IL-12R, and an IL-23R–specific chain (30, 31).

Using immunofluorescence microscopy, we previously showed 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), and 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.

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

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-tribromoethanol, and anesthesia was maintained by s.c. injection of 36 mg/kg pentobarbital or 125 mg/kg 2,2,2-tribromoethanol. 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 Instrument, Rockville, MD) delivering ∼8.5 × g of pressure. The clip was removed after 30 min of ischemia, and the intestines were reperfused for 2 h. The laparotomy incision was sutured closed using 4.0 Prolene suture, and the mice were resuscitated with 1 ml of warm PBS injected s.c. 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.

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 H&E. Intestinal H&E-stained 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 (M.G.T.).

Formalin-fixed paraffin sections of small intestine were subjected to rehydration, and endogenous peroxidase activity was quenched with 3% H₂O₂. Then Ag 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 Ab. The primary Ab rat anti-mouse Ly-6B.2 (Gr1) clone 7/4 (1/250 dilution; Bio-Rad, Hercules, CA) was used to stain for neutrophils. Primary Ab or isotype-control Ab prepared in 10% BSA/PBS was applied overnight at 4°C. The slides were incubated with HRP-conjugated secondary Ab 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 Fisher Scientific, Waltham, MA) and evaluated with a Nikon Eclipse 80i microscope (Nikon, Melville, NY). Images were analyzed using Nikon NIS-Elements software. For neutrophil infiltration, positive-staining cells in the area of injury were counted in 20 high-power fields (HPFs) at 200×, and the average was calculated and expressed as the number of neutrophils per HPF.

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

Serum from B6.lpr mice was obtained by cardiac puncture. The Melon Gel IgG Purification Kit (Thermo Fisher Scientific) was used to isolate and purify IgG. A buffer exchange to PBS was performed on the eluted material. A total of 200 μg of IgG was injected i.v. into Rag2^−/− or Rag2^−/−γc^−/− mice 30 min prior to ischemia or sham operation, as previously described (49).

Eight-week-old recipient CD45.2⁺ WT and Il17a^−/− mice were lethally irradiated (1100 rad delivered in two doses of 550 rad each with a 3-h interval) 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 wk after BM reconstitution using FACS analysis for the CD45.1 and CD45.2 markers on blood after RBC lysis.

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 the manufacturer’s protocol. Quantitative RT-PCR was performed (Light Cycler 480; Roche) for Il17a, Il23, Cxcl1, Cxcl2, 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 Brilliant I Master Mix (Stratagene, La Jolla, CA) with the appropriate primers for Il17a, Il23, and CycloA. Ct values were determined 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.

Numeric data are presented as mean ± SD or mean ± SEM. Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad, San Diego, CA). The ordinal values of the injury scores and polymorphonuclear cells per HPF were analyzed by the nonparametric Mann–Whitney U test. The two-tailed Student t test for unpaired samples was used to compare the means of two groups. A p value <0.05 was considered statistically significant.

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

Mesenteric IR injury in WT mice was associated with a significant increase in Il17a mRNA levels in the small intestine compared with sham-operated mice, as determined by quantitative RT-PCR (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 to mesenteric IR injury compared with 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 compared with WT controls (Fig. 1F).

Natural Abs (7), which 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 is due to defective production of natural Abs. 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 (Supplemental Fig. 1). 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 IL-17A 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 Abs, which play an essential role in intestinal IR injury (49, 52–55). To circumvent these limitations, we examined the effect of neutralizing IL-17A on intestinal IR injury in WT mice. WT mice were treated with a neutralizing IgG Ab to IL-17A or with IgG isotype-control Ab on days 4, 2, and 1 prior to IR injury. WT mice treated with neutralizing anti–IL-17A Ab developed significantly lower intestinal injury scores after IR (Supplemental Fig. 2A) and significantly diminished neutrophil infiltration in the lamina propria (Supplemental Fig. 2B) compared with mice treated with IgG isotype control. Collectively, these results confirm that IL-17A plays a critical role in 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 with WT controls, as assessed by injury scores and the numbers of infiltrating neutrophils in the small intestine (Fig. 2). These results indicate that RORγt plays an essential role in intestinal IR injury.

IL-23 plays an important role in IL-17A production and is primarily expressed by epithelial cells and DCs (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 (Supplemental Fig. 3). Nevertheless, intestinal damage, neutrophil infiltration, and expression of Cxcl1 and Cxcl2 mRNA after intestinal IR injury were significantly, although partially, attenuated in Il23r^−/− mice compared with WT controls (Fig. 3A–E).

To confirm the importance of IL-23 in IR injury, WT mice were treated with neutralizing IgG Ab to IL-23 or with IgG isotype control on days 4, 2, and 1 prior to IR. WT mice treated with neutralizing IL-23 Ab developed significantly less small intestinal damage after IR injury than did mice treated with IgG isotype control, as evidenced by significantly lower injury scores (Supplemental Fig. 4A) and significantly lower numbers of infiltrating neutrophils (Supplemental Fig. 4B). These results suggest that IL-23 constitutively expressed in the small intestine plays an important role in 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 they produce IL-17A or simply store and release IL-17A made by other cells. We used BM chimeras to ascertain whether the IL-17A in IR injury is of hematopoietic or nonhematopoietic 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 nonhematopoietic cells, BM from CD45.2⁺ WT Il17a^−/− donors was used to reconstitute lethally irradiated CD45.1⁺ WT recipients or CD45.2⁺ Il17a^−/− recipients. FACS analysis 8 wk after BM reconstitution revealed that >91% of the blood cells in 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, because 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 the numbers of infiltrating neutrophils in the small intestine (Fig. 4D, 4E). In contrast, minimal intestinal injury after IR was observed in Il17a^−/− → WT and Il17a^−/− → Il17a^−/− BM chimeras (Fig. 4D, 4E). These results indicate that cells of hematopoietic origin are the major source of IL-17A important for intestinal damage after intestinal IR.

The intestine is rich in TCRγδ cells and ILC3s, 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 IR injury. Intestinal damage and neutrophil infiltration were not significantly different in Tcrd^−/− mice compared with WT controls (Fig. 5). These results indicate that TCRγδ cells do not contribute significantly to intestinal IR injury.

It has been previously demonstrated that intestinal IR injury depends on the presence of natural Abs 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 mature T and B cells.

We first verified that the batch of IgG that we used restores IR injury in Rag2^−/− mice. As previously reported (13, 51, 55), Rag2^−/− mice had minimal intestinal IR injury compared with WT controls and neutrophil infiltration (Fig. 6A, 6B). 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, 6B). 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, coadministration of anti–IL-17A neutralizing Ab prevented the restoration of intestinal IR injury in Rag2^−/− mice treated with IgG from B6.lpr mice (Fig. 6A, 6B), demonstrating that this restoration depended on IL-17A.

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, 7B). It also failed to induce Il17a, Cxcl1, or 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.

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, 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 attenuated in Il17a^−/− 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 natural Abs, which are 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 Abs 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 that is 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, to our knowledge, that intestinal IR injury was significantly attenuated in Il23r^−/− mice, as evidenced by decreased injury scores and 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 Ab 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. Because 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 with Il17a^−/− mice and in WT mice treated with anti–IL-23 neutralizing Ab compared with WT mice treated with anti–IL-17A neutralizing Ab. In addition, pathways independent of the 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 that is normally produced by a cell of hematopoietic origin and, thereby, contribute to intestinal IR injury. The 8–9-wk 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.

TCRγδ 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 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 in 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 Ab. 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 Abs that cross-react 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.

We thank Stella Kourembanas (Division of Newborn Medicine, Children’s Hospital) and Paul H. Lerou (Division of Newborn Medicine, Massachusetts General Hospital) for support and Drs. Rene De Waal Malefyt and J. Kolls for providing Il23r^−/− and Il7a^−/− mice.

The authors have no financial conflicts of interest.