The Journal of Immunology

Heightened Immune Activation in Fetuses with May Be Blocked by Targeting IL-5

Michela Frascoli,*,† Cerine Jeanty,*,† Shannon Fleck,‡ Patriss W. Moradi,*,† Sheila Keating,x Aras N. Mattis,*,{ Qizhi Tang,† and Tippi C. MacKenzie*,†,‡

The development of the fetal immune system during is a well-orchestrated process with important consequences for fetal and neonatal health, but prenatal factors that affect immune activation are poorly understood. We hypothesized that chronic fetal inflammation may lead to alterations in development of the fetal immune system. To test this hypothesis, we examined ne- onates with gastroschisis, a congenital abdominal wall defect that leads to exposure of the fetal intestines to amniotic fluid, with resultant intestinal inflammation. We determined that patients with gastroschisis show high systemic levels of inflammatory cy- tokines and chemokines such as eotaxin, as well as earlier activation of CD4+ and CD8+ effector and memory T cells in the cord blood compared with controls. Additionally, increased numbers of T cells and eosinophils infiltrate the serosa and mucosa of the inflamed intestines. Using a mouse model of gastroschisis, we observed higher numbers of eosinophils and both type 2 and type 3 innate lymphoid cells (ILC2 and ILC3), specifically in the portion of organs exposed to the amniotic fluid. Given the role of IL-5 produced by ILC2 in regulating eosinophil development and survival, we determined that maternal or fetal administration of the anti–IL-5 neutralizing Ab, or a depleting Ab against ILCs, can both effectively reduce intestinal eosinophilia. Thus, a congenital anomaly causing chronic inflammation can alter the composition of circulating and tissue-resident fetal immune cells. Given the high rate of prenatal and neonatal complications in these patients, such changes have clinical significance and might become targets for fetal therapy. The Journal of Immunology, 2016, 196: 4957–4966.

uman fetal immune development is a critical component ongoing inflammation by producing chemokines such as IL-8 (3). of pre- and postnatal health, yet the environmental factors However, whether and how fetal immune development is altered H governing normal immune ontogeny remain poorly un- in patients with birth defects that change this developmental mi- derstood. There is a growing understanding of prenatal factors that lieu are not understood. may impact immune development. For example, exposure to ret- Gastroschisis is a congenital defect of the abdominal wall that inoic acid is important in the development of lymphoid tissue results in exposure of the intestines to amniotic fluid during de- inducer cells (1). Furthermore, increasing evidence indicates that velopment. Although the etiology is not known, gastroschisis is the fetal adaptive immune system is more sophisticated than characterized by inflammation of the exposed intestines, with re- previously recognized. For example, the neonatal intestine con- sultant bowel damage (4). Because this defect develops early in tains memory T cells that may facilitate mother-to-child HIV gestation, it represents a natural model in which to examine the transmission (2), and circulating neonatal T cells can contribute to consequences of chronic inflammation on the development of systemic and tissue-resident fetal immune cells. Several groups

*Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, have examined amniotic fluid from these and found University of California San Francisco, San Francisco, CA 94143; †Department of higher levels of IL-6 and IL-8, with concomitant increases in Surgery, University of California San Francisco, San Francisco, CA 94143; mononuclear cells and neutrophils in patients with gastroschisis ‡Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143; xBlood Systems Research Institute, San Francisco, CA 94118; compared with controls (5, 6). The current understanding of this { and Department of Pathology, University of California San Francisco, San Fran- disease is that exposure to the inflammatory cytokines in amniotic cisco, CA 94143 fluid, in addition to possible ischemic damage from a small ab- ORCIDs: 0000-0002-5607-6819 (S.F.); 0000-0002-8324-3694 (S.K.); 0000-0002- 5813-164X (A.N.M.); 0000-0001-7313-3574 (Q.T.). dominal wall defect, activates an innate immune response in the serosa of the exposed intestine. However, systemic immune changes Received for publication December 15, 2015. Accepted for publication April 18, 2016. in the fetus have not yet been examined. This work was supported by grants from the March of Dimes (to T.C.M.), National Studying immune changes in gastroschisis has important clinical Institutes of Health/National Institute of Allergy and Infectious Diseases Grant relevance. Pregnancies affected with fetal gastroschisis can have K08AI085042 (to T.C.M.), and by an American College of Surgeons resident re- search scholarship (to C.J.). multiple complications such as low fetal weight, in utero fetal demise, or preterm delivery (7, 8). Although the intestines can be placed back Address correspondence and reprint requests to Dr. Tippi C. MacKenzie, University of California San Francisco, 35 Medical Center Way, Box 0665, Room 903D, San into the after birth, neonates with gastroschisis often suffer Francisco, CA 94143-0665. E-mail address: [email protected] from poor intestinal motility after this operation and spend at least The online version of this article contains supplemental material. several weeks in the neonatal intensive care unit until the bowel Abbreviations used in this article: ACLP, aortic carboxypeptidase-like protein; E, recovers enough to tolerate enteral feeds. It is possible that alter- embryonic day; EM, effector memory; EMRA, terminally differentiated EM; IEL, intraepithelial lymphocyte; ILC, innate lymphoid cell; LPL, lamina propria lymphocyte; ations in fetal immune development may play a role in these related MDC, macrophage-derived chemokine; PTL, preterm labor; ROR, retinoid-related or- problems and, if so, targeted treatments could be developed. phan receptor; sPCA, supervised principal component analysis; sPTL, spontaneous To explore the potential roles of prenatal inflammation and fetal preterm labor. immune activation in gastroschisis, we examined cytokine profiles Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 and relevant cell types in cord blood and intestines of neonates with www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502587 4958 FETAL IMMUNE ACTIVATION IN CHRONIC INFLAMMATION this disease. We also used a genetic mouse model of gastroschisis case of fetal intervention, fetuses were injected with the same Abs on (9) to confirm these findings and to test fetal treatment strategies. E13.5 using a fetal intrahepatic injection as previously described (15–17). Our results indicate significant changes in both innate and adap- Briefly, mothers were anesthetized and the uterus was exposed by per- forming a laparotomy. The fetuses were injected individually, through the tive immune cell development in gastroschisis, which may impact translucent intact uterus, with the neutralizing Ab; controls were injected downstream complications in these patients. Importantly, we dem- with the isotype control Ab or PBS in a volume of 5 ml, using pulled glass onstrate that some of these changes can be reversed with fetal therapy. micropipettes. The laparotomy was closed in layers and the fetuses were analyzed on E18.5. Materials and Methods Ab-mediated depletion of fetal innate lymphoid cells Subjects ACLP+/2 pregnant females were bred to ACLP+/2 males and the fetuses Mothers carrying unaffected fetuses, those with gastroschisis, or those with were injected on E13.5 with an anti-Thy1.2 Ab (30H12; Bio X Cell) or a large omphalocele (an abdominal wall defect in which the intestines are isotype control (LTF-2; Bio X Cell) directly into the fetal as described covered with amnion and do not exhibit inflammation) were prospectively above. The pups were harvested on E18.5 and intestines were processed enrolled between November 2009 and December 2012. The Institutional and stained for flow cytometry. Review Board at the University of California approved the study and written informed consent was obtained. Flow cytometry analysis Human cells. We stained human PBMCs with mAbs to CD3 (UCHT1; BD Human blood sample processing Biosciences), CD4 (SK3; BD Biosciences), CD8 (SK1; BD Biosciences), Samples of cord blood were collected at the time of birth and maternal blood CD25 (M-A251; BD Biosciences), CD45RA (ALB11; Beckman Coulter), was collected within 24 h of delivery. Plasma was frozen and stored for batch CCR7 (MAB197; R&D Systems), Helios (22F6; BioLegend), Foxp3 (236A/ analysis of cytokines. PBMCs were isolated by Ficoll-Paque Plus density E7; eBioscience), IL-4 (MP4-25D2; BD Biosciences), IL-17 (eBio64DEC17; gradient centrifugation, frozen, and analyzed in batches during the study period. eBioscience), IFN-g (B27; BD Biosciences), and Fixable Viability Dye eFluor 780 (eBioscience). The cytokine-producing capacity of T cell Cytokine assay subsets was assessed after PBMCs were stimulated (at a density of 5 3 105 cells ml) for 5 h with 50 ng/ml PMA (Sigma-Aldrich) and 2 mg/ml Cytokine profiles in the maternal and cord blood plasma samples were assayed ionomycin (Sigma-Aldrich) in the presence of 10 mg/ml brefeldin A using the standard-sensitivity Milliplex Map kit (Millipore) as previously (Sigma-Aldrich). Cells were fixed and made permeable with the Foxp3/ reported (10). Samples were acquired and analyzed on a LABScan 100 transcription factor staining buffer set (eBioscience) according to the analyzer (Luminex) using Bio-Plex manager 6.0 software (Bio-Rad). manufacturer’s instructions. For flow cytometric analysis of human intes- tine, IELs and LPLs were stained with mAbs to CD3 (UCHT1; BD Bio- Preparation of human intestinal samples sciences), CD4 (SK3; BD Biosciences), CD8 (SK1; BD Biosciences), The University of California San Francisco pathology database was queried IL-7Ra (HIL-7R-M21; BD Pharmingen), and c-Kit (104D2; BD Biosciences). after Institutional Review Board approval to identify patients with gas- Mouse cells. We stained mouse leukocytes with mAbs to CD3 (17A2; troschisis who underwent bowel resection between February 2004 and April BioLegend), TCRb (H57-597; eBioscience), TCRg/d (GL3; BioLegend), 2012. Patients who underwent surgical resection secondary to intestinal CD5 (53-7.3; eBioscience), CD19 (6D5; BioLegend), CD11b (M1/70; BD obstruction or atresia (and not bowel necrosis) and had a lymphocytic Pharmingen), CD11c (N418; eBioscience), NK1.1 (PK136; eBioscience), infiltrate on H&E stain were identified for further analysis and immuno- CD45.2 (104; eBioscience), Thy1.2 (53-2.1; BioLegend), CD8a (53-6.7, staining. Control specimens were obtained from patients undergoing au- BD Pharmingen), CD4 (RM4-5; BD Pharmingen), retinoid-related orphan topsy for birth asphyxia or surgical resection specimens from patients receptor (ROR)gt (B2D; eBioscience), Gata-3 (TWAJ; eBioscience), c-Kit without gastroschisis, taking unaffected portions of intestine. Sections (2B8; eBioscience), IL-7Ra (A7R34; eBioscience), Siglec-F (E50-2440; (5 mm) from formalin-fixed, paraffin-embedded tissues were stained using BD Pharmingen), and Fixable Viability Dye eFluor 780 (eBioscience). All an Ab against human CD3 (F7.2.38; Dako) and counterstained with he- samples were acquired with an LSR II (Becton Dickinson) flow cytometer matoxylin to identify T cells. Eosinophils were identified using H&E and analyzed with FlowJo software (Tree Star). staining (11). Cell counts were performed on random high-power fields, excluding areas of lymphoid follicles, by a pathologist (A.N.M.) blinded Reagents to patient group. Fresh bowel specimens were collected from surgical procedures in three gastroschisis patients and four controls. The intraepithelial Reagents included Ficoll-Paque Plus (GE Healthcare), ACK lysing buffer lymphocytes (IELs) and lamina propria lymphocytes (LPLs) were isolated (Lonza), DL-DTT (AllStar Scientific), EDTA (Corning cellgro), collagenase from the mucosa and submucosa, respectively, as previously described (from Clostridium histolyticum; Sigma-Aldrich), Dispase (Life Technolo- (12). The mucosa was dissected from the muscular layer and washed with gies), and DNase I (bovine pancreas; Roche). 1mMDL-DTT followed by 5 mM EDTA to isolate IELs. The remaining Statistical analysis tissue was mechanically dissociated and then digested with 0.1 mg/ml collagenase to harvest LPLs. Statistical analysis and graphs were generated using Prism 5 software (GraphPad Software) or Stata version 12 (StataCorp, College Station, TX). Mice Data were analyzed using the Mann–Whitney U test (for nonparametric 2 +/2 t x Aortic carboxypeptidase-like protein (ACLP) mice (9) were obtained data), an unpaired test (for normally distributed data), or a test (to from Dr. Matthew Layne (Harvard Medical School). Mice were bred and assess the differences in proportion between groups). For multiple cate- gories, one-way ANOVA with a Tukey posttest was applied. For cytokine maintained in a specific pathogen-free facility at the University of Cal- analysis, a supervised principal component analysis (sPCA) was per- ifornia San Francisco. All mouse experiments were performed according to formed, which used the subset of cytokines that were most correlated with the University of California San Francisco Institutional Animal Care and +/2 the outcome (Spearman correlation coefficient . 0.25). The Kendall tau-c Use Committee–approved protocol. ACLP mice were bred to each other statistic was also calculated to test the magnitude and direction of increases and all pups were harvested at embryonic day (E)18.5. Lymphocytes were isolated from spleens, , and intestines of ACLP2/2 pups with gastro- or decreases in cytokine levels across disease severity groups. A multiple schisis and unaffected littermate controls. Liver was lysed using ACK regression analysis was performed to account for the presence of sponta- neous preterm labor and maternal (data on smoking was not lysing buffer. Intestine was mechanically dissociated and digested first with recorded in the medical record for 2 of 30 controls and 4 of 26 patients 10 mM DTT and then with 1 mg/ml dispase and 100 U/ml DNase I solution. with gastroschisis; these patients were analyzed as nonsmokers). A p value Fetal blockade of IL-5 ,0.05 was considered statistically significant. ACLP+/2 pregnant females were injected i.v. with a neutralizing Ab against IL-5, (TRFK5; BD Pharmingen) or an isotype control (R3-34; BD Results Pharmingen) for 5 consecutive days, using a dose of 100 mg on E13.5– Levels of inflammatory mediators are increased in the cord E14.5 and 150 mg on E15.5–E17.5 fetuses. The amount of Ab was cal- blood of patients with gastroschisis culated to achieve a fetal dose of 10 mg/kg (the same dose that is typically used in an adult mouse) (13) and an estimated maternal–fetal transmission We prospectively enrolled 27 patients with gastroschisis and 23 of 2–3% (14). Fetal intestines and liver were analyzed on E18.5. In the healthy term controls for collection of maternal and cord blood at The Journal of Immunology 4959 the time of delivery (Table I). There were no significant differ- which can control the differentiation of T cells to Th17 cells (21). ences in nulliparity, mode of delivery, and fetal gender between However, we did not observe a difference in the percentages of IL- mothers of healthy controls and those of fetuses with gastroschisis. 4–producing (Th2) fetal T cells (Supplemental Fig. 1A). We also Mothers of fetuses with gastroschisis were more likely to be examined the percentages of naive (CD45RA+CCR7+), central younger, experience preterm labor, deliver earlier, and have memory (CD45RA2CCR7+), effector memory (EM, CD45RA2 with lower birth weights than were mothers with unaffected preg- CCR72), and terminally differentiated EM (EMRA, CCR72 nancies, as previously reported (7). Other clinical characteristics of CD45RA+) cells (22) and found increased percentages of CD4+ patients with gastroschisis are shown in Supplemental Table I. EM and CD8+ EMRA T cells in patients with gastroschisis; once The amniotic fluid in patients with gastroschisis has increased again, the increases were most pronounced (and likely biologi- levels of inflammatory cytokines (5, 6). To determine whether there cally relevant) among CD8+ T cells (Fig. 1F, 1G). When we ad- is evidence of fetal systemic inflammation in this condition, we justed for the presence of sPTL, the changes in CD4 and CD8 cells analyzed serum cytokines in cord blood. We found that fetuses with producing IFN-g and CD8 EM and CD8 EMRA cells remained gastroschisis had significantly elevated levels of eotaxin-1 (CCL11), significant. Finally, given previous reports of the propensity of fetal CXCL1, IL-1Ra, IL-6, IL-8, soluble IL-2Ra, and MCP-1 and de- T cells to differentiate into regulatory T cells (23), we compared creased levels of macrophage-derived chemokine (MDC) in the percentages of Foxp3+Helios+ regulatory T cells in patients and cord blood compared with controls (Fig. 1A; results for all cytokines controls and found no differences (Supplemental Fig. 1B, 1C). tested and all relevant comparisons are shown in Supplemental Thus, chronic inflammation in gastroschisis leads to the production Table II). We also performed an sPCA using the plasma biomarkers of effector T cells without a compensatory regulatory response. that were correlated with gastroschisis (those with Spearman cor- Immune alteration in gastroschisis is manifested predominantly relation coefficient . 0.25; namely, eotaxin, CXCL1, IL-1Ra,IL-6, on the fetal, not maternal, side IL-8, soluble IL-2Ra,MCP-1,MDC,IL-12[p40],andTGF-a). The first principal component predominantly comprised eotaxin, To determine whether there were maternal immune changes in IL-1Ra, IL-6, IL-8, and MCP-1, and it differed the most between pregnancies affected with gastroschisis, we examined plasma cy- patients and controls (Fig. 1B). Of note, we also analyzed two tokine and blood T cell phenotypes in matched maternal samples patients who had a large omphalocele and their cytokine profiles collected at the time of delivery. We detected a generalized de- were within the control distribution (Fig. 1B). Thus, there is crease in cytokine, chemokine, and growth factor levels in women evidence of systemic inflammation in fetuses with gastroschisis. carrying fetuses with gastroschisis compared with controls, with Pregnancies with gastroschisis have a higher incidence of spon- significant decreases in levels of fractalkine, GM-CSF, and IL-12 taneous preterm labor (sPTL) and maternal smoking. To determine (p70) (Supplemental Table III). In examining circulating T whether the observed changes in cytokines were secondary to PTL or cell profiles, we detected a significant increase in Th17 cells maternal smoking, we performed a multiple regression analysis and (Supplemental Fig. 1D), but no differences in the production of adjusted for these variables. All of the observed cord blood cytokine IFN-g by CD4+ or CD8+ T cells (Supplemental Fig. 1E, 1F) in changes remained statistically significant even when controlling for mothers of gastroschisis patients. However, the increase in Th17 PTL and maternal smoking, suggesting these changes are secondary cells was not significant after adjusting for sPTL. We also observed to the underlying disease. that maternal CD4+ EM T cells increased slightly, but CD8+ EM and EMRAT cells did not change in pregnancies with gastroschisis Early development of effector T cells in patients with (Supplemental Fig. 1G). Thus, immune activation in this disease gastroschisis occurs predominantly on the fetal side. We next asked whether fetal immune development was altered in the context of systemic inflammation. We focused first on T cells, Infiltration of the intestine by T cells and eosinophils in given their role in promoting intestinal inflammation in other patients with gastroschisis settings (18–20). We found that patients with gastroschisis have The systemic increases in activated T cells led us to evaluate significantly increased percentages of CD4+ and CD8+ T cells that whether similar changes could be found locally in the inflamed produce IFN-g compared with controls (Fig. 1C, 1D); the increase intestines. Neonates with gastroschisis have an inflammatory peel was most pronounced in CD8+ T cells. The numbers of Th17 cells surrounding the intestine, characterized by subserosal edema, fi- in patients with gastroschisis also increased (Fig. 1E), although brosis, macrophage infiltration, capillary proliferation, and epi- their concentration in the blood is usually relatively low (21). thelial cell deposition, which develops after 30 wk of gestation (24), These results are consistent with the systemic increase in IL-6, but T cell infiltration has not been evaluated. We examined surgical

Table I. Demographic characteristics of healthy controls and patients with gastroschisis

Control (n = 30) Gastroschisis (n = 27) p Value Maternal age (y) 32 (18–43) 23 (15–34) <0.005 Maternal smoking 0 (0%) 6 (22.2%) 0.004 Nulliparous 9 (30.0%) 14 (51.9%) 0.09 Mode of delivery Vaginal 21 (70.0%) 23 (85.2%) 0.17 Cesarean section 9 (30.0%) 4 (14.8%) 0.17 Presence of labor 26 (86.7%) 27 (100.0%) 0.05 Gestational age (wk) 39.7 (37.1–41.6) 36.3 (34.7–37.4) <0.005 Male fetus 15 (50.0%) 18 (66.7%) 0.20 Birth weight (g) 3535 (2680–4440) 2495 (1730–3130) <0.005 Preterm labor 0 (0%) 9 (33.3%) 0.001 Data are presented as average (range) or n (%). The p values were calculated using a Student t test or x2 test. The p values #.05 (shown in bold) were determined to be significant. 4960 FETAL IMMUNE ACTIVATION IN CHRONIC INFLAMMATION

FIGURE 1. Elevated inflammatory markers and T cell activation in the cord blood of patients with gastroschisis. (A) Comparison of cytokine levels in unaffected controls (open bars, n = 19) and patients with gastroschisis (filled bars, n = 21). The horizontal line represents the median for each group. *p =0.05, **p , 0.01, ***p , 0.001 by Mann–Whitney rank sum test. (B) sPCA of the cord blood biomarkers most significantly correlated with gastroschisis in healthy controls (n = 19; blue), patients with gastroschisis (n = 21; green), and patients with giant omphalocele (n = 2; orange). Ellipsoids represent a 50% concentration of observations for controls and patients with gastroschisis. Eotaxin-1, IL-1Ra, IL-6, IL-8, and MCP-1 had the highest factor loadings on principal component (PC) 1. (C–E) Cord blood PBMCs were stimulated with PMA and ionomycin and stained with Abs to CD3, CD4, CD8, IL-17, and IFN-g to determine the percentage of T cells producing each cytokine. (C)IFN-g production by CD4+ Tcells.(D)IFN-g production by CD8+ Tcells.(E) IL-17 production by CD4+ T cells. Each symbol represents a single patient; small horizontal bars indicate the mean. (F and G)Theleft panels show representative flow cytometric analysis of purified maternal and cord blood PBMCs from healthy controls and patients with gastroschisis stained with Abs to CD3, CD4, CD8, CD45RA and CCR7 to detect naive (N, CCR7+CD45RA+), central memory (CM, CCR7+CD45RA2), EM (CCR72CD45RA2), and EMRA (CCR72CD45RA+)cells.Theright panels show compiled analysis for cord blood (F)CD4+ EM T cells and (G)CD8+ EM T cells and CD8+ EMRAT cells. Each symbol represents a single patient; small horizontal bars indicate the mean. Control, n = 20; gastroschisis, n = 21. *p , 0.05, **p , 0.01, ***p , 0.001 by Mann–Whitney test. specimens of nonnecrotic intestine in patients with gastroschisis and Because eotaxin-1 was increased in cord blood plasma of pa- age-matched controls using H&E, and stained for T cells using an tients with gastroschisis, we also examined eosinophils in bowel Ab against CD3. We confirmed that the intestine of patients with samples of these neonates. Activated eosinophils have been im- gastroschisis is characterized by a marked thickening of all layers plicated in intestinal inflammation and dysfunction in other clinical and serosal vascular proliferation, suggesting chronic ischemic in- settings (25) but have not been examined previously in this dis- jury (Fig. 2A). T cells were present in the intestinal serosa (Fig. 2B, ease. We found that eosinophils were dramatically increased in the 2C) and lymphoid follicles in patients with gastroschisis (Fig. 2D), intestinal mucosa of patients with gastroschisis compared with with infiltration beyond the boundaries of these follicles into the age-matched controls (Fig. 2F). submucosa and mucosa, which was not seen in healthy controls. Overall, patients with gastroschisis showed significantly higher Altered immune development in fetal mice with gastroschisis numbers of T cells in the intestinal mucosa as well as the serosa Given the interesting changes in fetal immune development in (Fig. 2E). Thus, histologic changes in the intestine extend well beyond patients with an abdominal wall defect, we next turned to an ap- the serosal surface that is exposed to amniotic fluid and are more propriate mouse model to validate these findings and test prenatal consistent with systemic immune alterations. treatment strategies. Mice lacking ACLP (9) have a spontaneous The Journal of Immunology 4961

FIGURE 2. Infiltration of T cells and eosinophils into the intestine of patients with gastroschisis. Histological analysis of bowel specimens from healthy controls (left panels) and patients with gastroschisis (right panels) is shown. (A) H&E staining of appendix sections shows the presence of numerous vessels in the serosa in patients with gastroschisis. Lower magnification scale bars, 500 mm; higher magnification scale bars, 100 mm. (B and C) Immunohisto- chemical staining of (B) appendix and (C) colon sections shows infiltration of CD3+ T cells (brown) in the serosa of specimens from patients with gas- troschisis. Lower magnification scale bars, 500 mm; higher magnification scale bars, 100 mm and insets,20mm. (D) Enlarged lymphoid follicles and infiltration of CD3+ T cells (brown) in the mucosa of colon in patients with gastroschisis. Lower magnification scale bars, 500 mm; higher magnification bars, 100 mm. (E) Numbers of CD3+ T cells infiltrating the intestine per field of view (FOV), excluding lymphoid follicles. Control, n = 5; gastroschisis, n =6.(F) H&E staining shows the presence of eosinophils (indicated by the arrows) infiltrating the mucosa in colon of patients with gastroschisis. Control, n = 7; gastroschisis, n = 6. Lower magnification scale bars, 200 mm; higher magnification scale bars, 50 mm and inset,20mm. The graph shows the numbers of eosinophils infiltrating the intestine. *p , 0.5, **p , 0.01 by Mann–Whitney U test. abdominal wall defect with exteriorized bowel and liver that lyzed ILC3 and found that both the percentages and absolute results in decreased numbers of enteric neurons and interstitial numbers of ILC3 (defined as CD45+Lin2CD32CD4+c-Kit+IL- cells of Cajal (26), similar to human gastroschisis (Fig. 3A). To 7Ra+RORgt+) (32) were also increased in the exposed intestine characterize the fetal immune response in this mouse model, (Fig. 4C) and livers (Fig. 4D) of ACLP2/2 mice compared with we examined cells of both the adaptive and innate immune littermate controls. We also examined ILCs in intestines of neo- systems in affected fetuses (ACLP2/2) and their unaffected lit- nates with gastroschisis. Our numbers were limited by the rarity of termates (ACLP+/2 and ACLP+/+) at E18.5. We detected elevated intestinal resection, and therefore of fresh tissues, in these pa- eosinophils (both the percentages and absolute numbers) in the tients, so we performed a general stain for ILCs that includes the fetal mouse intestine and liver (Fig. 3B, 3C). There were also different subsets (CD32CD42c-Kit+IL-7Ra+) (32) after isolating increased NK cells in the exteriorized liver (Fig. 3D). Impor- intraepithelial and lamina propria lymphocytes. We detected in- tantly, the changes in the affected mice were observed predomi- creased numbers of ILCs in the intestines of patients with gas- nantly in herniated organs exposed to amniotic fluid and were not troschisis compared with controls (Fig. 4E). seen in nonherniated organs (Fig. 3C, 3D) or in the spleen (data Fetal therapy with a neutralizing Ab against IL-5 can reverse not shown). T cells were unchanged (data not shown), likely 2 2 the inflammatory infiltrate in ACLP / mice secondary to their delayed maturation in mice compared with humans. Despite differences in the timing of fetal immune de- We next sought to define a treatment that could address some of the velopment between mice and humans, the finding of altered im- immunologic changes seen in the mouse model of gastroschisis. mune maturation in the mouse indicates that the ACLP2/2 mouse Because eosinophils and ILC2 were increased in this model, we is a suitable model for mechanistic studies on bowel damage in targeted IL-5 signaling and tested both direct fetal injection and gastroschisis. maternal administration of a blocking Ab. We first tested direct fetal administration by injecting fetal mice with TRFK5, a neu- Innate lymphoid cells accumulate in the intestines of patients tralizing Ab against IL-5, or either isotype control or PBS on and mice with gastroschisis E13.5–E14.5, using our established fetal intrahepatic injection Innate lymphoid cells (ILCs) are crucial determinants of fetal method (15–17). We then harvested the pups on E18.5 and ana- immune development (27, 28), especially in the fetal intestine lyzed the immune cells in exposed and nonexposed organs (29). In particular, eosinophil development and survival are me- (Fig. 5A). These experiments showed that all fetuses injected with diated by IL-5, the serum levels of which are maintained by tissue- the isotype control or PBS had high levels of eosinophils, indi- resident ILC2 (30). Because eosinophils were increased in mice cating that sterile inflammation from the surgery results in a fetal with gastroschisis, we examined ILC2 in the intestines of ACLP2/2 immune response. However, this rise in eosinophils was mitigated mice and found that both percentages and absolute numbers of effectively by blocking IL-5 in both the intestine and the liver ILC2 are increased in the exteriorized intestine compared with (Fig. 5B, 5C). littermate controls (Fig. 4A, 4B). Another subset of ILCs, ILC3, We next examined maternal administration of TRFK5, because has been shown to contribute to the inflammatory state of the this Ab can cross the placenta. Maternal i.v. injections of TRFK5 intestine during pathological conditions (31). Therefore, we ana- (or isotype control) were given daily between E13.5 and E17.5, and 4962 FETAL IMMUNE ACTIVATION IN CHRONIC INFLAMMATION

FIGURE 3. Increased eosinophils and NK cells in the exteriorized intestines and livers of ACLP2/2 mice. (A) E18.5 ACLP2/2 mouse exhibiting a defect in the anterior abdominal wall with exteriorized liver and intestine. (B) Representative flow cytometric analysis of NK cells (Nk1.1+) and eosinophils (Siglec-F+) from the intestine of E18.5 littermate controls (control) and ACLP2/2 mice after first gating on CD45.2+ leukocytes. (C and D) Histograms showing the percentages and absolute cell number per milligram tissue of (C) eosinophils and (D) NK cells in the exposed (out) or nonexposed (in) intestine and fetal liver. Each symbol represents a single fetus; small horizontal bars indicate the mean. Control, n = 13; ACLP2/2, n =9.*p , 0.05, **p , 0.01, ***p , 0.001 by one-way ANOVA with Tukey posttests. fetal intestine and liver were harvested on E18.5 (Fig. 5D). This mirrored in the mouse model of gastroschisis and could be ame- treatment led to a significant decrease in eosinophils compared liorated by administration of an Ab against IL-5 or by depleting with isotype control in both the intestine (Fig. 5E) and liver ILC2. Systemic changes in prenatal immune development as a (Fig. 5F). The treatment was effective in reducing eosinophil result of chronic prenatal inflammation are a novel finding; our counts in both gastroschisis and control mice; ILC2s were reduced work suggests that these changes have consequences for the health as well (Fig. 5G). of the fetus and neonate but could be reversed with in utero therapy. Our findings add to the growing literature on human fetal im- Depletion of ILCs can ameliorate intestinal eosinophilia in mune development and indicate that the presence of external ACLP2/2 mice stressors prenatally can significantly affect the fetal immune sys- ILC2s have been implicated in the onset of eosinophilia by pro- tem. It has been suggested that the initial insult in gastroschisis is ducing IL-5 (33). To determine whether there is a mechanistic link ischemic, secondary to a narrow abdominal wall defect (34), and between ILC2 and eosinophilia in our model, we depleted fetal the cytokine profile we obtained is consistent with the changes ILCs by injecting fetuses with a depleting Ab against Thy1.2 on reported in the setting of intestinal ischemia-reperfusion injury E13.5 and harvested the pups on E18.5 (Fig. 6A). We detected a (35). We speculate that the ischemic insult initiates an inflam- significant decrease in the percentages and absolute numbers of matory environment, leading to recruitment and activation of ILC2 and eosinophils in the intestines of treated pups compared T cells and eosinophils and ultimately resulting in bowel damage. with those treated with an isotype control (Fig. 6B, 6C). Thus, T cells have been found to infiltrate ischemic tissues and produce ILC2 may be involved in recruiting eosinophils to the intestine in IL-17, which may mediate tissue damage (36), and T cell deple- gastroschisis. Collectively, our results indicate that targeted pre- tion can ameliorate intestinal damage in an intestinal ischemia- natal therapies may limit ongoing fetal inflammation and aberrant reperfusion injury model (37). ILC3 and gd T cells may also immune development during prenatal inflammation. contribute to the production of IL-17, although their low numbers in circulation limited our ability to directly examine these cell Discussion types (38–40). Our data indicate that IL-5 production by ILC2 is In this study, we examined fetal immune development in the involved in the recruitment of eosinophils in this context, but that context of ongoing systemic inflammation, using a congenital prenatal blockade of IL-5 may dampen the fetal immune response. abdominal wall defect as a model system. We demonstrate that Epithelial cells contribute to the cytokine milieu that stimulates patients with gastroschisis have increased systemic levels of in- ILC2 (41-44): thus, it is possible that the intestinal inflammation flammatory cytokines and specific changes in the development of triggered by gastroschisis induces the expansion of ILC2s. Our effector T cell subsets. We also characterized changes in mucosal data in the mouse model suggest that ILC2 can, in turn, contribute immunity in this disease, including local infiltration of T cells, to increased intestinal eosinophilia. However, the role of ILCs in eosinophils, and ILCs in the affected intestine. These changes were intestinal development is complex and there have also been re- The Journal of Immunology 4963

FIGURE 4. Increased ILCs in the exteriorized organs of ACLP2/2 mice and patients with gastroschisis. (A) Sequential gating strategy to identify ILC2 (defined as CD45+Lin2CD32Thy1.2+IL-7Ra+GATA-3+) in the intestine of a representative ACLP2/2 mouse. (B) Representative flow cytometric plot of ILC2 and graphs showing the percentages and absolute cell number per milligram tissue in the exposed (out) or nonexposed (in) intestine in littermate controls and ACLP2/2 mice. Each symbol represents a single fetus; small horizontal bars indicate the mean. Control, n = 18; ACLP2/2, n = 12. *p , 0.05, **p , 0.01, ***p , 0.001 by one-way ANOVA. (C and D) Gating strategy to identify ILC3 (defined as CD45+Lin2CD32CD4+IL-7Ra+ c-Kit+RORgt+) from E18.5 ACLP2/2 mouse (example shown) and graphs showing the percentages (on total CD45+ cells) and absolute cell number per milligram tissue in the exposed (out) or nonexposed (in) (C) intestine and (D) fetal liver in littermate controls and ACLP2/2 mice. In this set of experiments, the entire intestine was exteriorized in the affected mice. Control, n =6;ACLP2/2, n =7.*p , 0.05, **p , 0.01, ***p , 0.001 by one- way ANOVA. (E) Flow cytometric analysis of ILCs (defined as CD32CD42IL-7Ra+c-Kit+) and compiled analysis of ILCs of lamina propria and intraepithelial lymphocytes isolated from intestine of healthy controls (cnt, n = 6 samples from four patients) and patients (Figure legend continues) 4964 FETAL IMMUNE ACTIVATION IN CHRONIC INFLAMMATION

FIGURE 5. Decreased eosinophil infiltration and ILC2 in ACLP2/2 mice after treatment with anti-IL-5 Ab. (A) Schematic layout of the experiment. Fetal intestine and liver are harvested 5 d after fetal liver injection of anti-IL-5 or isotype control/PBS. (B and C) Histograms showing the absolute cell number per milligram tissue of eosinophils in the intestine (B) and fetal liver (C). Each symbol represents a single fetus; small horizontal bars indicate the mean. Control, n = 43; ACLP2/2, n = 23. ***p , 0.001 by Student t test. (D) Schematic layout of the experiment. Maternal i.v. injection of anti-IL-5 or isotype control daily between E13.5 and E18.5 and harvest of fetal intestine and liver on E18.5. (E and F) Histograms showing the percentages and absolute cell number per milligram tissue of eosinophils in the intestine (E) and fetal liver (F). (G) Histograms showing the percentages and absolute cell number per milligram tissue of ILC2 in the intestine of littermate control and ACLP2/2 mice. Each symbol represents a single fetus; small horizontal bars indicate the mean. Control, n = 14; ACLP2/2, n =8.*p , 0.05, **p , 0.01, ***p , 0.001 by Student t test. ports that ILC2s promote tissue repair and homeostasis during pathways leading to intestinal damage. For example, activated inflammation (42, 45). It is possible that in patients with gastro- eosinophils and their granule proteins have been implicated in the schisis, ILCs may also serve to quell inflammation, particularly pathogenesis of intestinal inflammation in inflammatory bowel because one of the hallmarks of this disease is spontaneous res- disease, with increased levels correlating with worsening disease olution of inflammation over several weeks/months after birth. severity (48, 49). In addition to serving as a chemotactic factor for Finally, because ILCs regulate T cell responses to commensal eosinophils, eotaxin may be involved in recruiting T cells that flora (46), alterations in their development may affect early mi- express the eotaxin receptor CCR3 to the intestine (50) and may crobial colonization. Of note, patients with gastroschisis are par- adversely affect bowel motility (51). Locally, T cell infiltration ticularly susceptible to necrotizing enterocolitis, which has been and inflammatory cytokine production in the intestines may con- associated with changes in the microbiome (47) as well as dys- tribute to bowel dysmotility by multiple mechanisms, including regulation of intestinal T cells (12). damaging the interstitial cells of Cajal (52), which have been The present study also adds to our understanding of the patho- shown to be developmentally delayed in gastroschisis (53). physiology of intestinal damage in utero. Increases in tissue-resident In addition to implications for the pathophysiology of bowel eosinophils and T cells have been implicated in intestinal inflam- damage, the immune changes we observed may have implications mation in other conditions, and there may be similarities in the for the health of the pregnancy. Patients with gastroschisis are

with gastroschisis (GS, n = 6 samples from three patients). Among gastroschisis samples, each symbol (square, diamond, and circle) represents a different patient; small horizontal bars indicate the mean. *p , 0.05 by Mann–Whitney U test. The Journal of Immunology 4965

Although fetuses with gastroschisis are often diagnosed pre- natally, there is currently no fetal therapy to decrease intestinal damage. Serial exchange of amniotic fluid has been proposed as a prenatal therapy to relieve bowel inflammation, without robust results (55), likely because there is ongoing fetal production of inflammatory mediators that act at the intestinal level to promote tissue damage. However, systemic treatments targeted at reducing particular cell types in utero may be a more effective option, as demonstrated by our mouse experiments. In this regard, further examinations into the role of IL-5 signaling in the prenatal period may yield clinically relevant therapeutic approaches for this disease.

Acknowledgments We thank L. Coniglio, G. Bautista, and S. Trivedi for assistance with sam- ple collection and processing, and our patients for gracious participation in this study. We thank M. Layne (Department of Biochemistry, Boston Uni- versity School of Medicine, Boston, MA) for providing the ACLP+/2 mice, P. Ursell (Department of Pathology, University of California San Francisco) for providing pathology specimens, and R. Locksley, J. Bando, M. McCune, C. Iqbal, and S. Fisher (University of California San Francisco) for helpful discussions.

Disclosures The authors have no financial conflicts of interest.

References 1. van de Pavert, S. A., M. Ferreira, R. G. Domingues, H. Ribeiro, R. Molenaar, L. Moreira-Santos, F. F. Almeida, S. Ibiza, I. Barbosa, G. Goverse, et al. 2014. Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity. Nature 508: 123–127. 2. Bunders, M. J., C. M. van der Loos, P. L. Klarenbeek, J. L. van Hamme, K. Boer, J. C. H. Wilde, N. de Vries, R. A. W. van Lier, N. Kootstra, S. T. Pals, and T. W. Kuijpers. 2012. Memory CD4+CCR5+ T cells are abundantly present in the gut of newborn infants to facilitate mother-to-child transmission of HIV-1. Blood 120: 4383–4390. 3. Gibbons, D., P. Fleming, A. Virasami, M.-L. Michel, N. J. Sebire, K. Costeloe, R. Carr, N. Klein, and A. Hayday. 2014. Interleukin-8 (CXCL8) production is a signatory T cell effector function of human newborn infants. Nat. Med. 20: 1206–1210. 4. Christison-Lagay, E. R., C. M. Kelleher, and J. C. Langer. 2011. Neonatal ab- dominal wall defects. Semin. Fetal Neonatal Med. 16: 164–172. FIGURE 6. Decreased eosinophil infiltration in ACLP2/2 mice after 5. Guibourdenche, J., D. Berrebi, E. Vuillard, P. de Lagausie, Y. Aigrain, J.-F. Oury, fetal treatment with anti-Thy1.2 Ab to deplete ILCs. (A) Schematic layout and D. Luton. 2006. Biochemical investigations of bowel inflammation in gas- troschisis. Pediatr. Res. 60: 565–568. of the experiment. Fetal mice are injected with anti-Thy1.2 or isotype 6. Morrison, J. J., N. Klein, L. S. Chitty, G. Kocjan, D. Walshe, M. Goulding, control Ab and harvested on E18.5. (B) Histograms showing the depletion M. P. Geary, A. Pierro, and C. H. Rodeck. 1998. Intra-amniotic inflammation in of ILC2 (percentage and absolute cell number per milligram tissue) in the human gastroschisis: possible aetiology of postnatal bowel dysfunction. Br. J. intestine. (C) Histograms showing the reduction of eosinophils (percentage Obstet. Gynaecol. 105: 1200–1204. 7. Barseghyan, K., P. Aghajanian, and D. A. Miller. 2012. The prevalence of pre- and absolute cell number per milligram tissue) in the intestine. Each term births in pregnancies complicated with fetal gastroschisis. Arch. Gynecol. symbol represents a single fetus; small horizontal bars indicate the mean. Obstet. 286: 889–892. Control, n = 56; ACLP2/2, n = 11. *p , 0.05, **p , 0.01, ***p , 0.001 8. Overcash, R. T., D. A. DeUgarte, M. L. Stephenson, R. M. Gutkin, M. E. Norton, by Student t test. S. Parmar, M. Porto, F. R. Poulain, and D. B. Schrimmer, University of Cal- ifornia Fetal Consortium. 2014. Factors associated with gastroschisis outcomes. Obstet. Gynecol. 124: 551–557. 9. Layne, M. D., S. F. Yet, K. Maemura, C. M. Hsieh, M. Bernfield, M. A. Perrella, and M. E. Lee. 2001. Impaired abdominal wall development and deficient wound susceptible to pregnancy complications such as preterm labor and healing in mice lacking aortic carboxypeptidase-like protein. Mol. Cell. Biol. 21: intrauterine growth restriction and such complications may con- 5256–5261. tribute to the cytokine and T cell profiles we observed in cord 10. Fleck, S., G. Bautista, S. M. Keating, T.-H. Lee, R. L. Keller, A. J. Moon-Grady, K. Gonzales, P. J. Norris, M. P. Busch, C. J. Kim, et al. 2013. Fetal production of blood. Interestingly, all of the cytokine changes and some of the growth factors and inflammatory mediators predicts pulmonary hypertension in T cell changes remained significantly different when controlling congenital diaphragmatic . Pediatr. Res. 74: 290–298. 11. Brown, I. S., J. Smith, and C. Rosty. 2012. Gastrointestinal pathology in celiac for the presence of sPTL. It is possible that the underlying in- disease: a case series of 150 consecutive newly diagnosed patients. Am. J. Clin. flammation in gastroschisis (which is likely sterile, given the low Pathol. 138: 42–49. rate of clinical chorioamnionitis observed) results in precocious 12. Weitkamp, J.-H., T. Koyama, M. T. Rock, H. Correa, J. A. Goettel, P. Matta, K. Oswald-Richter, M. J. Rosen, B. G. Engelhardt, D. J. Moore, and D. B. Polk. activation of the fetal immune system and contributes to the onset 2013. Necrotising enterocolitis is characterised by disrupted immune regulation of PTL (54). It will be interesting to determine the Ag specificity and diminished mucosal regulatory (FOXP3)/effector (CD4, CD8) T cell ratios. of activated fetal T cells to understand their functional signi- Gut 62: 73–82. 13. Kobayashi, T., K. Iijima, and H. Kita. 2003. Marked airway eosinophilia pre- ficance. Understanding the consequences of specific fetal im- vents development of airway hyper-responsiveness during an allergic response in mune responses secondary to sterile inflammation (seen with this IL-5 transgenic mice. J. Immunol. 170: 5756–5763. 14. Medesan, C., P. Cianga, M. Mummert, D. Stanescu, V. Ghetie, and E. S. Ward. condition) or during prenatal infections could lead to valuable 1998. Comparative studies of rat IgG to further delineate the Fc:FcRn interaction insights into reproductive immunology. site. Eur. J. Immunol. 28: 2092–2100. 4966 FETAL IMMUNE ACTIVATION IN CHRONIC INFLAMMATION

15. Nijagal, A., T. Lee, M. Wegorzewska, and T. C. Mackenzie. 2011. A mouse 36. Edgerton, C., J. C. Crispı´n, C. M. Moratz, E. Bettelli, M. Oukka, M. Simovic, model of in utero transplantation. J. Vis. Exp. 2011(47): pii: 2303 doi:10.3791/ A. Zacharia, R. Egan, J. Chen, J. J. Dalle Lucca, et al. 2009. IL-17 producing 2303. CD4+ T cells mediate accelerated ischemia/reperfusion-induced injury in 16. Nijagal, A., C. Derderian, T. Le, E. Jarvis, L. Nguyen, Q. Tang, and autoimmunity-prone mice. Clin. Immunol. 130: 313–321. T. C. Mackenzie. 2013. Direct and indirect antigen presentation lead to deletion 37. Watson, M. J., B. Ke, X.-D. Shen, F. Gao, R. W. Busuttil, J. W. Kupiec- of donor-specific T cells after in utero hematopoietic cell transplantation in mice. Weglinski, and D. G. Farmer. 2012. Treatment with antithymocyte globulin Blood 121: 4595–4602. ameliorates intestinal ischemia and reperfusion injury in mice. Surgery 152: 17. Derderian, S. C., P. P. Togarrati, C. King, P. W. Moradi, D. Reynaud, 843–850. A. Czechowicz, I. L. Weissman, and T. C. MacKenzie. 2014. In utero depletion 38. Mjo¨sberg, J. M., S. Trifari, N. K. Crellin, C. P. Peters, C. M. van Drunen, B. Piet, of fetal hematopoietic stem cells improves engraftment after neonatal trans- W. J. Fokkens, T. Cupedo, and H. Spits. 2011. Human IL-25- and IL-33- plantation in mice. Blood 124: 973–980. responsive type 2 innate lymphoid cells are defined by expression of CRTH2 18. Powrie, F., M. W. Leach, S. Mauze, S. Menon, L. B. Caddle, and R. L. Coffman. and CD161. Nat. Immunol. 12: 1055–1062. 1994. Inhibition of Th1 responses prevents inflammatory bowel disease in scid 39. Hazenberg, M. D., and H. Spits. 2014. Human innate lymphoid cells. Blood 124: mice reconstituted with CD45RBhi CD4+ T cells. Immunity 1: 553–562. 700–709. 19. Cho, J. H. 2008. The genetics and immunopathogenesis of inflammatory bowel 40. Groh, V., S. Porcelli, M. Fabbi, L. L. Lanier, L. J. Picker, T. Anderson, disease. Nat. Rev. Immunol. 8: 458–466. R. A. Warnke, A. K. Bhan, J. L. Strominger, and M. B. Brenner. 1989. 20. Kleinschek, M. A., K. Boniface, S. Sadekova, J. Grein, E. E. Murphy, Human lymphocytes bearing T cell receptor g/d are phenotypically diverse and S. P. Turner, L. Raskin, B. Desai, W. A. Faubion, R. de Waal Malefyt, et al. 2009. evenly distributed throughout the lymphoid system. J. Exp. Med. 169: 1277– Circulating and gut-resident human Th17 cells express CD161 and promote 1294. intestinal inflammation. J. Exp. Med. 206: 525–534. 41. Moro, K., T. Yamada, M. Tanabe, T. Takeuchi, T. Ikawa, H. Kawamoto, 21. Acosta-Rodriguez, E. V., G. Napolitani, A. Lanzavecchia, and F. Sallusto. 2007. J. Furusawa, M. Ohtani, H. Fujii, and S. Koyasu. 2010. Innate production of TH2 Interleukins 1b and 6 but not transforming growth factor-b are essential for the cytokines by adipose tissue-associated c-Kit+Sca-1+ lymphoid cells. Nature 463: differentiation of interleukin 17-producing human T helper cells. Nat. Immunol. 540–544. 8: 942–949. 42. Price, A. E., H.-E. Liang, B. M. Sullivan, R. L. Reinhardt, C. J. Eisley, D. J. Erle, 22. Sallusto, F., D. Lenig, R. Fo¨rster, M. Lipp, and A. Lanzavecchia. 1999. Two and R. M. Locksley. 2010. Systemically dispersed innate IL-13-expressing cells subsets of memory T lymphocytes with distinct homing potentials and effector in type 2 immunity. Proc. Natl. Acad. Sci. USA 107: 11489–11494. functions. Nature 401: 708–712. 43. Neill, D. R., S. H. Wong, A. Bellosi, R. J. Flynn, M. Daly, T. K. A. Langford, 23. Mold, J. E., J. Michae¨lsson, T. D. Burt, M. O. Muench, K. P. Beckerman, C. Bucks, C. M. Kane, P. G. Fallon, R. Pannell, et al. 2010. Nuocytes represent a M. P. Busch, T.-H. Lee, D. F. Nixon, and J. M. McCune. 2008. Maternal allo- new innate effector leukocyte that mediates type-2 immunity. Nature 464: 1367– antigens promote the development of tolerogenic fetal regulatory T cells in utero. 1370. Science 322: 1562–1565. 44. Sonnenberg, G. F., and D. Artis. 2015. Innate lymphoid cells in the initiation, 24. Tibboel, D., C. Vermey-Keers, P. Kluck,€ J. L. J. Gaillard, J. Koppenberg, and regulation and resolution of inflammation. Nat. Med. 21: 698–708. J. C. Molenaar. 1986. The natural history of gastroschisis during fetal life: de- 45. Sonnenberg, G. F., L. A. Monticelli, T. Alenghat, T. C. Fung, N. A. Hutnick, velopment of the fibrous coating on the bowel loops. Teratology 33: 267–272. J. Kunisawa, N. Shibata, S. Grunberg, R. Sinha, A. M. Zahm, et al. 2012. Innate 25. Bischoff, S. C., J. Mayer, Q. T. Nguyen, M. Stolte, and M. P. Manns. 1999. lymphoid cells promote anatomical containment of lymphoid-resident com- Immunnohistological assessment of intestinal eosinophil activation in patients mensal bacteria. Science 336: 1321–1325. with eosinophilic gastroenteritis and inflammatory bowel disease. Am. J. Gas- 46. Hepworth, M. R., L. A. Monticelli, T. C. Fung, C. G. K. Ziegler, S. Grunberg, troenterol. 94: 3521–3529. R. Sinha, A. R. Mantegazza, H.-L. Ma, A. Crawford, J. M. Angelosanto, et al. 26. Danzer, E., M. D. Layne, F. Auber, S. Shegu, P. Kreiger, A. Radu, M. Volpe, 2013. Innate lymphoid cells regulate CD4+ T-cell responses to intestinal com- N. S. Adzick, and A. W. Flake. 2010. Gastroschisis in mice lacking aortic mensal bacteria. Nature 498: 113–117. carboxypeptidase-like protein is associated with a defect in neuromuscular de- 47. de la Cochetiere, M. F., H. Piloquet, C. des Robert, D. Darmaun, J. P. Galmiche, velopment of the eviscerated intestine. Pediatr. Res. 68: 23–28. and J. C. Roze. 2004. Early intestinal bacterial colonization and necrotizing 27. Mebius, R. E., P. Rennert, and I. L. Weissman. 1997. Developing lymph nodes enterocolitis in premature infants: the putative role of Clostridium. Pediatr. Res. collect CD4+CD32 LTb+ cells that can differentiate to APC, NK cells, and 56: 366–370. follicular cells but not T or B cells. Immunity 7: 493–504. 48. Saitoh, O., K. Kojima, K. Sugi, R. Matsuse, K. Uchida, K. Tabata, K. Nakagawa, 28. Eberl, G., S. Marmon, M.-J. Sunshine, P. D. Rennert, Y. Choi, and D. R. Littman. M. Kayazawa, I. Hirata, and K. Katsu. 1999. Fecal eosinophil granule-derived 2004. An essential function for the nuclear receptor RORgt in the generation of proteins reflect disease activity in inflammatory bowel disease. Am. J. Gastro- fetal lymphoid tissue inducer cells. Nat. Immunol. 5: 64–73. enterol. 94: 3513–3520. 29. Bando, J. K., H.-E. Liang, and R. M. Locksley. 2015. Identification and distri- 49. Raab, Y., K. Fredens, B. Gerdin, and R. Ha¨llgren. 1998. Eosinophil activation in bution of developing innate lymphoid cells in the fetal mouse intestine. Nat. ulcerative colitis: studies on mucosal release and localization of eosinophil Immunol. 16: 153–160. granule constituents. Dig. Dis. Sci. 43: 1061–1070. 30. Nussbaum, J. C., S. J. Van Dyken, J. von Moltke, L. E. Cheng, A. Mohapatra, 50. Gerber, B. O., M. P. Zanni, M. Uguccioni, M. Loetscher, C. R. Mackay, A. B. Molofsky, E. E. Thornton, M. F. Krummel, A. Chawla, H.-E. Liang, and W. J. Pichler, N. Yawalkar, M. Baggiolini, and B. Moser. 1997. Functional ex- R. M. Locksley. 2013. Type 2 innate lymphoid cells control eosinophil ho- pression of the eotaxin receptor CCR3 in T lymphocytes co-localizing with meostasis. Nature 502: 245–248. eosinophils. Curr. Biol. 7: 836–843. 31. Buonocore, S., P. P. Ahern, H. H. Uhlig, I. I. Ivanov, D. R. Littman, K. J. Maloy, 51. Smyth, C. M., N. Akasheh, S. Woods, E. Kay, R. K. Morgan, M. A. Thornton, and F. Powrie. 2010. Innate lymphoid cells drive interleukin-23-dependent innate A. O’Grady, R. Cummins, O. Sheils, P. Smyth, et al. 2013. Activated eosinophils intestinal pathology. Nature 464: 1371–1375. in association with enteric nerves in inflammatory bowel disease. PLoS One 8: 32. Spits, H., D. Artis, M. Colonna, A. Diefenbach, J. P. Di Santo, G. Eberl, e64216. S. Koyasu, R. M. Locksley, A. N. J. McKenzie, R. E. Mebius, et al. 2013. Innate 52. Kiyosue, M., M. Fujisawa, K. Kinoshita, M. Hori, and H. Ozaki. 2006. Different lymphoid cells—a proposal for uniform nomenclature. Nat. Rev. Immunol. 13: susceptibilities of spontaneous rhythmicity and myogenic contractility to intes- 145–149. tinal muscularis inflammation in the hapten-induced colitis. Neurogastroenterol. 33. Van Gool, F., A. B. Molofsky, M. M. Morar, M. Rosenzwajg, H.-E. Liang, Motil. 18: 1019–1030. D. Klatzmann, R. M. Locksley, and J. A. Bluestone. 2014. Interleukin-5- 53. Midrio, P., M. G. Vannucchi, L. Pieri, R. Alaggio, and M. S. Faussone-Pellegrini. producing group 2 innate lymphoid cells control eosinophilia induced by 2008. Delayed development of interstitial cells of Cajal in the ileum of a human interleukin-2 therapy. Blood 124: 3572–3576. case of gastroschisis. J. Cell. Mol. Med. 12: 471–478. 34. Houben, C., M. Davenport, N. Ade-Ajayi, N. Flack, and S. Patel. 2009. Closing 54. Gomez, R., R. Romero, F. Ghezzi, B. H. Yoon, M. Mazor, and S. M. Berry. 1998. gastroschisis: diagnosis, management, and outcomes. J. Pediatr. Surg. 44: 343–347. The fetal inflammatory response syndrome. Am. J. Obstet. Gynecol. 179: 194– 35. Kadry, Z., P. Roux-Lombard, J. M. Dayer, J. Lieberman, W. H. Marks, D. Dafoe, 202. N. Cretin, G. Cighetti, L. Buhler, P. Charbonnet, and P. Morel. 2000. Impact of 55. Midrio, P., G. Stefanutti, M. Mussap, D. D’Antona, E. Zolpi, and P. Gamba. intestinal ischemia-reperfusion on cytokine profile and enterocyte viability in 2007. Amnioexchange for fetuses with gastroschisis: is it effective? J. Pediatr. human syngeneic intestinal transplantation. Transplant. Proc. 32: 1292–1293. Surg. 42: 777–782.