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Cell Reports Report Tissue-Expressed B7-H1 Critically Controls Intestinal Inflammation Lisa Scandiuzzi,1 Kaya Ghosh,1 Kimberly A. Hofmeyer,1 Yael M. Abadi,1 Eszter Lázár-Molnár,1 Elaine Y. Lin,3 Qiang Liu,4 Hyungjun Jeon,1 Steven C. Almo,5 Lieping Chen,6 Stanley G. Nathenson,1,2 and Xingxing Zang1,* 1Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA 3Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA 4Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461, USA 5Department of Biochemistry, Physiology, and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA 6Department of Immunobiology and Yale Comprehensive Cancer Center, Yale University, New Haven, CT 06519, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2014.01.020 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2Department SUMMARY B7-H1 (PD-L1) on immune cells plays an important role in T cell coinhibition by binding its receptor PD-1. Here, we show that both human and mouse intestinal epithelium express B7-H1 and that B7H1-deficient mice are highly susceptible to dextran sodium sulfate (DSS)- or trinitrobenzenesulfonic acid (TNBS)-induced gut injury. B7-H1 deficiency during intestinal inflammation leads to high mortality and morbidity, which are associated with severe pathological manifestations in the colon, including loss of epithelial integrity and overgrowth of commensal bacteria. Results from bone marrow chimeric and knockout mice show that B7-H1 expressed on intestinal parenchyma, but not on hematopoietic cells, controls intestinal inflammation in an adaptive immunity-independent fashion. Finally, we demonstrate that B7-H1 dampened intestinal inflammation by inhibiting tumor necrosis factor a (TNF-a) production and by stimulating interleukin 22 secretion from CD11c+CD11b+ lamina propria cells. Thus, our data uncover a mechanism through which intestinal tissue-expressed B7-H1 functions as an essential ligand for innate immune cells to prevent gut inflammation. rant activation of mucosal innate and adaptive immunity, can result in gut injury, inflammation, and inflammatory bowel disease (IBD). However, the fundamental pathophysiologic mechanisms underlying IBD are still unclear. The interaction between the B7 family members and their receptors provides critical costimulation and coinhibition, which regulate T cell function. In addition to the long-established pathway of B7-1/B7-2-CD28/CTLA-4, the interaction between B7-H1 (PD-L1) (Dong et al., 1999; Freeman et al., 2000), a member of the B7 family, and its receptor PD-1, a member of the CD28 family, has a major role in inhibiting T cell responses (Freeman et al., 2000) and in inducing CD8 T cell exhaustion during viral infections (Day et al., 2006). Given that B7-H1 is mainly expressed on immune cells and PD-1 is expressed on activated T cells, research on this pathway has primarily focused on T cell coinhibition; nonetheless, the role of the B7-H1/PD-1 pathway in T cell-mediated intestinal inflammation and IBD remains largely unknown. A few earlier works report that the administration of anti-B7-H1 suppresses intestinal inflammation (Kanai et al., 2003), whereas the loss of PD-1/ PD-L1 signaling leads to expansion of gut antigen-specific CD8 T cells (Reynoso et al., 2009), and the PD-1 blockade in simian-immunodeficiency-virus-infected monkeys enhances repair of gut-associated junctions (Dyavar Shetty et al., 2012). B7-H1 can also be detected on some tissue cells (Liang et al., 2003), but its function is largely unexplored. Here, we identified epithelium-expressed B7-H1 as a key regulator of intestinal inflammation and colitis by inhibiting innate immune cells. INTRODUCTION RESULTS The gut lumen hosts 90% of the microorganisms in the human body. This microbiota community benefits the host by extracting energy and nutrients from food, by preventing colonization of pathogenic species (Hooper and Gordon, 2001), and by regulating immune cell function (Maloy and Powrie, 2011). The epithelial intestinal barrier is an essential boundary that precludes bacterial entry and maintains mucosal homeostasis. Deregulation of intestinal homeostasis, with concomitant aber- B7-H1 Protects from Mortality and Morbidity in Two Models of Intestinal Injury To investigate B7-H1 function in gut immunity, we chose a chemical model of intestinal injury utilizing oral administration of dextran sodium sulfate (DSS) that injures the colonic epithelium (Okayasu et al., 1990) and triggers potent inflammatory responses (Rakoff-Nahoum et al., 2004). B7-H1-deficient Cell Reports 6, 625–632, February 27, 2014 ª2014 The Authors 625 Figure 1. B7-H1/ Mice Are Hypersensitive to DSS-Induced Intestinal Inflammation (A) Wild-type (WT) and B7-H1/ mice were fed with 2% DSS in drinking water for 7 days. Survival, percentage of original weight loss, anal bleeding, diarrhea, anal erosion, and colonic blood scores were monitored. Survival data are represented as Kaplan-Meier curves, ***p < 0.001; log rank test. Data were pooled from two independent experiments (n = 15/group). *p < 0.05; **p < 0.01; ***p < 0.001; Student’s t test. (B) H&E-stained colon tissue of DSS-treated WT and B7-H1/ mice (D0, D2, and D6). (C and D) Histopathological scoring of infiltrating leukocytes, edema, ulcerationl, and MALT area (n = 20 MALTs/mouse) in WT and B7-H1/ mice (D6; scale bars represent 100 mm; arrows indicate MALTs). ***p < 0.001; Student’s t test. (E) DSS-fed mice were gavaged (D0 and D6) with FITC-dextran. Three hours later, FITC fluorescence was determined in sera. Data are pooled from two independent experiments (n = 6–9). *p < 0.05; Student’s t test. (F) CFUs in feces and colon of DSS-fed mice (D0, D2, and D6); *p < 0.05; Mann-Whitney U test. (G) Quantitative PCR analysis of 16S rDNA copies for the whole bacterial kingdom in the feces of DSS-treated (D0 and D6) WT and B7-H1/ mice. Data were pooled from two independent experiments, n = 3–4. *p < 0.05; Student’s t test. (B7-H1/) mice showed much higher mortality and morbidity (weight loss, anal bleeding, diarrhea, and anal erosion scores) upon DSS administration (2%; w/v) for 6 days (Figure 1A) than wild-type (WT) mice. Although less than 20% of B7-H1/ mice survived, more than 70% of WT mice remained alive. Higher DSS concentration (4%) for a shorter time (5 days) provided similar results with an earlier onset of disease (data not shown). We extended studies to a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis model and obtained similar results (Figure S1A). Thus, B7-H1 is critical for controlling intestinal epithelial injury and inflammation. 626 Cell Reports 6, 625–632, February 27, 2014 ª2014 The Authors We dissected the causes of death and morbidity in B7-H1/ mice by histopathological analyses of colon tissues. Although colonic bleeding in B7-H1/ mice occurred earlier than in WT mice (Figure 1A), both groups of mice became anemic at day 6 of DSS treatment (data not shown). Hematoxylin and eosin (H&E) analyses confirmed that B7-H1/ mice displayed more severe ulceration, extensive epithelium erosion, and cellular infiltration (Figures 1B and 1C). In addition, we found an enlargement of distinct lymphoid aggregations known as ‘‘mucosal associated lymphoid tissues’’ (MALTs) beneath the epithelium in B7H1/ mice (Figures 1B and 1D), which correlates with severe pathological changes (Laukoetter et al., 2007). Neither naive B7-H1/ nor naive WT mice showed signs of colonic inflammation or tissue damage. Augmented mucosal permeability and intestinal barrier dysfunction are associated with colitis development in mice (Garrett et al., 2007) and IBD patients (Cobrin and Abreu, 2005). Therefore, we investigated the role of B7-H1 in maintaining gut epithelial integrity by orally gavaging mice with fluorescein isothiocyanate (FITC)-dextran (Garrett et al., 2007). We found a 4-fold increase of FITC-dextran levels in sera of B7H1/ mice at day 6 of DSS treatment (Figure 1E), suggesting that B7-H1 is important for regulation of intestinal permeability during gut injury. Intestinal microbial populations vary enormously between IBD patients and healthy individuals (Hooper and Gordon, 2001); therefore, we analyzed their concentration and composition. After DSS administration, B7-H1/ mice had higher bacteria colony-forming units (CFUs) not only in feces and colon (Figure 1F), but also in mesenteric lymph nodes (MLNs), spleen, and liver (data not shown) than WT mice, suggesting the presence of a bacterial systemic dissemination in B7-H1/ mice. Quantitative analysis of 16S rDNA confirmed that B7-H1/ mice had an increased number of total bacteria than WT mice (Figure 1G), but none of the specific intestinal microbiota groups analyzed accounted for this increase (Figure S1B). Altogether, our findings demonstrate that B7-H1 is required to maintain gastrointestinal integrity during gut inflammation and to avoid commensal bacteria overgrowth. B7-H1 Expression on Intestinal Parenchyma Confers Protection B7-H1 is constitutively expressed on a wide range of immune and tissue cells and is upregulated after activation (Freeman et al., 2000; Liang et al., 2003). As the colon is the site of DSSinduced disease, we examined B7-H1 expression in the colon. Single-cell suspensions from colon of naive and DSS-treated mice were analyzed by flow cytometry to detect hematopoietic cells (cytokeratinCD45+) and parenchymal cells such as epithelial cells (cytokeratin+CD45) or myofibroblasts and pericytes (cytokeratinCD45) (Mifflin et al., 2011). We found B7-H1 was expressed on both colonic parenchymal and hematopoietic cells from naive and DSS-treated WT mice, and it was moderately upregulated on cytokeratinCD45+ cells at D6 of DSS treatment (Figure 2A). B7-H1 expression was also increased on colon of IBD patients (Figure 2B). PD-1, the receptor for B7-H1, is not expressed on parenchymal cells but is mainly expressed on activated T and B cells (Agata et al., 1996), on exhausted CD8 T cells (Barber et al., 2006; Day et al., 2006) and on dendritic cells (DCs) (Yao et al., 2009) or monocytes or macrophages (Huang et al., 2009) during infection. We detected PD-1 on colonic hematopoietic cells of both WT and B7-H1/ mice before and after DSS administration, respectively (Figure 2A). We next asked whether B7-H1 on hematopoietic cells and/or parenchymal cells was required to inhibit the pathogenic process during DSS-induced disease. We generated B7-H1 bone marrow (BM) chimera mice with control groups (Figure S2A). Upon DSS administration, B7-H1/ mice reconstituted with WT BM developed significantly worse clinical symptoms as compared to WT mice reconstituted with B7-H1/ BM or with WT BM (Figure 2C). Only 10% of mice survived in the first group versus 70%–80% in the other two groups. The first group also showed severe histopathological changes in colon tissue sections stained with H&E (Figure 2D). These findings, together with the B7-H1 expression pattern on colonic cell populations, suggest that B7-H1 on parenchyma, but not on hematopoietic cells, confers protection from intestinal injury and inflammation. Effect of B7-H1 on Intestinal Epithelium Homeostasis To understand how intestinal epithelium-expressed B7-H1 regulates intestinal injury and inflammation, we considered two possible mechanisms: cell-intrinsic alterations and cell-extrinsic interactions. A possible cell-intrinsic mechanism could be a homeostatic imbalance of intestinal epithelium in the absence of B7-H1. We hypothesized that intestinal epithelium-expressed B7-H1 controls intestinal injury by affecting proliferation or by impairing apoptosis of intestinal epithelial cells. To assess the proliferation, we used an immunofluorescence staining for Ki67 and the 5-bromo-20 -deoxyuridine (BrdU) assay. Our data showed a slightly higher baseline level (D0) of proliferation in B7-H1/ mice (Figure S2B), which quickly disappeared at D2 and did not correlate with an alteration of the intestinal epithelial permeability (Figure 1E) or with colonic hyperplasia (data not shown). However, upon inflammation (D6), both B7-H1/ and WT colonic cells exhibited a similar epithelial proliferative capacity (Figure S2B). Another possible cell-intrinsic mechanism accounting for the dysregulation of intestinal epithelial cells in B7-H1/ mice is increased cell death. Analyses of apoptosis and necrosis in colon epithelial cells showed an increased number of both apoptotic and necrotic epithelial cells in B7-H1/ mice at D6 of DSS-treatment (Figures 3A and 3B). Altogether, these results demonstrated that B7-H1 did not affect tissue repair processes after an injurious insult, but it prevents apoptosis and necrosis of intestinal epithelial cells during gut inflammation. Protective Function of B7-H1 Is Not Dependent on Adaptive Immunity We next explored a cell-extrinsic mechanism by testing the hypothesis that intestinal parenchymal cells expressed B7-H1 could interact with immune cells and, as a consequence, reduce intestinal inflammation. Both innate and adaptive immunity contribute to intestinal inflammation (Maloy and Powrie, 2011). Therefore, we analyzed immune cell populations in the colon and MLN and found no difference in the number of T and B cells, NK/NKT cells, macrophages, DCs, neutrophils, myeloid, and innate lymphoid cells (Figure S3A). To assess if components of the adaptive immune system including T and B cells could participate in the B7-H1-mediated protection, we generated Rag-1/B7-H1/ mice and found that these mice exhibited significant higher mortality and morbidity than Rag-1/ mice after DSS administration (Figure 3C). Compared to WT mice reconstituted with WT BM, WT mice reconstituted with PD-1/ BM seemed to be slightly more sensitive to DSS-induced colitis without reaching significant difference (Figure 3D), which correlates with no significant difference between PD-1/ mice and Cell Reports 6, 625–632, February 27, 2014 ª2014 The Authors 627 Figure 2. B7-H1 Is Expressed in Inflamed Colon and Is Required on the Parenchyma for Protection against DSS-Induced Colitis (A) Fluorescence-activated cell sorting (FACS) plots of single-cell suspensions from colons of DSS-fed mice (D0 and D6) representing hematopoietic cells (cytokeratinCD45+), epithelial cells (cytokeratin+CD45), and myofibroblast or pericytes (cytokeratinCD45) to detect B7-H1 and PD-1 expression (open histograms), whereas shaded histograms indicate isotype control staining, n = 4. (B) Immunohistochemistry staining for B7-H1 in human colon tissue from healthy donors and IBD patients and relative quantification. Isotype control was used. Arrows and stars indicate focal expression of B7-H1; scale bars represent 200, 50, and 25 mm. Quantification is represented as amount of pixels positively stained for B7-H1 by Velocity software. Data are representative of two independent experiments, n = 3. **p < 0.01; ***p < 0.001; Student’s t test. (C) Three groups of bone-marrow chimera mice (B7-H1/ mice reconstituted with WT BM [WT BM/B7-H1/], WT mice reconstituted with B7-H1/ BM [B7-H1/BM/WT], or WT BM [WT BM/WT]) were treated with 3% DSS in drinking water for 6 days. Survival, percentage of original weight loss, anal bleeding, diarrhea, and anal erosion score were monitored. Data represent one of three independent experiments, n = 7–10. Survival data are represented as Kaplan-Meier curve (*p < 0.05; log rank test). *p < 0.05; **p < 0.01; Student’s t test. (D) H&E staining of colon tissue sections of chimera colons (D0 and D6). Scale bars represent 100 and 50 mm. WT mice during DSS treatment (Figure 3E). However, we cannot exclude the involvement of PD-1 on radioresistant PD-1 positive tissue resident innate cells. These unexpected findings demonstrated that B7-H1 delivered protection from intestinal injury and inflammation in the absence of adaptive immunity. B7-H1 Inhibits Tumor Necrosis Factor a Production and Enhances Interleukin 22 Production from CD11c+CD11b+ LP Inflamed Cells Tumor necrosis factor a (TNF-a) has been shown to have a major role in the pathogenesis of IBD (Dharmani et al., 2011). Thus, we measured cytokines in the colon and found B7-H1/ colons 628 Cell Reports 6, 625–632, February 27, 2014 ª2014 The Authors produced about 3-fold more TNF-a than WT colons at D6 of DSS treatment (Figure 4A), whereas the levels of cytokines interleukin 6 (IL-6), IL-2p70, IL-4, IFN-g, and MCP-1 were comparable (Figure S3B). Next, we evaluated innate immune cell activity in the colon. Intracellular cytokine staining of inflamed LP cells (D6) revealed that CD11c+CD11b+ cells in B7-H1/ mice significantly enhanced TNF-a production (Figure S4A) despite a similar expression of surface activation markers in both groups of mice (data not shown). Treatment with anti-TNF-a neutralizing antibody reduced by 50% the mortality in B7-H1/ mice as compared to isotype-treated mice (Figure 4B). These data indicate that TNF-a is one of the major causes of mortality in Figure 3. B7-H1 Prevents Intestinal Cell Death, and Adaptive Immunity Is Not Needed for B7-H1 Function (A) FACS plots for Annexin V+ epithelial cells and quantification of apoptotic epithelial cells in DSS-fed (D6) WT and B7-H1/ mice. *p < 0.05; Student’s t test. (B) Dot plots and quantification of necrotic epithelial cells double positive for Annexin V and LIVE/DEAD Violet marker from DSS-fed (D6). Data represent one of three independent experiments; n = 3/group. *p < 0.05; Student’s t test. (C) B7-H1/Rag1/ and Rag1/ were treated with 3% DSS for 6 days and monitored for survival, body weight loss, anal bleeding, and diarrhea scores. Data are pooled from two independent experiments, n = 17. (D) WT mice were reconstituted with PD-1/ (PD-1/BM/WT) or WT bone marrow cells (WT BM/WT) and then treated with 3% DSS for 6 days. Survival, percentage of weight loss, anal bleeding, and diarrhea scores were monitored. Data are representative of two independent experiments, n = 6–8. (E) WT and PD-1/ mice were fed with 3% DSS in drinking water for 6 days and monitored for survival, body weight loss, anal bleeding, and diarrhea score. Data are representative of four independent experiments, n = 9. Survival data are showed as Kaplan-Meier curves (*p < 0.05; log rank test). Data represent means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; Student’s t test. B7-H1/ mice, suggesting a cell-extrinsic mechanism where B7-H1 might inhibit TNF-a production from CD11c+CD11b+ LP cells. To test this possibility, we generated a B7-H1-Ig fusion protein and found that plate-bound B7-H1-Ig protein, but not control Ig protein, suppressed TNF-a production from both WT and PD-1/B7-1/ inflamed LP cells (Figure 4C) as B7-1 has been found as an additional binding molecule (Butte et al., 2007). These data indicate the PD-1/B7-H1 pathway alone or the B7-1/B7-H1 pathway alone may not significantly contribute to the disease onset in B7-H1/ mice. IL-22 induces innate immune response during mucosal infections (Aujla et al., 2008) and promotes colonic epithelial cell restitution including induction of antimicrobial peptides such as Reg3-g (Zheng et al., 2008) and b-defensins (Wolk et al., 2004). We investigated whether the cell-extrinsic mechanism so far observed could promote a release of protective factors like IL-22 and TGF-b. We found WT colon supernatants had higher levels of IL-22 than B7-H1/ samples, but no difference for TGF-b was detected (Figure 4A). Interestingly, we discovered IL-22 production was strongly produced by WT Cell Reports 6, 625–632, February 27, 2014 ª2014 The Authors 629 Figure 4. TNF-a Blockade Rescues B7-H1/ Mice from Intestinal Inflammation, and B7-H1-Expressing IECs Promote IL-22 Production (A) Production of TNF, IL-22, and TGF-b in supernatant of colons from DSS-fed (D0, D2, and D6) mice. Data are pooled from three independent experiments (n = 4). (B) WT and B7-H1/ mice were intraperitoneally injected with anti-TNF-a blocking antibody or isotype control or PBS starting at D0 of 3% DSS treatment and every other day until D20. Survival, percentage of original weight loss, anal bleeding, diarrhea, and anal erosion were monitored. Data were pooled from two independent experiments (n = 8/group). Survival data are represented as Kaplan-Meier curves. **p < 0.01; ***p < 0.001; log rank test; *p < 0.05; **p < 0.01; Student’s t test. (C) LP cells were isolated from WT or PD-1/B7-1/ mice on day 6 after DSS treatment and incubated with plate-bound B7-H1-Ig or control Ig proteins for 24 hr. LP cells were stained for CD11c+CD11b+ cells to detect percentage of intracellular TNF-a production by flow cytometry. Data are pooled from at least two independent experiments. Each symbol represents an individual mouse. Data represent means ± SEM. *p < 0.05; Student’s t test. (D) FACS plots and numbers of CD11b+CD103+ and CD11b+CD103 LP cells producing IL-22 from WT and B7-H1/ mice at day 6 of DSS treatment. Data represent one of two independent experiments, n = 3. Data represent means ± SEM. *p < 0.05; Student’s t test. (E) FACS plots and quantification of intracellular IL-22 production from CD11c+CD11b+ inflamed LP cells. LP cells were isolated from WT mice (D6 of DSS treatment) and cocultured with naive IECs of WT or B7-H1/ mice for 24 hr. Numbers above bracketed lines indicate percentage of IL-22-producing cells. Data are pooled from two independent experiments (n = 3). *p < 0.05; Student’s t test. CD11b+CD103+ dendritic cells, which have a tolerogenic phenotype (Maloy and Powrie, 2011), but not from CD11b+CD103 DCs (Figure 4D). Finally, we found, in a coculture assay, WT primary intestinal epithelial cells (IECs) were able to stimulate IL-22 production from WT CD11c+CD11b+ LP cells significantly better than B7-H1/ IECs (Figure 4E). These data proved B7-H1 is required on the intestinal epithelium to dampen the inflammation by CD11c+CD11b+ LP cells in this disease model. Our results provide a mechanism by which B7-H1 expressed on parenchyma colon reduces inflam630 Cell Reports 6, 625–632, February 27, 2014 ª2014 The Authors mation through inhibition of TNF-a and by promoting IL-22 production from CD11c+CD11b+ LP cells. Altogether, our results demonstrate that tissue-expressed B7-H1 is an essential regulator in the control of intestinal inflammation. DISCUSSION Beside the classical coinhibitory function of the B7-H1/PD-1 pathway in T cell activation and immune pathology (Keir et al., 2008), very few data are available on the role of tissue-expressed B7-H1. Here, we reported a role for B7-H1 in reducing tissue pathology during gut injury as demonstrated by severe morbidity and mortality in B7-H1/ mice upon DSS or TNBS treatment. We revealed two unexpected functions for B7-H1. First, B7H1-mediated protection during intestinal inflammation minimally required the expression of PD-1 on hematopoietic cells because the morbidity and mortality observed in PD1/ chimera mice were slightly increased but were not statistically different from that of control chimera mice. Second, B7-H1 expressed on tissue cells, but not on hematopoietic cells, was essential for reducing gut pathogenesis because chimera mice lacking B7H1 expression on parenchyma exhibited an increased mortality and morbidity during DSS-induced colitis, whereas chimera mice lacking B7-H1 expression on hematopoietic cells showed minimal signs of disease. These results, together with other reports in type 1 diabetes (Keir et al., 2006) and LCMV infection (Mueller et al., 2010), highlight an emerging role for tissueexpressed B7-H1 in the control of peripheral immune responses. In addition, we found that adaptive immune response was not required for B7-H1 protection, because Rag-1/B7-H1/ mice developed more severe gut pathogenesis than control Rag-1/ mice. These data suggest that B7-H1 controls intestinal inflammation through innate immunity, which is in line with previous reports on the role of innate immunity in DSS-induced colitis (Garrett et al., 2007; Rakoff-Nahoum et al., 2004; Zaki et al., 2010). TNF-a has been recognized as a master cytokine in the onset of DSS-induced colitis (Dharmani et al., 2011), and anti-TNF-a therapy inhibits IEC apoptosis in patients with IBD (Marini et al., 2003). The increased TNF-a production observed in inflamed B7-H1/ colons corresponded with the peak of mortality and intestinal epithelial apoptosis, and the disease was successfully ameliorated by TNF-a blockade, indicating that B7-H1 is one of the major inhibitors of the TNF-a production in this disease model. An in vitro evidence of the B7-H1 inhibitory effect was provided using B7-H1-Ig fusion protein, which impaired the TNF-a production of inflamed CD11c+CD11b+ LP cells isolated from WT or PD-1/B7-1/ mice. These data showed not only that B7-H1 inhibited innate cells, but also that the B7H1/PD-1 pathway alone or the B7-H1/B7-1 pathway alone might not be primarily involved. In addition to PD-1 and B7-1, new studies also demonstrate unidentified receptor(s) for B7-H1 (Xu et al., 2013). Given that the multiple interactions of those molecules could raise some difficulties for the data interpretation of these different pathways, further investigations on the specific functions of those molecules are warranted to better understand their specific roles in the maintenance of intestinal peripheral tolerance. Different subsets of intestinal dendritic cells are known to play crucial roles in the maintenance of intestinal tolerance by inducing tolerogenic T cell responses including increasing TGF-b and retinoic acid levels (Maloy and Powrie, 2011). Our data showed that, although TGF-b was not differentially regulated, IL-22 levels were significantly increased in mice expressing B7-H1. We further proved that IL-22 production was strongly impaired when B7-H1/ IECs were cultured with DSS-inflamed LP. These data revealed a role for B7-H1 expressed on IECs by regulating IL-22 production for the epithelial restoration process. Thus, a lack of B7-H1 could skew the mucosal intestinal responses toward a proinflammatory phenotype leading to increased inflammation and injury. Strategies that specifically enhance the activation of B7-H1 in the parenchyma would be beneficial for therapeutic control of IBD. EXPERIMENTAL PROCEDURES Mice B7-H1/ and PD-1/ mice were previously described (Dong et al., 2004). C57BL/6, Rag1/ on C57BL/6 background, and B7-1/ on C57BL/6 background mice were purchased from the Jackson Laboratory and bred at the Albert Einstein Animal Facility. Rag1/B7-H1/ and PD-1/B7-1/ mice were obtained by intercrossing Rag1/ with B7-H1/ and PD-1/ with B7-1/ mice, respectively. Mice were housed in the same room in a specific pathogen-free facility and used for studies at 7–9 weeks old under protocols approved by the Institutional Animal Care and Use Committee. Experimental Colitis Models DSS-mediated colitis was induced by oral administration of 2%, 3%, and 4% (w/v) of DSS (molecular weight 36,000–50,000, MP Biomedicals) in drinking water for 7, 6, and 5 days, respectively, ad libitum; then, water was replaced until day 20. TNBS-mediated colitis was induced by intrarectal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS) at 15 mg/kg body weight diluted in 50% ethanol at day 0. The intensity of colitis for both models was monitored daily and clinical parameters were determined as follows: anal erosion (score 0–3; 0 = normal; 1 = mild; 2 = moderate; 3 = severe), anal bleeding (score 0–3: 0 = normal; 1 = mild; 2 = moderate; 3 = severe), diarrhea (score 0–3; 0 = normal; 1 = mild; 2 = moderate; 3 = severe), and percentage of body weight loss. Statistical Analysis Data were expressed as mean ± SEM. Student’s t test or Mann-Whitney test were performed, and p values < 0.05 using a 95% confidence interval were considered significant. Survival graphs were represented as Kaplan-Meier curves and analyzed with log rank test. The GraphPad Prism statistical software program (GraphPad Software) was used for all analyses. Other detailed experimental procedures can be found in the Supplemental Experimental Procedures. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and four figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.celrep.2014.01.020. AUTHOR CONTRIBUTIONS L.S. designed the study, performed experiments, analyzed data, and wrote the manuscript. K.G., K.A.H., Y.M.A., and H.J. contributed to discussion. E.L.-M., S.C.A., S.G.N., and L.C. provided knockout mice. E.Y.L and Q.L. provided human colon tissue. X.Z. supervised the study and wrote the manuscript. ACKNOWLEDGMENTS We thank the Flow Cytometry Core and the Histotechnology and Comparative Pathology Facility of Albert Einstein College of Medicine, Vera Des Marais for the assistance on Volocity software, and Jordan Chinai for reading the manuscript. This work was supported by NIH DP2DK083076, GM094665, AI007289, DOD PC094137, NIH P30CA013330, P60DK020541, AI51519, P30AG038072, T32DK007218, T32DK007513, and T32GM007491. Received: June 19, 2012 Revised: November 26, 2013 Accepted: January 15, 2014 Published: February 13, 2014 Cell Reports 6, 625–632, February 27, 2014 ª2014 The Authors 631 REFERENCES Agata, Y., Kawasaki, A., Nishimura, H., Ishida, Y., Tsubata, T., Yagita, H., and Honjo, T. (1996). Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. Int. Immunol. 8, 765–772. 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Med. 14, 282–289. Tissue-expressed B7-H1 Critically Controls Intestinal Inflammation Lisa Scandiuzzi, Kaya Ghosh, Kimberly A. Hofmeyer, Yael M. Abadi, Eszter Lázár-molnár, Elaine Y. Lin, Qiang Liu, Hyungjun Jeon, Steven C. Almo, Lieping Chen, Stanley G. Nathenson and Xingxing Zang A BALL NORMALIZED BY TOTAL BACTERIA GROUP 10 Bleeding score 100 80 60 40 20 0 3 ** 2 ** 4 6 8 10 ** ** ** ** ** Relative quantity 16S rDNA % survival 10000 4 Gram+ Gram- WT d0 PD-L1-/- D0 WT D6 PD-L1-/- D6 10 10003 10 1002 10 10 11 0.1 D0 D6 D0 D6 D0 D6 D0 D6 Clost. SFB Lact. Bact. 2 1 0 0 1 2 3 4 5 6 7 Days after TNBS administration WT B7-H1-/- Figure S1. Mortality and morbidity of B7-H1-/- mice upon TNBS treatment and microbiota groups after DSS treatement (A) TNBS treatment in WT and B7-H1-/- mice. Wild-type (n=18) and B7-H1-/- mice (n=17) were intrarectally instilled with TNBS at day 0. Survival and bleeding score were monitored after TNBS administration. Data were pooled from two independent experiments. Survival data are represented as Kaplan-Meier curves (** P < 0.01; *** P < 0.001; log-rank test). Data represent means ± S.E.M. (* P < 0.05, ** P < 0.01, *** P < 0.001; Student’s t test). (B) qPCR analysis of 16S rDNA for microbiota groups during DSS-treatment normalized with values obtained from untreated mice. Data are pooled from two independent experiments with (n=3-4). Data represent means ± S.E.M. Clost. = Clostridiales; SFB = Segmented Filamentous Bacteria; Lact. = Lactobacillaceae; Bact. = Bacteroides. Diarrhea score * 75 50 25 0 3 % DSS 3 WT BM WT B7-H1-/- BM B7-H1-/- 2 D6 Cytokeratin Ki67 DAPI WT B7-H1-/- 100 µm BrdU (D6) 50µm WT B7-H1-/- * 6 7 8 0 2 4 6 8 10 12 14 16 18 20 1 2 3 4 Days after DSS administration B * 1 2.5 2.0 1.5 1.0 0.5 0 BrdU+ cells/crypt % survival 100 Number of pixels for Ki67+ cells (x104) A 10 8 6 4 2 0 5 ** n.s. WT B7-H1-/- D0 * D2 D6 n.s. WT B7-H1-/- D0 D6 Figure S2. Chimera control mice and epithelial cell proliferation during intestinal inflammation. (A) Survival and morbidity in chimera control mice. Wild-type (WT) mice reconstituted with WT bone marrow (BM) cells and B7-H1-/- mice reconstituted with B7-H1-/- bone marrow cells were fed with 3% DSS in drinking water for 6 days. Survival and diarrhea score were monitored after the beginning of DSS. Data are representative of two experiments with at least 5 mice per group. Survival data are represented as Kaplan-Meier curves (*P < 0.01; log-rank test). (B) Cell proliferation analysis. Upper panel: IF staining on colon tissues from DSS-fed (D6) WT and B7-H1-/- mice for Cytokeratin+ (green) cells, Ki-67+ cells (magenta) and nuclei; scale bar, 100µm. Quantification of Ki-67+ cells was calculated with Volocity® software. 10 fields/tissues sections were quantified per mouse. Lower panel: IHC staining for BdrU on colon section from WT and B7-H1-/- mice during DSS-treatment (D6) and quantification of BrdU+ cells (scale bars, 50µm) at D0 and D6. At least 20 intact crypt/time point were counted. Small horizontal bars indicate the mean. Data represent means ± S.E.M. ( * P < 0.05; n.s.=not significant; Student’s t test). Cell number (x105) 10.0 B LP 4 pg/ml (x103) A 0.5 1.0 0.5 3 2 1 0 Cell number (x106) ILC MLN CD11c+ CD11b+ Myeloids NK cells Macroph. Neutr. B cells Tregs CD8+ CD4+ 4 WT D0 B7-H1-/- D0 WT D2 B7-H1-/- D2 WT D6 B7-H1-/- D6 IL-6 IL-12p70 IL-4 IFN-γ MCP-1 WT B7-H1-/- 2 1 CD11c+ CD11b+ Macroph. NK cells Neutr. B cells Tregs CD8+ CD4+ Figure S3. Cell infiltration in lamina propria (LP) and mesenteric lymphonode (MLN) and inflammatory cytokines in colon supernatant. (A) Single cell suspension of cells isolated from lamina propria and mesenteric lymph node were analyzed at day 6 of DSS-mediated inflammation for cell surface markers to detect immune cell populations. Data represent means ± S.E.M. ILC= innate lymphoid cells. (B) Production of IL-6, IL-12p70, IL4, IFN-γ and MCP-1 in supernatant of colons from WT and B7-H1-/- mice at D0, D2 and D6 of DSS-treatment. Data are representative of three independent experiments (n=4). B 4 7.8 7.8 10 2 0 5 10 4 <PE-Cy7-YG-A> 10 0 10 2 3 10 10 <PE-YG-A> 4 10 5 103 28.1 28.1 10 2 10 5 0 WT B7-H1-/B7-H1-Ig 4 0.5 Human-Ig 3 10 10 <PE-YG-A> 1.0 Marker 2 1.5 Pos. Ctr. 10 TNF- α 1 2.0 B7-H1-Ig 0 2 Human-Ig 0 B7-H1-/- CD11c WT WT 103 2.5 EU/mg 10 <PE-Cy7-YG-A> 5 Number of CD11c+CD11b+ TNF- α producing cells (x104) Gated on 10 CD11b+ ** A Figure S4. TNF production from CD11c+CD11b+ LP cells and quality check of fusion proteins. (A) FACS plots and number of TNF-α-producing CD11c+CD11b+ cells isolated from DSS-fed (D6) mice. Numbers above bracketed lines indicate percentage of TNF-α + cells. Data are pooled from at least two independent experiments (n=4). Data represent means ± S.E.M. ( ** P < 0.01; Student’s t test). (B) Left panel: Bacterial endotoxins (EU) quantification per mg of protein in Human-Ig and B7-H1-Ig as compared to a positive control. Measurements were performed using an ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit (GenScript). Right panel: Coomassie stained gel of Human-Ig and B7-H1-Ig. Extended Experimental Procedures Histological analysis, immunohistochemistry and immunofluorescence Colon tissues were isolated, washed in PBS and either fixed with Zinc Fixative or embedded in OCT compound. Paraffin-embedded sections (5µm) were stained for eosin-hematoxylin (HE) by the Histotechnology and Comparative Pathology Facility. HE stained tissues were blindly analyzed on an AxioCam MRC Zeiss microscope. Each section was evaluated for infiltration, edema and ulceration. A semi-quantitative criterion-based method was used ranging from 0 to 3 where 0=within normal limits or absent; 1=mild changes; 2= moderate changes; 3:=severe changes. Mucosa associated lymphoid tissues (MALTs) area was quantified by matching the size of each MALT to a graduated scale of circles, each with a numerical value. The final values were expressed in µm2. For immunohistochemistry of human colon tissue, frozen-section slides were used and stained with anti-human B7-H1 biotinylated antibody (R&D). Immunoperoxidase method was used for detection. For immunofluorescence staining we used an anti-FITC-Pan Cytokeratin and an anti-Ki67 antibodies followed by a secondary antibody. DAPI (Invitrogen) was used to detect nucleus. At least 10 fields/mouse were blindly analyzed with an Olympus BX61. Blockade of TNF-α 100 µg of monoclonal anti-mouse TNF-α blocking antibody (BioXCell, BE0058) or an isotype-matched control rat IgG1 (BioXCell, BE0088) were injected i.p into mice every other day for up to 20 days of DSS treatment starting at day 0. Analysis of red blood cells Red blood cell (RBC) concentration and hematocrit were determined by standard hematological analysis in the Histotechnology and Comparative Pathology Facility of Albert Einstein College of Medicine. Cytokine measurement All cytokines, except for TGF-β and IL-22, were measured in the colon supernatants using Cytometric Beads Array (BD Biosciences) according to the manufacturer’s instructions. Data were acquired on FACscalibur (BD Biosciences) and analyzed with FlowJo software (version 8.8.4). TGF-β was detected using Single Plex Flex Set CBA (BD, Bioscience) according to manufacturer’s instructions. IL-22 was detected by ELISA assay using AAM65 (AbDSerotec) as capture antibody and AAM65B (AbDSerotec) as detection antibody according to manufacturer’s instruction. Quantification of B7-H1+ on human samples and of Ki67+ cells on mouse samples B7-H1 expression on human samples was quantified using Volocity® software based on a 2 steps protocol. Briefly, two images in .tiff format were imported into Volocity® as follows: one isotype control image as negative control and one image positively stained for the antibody of interest as positive control. The object of interest was found using RGB with the following intensity thresholds (first channel: lower 10, upper 137; second channel: lower 27, upper 133; third channel: lower 25, upper 101) and then either excluded or retained based upon color and size (< 2 µm2). A uniform filter was used to remove noise from the system and to identify positive cells. A similar protocol has been used to identify Ki67+ cells. Flow cytometry reagents Single-cell suspensions isolated from colons were washed in FACS buffer (icecold 0.5% BSA in PBS) and stained with specific antibodies from eBioscience: FITC-CD62L (MEL-14), APC-CD45R (RA3-6B2), PE-CD11b (M1/70), APC-F4/80 (BM8), alexa fluor 647-F4/80, FITC-B220, FITC-B7-2 (GL1), Alexa-fluor 700-CD3 (17A2), eFluor405- CD45 (2D1), PE-B7-H1 (MIH5), biotin-CD11c (N418), biotinNK1.1 (PK136), CD49d and PE-Cy7 CD90.2 (53-2.1); three Abs from BD: FITCLy-6G (1A8), Annexin V and CD40 (3/23); one Ab from Abd Serotec: F4/80-alexa fluor 647; one antibody was from Sigma-Aldrich: FITC-conjugated anti-Pan Cytokeratin monoclonal (C-11) and one from Abcam: anti-Ki67 (ab15580). Biotinconjugated Abs were followed by APC-, PE-, FITC- or PE-Cy7- conjugated streptavidin (eBioscience) staining. For intracellular staining we used PEconjugated anti-mouse/rat Foxp3 (FJK-16s, Ebioscience), anti-mouse TNF-α (MP6-XT22, Ebioscience) and an anti-mouse IL-22 (Poly5164, BioLegend). To prevent non-specific binding, all samples were pre-incubated with Fc-Block (eBioscience) and isotype-matched antibodies were used. Data were acquired using BD™LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Version 8.8.4). Scoring of colonic bleeding Colons isolated from mice at different time points during DSS-treatment were scored as follows: 0= lack of any gross blood visible throughout the entire colon; 1= gross blood present in <1/3 of the colon; 2= <2/3; 3= >2/3 of the colon. Bacterial culture Samples of feces, colon, mesenteric lymph nodes (MLN), liver and spleen were collected in 0.01% Triton X-100/PBS and homogenized. Different dilutions of the obtained homogenate were plated on blood agar-plates and incubated for 24 h. Bacterial count was determined by colony-forming assay. Isolation of epithelial and LP cells Colons were washed in PBS and chopped into 0.5 cm pieces. Tissues were incubated in 2 mM EDTA with PBS for 30 min at 37°C while shaking at 200 rpm. Samples were filtered in a 70-µm strainer, centrifuged for 15’ at 1700 rpm in 30% Percoll to isolate epithelial cells which were then used for FACS analysis or coculture assay. The remaining pieces of colons were further digested in 2 mg/ml of collagenase-IV, 5% FBS, 1 mg/ml DNaseI in PBS for 30 min at 37°C shaking at 200 rpm. Samples were passed through 40-µm strainers. LP cells were then separated in a 30% Nycoprep gradient and used for experiments. qPCR To analyze commensal bacteria, stool from colon was collected and DNA was extracted with Qiagen DNA isolation kit and quantitative PCR for 16S rDNA was performed. Absolute copy numbers of bacterial 16S rDNA were determined from standard curves established by qPCR of serial dilutions of reference samples harboring the 16S rDNA gene from each of the bacteria strains analyzed. All reactions were performed in 10µl using Power SYBR Green Master Mix (Applied Biosystems) with 1 µM concentration of each primer using the following steps: 90˚C for 3 minutes, 25 cycles of 95˚C for 40 seconds, 60˚C for 40 seconds, and 60˚ for 4 minutes on a ABI-PRISM 7900 (Applied Biosystem). Primer sequences for bacteria groups are the followed: Total Bacteria Fwd: 5’ - ACTCCTACGGGAGGCAGCAGT- 3’; Rev: 5’ -ATTACCGCGGCTGCTGGC- 3’. Clostridiales Fwd: 5’ -ACTCCTACGGGAGGCAGC- 3’ Rev: 5’ - GCTTCTTAGTCAGGTACCGTCAT- 3’; SFB (Segmented Filamentous Bacteria) Fwd: 5’ -GACGCTGAGGCATGAGAGCAT- GACGGCACGGATTGTTATTCA- 3’; 3’, Rev: Lactobacillaceae Fwd: 5’ - 5’ - AGCAGTAGGGAATCTTCCA- 3’, Rev: 5’ -CACCGCTACACATGGAG- 3’; Bacteroides Fwd: 5’ -GGTTCTGAGAGGAAGGTCCC- 3, Rev: 5’ - GCTGCCTCCCGTAGGAGT- 3’. In vivo epithelial permeability assay Mice were deprived of water and food for 3 h and then gavaged with FITCdextran (MW 40,000; Sigma-Aldrich) at 0.6mg/g body weight. 3h later fluorescence was measured in sera using a spectrophotometer (Synergy H4, BioTek). FITC-dextran concentration was determined from standard curves generated by serial dilution of FITC-dextran. Production of B7-H1-Ig Fusion Protein and Functional Assay B7-H1-Ig fusion protein was produced in an inducible secreted serum-free Drosophila expression system. The coding region of the extracellular domain of B7-H1 was fused to a human Ig-G1 Fc tag of plasmid pMT/BiP. The construct was co-transfected into Drosophila cell line S2 with a hygromycin resistance plasmid. The stable transfected cell line was induced with CuSO4 to secrete B7H1 in Drosophila serum-free medium (Invitrogen). B7-H1-Ig was purified on an ImmunoPure Plus protein G column. The control human IgG1 Fc protein was produced in the same way. The purity of fusion proteins was confirmed by Coomassie blue staining (Figure S4B), immunoblotting with antibodies against human IgG Fc (Jackson Imm. Res.) and by checking the bacterial endotoxin levels using an ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit (GenScript) (Figure S4B). B7-H1-Ig or control Ig (3µg/ml) in PBS were incubated in a 96-well plate overnight at 4°C. After washing the wells twice with PBS, 2 × 104 LP cells isolated from DSS-treated wild-type or PD1-/-B7-1/-/- mice were added and incubated for 24h at 37°C in 10% FBS, 100 units/ml penicillin and 100 µg/ml streptomycin in DMEM-F12 medium. Cells were collected and used for flow cytometry analyses. Co-culture assay Isolated IEC cells from naïve wild-type or B7-H1-/- mice were plated on a thin layer of matrigel (BD) in a 96-well plate at 1 x 105/well in complete RPMI. LP cells, isolated as previously described from different mice at different time points during DSS-treatment, were added on the top at 1 x 105/well in complete RMPI plus GolgiStop reagents (BD). 24h after LP cells were collected and processed for FACS analysis. Colon organ culture Colons were washed in PBS supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin, cut in 0.5-cm pieces and cultured in 24-well-flat-bottom plates in serum-free RPMI-1640 medium supplemented with 100U/ml penicillin and 100 µg/ml streptomycin, L-glutamine and nonessential amino acids. 24h later supernatant was harvested, centrifuged at 13,000 g for 10 minutes at 4°C and stored at -20°C until analyzed.