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INTRODUCTION: Iron deficiency and vitamin D deficiency are common comorbidities in inflammatory bowel disease (IBD). Accumulating evidence indicates that active 1,25-dihydroxyvitamin D (1,25(OH)D) may enhance iron absorption by suppressing hepcidin. We investigated the influence of vitamin D on iron metabolism in patients with IBD and on the expression of genes facilitating intestinal epithelial iron absorption. METHODS: Iron parameters and serum levels of 25-hydroxyvitamin D (25(OH)D), 1,25(OH)D, and hepcidin were measured in 104 adult patients with IBD (67 with Crohn's disease and 37 with ulcerative colitis). Genes involved in iron absorption were tested for induction by 1,25(OH)D in Caco-2 cells, which resemble the small intestinal epithelium. RESULTS: In multiple regression models controlling for age, sex, body mass index, smoking status, disease activity, and C-reactive protein levels, low 25(OH)D levels were associated with iron deficiency in patients with IBD (β [SE] = −0.064 [0.030], P = 0.029). Vitamin D sufficiency was associated with increased levels of ferritin (β [SE] = 0.25 [0.11], P = 0.024) and transferrin saturation (β [SE] = 8.41 [4.07], P = 0.044). Higher 1,25(OH)D:25(OH)D ratios were associated with lower hepcidin levels (β [SE] = −4.31 [1.67], P = 0.012). Especially in Crohn's disease, increased 1,25(OH)D correlated with higher transferrin saturation (β [SE] = 0.43 [0.18], P = 0.027). Furthermore, 1,25(OH)D strongly induced the expression of the ferroxidase ceruloplasmin in Caco-2 cells. DISCUSSION: Low vitamin D levels in IBD correlate with iron deficiency. Vitamin D may ameliorate iron deficiency, potentially by downregulating hepcidin and upregulating ceruloplasmin, enhancing intestinal iron absorption.

BackgroundNOD2 variants are the strongest genetic predictors for susceptibility to Crohn's disease (CD). However, the clinical value of NOD2 on an individual patient level remains controversial. We aimed to define the predictive power of the major NOD2 mutations regarding complicated CD in a large single center cohort.Methods1076 CD patients were prospectively genotyped for the three common CD-associated NOD2 mutations rs2066844, rs2066845, and rs2066847, followed by detailed genotype-phenotype analyses.ResultsOverall, 434 CD patients (40.3%) carried at least one of the three main NOD2 mutations. A significantly higher minor allele frequency (15.6%) of the NOD2 frameshift mutation p.Leu1007fsX1008 (rs2066847) was seen in patients with aggressive disease compared to 8.2% in patients with mild disease (p = 2.6 x 10-5). Moreover, a total of 54 CD patients (5.0%) were homozygous for this NOD2 frameshift mutation. 100% of these patients had ileal disease compared to 82% of NOD2 wild-type carriers (pConclusionHomozygosity for Leu1007fsX1008 is an excellent biomarker for predicting complicated CD on an individual patient level. Active smoking and homozygosity for this mutation is associated with a 100% risk for developing ileal stenosis requiring CD-related surgery. In these patients, smoking cessation and early initiation of immunosuppressive strategies may be beneficial.

Invariant natural killer T (iNKT) cells recognize lipid antigens presented by CD1d and play a central role in regulating immunity and inflammation in peripheral tissues. However, the mechanisms which govern iNKT cell homeostasis after thymic emigration are incompletely understood. Here we demonstrate that microsomal triglyceride transfer protein (MTP), a protein involved in the transfer of lipids onto CD1d, regulates liver iNKT cell homeostasis in a manner dependent on hepatocyte CD1d. Mice with hepatocyte-specific loss of MTP exhibit defects in the function of CD1d and show increased hepatic iNKT cell numbers as a consequence of altered iNKT cell apoptosis. Similar findings were made in mice with hepatocyte-specific loss of CD1d, confirming a critical role of CD1d in this process. Moreover, increased hepatic iNKT cell abundance in the absence of MTP is associated with susceptibility to severe iNKT cell-mediated hepatitis, thus demonstrating the importance of CD1d-dependent control of liver iNKT cells in maintaining immunological homeostasis in the liver. Together, these data demonstrate an unanticipated role of parenchymal cells, as shown here for hepatocytes, in tissue-specific regulation of CD1d-restricted immunity and further suggest that alterations in lipid metabolism may affect iNKT cell homeostasis through effects on CD1d-associated lipid antigens.

OBJECTIVES: Recent experimental evidence suggests that environmental microbial factors early in life determine susceptibility to allergic diseases through inappropriate chemotaxis and local activation of CD1d-restricted, invariant chain natural killer T (iNKT) cells. In this study, we analyzed the involvement of these pathways in pediatric patients with eosinophilic esophagitis (EoE) before and after dietary allergen elimination.METHODS: mRNA expression levels of components of the C-X-C motif chemokine ligand 16 (CXCL16)-iNKT-CD1d axis were compared in esophageal biopsies from EoE patients vs. normal or inflammatory controls and before and after treatment.RESULTS: CXCL16, iNKT cell-associated cell marker Vα24, and CD1d were significantly upregulated in esophageal biopsies from EoE patients and correlated with the expression of inflammatory mediators associated with allergy. Upregulation of each of these factors was significantly more pronounced in patients aged CONCLUSIONS: Our observations place iNKT cells at the center of allergic inflammation associated with EoE, which could have profound implications for our understanding, treatment and prevention of this and other human allergic diseases.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed on activated natural killer (NK) cells wherein it inhibits lysis of CEACAM1-bearing tumor cell lines. The mechanism for this is unknown. Here, we show that interleukin-2-induced expression of CEACAM1 on both mouse and primary human NK cells impairs the ability of NK gene complex group 2 member D (NKG2D) to stimulate cytolysis of CEACAM1-bearing cells. This process requires the expression of CEACAM1 on the NK cells and on the tumor cells, which is consistent with the involvement of trans-homophilic interactions between CEACAM1. Mechanistically, co-engagement of NKG2D and CEACAM1 results in a biochemical association between these two surface receptors and the recruitment of Src homology phosphatase 1 by CEACAM1 that leads to dephosphorylation of the guanine nucleotide exchange factor Vav1 and blockade of downstream signaling that is associated with the initiation of cytolysis. Thus, CEACAM1 on activated NK cells functions as an inhibitory receptor for NKG2D-mediated cytolysis, which has important implications for understanding the means by which CEACAM1 expression adversely affects tumor immunity.

The role of the Th17 cell inhibiting cytokine IL-27 in the pathogenesis of inflammatory bowel disease is contradictory. Its effects on the intestinal barrier have so far not been investigated, which was the aim of this study. We show that intestinal epithelial cells (IEC) express both IL-27 receptor subunits IL-27RA and gp130. The IL-27 receptor expression is up-regulated in intestinal inflammation and during bacterial infection. IL-27 activates ERK and p38 MAPKs as well as Akt, STAT1, STAT3, and STAT6 in IEC. IL-27 significantly enhances cell proliferation and IEC restitution. These functions of IL-27 are dependent on the activation of STAT3 and STAT6 signaling pathways. As analyzed by microarray, IL-27 modulates the expression of 428 target genes in IEC (316 up and 112 down; p < 0.05). IL-27 as well as its main target genes are up-regulated in colonic tissue and IEC isolated from mice with dextran sulfate sodium (DSS)-induced colitis. The IL-27-induced expression of the anti-bacterial gene deleted in malignant brain tumor 1 (DMBT1) is mediated by p38 and STAT3 signaling, whereas the activation of the anti-inflammatory and anti-bacterial gene indoleamine 2,3-dioxygenase (IDO1) is dependent on STAT1 signal transduction. IL-27-induced indoleamine 2,3-dioxygenase enzymatic activity leads to growth inhibition of intestinal bacteria by causing local tryptophan depletion. For the first time, we characterize IL-27 as a mediator of intestinal epithelial barrier protection mediated via transcriptional activation of anti-inflammatory and antibacterial target genes.

The IL-10-like cytokine IL-22 is produced by activated T cells. In this study, we analyzed the role of this cytokine system in hepatic cells. Expression studies were performed by RT-PCR and quantitative PCR. Signal transduction was analyzed by Western blot experiments and ELISA. Cell proliferation was measured by MTS and [3H]thymidine incorporation assays. Hepatocyte regeneration was studied in in vitro restitution assays. Binding of IL-22 to its receptor complex expressed on human hepatic cells and primary human hepatocytes resulted in the activation of MAPKs, Akt, and STAT proteins. IL-22 stimulated cell proliferation and migration, which were both significantly inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin. IL-22 increased the mRNA expression of suppressor of cytokine signaling (SOCS)-3 and the proinflammatory cytokines IL-6, IL-8, and TNF-α. SOCS-1/3 overexpression abrogated IL-22-induced STAT activation and decreased IL-22-mediated liver cell regeneration. Hepatic IL-22 mRNA expression was detectable in different forms of human hepatitis, and hepatic IL-22 mRNA levels were increased in murine T cell-mediated hepatitis in vivo following cytomegalovirus infection, whereas no significant differences were seen in an in vivo model of ischemia-reperfusion injury. In conclusion, IL-22 promotes liver cell regeneration by increasing hepatic cell proliferation and hepatocyte migration through the activation of Akt and STAT signaling, which is abrogated by SOCS-1/3 overexpression.

Background Currently, limited data of the outcome of inflammatory bowel disease (IBD) in patients after solid organ transplantation (SOT) are available. We aimed to analyze effects of SOT on the IBD course in a large IBD patient cohort. Methods Clinical data from 1537 IBD patients were analyzed for patients who underwent SOT (n = 31) between July 2002 and May 2014. Sub-analyses included SOT outcome parameters, IBD activity before and after SOT, and efficacy of IBD treatment. Results 4.74% of patients with ulcerative colitis (UC) and 0.84% of patients with Crohn’s disease (CD) underwent SOT (p = 2.69 x 10−6, UC vs. CD). 77.4% of patients with SOT underwent liver transplantation (LTx) with tacrolimus-based immunosuppressive therapy after SOT. All LTx were due to primary sclerosing cholangitis (PSC) or PSC overlap syndromes. Six patients (19.4%) required renal transplantation and one patient (3.2%) heart transplantation. A survival rate of 83.9% after a median follow-up period of 103 months was observed. Before SOT, 65.0% of patients were in clinical remission and 5 patients received immunosuppressive therapy (16.1%). After SOT, 61.0% of patients were in remission (p = 1.00 vs. before SOT) and 29.0% required IBD-specific immunosuppressive or anti-TNF therapy (p = 0.54 vs. before SOT). 42.9% of patients with worsening of IBD after SOT were at higher risk of needing steroid therapy for increased IBD activity (p = 0.03; relative risk (RR): 10.29; 95% CI 1.26–84.06). Four patients (13.0%) needed anti-TNF therapy after SOT (response rate 75%). Conclusions SOT was more common in UC patients due to the higher prevalence of PSC-related liver cirrhosis in UC. Despite mainly tacrolimus-based immunosuppressive regimens, outcome of SOT and IBD was excellent in this cohort. In this SOT cohort, concomitant immunosuppressive therapy due to IBD was well tolerated.

IL-22 is produced by activated T cells and signals through a receptor complex consisting of IL-22R1 and IL-10R2. The aim of this study was to analyze IL-22 receptor expression, signal transduction, and specific biological functions of this cytokine system in intestinal epithelial cells (IEC). Expression studies were performed by RT-PCR. Signal transduction was analyzed by Western blot experiments, cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and Fas-induced apoptosis by flow cytometry. IEC migration was studied in wounding assays. The IEC lines Caco-2, DLD-1, SW480, HCT116, and HT-29 express both IL-22 receptor subunits IL-22R1 and IL-10R2. Stimulation with TNF-α, IL-1β, and LPS significantly upregulated IL-22R1 without affecting IL-10R2 mRNA expression. IL-22 binding to its receptor complex activates STAT1/3, Akt, ERK1/2, and SAPK/JNK MAP kinases. IL-22 significantly increased cell proliferation ( P = 0.002) and phosphatidylinsitol 3-kinase-dependent IEC cell migration ( P < 0.00001) as well as mRNA expression of TNF-α, IL-8, and human β-defensin-2. IL-22 had no effect on Fas-induced apoptosis. IL-22 mRNA expression was increased in inflamed colonic lesions of patients with Crohn’s disease and correlated highly with the IL-8 expression in these lesions ( r = 0.840). Moreover, IL-22 expression was increased in murine dextran sulfate sodium-induced colitis. IEC express functional receptors for IL-22, which increases the expression of proinflammatory cytokines and promotes the innate immune response by increased defensin expression. Moreover, our data indicate intestinal barrier functions for this cytokine-promoting IEC migration, which suggests an important function in intestinal inflammation and wound healing. IL-22 is increased in active Crohn’s disease and promotes proinflammatory gene expression and IEC migration.

The mechanisms by which mucosal homeostasis is maintained are of central importance to inflammatory bowel disease. Critical to these processes is the intestinal epithelial cell (IEC), which regulates immune responses at the interface between the commensal microbiota and the host1,2. CD1d presents self and microbial lipid antigens to natural killer T (NKT) cells, which are involved in the pathogenesis of colitis in animal models and human inflammatory bowel disease3–8. As CD1d crosslinking on model IECs results in the production of the important regulatory cytokine interleukin (IL)-10 (ref.9), decreased epithelial CD1d expression—as observed in inflammatory bowel disease10,11—may contribute substantially to intestinal inflammation. Here we show in mice that whereas bone-marrow-derived CD1d signals contribute to NKT-cell-mediated intestinal inflammation, engagement of epithelial CD1d elicits protective effects through the activation of STAT3 and STAT3-dependent transcription of IL-10, heat shock protein 110 (HSP110; also known as HSP105), and CD1d itself. All of these epithelial elements are critically involved in controlling CD1d-mediated intestinal inflammation. This is demonstrated by severe NKT-cell-mediated colitis upon IEC-specific deletion of IL-10, CD1d, and its critical regulator microsomal triglyceride transfer protein (MTP)12,13, as well as deletion of HSP110 in the radioresistant compartment. Our studies thus uncover a novel pathway of IEC-dependent regulation of mucosal homeostasis and highlight a critical role of IL-10 in the intestinal epithelium, with broad implications for diseases such as inflammatory bowel disease.

SummaryCoevolution of beneficial microorganisms with the mammalian intestine fundamentally shapes mammalian physiology. Here, we report that the intestinal microbe Bacteroides fragilis modifies the homeostasis of host invariant natural killer T (iNKT) cells by supplementing the host’s endogenous lipid antigen milieu with unique inhibitory sphingolipids. The process occurs early in life and effectively impedes iNKT cell proliferation during neonatal development. Consequently, total colonic iNKT cell numbers are restricted into adulthood, and hosts are protected against experimental iNKT cell-mediated, oxazolone-induced colitis. In studies with neonatal mice lacking access to bacterial sphingolipids, we found that treatment with B. fragilis glycosphingolipids—exemplified by an isolated peak (MW = 717.6) called GSL-Bf717—reduces colonic iNKT cell numbers and confers protection against oxazolone-induced colitis in adulthood. Our results suggest that the distinctive inhibitory capacity of GSL-Bf717 and similar molecules may prove useful in the treatment of autoimmune and allergic disorders in which iNKT cell activation is destructive.PaperClip

Natural Killer T (NKT) cells are a subset of T lymphocytes that recognize a wide variety of lipid antigens presented by CD1 molecules. NKT cells exhibit rapid activation after recognition of cognate antigens, secrete abundant amounts of T helper (Th) 1, Th2, and Th17 cytokines within hours of activation and shape the immune response through subsequent activation of dendritic, NK, T and B cells. NKT cells therefore play central roles in antimicrobial and anticancer immunity and in modulation of various autoimmune disorders. Consequently, recent research has focused on the discovery of microbial and self-antigens involved in NKT cell activation. In this chapter, we discuss different strategies for studying antigen recognition by NKT cells including CD1d tetramer-based approaches and in vitro assays characterizing NKT cell activation in response to lipid antigen presentation. While toll-like receptor (TLR) agonists and cytokines such as IL-12 are critical for NKT cell activation in vivo, particularly in the context of microbial infection, methods for detection of TLR- and cytokine-dependent NKT cell activation will not be discussed in this section.

Microbes: Early and Often Epidemiological studies have suggested that the increase in the incidence of asthma and other inflammatory diseases seen in many parts of the world may be due to a reduced exposure to microbes during early childhood. Olszak et al. (p. 489, published online 22 March) now show that commensal microflora help to regulate the numbers and functions of natural killer T (NKT) cells in the colon and lung in mice. Germ-free mice had elevated numbers of NKT cells in these tissues and were more susceptible to chemically induced colitis and allergic asthma. Neonatal recolonization of germ-free mice with microflora prevented enhanced colitis and asthma sensitivity; however, exposure of adult mice to these conditions was not effective. Thus, early exposure to microbes has important, lasting effects on the immune system's sensitivity to inflammation.

Background Genome-wide association studies identified a PTGER4 expression-modulating region on chromosome 5p13.1 as Crohn's disease (CD) susceptibility region. The study aim was to test this association in a large cohort of patients with inflammatory bowel disease (IBD) and to elucidate genotypic and phenotypic interactions with other IBD genes. Methodology/Principal Findings A total of 7073 patients and controls were genotyped: 844 CD and 471 patients with ulcerative colitis and 1488 controls were analyzed for the single nucleotide polymorphisms (SNPs) rs4495224 and rs7720838 on chromosome 5p13.1. The study included two replication cohorts of North American (CD: n = 684; controls: n = 1440) and of German origin (CD: n = 1098; controls: n = 1048). Genotype-phenotype, epistasis and transcription factor binding analyses were performed. In the discovery cohort, an association of rs4495224 (p = 4.10×10−5; 0.76 [0.67–0.87]) and of rs7720838 (p = 6.91×10−4; 0.81 [0.71–0.91]) with susceptibility to CD was demonstrated. These associations were confirmed in both replication cohorts. In silico analysis predicted rs4495224 and rs7720838 as essential parts of binding sites for the transcription factors NF-κB and XBP1 with higher binding scores for carriers of the CD risk alleles, providing an explanation of how these SNPs might contribute to increased PTGER4 expression. There was no association of the PTGER4 SNPs with IBD phenotypes. Epistasis detected between 5p13.1 and ATG16L1 for CD susceptibility in the discovery cohort (p = 5.99×10−7 for rs7720838 and rs2241880) could not be replicated in both replication cohorts arguing against a major role of this gene-gene interaction in the susceptibility to CD. Conclusions/Significance We confirmed 5p13.1 as a major CD susceptibility locus and demonstrate by in silico analysis rs4495224 and rs7720838 as part of binding sites for NF-κB and XBP1. Further functional studies are necessary to confirm the results of our in silico analysis and to analyze if changes in PTGER4 expression modulate CD susceptibility.

By retaining NKG2D ligands within tumor cells, carcinoembryonic antigen–related cell adhesion molecule 1 (CEACAM1) facilitates tumor cell escape from NK cell–mediated cytolysis in vitro and in vivo., Although carcinoembryonic antigen (CEA)–related cell adhesion molecule 1 (CEACAM1) has been viewed as a tumor suppressor, increasing clinical evidence shows that high levels of CEACAM1 expression on tumors correlates with poor prognosis and high risk of metastasis. Here, we examined the consequences of CEACAM1 expression on tumor cells. We show that tumor cell–associated CEACAM1 causes intracellular retention of various NKG2D ligands in mouse and human tumor cells. CEACAM1-silenced tumor cells expressed more cell surface NKG2D ligands and exhibited greater sensitivity to natural killer cell–mediated cytolysis in vitro and rejection in vivo. Our studies reveal a novel mechanism through which CEACAM1-bearing tumor cells may escape immune-surveillance.

Sepsis remains the leading cause of death in critically ill patients, despite modern advances in critical care. Intestinal barrier dysfunction may lead to secondary bacterial translocation and the development of the multiple organ dysfunction syndrome during sepsis. Cyclooxygenase (COX)-2 is highly upregulated in the intestine during sepsis, and we hypothesized that it may be critical in the maintenance of intestinal epithelial barrier function during peritonitis-induced polymicrobial sepsis. COX-2−/− and COX-2+/+ BALB/c mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice chimeric for COX-2 were derived by bone marrow transplantation and underwent CLP. C2BBe1 cells, an intestinal epithelial cell line, were treated with the COX-2 inhibitor NS-398, PGD2, or vehicle and stimulated with cytokines. COX-2−/− mice developed exaggerated bacteremia and increased mortality compared with COX-2+/+ mice following CLP. Mice chimeric for COX-2 exhibited the recipient phenotype, suggesting that epithelial COX-2 expression in the ileum attenuates bacteremia following CLP. Absence of COX-2 significantly increased epithelial permeability of the ileum and reduced expression of the tight junction proteins zonula occludens-1, occludin, and claudin-1 in the ileum following CLP. Furthermore, PGD2 attenuated cytokine-induced hyperpermeability and zonula occludens-1 downregulation in NS-398–treated C2BBe1 cells. Our findings reveal that absence of COX-2 is associated with enhanced intestinal epithelial permeability and leads to exaggerated bacterial translocation and increased mortality during peritonitis-induced sepsis. Taken together, our results suggest that epithelial expression of COX-2 in the ileum is a critical modulator of tight junction protein expression and intestinal barrier function during sepsis.

BACKGROUND: IL12B encodes the p40 subunit of IL-12, which is also part of IL-23. Recent genome-wide association studies identified IL12B and IL23R as susceptibility genes for inflammatory bowel disease (IBD). However, the phenotypic effects and potential gene-gene interactions of IL12B variants are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed IL12B gene variants regarding association with Crohn's disease (CD) and ulcerative colitis (UC). Genomic DNA from 2196 individuals including 913 CD patients, 318 UC patients and 965 healthy, unrelated controls was analyzed for four SNPs in the IL12B gene region (rs3212227, rs17860508, rs10045431, rs6887695). Our analysis revealed an association of the IL12B SNP rs6887695 with susceptibility to IBD (p = 0.035; OR 1.15 [95% CI 1.01-1.31] including a trend for rs6887695 for association with CD (OR 1.41; [0.99-1.31], p = 0.066) and UC (OR 1.18 [0.97-1.43], p = 0.092). CD patients, who were homozygous C/C carriers of this SNP, had significantly more often non-stricturing, non-penetrating disease than carriers of the G allele (p = 6.8×10(-5); OR = 2.84, 95% CI 1.66-4.84), while C/C homozygous UC patients had less often extensive colitis than G allele carriers (p = 0.029; OR = 0.36, 95% CI 0.14-0.92). In silico analysis predicted stronger binding of the minor C allele of rs6887695 to the transcription factor RORα which is involved in Th17 differentiation. Differences regarding the binding to the major and minor allele sequence of rs6887695 were also predicted for the transcription factors HSF1, HSF2, MZF1 and Oct-1. Epistasis analysis revealed weak epistasis of the IL12B SNP rs6887695 with several SNPs (rs11889341, rs7574865, rs7568275, rs8179673, rs10181656, rs7582694) in the STAT4 gene which encodes the major IL-12 downstream transcription factor STAT4 (p

BACKGROUND: Osteopontin represents a multifunctional molecule playing a pivotal role in chronic inflammatory and autoimmune diseases. Its expression is increased in inflammatory bowel disease (IBD). The aim of our study was to analyze the association of osteopontin (OPN/SPP1) gene variants in a large cohort of IBD patients. METHODOLOGY/PRINCIPAL FINDINGS: Genomic DNA from 2819 Caucasian individuals (n = 841 patients with Crohn's disease (CD), n = 473 patients with ulcerative colitis (UC), and n = 1505 healthy unrelated controls) was analyzed for nine OPN SNPs (rs2728127, rs2853744, rs11730582, rs11739060, rs28357094, rs4754 = p.Asp80Asp, rs1126616 = p.Ala236Ala, rs1126772 and rs9138). Considering the important role of osteopontin in Th17-mediated diseases, we performed analysis for epistasis with IBD-associated IL23R variants and analyzed serum levels of the Th17 cytokine IL-22. For four OPN SNPs (rs4754, rs1126616, rs1126772 and rs9138), we observed significantly different distributions between male and female CD patients. rs4754 was protective in male CD patients (p = 0.0004, OR = 0.69). None of the other investigated OPN SNPs was associated with CD or UC susceptibility. However, several OPN haplotypes showed significant associations with CD susceptibility. The strongest association was found for a haplotype consisting of the 8 OPN SNPs rs2728127-rs2853744-rs11730582-rs11439060-rs28357094-rs112661-rs1126772-rs9138 (omnibus p-value = 2.07×10⁻⁸). Overall, the mean IL-22 secretion in the combined group of OPN minor allele carriers with CD was significantly lower than that of CD patients with OPN wildtype alleles (p = 3.66×10⁻⁵). There was evidence for weak epistasis between the OPN SNP rs28357094 with the IL23R SNP rs10489629 (p = 4.18×10⁻²) and between OPN SNP rs1126616 and IL23R SNP rs2201841 (p = 4.18×10⁻²) but none of these associations remained significant after Bonferroni correction. CONCLUSIONS/SIGNIFICANCE: Our study identified OPN haplotypes as modifiers of CD susceptibility, while the combined effects of certain OPN variants may modulate IL-22 secretion.

Background: The pregnane X receptor (PXR/NR1I2) is an important regulator of xenobiotic metabolism and intestinal integrity. However, there are controversial studies on the role of PXR/NR1I2 in inflammatory bowel disease (IBD). We therefore initiated the largest analysis to date on PXR/NR1I2 gene variants in IBD patients. Methods: Genomic DNA from 2823 individuals of Caucasian origin including 859 patients with Crohn's disease (CD), 464 patients with ulcerative colitis (UC), and 1500 healthy, unrelated controls was analyzed for eight PXR/NR1I2 single nucleotide polymorphisms (SNPs) (rs12721602 (−25564), rs3814055 (−25385), rs1523128 (−24756), rs1523127 (−24381), rs45610735 = p.Gly36Arg (+106), rs6785049 (+7635), rs2276707 (+8055), and rs3814057 (+11156)). In addition, detailed haplotype and genotype–phenotype analyses were performed. Results: The PXR/NR1I2 SNP rs2276707 was weakly associated with UC susceptibility (P = 0.01; odds ratio [OR] 1.27 [1.06–1.52]). None of the other PXR/NR1I2 SNPs were associated with UC or CD susceptibility. However, several rare PXR/NR1I2 haplotypes were highly associated with CD susceptibility. In CD, the strongest disease association was found for a haplotype consisting of the SNPs rs12721602-rs3814055-rs1523128-rs1523127-rs12721607-rs6785049-rs2276707-rs3814057 (omnibus P-value: 6.50 × 10−15) which was found in two separate cohorts (cohort I = discovery cohort: CD: n = 492, controls: n = 793; P = 4.51 × 10−17; Bonferroni corrected: P = 1.27 × 10−15; cohort II = replication cohort: CD: n = 367, controls: n = 707; P = 7.12 × 10−4; Pcorr = 1.99 × 10−2). Conclusions: Several PXR/NR1I2 haplotypes contribute to CD susceptibility, suggesting a role for PXR in the IBD pathogenesis of a certain patient subcohort. Given the accumulating evidence for an important role of PXR in intestinal inflammation, further analyses are required to investigate the functional and pharmacogenetic implications of these PXR/NR1I2 gene variants in IBD. (Inflamm Bowel Dis 2010)

PLoS one 5(12), e14466 (2010). doi:10.1371/journal.pone.0014466, Published by PLoS [u.a.], Lawrence, Kan.

Background & Aims: Interleukin (IL)-17F is a novel proinflammatory cytokine which is a member of the IL-17 cytokine family. IL-17F mediates signal transduction via the heterodimeric IL-17RA/IL-17RC receptor complex. IL-17F is closely related to IL-17A. Both cytokines are secreted by Th17 cells which play a major role in the pathogenesis of Crohn's disease (CD) and other autoimmune diseases. Previous studies demonstrated that IL-17RA, the specific receptor for IL-17A, is expressed in intestinal epithelial cells (IECs). We therefore analyzed expression, signal transduction and potential biological functions of IL-17F in IECs. Methods: Expression studies were performed by RT-PCR, Western blot analysis, and immunohistochemical analysis. IL-17F mediated signaling was analyzed in Western blot experiments. IEC restitution was analyzed by wounding assays in IEC monolayer cultures. Cell proliferation was measured in MTS assays. The effect of IL-17F on apoptosis was analyzed by flow cytometry. Results: Using RT-PCR, we demonstrated expression of IL17RA and IL-17RC in several colorectal cancer (CRC)-derived IEC lines. High IL-17RC expression was found especially in DLD-1 cells which was also confirmed by Western blot analysis and immunohistochemistry. DLD-1 cells were therefore used in the following experiments. IL-17F mediated the activation of ERK-1/2 and p38 MAP kinases and of Akt but had no significant effect on the phosphorylation levels of SAPK/JNK. IL-17F mediated signals resulted in significantly increased cell proliferation (p=0.01) and an increase of IEC restitution in wounding assays (p

Background & Aims: Interleukin (IL)-17F is a novel proinflammatory cytokine which is a member of the IL-17 cytokine family. IL-17F mediates signal transduction via the heterodimeric IL-17RA/IL-17RC receptor complex. IL-17F is closely related to IL-17A. Both cytokines are secreted by Th17 cells which play a major role in the pathogenesis of Crohn's disease (CD) and other autoimmune diseases. Previous studies demonstrated that IL-17RA, the specific receptor for IL-17A, is expressed in intestinal epithelial cells (IECs). We therefore analyzed expression, signal transduction and potential biological functions of IL-17F in IECs. Methods: Expression studies were performed by RT-PCR, Western blot analysis, and immunohistochemical analysis. IL-17F mediated signaling was analyzed in Western blot experiments. IEC restitution was analyzed by wounding assays in IEC monolayer cultures. Cell proliferation was measured in MTS assays. The effect of IL-17F on apoptosis was analyzed by flow cytometry. Results: Using RT-PCR, we demonstrated expression of IL17RA and IL-17RC in several colorectal cancer (CRC)-derived IEC lines. High IL-17RC expression was found especially in DLD-1 cells which was also confirmed by Western blot analysis and immunohistochemistry. DLD-1 cells were therefore used in the following experiments. IL-17F mediated the activation of ERK-1/2 and p38 MAP kinases and of Akt but had no significant effect on the phosphorylation levels of SAPK/JNK. IL-17F mediated signals resulted in significantly increased cell proliferation (p=0.01) and an increase of IEC restitution in wounding assays (p

Background: Very recently, a sub-analysis of genome-wide association scans revealed that the non-coding single nucleotide polymorphism (SNP) rs12212067 in the FOXO3A gene is associated with a milder course of Crohn's disease (CD) (Cell 2013; 155: 57-69). The aim of our study was to evaluate the clinical value of the SNP rs12212067 in predicting the severity of CD by correlating CD patient genotype status with the most relevant complications of CD such as stenoses, fistulas, and CD-related surgery. Methodology/Principal Findings: We genotyped 550 CD patients for rs12212067 (FOXO3A) and the three common CD-associated NOD2 mutations rs2066844, rs2066847, and rs2066847 and performed genotype-phenotype analyses. Results: No significant phenotypic differences were found between the wild-type genotype TT of the FOXO3A SNP rs12212067 and the minor genotypes TG and GG independently from NOD2 variants. The allele frequency of the minor G allele was 12.7%. Age at diagnosis, disease duration, body mass index, surgery rate, stenoses, fistula, need for immunosuppressive therapy, and disease course were not significantly different. In contrast, the NOD2 mutant p. Leu1007fsX1008 (rs2066847) was highly associated with penetrating CD (p = 0.01), the development of fistulas (p = 0.01) and stenoses (p = 0.01), and ileal disease localization (p = 0.03). Importantly, the NOD2 SNP rs2066847 was a strong separator between an aggressive and a mild course of CD (p = 2.99 x 10(-5)), while the FOXO3A SNP rs12212067 did not separate between mild and aggressive CD behavior in our cohort (p = 0.35). 96.2% of the homozygous NOD2 p. Leu1007fsX1008 carriers had an aggressive disease behavior compared to 69.3% of the patients with the NOD2 wildtype genotype (p = 0.007). Conclusion/Significance: In clinical practice, the NOD2 variant p. Leu1007fsX1008 (rs2066847), in particular in homozygous form, is a much stronger marker for a severe clinical phenotype than the FOXO3A rs12212067 SNP for a mild disease course on an individual patient level despite its important impact on the inflammatory response of monocytes.

The expression of CCL20 (MIP-3alpha), which chemoattracts leukocytes to sites of inflammation, has been shown in intestinal epithelial cells (IEC). Aim of this study was to analyze the role of the CCL20 receptor CCR6 in IEC and colorectal cancer (CRC) cells. Expression of CCR6 and CCL20 was analyzed by RT-PCR and immunohistochemistry. Signaling was investigated by Western blotting, proliferation by MTS assays and chemotactic cell migration by wounding assays. The effect of CCL20 on Fas-induced apoptosis was determined by flow cytometry. CCR6 and its ligand CCL20 are expressed in IEC. Moreover, CRC and CRC metastases express CCR6, which is upregulated during IEC differentiation. Stimulation of IEC with CCL20 and proinflammatory stimuli (TNF-alpha, IL-1beta, LPS) significantly upregulates CCL20 mRNA expression. CCL20 expression was significantly increased in inflamed colonic lesions in Crohn's disease and correlated significantly with the IL-8 mRNA expression in these lesions (r = 0.71) but was downregulated in CRC metastases. CCL20 activated Akt, ERK-1/2, and SAPK/JNK MAP kinases and increased IL-8 protein expression. The CCL20 mediated activation of these pathways resulted in a 2.6-fold increase of cell migration (P = 0.001) and in a significant increase of cell proliferation (P < 0.05) but did not influence Fas-induced apoptosis. In conclusion, IEC and CRC express CCL20 and its receptor CCR6. CCL20 expression is increased in intestinal inflammation, while CCR6 is upregulated during cell differentiation. CCR6 mediated signals result in increased IEC migration and proliferation suggesting an important role in intestinal homeostasis and intestinal inflammation by mediating chemotaxis of IEC but also in mediating migration of CRC cells.

Recently, we have shown that SOCS-1/3 overexpression in hepatic cells abrogates signaling of type I interferons (IFN) which may contribute to the frequently observed IFN resistance of hepatitis C virus (HCV). IFN-lambdas (IL-28A/B and IL-29), a novel group of IFNs, also efficiently inhibit HCV replication in vitro with potentially less hematopoietic side effects than IFN-alpha because of limited receptor expression in hematopoietic cells. To further evaluate the potential of IFN-lambdas in chronic viral hepatitis, we examined the influence of SOCS protein expression on IFN-lambda signaling. First, we show that hepatic cell lines express the IFN-lambda receptor complex consisting of IFN-lambdaR1 (IL-28R1) and IL-10R2. Whereas in mock-transfected HepG2 cells, IL-28A and IL-29 induced STAT1 and STAT3 phosphorylation, overexpression of SOCS-1 completely abrogated IL-28A and IL-29-induced STAT1/3 phosphorylation. Similarly, IL-28A and IL-29 induced mRNA expression of the antiviral proteins 2',5'-OAS and MxA was abolished by overexpression of SOCS-1. In conclusion, we assume that despite antiviral properties of IFN-lambdas, their efficacy as antiviral agents may have similar limitations as IFN-alpha due to inhibition by SOCS proteins.

Colorectal cancer (CRC) is characterized by a distinct metastatic pattern resembling chemokine-induced leukocyte trafficking. This prompted us to investigate expression, signal transduction and specific functions of the chemokine receptor CXCR4 in CRC cells and metastases. Using RT-PCR analysis and Western blotting, we demonstrated CXCR4 and CXCL12 expression in CRC and CRC metastases. Cell differentiation increases CXCL12 mRNA levels. Moreover, CXCR4 and its ligand are inversely expressed in CRC cell lines with high CXCR4 and low or not detectable CXCL12 expression. CXCL12 activates ERK-1/2, SAPK/JNK kinases, Akt and matrix metalloproteinase-9. These CXCL12-induced signals mediate reorganization of the actin cytoskeleton resulting in increased cancer cell migration and invasion. Moreover, CXCL12 increases vascular endothelial growth factor (VEGF) expression and cell proliferation but has no effect on CRC apoptosis. Therefore, the CXCL12/CXCR4 system is an important mediator of invasion and metastasis of CXCR4 expressing CRC cells.

Symbiotic bacteria intimately participate in host physiological processes and impact host health and disease, although details at the molecular level largely remain unknown. We discovered that symbiotic Bacteroides species produce sphingolipids to modify the host antigen environment of the invariant natural killer T (iNKT) cells, a key immune modulator recognizing CD1d-restricted lipids. Using gnotobiotic technology and analytical tools, we found that bacterial glycosphingolipids, specifically one molecular fraction called GSL-Bf717, block iNKT cell activation and inhibit cell proliferation in early life. As a result, the total colonic iNKT cell number is restricted in adulthood and the host is protected when challenged by iNKT cell-mediated oxazolone colitis. These results suggest that intestinal bacterial sphingolipids are critical for establishing colonic iNKT cell homeostasis and host tolerance to environmental challenges. In addition, we find that the regulation depends on exposure to these symbiotic molecules in a critical early-life window, which, if missed, has irreversible impacts on host. This study contributes to our understanding of the essential role microbes play in host health and disease with precious molecular details that are largely lacking. The unique inhibitive power of GSL-Bf717 can be harnessed to provide therapeutic interventions in human autoimmune and allergic disorders where iNKT cell activation is detrimental.

PLoS one 8(11), e77773 (2013). doi:10.1371/journal.pone.0077773, Published by PLoS [u.a.], Lawrence, Kan.

Background & Aims Genome-wide association studies identified the autophagy gene IRGM to be strongly associated with Crohn's disease (CD) but its impact in ulcerative colitis (UC), its phenotypic effects and potential epistatic interactions with other IBD susceptibility genes are less clear which we therefore analyzed in this study. Methodology/Principal Findings Genomic DNA from 2060 individuals including 817 CD patients, 283 UC patients, and 961 healthy, unrelated controls (all of Caucasian origin) was analyzed for six IRGM single nucleotide polymorphisms (SNPs) (rs13371189, rs10065172 = p.Leu105Leu, rs4958847, rs1000113, rs11747270, rs931058). In all patients, a detailed genotype-phenotype analysis and testing for epistasis with the three major CD susceptibility genes NOD2, IL23R and ATG16L1 were performed. Our analysis revealed an association of the IRGM SNPs rs13371189 (p = 0.02, OR 1.31 [95% CI 1.05–1.65]), rs10065172 = p.Leu105Leu (p = 0.016, OR 1.33 [95% CI 1.06–1.66]) and rs1000113 (p = 0.047, OR 1.27 [95% CI 1.01–1.61]) with CD susceptibility. There was linkage disequilibrium between these three IRGM SNPs. In UC, several IRGM haplotypes were weakly associated with UC susceptibility (p

BACKGROUND: Genome-wide association studies identified PTPN2 (protein tyrosine phosphatase, non-receptor type 2) as susceptibility gene for inflammatory bowel diseases (IBD). However, the exact role of PTPN2 in Crohn's disease (CD) and ulcerative colitis (UC) and its phenotypic effect are unclear. We therefore performed a detailed genotype-phenotype and epistasis analysis of PTPN2 gene variants. METHODOLOGY/PRINCIPAL FINDINGS: Genomic DNA from 2131 individuals of Caucasian origin (905 patients with CD, 318 patients with UC, and 908 healthy, unrelated controls) was analyzed for two SNPs in the PTPN2 region (rs2542151, rs7234029) for which associations with IBD were found in previous studies in other cohorts. Our analysis revealed a significant association of PTPN2 SNP rs2542151 with both susceptibility to CD (p = 1.95×10⁻⁵; OR 1.49 [1.34-1.79]) and UC (p = 3.87×10⁻², OR 1.31 [1.02-1.68]). Moreover, PTPN2 SNP rs7234029 demonstrated a significant association with susceptibility to CD (p = 1.30×10⁻³; OR 1.35 [1.13-1.62]) and a trend towards association with UC (p = 7.53×10⁻²; OR 1.26 [0.98-1.62]). Genotype-phenotype analysis revealed an association of PTPN2 SNP rs7234029 with a stricturing disease phenotype (B2) in CD patients (p = 6.62×10⁻³). Epistasis analysis showed weak epistasis between the ATG16L1 SNP rs2241879 and PTPN2 SNP rs2542151 (p = 0.024) in CD and between ATG16L1 SNP rs4663396 and PTPN2 SNP rs7234029 (p = 4.68×10⁻³) in UC. There was no evidence of epistasis between PTPN2 and NOD2 and PTPN2 and IL23R. In silico analysis revealed that the SNP rs7234029 modulates potentially the binding sites of several transcription factors involved in inflammation including GATA-3, NF-κB, C/EBP, and E4BP4. CONCLUSIONS/SIGNIFICANCE: Our data confirm the association of PTPN2 variants with susceptibility to both CD and UC, suggesting a common disease pathomechanism for these diseases. Given recent evidence that PTPN2 regulates autophagosome formation in intestinal epithelial cells, the potential link between PTPN2 and ATG16L1 should be further investigated.

However, an inflammatory response can be induced not only by PAMPs, but also intracellular alarm signals arising from non-physiological cell death, tissue damage, or stress, the so called DAMPs. Necrotic cell death releases several endogenous pro-inflammatory DAMPs, including proteins, DNA, RNA, extracellular matrix components and lipid mediators, all of which contribute to the pathogenesis of injury and exacerbate an initial insult. Moreover, sterile inflammation secondary to ongoing tissue damage is thought to contribute to many chronic diseases. We hypothesize that DAMPs may also play a role in the intestinal inflammatory response. Methods: Necrotic cell (NC) supernatants were prepared from HT-29 cells and freshly isolated human IEC, suspended at 108 cells/ml and subjected to 5 freeze/thaw cycles. Cells were then centrifuged and supernatants collected. Human Intestinal Fibroblasts (HIF) were used as targets of DAMPderived signals by exposing them to NC supernatants for 24 h. IL-6 and IL-8 levels in the cultures were measured by ELISA. Blockade with specific antibodies to TLR2, TLR4, RAGE, IL-1RI, IL-1a and IL-1b was performed by incubating these with HIF or NC supernatants. Results: HIF secreted large amounts of IL-6 and IL-8 (up to 120,000 pg/ml) in response to NC supernatants from HT29 cells or freshly isolated IEC. Neutralization studies revealed that a functional IL-1RI, but not TLR2, TLR4 or RAGE, was critical for IL-6 and IL-8 production by HIF. Both IL-1RI agonists, IL-1a and IL-1b, could trigger IL-6 and IL-8 production but only IL-1a, and not IL-1b, was obligatory for the induction of this effect with NC. Control experiments excluded a role of histones, chromatin, mitochondria, uric acid, HMGB1, IL-33, caspase-1 and ER stress in the response to necrotic IEC. Conclusions: These results show the unexpected, highly selective and potent pro-inflammatory activity of IEC-derived IL-1a, an intracellular non-secreted cytokine that belongs to the “alarmin” group of DAMPs. Thus, IL-1a emerges as a potential major inducer of mucosal inflammation in all gastrointestinal diseases where IEC death occurs, such as IBD.

Background: To evaluate the demographics, resource use and cost determinants associated with hospitalization of Crohn’s disease patients. Methods: All patients hospitalized at our institution from 01.01.2010 to 31.12.2010 were analyzed. We extracted all inpatient records whose hospital discharge abstracts included ICD-10 code K50 consistent with the diagnosis of Crohn’s disease. Statistical analysis was performed using the chi-square test. Results: Of 2048 all hospital admissions 219 belonged to Crohn’s disease. There were 109 females and 110 males, mean patient age was 36.7 years. 38 (27.5%) patients were hospitalized more than once therefore the total number of patients hospitalized was 138. The mean length of stay was 4.9 days. There were 40 hospitalizations in order to administer anti-TNF drug; these were excluded from further analysis. The distribution of resource use was as follows: plain abdominal radiograph (6.1% of hospitalizations), small bowel pass through (8.4%), chest x-ray (31.3%), abdominal ultrasound (72.6%), CT (6.1%), CT enteroclysis (31.8%), MRI (2.8%), upper gastrointestinal endoscopy (56.4%), colonoscopy (54.7%). The drug use was as follows: sulfasalazine (33%), mesalamine (45.2%), azathioprine (15.6%), antibiotics (23%). 7 cases required total parenteral nutrition. The length of stay, resource and drug use were dependent on disease activity. 67% patients with CRP 10.0mg/dl were hospitalized for not more than 5 days. Patients in whom antibiotics were used accounted for 23% of cases and 39.7% of costs. Conclusions: This is the first time that the structure of inpatient treatment costs for CD has been demonstrated for a Polish hospital. Genetics

Background & Aims: Recent studies have shown that OLFM4 is a robust marker for intestinal epithelial stem cells. However, the precise distribution of OLFM4-expressing cells within the inflamed human intestinal mucosa has never been described. Also, the molecular mechanism regulating its expression within intestinal epithelial cells (IECs), with regard to the inflammatory environment, remains largely unknown. Our previous studies have shown that NotchHes1 pathway is constitutively activated in IECs of the inflamed intestinal mucosa, and function as a key pathway for lineage-specific gene expression. Thus, we planned the present study to identify the distribution of OLFM4-expressing IECs, and also to reveal the underlying molecular mechanism regulating expression of OLFM4, under such an inflammatory environment. Methods: Immunostaining using human intestinal tissues were performed to determine the distribution of OLFM4-expressing cells. The expression level of OLFM4 by IECs in response to various inflammatory cytokines was analyzed by RT-PCR or western blot. Also, tetracycline inducible over-expression of Notch1 intracellular domain (NICD1), or Hes1, was employed to examine the involvement of Notch-Hes1 pathway upon OLFM4 expression. Finally, a series of promoter assay was performed to analyze the transcriptional regulation of the human OLFM4 gene. Results: Immunostaining of normal small intestinal and colonic tissues clearly showed the distribution of OLFM4-positive IECs at the lowest part of the crypt, including the crypt base columnar cells. However, in inflamed intestinal tissues of inflammatory bowel disease patients, increased number of OLFM4-expressing IECs was observed, expanding its distribution to the upper part of the crypt. In LS174T cells, stimulation by TNF-α, but not by IL1-β and IFN-γ, significantly up-regulated the mRNA expression of OLFM4. Also, forced expression of both NICD1 and Hes1 significantly up-regulated mRNA and protein expression of OLFM4. Surprisingly, combination of NICD1 or Hes1 over-expression with TNF-α stimulation had synergistic effect upon up-regulation of OLFM4 mRNA expression, reaching up to 2500 fold increase in LS174T cells. Promoter assays using 5' flanking region of the human OLFM4 gene revealed that such a synergistic effect of TNF-α and Notch-Hes1 pathway is mediated through transcriptional regulation, depending on the proximal NF-κB binding site. Consistently, TNF-αmediated up-regulation of NF-κB-dependent transcriptional activity was significantly enhanced by both NICD1 and Hes1 over-expression in LS174T cells. Conclusion: In the inflamed human intestinal mucosa, TNF-α and Notch-Hes1 pathway synergistically up-regulate expression of OLFM4 in IECs. Such an up-regulation of a stem-cell specific genemight be required to promote the regenerative response of the inflamed intestinal environment.