Jan Söderman

Interleukin 34: a new modulator of human and experimental inflammatory bowel disease

IBD (inflammatory bowel disease), where CD (Crohns disease) and UC (ulcerative colitis) represent the two main forms, are chronic inflammatory conditions of the intestine. Macrophages play a central role in IBD pathogenesis and are regulated by major differentiation factors such as CSF-1 (colony-stimulating factor 1) in homoeostasis and inflammation. IL (interleukin)-34 has recently been discovered as a second ligand for CSF-1R (CSF-1 receptor). However, expression and involvement of IL-34 in IBD remain unknown. In the present paper, we investigated the expression of IL34, CSF1 and their shared receptor CSF1R in normal human ileum and colon, in inflamed and non-inflamed tissues of CD and UC patients, and in a mouse model of experimental colitis. We found distinct expression patterns of IL34 and CSF1 in ileum and colon, with higher IL34 in ileum and, in contrast, higher CSF1 in colon. Furthermore, IL34 and CSF1 expression was increased with inflammation in IBD patients and in experimental colitis. In humans, infiltrating cells of the lamina propria and intestinal epithelial cells expressed IL-34, and TNF-α (tumour necrosis factor α) regulated IL-34 expression in intestinal epithelial cells through the NF-κB (nuclear factor κB) pathway. These data demonstrate the expression pattern of IL-34 in ileum and colon and suggest IL-34 as a new modulator of inflammation in IBD.

The Role of Inosine-5'-Monophosphate Dehydrogenase in Thiopurine Metabolism in Patients With Inflammatory Bowel Disease.

Background: There is a large interindividual variability in thiopurine metabolism. High concentrations of methylthioinosine-5′-monophosphate (meTIMP) and low concentrations of 6-thioguanine nucleotides (6-TGNs) have been associated with a lower response rate and an increased risk of adverse events. In this study, the role of inosine-5′-monophosphate dehydrogenase (IMPDH) for differences in metabolite patterns of thiopurines was investigated. Methods: IMPDH activity and thiopurine metabolite concentrations were determined in patients with inflammatory bowel disease and a normal thiopurine methyltransferase (TPMT) phenotype and meTIMP/6-TGN concentration ratio > 20 (n = 26), in patients with a metabolite ratio ≤20 (n = 21), in a subgroup with a metabolite ratio <4 (n = 6), and in 10 patients with reduced TPMT activity. In vitro studies were conducted on human embryonic kidney cells (HEK293) with genetically engineered IMPDH and TPMT activities. Results: Patients with metabolite ratios >20 had lower IMPDH activity than those with ratios ≤20 (P < 0.001). Metabolite ratios >20 were only observed in patients with normal TPMT activity. Downregulation of IMPDH activity in HEK293 cells was associated with an increase in the concentration of meTIMP (fold change: 17 up to 93, P < 0.001) but, unexpectedly, also of 6-thioguanosine monophosphate (fold change: 2.6 up to 5.0, P < 0.001). Conclusions: These data question the general view of IMPDH as the rate-limiting enzyme in the phosphorylation of thiopurines. Investigations of other mechanisms are needed to more fully explain the various metabolite patterns and outcomes in patients under treatment.

Gene Expression Profiling of Duodenal Biopsies Discriminates Celiac Disease Mucosa From Normal Mucosa

Celiac disease (CD) is identified by histopathologic changes in the small intestine which normalize during a gluten-free diet. The histopathologic assessment of duodenal biopsies is usually routine but can be difficult. This study investigated gene expression profiling as a diagnostic tool. A total of 109 genes were selected to reflect alterations in crypt-villi architecture, inflammatory response, and intestinal permeability and were examined for differential expression in normal mucosa compared with CD mucosa in pediatric patients. Biopsies were classified using discriminant analysis of gene expression. Fifty genes were differentially expressed, of which eight (APOC3, CYP3A4, OCLN, MAD2L1, MKI67, CXCL11, IL17A, and CTLA4) discriminated normal mucosa from CD mucosa without classification errors using leave-one-out cross-validation (n = 39) and identified the degree of mucosal damage. Validation using an independent set of biopsies (n = 27) resulted in four discrepant cases. Biopsies from two of these cases showed a patchy distribution of lesions, indicating that discriminant analysis based on single biopsies failed to identify CD mucosa. In the other two cases, serology support class according to discriminant analysis and histologic specimens were judged suboptimal but assessable. Gene expression profiling shows promise as a diagnostic tool and for follow-up of CD, but further evaluation is needed.

Gene expression and thiopurine metabolite profiling in inflammatory bowel disease - novel clues to drug targets and disease mechanisms?

Background and Aims Thiopurines are effective to induce and maintain remission in inflammatory bowel disease (IBD). The methyl thioinosine monophosphate (meTIMP)/6-thioguanine nucleotide (6-TGN) concentration ratio has been associated with drug efficacy. Here we explored the molecular basis of differences in metabolite profiles and in relation to disease activity. Methods Transcriptional profiles in blood samples from an exploratory IBD-patient cohort (n = 21) with a normal thiopurine S-methyltransferase phenotype and meTIMP/6-TGN ratios >20, 10.0–14.0 and ≤4, respectively, were assessed by hybridization to microarrays. Results were further evaluated with RT qPCR in an expanded patient cohort (n = 54). Additionally, 30 purine/thiopurine related genes were analysed separately. Results Among 17 genes identified by microarray-screening, there were none with a known relationship to pathways of purines/thiopurines. For nine of them a correlation between expression level and the concentration of meTIMP, 6-TGN and/or the meTIMP/6-TGN ratio was confirmed in the expanded cohort. Nine of the purine/thiopurine related genes were identified in the expanded cohort to correlate with meTIMP, 6-TGN and/or the meTIMP/6-TGN ratio. However, only small differences in gene expression levels were noticed over the three different metabolite profiles. The expression levels of four genes identified by microarray screening (PLCB2, HVCN1, CTSS, and DEF8) and one purine/thiopurine related gene (NME6) correlated significantly with the clinical activity of Crohn’s disease. Additionally, 16 of the genes from the expanded patient cohort interacted in networks with candidate IBD susceptibility genes. Conclusions Seventeen of the 18 genes which correlated with thiopurine metabolite levels also correlated with disease activity or participated in networks with candidate IBD susceptibility genes involved in processes such as purine metabolism, cytokine signaling, and functioning of invariant natural killer T cells, T cells and B cells. Therefore, we conclude that the identified genes to a large extent are related to drug targets and disease mechanisms of IBD.

Analysis of single nucleotide polymorphisms in the region of CLDN2-MORC4 in relation to inflammatory bowel disease

AIM To investigate a possible genetic influence of claudin (CLDN)1, CLDN2 and CLDN4 in the etiology of inflammatory bowel disease. METHODS Allelic association between genetic regions of CLDN1, CLDN2 or CLDN4 and patients with inflammatory bowel disease, Crohns disease (CD) or ulcerative colitis were investigated using both a case-control study approach (one case randomly selected from each of 191 Swedish inflammatory bowel disease families and 333 controls) and a family-based study (463 non-Swedish European inflammatory bowel disease -families). A nonsynonymous coding single nucleotide polymorphism in MORC4, located on the same linkage block as CLDN2, was investigated for association, as were two novel CLDN2 single nucleotide polymorphism markers, identified by resequencing. RESULTS A single nucleotide polymorphism marker (rs12014762) located in the genetic region of CLDN2 was significantly associated to CD (case-control allelic OR = 1.98, 95%CI: 1.17-3.35, P = 0.007). MORC4 was present on the same linkage block as this CD marker. Using the case-control approach, a significant association (case control allelic OR = 1.61, 95%CI: 1.08-2.41, P = 0.018) was found between CD and a nonsynonymous coding single nucleotide polymorphism (rs6622126) in MORC4. The association between the CLDN2 marker and CD was not replicated in the family-based study. Ulcerative colitis was not associated to any of the single nucleotide polymorphism markers. CONCLUSION These findings suggest that a variant of the CLDN2-MORC4 region predisposes to CD in a Swedish population.

FNDC4 acts as an anti-inflammatory factor on macrophages and improves colitis in mice

FNDC4 is a secreted factor sharing high homology with the exercise-associated myokine irisin (FNDC5). Here we report that Fndc4 is robustly upregulated in several mouse models of inflammation as well as in human inflammatory conditions. Specifically, FNDC4 levels are increased locally at inflamed sites of the intestine of inflammatory bowel disease patients. Interestingly, administration of recombinant FNDC4 in the mouse model of induced colitis markedly reduces disease severity compared with mice injected with a control protein. Conversely, mice lacking Fndc4 develop more severe colitis. Analysis of binding of FNDC4 to different immune cell types reveals strong and specific binding to macrophages and monocytes. FNDC4 treatment of bone marrow-derived macrophages in vitro results in reduced phagocytosis, increased cell survival and reduced proinflammatory chemokine expression. Hence, treatment with FNDC4 results in a state of dampened macrophage activity, while enhancing their survival. Thus, we have characterized FNDC4 as a factor with direct therapeutic potential in inflammatory bowel disease and possibly other inflammatory diseases.

Single Nucleotide Polymorphisms in MORC4, CD14, and TLR4 Are Related to Outcome of Allogeneic Stem Cell Transplantation

BACKGROUND Non-HLA genes may contribute to the prognosis after hematopoietic stem cell transplantation. We investigated associations between single nucleotide polymorphisms in regions of MORC4, CD14, TLR4, NOD2, SLC22A4, SLC22A5, CARD8, NLRP3, and CLDN2 and the outcomes of patients undergoing allogeneic stem cell transplantation. MATERIAL AND METHODS Single nucleotide polymorphisms in selected regions were determined and analyzed for putative associations with overall mortality and acute graft-versus-host disease. Significant associations were further explored by logistic regression, controlling for additional variables. RESULTS A significant association was identified between overall mortality among recipients and a nonsynonymous coding variant of MORC4 (rs6622126) in the recipient genetic makeup (P=0.029). Since MORC4 is located on the X-chromosome, the results were also analyzed separately for males and females. The association between overall mortality for recipients and the risk allele (rs6622126; A) was confirmed for males with respect to genetic makeup of recipients (P=0.012), donor genetic makeup (P=0.004), and the combined allele composition of the donor and recipient (P=0.001). A significant association was also identified between overall mortality and the recipient risk allele of CD14 (rs2569190; P=0.031), TLR4 (rs4986790; P=0.043), and NOD2 (carriage of at least 1 mutant allele of rs2066844, rs2066845, or rs2066847; P=0.048). Among the investigated genes, only the CD14 (rs2569190) recipient risk allele was significantly associated with acute graft-versus-host disease (P=0.023). Logistic regression models confirmed these findings, except for NOD2, and also identified a significant contribution by age at stem cell transplantation (MORC4, CD14, TLR4), diagnosis (CD14, TLR4), and prophylaxis (MORC4). CONCLUSIONS Genetic variation in MORC4, CD14, and TLR4 may affect the outcome of allogeneic stem cell transplantation.

Potential blood-based markers of celiac disease

BackgroundBlood-based diagnostics has the potential to simplify the process of diagnosing celiac disease (CD). Although high levels of autoantibodies against tissue transglutaminase (anti-TG2) are strongly indicative of active CD, several other scenarios involve a need for additional blood-based CD markers.MethodsWe investigated the levels of messenger RNA (mRNA) in whole blood (n = 49) and protein in plasma (n = 22) from cases with active CD (n = 20), with confirmed CD and normalized histology (n = 15), and without a CD diagnosis (n = 14). Group differences were analyzed using Kruskal-Wallis one-way analysis of variance by ranks. We also investigated correlations between levels of potential markers, histopathology according to the modified Marsh scale, and CD risk gradient based on HLA type, using Spearman rank correlation. The relation between HLA-DQ2 gene dose effect and the expression levels of selected blood-based markers was investigated using the Mann–Whitney U test. Finally, the diagnostic performance of anti-TG2, potential blood-based CD markers, and logistic regression models of combined markers was evaluated using receiver operating characteristic (ROC) curve analysis.ResultsCXCL11 protein levels and TNFRSF9 and TNFSF13B mRNA levels were identified as potential CD markers. These are all affected by or involved in the regulation of the NF-κB complex. CXCL11 protein levels and IL21 and IL15 mRNA levels were correlated with histopathology according to the modified Marsh scale, as were the established CD markers. HLA genotype risk and HLA-DQ2 gene dose effect did not show any significant relations with either the potential CD markers or the established CD markers. ROC curve analysis revealed a slight, non-significant increase in the area under the curve for the combined use of anti-TG2 and different constellations of potential blood-based CD markers compared to anti-TG2 alone.ConclusionsThe CD markers identified in this study further emphasize the significance of components related to NF-κB regulation in relation to CD. However, the relevance of CXCL11, TNFSF13B, TNFRSF9, and other NF-κB interacting proteins recognized by pathway analysis, needs to be further investigated in relation to diagnosis and monitoring of CD.

Gene Expression-Genotype Analysis Implicates GSDMA, GSDMB, and LRRC3C as Contributors to Inflammatory Bowel Disease Susceptibility.

To investigate the biological foundation of the inflammatory bowel disease (IBD), ulcerative colitis and Crohns disease, susceptibility locus rs2872507, we have investigated the expression of 13 genes using ileal and colonic biopsies from patients with IBD (inflamed and noninflamed mucosa) or from individuals without IBD (noninflamed mucosa). The susceptibility allele was consistently associated with reduced expression of GSDMB (P = 4.1 × 10−3–7.2 × 10−10). The susceptibility allele was also associated with the increased expression of GSDMA (P = 1.6 × 10−4) and LRRC3C (P = 7.8 × 10−6) in colon tissue from individuals without IBD and with the reduced expression of PGAP3 (IBD; P = 2.0 × 10−3) and ZPBP2 (Crohns disease; P = 7.7 × 10−4) in noninflamed ileum. Inflammation resulted in the reduced colonic expression of ERBB2, GRB7, MIEN1, and PGAP3 (P = 1.0 × 10−4–1.0 × 10−9) and the increased colonic expression of IKZF3 and CSF3 (P = 2.4 × 10−7–3.5 × 10−8). Based on our results and published findings on GSDMA, GSDMB, LRRC3C, and related proteins, we propose that this locus in part affects IBD susceptibility via effects on apoptosis and cell proliferation and believe this hypothesis warrants further experimental investigation.

Genetic variation and expression levels of tight junction genes identifies association between MAGI3 and inflammatory bowel disease

BackgroundInflammatory bowel disease (IBD) is associated with increased intestinal permeability, which involves paracellular passage regulated through tight junctions (TJ). The aim of the study was to investigate single nucleotide polymorphisms (SNP) located in genes encoding interacting TJ proteins and corresponding expressions, in relation to IBD.MethodsAllelic associations between TJ-related genes (F11R, MAGI1, MAGI2, MAGI3, PARD3, PTEN, and TJP1) and IBD, Crohn’s disease (CD), or ulcerative colitis (UC) were investigated. PTPN22 was included since it’s located in the same genetic region as MAGI3. Gene expression levels were investigated in relation to genotype, inflammatory status, phenotype, and medical treatment.ResultsThe two strongest allelic associations were observed between IBD and SNPs in MAGI2 (rs6962966) and MAGI3 (rs1343126). Another MAGI3 SNP marker (rs6689879) contributed to increased ileal MAGI3 expression level in non-IBD controls. Furthermore, association between inflammation and decreased expression levels of MAGI3, PTEN, and TJP1 in colonic IBD as well as UC mucosa, and between inflammation and increased expression of PTPN22 in colonic IBD mucosa, was observed.ConclusionsOur findings lend support to a genetic basis for modulation of intestinal epithelial barrier in IBD, and we have identified MAGI3 as a new candidate gene for IBD.