Sezin Günaltay

Differential expression of interleukin-1/Toll-like receptor signaling regulators in microscopic and ulcerative colitis

AIM To investigate Toll-like receptor (TLR) signaling regulators in microscopic and ulcerative colitis patients. METHODS Total RNA and microRNA were isolated from fresh frozen colonic biopsies of non-inflamed controls and patients with active or in-remission collagenous colitis (CC), lymphocytic colitis (LC), or ulcerative colitis (UC). We compared expressions of interleukin-1 receptor-associated kinase (IRAK)-2, IRAK-M, interleukin (IL)-37, microRNA (miR)-146a, miR-155, and miR-21 using quantitative real time reverse transcription polymerase chain reaction. RESULTS IRAK-M expression was increased in LC patients with active disease in histopathological remission (LC-HR; P = 0.02) and UC patients (P = 0.01), but no differences in IRAK-2 expression were detected compared to controls. miR-146a, -155 and -21 expressions were increased in LC-HR (P = 0.04, 0.07, and 0.004) and UC (P = 0.02, 0.04 and 0.03) patients. miR-146a and miR-21 expressions were significantly enhanced in UC patients compared to UC remission (UC-R; P = 0.01 and 0.04). Likewise, active CC patients showed significantly increased expression of miR-155 (P = 0.003) and miR-21 (P = 0.006). IL-37 expression was decreased in both CC (P = 0.03) and LC (P = 0.04) patients with a similar trend in UC patients but not statistically significant, whilst it was increased in UC-R patients compared to controls (P = 0.02) and active UC (P = 0.001). CONCLUSION The identification of differentially expressed miRNAs, IL-37, and IRAK-M suggests different pathophysiologic mechanisms in various disease stages in LC, CC, and UC.

Enhanced Levels of Chemokines and Their Receptors in the Colon of Microscopic Colitis Patients Indicate Mixed Immune Cell Recruitment

Microscopic colitis (MC), comprising collagenous colitis (CC) and lymphocytic colitis (LC), is a common cause of chronic diarrhea. Various immune cell infiltrations in the epithelium and lamina propria are seen in MC immunopathology. We compared gene and protein expressions of different immune cell attracting chemokines and their receptors in colon biopsies from MC patients in active disease or histopathological remission (CC/LC-HR) with controls, using qRT-PCR and Luminex, respectively. CC and LC patients with active disease demonstrated a mixed chemokine profile with significantly enhanced gene and/or protein expressions of the chemokines CCL2, CCL3, CCL4, CCL5, CCL7, CCL22, CXCL8, CXCL9, CXCL10, CXCL11, and CX3CL1 and the receptors CCR2, CCR3, CCR4, CXCR1, CXCR2, and CX3CR1. Enhanced chemokine/chemokine receptor gene and protein levels in LC-HR patients were similar to LC patients, whereas CC-HR patients demonstrated almost normalized levels. These findings expand the current understanding of the involvement of various immune cells in MC immunopathology and endorse chemokines as potential diagnostic markers as well as therapeutic candidates. Moreover, this study further supports the hypothesis that CC and LC are two different entities due to differences in their immunoregulatory responses.

Clinical and immunologic effects of faecal microbiota transplantation in a patient with collagenous colitis

One to six percent of patients with microscopic colitis are refractory to medical treatment. The effect of faecal microbiota transplantation (FMT) in active collagenous colitis (CC) has, to the best of our knowledge, never been reported before. Here, we report the effect of repeated FMT in a patient with CC. The patient presented with severe symptoms including profuse diarrhea and profound weight loss. Although she responded to budesonide in the beginning, she became gradually refractory to medical treatment, and was therefore treated with FMT. The patient remained in remission for 11 mo after the third faecal transplantation. The immunomodulatory effect of the therapy was evaluated using flow cytometry, which showed alterations in the profile of intraepithelial and lamina propria lymphocyte subsets after the second transplantation. Our observations indicate that FMT can have an effect in CC, which support the hypothesis that luminal factors, influencing the intestinal microbiota, are involved in the pathogenesis of CC.

Reduced IL-37 Production Increases Spontaneous Chemokine Expressions in Colon Epithelial Cells

Background and AimMicroscopic colitis, comprising collagenous colitis and lymphocytic colitis, is a common cause of chronic diarrhea. Previously, we showed enhanced chemokine productions in microscopic colitis patients, indicating dysregulated immune cell chemotaxis in the immunopathogenesis. We also showed decreased mRNA of IL-37, mainly regarded as an anti-inflammatory cytokine, in the colonic mucosa of these patients, potentially an important factor for the chronicity of the colitis. Our aim in this study was to understand the possible role of IL-37 in chemokine production using a cell line model.MethodsA colon epithelial cell line, T84, was stimulated with the TLR5 ligand flagellin. IL-37 protein production was reduced 20% using the CRISPR/Cas9 system, and the changes in chemokine mRNA and protein expressions were compared to cells transfected with empty plasmid.ResultsThe 20% reduction in IL-37 protein levels spontaneously increased CCL5, CXCL8, CXCL10, and CXCL11 mRNA and protein expressions. CCL2 mRNA and protein levels were enhanced upon TLR5 stimulation. CCL3, CCL20, and CX3CL1 mRNA expressions were increased either spontaneously or following TLR5 stimulation, whereas CCL4 and CCL22 mRNA expressions were significantly decreased.ConclusionsEven a minor decrease in the ability of colon epithelial cells to produce IL-37 results in altered chemokine expression, mainly an increase in the production of several chemokines. Our results indicate that a decreased IL-37 expression by colon epithelial cells may be an important factor for increasing the recruitment of immune cells and subsequently developing microscopic colitis.

PKC, ERK/p38 MAP kinases and NF-κB targeted signalling play a role in the expression and release of IL-1β and CXCL8 in Porphyromonas gingivalis-infected THP1 cells.

Porphyromonas gingivalis is a keystone pathogen in periodontitis and is gaining importance in cardiovascular pathogenesis. Protease‐activated receptors (PARs), toll‐like receptors (TLRs) and nucleotide‐binding oligomerization domain (NOD) on monocytes recognize the structural components on P. gingivalis, inducing inflammatory intermediates. Here, we elucidate the modulation of PARs, TLRs, NODs, and the role of MAPK and NF‐κB in IL‐1β and CXCL8 release. THP1 cells were stimulated with P. gingivalis wild‐type W50 and its isogenic gingipain mutants: Rgp mutant E8 and Kgp mutant K1A. We observed modulation of PARs, TLRs, NOD, IL‐1β and CXCL8 expression by P. gingivalis. Gingipains hydrolyse IL‐1β and CXCL8, which is more evident for IL‐1β accumulation at 24 h. Inhibition of PKC (protein kinase C), p38 and ERK (extracellular signal‐regulated kinases) partially reduced P. gingivalis‐induced IL‐1β at 6 h, whereas PKC and ERK reduced CXCL8 at both 6 and 24 h. Following NF‐κB inhibition, P. gingivalis‐induced IL‐1β and CXCL8 were completely suppressed to basal levels. Overall, TLRs, PARs and NOD possibly act in synergy with PKC, MAPK ERK/p38 and NF‐κB in P. gingivalis‐induced IL‐1β and CXCL8 release from THP1 cells. These pro‐inflammatory cytokines could affect leucocytes in circulation and exacerbate other vascular inflammatory conditions such as atherosclerosis.

Oligoclonal T-cell Receptor Repertoire in Colonic Biopsies of Patients with Microscopic Colitis and Ulcerative Colitis

Background: Microscopic colitis (MC), comprising collagenous colitis (CC) and lymphocytic colitis (LC), is a type of variation of inflammatory bowel diseases. Local T-cell infiltration in the mucosa plays a major role in MC immunopathology. Methods: To understand diversity and clonality of infiltrating T cells, we analyzed the T-cell receptor beta (TCR&bgr;) chains in colonic biopsies of MC, ulcerative colitis (UC), and their remission counterparts (CC/LC-HR [histological remission] or UC-R [remission]) compared with patients with noninflamed colons using next-generation sequencing. Results: Compared with controls and patients with CC, patients with LC had significantly lower diversity with significantly lower evenness and richness in TCRV&bgr;-J&bgr; gene segments. Similarly, patients with LC-HR had lower diversity because of significantly lower TCRV&bgr;-J&bgr; clone richness. Patients with UC and UC-R showed significantly higher diversity and richness. Univariate and multivariate analyses were performed to identify TCRV&bgr;-J&bgr; gene segments differentiating disease types from controls or their remission counterparts. Patients with LC were discriminated from controls by 12 clones and from patients with CC by 8 clones. Neither univariate nor multivariate analyses showed significance for patients with CC or CC-HR compared with controls. Patients with UC and UC-R had 16 and 14 discriminating clones, respectively, compared with controls. Conclusions: Altogether, patients with MC and UC showed an oligoclonal TCR&bgr; distribution. TCRV&bgr;-J&bgr; clone types and their diversity were distinctive between patients with CC and LC, as well as for patients with UC, suggesting different pathophysiological mechanisms according to disease type and stage. This study suggests that CC and LC are different entities because of differences in immunoregulatory responses, as mirrored by their T-cell repertoire.