Cytokine Receptor Profiling in Human Colonic Subepithelial Myofibroblasts: A Differential Effect of Th Polarization-Associated Cytokines in Intestinal Fibrosis.

Background: Colonic subepithelial myofibroblasts (cSEMFs) are mesenchymal cells with a pivotal role in the pathophysiology of Crohn's disease (CD) fibrosis. Here, we demonstrate for the first time a complete expression mapping of cytokine receptors, implicated in inflammatory bowel diseases, in primary human cSEMFs and how pro-inflammatory cytokines regulate this expression. Furthermore, we show the effect of Th1-, Th2-, Th17- and Treg-related cytokines on a fibrosis-related phenotype of cSEMFs. Methods: Colonic subepithelial myofibroblasts were isolated from healthy individuals' colonic biopsies. Interleukin (IL)-1α- and/or tumor necrosis factor (TNF)-α-induced mRNA and protein expression of cytokine receptors was assayed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence, respectively. Th-related cytokine effects on mRNA and protein profibrotic factor expression were analyzed by qRT-PCR and/or colorimetric assays and on the wound-healing capacity of cSEMFs by scratch test. Results: In cSEMFs, we observed basal cytokine receptor expression, which was modified by IL-1α and TNF-α. Th1-related cytokines upregulated tissue factor (TF), collagen, fibronectin and matrix metalloproteinase (MMP)-1 and downregulated α-smooth muscle actin (α-SMA), MMP-9, and wound healing rate. Th2-related cytokines upregulated collagen, TF, α-SMA, MMP-1, and wound healing rate and downregulated fibronectin and MMP-9. IL-17 and IL-23 upregulated fibronectin, and IL-22 downregulated TF. IL-17 and IL-22 decreased wound healing rate. Similar to TGF-ß, IL-23 upregulated MMP-1, tissue inhibitor of metalloproteinases-1, collagen expression, and wound healing rates. Conclusions: Our results suggest that cSEMFs have a central role in inflammation and fibrosis, as they express a great variety of Th-related cytokine receptors, making them responsive to pro-inflammatory cytokines, abundant in the inflamed mucosa of CD patients.

Crohn's disease-associated mucosal factors regulate the expression of TNF-like cytokine 1A and its receptors in primary subepithelial intestinal myofibroblasts and intestinal epithelial cells.

Intestinal subepithelial myofibroblasts (SEMFs) exert a profibrotic role in Crohn's disease (CD). Tumor necrosis factor-like cytokine 1A (TL1A) and its receptors, death-domain receptor 3 (DR3) and decoy receptor 3 (DcR3), are mucosal factors with significant involvement in experimental inflammation and CD. We aimed to determine the regulation of expression of this system of proteins in SEMFs and intestinal epithelial cells. The relative amount of mRNA transcripts for TL1A, DR3, and DcR3 was measured by real-time reverse transcription polymerase chain reaction in cultured primary SEMFs, colonic myofibroblast cell line 18CO, and epithelial cell line HT29. Protein expression was determined by immunofluorescence. The effect of various proinflammatory stimuli in mRNA and protein expression was studied. TL1A mRNA and protein expression in primary SEMFs (and 18CO cells) was significantly upregulated after stimulation with interleukin 1-alpha and/or tumor necrosis factor alpha (TNF-α) (32- to 44-fold increase, P < 0.05 vs unstimulated). Following stimulation with interleukin 1-alpha + TNF-α + IFN-γ, HT-29 cells highly expressed DR3 (4.1-fold over unstimulated, P = 0.008) and DcR3 (56-fold, P = 0.009) and secreted soluble factors that led to induction of TL1A mRNA in primary SEMFs (28-fold, P = 0.008). Activated epithelial cells significantly upregulated IL-8 expression in response to stimulation with recombinant TL1A. Supernatants from mucosal cultures of patients with CD were able to stimulate the expression of TL1A in cultured primary SEMFs, in comparison to supernatants from healthy controls (3.8-fold increase, P < 0.05) or culture media alone (P < 0.05). In conclusion, we found that proinflammatory cytokines are important regulators of the expression of TL1A in SEMFs and of its receptors in intestinal epithelial cells. Our results raise the possibility for involvement of TL1A/DR3/DR3-mediated mechanisms in epithelial-mesenchymal interactions and the development of inflammation-induced intestinal fibrosis in CD.

Anemia of chronic disease in rheumatoid arthritis is associated with increased apoptosis of bone marrow erythroid cells: improvement following anti-tumor necrosis factor-alpha antibody therapy.

Circumstantial evidence has implicated tumor necrosis factor alpha (TNF-alpha) in the pathogenesis of anemia of chronic disease (ACD) in rheumatoid arthritis (RA). We investigated the role of TNF-alpha in erythropoiesis of patients with active RA (n = 40) and the effect of anti-TNF-alpha antibody administration (cA2). Patients with RA had lower numbers of CD34+/CD71+ and CD36-/glycophorin A+ (glycoA+) bone marrow (BM) cells and increased proportions of apoptotic cells within the CD34+/CD71+ and CD36+/glycoA+ cell compartments, compared to healthy controls (n = 24). Erythroid burst-forming units (BFU-Es) obtained by BM mononuclear or purified CD34+ cells were significantly lower in RA patients compared to controls. These abnormalities were more pronounced among patients with ACD. Increased TNF-alpha levels in patient long-term BM culture supernatants inversely correlated with BFU-Es and hemoglobin levels and positively with the percentage of apoptotic CD34+/CD71+ and CD36+/glycoA+ cells. Following cA2 therapy, a normalization was documented in the number of CD34+/CD71+ and CD36-/glycoA+ cells, the number of BFU-Es, and the proportion of apoptotic CD34+/CD71+ and CD36+/glycoA+ cells, which was associated with a significant increase in hemoglobin levels compared to baseline. Recovery from anemia was more prominent in patients with ACD. The exogenous addition of an anti-TNF-alpha antibody in the cultures increased BFU-E number in patients prior to cA2 treatment but not after treatment, further substantiating the inhibitory role of TNF-alpha on patients' erythropoiesis. We conclude that TNF-alpha-mediated apoptotic depletion of BM erythroid cells may account for ACD in RA and that cA2 administration may ameliorate ACD in these patients by down-regulating the apoptotic mechanisms involved in erythropoiesis.