Inflammatory Response Of Fibroblasts Research Articles

P044 Ex vivo effects of infliximab on the long non-coding RNAs expression levels in Crohn′s disease

Abstract Background In recent years, evidence shows that long non-coding RNAs (lncRNAs) are key regulators of gene transcription and play important roles in the pathogenesis of inflammatory bowel diseases. LncRNA are involved in regulation of intestinal epithelial cell apoptosis, cell-cell interactions, enhancing inflammation, among others. Biological therapies, which are considered the most potent for disease control, only benefit one-third of patients. For this reason, a deeper understanding regarding the mechanisms by which biological drugs elicit their effect on intestinal mucosal is needed. Hence, we aimed to unravel the ex vivo modulator effect of infliximab on the lncRNAs expression in intestinal biopsies from patients with Crohn′s Disease (CD). Methods We performed an unbiased transcriptomic analysis of intestinal biopsies from the ileum and colon from 30 patients [active CD = 10, quiescent CD = 10, healthy controls (HC) = 10] to identify lncRNA differentially expressed in the setting of infliximab modulation (Figure 1). Endoscopic biopsies were cultured with or without infliximab, and the transcriptome was determined by stranded total-RNAseq (reagents from Illumina Inc.). We used different databases (Ensembl Biomart, RNAcentral and ToppGene) to search for non-annotated lncRNA information and data on the location (cellular component), biological process and molecular function of differentially expressed lncRNAs. Results Transcriptomic results revealed a widespread dysregulation of lncRNAs in ileum biopsies from patients with active CD, quiescent CD and HC compared to the colon at baseline and after infliximab culture (Figure 2). These differentially expressed lncRNAs were enriched in such pathways as proliferation, apoptosis, migration, inflammatory response of fibroblasts, response to wounding, posttranscriptional regulation of inflammatory genes and activation of the mitogen-activated protein kinase signaling pathways. Regarding the effect of infliximab according to intestinal location, presence of disease and activity, no significant lncRNAs were identified in the different study comparatives. Conclusion We have characterized the basal transcriptomic landscape of lncRNAs in patients with CD (active and quiescent) and HC both in ileum and left colon. However, we have not found differential lncRNA expression due to the effect of infliximab, suggesting that the location (ileum or colon) is more relevant when analyzing differences in lncRNA expression in the intestinal tissue.

LncRNA TM1-3P Regulates Proliferation, Apoptosis and Inflammation of Fibroblasts in Osteoarthritis through miR-144-3p/ONECUT2 Axis.

This study explores LncRNA TM1-3P effects on the proliferation, apoptosis, and inflammatory response of fibroblasts in osteoarthritis (OA) and its underlying mechanism. Bioinformatics was performed to analyze OA disease-related genes, miRNA profiles, and function. The targeted regulation of LncRNA TM1-3P and miR-144-3p, ONECUT2 and miR-144-3p were analyzed by dual luciferase reporter gene assay, RNA Binding Protein Immunoprecipitation (RIP), and RNA pull down. Histopathological morphology of the knee joint was observed by hematoxylin-eosin (HE) and Annona Red O/Fast Green. The expressions of mRNAs and proteins were detected by RT-qPCR, Western blot, and immunohistochemistry. Unpaired T test was used between groups, and the one-way analysis of variance of repeated measurement data was applied for multi-group comparison, following Tukey's post-test. ONECUT2 and Smurf2 genes were significantly elevated in the osteoarthritis group compared with the normal group (P < 0.001, P < 0.001). Expressions of ONECUT2 and LncRNA TM1-3P were increased, and expression of miR-144-3p was decreased in interleukin (IL)-1β-induced human fibroblast synovial cells (hFSCs) (mRNA: 1.06 ± 0.24 vs. 3.29 ± 0.73, proteins: 0.22 ± 0.03 vs. 0.46 ± 0.22, 1.23 ± 0.22 vs. 3.76 ± 0.73, 1.06 ± 0.25 vs. 0.37 ± 0.13, P < 0.01, P < 0.001, P < 0.01, P < 0.05). Overexpression of miR-144-3p down-regulated the ONECUT2 expression, reduced cell proliferation, promoted apoptosis in hFSCs induced by IL-1β (mRNA: 0.89 ± 0.14 vs. 0.15 ± 0.01, P < 0.05; proteins: 0.46 ± 0.01 vs. 0.23 ± 0.01, P < 0.001; CCK8: 1.88 ± 0.07 vs. 1.65 ± 0.07; P < 0.05; EDU: 55.82 ± 1.44 vs 40.57 ± 2.24, P < 0.05; apoptosis: 10.57 ± 0.79 vs 16.36 ± 0.35, P < 0.0001). Overexpression of LncRNA TM1-3P up-regulated the expression of ONECUT2, promoted cell proliferation, and inhibited apoptosis (mRNA: 0.9 ± 0.09 vs 1.94 ± 0.12, P < 0.05; proteins: 0.61 ± 0.05 vs 0.76 ± 0.03, P > 0.05; CCK8: 2.07 ± 0.05 vs 2.47 ± 0.06; P < 0.01; EDU: 52.67 ± 1.17 vs 60.06 ± 3.24, P < 0.05; apoptosis: 10.57 ± 0.79 vs 16.36 ± 0.35, P < 0.001), which were reversed by the overexpression of miR-144-3p treatment (mRNA: 1.82 ± 0.07 vs 0.31 ± 0.07, P < 0.0001; proteins: 0.74 ± 0.02 vs 0.35 ± 0.01, P < 0.01; CCK8: 2.41 ± 0.01 vs 1.67 ± 0.02; P < 0.0001; EDU: 66.85 ± 2.86 vs 44.68 ± 1.97, P < 0.0001; apoptosis: 7.19 ± 0.19 vs 13.36 ± 0.53, P < 0.0001). Silencing LncRNA TM1-3P attenuated the injury of knee joint tissue, down-regulated the expression of ONECUT2, Smurf2, IL-1β, IL-6, TNF-α, and improved the expression of Rap1 in rats (0.71 ± 0.04 vs 0.48 ± 0.02, 0.68 ± 0.06 vs 0.36 ± 0.02, 0.74 ± 0.03 vs 0.49 ± 0.04, 0.78 ± 0.01 vs 0.54 ± 0.03, 0.68 ± 0.02 vs 0.4 ± 0.04, 0.24 ± 0.01 vs 0.4 ± 0.03, P < 0.05, P < 0.05, P < 0.05, P < 0.01, P < 0.01, P < 0.05). LncRNA TM1-3P improved inflammation and damage of knee joints in OA rats through miR-144-3p/ONECUT2 axis, providing a new theoretical basis for gene therapy of OA.

Dysregulation of Transcription Factor Activity During Formation of Cancer-Associated Fibroblasts.

The reciprocal interactions between cancer cells and the quiescent fibroblasts leading to the activation of cancer-associated fibroblasts (CAFs) serve an important role in cancer progression. Here, we investigated the activation of transcription factors (TFs) in prostate fibroblasts (WPMY cell line) co-cultured with normal prostate or tumorous cells (RWPE1 and RWPE2 cell lines, respectively). After indirect co-cultures, we performed mRNA-seq and predicted TF activity using mRNA expression profiles with the Systems EPigenomics Inference of Regulatory Activity (SEPIRA) package and the GTEx and mRNA-seq data of 483 cultured fibroblasts. The initial differential expression analysis between time points and experimental conditions showed that co-culture with normal epithelial cells mainly promotes an inflammatory response in fibroblasts, whereas with the cancerous epithelial, it stimulates transformation by changing the expression of the genes associated with microfilaments. TF activity analysis revealed only one positively regulated TF in the RWPE1 co-culture alone, while we observed dysregulation of 45 TFs (7 decreased activity and 38 increased activity) uniquely in co-culture with RWPE2. Pathway analysis showed that these 45 dysregulated TFs in fibroblasts co-cultured with RWPE2 cells may be associated with the RUNX1 and PTEN pathways. Moreover, we showed that observed dysregulation could be associated with FER1L4 expression. We conclude that phenotypic changes in fibroblast responses to co-culturing with cancer epithelium result from orchestrated dysregulation of signaling pathways that favor their transformation and motility rather than proinflammatory status. This dysregulation can be observed both at the TF and transcriptome levels.

MTOR Senses Environmental Cues to Shape the Fibroblast-like Synoviocyte Response to Inflammation

SummaryAccumulating evidence suggests that metabolic master regulators, including mTOR, regulate adaptive and innate immune responses. Resident mesenchymal tissue components are increasingly recognized as key effector cells in inflammation. Whether mTOR also controls the inflammatory response in fibroblasts is insufficiently studied. Here, we show that TNF signaling co-opts the mTOR pathway to shift synovial fibroblast (FLS) inflammation toward an IFN response. mTOR pathway activation is associated with decreased NF-κB-mediated gene expression (e.g., PTGS2, IL-6, and IL-8) but increased STAT1-dependent gene expression (e.g., CXCL11 and TNFSF13B). We further demonstrate how metabolic inputs, such as amino acids, impinge on TNF-mTORC1 signaling to differentially regulate pro-inflammatory signaling circuits. Our results define a critical role for mTOR in the regulation of the pro-inflammatory response in FLSs and unfold its pathogenic involvement in TNF-driven diseases, such as rheumatoid arthritis (RA).

Regulation of the innate immune response by fibronectin: synergism between the III-1 and EDA domains.

Fibronectin is a critical component of the extracellular matrix and alterations to its structure will influence cellular behavior. Matrix fibronectin is subjected to both mechanical and biochemical regulation. The Type III domains of fibronectin can be unfolded in response to increased cellular contractility, included or excluded from the molecule by alternative splicing mechanisms, or released from the matrix by proteolysis. Using Inflammatory Cytokine microarrays we found that the alternatively spliced fibronectin Type III domain, FnEDA, and the partially unfolded III-1 domain, FnIII-1c, induced the expression of a multitude of pro-inflammatory cytokines in human dermal fibroblasts, most notably CXCL1-3, IL-8 and TNF-α. FnIII-1c, a peptide representing an unfolded intermediate structure of the first Type III domain has been shown to initiate the toll-like receptor-4 (TLR4)-NFκB-dependent release of cytokines from human dermal fibroblasts (You, et al., J. Biol. Chem., 2010). Here we demonstrate that FnIII-1c and the alternatively spliced FnEDA domain induce a TLR4 dependent activation of p38 MAP kinase and its downstream effector, MAPKAP Kinase-2 (MK-2), to regulate cytokine expression in fibroblasts. RT-qPCR analysis indicated that the p38-MK-2 pathway regulates IL-8 mRNA stability. Interestingly, addition of FnIII-1c and FnEDA synergistically enhanced TLR4-dependent IL-8 release. These data indicate that Fn contains two Type III domains which can activate TLR signaling to induce an inflammatory response in fibroblasts. Furthermore, our data identifies the NF-κB and p38/MK2 signaling pathways as transducers of signals initiated in response to structural changes in fibronectin.

Inflammatory response of fibroblasts stimulated by dental materials

for the push-out test in an Instron universal testing machine at a crosshead speed of 0.5mm/min. After the push-out test, the specimens were examined with an ISM-PM200S stereomicroscope (50×) for analysis of the failure modes (adhesive, cohesive and mixed). Data were analyzed by ANOVA and Tukey’s test (a=5%). Results: In the fluid filtration test, there was a statistically significant difference (p 0.05), which showed significantly higher bond strength than 1% NaOCl (0.12± 0.04MPa) (p<0.05). The coronal third presented the highest bond strength and was significantly different from the other thirds (p<0.05). There was a predominance of adhesive failures in all groups. Conclusion: It may be concluded that the 0.2% chitosan solution presented values similar to those of 17% EDTA solution for apical seal and bond strength of the filling material in endodontically treated teeth.

Potential of human γD‐crystallin for hair damage repair: insights into the mechanical properties and biocompatibility

The objective of this work was to develop a new strategy to physically 'repair' chemically damaged hair. Hence the human eye γD-crystallin, a protein from the superfamily characterized structurally by the Greek key motif, was studied. The human γD-crystallin was chosen based on the ability of proteins belonging to this superfamily to be involved in the coating of specific structures. Two crystallins were used on the study, the wild type (Protein Data Bank ID: 1HK0) and the mutant protein. The mutant form was intended to induce a strong and quick protein polymerization as well to have new possible points of anchorage to hair. The ability of both crystallins to bind to damaged hair and even penetrate into its cortex was checked by fluorescence microscopy, confocal microscopy and scanning electron microscopy. Furthermore the reinforcement of hair mechanical resistance, the potential cytotoxic/inflammatory effect of crystallins were studied in order to have a fully comprehension about the protein based formulation. Although the chemical over-bleaching treatment induced a decrease of 20% on the resistance of the hair, the crystallins which bind and penetrate the hair fibre were able to recover and even to improve its mechanical properties when compared to the virgin hair. Moreover none of the crystallins displayed a toxic effect in fibroblasts for all the range of tested concentrations upon 72 h of exposure. The active aggregation process of mutant crystallin induced an inflammatory response in fibroblasts in the first 24 h of contact, measured by the amount of released pro-inflammatory cytokine IL-6 to the medium. In contrast contact with wild type crystallin did not lead to significant inflammation. Outcome from protein formulation characterization supports the hypothesis that the γD-crystallin it is able to recover and improve the mechanical properties of chemical damaged hair. Therefore it can be considered as a very promising strengthening agent for the development of new restorative hair care products.

A role for specific collagen motifs during wound healing and inflammatory response of fibroblasts in the teleost fish gilthead seabream

Specific sites and sequences in collagen to which cells can attach, either directly or through protein intermediaries, were identified using Toolkits of 63-amino acid triple-helical peptides and specific shorter GXX′GEX″ motifs, which have different intrinsic affinity for integrins that mediate cell adhesion and migration. We have previously reported that collagen type I (COL-I) was able to prime in vitro the respiratory burst and induce a specific set of immune- and extracellular matrix-related molecules in phagocytes of the teleost fish gilthead seabream (Sparus aurata L.). It was also suggested that COL-I would provide an intermediate signal during the early inflammatory response in gilthead seabream. Since fibroblasts are highly involved in the initiation of wound repair and regeneration processes, in the present study SAF-1 cells (gilthead seabream fibroblasts) were used to identify the binding motifs in collagen by end-point and real-time cell adhesion assays using the collagen peptides and Toolkits. We identified the collagen motifs involved in the early magnesium-dependent adhesion of these cells. Furthermore, we found that peptides containing the GFOGER and GLOGEN motifs (where O is hydroxyproline) present high affinity for SAF-1 adhesion, expressed as both cell number and surface covering, while in cell suspensions, these motifs were also able to induce the expression of the genes encoding the proinflammatory molecules interleukin-1β and cyclooxygenase-2. These data suggest that specific collagen motifs are involved in the regulation of the inflammatory and healing responses of teleost fish.