Objective: Fibroblasts play a major role in the development of systemic sclerosis (SSc), and the occurrence of serum autoantibodies reacting with fibroblast plasma membrane antigens in SSc has been reported. This study was undertaken to investigate whether IgG from SSc sera that react with human fibroblasts modulates the fibroblasts' function.Methods: Sera from 69 patients with SSc (28 with limited cutaneous SSc [lcSSc] and 41 with diffuse cutaneous SSc [dcSSc]), 30 patients with sarcoidosis, and 50 matched healthy controls were examined. We evaluated antibody binding to human skin and lung fibroblasts by cell‐based enzyme‐linked immunosorbent assay (ELISA), indirect immunofluorescence, and flow cytometry. We further investigated the ability of purified IgG to modulate 1) intercellular adhesion molecule 1 (ICAM‐1) expression, 2) U937 cell adhesion to fibroblasts, and 3) fibroblast steady‐state messenger RNA (mRNA) levels of interleukin‐1α (IL‐1α), IL‐β, and IL‐6, and IL‐6 protein production.Results: Of 69 SSc sera tested by cell‐based ELISA, 58% bound to normal skin and lung fibroblasts. The prevalence of binding was significantly higher in dcSSc than in lcSSc (P < 0.05). Only IgG from SSc sera that were positive for antifibroblast antibody (AFA) induced a dose‐dependent up‐regulation of ICAM‐1 expression and IL‐6 production, enhancement of U937 cell adhesion, and increased levels of IL‐1α, IL‐1β, and IL‐6 mRNA in fibroblasts. Up‐regulation of ICAM‐1 mediated by AFA IgG was inhibited by the addition of IL‐1 receptor antagonist, indicating an autocrine activation loop.Conclusion: Our findings confirm the presence of AFAs in SSc sera and demonstrate, for the first time, that autoantibodies reacting with fibroblast surface molecules act as an extrinsic stimulus inducing fibroblast activation in vitro.

Rationale: Given the paucity of effective treatments for idiopathic pulmonary fibrosis (IPF), new insights into the deleterious mechanisms controlling lung fibroblast activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies. TGF-β (transforming growth factor-β) is the main profibrotic factor, but its inhibition is associated with severe side effects because of its pleiotropic role. Objectives: To determine if downstream noncoding effectors of TGF-β in fibroblasts may represent new effective therapeutic targets whose modulation may be well tolerated. Methods: We investigated the whole noncoding fraction of TGF-β–stimulated lung fibroblast transcriptome to identify new genomic determinants of lung fibroblast differentiation into myofibroblasts. Differential expression of the long noncoding RNA (lncRNA) DNM3OS (dynamin 3 opposite strand) and its associated microRNAs (miRNAs) was validated in a murine model of pulmonary fibrosis and in IPF tissue samples. Distinct and complementary antisense oligonucleotide–based strategies aiming at interfering with DNM3OS were used to elucidate the role of DNM3OS and its associated miRNAs in IPF pathogenesis. Measurements and Main Results: We identified DNM3OS as a fibroblast-specific critical downstream effector of TGF-β–induced lung myofibroblast activation. Mechanistically, DNM3OS regulates this process in trans by giving rise to three distinct profibrotic mature miRNAs (i.e., miR-199a-5p/3p and miR-214-3p), which influence SMAD and non-SMAD components of TGF-β signaling in a multifaceted way. In vivo, we showed that interfering with DNM3OS function not only prevents lung fibrosis but also improves established pulmonary fibrosis. Conclusions: Pharmacological approaches aiming at interfering with the lncRNA DNM3OS may represent new effective therapeutic strategies in IPF.

Objective: Previous studies have revealed the presence of IgG antifibroblast antibodies (AFAs) capable of binding to the surface of fibroblasts in systemic sclerosis (SSc) sera. Since chemokines may directly or indirectly affect the development of fibrosis, this study was undertaken to investigate the production of chemokines induced by AFAs in fibroblasts, and to characterize the signaling pathways and surface molecules involved.Methods: AFA‐positive and AFA‐negative IgG were tested on fibroblasts. Chemokine messenger RNA expression was screened by microarray and quantitative reverse transcription–polymerase chain reaction. Production of CCL2, CXCL8, and CXCL10 proteins was assessed by enzyme‐linked immunosorbent assay. Pharmacologic inhibitors were used to study signal transduction, with results assessed by Western blotting and immunofluorescence analysis. Fibroblasts with defective expression of Toll‐like receptors (TLRs) and anti‐TLR monoclonal antibodies (mAb) were used to assess AFA specificity.Results: In human fibroblasts, AFA‐positive IgG induced the preferential transcription of chemokines with profibrotic and proangiogenic potential, including, but not exclusively, CCL2, CXCL1, CXCL8, CKLF, and ECGF1, which were distinctly different from those induced by interferon‐γ. Levels of CCL2 and CXCL8 proteins were increased in AFA‐stimulated fibroblast culture supernatants. AFA binding to fibroblasts resulted in concomitant activation of ERK‐1/2, c‐Jun, and NF‐κB. CCL2 production was sensitive to inhibition of both proteasome and JNK, while CXCL8 production was sensitive only to inhibition of proteasome. AFAs failed to up‐regulate CCL2 expression in TLR‐4–deficient fibroblasts but not in TLR‐6– or TLR‐2–deficient fibroblasts. Moreover, anti–TLR‐4 mAb, but not anti–TLR‐2 mAb, partially inhibited the production of CCL2 induced by AFAs in human fibroblasts.Conclusion: Autoantibodies that bind to the surface of fibroblasts may contribute to the pathogenesis of SSc by up‐regulating the fibroblast production of profibrotic and proangiogenic chemokines, in a proteasome‐ and TLR‐4–dependent manner.

The mechanisms that regulate keratinocyte migration and proliferation in wound healing remain largely unraveled, notably regarding possible involvements of microRNAs (miRNAs). Here we disclose up‐regulation of miR‐483‐3p in 2 distinct models of wound healing: scratch‐injured cultures of human keratinocytes and wounded skin in mice. miR‐483‐3p accumulation peaks at the final stage of the wound closure process, consistent with a role in the arrest of “healing” progression. Using an in vitro wound‐healing model, videomicroscopy, and 5‐bromo‐2'‐uridine incorporation, we observed that overexpression of miR‐483‐3p inhibits keratinocyte migration and proliferation, whereas delivery of anti‐miR‐483‐3p oligonucleotides sustains keratinocyte proliferation beyond the closure of the wound, compared with irrelevant anti‐miR treatment. Expression profiling of keratinocytes transfected with miR‐483‐3p identified 39 transcripts that were both predicted targets of miR‐483‐3p and down‐regulated after miR‐483‐3p overexpression. Luciferase reporter assays, Western blot analyses, and silencing by specific siRNAs finally established that kinase MK2, cell proliferation marker MKI67, and transcription factor YAP1 are direct targets of miR‐483‐3p that control keratinocyte proliferation. miR‐483‐3p‐mediated down‐regulation of MK2, MKI67, and YAP1 thus represents a novel mechanism controlling keratinocyte growth arrest at the final steps of reepithelialization.—Bertero, T., Gastaldi, C., Bourget‐Ponzio, I., Imbert, V., Loubat, A., Selva, E., Busca, R., Mari, B., Hofman, P., Barbry, P., Meneguzzi, G., Ponzio, G., Rezzonico, R. miR‐483‐3p controls proliferation in wounded epithelial cells. FASEB J. 25, 3092‐3105 (2011). www.fasebj.org

Chemokines and adhesion molecules such as integrins play a major part in the trafficking, extravasation, and recruitment of leukocytes to inflammatory sites. This study investigated the effects of β2 integrin engagement on chemokine production by freshly isolated human monocytes. We found that ligation of CD11b or CD11c but not CD11a α chains of β2 integrins by antibodies or soluble CD23 (sCD23) fusion proteins rapidly induced transcription and secretion of interleukin 8, macrophage inflammatory protein (MIP) 1α, and MIP-1β. Because the promoters of these chemokine genes contain κB binding sites, we assessed the possible role of nuclear factor–κB (NF-κB) in controlling induction of the genes through β2 integrin engagement. Electrophoretic mobility shift assays showed that sCD23 or antibodies to CD11b or to CD11c up-regulated DNA-binding activity of NF-κB. Activation of NF-κB was accompanied by degradation of its cytosolic inhibitor IκB-α. Blockade of depletion of IκB-α by proteasome inhibitors (proteasome inhibitor I or acetyl-leucinyl-leucinyl-norleucinal) led to concomitant inhibition of NF-κB DNA-binding activity and expression of MIP-1α and MIP-1β messenger RNA induced by β2 integrin ligation. These results suggest that triggering of CD11b or CD11c β2 integrin on primary human monocytes provides activation signals leading to nuclear translocation of NF-κB and subsequent secretion of MIP-1α and MIP-1β that may have an important role in recruitment of other inflammatory cells during initiation of an inflammatory response.