The Study On Autoimmune Features Of Keloid And Polypyrimidine Tract Binding Protein As A Molecular Therapy Target

Background:The pathogenesis of keloid remains unclear, its high recurrence rate despite a variety of treatments makes keloid one of the major unsolved clinical challenges in wound healing. The pathology of keloid is characterized by excessive extracellular matrix deposition and a great number of infiltrated immune cells, so keloid could been regarded as a fibrotic skin disease. Meanwhile, in autoimmune diseases, triggering of the inflammation response can lead to tissue injury and subsequent organ fibrosis. When the skin is involved in autoimmune disease, skin fibrosis such as that seen in scleroderma can occur. In this study, we propose that keloids possess features of autoimmune disease and verified this hypothesis.Keloids behave like tumors as they grow beyond the boundaries of the original wound margin, invade the surrounding normal tissue, do not regress spontaneously, have a rich vascular supply and recur despite treatments. Polypyrimidine tract binding protein(PTB) is a57-kDa RNA binding protein and express in nucleus of various cells. Recently, a great number of studies have found PTB increased in a few of tumors and PTB can promote tumor cells proliferation and metastasis. Therefore, PTB was regarded as a therapeutic target for tumor. We think PTB is also a therapeutic target for keloid.Objectives:1. To verify whether keloid is a fibrotic disease mediated by autoimmune response, and attempt to find a laboratory indication for the diagnosis of keloid.2. To explore the role of PTB in keloid and confirm whether PTB be a potential molecular therapeutic target for keloid.Methods and Results:1. Study on keloid patients have some features of autoimmune diseaseMethods:Routine anti-nuclear antibody profile was detected in sera from28keloid patients and28healthy controls, using indirect immunofluorescence and line immunoassay. The anti-hnRNPA2B1autoantibody and hnRNPA2B1protein concentration in sera were evaluated by ELISA, while western bloting was used to examine hnRNPA2B1protein expression in keloid and normal tissues. Immune complex deposits in keloid and normal skin were evaluated by direct immunofluorescence. Lastly, immune cell infiltration in keloid tissue was detected with immunohistochemical staining.Results:A very small number of patients or healthy controls were positive for ANA or a specific ANA, which did not reach statistical significance. Anti-hnRNPA2Bl levels in sera from keloid patients and hnRNPA2B1protein in keloid tissues were elevated, while IgA, IgM, C3and Clq deposits were found in keloid tissues but not normal skin. The number of Langerhans cells, macrophages, B lymphocytes and T lymphocytes were increased in keloids compared to normal skin.2. Study on polypyrimidine tract binding protein(PTB) as a therapeutic target for keloid2.1The expression and function of PTB in keloid tissue and fibroblastMethods:The histology of keloid and normal skin was observed using HE staining and sirius red staining. Immunohistochemistry for Ki-67and PCNA was performed to determine cell proliferation, while immunohistochemistry for PTB was also done. The mRNA of PTB and some genes coding extracellular matrix was detected by real time PCR. Fibroblasts isolated from keloid and normal skin were cultured in normal culture condition and PTB expression as well as extracellular matrix synthesis were valuated. PTB was knockdown using siRNA, cell proliferation and extracelluar matrix synthesis were determined following PTB knockdown.Results:Histologically, there were excessive extracellular matrix and infiltrated cells in keloid and different tissue layers presented different histological features. In keloid tissue, the number of Ki-67and PCNA positive cells was increased and the expression of COL1A1, COL3A1and FN1was upregulated, which were accompanied by PTB increasing. In fibroblasts from keloid tissues, the expression of PTB, FN1and ACTA2were upregulated, but not COL1A1and COL3A1. In normal culture condition, PTB knockdown suppressed cell proliferation and changed the alternative splicing of some gene related to cell proliferation, however, extracellular matrix expression was not influenced.2.2The influence of PTB on TGF-β1promoting the expression of genes coding extracellular matrixMethods:To simulate keloid microenvironment, fibroblasts were cultured in medium containing a different concentration of TGF-β1(Ong/ml,5ng/ml,10ng/ml,20ng/ml). At0,2,6,12,24,48h following TGF-β1treatment, the expression of PTB and extracellular matrix was determined. In the condition of TGF-β1, PTB was also knockdown and extracellular matrix was detected following PTB knockdown. Results:In fibroblasts from keloid tissues, TGF-β1promoted PTB, COL1A1, FN1and ACTA2expression, as well as the function of TGF-β1was concentration and time dependent. In fibroblasts from normal skin, TGF-β1promoted PTB, COL1A1, FN1and ACTA2expression, however which was not obvious as that in fibroblasts from keloid tissues. In TGF-β1culture condition, PTB knockdown suppressed FN1expression.2.3The influence of PTB knockdown on transplanted keloidMethods:To confirm whether PTB is a potential therapeutic target for keloid, we transplanted keloid tissues into nude mice to establish an animal model for keloid. PTB siRNA and control siRNA were injected into transplants, twice a week, for four weeks. After four weeks, transplants were harvested and weighed. The histology of transplants was observed, while PTB and extracelluar matrix expression were valuated.Results:The weight of transplants from PTB siRNA group was less than control siRNA group. Histologically, there was less cells and III type collagen fibres in transplants from PTB siRNA group. Real time PCR results show PTB, COL3A1and FN1expression were downregulated in PTB siRNA group.Conclusion:1. The anti-hnRNPA2Bl titer was firstly found elevated in sera from keloid patients, with immune complex deposits and CD20+B lymphocyte based on immune cell infiltration detected in keloid tissues. These findings suggest that keloid is a fibrotic skin disease mediated by autoimmune responses.2. PTB increased in tissues and fibroblasts from keloid. PTB knockdown suppressed fibroblasts proliferation in normal culture condition and downregulated FN1expression in TGF-β1culture condition. In keloid transplanted nude mice model, PTB knockdown suppressed COL3A1and FN1expression and accelerated transplants regression. These results suggest PTB may involve in keloid development and been a potential therapeutic target for keloids.