The role of cytoskeletal protein flightless I (FLII) in diabetic wound healing.

Skin lesions and ulcerations are common and severe complications of diabetes. A significant proportion of these wounds fail to respond to conventional treatment, hence amputation is a feared outcome of diabetes. Overexpression of Flightless (Flii) inhibits wound healing and ablation of Flii using specific neutralising monoclonal antibodies (FnAb) enhances cellular proliferation and migration. It was therefore hypothesized that decreasing Flii expression in diabetic wounds would create a permissive environment for cellular proliferation, enhanced neovascularization, and improved healing outcomes. The aim of this study was to determine whether genetic Flii gene knockdown or treatment with FnAb were effective in improving diabetic wound repair. A mouse model of diabetes was used in which type 1 diabetes was induced using streptozotocin. Diabetes was subsequently induced in low (Flii⁺ʹ⁻), normal (WT) and high (FliiTg/Tg) [Tg/Tg in superscript] mice. Full-thickness dorsal wounds were created and it was found that these wounds healed more rapidly when Flii gene expression was decreased. Further studies revealed that this improved healing was accompanied by a robust pro-angiogenic response with significantly elevated von Willebrand factor and VEGF positive endothelial cell infiltration. In a separate study, wounds in WT diabetic mice were injected intradermally with FnAb and here too improved healing was observed with significantly increased rate of re-epithelialisation compared with placebo control. We investigated the angiogenic response of FnAb both in vitro and in vivo. FnAb enhanced capillary tube formation in human umbilical vein endothelial cells (HUVEC) and promoted formation of functional neovasculature in vivo. Mice with reduced Flii also showed increased numbers of mature blood vessels using an in vivo Matrigel plug assay with increased recruitment of α-SMA positive cells and improved tight junction aiding cell to cell attachments. In conclusion, reducing Flii levels in wounds either genetically or using neutralising antibodies promotes wound healing in diabetic mice by enhancing epithelialisation and improving angiogenic processes. Manipulating Flightless I may therefore be a potential approach for therapeutic intervention in the treatment of the diabetic foot.Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 201

Lysosomal secretion of Flightless I upon injury has the potential to alter inflammation

Intracellular Flightless I (Flii), a gelsolin family member, has been found to have roles modulating actin regulation, transcriptional regulation and inflammation. In vivo Flii can regulate wound healing responses. We have recently shown that a pool of Flii is secreted by fibroblasts and macrophages, cells typically found in wounds, and its secretion can be upregulated upon wounding. We show that secreted Flii can bind to the bacterial cell wall component lipopolysaccharide and has the potential to regulate inflammation. We now show that secreted Flii is present in both acute and chronic wound fluid

Combined effect of genetic background and gender in a mouse model of bleomycin-induced skin fibrosis

International audienceSystemic sclerosis (SSc) is a connective tissue disorder characterised by the development of skin fibrosis. Our current understanding of the disease pathogenesis is incomplete and the study of SSc is hindered, at least partially, by a lack of animal models that fully replicate the complex state of human disease. Murine model of bleomycin-induced dermal fibrosis encapsulates important events that take place early in the disease course.METHODS:To characterise the optimum in vivo parameters required for the successful induction of dermal fibrosis we subjected three commonly used mouse strains to repeated subcutaneous bleomycin injections. We aimed to identify the effects of genetic background and gender on the severity of skin fibrosis. We used male and female Balb/C, C57BL/6, and DBA/2 strains and assessed their susceptibility to bleomycin-induced fibrosis by measuring dermal thickness, hydroxyproline/collagen content and number of resident myofibroblasts, all of which are important indicators of the severity of skin fibrosis. All data are expressed as mean values ± SEM. The Mann-Whitney U test was used for statistical analysis with GraphPad Prism 6.04 software.RESULTS:Dermal fibrosis was most severe in Balb/C mice compared to C57BL/6 and DBA/2 suggesting that Balb/C mice are more susceptible to bleomycin-induced fibrosis. Analysis of the effect of gender on the severity of fibrosis showed that male Balb/C, C57BL/6, DBA/2 mice had a tendency to develop more pronounced fibrosis phenotype than female mice. Of potential importance, male Balb/C mice developed the most severe fibrosis phenotype compared to male C57BL/6 and male DBA/2 as indicated by significantly increased number of dermal myofibroblasts.CONCLUSION:Our study highlights the importance of genetic background and gender in the induction of murine dermal fibrosis. Robust and reproducible animal models of fibrosis are important research tools used in pharmacological studies which may lead to better understanding of the pathogenesis of fibrotic diseases and assist in identification of new drugs

The nuclear receptor constitutive androstane receptor/NR1I3 enhances the profibrotic effects of transforming growth factor β and contributes to the development of experimental dermal fibrosis

OBJECTIVE Nuclear receptors regulate cell growth, differentiation, and homeostasis. Selective nuclear receptors promote fibroblast activation, which leads to tissue fibrosis, the hallmark of systemic sclerosis (SSc). This study was undertaken to investigate the effects of constitutive androstane receptor (CAR)/NR1I3, an orphan nuclear receptor, on fibroblast activation and experimental dermal fibrosis. METHODS CAR expression was quantified by quantitative polymerase chain reaction, Western blotting, immunohistochemistry, and immunofluorescence. CAR expression was modulated by small molecules, small interfering RNA, forced overexpression, and site-directed mutagenesis. The effects of CAR activation were analyzed in cultured fibroblasts, in bleomycin-induced dermal fibrosis, and in mice overexpressing a constitutively active transforming growth factor β (TGFβ) receptor type I (TβRI-CA). RESULTS Up-regulation of CAR was detected in the skin and in dermal fibroblasts in SSc patients. Stimulation of healthy fibroblasts with TGFβ induced the expression of CAR messenger RNA and protein in a Smad-dependent manner. Pharmacologic activation or overexpression of CAR in healthy fibroblasts significantly increased the stimulatory effects of TGFβ on collagen synthesis and myofibroblast differentiation, and amplified the stimulatory effects of TGFβ on COL1A2 transcription activity. Treatment with CAR agonist increased the activation of canonical TGFβ signaling in murine models of SSc and exacerbated bleomycin-induced and TβRI-CA-induced fibrosis with increased dermal thickening, myofibroblast counts, and collagen accumulation. CONCLUSION Our findings indicate that CAR is up-regulated in SSc and regulates TGFβ signaling. Activation of CAR increases the profibrotic effects of TGFβ in cultured fibroblasts and in different preclinical models of SSc. Thus, inactivation of CAR might be a novel approach to target aberrant TGFβ signaling in SSc and in other fibrotic diseases

Identification of NF-κB and PLCL2 as new susceptibility genes and highlights on a potential role of IRF8 through interferon signature modulation in systemic sclerosis

International audienceIdentification of NF-κB and PLCL2 as new susceptibility genes and highlights on a potential role of IRF8 through interferon signature modulation in systemic sclerosis Abstract Introduction: Systemic sclerosis (SSc) and primary biliary cirrhosis (PBC) are rare polygenic autoimmune diseases (AIDs) characterized by fibroblast dysfunction. Furthermore, both diseases share some genetic bases with other AIDs, as evidenced by autoimmune gene pleiotropism. The present study was undertaken to investigate whether single-nucleotide polymorphisms (SNPs) identified by a large genome-wide association study (GWAS) in PBC might contribute to SSc susceptibility. Methods: Sixteen PBC susceptibility SNPs were genotyped in a total of 1,616 patients with SSc and 3,621 healthy controls from two European populations (France and Italy). Results: We observed an association between PLCL2 rs1372072 (odds ratio (OR) = 1.22, 95% confidence interval (CI) 1.12 to 1.33, P adj = 7.22 × 1

IRF8 Gene Contributes to Disease Susceptibility and Interacts with NF-KB By Modulating Interferon Signature in Patients with Systemic Sclerosis

Background/Purpose: Systemic Sclerosis (SSc) is a polygenic autoimmune disease (AID) characterized by fibroblast dysregulation. It shares some genetic bases with other AIDs, as evidenced by autoimmune gene pleiotropism. Fibroblast dysregulation can be also observed in Primary Biliary Cirrhosis (PBC), another polygenic AID, which can be associated with SSc in the so called Reynold’s Syndrome. The present study was undertaken to investigate whether single nucleotide polymorphisms (SNPs) identified by a large GWAS in PBC might contribute to SSc susceptibility by a cross-disease approach. Methods: Sixteen PBC susceptibility SNPs were genotyped in a total of 1,616 SSc patients and 3,621 healthy controls all of whom were of European Caucasian origin. Results: We observed an association between PLCL2 rs1372072 (OR 1.23 [95% CI 1.12–1.33] Padj 7.2210 5 , NF-kB rs7665090 OR 1.16 [95% CI 1.06–1.25], Padj 0.01, and IRF8 rs11117432, OR 0.75 [95% CI 0.67–0.86], Padj 2.5010 4 with SSc susceptibility. We subsequently queried associations according to the main subtypes and found that S496 Monday, November 17 rs1372072 and rs11117432 were associated with the limited cutaneous subgroup (Padj 0.001 and Padj 0.003, respectively) and that rs7665090 was conversely associated with the diffuse cutaneous subset (Padj 0.007). We then looked for genotype – phenotype correlations by measuring mRNA expression of PBMC, obtained from patients (n 39) and controls (n 24), and observed that the IRF8 protective allele was associated with decreased IFIT1 expression reflecting type 1 interferon signature. We investigated gene interactions between the 3 associated SNPs that revealed an epistatic interaction between NF-kB and IRF8 SNPs (OR 0.56 [95% CI 0.00–0.74], P 410 4 ). Interestingly, we observed that the effects of IRF8 and NF-kB were only observed in patients carrying the susceptibility allele from both genes. Conclusion. By a cross disease approach querying pleiotropic genes, we identified 2 new susceptibility genes for SSc and confirmed IRF8 locus. We also identified functional effects of IRF8 variant affecting interferon signature and that an interaction between IRF8 and NF-kB genes might play a role in SSc susceptibility

Topically applied flightless I neutralizing antibodies improve healing of blistered skin in a murine model of Epidermolysis Bullosa Acquisita

Epidermolysis bullosa (EB) is a chronic inheritable disease that leads to severe blistering and fibrosis. Previous studies have shown that the actin cytoskeletal protein flightless I (Flii) impairs wound healing associated with EB. Using a mouse model of EB acquisita (EBA), the effect of ‘‘mopping up’’ Flii using Flii-neutralizing antibodies (FnAbs) before, during, and after blister formation was determined. FnAbs, incorporated into a cream vehicle and applied topically to the skin, penetrated into the basal epidermis and upper papillary dermis but were not detected in serum or other organs and did not alter neutrophil or macrophage infiltration into the blistered skin. Histological assessment of blister severity showed that treatment of early-stage blisters with FnAb cream reduced their severity and improved their rate of healing. Treatment of established blisters with FnAb cream also improved healing and restored the skin’s tensile strength toward that of normal skin. Repeated application of FnAbs to EBA skin before the onset of blistering reduced the severity of skin blistering. Independent of when the FnAbs were applied, skin barrier function and wound healing were improved and skin fragility was reduced, suggesting that FnAbs could potentially improve healing of patients with EB.Zlatko Kopecki, Nadira Ruzehaji, Christopher Turner, Hioraki Iwata, Ralf J. Ludwig, Detlef Zillikens, Dedee F. Murrell and Allison J. Cowi