Autologous protein solution inhibits MMP‐13 production by IL‐1β and TNFα‐stimulated human articular chondrocytes

Catabolic inflammatory cytokines are prevalent in osteoarthritis (OA). The purpose of this study was to evaluate an autologous protein solution (APS) as a potential chondroprotective agent for OA therapy. APS was prepared from platelet‐rich plasma (PRP). The APS solution contained both anabolic (bFGF, TGF‐β1, TGF‐β2, EGF, IGF‐1, PDGF‐AB, PDGF‐BB, and VEGF) and anti‐inflammatory (IL‐1ra, sTNF‐RI, sTNF‐RII, IL‐4, IL‐10, IL‐13, and IFNγ) cytokines but low concentrations of catabolic cytokines (IL‐1α, IL‐1β, TNFα, IL‐6, IL‐8, IL‐17, and IL‐18). Human articular chondrocytes were pre‐incubated with the antagonists IL‐1ra, sTNF‐RI, or APS prior to the addition of recombinant human IL‐1β or TNFα. Following exposure to inflammatory cytokines, the levels of MMP‐13 in the culture medium were evaluated by ELISA. MMP‐13 production stimulated in chondrocytes by IL‐1β or TNFα was reduced by rhIL‐1ra and sTNF‐RI to near basal levels. APS was also capable of inhibiting the production of MMP‐13 induced by both IL‐1β and TNFα. The combination of anabolic and anti‐inflammatory cytokines in the APS created from PRP may render this formulation to be a potential candidate for the treatment of inflammation in patients at early stages of OA. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1320–1326, 2011     

MicroRNA 26a prolongs skin allograft survival and promotes regulatory T cell expansion in mice

MicroRNA 26a (Mir‐26a) has been reported to play functions in cellular differentiation, cell growth, cell apoptosis, and metastasis. However, the role of Mir‐26a in transplant rejection has never been investigated. Full‐thickness skin grafts 1–2 cm in diameter were obtained from the tail‐skin CBA/J donor mice and transplanted onto the back of wild‐type C57Bl/6 recipient mice. Vectors encoding pre‐Mir‐26a (LV‐26a) and an empty lentiviral vector (LV‐Con) delivered approximately 2 × 10⁷ transforming units of recombinant lentivirus were injected to mice once through the tail vein. Mir‐26a overexpression results in prolonged skin allograft survival (MST = 9.5 days in LV‐Con mice; MST = 22 days in LV‐26a mice. P < 0.01) and promoted regulatory T cells (Tregs) expansion. The prolonged skin allograft survival induced by LV‐26a was abrogated by depletion of Tregs with anti‐CD25 antibodies. Mir‐26a significantly promoted IL‐10 expression and suppressed the expression of IL‐6, IL‐17, and IFN‐γ. Furthermore, IL‐6 overexpression led to complete suppression of the Mir‐26a‐induced upregulation of Foxp3. The prolonged allograft survival induced by LV‐Mir‐26a was also completely abrogated by IL‐6 overexpression. In conclusion, Mir‐26a prolongs skin allograft survival and promotes Tregs expansion in part through inhibition of IL‐6 expression.     

Osteoblast‐Derived TGF‐β1 Stimulates IL‐8 Release Through AP‐1 and NF‐κB in Human Cancer Cells

Introduction: The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption. It has been reported that the chemokine interleukin (IL)‐8 is a potent and direct activator of osteoclastic differentiation and bone resorption. However, the effect of bone‐derived growth factors on the IL‐8 production in human cancer cells and the promotion of osteoclastogenesis are largely unknown. The aim of this study was to investigate whether osteoblast‐derived TGF‐β1 is associated with osteolytic bone diseases. Materials and Methods: IL‐8 mRNA levels were measured using RT‐PCR analysis. MAPK phosphorylation was examined using the Western blot method. siRNA was used to inhibit the expression of TGF‐β1, BMP‐2, and IGF‐1. DNA affinity protein‐binding assay and chromatin immunoprecipitation assays were used to study in vitro and in vivo binding of c‐fos, c‐jun, p65, and p50 to the IL‐8 promoter. A transient transfection protocol was used to examine IL‐8, NF‐κB, and activator protein (AP)‐1 activity. Results: Osteoblast conditioned medium (OBCM) induced activation of IL‐8, AP‐1, and NF‐κB promoter in human cancer cells. Osteoblasts were transfected with TGF‐β1, BMP‐2, or IGF‐1 small interfering RNA, and the medium was collected after 48 h. TGF‐β1 but not BMP‐2 or IGF‐1 siRNA inhibited OBCM‐induced IL‐8 release in human cancer cells. In addition, TGF‐β1 also directly induced IL‐8 release in human cancer cells. Activation of AP‐1 and NF‐κB DNA‐protein binding and MAPKs after TGF‐β1 treatment was shown, and TGF‐β1–induced IL‐8 promoter activity was inhibited by the specific inhibitors of MAPK cascades. Conclusions: In this study, we provide evidence to show that the osteoblasts release growth factors, including TGF‐β1, BMP‐2, and IGF‐1. TGF‐β1 is the major contributor to the activation of extracellular signal‐related kinase (ERK), p38, and c‐Jun N‐terminal kinase (JNK), leading to the activation of AP‐1 and NF‐κB on the IL‐8 promoter and initiation of IL‐8 mRNA and protein release, thereby promoting osteoclastogenesis.     

Effects of Curcumin on Tumour Necrosis Factor‐α and Interleukin‐6 in the Late Phase of Experimental Acute Pancreatitis

Inflammatory cytokines have been demonstrated to play an important role in the induction and severity of acute pancreatitis (AP) in the recent studies. The aim of this study was to investigate the effects of curcumin on inflammatory cytokines, such as tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 in the late phase of AP. The study was conducted on 40 male Wistar Albino rats. The animals were divided randomly into four equal groups. AP was induced by the infusion of 3% sodium taurocholate into the biliopancreatic duct (in groups I and II). Starting on day 20 prior to the induction of AP, rats in group I received daily dose of 100 mg/kg of curcumin, dissolved in 9% ethanol via an intragastric tube. The same procedure was repeated for 6 days following the onset of AP. Group III was infused only on saline solution. Group IV (curcumin control group) received 9% ethanol via an intragastric tube, during the experimental period (totally 26 days). All the animals were sacrificed on day 6 after the collection of blood samples and serum TNF‐α and IL‐6 levels were determined. Tissue samples were taken from pancreas, mesenteric lymph nodes, liver, lungs, spleen and the kidneys for histopathological evaluation. Serum TNF‐α and IL‐6 levels in the group, which received curcumin (group I), were determined to be significantly lower than those of the untreated group (group II) (P<0.05). No statistically significant difference was detected in terms of total histopathological scores in the treatment group versus untreated group. Curcumin has been shown to markedly reduce serum TNF‐α and IL‐6 levels in the late phase of AP, but failed in the prevention of tissue injury.     

Epithelial–mesenchymal transition in keloid tissues and TGF‐β1–induced hair follicle outer root sheath keratinocytes

Keloid is a skin fibrotic disease with the characteristics of recurrence and invasion, its pathogenesis still remains unrevealed. The epithelial–mesenchymal transition (EMT) is critical for wound healing, fibrosis, recurrence, and invasion of cancer. We sought to investigate the EMT in keloid and the mechanism through which the EMT regulates keloid formation. In keloid tissues, the expressions of EMT‐associated markers and transforming growth factor (TGF)‐β1/Smad3 signaling were examined by immunohistochemistry. In the keloid epidermis and dermal tissue, the expressions of genes related to the regulation of skin homeostasis, fibroblast growth factor receptor 2 (FGFR2) and p63, were analyzed using quantitative real‐time polymerase chain reaction. The results showed that accompanying the loss of the epithelial marker E‐cadherin and the gain of the mesenchymal markers fibroblast‐specific protein 1 (FSP1) and vimentin in epithelial cells from epidermis and skin appendages, and in endothelial cells from dermal microvessels, enhanced TGF‐β1 expression and Smad3 phosphorylation were noted in keloid tissues. Moreover, alternative splicing of the FGFR2 gene switched the predominantly expressed isoform from FGFR2‐IIIb to ‐IIIc, concomitant with the decreased expression of ΔNp63 and TAp63, which changes might partially account for abnormal epidermis and appendages in keloids. In addition, we found that TGF‐β1‐induced hair follicle outer root sheath keratinocytes (ORSKs) and normal skin epithelial cells underwent EMT in vitro with ORSKs exhibiting more obvious EMT changes and more similar expression profiles for EMT‐associated and skin homeostasis‐related genes as in keloid tissues, suggesting that ORSKs might play crucial roles in the EMT in keloids. Our study provided insights into the molecular mechanisms mediating the EMT pathogenesis of keloids.     

Role of Toll‐like receptor 4 signaling in cutaneous chronic graft‐versus‐host disease

Cutaneous damage is one of the characterized manifestations in chronic graft‐versus‐host disease (cGVHD). When local effective immunity in the skin is altered to a dysimmune reaction, cutaneous injuries occur. Toll‐like receptor 4 signaling is regarded as a central mediator of inflammation and organ injury. In this study, we found that TLR4 mRNA in peripheral blood from patients with cutaneous cGVHD was markedly increased compared with that from non‐GVHD patients and healthy controls. In addition, NF‐κB expression, TLR4 downstream signaling, and TLR4‐mediated cytokines, including IL‐6 and ICAM‐1, were upregulated. Moreover, ICAM‐1 was widely distributed in skin biopsies from patients with cutaneous cGVHD. We also found that LPS induced TLR4‐mediated NF‐κB activation and IL‐6 and ICAM‐1 secretion in human fibroblasts in vitro. Thus, TLR4, NF‐κB, IL‐6, and ICAM‐1 contribute to the inflammatory response that occurs in cutaneous cGVHD, indicating the TLR4 pathway may be a novel target for cutaneous cGVHD therapy.     

Oxidative stress in subsynovial connective tissue of idiopathic carpal tunnel syndrome

Ischemic‐reperfusion injury is thought to be a cause of idiopathic carpal tunnel syndrome (CTS). The purpose of this study was to determine whether oxidative stress caused by ischemia‐reperfusion injury in subsynovial connective tissue is associated with idiopathic CTS and its symptoms. Bioptic samples of tenosynovial tissue were collected from 20 idiopathic CTS patients during surgery. Control specimens of tenosynovial tissue were collected from eight non‐CTS patients. Analysis included histological and immunohistochemical examination for the distribution of endothelial nitric oxide synthase (eNOS), nuclear factor (NF)‐κβ, and transforming growth factor (TGF)‐β RI in subsynovial connective tissues. Histological examinations showed a marked increase in fibroblast density and vascular proliferation in specimens from CTS patients. The expressions of eNOS, NF‐κβ, and TGF‐β RI in fibroblasts and vascular endothelial cells of subsynovial connective tissues of patients were significantly higher than in those of controls. A significant positive correlation was found between the subjective symptom severity of CTS, and the immunoreactivities of eNOS and NF‐κβ. This study suggests that oxidative stress in subsynovial connective tissue is related to CTS and its symptoms. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1463–1468, 2010     

Effect of pro‐inflammatory and immunoregulatory cytokines on human tenocytes

Tendon injury induces a local inflammatory response, characterized by the induction of pro‐inflammatory cytokines. The aim of the present study was to analyze the effects of TNFα, IL‐6 and IL‐10 on key parameters of tendon homeostasis. Cultured primary human tenocytes were treated with the recombinant cytokines IL‐6, IL‐10, TNFα, or combinations of TNFα with IL‐6 and IL‐10 (10 ng/mL, 6, 24 h). Expression of type I collagen, elastin, MMP‐1, TNFα, IL‐1β, IL‐6, IL‐10, and suppressors of cytokine signaling (SOCS1, 3) was analyzed with the use of RTD‐PCR, immunocytochemistry, and Western blot analysis. In response to TNFα, tenocytes reduced their type I collagen deposition but increased their elastin gene expression and highly upregulated their expression for MMP‐1, pro‐inflammatory (TNFα, IL‐1β) and immunoregulatory (IL‐6, IL‐10) cytokines. TNFα stimulation augmented SOCS1, whereas SOCS3 expression in tenocytes was also induced by IL‐6. The treatment of tenocytes with IL‐6 and IL‐10 had no effect on cytokine expression. Neither IL‐6 nor IL‐10 modulated the observed effects of TNFα significantly. These results indicate that TNFα strongly activates the tenocytes to amplify their own TNFα expression and, subsequently, that of other regulatory cytokines and matrix degrading enzymes. However, the impact of IL‐6 and IL‐10 on tenocytes remains unclear. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1071–1077, 2010     

Obesity-induced renal impairment is exacerbated in interleukin-6-knockout mice

Aim: Interleukin‐6 (IL‐6) is secreted from adipose tissue and thought to contribute to obesity‐related disorders. The aim of this study was to assess if IL‐6‐knockout (IL‐6‐/‐) mice would develop obesity‐induced renal impairment. Methods: Wild‐type (WT) and IL‐6‐/‐ mice were high‐fat fed (HFF) for 16 weeks to induce obesity. At the end of the study, renal function was measured via albumin/creatinine ratio and serum creatinine levels, using enzyme‐linked immunosorbent assay (ELISA) and high‐performance liquid chromatography (HPLC). Glomerulosclerotic index (GSI) was scored in periodic acid Schiff‐stained sections and collagen IV accumulation was assessed by immunohistochemistry. Renal cortical tumour growth factor beta (TGF‐β₁) activity and monocyte chemotactic protein‐1 (MCP‐1) levels were measured via ELISA. Results: Renal IL‐6 concentrations were increased with obesity. Although both WT HFF and IL‐6‐/‐ HFF mice exhibited renal impairment as measured by increased serum creatinine and urinary albumin/creatinine ratios, this was exacerbated in IL‐6‐/‐ mice. Obese mice had renal activation of cortical TGF‐β₁, which was also higher in IL‐6‐/‐ mice. Collagen IV staining was not affected by obesity. GSI was increased with obesity in both WT and IL‐6‐/‐ mice. Conclusion: Obese IL‐6‐/‐ mice demonstrated renal functional and structural abnormalities above that seen in obese WT mice. We suggest that absence or low IL‐6 levels may be an important accelerating factor implicated in the development and progression of obesity‐induced renal disease.     

Comparison of the efficacy and safety of povidone‐iodine foam dressing (Betafoam), hydrocellular foam dressing (Allevyn), and petrolatum gauze for split‐thickness skin graft donor site dressing

We evaluated the efficacy and safety of a povidone‐iodine (PVP‐I) foam dressing (Betafoam) for donor site dressing versus a hydrocellular foam dressing (Allevyn) and petrolatum gauze. This prospective Phase 4 study was conducted between March 2016 and April 2017 at eight sites in Korea. A total of 106 consenting patients (aged ≥ 19 years, scheduled for split‐thickness skin graft) were randomised 1:1:1 to PVP‐I foam, hydrocellular, or petrolatum gauze dressings for up to 28 days after donor site collection. We assessed time to complete epithelialisation, proportion with complete epithelialisation at Day 14, and wound infection. Epithelialisation time was the shortest with PVP‐I foam dressing (12.74 ± 3.51 days) versus hydrocellular foam dressing (16.61 ± 4.45 days; P = 0.0003) and petrolatum gauze (15.06 ± 4.26 days, P = 0.0205). At Day 14, 83.87% of PVP‐I foam dressing donor sites had complete epithelialisation, versus 36.36% of hydrocellular foam dressing donor sites (P = 0.0001) and 55.88% of petrolatum gauze donor sites (P = 0.0146). There were no wound infections. Incidence rates of adverse events were comparable across groups (P = 0.1940). PVP‐I foam dressing required less time to complete epithelialisation and had a good safety profile.     

Nedd4 Deficiency in Vascular Smooth Muscle Promotes Vascular Calcification by Stabilizing pSmad1

The nonosseous calcification process such as atherosclerosis is one of the major complications in several types of metabolic diseases. In a previous study, we uncovered that aberrant activity of transforming growth factor β (TGF‐β) signaling pathway could contribute to the vascular smooth muscle cells’ (VSMCs) calcification process. Also, we identified NEDD4 E3 ligase as a key suppressor of bone morphogenetic protein (BMP)/Smad pathway via a polyubiquitination‐dependent selective degradation of C‐terminal phosphorylated Smad1 (pSmad1) activated by TGF‐β. Here, we further validated and confirmed the role of Nedd4 in in vivo vascular calcification progression. First, Nedd4 deletion in SM22α‐positive mouse tissues (Nedd4^fl/fl;SM22α‐Cre) showed deformed aortic structures with disarranged elastin fibers at 24 weeks after birth. Second, vitamin D–induced aorta vascular calcification rate in Nedd4^fl/fl;SM22α‐Cre mice was significantly higher than their wild‐type littermates. Nedd4^fl/fl;SM22α‐Cre mice showed a development of vascular calcification even at very low‐level injection of vitamin D, but this was not exhibited in wild‐type littermates. Third, we confirmed that TGF‐β1–induced pSmad1 levels were elevated in Nedd4‐deficient primary VSMCs isolated from Nedd4^fl/fl;SM22α‐Cre mice. Fourth, we further found that Nedd4^fl/fl;SM22α‐Cre mVSMCs gained mesenchymal cell properties toward osteoblast‐like differentiation by a stable isotope labeling in cell culture (SILAC)‐based proteomics analysis. Finally, epigenetic analysis revealed that methylation levels of human NEDD4 gene promoter were significantly increased in atherosclerosis patients. Collectively, abnormal expression or dysfunction of Nedd4 E3 ligase could be involved in vascular calcification of VSMCs by activating bone‐forming signals during atherosclerosis progression. © 2016 American Society for Bone and Mineral Research.     

Honokiol,a low molecular weight natural product,prevents inflammatory response and cartilage matrix degradation in human osteoarthritis chondrocytes

Proinflammatory cytokine interleukin‐1β (IL‐1β) stimulates several mediators of cartilage degradation and plays an important role in the pathogenesis of osteoarthritis (OA). Honokiol, a low molecular weight natural product isolated from the Magnolia officinalis, has been shown to possess anti‐inflammatory effect. Here, we used an in vitro model of cartilage inflammation to investigate the therapeutic potential of honokiol in OA. Human OA chondrocytes were cultured and pretreated with honokiol (2.5–10 µM) with or without IL‐1β (10 ng/ml). Nitric oxide (NO) production was quantified by Griess reagent. Prostaglandin (PG)E₂, metalloproteinase‐13 (MMP‐13), and interleukin‐6 (IL‐6) productions were quantified by enzyme‐linked immunosorbent assay. The expressions of collagen II, cyclooxygenase‐2 (COX‐2), inducible nitric oxide synthase (iNOS), and nuclear factor κB (NF‐κB)‐related signaling molecules were determined by Western blotting. Our data showed that IL‐1β markedly stimulated the expressions of iNOS and COX‐2 and the productions of NO, PGE₂, and IL‐6, which could be significantly reversed by honokiol. Honokiol could also suppress the IL‐1β‐triggered activation of IKK/IκBα/NF‐κB signaling pathway. Moreover, honokiol significantly inhibited the IL‐1β‐induced MMP‐13 production and collagen II reduction. Taken together, the present study suggests that honokiol may have a chondroprotective effect and may be a potential therapeutic choice in the treatment of OA patients. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:573–580, 2014.     

IL‐6 Induced by Staphylococcus aureus Infection Prevents the Induction of Skin Allograft Acceptance in Mice

Clinical correlations between bacterial infections and rejection suggest a hypothesis that innate immune stimulation by bacterial infections results in the production of inflammatory cytokine that facilitate bystander T‐cell activation, increased alloreactivity and inhibition of tolerance induction. Previous studies demonstrated that IFNβ produced during an infection with a model bacterium, Listeria monocytogenes, prevented the induction of transplantation tolerance in mice with anti‐CD154 and donor‐specific transfusion (DST) ( 1 ). We investigated the impact of two clinically relevant bacterial infections at the time of transplantation on the ability of anti‐CD154 and DST to induce skin allograft acceptance in mice. Staphylococcus aureus (SA) infection prevented skin allograft acceptance whereas maximally tolerated doses of Pseudomonas aeruginosa infection had no effect. SA induced an acute production of IL‐6, which was necessary and sufficient for the prevention of skin allograft acceptance. Furthermore, a single pulse of methylprednisolone modulated IL‐6 production during SA infection and facilitated skin allograft acceptance in SA‐infected recipients. Taken together, our results suggest that bacterial infections elicit specific proinflammatory cytokines signatures that can serve as barriers to tolerance induction, and that inhibiting the production of or neutralizing these inflammatory cytokines can synergize with costimulatory blockade‐based therapies to facilitate the development of transplantation tolerance.     

The effects of caffeine on wound healing

The purine alkaloid caffeine is a major component of many beverages such as coffee and tea. Caffeine and its metabolites theobromine and xanthine have been shown to have antioxidant properties. Caffeine can also act as adenosine‐receptor antagonist. Although it has been shown that adenosine and antioxidants promote wound healing, the effect of caffeine on wound healing is currently unknown. To investigate the effects of caffeine on processes involved in epithelialisation, we used primary human keratinocytes, HaCaT cell line and ex vivo model of human skin. First, we tested the effects of caffeine on cell proliferation, differentiation, adhesion and migration, processes essential for normal wound epithelialisation and closure. We used 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) proliferation assay to test the effects of seven different caffeine doses ranging from 0·1 to 5 mM. We found that caffeine restricted cell proliferation of keratinocytes in a dose‐dependent manner. Furthermore, scratch wound assays performed on keratinocyte monolayers indicated dose‐dependent delays in cell migration. Interestingly, adhesion and differentiation remained unaffected in monolayer cultures treated with various doses of caffeine. Using a human ex vivo wound healing model, we tested topical application of caffeine and found that it impedes epithelialisation, confirming in vitro data. We conclude that caffeine, which is known to have antioxidant properties, impedes keratinocyte proliferation and migration, suggesting that it may have an inhibitory effect on wound healing and epithelialisation. Therefore, our findings are more in support of a role for caffeine as adenosine‐receptor antagonist that would negate the effect of adenosine in promoting wound healing.     

Erythropoietin and its non‐erythropoietic derivative: Do they ameliorate renal tubulointerstitial injury in ureteral obstruction?

Objectives: Pleiotropic effects of recombinant human erythropoietin (EPO) have recently been discovered in many non‐renal animal models. The renoprotective effects of EPO and carbamylated‐erythropoietin (CEPO), a novel EPO which has a small stimulatory effect on hemoglobin, have never been explored in unilateral ureteral obstruction (UUO), a chronic tubulointerstitial (TI) disease model which is independent of systemic factors. Methods: In order to examine the effects of EPO and CEPO treatments on renal TI injury, 36 male Sprague‐Dawley rats, weighing 250–320 g, underwent: UUO without treatment (group 1, n = 12), UUO with EPO (groups 2, n = 12), and UUO with CEPO (group 3, n = 12). EPO and CEPO were injected subcutaneously at a dose of 5000 u/kg to each respective rat at 1 day pre‐UUO and at day 3, 7 and 10 post‐UUO. After days 3, 7, and 14 of UUO, TI injury, collagen, α‐smooth muscle actin (α‐SMA) positive cell, ED1‐positive cell, terminal deoxynucleotidyl transferase (TdT) mediated nick‐end labeling (TUNEL)‐positive cell, and transforming growth factor‐β1 (TGF‐β1) messenger ribonucleic acid (mRNA) were determined. Bcl‐2 expression was also assessed to verify the mechanism of apoptosis. Results: At day 14 UUO caused severe TI injury with a significant increase in collagen, α‐SMA, ED1‐positive cell, TUNEL‐positive cell, and TGF‐β1 mRNA expression. Administration of EPO and CEPO significantly attenuated TI injury, collagen, ED1‐positive cells, and TUNEL‐positive cells. Only CEPO‐treated rats had decreased α‐SMA positive cells and TGF‐β1 mRNA. The expression of Bcl‐2 was demonstrated only in EPO‐treated rats. The hematocrit levels in EPO‐treated rats were higher than the control and CEPO‐treated rats. Conclusions: EPO and CEPO can limit 14‐day UUO‐induced TI injury by reducing inflammation, interstitial fibrosis, and tubular apoptosis.     

The protective role of interleukin‐18 binding protein in a murine model of cardiac ischemia/reperfusion injury

IL‐18, a proinflammatory cytokine, is produced by macrophages, epithelial cells, T cells, neutrophils, NK‐T cells, and B cells, and has been implicated in the pathophysiology of a variety of inflammatory diseases including ischemia/reperfusion (IR) injury, transplant rejection, and autoimmune disease. Recent study indicated that neutralization of IL‐18 with anti‐IL‐18 antibody or IL‐18‐binding protein (IL‐18BP) ameliorates IR‐induced myocardial injury. However, the mechanism needs to be further investigated. In our current study, syngeneic heterotopic heart transplantation was performed by a modified non‐suture cuff technique. We found that IL‐18BP treatment ameliorated cardiomyocyte necrosis and infiltration of CD4⁺ T cells, neutrophils, and macrophages. IL‐18BP‐treated mice exhibited decreased expression of inflammatory cytokines including IL‐1β, IL‐23, IL‐18, and IL‐17. IL‐18BP treatment suppressed Th17 differentiation in vivo and in vitro. Adoptive transfer of T cells from IL‐18BP‐treated mice showed alleviated cardiac IR injury when compared with that transferred from control mice. Furthermore, the decreased infiltration of mononuclear cells and production of troponin T (TnT) induced by IL‐18BP treatment were both abrogated by additional administration of recombinant mouse IL‐17 (rmIL‐17). These data revealed a protective role of IL‐18BP in cardiac IR injury. Above all, IL‐18BP ameliorates cardiac IR injury in part through suppression of Th17 differentiation.     

Distinct Inflammatory Signals Have Physiologically Divergent Effects on Epigenetic Regulation of Foxp3 Expression and Treg Function

Foxp3 expression in regulatory T cells (Treg) is required for their development and suppressive function. How different inflammatory signals affect Foxp3 chromatin structure, expression and Tregs plasticity are not completely known. In the present study, the Toll‐like receptor 2 (TLR2) ligand peptidoglycan inhibited Foxp3 expression in both natural Treg (nTreg) and TGFβ‐driven adaptive Treg (aTreg). Inhibition was independent of paracrine Th1, Th2 and Th17 cytokines. PGN‐induced T cell‐intrinsic TLR2‐Myd88‐dependent IFR1 expression and induced IRF1 bound to IRF1 response elements (IRF‐E) in the Foxp3 promoter and intronic enhancers, and negatively regulated Foxp3 expression. Inflammatory IL‐6 and TLR2 signals induced divergent chromatin changes at the Foxp3 locus and regulated Treg suppressor function, and in an islet transplant model resulted in differences in their ability to prolong graft survival. These findings are important for understanding how different inflammatory signals can affect the transplantation tolerance and immunity.     

Endothelial Cell Apoptosis Induces TGF‐β Signaling‐Dependent Host Endothelial–Mesenchymal Transition to Promote Transplant Arteriosclerosis

Endothelial cells (ECs) apoptosis is an initial event in transplant arteriosclerosis (TA), resulting in allograft function loss. To elucidate the precise mechanisms of ECs apoptosis leading to neointimal smooth muscle cells (SMCs) accumulation during TA. We induced apoptosis in cultured ECs by overexpressing p53 through lentivirus‐mediated transfection. ECs apoptosis induced the production of transforming growth factor (TGF)‐β1 in both apoptotic and neighboring viable cells, leading to increased TGF‐β1 in the culture media. Conditioned media from Ltv‐p53‐transfected ECs further promoted transition of cultured ECs to SM‐like cells by activating TGF‐β/Smad3, PI3K/Akt/mTOR, and MAPK/ERK signaling in a TGF‐β‐dependent manner. In transgenic rat aorta transplantation models, inhibition of ECs apoptosis in Bcl‐xL ^+/+ knock‐in rat aortic allografts significantly reduced TGF‐β1 production both in allograft endothelia and in blood plasma, which in turn decreased accumulation of SM22α+ cells from transgenic recipient ECs originally marked with EGFP knock‐in in neointima and alleviated TA. Systemic treatment with SIS3, AP23573, or PD98059 also prevented recipient ECs‐originated SM‐like cells accumulation and intima hyperplasia in aortic allografts. These data suggest that allograft EC apoptosis induced recipient endothelial–mesenchymal (smooth muscle) transition via TGF‐β signaling, resulting in recipient EC‐derived SMC accumulation as a major mechanism of vascular remodeling during TA.     

Connective Tissue Growth Factor Promotes Fibrosis Downstream of TGFβ and IL‐6 in Chronic Cardiac Allograft Rejection

Cardiac transplantation is an effective treatment for multiple types of heart failure refractive to therapy. Although immunosuppressive therapeutics have increased survival rates within the first year posttransplant, chronic rejection (CR) remains a significant barrier to long‐term graft survival. Indicators of CR include patchy interstitial fibrosis, vascular occlusion and progressive loss of graft function. Multiple factors have been implicated in the onset and progression of CR, including TGFβ, IL‐6 and connective tissue growth factor (CTGF). While associated with CR, the role of CTGF in CR and the factors necessary for CTGF induction in vivo are not understood. To this end, we utilized forced expression and neutralizing antibody approaches. Transduction of allografts with CTGF significantly increased fibrotic tissue development, though not to levels observed with TGFβ transduction. Further, intragraft CTGF expression was inhibited by IL‐6 neutralization whereas TGFβ expression remained unchanged, indicating that IL‐6 effects may potentiate TGFβ‐mediated induction of CTGF. Finally, neutralizing CTGF significantly reduced graft fibrosis without reducing TGFβ and IL‐6 expression levels. These findings indicate that CTGF functions as a downstream mediator of fibrosis in CR, and that CTGF neutralization may ameliorate fibrosis and hypertrophy associated with CR.