Recombinant IL-17A Research Articles

IL17A impairs blood–testis barrier integrity and induces testicular inflammation

Experimental autoimmune orchitis is a useful model for studying testicular inflammation and germ/immune cell interactions. Th17 cells and their hallmark cytokine IL17A were reported to be involved in the development of autoimmune orchitis. The aim of the present work is to investigate the pathogenic role of IL17A in rat testis. In vitro experiments were performed in order to analyze effects of IL17A on Sertoli cell tight junctions. The addition of IL17A to normal rat Sertoli cell cultures induced a significant decline in transepithelial electrical resistance and a reduction of occludin expression and redistribution of occludin and claudin 11, altering the Sertoli cell tight junction barrier. Intratesticular injection of 1 μg of recombinant rat IL17A to Sprague-Dawley rats induced increased blood-testis barrier permeability, as shown by the presence of biotin tracer in the seminiferous tubule adluminal compartment, and delocalization of occludin and claudin 11. Results showed that IL17A induced focal inflammatory cell infiltration in the interstitium and germ cell sloughing in adjacent seminiferous tubules. Moreover, an increase in TUNEL+ apoptotic germ cells was also observed. Inflammatory ED1+ macrophages were the main population infiltrating the interstitium following IL17A injection. This correlated with an increase in mRNA expression of the monocyte chemoattractant protein Ccl2, its receptor Ccr2 and the vascular cell adhesion molecule Vcam1. Overall results suggest a relevant role of IL17A in the development of testicular inflammation, facilitating the recruitment of immune cells to the testicular interstitium and inducing impairment of blood-testis barrier function.

Glycyrrhizin protects brain against ischemia–reperfusion injury in mice through HMGB1-TLR4-IL-17A signaling pathway

High mobility group box 1 (HMGB1)-Toll-like receptor 4 (TLR4) signaling has been recently found to induce interleukin (IL)-17A secretion in drug-induced hepatitis and myocardial I/R injury. The purpose of this study is to evaluate whether HMGB1-TLR4 signaling could induce IL-17A secretion and lead to brain I/R injury. We also sought to investigate whether glycyrrhizin elucidated its neuroprotective effects through HMGB1-TLR4-IL-17A signaling pathway. Various biochemical estimations, neurological status, and assessment of cerebral infarct size were carried out 72h after middle cerebral artery occlusion (MCAO) stroke. Apoptotic cells were assessed using a terminal deoxynucleotidyl transferase, dUTP nick and labeling (TUNEL) kit. The expression of HMGB1, IL-17A, bcl-2, bax and cleaved caspase-3, were determined by Western blot assay. In the present study we found that glycyrrhizin significantly decreased HMGB1 protein expression. Glycyrrhizin markedly reduced whereas recombinant HMGB1 (rHMGB1) increased IL-17A expression. HMGB1 induced increase of IL-17A was significantly diminished in TLR4-mutant C3H/HeJ mice. Brain injury and neurological deficits were largely abrogated by glycyrrhizin or IL-17A knockout. In contrast, rHMGB1 or recombinant mouse IL-17A markedly increased I/R injury. However, rHMGB1 had no effects on infarct size and neurological deficits in Il17a−/− mice following brain I/R injury. In addition, IL-17A knockout mice significantly increased bcl-2 protein expression and had fewer apoptotic cells, whereas recombinant IL-17A-treated mice significantly increased bax and cleaved caspase-3 protein expression and had more apoptotic cells. Together these results indicate that glycyrrhizin has neuroprotective efficacy in the postischemic brain through HMGB1-TLR4-IL-17A signaling pathway.

IL-17A is a novel player in dialysis-induced peritoneal damage

The classical view of the immune system has changed by the discovery of novel T-helper (Th) subsets, including Th17 (IL-17A-producing cells). IL-17A participates in immune-mediated glomerulonephritis and more recently in inflammatory pathologies, including experimental renal injury. Peritoneal dialysis patients present chronic inflammation and Th1/Th2 imbalance, but the role of the Th17 response in peritoneal membrane damage has not been investigated. In peritoneal biopsies from dialyzed patients, IL-17A immunostaining was found mainly in inflammatory areas and was absent in the healthy peritoneum. IL-17A-expressing cells included lymphocytes (CD4+ and γδ), neutrophils, and mast cells. Elevated IL-17A effluent concentrations were found in long-term peritoneal dialysis patients. Studies in mice showed that repeated exposure to recombinant IL-17A caused peritoneal inflammation and fibrosis. Moreover, chronic exposure to dialysis fluids resulted in a peritoneal Th17 response, including elevated IL-17A gene and protein production, submesothelial cell infiltration of IL-17A-expressing cells, and upregulation of Th17 differentiation factors and cytokines. IL-17A neutralization diminished experimental peritoneal inflammation and fibrosis caused by chronic exposure to dialysis fluids in mice. Thus, IL-17A is a key player of peritoneum damage and it may be a good candidate for therapeutic intervention in peritoneal dialysis patients.

IL-17A contributes to brain ischemia reperfusion injury through calpain-TRPC6 pathway in mice

Interleukin (IL)-17A plays an important role in the cerebral ischemia/reperfusion (I/R) injury. However, the mechanisms are still largely unknown. Calpain-transient receptor potential canonical (subtype) 6 (TRPC6) signaling pathway has been recently found to be implicated in brain I/R injury. However, their relationships with IL-17A remain unknown. This study aims to test whether this important signaling has correlation with IL-17A and how they led to the neuronal damage in I/R injury. In the present study, mice were subjected to middle cerebral artery occlusion (60min) followed by reperfusion for different times. Infarct volumes and neurological deficits were examined. Real-time PCR (RT-PCR) and Western blotting were conducted to detect IL-17A expression in the penumbral brain tissue. Activation of calpain and expression of TRPC6 were also studied. We found that cerebral I/R significantly increased the levels of IL-17A at 1, 3 and 6days after reperfusion in the penumbral area. IL-17A knockout or anti-IL-17A monoclonal antibody (mAb) significantly reduced whereas recombinant mouse-IL-17A (rIL-17A) increased the activation of calpain at 3days after reperfusion. The calpain specific inhibitor calpeptin significantly increased TRPC6 expression. Brain injury and neurological deficits were largely abrogated by IL-17A knockout, anti-IL-17A mAb or calpeptin. Recombinant IL-17A treatment markedly increased I/R injury. In conclusion, IL-17A may promote brain I/R injury through the increase of calpain-mediated TRPC6 proteolysis. These results further outline a novel neuroprotective strategy with increased effectiveness for the inhibition of excess brain IL-17A in cerebral I/R injury.

OP0226 Human Mast Cells Engulf and Store Exogenous IL-17A

BackgroundIL-17A plays an important role in rheumatic diseases, like rheumatoid arthritis and spondyloarthritis. Direct analysis of inflamed synovial tissue revealed an abundant presence of IL-17A-positive mast cells.ObjectivesAs mast cells are...

IL-17A produced by γδ T cells promotes tumor growth in hepatocellular carcinoma.

Interleukin (IL)-17A is expressed in the tumor microenvironment where it appears to contribute to tumor development, but its precise role in tumor immunity remains controversial. Here, we report mouse genetic evidence that IL-17A is critical for tumor growth. IL-17A-deficient mice exhibited reduced tumor growth, whereas systemic administration of recombinant mouse IL-17A promoted the growth of hepatocellular carcinoma. The tumor-promoting effect of IL-17A was mediated through suppression of antitumor responses, especially CD8(+) T-cell responses. Furthermore, we found that IL-17A was produced mainly by Vγ4 γδ T cells, insofar as depleting Vγ4 γδ T cells reduced tumor growth, whereas adoptive transfer of Vγ4 γδ T cells promoted tumor growth. Mechanistic investigations showed that IL-17A induced CXCL5 production by tumor cells to enhance the infiltration of myeloid-derived suppressor cells (MDSC) to tumor sites in a CXCL5/CXCR2-dependent manner. IL-17A also promoted the suppressive activity of MDSC to reinforce suppression of tumoral immunity. Moreover, we found that MDSC could induce IL-17A-producing γδ T cells via production of IL-1β and IL-23. Conversely, IL-17A could also enhance production of IL-1β and IL-23 in MDSC as a positive feedback. Together, our results revealed a novel mechanism involving cross-talk among γδ T cells, MDSCs, and tumor cells through IL-17A production. These findings offer new insights into how IL-17A influences tumor immunity, with potential implications for the development of tumor immunotherapy.

Increased expression of IL17A in human gastric cancer and its potential roles in gastric carcinogenesis

Inflammatory cytokines modulate immune responses in the tumor microenvironment during progression. The role of interleukin (IL) 17A in cancer is currently under debate. We aim to investigate the expression of IL17A in situ tumors as well as in nontumor gastric mucosa tissues and further explore the functional significance of IL17A on gastric cancer cells in vitro. We found that compared with nontumor regions, the expression of IL17A were increased significantly in tumors of gastric cancer patients (P=0.007). The immunoreactivity for IL17A was found only in cytoplasm of inflammatory cells as well as vascular endothelial cells but not in tumor cells. Consistently, IL17A transcription was silenced in a variety of gastric cancer cell lines. In vitro, recombinant human IL17A protein promotes cell proliferation and monolayer wound healing of both AGS and SGC7901cells, in a dose-dependent manner. Besides, IL17A inhibits H2O2-induced cell apoptosis. Expression of IL6 and MMP13 mRNA was increased significantly after IL17A stimulation. These data suggest that accumulation of intratumoral IL17A-producing cells may promote gastric cancer progression directly or by inducing key signal transduction pathways implicated in gastric carcinogenesis.

IL-17A signaling in colonic epithelial cells inhibits pro-inflammatory cytokine production by enhancing the activity of ERK and PI3K.

Our previous data suggested that IL-17A contributes to the inhibition of Th1 cell function in the gut. However, the underlying mechanisms remain unclear. Here we demonstrate that IL-17A signaling in colonic epithelial cells (CECs) increases TNF-α-induced PI3K-AKT and ERK phosphorylation and inhibits TNF-α induced expression of IL-12P35 and of a Th1 cell chemokine, CXCL11 at mRNA level. In a co-culture system using HT-29 cells and PBMCs, IL-17A inhibited TNF-ãinduced IL-12P35 expression by HT-29 cells and led to decreased expression of IFN-γ and T-bet by PBMCs. Finally, adoptive transfer of CECs from mice with Crohn's Disease (CD) led to an enhanced Th1 cell response and exacerbated colitis in CD mouse recipients. The pathogenic effect of CECs derived from CD mice was reversed by co-administration of recombinant IL-17A. Our data demonstrate a new IL-17A-mediated regulatory mechanism in CD. A better understanding of this pathway might shed new light on the pathogenesis of CD.

IL-17A is produced by breast cancer TILs and promotes chemoresistance and proliferation through ERK1/2

The proinflammatory cytokine Interleukin 17A (hereafter named IL–17A) or IL-17A producing cells are elevated in breast tumors environment and correlate with poor prognosis. Increased IL-17A is associated with ER(−) or triple negative tumors and reduced Disease Free Survival. However, the pathophysiological role of IL-17A in breast cancer remains unclear although several studies suggested its involvement in cancer cell dissemination. Here we demonstrated that a subset of breast tumors is infiltrated with IL-17A-producing cells. Increased IL-17A seems mainly associated to ER(−) and triple negative/basal-like tumors. Isolation of tumor infiltrating T lymphocytes (TILs) from breast cancer biopsies revealed that these cells secreted significant amounts of IL-17A. We further established that recombinant IL-17A recruits the MAPK pathway by upregulating phosphorylated ERK1/2 in human breast cancer cell lines thereby promoting proliferation and resistance to conventional chemotherapeutic agents such as docetaxel. We also confirmed here that recombinant IL-17A stimulates migration and invasion of breast cancer cells as previously reported. Importantly, TILs also induced tumor cell proliferation, chemoresistance and migration and treatment with IL-17A-neutralizing antibodies abrogated these effects. Altogether these results demonstrated the pathophysiological role of IL-17A-producing cell infiltrate in a subset of breast cancers. Therefore, IL-17A appears as potential therapeutic target for breast cancer.

Interleukin-17 Signalling in a Murine Model of Mild Chronic Asthma

Background: The role of Th17 cell-derived cytokines in the pathogenesis of airway inflammation and remodelling in mild asthma remains unclear. We investigated this in a mouse model which reproduces most of the features of the human disease. Methods: Systemically sensitised BALB/c mice were challenged via the airways with a low mass concentration of ovalbumin aerosol for 8 weeks to induce lesions of mild chronic asthma. Changes were compared with those in animals deficient in signalling via the interleukin (IL)-17 receptor A (IL-17R). Low-passage airway epithelial cells (AEC) and fibroblasts were cultured with IL-17A, or with media from Th17-polarised cells, to assess activation. Results: In CD4+ T cells from chronically challenged mice, expression of mRNA for Th17 cytokines IL-17A, IL-17F, IL-21 and IL-22 was significantly increased. Both recombinant IL-17A and media from Th17 cells significantly stimulated the production of various pro-inflammatory and pro-remodelling cytokines by AEC and fibroblasts. In the mouse model, abrogation of IL-17R signalling had no effect on the development of airway inflammation or on most changes of remodelling. However, numbers of mucus-producing cells and expression of mRNA for Gob-5 were attenuated in the absence of IL-17R signalling. Conclusions: Although IL-17A and Th17 cells stimulate cytokine production by structural cells of the airways, and Th17 cells are induced in our model of mild chronic asthma, signalling via IL-17R did not contribute significantly to the development of airway inflammation and most changes of remodelling in this model. However, in mild asthma, IL-17A appears to have a role in the goblet cell response in the airways.

Autologous solution protects bovine cartilage explants from IL‐1α‐ and TNFα‐induced cartilage degradation

Osteoarthritis (OA) is characterized by deterioration of articular cartilage driven by an imbalance of pro- and anti-inflammatory cytokines. To address the cartilage deterioration observed in OA, an autologous protein solution (APS) has been developed which has been shown to inhibit the production of destructive proteases and inflammatory cytokines from chondrocytes and monocytes, respectively. The purpose of this study was to determine the chondroprotective effect of APS on IL-1α- or TNFα-challenged bovine articular cartilage explants. Cartilage explants were cultured in the presence or absence of recombinant inflammatory cytokines, IL-1α and TNFα. Explants under equivalent inflammatory conditions were pretreated with recombinant antagonists IL-1ra, sTNF-RI, or APS to measure their inhibition of matrix degradation. Explants were further evaluated with Safranin-O, Masson's Trichrome, and Hematoxylin and Eosin histological staining. APS was more effective than recombinant antagonists in preventing cartilage matrix degradation and inhibited any measurable IL-1α-induced collagen release over a 21-day culture period. APS treatment reduced the degree of Safranin-O staining loss when cartilage explants were cultured with IL-1α or TNFα. Micrographs of APS treated cartilage explants showed an increase in observed cellularity and apparent cell division. APS may have the potential to prevent cartilage loss associated with early OA.

Modified Foxp3 mRNA protects against asthma through an IL-10–dependent mechanism

Chemically modified mRNA is capable of inducing therapeutic levels of protein expression while circumventing the threat of genomic integration often associated with viral vectors. We utilized this novel therapeutic tool to express the regulatory T cell transcription factor, FOXP3, in a time- and site-specific fashion in murine lung, in order to prevent allergic asthma in vivo. We show that modified Foxp3 mRNA rebalanced pulmonary T helper cell responses and protected from allergen-induced tissue inflammation, airway hyperresponsiveness, and goblet cell metaplasia in 2 asthma models. This protection was conferred following delivery of modified mRNA either before or after the onset of allergen challenge, demonstrating its potential as both a preventive and a therapeutic agent. Mechanistically, FOXP3 induction controlled Th2 and Th17 inflammation by regulating innate immune cell recruitment through an IL-10-dependent pathway. The protective effects of FOXP3 could be reversed by depletion of IL-10 or administration of recombinant IL-17A or IL-23. Delivery of Foxp3 mRNA to sites of inflammation may offer a novel, safe therapeutic tool for the treatment of allergic asthma and other diseases driven by an imbalance in helper T cell responses.

The Interleukin-1α Precursor is Biologically Active and is Likely a Key Alarmin in the IL-1 Family of Cytokines

Among the 11 members of the IL-1 family cytokines, the precursors of IL-1α, IL-1β, and IL-33 have relatively long N-terminal pro-sequences of approximately 100 amino acid residues prior to the N-terminus of the mature forms. Compared to the mature forms secreted from the cell, 80–90% of the primary translation product is in the intracellular compartment in the precursor form. However, the precursors are readily released from cells during infections but also with non-infectious conditions such a hypoxia and trauma. In this setting, the precursors act rapidly as “alarmins” in the absence of a processing mechanism to remove the pro-sequence and generate a mature form. In the case of IL-1α, the release of the precursor activates adjacent cells via receptor-mediated signaling. However, there are no data comparing the specific activity of the IL-1α precursor to the mature form. In the present study, we compared the precursor and mature forms of recombinant human IL-1α, IL-1β, and IL-33 proteins on the induction of cytokines from A549 cells as well as from human peripheral blood mononuclear cells (PBMC). Similar to the mature form, the IL-1α precursor was active in inducing IL-6 and TNFα, whereas the precursor forms of IL-1β and IL-33 were not active. On PBMC, precursor and mature IL-1α at 0.04 and 0.2 nM were equally active in inducing IL-6. Given the fact that during necrotic cell death, the IL-1α precursor is released intact and triggers IL-1 receptors on tissue macrophages, these data identify the precursor form of IL-1α as a key player in sterile inflammation.

Increased Expression Levels of Integrin α9β1 and CD11b on Circulating Neutrophils and Elevated Serum IL-17A in Elderly Aspiration Pneumonia

Background: Repeated aspiration pneumonia is a serious problem in the elderly. In aspiration pneumonia, neutrophils play an important role in acute lung injury, while CD18-independent neutrophil transmigration pathways have also been reported in acid-aspiration pneumonia animal models. However, the involvement of IL-17A and β1 integrin still remains unclear. The β1 integrin subfamily integrin α9β1 has been shown to be expressed on human neutrophils and to mediate adhesion to extracellular matrix proteins including the vascular cell adhesion molecule-1. Objectives: To elucidate the possible involvement of β1 integrin subfamily and IL-17A in aspiration pneumonia. Methods: We analyzed the expression levels of CD11b, CD18 and integrin α9β1 in circulating neutrophils and serum concentration of IL-17A, IL-22 and IL-23 in elderly aspiration pneumonia patients (n = 32, 14 males and 18 females, 78.8 ± 3.9 years old) at 2 time points (on the day of admission before starting antibiotics and the day after finishing antibiotics) and compared the results with those of a control group (n = 30, 13 males and 17 females, 76.1 ± 3.4 years old). Results: Recombinant IL-17A stimulated integrin α9β1 and CD11b expression levels in healthy human neutrophils in vitro. The expression levels of integrin α9β1 and CD11b in circulating neutrophils were significantly higher in pneumonia patients compared with the controls. In addition, serum IL-17A concentration was significantly increased in pneumonia patients. Integrin α9β1 levels positively correlated with serum IL-17A and CD18 expression levels. Conclusions: These findings suggest a potential role of integrin α9β1 expressed in neutrophils and elevated serum IL-17A in extravasation of neutrophils in cases of aspiration pneumonia.

Inflammatory Cytokines and Thrombosis

Abstract SCI-44Platelets circulate in abundance in a quiescent state, yet are readily activated in physiologic control of blood loss and support for vascular integrity, with excessive or prolonged responsiveness underlying a range of cardiovascular events. Platelet activation is initiated by soluble agonists, including thrombin, platelet-activating factor, and ADP, and is accompanied by adhesion to injured vascular walls or to other circulating inflammatory cells. Activated platelets produce thromboxane A2, ATP and ADP, growth factors, and cytokines such as CD40 ligand, and they shed highly thrombotic microparticles. Anucleate platelets cannot generate new RNA to alter their proteome, but they store factors for prompt release after stimulation. Despite the handicap of lacking a nucleus to direct RNA transcript formation, platelets do, in fact, produce a number of new proteins after appropriate stimulation. The questions, then, are: how do platelets respond to inflammatory signaling in the absence of transcription factors that integrate so much of inflammatory signaling? How are new proteins produced in the absence of stored mRNA? To what events do new platelet proteins contribute? The answers to these questions derive both from stimulated translation of stored mRNA in unactivated platelets and from the presence of unspliced heteronuclear RNA transcripts of select genes, accompanied by stimulated posttranscriptional splicing that removes introns from stored heteronuclear RNA to generate functional mRNA, followed by stimulated translation to produce new protein. These mechanisms are retained in murine platelets, suggesting a continued role for the unique process of posttranscriptional protein production by activated platelets. Thrombin stimulates mTOR and S6 kinase signaling to stimulate spliceosome activity, while platelets respond to lipopolysaccharide through their TLR4 receptor, ultimately to stimulate the AKT and JNK kinases and then spliceosome activation. Human and murine platelets store interleukin-1β heteronuclear RNA, and, upon activation, both produce new pro-IL-1β, but also process it via caspase-1 activity to active cytokine. Activated platelets release functional IL-1β, in association with shed microparticles, to active endothelial cells and naïve platelets themselves. Platelets express signaling type 1 receptors for IL-1, and recombinant IL-1α and IL-1β each activate IL-1β RNA processing and cytokine production. Accordingly, specific blockade of IL-1 signaling with IL-1Ra, in clinical use as Anakinra, suppressed platelet stimulation by soluble IL-1, and suppressed microparticles shed from activated platelets. IL-1 signaling additionally affects platelets’ structure and their interaction with immobile surfaces. Remarkably, IL-1Ra also abolished platelet stimulation by lipopolysaccharide, showing that platelets amplify TLR4 signaling by the IL-1 signaling axis. IL-1β accumulates in association with platelets within thrombi formed after FeCl3 damage to murine carotid arteries, not by captured or infiltrating mononuclear cells. The process of posttranscriptional RNA splicing and translation uniquely generates the paradigmatic inflammatory cytokine IL-1β in thrombi, and because this process can be tightly targeted by inhibition of posttranslational splicing, the contribution of platelets to vascular remodeling during and after thrombosis may be specifically addressed. Disclosures:No relevant conflicts of interest to declare.

Cloning and Characterization of Rainbow Trout Interleukin-17A/F2 (IL-17A/F2) and IL-17 Receptor A: Expression during Infection and Bioactivity of Recombinant IL-17A/F2

Lower vertebrates have been found to possess genes that have similar homology to both interleukin (IL)-17A and IL-17F, which have been termed IL-17A/F. In fish species, several of these genes can be present, but, to date, very little is known about their functional activity. This article describes the discovery and sequence analysis of a rainbow trout (Oncorhynchus mykiss) IL-17A/F2 molecule and an IL-17RA receptor. In addition, the bioactivity of the trout IL-17A/F2 is investigated for the first time in any species. The predicted IL-17A/F2 and IL-17RA proteins consist of 146 and 966 amino acids (aa), respectively, with both molecules containing conserved family motifs. Expression analysis revealed high constitutive expression of trout IL-17A/F2 in mucosal tissues from healthy fish, suggesting a potential role in mucosal immunity. When the modulation of IL-17A/F2 and IL-17RA in vitro was analyzed, it was observed that the two molecules were similarly affected. The expression of IL-17A/F2 was also induced in head kidney during bacterial, parasitic, and viral infections, revealing a possible function in defense against such pathogens. However, downregulation of IL-17RA was seen in some tissues and infections. The recombinant IL-17A/F2 protein was produced in Escherichia coli and was found to affect the expression of an antimicrobial peptide and the proinflammatory cytokines IL-6 and IL-8 in splenocytes. Consistent with mammalian IL-17 homologues, our expression and bioactivity results imply that trout IL-17A/F2 plays an important role in promoting inflammatory and host innate immune responses directed against different pathogen groups.

Potential roles of interleukin‐17A in the development of skin fibrosis in mice

Although transforming growth factor β (TGFβ) and connective tissue growth factor (CTGF) have been considered to play central roles in the pathogenesis of systemic sclerosis (SSc), other cytokines may also be crucial for the development of SSc. The aim of this study was to examine the roles of T helper cytokines in the development of skin fibrosis. To compare the roles of Th1, Th2, and Th17 cytokines, we examined bleomycin-induced SSc in mice deficient for interferon-γ (IFNγ), interleukin-4 (IL-4), and IL-17A. The mechanism by which IL-17A contributes to bleomycin-induced fibrosis was investigated in vivo and in vitro. The outcome of mice lacking IL-17A was also investigated in TSK-1 mice. The loss of IL-17A significantly attenuated bleomycin-induced skin fibrosis, whereas a deficiency of IFNγ or IL-4 did not. Leukocyte infiltration and the expression of TGFβ and CTGF messenger RNA in bleomycin-injected skin were significantly reduced in IL-17A-deficient mice compared with wild-type (WT) mice. Daily bleomycin injections induced the expression of IL-17A in the skin and potent IL-17A producers in splenic CD4+ T cells from WT mice. Furthermore, a skin fibroblast cell line expressed increased TGFβ, CTGF, and collagen after the addition of recombinant IL-17A. IL-17A deficiency also attenuated skin thickness in TSK-1 mice. This study demonstrates that IL-17A contributes to skin fibrosis in 2 mouse models of SSc. These findings suggest that inhibition of IL-17A represents a therapeutic target for antagonizing fibrotic skin disorders such as SSc.

Involvement of Interleukin-17A in Pancreatic Damage in Rat Experimental Acute Necrotizing Pancreatitis

Interleukin (IL)-17A is a proinflammatory cytokine, which has recently attracted much interest due to its pathogenic role in various inflammatory conditions such as ischemia/reperfusion injury, chronic inflammation, and autoimmune diseases, but the role of IL-17A in acute pancreatitis remains unclear. This study aimed to investigate the role of IL-17A in experimental acute necrotizing pancreatitis (ANP). We analyzed the expression of IL-17A during the pathogenesis of ANP in vivo induced by 3 % sodium taurocholate (NaTc), by microarray test, quantitative real-time PCR, Western blotting, enzyme-linked immunosorbent assay, and immunohistochemistry. The effects of IL-17A on pancreatic acinar cells and pancreatic stellate cells (PSCs) were further investigated in vitro using recombinant rat IL-17A (rIL-17A). Expression of IL-17A was significantly increased following experimental acute pancreatitis. In addition, rIL-17A induced rat pancreatic acinar cell necrosis and promoted expression of several target genes, including IL-6, IL-1β, CXCL1, CXCL2, and CXCL5, in acinar cells and PSCs. These findings suggest that IL-17A may be involved in pancreatic damage by regulating the expression of inflammatory cytokines and chemokines during experimental acute pancreatitis.

Epidermal IL-17A leads to bone loss through inhibition of osteoblast differentiation

The AP-1 transcription factor family is a central regulator of skin and bone homeostasis. We have previously shown that specific deletion of JunB/AP-1 in epidermis (JunBmice) results in skin inflammation,myeloproliferative disease, lupus-like disease and osteopenia. While upregulation of serum IL-6 and G-CSF are observed in this model, genetic deletion of these cytokines does not rescue osteopenia in JunB mice. Thus, we carried out a screen for other cytokines that are regulated by the loss of JunB in the epidermis. We have identified IL-17A as a cytokine expressed in JunB epidermis in vivo, and hypothesize that IL-17A leads to osteopenia in JunBmice. To test this,we carried out osteoblast and osteoclast differentiation assays in the presence of recombinant IL-17A or serum from JunB mice. Although there were no changes in osteoclast differentiation under these conditions, osteoblast differentiation, as visualized by Alizarin Red staining, was inhibited. Osteoblast differentiation markers were also downregulated in cultures in the presence of recombinant IL-17A or serum from JunB mice. To understand the mechanism by which IL-17A leads to inhibition of osteoblast differentiation, we stimulated osteoblasts with IL-17A in vitro, and observed upregulation in phospho-STAT3, phospho-NFkB andphospho-p38 levels. To determine if IL-17Aupregulation in the epidermis is sufficient to induce bone loss, we analyzed bones of mice ectopically expressing IL-17A in the epidermis. Thesemice displayed bone loss and had decreased levels of serumosteocalcin aswell as decreased osteoblast differentiationmarker expression in vivo. Therefore, thesedata suggest thatepidermal IL002D17A is able to inducebone loss through its action on osteoblast differentiation. These findings will likely be relevant for the treatment of patients with inflammatory diseases and skeletal involvement, such as psoriatic arthritis. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.078 OC08 Depletion of cytotoxic T cells at the time of bone injury enhances bone healing outcome K. Schmidt-Bleek⁎, I. Konnecke, A. Ellinghaus, H.-D. Volk, G.N. Duda, H. Schell Julius Wolff Institut, Charite-Universitatsmedizin Berlin, Berlin, Germany Institut for Medical Immunology, Charite-Universitatsmedizin Berlin,

Depletion of cytotoxic T cells at the time of bone injury enhances bone healing outcome

The AP-1 transcription factor family is a central regulator of skin and bone homeostasis. We have previously shown that specific deletion of JunB/AP-1 in epidermis (JunBmice) results in skin inflammation,myeloproliferative disease, lupus-like disease and osteopenia. While upregulation of serum IL-6 and G-CSF are observed in this model, genetic deletion of these cytokines does not rescue osteopenia in JunB mice. Thus, we carried out a screen for other cytokines that are regulated by the loss of JunB in the epidermis. We have identified IL-17A as a cytokine expressed in JunB epidermis in vivo, and hypothesize that IL-17A leads to osteopenia in JunBmice. To test this,we carried out osteoblast and osteoclast differentiation assays in the presence of recombinant IL-17A or serum from JunB mice. Although there were no changes in osteoclast differentiation under these conditions, osteoblast differentiation, as visualized by Alizarin Red staining, was inhibited. Osteoblast differentiation markers were also downregulated in cultures in the presence of recombinant IL-17A or serum from JunB mice. To understand the mechanism by which IL-17A leads to inhibition of osteoblast differentiation, we stimulated osteoblasts with IL-17A in vitro, and observed upregulation in phospho-STAT3, phospho-NFkB andphospho-p38 levels. To determine if IL-17Aupregulation in the epidermis is sufficient to induce bone loss, we analyzed bones of mice ectopically expressing IL-17A in the epidermis. Thesemice displayed bone loss and had decreased levels of serumosteocalcin aswell as decreased osteoblast differentiationmarker expression in vivo. Therefore, thesedata suggest thatepidermal IL002D17A is able to inducebone loss through its action on osteoblast differentiation. These findings will likely be relevant for the treatment of patients with inflammatory diseases and skeletal involvement, such as psoriatic arthritis. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.078 OC08 Depletion of cytotoxic T cells at the time of bone injury enhances bone healing outcome K. Schmidt-Bleek⁎, I. Konnecke, A. Ellinghaus, H.-D. Volk, G.N. Duda, H. Schell Julius Wolff Institut, Charite-Universitatsmedizin Berlin, Berlin, Germany Institut for Medical Immunology, Charite-Universitatsmedizin Berlin,