Tofacitinib Inhibits STAT Phosphorylation and Matrix Metalloproteinase-3, -9 and -13 Production by C28/I2 Human Juvenile Chondrocytes.

Purpose: This in vitro study was designed to determine the effect of the pan-Janus kinase inhibitor, Tofacitinib, on basal and interleukin-6 (IL-6)-induced signal transducers and activators of transcription (STAT) phosphorylation and matrix metalloproteinase (MMP) gene expression and MMP production by C28/I2 human chondrocytes. Methods: C28/I2 chondrocytes were grown to a confluent high-density and treated either with recombinant human IL-6 (rhIL-6; 10-20ng/mL) or maintained in the basal state for up to 60 min. MMP gene expression was determined using RT-PCR and MMP production by semi-quantitative immunohistochemistry. The effect of IL-6 with or without Tofacitinib on activation of STAT proteins was determined from quantitative Western blots. Results: C28/I2 chondrocytes produced STAT1, STAT3 and STAT5AB which were phosphorylated (p) following treatment with rhIL-6 for 30 min. Tofacitinib (2.5nM-100nM) decreased rhIL-6-induced activation of STAT1, STAT3, and STAT5AB as well as decreasing the expression of MMP3 and MMP13 but not MMP9, MMP1 or MMP2. In addition, Tofacitinib (50nM) reduced the number of rhIL-6-induced MMP3-, and MMP13- antibody-positive C28/I2 chondrocytes. However, Tofacitinib did decrease the number of MMP9-antibody-positive C28/I2 chondrocytes. Conclusion: Taken together, these data showed that Tofacitinib, a pan-JAK small molecule inhibitor employed for the medical therapy of rheumatoid arthritis was a potent inhibitor of rhIL-6-induced STAT phosphorylation that appeared to be coupled to the inhibition of MMP-3, -9 and -13 production by C28/I2 chondrocytes.

Extracellular Signal-Regulated Kinase: A Regulator of Cell Growth, Inflammation, Chondrocyte and Bone Cell Receptor-Mediated Gene Expression.

Extracellular signal-regulated kinase (ERK) is a member of the mitogen-activated protein kinase family of signaling molecules. ERK is predominantly found in two forms, ERK1 (p44) and ERK2 (p42), respectively. There are also several atypical forms of ERK, including ERK3, ERK4, ERK5 and ERK7. The ERK1/2 signaling pathway has been implicated in many and diverse cellular events, including proliferation, growth, differentiation, cell migration, cell survival, metabolism and transcription. ERK1/2 is activated (i.e., phosphorylated) in the cytosol and subsequently translocated to the nucleus, where it activates transcription factors including, but not limited to, ETS, c-Jun, and Fos. It is not surprising that the ERK1/2 signaling cascade has been implicated in many pathological conditions, namely, cancer, arthritis, chronic inflammation, and osteoporosis. This narrative review examines many of the cellular events in which the ERK1/2 signaling cascade plays a critical role. It is anticipated that agents designed to inhibit ERK1/2 activation or p-ERK1/2 activity will be developed for the treatment of those diseases characterized by dysregulated gene expression through ERK1/2 activation.

Inhibition of MMPs and ADAM/ADAMTS.

Matrix metalloproteinases (MMPs), A Disintegrin and Metalloproteinase (ADAM) and A Disintegrin and Metalloproteinase with Thrombospondin Motif (ADAMTS) are zinc-dependent endopeptidases that play a critical role in the destruction of extracellular matrix proteins and, the shedding of membrane-bound receptor molecules in various forms of arthritis and other diseases. Under normal conditions, MMP, ADAM and ADAMTS gene expression aids in the maintenance of homeostasis. However, in inflamed synovial joints characteristic of rheumatoid arthritis and osteoarthritis. MMP, ADAM and ADAMTS production is greatly increased under the influence of pro-inflammatory cytokines. Analyses based on medicinal chemistry strategies designed to directly inhibit the activity of MMPs have been largely unsuccessful when these MMP inhibitors were employed in animal models of rheumatoid arthritis and osteoarthritis. This is despite the fact that these MMP inhibitors were largely able to suppress pro-inflammatory cytokine-induced MMP production in vitro. A focus on ADAM and ADAMTS inhibitors has also been pursued. Thus, recent progress has identified the "sheddase" activity of ADAMs as a viable target and the development of GW280264X is an experimental ADAM17 inhibitor. Of note, a monoclonal antibody, GLPG1972, developed as an ADAMTS-5 inhibitor, entered a Phase I OA clinical trial. However, the failure of many of these previously developed inhibitors to move beyond the preclinical testing phase has required that novel strategies be developed that are designed to suppress both MMP, ADAM and ADAMTS production and activity.

Progesterone and cAMP synergize to inhibit responsiveness of myometrial cells to pro-inflammatory/pro-labor stimuli.

Progesterone (P4) acting through the P4 receptor (PR) isoforms, PR-A and PR-B, promotes uterine quiescence for most of pregnancy, in part, by inhibiting the response of myometrial cells to pro-labor inflammatory stimuli. This anti-inflammatory effect is inhibited by phosphorylation of PR-A at serine-344 and -345 (pSer344/345-PRA). Activation of the cyclic adenosine monophosphate (cAMP) signaling pathway also promotes uterine quiescence and myometrial relaxation. This study examined the cross-talk between P4/PR and cAMP signaling to exert anti-inflammatory actions and control pSer344/345-PRA generation in myometrial cells. In the hTERT-HMA/B immortalized human myometrial cell line P4 inhibited responsiveness to interleukin (IL)-1ß and forskolin (increases cAMP) and 8-Br-cAMP increased this effect in a concentration-dependent and synergistic manner that was mediated by activation of protein kinase A (PKA). Forskolin also inhibited the generation of pSer344/345-PRA and expression of key contraction-associated genes. Generation of pSer344/345-PRA was catalyzed by stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Forskolin inhibited pSer344/345-PRA generation, in part, by increasing the expression of dual specificity protein phosphatase 1 (DUSP1), a phosphatase that inactivates mitogen-activated protein kinases (MAPKs) including SAPK/JNK. P4/PR and forskolin increased DUSP1 expression. The data suggest that P4/PR promotes uterine quiescence via cross-talk and synergy with cAMP/PKA signaling in myometrial cells that involves DUSP1-mediated inhibition of SAPK/JNK activation.

Defective T-Cell Apoptosis and T-Regulatory Cell Dysfunction in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic, progressive, systemic autoimmune disease that mostly affects small and large synovial joints. At the molecular level, RA is characterized by a profoundly defective innate and adaptive immune response that results in a chronic state of inflammation. Two of the most significant alterations in T-lymphocyte (T-cell) dysfunction in RA is the perpetual activation of T-cells that result in an abnormal proliferation state which also stimulate the proliferation of fibroblasts within the joint synovial tissue. This event results in what we have termed "apoptosis resistance", which we believe is the leading cause of aberrant cell survival in RA. Finding therapies that will induce apoptosis under these conditions is one of the current goals of drug discovery. Over the past several years, a number of T-cell subsets have been identified. One of these T-cell subsets are the T-regulatory (Treg) cells. Under normal conditions Treg cells dictate the state of immune tolerance. However, in RA, the function of Treg cells become compromised resulting in Treg cell dysfunction. It has now been shown that several of the drugs employed in the medical therapy of RA can partially restore Treg cell function, which has also been associated with amelioration of the clinical symptoms of RA.

Red Cell Distribution Width Is Positively Correlated with Atherosclerotic Cardiovascular Disease 10-Year Risk Score, Age, and CRP in Spondyloarthritis with Axial or Peripheral Disease.

BACKGROUND: Red blood cell distribution width (RDW) is a routine hematologic parameter that is a predictor of cardiovascular disease (CVD) events and is independent of combined traditional risk factor scoring systems. The RDW has also been associated with rheumatic disease activity. Whether RDW is associated with traditional CVD risk factors or Atherosclerotic Cardiovascular Disease (ASCVD) 10-year CVD risk score in patients with seronegative spondyloarthritis with axial or peripheral disease has not been previously determined. METHODS: We performed a retrospective, chart review study evaluating the relationship between RDW, albumin, hemoglobin, C-reactive protein (CRP), absolute lymphocyte count (ALC), and ASCVD scoring parameters [age, hypertension status, diabetes mellitus (DM) status, lipid profile, and smoking status] in a cohort of spondyloarthritis patients, taking into consideration their HLA-B27 status, race, and treatment status. RESULTS: RDW was found to positively correlate with ASCVD 10-year score and age, and ASCVD score did not change over time after patients were treated for spondyloarthritis. Albumin was found to negatively correlate with ASCVD 10-year risk score. Both RDW and albumin correlated with CRP. ALC failed to correlate with ASCVD 10-year score but did show a tendency to be associated with CVD, CVD events, and cardiac conduction abnormalities. CONCLUSIONS: These data indicate that further study is warranted to evaluate RDW, albumin level, and ALC as potential predictors of CVD in the spondyloarthritis patient population.

MicroRNAs and Osteoarthritis.

An imbalance in gene expressional events skewing chondrocyte anabolic and catabolic pathways toward the latter causes an aberrant turnover and loss of extracellular matrix proteins in osteoarthritic (OA) articular cartilage. Thus, catabolism results in the elevated loss of extracellular matrix proteins. There is also evidence of an increase in the frequency of chondrocyte apoptosis that compromises the capacity of articular cartilage to undergo repair. Although much of the fundamental OA studies over the past 20 years identified and characterized many genes relevant to pro-inflammatory cytokines, apoptosis, and matrix metalloproteinases (MMPs)/a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS), more recent studies focused on epigenetic mechanisms and the associated role of microRNAs (miRs) in regulating gene expression in OA cartilage. Thus, several miRs were identified as regulators of chondrocyte signaling pathways, apoptosis, and proteinase gene expression. For example, the reduced expression of miR-146a was found to be coupled to reduced type II collagen (COL2) in OA cartilage, whereas MMP-13 levels were increased, suggesting an association between MMP-13 gene expression and COL2A1 gene expression. Results of these studies imply that microRNAs could become useful in the search for diagnostic biomarkers, as well as providing novel therapeutic targets for intervention in OA.

The role of the JAK/STAT signal pathway in rheumatoid arthritis.

Proinflammatory cytokine activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signal transduction pathway is a critical event in the pathogenesis and progression of rheumatoid arthritis. Under normal conditions, JAK/STAT signaling reflects the influence of negative regulators of JAK/STAT, exemplified by the suppressor of cytokine signaling and protein inhibitor of activated STAT. However, in rheumatoid arthritis (RA) both of these regulators are dysfunctional. Thus, continuous activation of JAK/STAT signaling in RA synovial joints results in the elevated level of matrix metalloproteinase gene expression, increased frequency of apoptotic chondrocytes and most prominently 'apoptosis resistance' in the inflamed synovial tissue. Tofacitinib, a JAK small molecule inhibitor, with selectivity for JAK2/JAK3 was approved by the United States Food and Drug Administration (US FDA) for the therapy of RA. Importantly, tofacitinib has demonstrated significant clinical efficacy for RA in the post-US FDA-approval surveillance period. Of note, the success of tofacitinib has spurred the development of JAK1, JAK2 and other JAK3-selective small molecule inhibitors, some of which have also entered the clinical setting, whereas other JAK inhibitors are currently being evaluated in RA clinical trials.

A Role for Soluble IL-6 Receptor in Osteoarthritis.

Interleukin-6 (IL-6) is one of several pro-inflammatory cytokines present at elevated levels in the synovial fluid of individuals with confirmed clinical diagnosis of rheumatoid arthritis (RA) and osteoarthritis (OA). The mechanism of action of IL-6 was shown to involve its capacity to interact with a membrane-bound IL-6 receptor (mIL-6Rα), also known as the "classical" IL-6 pathway, or through its interaction with a soluble IL-6 receptor (sIL-6R) termed the "trans-signaling" pathway. Activation of downstream signaling is transduced via these IL-6 receptors and principally involves the Janus Kinase/Signal Transduction and Activators of Transcription (JAK/STAT) signaling pathway that is further regulated by glycoprotein-130 (gp130) interacting with the IL-6/mIL-6R complex. Phosphorylation of STAT proteins via JAK activation facilitates STAT proteins to act as transcription factors in inflammation. However, the biological function(s) of the sIL-6R in human chondrocytes requires further elucidation, although we previously showed that exogenous sIL-6R significantly suppressed the synthesis of neutrophil gelatinase-associated lipocalin (NGAL) in the immortalized line of human chondrocytes, C28/I2. NGAL was shown to regulate the activity of matrix metalloproteinase-9 (MMP-9), whose activity is crucial in OA for the destruction of articular cartilage. The "shedding" of sIL-6R from the plasma membrane is carried out by a family of enzymes known as A Distintegrin and Metalloproteinase (ADAM), which are also elevated in OA. In this paper, we have systematically reviewed the role played by IL-6 in OA. We have proposed that sIL-6R may be an important target for future drug development in OA by ameliorating cartilage extracellular protein degradation.

Phosphorylation of STAT proteins by recombinant human IL-6 in immortalized human chondrocyte cell lines, T/C28a2 and C28/I2.

Two immortalized human juvenile chondrocyte cell lines, T/C28a2 and C28/I2, were employed to determine the extent to which recombinant human (rh) IL-6, a known cytokine activator of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway in many cell types, caused STAT proteins to be phosphorylated. The results showed that STAT3 was constitutively phosphorylated in the absence of rhIL-6 in T/C28a2 chondrocytes. However, C28/I2 chondrocytes treated with rhIL-6 caused STAT1, STAT3, and STAT5 to be phosphorylated without altering total unphosphorylated STAT proteins. STAT3 phosphorylation in response to rhIL-6 in T/C28a and C28/I2 chondrocytes was efficiently blocked by the JAK3-selective inhibitor WHI-P131 (Janex-1) and by soluble IL-6 receptor-α (sIL-6R). However, the combination of rhIL-6 and ruxolitinib, a JAK1/JAK2-selective inhibitor, was a less effective inhibitor of STAT protein activation. These findings showed that rhIL-6 activated STAT proteins in the C28/I2 chondrocyte cell line. STAT protein phosphorylation could be blocked by a JAK3-selective inhibitor or by the combination of rhIL-6 and sIL-6R.

Matrix Metalloproteinases and Synovial Joint Pathology.

Matrix metalloproteinases (MMPs) are zinc-dependent enzymes. These enzymes play a critical role in the destruction of articular cartilage in rheumatoid arthritis (RA), osteoarthritis (OA), psoriatic arthritis (PsA), and the spondyloarthropathies. MMP gene expression is upregulated in these synovial joint pathologies in response to elevated levels of proinflammatory cytokines and soluble mediators such as tumor necrosis factor-α, interleukin-1 (IL-1), IL-6, IL-17, and interferon-γ. These molecules are capable of activating the mitogen-activated protein kinase and Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways by binding the cytokine to their respective receptors on immune cells, macrophages, chondrocytes, synoviocytes, and osteocytes leading to increased synthesis of MMPs. Biologic drugs and/or small-molecule inhibitors designed to block cytokine to cytokine receptor interactions or to selectively inhibit JAKs have clinical efficacy in RA, PsA, and ankylosing spondylitis which correlated with a reduction in MMPs. Although there are currently no OA-selective drugs, it is likely that such a drug would have to reduce MMP gene expression to have clinical efficacy.

Idiopathic Inflammatory Myopathies: A Review of the Classification and Impact of Pathogenesis.

Idiopathic inflammatory myopathies (IIMs) are a group of autoimmune muscle diseases with significant morbidity and mortality. This review details and updates the pathogenesis and emerging importance of myositis-specific antibodies in the development of IIMs. An increase in the understanding of how these myositis-specific antibodies play a role in IIMs has led to the further categorization of IIMs from the traditional polymyositis versus dermatomyositis, to additional subcategories of IIMs such as necrotizing autoimmune myositis (NAM). The diagnosis of IIMs, including manual muscle testing, laboratory studies, and non-invasive imaging have become important in classifying IIM subtypes and for identifying disease severity. Treatment has evolved from an era where glucocorticoid therapy was the only option to a time now that includes traditional steroid-sparing agents along with immunoglobulin therapy and biologics, such as rituximab.

Negative Regulators of JAK/STAT Signaling in Rheumatoid Arthritis and Osteoarthritis.

Elevated levels of pro-inflammatory cytokines are generally thought to be responsible for driving the progression of synovial joint inflammation in rheumatoid arthritis (RA) and osteoarthritis (OA). These cytokines activate several signal transduction pathways, including the Janus kinase/Signal Transducers and Activators of Transcription (JAK/STAT), Stress-Activated/Mitogen-Activated Protein Kinase (SAPK/MAPK) and phosphatidylinositol-3-kinase/Akt/mechanistic target of rapamycin (PI3K/Akt/mTOR) pathways which regulate numerous cellular responses. However, cytokine gene expression, matrix metalloproteinase gene expression and aberrant immune cell and synoviocyte survival via reduced apoptosis are most critical in the context of inflammation characteristic of RA and OA. Negative regulation of JAK/STAT signaling is controlled by Suppressor of Cytokine Signaling (SOCS) proteins. SOCS is produced at lower levels in RA and OA. In addition, gaining further insight into the role played in RA and OA pathology by the inhibitors of the apoptosis protein family, cellular inhibitor of apoptosis protein-1, -2 (c-IAP1, c-IAP2), X (cross)-linked inhibitor of apoptosis protein (XIAP), protein inhibitor of activated STAT (PIAS), and survivin (human) as well as SOCS appears to be a worthy endeavor going forward.

Human Parturition Involves Phosphorylation of Progesterone Receptor-A at Serine-345 in Myometrial Cells.

The hypothesis that phosphorylation of progesterone receptor (PR) isoforms, PR-A and PR-B, in myometrial cells affects progesterone action in the context of human parturition was tested. Immunodetection of phosphoserine (pSer) PR forms in term myometrium revealed that the onset of labor is associated with increased phosphorylation of PR-A at serine-345 (pSer345-PRA) and that pSer345-PRA localized to the nucleus of myometrial cells. In explant cultures of term myometrium generation of pSer345-PRA was induced by interleukin-1ß and dependent on progesterone, suggesting that pSer345-PRA generation is induced by a proinflammatory stimulus. In the hTERT-HMA/B human myometrial cell line, abundance of pSer345-PRA was induced by progesterone in a dose- (EC50 ∼1 nM) and time-dependent manner. Prevention of pSer345 (by site-directed mutagenesis) abolished the capacity for PR-A to inhibit anti-inflammatory actions of progesterone mediated by PR-B but had no effect on the transrepressive activity of PR-A at a canonical progesterone response element. Taken together, the data show that human parturition involves the phosphorylation of PR-A at serine-345 in myometrial cells and that this process is ligand dependent and induced by a proinflammatory stimulus. We also found that in myometrial cells, pSer345 activates the capacity for PR-A to inhibit antiinflammatory actions of progesterone mediated by PR-B. Phosphorylation of PR-A at serine-345 may be an important functional link between tissue-level inflammation and PR-A-mediated functional progesterone withdrawal to trigger parturition.

Matrix Metalloproteinase-9 Production by Immortalized Human Chondrocyte Lines.

We reported at the Keynote Forum of Immunology Summit-2015 that recombinant human (rh) TNF-α or rhIL-6 stimulated production of matrix metalloproteinase-9 (MMP-9) in the T/C28a2 and C-28/I2 human immortalized chondrocyte cell lines. Furthermore, we reported that tocilizumab (TCZ), a fully humanized monoclonal antibody which neutralizes IL-6-mediated signaling, inhibited the rhIL-6-mediated increase in the production of MMP-9. IL-6 is also a known activator of the JAK/STAT signaling pathway. In that regard, we evaluated the effect of rhIL-6 on total and phosphorylated Signal Transducer and Activator of Transcription by these chondrocyte lines which showed that whereas STAT3 was constitutively phosphorylated in T/C28a2 chondrocytes, rhIL-6 activated STAT3 in C-28/I2 chondrocytes. The finding that rhIL-6 increased the production of MMP-9 by human immortalized chondrocyte cell lines may have important implications with respect to the destruction of articular cartilage in rheumatoid arthritis and osteoarthritis. Thus, the markedly elevated level of IL-6 in rheumatoid arthritis and osteoarthritis sera and synovial fluid would be expected to generate significant MMP-9 to cause the degradation of articular cartilage extracellular matrix proteins. The finding that TCZ suppressed rhIL-6-mediated MMP-9 production suggests that TCZ, currently employed in the medical therapy of rheumatoid arthritis, could be considered as a drug for osteoarthritis.

Recent strategies for drug development in fibromyalgia syndrome.

INTRODUCTION: The US Federal Drug Administration (FDA) approved 3 medications for treating fibromyalgia syndrome (FMS). There have been no additional FDA approvals since January 2009 and the efficacy of the FDA-approved medications for FMS has been questioned. Areas covered: The "search for studies" tool using clinicaltrials.gov and PubMed were employed. The term, "fibromyalgia" was used for clinicaltrials.gov. The terms employed for PubMed were "Name-of-Drug Fibromyalgia", "Fibromyalgia Treatment" or "Fibromyalgia Drug Treatment." Clinical trials were reviewed if they were prospective and blinded, and if they employed a comparator, either placebo or another pharmaceutical. Expert commentary: Progress toward standardizing the outcome measures for FMS clinical trials have been made but challenges remain. Several pharmaceutical candidates for FMS have been tested since 2009. The results of these studies with potential novel targets for drug development for FMS were reviewed including the results of trials with sodium oxybate, quetiapine, esreboxetine, nabilone, memantine, naltrexone, and melatonin.

The Relationship between NALP3 and Autoinflammatory Syndromes.

The nucleotide-binding domain, leucine-rich repeat/pyrin domain-containing-3 (NALP3) inflammasome, which is required for synthesis of interleukin-1ß, has been implicated in the pathogenesis of several autoinflammatory syndromes. This review of the literature summarizes the interconnectedness of NALP3 inflammasome with some of these disorders. Familial Mediterranean fever results from a mutation in the Mediterranean fever (MEFV) gene, which encodes the pyrin protein. Previous study results suggest that pyrin suppresses caspase-1 activation, perhaps by competing for the adaptor protein, termed, pyrin domain of apoptosis/speck-like protein containing a caspase-recruitment domain (ACS) which therefore interferes with NALP3 inflammasome activation. The nucleotide-binding domain, leucine-rich repeat/pyrin domain-containing-3 (NALP3) inflammasome is constitutively activated in cryopyrin-associated periodic syndromes due to gain-of-function mutations resulting from point mutations within the neuronal apoptosis inhibitor protein/class 2 transcription factor/heterokaryon incompatibility/telomerase-associated protein-1 (NACHT) domain of the NALP3 protein. Pyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome is caused by mutations in the genes encoding proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1). These PSTPIP1 mutants are thought to bind to pyrin causing an increase in the pyrin domain of apoptosis/speck-like protein containing a caspase-recruitment domain (ASC) pyroptosome assembly leading to procaspase-1 recruitment and therefore its activation. Hyperimmunoglublinemia D syndrome is caused by mevalonate kinase (MVK) deficiency, which may be affected by protein accumulation that leads to NALP3 inflammasome activation. Tumor necrosis factor receptor-associated periodic syndrome is associated with mutations in the tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A) gene which decreases the level of soluble tumor necrosis factor receptor-1 (TNFR1) leading to neutralization of tumor necrosis factor (TNF)-α. In general, these autoinflammatory disorders have shown a clinical response to interleukin-1 (IL-1) antagonists, suggesting that the NALP3 inflammasome serves a critical role in their pathogenesis.

JAK/STAT regulation of Aspergillus fumigatus corneal infections and IL-6/23-stimulated neutrophil, IL-17, elastase, and MMP9 activity.

IL-6 and IL-23 (IL-6/23) induce IL-17A (IL-17) production by a subpopulation of murine and human neutrophils, resulting in autocrine IL-17 activation, enhanced production of reactive oxygen species, and increased fungal killing. As IL-6 and IL-23 receptors trigger JAK1, -3/STAT3 and JAK2/STAT3 phosphorylation, respectively, we examined the role of this pathway in a murine model of fungal keratitis and also examined neutrophil elastase and gelatinase (matrix metalloproteinase 9) activity by IL-6/23-stimulated human neutrophils in vitro. We found that STAT3 phosphorylation of neutrophils in Aspergillus fumigatus-infected corne as was inhibited by the JAK/STAT inhibitor Ruxolitinib, resulting in impaired fungal killing and decreased matrix metalloproteinase 9 activity. In vitro, we showed that fungal killing by IL-6/23-stimulated human peripheral blood neutrophils was impaired by JAK/STAT inhibitors Ruxolitinib and Stattic, and by the retinoic acid receptor-related orphan receptor γt inhibitor SR1001. This was also associated with decreased reactive oxygen species, IL-17A production, and retinoic acid receptor-related orphan receptor γt translocation to the nucleus. We also demonstrate that IL-6/23-activated neutrophils exhibit increased elastase and gelatinase (matrix metalloproteinase 9) activity, which is inhibited by Ruxolitinib and Stattic but not by SR1001. Taken together, these observations indicate that the regulation of activity of IL-17-producing neutrophils by JAK/STAT inhibitors impairs reactive oxygen species production and fungal killing activity but also blocks elastase and gelatinase activity that can cause tissue damage.

STAT1 is Constitutively Activated in the T/C28a2 Immortalized Juvenile Human Chondrocyte Line and Stimulated by IL-6 Plus Soluble IL-6R.

T/C28a2 immortalized juvenile human chondrocytes were employed to determine the extent to which activation of Signal Transducers and Activators of Transcription-1 (STAT1) occurred in response to recombinant human interleukin-6 (rhIL-6) or rhIL-6 in combination with the soluble IL-6 receptor (sIL-6R). Two forms of STAT1, STAT1A and STAT1B, were identified on SDS-PAGE and western blotting with anti-STAT1 antibody. Western blotting revealed that STAT1 was constitutively phosphorylated (p-STAT1). Although incubation of T/C28a2 chondrocytes with rhIL-6 (50 ng/ml) increased p-STAT1A by Δ=22.3% after 30 min, this percent difference failed to reach significance by Chi-square analysis. Similarly, no effect of rhIL-6 (Δ=+10.7%) on p-STAT1B was seen at 30 min. In contrast, although the combination of rhIL-6 plus sIL-6R had no effect on p-STAT1A, rhIL-6 plus sIL-6R increased p-STAT1B by Δ=73.3% (p<0.0001) after 30 min compared to the control group and by Δ=56.7% (p<0.0001) compared to rhIL-6 alone. Janex-1, a Janus kinase-3-specific inhibitor (100 µM) partially reduced the effect of rhIL-6 on p-STAT1B by Δ=27.7% (p<0.05). The results of this study showed that STAT1A/STAT1B was constitutively activated in T/C28a2 chondrocytes. Although rhIL-6 increased p-STAT1B to a small extent, the combination of rhIL-6 plus sIL-6R was far more effective in stimulating STAT1B phosphorylation compared to controls or rhIL-6 alone. These data support the likelihood that although JAK3-mediated activation of STAT1 in T/C28a2 chondrocytes may involve the IL-6/IL-6R/gp130 pathway, these results indicated that STAT1 activation in response to IL-6 preferentially involved IL-6 trans-signaling via sIL-6R.