Cadherin-11 targeted cell-specific liposomes enabled skin fibrosis treatment by inducing apoptosis.

Continuous and aberrant activation of myofibroblasts is the hallmark of pathological fibrosis (e.g., abnormal wound healing). The deposition of excessive extracellular matrix (ECM) components alters or increases the stiffness of tissue and primarily accounts for multiple organ dysfunctions. Among various proteins, Cadherin-11 (CDH11) has been reported to be overexpressed on myofibroblasts in fibrotic tissues. Anti-apoptotic proteins such as (B cell lymphoma-2) (BCL-2) are also upregulated on myofibroblasts. Therefore, we hypothesize that CDH11 could be a targeted domain for cell-specific drug delivery and targeted inhibition of BCL-2 to ameliorate the development of fibrosis in the skin. To prove our hypothesis, we have developed liposomes (LPS) conjugated with CDH11 neutralizing antibody (antiCDH11) to target cell surface CDH11 and loaded these LPS with a BCL-2 inhibitor, Navitoclax (NAVI), to induce apoptosis of CDH11 expressing fibroblasts. The developed LPS were evaluated for physicochemical characterization, stability, in vitro therapeutic efficacy using dermal fibroblasts, and in vivo therapeutic efficacy in bleomycin-induced skin fibrosis model in mice. The findings from in vitro and in vivo studies confirmed that selectivity of LPS was improved towards CDH11 expressing myofibroblasts, thereby improving therapeutic efficacy with no indication of adverse effects. Hence, this novel research work represents a versatile LPS strategy that exhibits promising potential for treating skin fibrosis.

Factors Associated With Adherence of Cervical Cancer Screening in Women With Systemic Lupus Erythematosus.

OBJECTIVE: The objective is to determine cervical cancer screening rates and factors associated with decreased cervical cancer screening in women with systemic lupus erythematosus (SLE). METHODS: We conducted a cross-sectional study that enrolled consecutive women (age 21-64 years) with SLE. We collected demographics, clinical characteristics, constructs of the Health Beliefs Model (HBM) (ie, susceptibility, severity, barriers, benefits, cues to action, and self-efficacy), and self-reported cervical cancer screening (confirmed with the electronic medical record). The primary outcome was adherence to cervical cancer screening according to current guidelines. Multivariable logistic regression models were used to examine the association between SLE disease activity and cervical cancer screening and explore mediation effects from HBM constructs. RESULTS: We enrolled 130 women with SLE. The median age was 42 years (interquartile range 32-52 years). The cervical cancer screening adherence rate was 61.5%. Women with high SLE disease activity were less likely to have cervical cancer screening versus those with low disease activity (odds ratio 0.59, 95% confidence interval [CI] 0.39-0.89; P = 0.01), which remained statistically significant after adjusting for baseline demographics and drug therapy in a multivariable model (odds ratio 0.25, 95% CI 0.08-0.79; P = 0.02). Regarding the HBM constructs, increased perceived barriers to cervical cancer screening (r = -0.30, P < 0.01) and decreased self-efficacy (r = -0.21, P = 0.02) correlated with decreased cervical cancer screening. CONCLUSION: Patients with SLE with high disease activity undergo cervical cancer screening less frequently than those with low disease activity. Perceived barriers to cervical cancer screening are moderately correlated with decreased screening. These data highlight the need to develop strategies to increase cervical cancer screening in this high-risk patient population.

CADHERIN-11 regulation of myeloid phagocytes and autoimmune inflammation in murine lupus.

BACKGROUND AND OBJECTIVE: Understanding the regulation of efferocytosis by myeloid phagocytes is important in identifying novel targets in systemic lupus erythematosus (SLE). Cadherin-11 (CDH11), a cell adhesion molecule, is implicated in inflammatory arthritis and fibrosis and recently been shown to regulate macrophage phagocytosis. The extent and mechanism of this regulation is unknown. Our objective was to examine the extent to which CDH11 regulates myeloid phagocytes and contributes to autoimmunity and tissue inflammation. METHODS: We analyzed efferocytosis in macrophages and dendritic cells (DCs) from WT and Cdh11-/- mice and investigated the mechanisms in vitro. We investigated the role of CDH11 in disease development in vivo using the pristane induced lupus model. To translate the clinical relevance of CDH11 in human disease, we measured serum CDH11 levels in two independent pediatric SLE (pSLE) cohorts and healthy controls. RESULTS: Using bone marrow derived macrophages (BMDMs) and DCs (BMDCs), we found impaired efferocytosis in phagocytes from Cdh11-/- mice, mediated by downregulated efferocytosis receptor expression and RhoGTPase activation. Specifically, loss of CDH11 downregulated Mertk expression and Rac1 activation in BMDMs, and integrin αVß3 expression and Cdc42 activation in BMDCs, highlighting distinct pathways. In vivo, Cdh11-/- mice displayed defective efferocytosis and increased accumulation of apoptotic debris in pristane-induced lupus. Further, Cdh11-/- mice had enhanced systemic inflammation and autoimmune inflammation with increased anti-dsDNA autoantibodies, splenomegaly, type I interferons, and inflammatory cytokines. Paradoxically, at the tissue level, Cdh11-/- mice were protected against glomerulonephritis, indicating a dual role in murine lupus. Finally, SLE patients had increased serum CDH11 compared to controls. CONCLUSION: This study highlights a novel role of CDH11 in regulating myeloid cells and efferocytosis and its potential as a contributor to development in autoimmunity murine lupus. Despite the increase in autoimmunity, Cdh11-/- mice developed decreased tissue inflammation and damage.

Risk of incident cardiovascular events with disease-modifying anti-rheumatic drugs among adults with rheumatoid arthritis: a nested case-control study.

OBJECTIVE: This study examined the risk of cardiovascular disease (CVD) associated with the disease-modifying anti-rheumatic drugs (DMARDs) in rheumatoid arthritis (RA). METHOD: This nested case-control study used the MarketScan database (2012-2014), involving adult RA patients (aged ≥18 years) initiating either a conventional synthetic (cs) DMARD, biologic DMARD, or targeted synthetic (ts) DMARD between January 1, 2013 and December 31, 2014 (cohort entry) and had no CVD history. Cases were individuals with incident CVD identified using diagnosis codes or procedure codes from medical claims. For each case, 10 age- and sex-matched controls were selected using the incident density sampling with replacement. Prescriptions of DMARDs were measured 90 days before the event date. Conditional logistic regression examined the association of risk of CVD with DMARDs in combination treatment or individual use, with reference to methotrexate (MTX) monotherapy, adjusting for baseline confounders. Subgroup analyses were performed separately in DMARD combination therapy users or individual DMARD users, respectively. RESULTS: In total, 270 cases of incident CVD and 2700 controls were included (mean [standard deviation (SD)] age: 54 [1]; 75.6% women). The commonly prescribed DMARD therapies were csDMARD monotherapy (n = 795, 27.04%), followed by  tumor necrosis factor inhibitors (TNFi) monotherapy (n = 367, 12.48%), and TNFi in combination with MTX (n = 314, 10.68%). Compared with MTX monotherapy, overall use of DMARD agents was not associated with the differential risk of CVD, including various types of DMARD combination regimens. The findings were similar across subgroup analyses. CONCLUSIONS: The study found no differential risk of CVD with DMARDs in combination therapy or monotherapy compared to MTX monotherapy in patients with RA. Key Points • This study evaluated the risk of cardiovascular disease (CVD) associated with the disease-modifying anti-rheumatic drugs (DMARDs) in rheumatoid arthritis (RA). • Findings suggest no differential CVD risk with DMARDs in combination with MTX or used individually compared with MTX monotherapy in patients with early RA. • Further efforts should focus on a better understanding of the mechanism of DMARD combination treatments with MTX in modifying CV risk.

Real-world Comparative Effectiveness of Methotrexate-based Combinations for Rheumatoid Arthritis: A Retrospective Cohort Study.

PURPOSE: Guidelines recommend using disease-modifying antirheumatic drugs (DMARDs) in combination with methotrexate (MTX) for patients with rheumatoid arthritis (RA) after monotherapy. Little is known about the real-world comparative effectiveness of these MTX-DMARD combinations. This study compared the effectiveness of various MTX-based DMARD combinations for patients with RA initiating MTX-DMARD combination therapy using administrative claims database. METHODS: This retrospective cohort study included adults (aged ≥18 years) with RA who initiated MTX combination treatment with conventional synthetic DMARDs (csDMARDs), tumor necrosis factor inhibitor (TNFi) biologic DMARDs (bDMARDs), non-TNFi bDMARDs, or targeted synthetic DMARDs (tsDMARDs) between July 1, 2012, and December 31, 2013 (index date), from the MarketScan Commercial Claims Data. Patients had continuous enrollment from the 6 months of preindex period until the 12 months of postindex period. The MTX-based DMARD combination therapy cohort was defined as ≥1 MTX prescription in the first 30 days from the index date and ≥14 days overlapping use of the prescription fills of the MTX and the index DMARD. Effectiveness was measured by using the claims algorithm (dosing, switching, addition, oral glucocorticoid use, or multiple glucocorticoid injection). Propensity score analysis with the inverse probability of treatment weighting (PS-IPTW), estimated by using the generalized boosted machine learning method, was used to balance the distribution of baseline variables between the combination groups. Multivariable logistic regression using PS-IPTW was conducted to compare the effectiveness of the combination groups. Sensitivity analysis evaluated the modified effectiveness algorithms or the time to the first treatment failure. FINDINGS: A total of 3174 adult patients with RA starting an MTX-DMARD combination therapy were identified (mean [SD] age, 50 [9] years), including 1568 (49%) initiating a csDMARD + MTX, 1343 (42%) initiating TNFi + MTX, and 240 (8%) initiating non-TNFi bDMARD + MTX, and 23 (1%) initiating tsDMARD + MTX. Owing to the small sample, the tsDMARD combination group was not included in the comparative analysis. Algorithm-based therapy effectiveness was found in 9.95% of the csDMARD + MTX, 20.48% of the TNFi + MTX, and 20.83% of the non-TNFi + MTX groups. PS-IPTW showed that the csDMARD combination is less effective (adjusted odds ratio, 0.422; 95% CI, 0.341-0.524) than the TNFi combination; however, the non-TNFi biologic combination had similar effectiveness (aOR, 1.063; 95% CI, 0.680-1.662) compared to the TNFi combination. Sensitivity analyses confirmed the main results. IMPLICATIONS: Among RA patients initiating MTX-DMARD combinations, both non-TNFi biologics and TNFi-based combinations with MTX were equally effective, but csDMARD + MTX was less effective than the TNFi plus MTX.

Factors influencing prescribing the first add-on disease-modifying antirheumatic drugs in patients initiating methotrexate for rheumatoid arthritis.

Background: Advances in Disease-Modifying Antirheumatic Drugs (DMARDs) have expanded the treatment landscape for Rheumatoid Arthritis (RA). Guidelines recommend adding either conventional synthetic (cs), biologic (b), or targeted synthetic (ts) DMARDs to methotrexate (MTX) for managing RA. Limited evidence exists regarding the factors that contribute to adding a DMARD agent to the MTX regimen. This study examined the factors associated with adding the first DMARD in RA patients initiating MTX. Methods: This retrospective cohort study utilized the MarketScan data (2012-2014) involving adults (aged ≥18) with RA initiating an MTX (index date) between Jul 1, 2012 and Dec 30, 2013, and with continuous enrollment for the 6-month pre-index period. The combination therapy users received the first treatment addition of DMARD starting from day 30 after the index MTX over one year period. The study focused on the addition of csDMARDs, Tumor Necrosis Factor Inhibitors (TNFi) bDMARDs, non-TNFi bDMARDs, or tsDMARDs. Baseline covariates were measured in the 6-month pre-index and grouped into predisposing, enabling, and need factors, as per the Andersen Behavior Model. Multivariable logistic regression examined the factors associated with the addition of TNFi compared to adding a csDMARD. An additional regression model evaluated the factors associated with adding any biologic (combining TNFi and non-TNFi biologics). Results: Among 8350 RA patients starting MTX, 31.92% (n = 2665) initiated any DMARD within the 1-year post-index period. Among RA patients initiating a DMARD prescription after starting MTX, 945 (11.32%) received combination therapy with treatment addition of a DMARD to MTX regimen; majority added TNFi (550, 58%), followed by csDMARD (352, 37%); non-TNF biologic (40, 4%), or tsDMARD (3, 0.3%). The tsDMARD group was limited and was not included for further analysis. The multivariable model found Preferred Provider Organization insurance coverage (odds ratio [OR], 1.43; 95% confidence interval (CI), 1.06-1.93), chronic pulmonary disease (OR, 1.98; 95% CI, 1.14-3.44), liver disease (OR, 5.24; 95% CI, 1.77-15.49), and Elixhauser score (OR, 0.91; 95% CI, 0.86-0.97) were significantly associated with the addition of TNF-α inhibitors. The separate multivariable model additionally found that patients from metropolitan areas (OR, 1.50; 95% CI, 1.04-2.16) were positively associated with adding any biological agent. Conclusions: TNFi are often added to MTX for managing RA. Enabling and need factors contribute to the prescribing of a TNFi add-on therapy in RA. Future research should examine the impact of these combination therapies on RA management.

Oral delivery of RNAi for cancer therapy.

Cancer is a major health concern worldwide and is still in a continuous surge of seeking for effective treatments. Since the discovery of RNAi and their mechanism of action, it has shown promises in targeted therapy for various diseases including cancer. The ability of RNAi to selectively silence the carcinogenic gene makes them ideal as cancer therapeutics. Oral delivery is the ideal route of administration of drug administration because of its patients' compliance and convenience. However, orally administered RNAi, for instance, siRNA, must cross various extracellular and intracellular biological barriers before it reaches the site of action. It is very challenging and important to keep the siRNA stable until they reach to the targeted site. Harsh pH, thick mucus layer, and nuclease enzyme prevent siRNA to diffuse through the intestinal wall and thereby induce a therapeutic effect. After entering the cell, siRNA is subjected to lysosomal degradation. Over the years, various approaches have been taken into consideration to overcome these challenges for oral RNAi delivery. Therefore, understanding the challenges and recent development is crucial to offer a novel and advanced approach for oral RNAi delivery. Herein, we have summarized the delivery strategies for oral delivery RNAi and recent advancement towards the preclinical stages.

Cadherin-11 and Its Role in Tissue Fibrosis.

Fibrosis is the excessive deposition of extracellular matrix that results from chronic inflammation and injury, leading to the loss of tissue integrity and function. Cadherins are important adhesion molecules that classically mediate calcium-dependent cell-to-cell adhesion and play important roles in tissue development and cellular migration but likely have functions beyond these important roles. Cadherin-11 (CDH11), a member of the cadherin family, has been implicated in several pathological processes including cancer. More recent evidence suggests that CDH11 is a central mediator of tissue fibrosis. CDH11 expression is increased in patients with fibrotic diseases such as idiopathic pulmonary fibrosis and systemic sclerosis. CDH11 expression is increased in mouse models of lung, skin, liver, cardiac, renal, and intestinal fibrosis. Targeting CDH11 in murine models of fibrosis clearly demonstrates that CDH11 is a common mediator of fibrosis across multiple tissues. Insight into potential mechanisms at the cellular and molecular level is emerging. In this review, we present the evolving evidence for the involvement of CDH11 in tissue fibrosis. We also discuss some of the proposed mechanisms and highlight the potential of CDH11 as a common therapeutic target and biomarker in different fibrotic pathologies.

Retinoic Acid Receptor Activation Reduces Metastatic Prostate Cancer Bone Lesions by Blocking the Endothelial-to-Osteoblast Transition.

Metastatic prostate cancer in the bone induces bone-forming lesions that contribute to progression and therapy resistance. Prostate cancer-induced bone formation originates from endothelial cells (EC) that have undergone endothelial-to-osteoblast (EC-to-OSB) transition in response to tumor-secreted BMP4. Current strategies targeting prostate cancer-induced bone formation are lacking. Here, we show that activation of retinoic acid receptor (RAR) inhibits EC-to-OSB transition and reduces prostate cancer-induced bone formation. Treatment with palovarotene, an RARγ agonist being tested for heterotopic ossification in fibrodysplasia ossificans progressiva, inhibited EC-to-OSB transition and osteoblast mineralization in vitro and decreased tumor-induced bone formation and tumor growth in several osteogenic prostate cancer models, and similar effects were observed with the pan-RAR agonist all-trans-retinoic acid (ATRA). Knockdown of RARα, ß, or γ isoforms in ECs blocked BMP4-induced EC-to-OSB transition and osteoblast mineralization, indicating a role for all three isoforms in prostate cancer-induced bone formation. Furthermore, treatment with palovarotene or ATRA reduced plasma Tenascin C, a factor secreted from EC-OSB cells, which may be used to monitor treatment response. Mechanistically, BMP4-activated pSmad1 formed a complex with RAR in the nucleus of ECs to activate EC-to-OSB transition. RAR activation by palovarotene or ATRA caused pSmad1 degradation by recruiting the E3-ubiquitin ligase Smad ubiquitination regulatory factor1 (Smurf1) to the nuclear pSmad1/RARγ complex, thus blocking EC-to-OSB transition. Collectively, these findings suggest that palovarotene can be repurposed to target prostate cancer-induced bone formation to improve clinical outcomes for patients with bone metastasis. SIGNIFICANCE: This study provides mechanistic insights into how RAR agonists suppress prostate cancer-induced bone formation and offers a rationale for developing RAR agonists for prostate cancer bone metastasis therapy. See related commentary by Bhowmick and Bhowmick, p. 2975.

The utility of erythrocyte sedimentation rate, C-reactive protein, and procalcitonin in detecting infections in patients with systemic lupus erythematosus: A systematic review.

OBJECTIVES: We conducted a systematic review with metanalysis to investigate the utility of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and procalcitonin (PCT) in diagnosing infections in hospitalized patients with SLE. METHODS: We searched Medline, Embase, Web of Science, ClinicalTrials.gov, and Cochrane Central Register of Controlled Trials (CENTRAL) with a search strategy developed by a medical librarian. We included retrospective, cross-sectional, case-control, and prospective studies in our analysis. We used the Quality Assessment of Diagnostic Studies (QUADAS-2) to assess for bias and applicability. We obtained mean differences, sensitivities, and specificities in our analysis. RESULTS: We included 26 studies in our analysis. Most studies had an unclear or high risk of bias and our results were widely heterogenous. For the diagnosis of infections, the CRP had a pooled sensitivity of 0.75 (95%CI 0.57-0.94) and specificity of 0.72 (0.59-0.85), PCT had a pooled sensitivity of 0.68 (95% CI 0.0.59-0.77) and specificity of 0.75 (0.59-0.90), and for ESR pooled estimates were not calculated but sensitivity ranged from 50 to 69.8 and specificity from 38.5 to 55.6. Modifying cut-offs improved sensitivities and specificities. The ESR, CRP, and PCT mean differences were all greater in infection groups versus non-infection (10.1, 95% CI 3.2-17.0; 46.8, 95% CI 36.5-57.0; 0.53, 95% CI 0.26-0.80; respectively). DISCUSSION: Poor sensitivities and specificities were observed for the evaluated biomarkers with substantial heterogeneity in the cut-offs used to determine infection. Although mean biomarker values were increased in the infection group compared with the non-infection, our findings do not support the widespread use of ESR, CRP, or PCT in diagnosing infection in hospitalized patients with SLE due to increased heterogeneity and risk of bias. Further investigation is needed.

Cadherin-11 Regulates Macrophage Development and Function.

Cadherin-11 (CDH11) is a cell-cell adhesion protein that has previously been reported to play an important role in the pathogenesis of pulmonary fibrosis. It is expressed on macrophages in the fibrotic lung. However, the role of CDH11 on macrophage biology has not yet been studied. We show using immunophenotypic analyses that Cdh11-/- mice have fewer recruited monocyte-derived macrophages and Ly6Chi monocytes in the lungs compared to wild-type mice in the intraperitoneal bleomycin-induced pulmonary fibrosis model. Additionally, fewer Ly6Chi monocytes were detected in the bone marrow and peripheral blood of naive Cdh11-/- mice. Given that macrophages are derived from monocytes, we investigated the precursors of the monocyte/macrophage lineage in the bone marrow. We found increased numbers of CMPs and reduced numbers of GMPs and MPs/cMoPs in Cdh11-/- mice compared to wild-type mice, suggesting decreased differentiation towards the myeloid lineage in Cdh11-/- mice. Furthermore, we show using bone marrow cells that loss of CDH11 impaired monocyte to macrophage differentiation. We also demonstrate that CDH11 deficiency repressed the M2 program and impaired the phagocytic function of bone marrow-derived macrophages. Overall, our findings demonstrate a role for CDH11 in macrophage development, M2 polarization, and phagocytic function.

Macrophages and cadherins in fibrosis and systemic sclerosis.

PURPOSE OF REVIEW: Macrophages are key players in systemic sclerosis (SSc) and fibrosis. The mechanism by which macrophages regulate fibrogenesis is unclear and understanding the origin and function of macrophages is critical to developing effective therapeutics. Novel targets on macrophages are under investigation and recently, cadherins have emerged as a potential therapeutic target on macrophages. The current review will discuss the importance of macrophages in SSc and fibrosis and summarize recent studies on the role of cadherin-11 (Cdh11) on macrophages and fibrosis. RECENT FINDINGS: Genome-wide expression studies demonstrate the importance of macrophages in SSc and fibrosis. Although M2 macrophages are associated with fibrosis, the presence of a mixed M1/M2 phenotype in fibrosis has recently been reported. Several studies aiming to identify macrophage subsets involved in fibrogenesis suggest that monocyte-derived alveolar macrophages are key players in the development of murine lung fibrosis. Recent functional studies show that Cdh11 regulates macrophages, fibroblast invasion, and adhesion of macrophages to myofibroblasts. SUMMARY: Macrophages play an important role in SSc and fibrosis. New insights into the mechanisms by which macrophages regulate fibrogenesis have been discovered on the basis of Cdh11 studies and suggest that targeting Cdh11 may be an effective target to treat fibrosis.

Beyond Autoantibodies: Biologic Roles of Human Autoreactive B Cells in Rheumatoid Arthritis Revealed by RNA-Sequencing.

OBJECTIVE: To obtain the comprehensive transcriptome profile of human citrulline-specific B cells from patients with rheumatoid arthritis (RA). METHODS: Citrulline- and hemagglutinin-specific B cells were sorted by flow cytometry using peptide-streptavidin conjugates from the peripheral blood of RA patients and healthy individuals. The transcriptome profile of the sorted cells was obtained by RNA-sequencing, and expression of key protein molecules was evaluated by aptamer-based SOMAscan assay and flow cytometry. The ability of these proteins to effect differentiation of osteoclasts and proliferation and migration of synoviocytes was examined by in vitro functional assays. RESULTS: Citrulline-specific B cells, in comparison to citrulline-negative B cells, from patients with RA differentially expressed the interleukin-15 receptor α (IL-15Rα) gene as well as genes related to protein citrullination and cyclic AMP signaling. In analyses of an independent cohort of cyclic citrullinated peptide-seropositive RA patients, the expression of IL-15Rα protein was enriched in citrulline-specific B cells from the patients' peripheral blood, and surprisingly, all B cells from RA patients were capable of producing the epidermal growth factor ligand amphiregulin (AREG). Production of AREG directly led to increased migration and proliferation of fibroblast-like synoviocytes, and, in combination with anti-citrullinated protein antibodies, led to the increased differentiation of osteoclasts. CONCLUSION: To the best of our knowledge, this is the first study to document the whole transcriptome profile of autoreactive B cells in any autoimmune disease. These data identify several genes and pathways that may be targeted by repurposing several US Food and Drug Administration-approved drugs, and could serve as the foundation for the comparative assessment of B cell profiles in other autoimmune diseases.

The Antifibrotic Effect of A2B Adenosine Receptor Antagonism in a Mouse Model of Dermal Fibrosis.

OBJECTIVE: Systemic sclerosis (SSc; scleroderma) is a chronic disease that affects the skin and various internal organs. Dermal fibrosis is a major component of this disease. The mechanisms that promote dermal fibrosis remain elusive. Elevations in tissue adenosine levels and the subsequent engagement of the profibrotic A2B adenosine receptor (ADORA2B) have been shown to regulate fibrosis in multiple organs including the lung, kidney, and penis; however, the role of ADORA2B in dermal fibrosis has not been investigated. We undertook this study to test our hypothesis that elevated expression of ADORA2B in the skin drives the development of dermal fibrosis. METHODS: We assessed the involvement of ADORA2B in the regulation of dermal fibrosis using a well-established mouse model of dermal fibrosis. Using an orally active ADORA2B antagonist, we demonstrated how inhibition of ADORA2B results in reduced dermal fibrosis in 2 distinct experimental models. Finally, using human dermal fibroblasts, we characterized the expression of adenosine receptors. RESULTS: We demonstrated that levels of ADORA2B were significantly elevated in dermal fibrosis and that the therapeutic blockade of this receptor in vivo using an ADORA2B antagonist could reduce the production of profibrotic mediators in the skin and attenuate dermal fibrosis. Antagonism of ADORA2B resulted in reduced numbers of arginase-expressing macrophages and myofibroblasts and in reduced levels of the extracellular matrix proteins fibronectin, collagen, and hyaluronan. CONCLUSION: These findings identify ADORA2B as a potential profibrotic regulator in dermal fibrosis and suggest that ADORA2B antagonism may be a useful approach for the treatment of SSc.

Vitamin D Levels, Vitamin D Receptor Polymorphisms, and Inflammatory Cytokines in Aromatase Inhibitor-Induced Arthralgias: An Analysis of CCTG MA.27.

BACKGROUND: Approximately half of women taking aromatase inhibitor (AI) therapy develop AI-induced arthralgia (AIA), and many might discontinue AI therapy because of the pain. Using plasma samples from the MA.27 study, we assessed several factors potentially associated with AIA. PATIENTS AND METHODS: MA.27 is a phase III adjuvant trial comparing 2 AIs, exemestane versus anastrozole. Within an 893-participant nested case-control AIA genome-wide association study, we nested a 72 AIA case-144 control assessment of vitamin D plasma concentrations, corrected for seasonal and geographic variation. We also examined 9 baseline inflammatory cytokines: interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, interferon (IFN)γ, IL-10, IL-12p70, IL-17, IL-23, and chemokine ligand (CCL)-20. Finally, we analyzed the multivariate effects of baseline factors: vitamin D level, previously identified musculoskeletal single nucleotide polymorphisms, age, body mass index, and vitamin D receptor (VDR) Fok-I variant genotype on AIA development. RESULTS: Changes in vitamin D from baseline to 6 months were not significantly different between cases and controls. Elevated inflammatory cytokine levels were not associated with development of AIA. The multivariate model included no clinical factors associated with AIA. However, women with the VDR Fok-I variant genotype were more likely to have a lower IL-1ß level (P = .0091) and less likely to develop AIA after 6 months of AI compared with those with the wild type VDR (P < .0001). CONCLUSION: In this nested case-control correlative study, vitamin D levels were not significantly associated with development of AIA; however, patients with the Fok-I VDR variant genotype were more likely to have a significant reduction in IL-1ß level, and less likely to develop AIA.

Role of STAT3 in skin fibrosis and transforming growth factor beta signalling.

Objective: SSc is an autoimmune disease characterized by progressive fibrosis of the skin and internal organs. IL-6 and related cytokines that signal through STAT3 have been implicated in the pathogenesis of SSc and mouse models of fibrosis. The aim of this study was to investigate the efficacy of inhibiting STAT3 in the development of fibrosis in two mouse models of skin fibrosis. Methods: Biopsy samples of skin from SSc patients and healthy control subjects were used to determine the expression pattern of phosphotyrosyl (pY705)-STAT3. C188-9, a small molecule inhibitor of STAT3, was used to treat fibrosis in the bleomycin-induced fibrosis model and Tsk-1 mice. In vitro studies were performed to determine the extent to which STAT3 regulates the fibrotic phenotype of dermal fibroblasts. Results: Increased STAT3 and pY705-STAT3 was observed in SSc skin biopsies and in both mouse models of SSc. STAT3 inhibition with C188-9 resulted in attenuated skin fibrosis, myofibroblast accumulation, pro-fibrotic gene expression and collagen deposition in both mouse models of skin fibrosis. C188-9 decreased in vitro dermal fibroblast production of fibrotic genes induced by IL-6 trans-signalling and TGF-ß. Finally, TGF-ß induced phosphotyrosylation of STAT3 in a SMAD3-dependent manner. Conclusion: STAT3 inhibition decreases dermal fibrosis in two models of SSc. STAT3 regulates dermal fibroblasts function in vitro and can be activated by TGF-ß. These data suggest that STAT3 is a potential therapeutic target for dermal fibrosis in diseases such as SSc.

Glycolytic requirement for NK cell cytotoxicity and cytomegalovirus control.

NK cell activation has been shown to be metabolically regulated in vitro; however, the role of metabolism during in vivo NK cell responses to infection is unknown. We examined the role of glycolysis in NK cell function during murine cytomegalovirus (MCMV) infection and the ability of IL-15 to prime NK cells during CMV infection. The glucose metabolism inhibitor 2-deoxy-ᴅ-glucose (2DG) impaired both mouse and human NK cell cytotoxicity following priming in vitro. Similarly, MCMV-infected mice treated with 2DG had impaired clearance of NK-specific targets in vivo, which was associated with higher viral burden and susceptibility to infection on the C57BL/6 background. IL-15 priming is known to alter NK cell metabolism and metabolic requirements for activation. Treatment with the IL-15 superagonist ALT-803 rescued mice from otherwise lethal infection in an NK-dependent manner. Consistent with this, treatment of a patient with ALT-803 for recurrent CMV reactivation after hematopoietic cell transplant was associated with clearance of viremia. These studies demonstrate that NK cell-mediated control of viral infection requires glucose metabolism and that IL-15 treatment in vivo can reduce this requirement and may be effective as an antiviral therapy.

Targeting of cadherin-11 decreases skin fibrosis in the tight skin-1 mouse model.

OBJECTIVE: Systemic sclerosis (SSc) is an autoimmune disease clinically manifesting as progressive fibrosis of the skin and internal organs. Cadherin-11 (CDH11) expression is increased in fibrotic skin and lung tissue. Targeting CDH11 may be an effective approach to treating fibrosis. We hypothesize that targeting CDH11 will decrease fibrosis in the tight skin-1 (Tsk-1) mouse model. METHODS: CDH11 expression was determined in the Tsk-1 mouse model using quantitative real time PCR and immunofluorescence (IF). Inhibitory anti- CDH11 monoclonal antibodies were tested in Tsk-1 mice for their ability to decrease hypodermal fibrosis. RESULTS: Expression of CDH11 was increased in fibrotic skin from Tsk-1 mice compared to pallid controls. IF staining demonstrated that CDH11 expression localized to fibroblasts within the hypodermis of fibrotic skin. Treatment with inhibitory anti-CDH11 monoclonal antibodies decreased hypodermal thickness and fibrotic mediators in Tsk-1 mice compared to control antibodies. CONCLUSIONS: These data demonstrate an important role for CDH11 in the development of skin fibrosis in Tsk-1 mice. These data add to the growing evidence for the important role of CDH11 in tissue fibrosis and fibrotic disease such as systemic sclerosis.

CCL2 in the Circulation Predicts Long-Term Progression of Interstitial Lung Disease in Patients With Early Systemic Sclerosis: Data From Two Independent Cohorts.

OBJECTIVE: There are few clinical predictors of the progression of systemic sclerosis (SSc)-related interstitial lung disease (ILD). The purpose of this study was to examine the predictive significance of key cytokines for long-term progression of ILD and survival in 2 independent cohorts of patients with early SSc. METHODS: Plasma levels of 11 Th1/Th2 cytokines (interleukin-1ß [IL-1ß], IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, tumor necrosis factor, CCL2, interferon-inducible T cell α chemoattractant, and interferon-γ-inducible 10-kd protein) were measured in 266 patients with early SSc in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) discovery cohort. Levels of CCL2, IL-10, and IL-6 were measured in 171 patients with early SSc in the Canadian Scleroderma Research Group (CSRG) replication cohort. The primary outcome measure was a decline in the forced vital capacity percent predicted (FVC%) value over time. A joint analysis of longitudinal FVC% values and survival was performed. RESULTS: After adjustment for age, sex, and ethnicity, CCL2 and IL-10 were found to be significant predictors of ILD progression in the discovery cohort. Higher CCL2 levels predicted a faster decline in FVC% values (b = -0.57, P = 0.032), while higher IL-10 levels predicted a slower decline (b = 0.26, P = 0.01). A higher CCL2 value was also predictive of poorer survival (hazard ratio 1.76, P = 0.030). In the CSRG replication cohort, higher CCL2 levels predicted a faster decline in FVC% values (b = -0.58, P = 0.038), but neither IL-10 nor IL-6 had predictive significance. A higher CCL2 level also predicted poorer survival (hazard ratio 3.89, P = 0.037). CONCLUSION: Higher CCL2 levels in the circulation were predictive of ILD progression and poorer survival in patients with early SSc, findings that support the notion that CCL2 has a role as a biomarker and potential therapeutic target.

Cadherin-11 is a novel regulator of extracellular matrix synthesis and tissue mechanics.

We discovered that Cadherin-11 (CDH11) regulates collagen and elastin synthesis, both affecting the mechanical properties and contractile function of animal tissues. Using a Cdh11-null mouse model, we observed a significant reduction in the mechanical properties [Youngs' modulus and ultimate tensile strength (UTS)] of Cdh11(-/-) as compared to wild-type (WT) mouse tissues, such as the aorta, bladder and skin. The deterioration of mechanical properties (Youngs' modulus and UTS) was accompanied by reduced collagen and elastin content in Cdh11(-/-) mouse tissues as well as in cells in culture. Similarly, knocking down CDH11 abolished collagen and elastin synthesis in human cells, and consequently reduced their ability to generate force. Conversely, engagement of CDH11 through homophilic interactions, led to swift activation of the TGF-ß and ROCK pathways as evidenced by phosphorylation of downstream effectors. Subsequently, activation of the key transcription factors, MRTF-A (also known as MKL1) and MYOCD led to significant upregulation of collagen and elastin genes. Taken together, our results demonstrate a novel role of adherens junctions in regulating extracellular matrix (ECM) synthesis with implications for many important biological processes, including maintenance of tissue integrity, wound healing and tissue regeneration.