CG223, a novel BET inhibitor, exerts TGF-ß1-mediated antifibrotic effects in a murine model of bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis is a progressive disease with poor prognosis and limited therapeutic options. In this study, we evaluated the potential therapeutic effects of CG223, a novel inhibitor of bromodomain and extra-terminal motif (BET) proteins, on pulmonary fibrosis by focusing on the transforming growth factor-ß1 (TGF-ß1) pathway. In a murine model of bleomycin-induced pulmonary fibrosis, CG223 attenuated fibrosis while reducing the infiltration of inflammatory cells into the lungs. Fibroblasts expressing BRD4, a member of the BET protein family, were enriched in the tissue regions corresponding to bleomycin-induced fibrotic lesions. Additionally, pulmonary fibroblasts isolated from bleomycin-instilled mice showed a significantly increased association of BRD4 with the promoters of two pro-fibrotic genes linked to the entry into the TGF-ß1 autocrine/paracrine loop, thrombospondin 1 (Thbs1) and integrin ß3 (Itgb3), as well as with the promoter of a myofibroblast marker gene, actin alpha 2 (Acta2). Subsequent in vitro studies with murine primary lung fibroblasts showed that the mRNA induction of Thbs1, Itgb3, and Acta2 by TGF-ß1 can be inhibited by CG223 in a dose-dependent manner. Taken together, CG223-induced BRD4 inhibition suppressed lung fibrogenesis by affecting multiple genes, including those involved in the triggering of the TGF-ß1 autocrine/paracrine loop.

Risk factors for cytomegalovirus disease with cytomegalovirus re-activation in patients with rheumatic disease.

Objective: To assess risk factors for cytomegalovirus (CMV) disease with CMV re-activation in patients with rheumatic disease.Methods: The clinical data of consecutive patients with rheumatic disease who experienced CMV re-activation were examined. We evaluated the difference in various baseline factors at the first detection of CMV pp65 antigenemia on the development of CMV disease using logistic regression models. The changes of laboratory data in the 4 weeks before CMV re-activation were also assessed.Results: We identified 80 patients (median age [interquartile range] = 65.0 years [51.5-74.0]) with CMV re-activation. Oral candidiasis, serum albumin ≤30 g/L, and CMV pp65-positive cell count >5.6/105 polymorphonuclear leukocyte cells were found to be associated with CMV disease (odds ratio [OR] [95% CI] = 9.99 [2.02-49.50], 11.4 [1.94-67.40] and 6.80 [1.63-28.30], respectively). Moreover, decreases in serum albumin level and blood lymphocyte count in the 4 weeks before CMV re-activation also predicted CMV disease (OR [95% CI] = 2.02 [1.07-3.8] and 1.96 [1.09-3.54], respectively).Conclusion: In CMV re-activation patients with rheumatic disease, the presence of oral candidiasis, high CMV pp65 positive cell count, and hypoalbuminemia are possible risk factors for CMV disease.

Utility of Coagulation Markers for the Prediction of Rapidly Progressive Interstitial Lung Disease in Patients with Dermatomyositis.

We aimed to evaluate the utility of coagulation markers for the prediction of rapidly progressive interstitial lung disease (RP-ILD) in patients with dermatomyositis (DM). In this retrospective study, 29 patients with DM-associated ILD were analyzed. The number of patients with RP-ILD was 15 (52%). The baseline clinical and demographic data and laboratory markers were analyzed to identify predictive factors for RP-ILD.The univariate logistic regression analysis demonstrated that in addition to well-known laboratory markers, such as serum ferritin, KL-6, and lymphocyte counts, a prolonged activated partial thromboplastin time (aPTT) ratio at the time of DM-associated ILD diagnosis was useful for predicting RP-ILD. Moreover, the logistic regression model and receiver operating characteristic curve analysis showed that combinations of the aPTT ratio and well-known laboratory markers were significantly effective in predicting RP-ILD. This study suggested that an association between RP-ILD and the coagulation system exists.

Roles of high-mobility group box 1 and thrombin in murine pulmonary fibrosis and the therapeutic potential of thrombomodulin.

Cross talk between inflammation and coagulation plays important roles in acute or subacute progressive pulmonary fibrosis characterized by diffuse alveolar damage. Thrombomodulin is a physiological inhibitor of high-mobility group box 1 (HMGB1), and thrombin and may be effective for this condition. This study investigated the roles of HMGB1 and thrombin in the pathophysiology of bleomycin-induced pulmonary fibrosis and the efficacy of recombinant human soluble thrombomodulin (rhTM). Pulmonary fibrosis was induced in wild-type C57BL/6 mice by intratracheal instillation of bleomycin. We first assessed HMGB1, thrombin, transforming growth factor (TGF)-ß1, and α-smooth muscle actin (SMA) levels in bronchoalveolar lavage fluid and lung tissue sections over time. Expression of HMGB1 and thrombin was elevated before that of TGF-ß1 and α-SMA and remained high during the fibrotic phase after bleomycin instillation. We next examined whether in vitro stimulation with HMGB1 and thrombin induced expression of TGF-ß1 and α-SMA in cultured alveolar macrophages and lung fibroblasts, respectively, by performing quantitative PCR, enzyme-linked immunosorbent assay, Western blot, and immunofluorescence analyses. HMGB1 and thrombin stimulation induced TGF-ß1 production by alveolar macrophages, and thrombin stimulation also induced α-SMA expression in lung fibroblasts. Finally, we evaluated the effect of rhTM on bleomycin-induced pulmonary fibrosis. Compared with the vehicle control, both early and late-phase administration of rhTM suppressed the fibrotic process. Our results suggest that HMGB1 and thrombin were involved in the pathophysiology of pulmonary fibrosis via production of profibrotic proteins and that rhTM attenuated bleomycin-induced pulmonary fibrosis. rhTM may be a therapeutic option for acute or subacute pulmonary fibrosis.

Role of allograft inflammatory factor-1 in bleomycin-induced lung fibrosis.

Allograft inflammatory factor-1 (AIF-1) is a protein expressed by macrophages infiltrating the area around the coronary arteries in a rat ectopic cardiac allograft model. We previously reported that AIF-1 is associated with the pathogenesis of rheumatoid arthritis and skin fibrosis in sclerodermatous graft-versus-host disease mice. Here, we used an animal model of bleomycin-induced lung fibrosis to analyze the expression of AIF-1 and examine its function in lung fibrosis. The results showed that AIF-1 was expressed on lung tissues, specifically macrophages, from mice with bleomycin-induced lung fibrosis. Recombinant AIF-1 increased the production of TGF-ß which plays crucial roles in the mechanism of fibrosis by mouse macrophage cell line RAW264.7. Recombinant AIF-1 also increased both the proliferation and migration of lung fibroblasts compared with control group. These results suggest that AIF-1 plays an important role in the mechanism underlying lung fibrosis, and may provide an attractive new therapeutic target.

Myeloid-derived suppressor cells play crucial roles in the regulation of mouse collagen-induced arthritis.

Myeloid-derived suppressor cells (MDSCs) are of myeloid origin and are able to suppress T cell responses. The role of MDSCs in autoimmune diseases remains controversial, and little is known about the function of MDSCs in autoimmune arthritis. In this study, we clarify that MDSCs play crucial roles in the regulation of proinflammatory immune response in a collagen-induced arthritis (CIA) mouse model. MDSCs accumulated in the spleens of mice with CIA when arthritis severity peaked. These MDSCs inhibited the proliferation of CD4(+) T cells and their differentiation into Th17 cells in vitro. Moreover, MDSCs inhibited the production of IFN-γ, IL-2, TNF-α, and IL-6 by CD4(+) T cells in vitro, whereas they promoted the production of IL-10. Adoptive transfer of MDSCs reduced the severity of CIA in vivo, which was accompanied by a decrease in the number of CD4(+) T cells and Th17 cells in the draining lymph nodes. However, depletion of MDSCs abrogated the spontaneous improvement of CIA. In conclusion, MDSCs in CIA suppress the progression of CIA by inhibiting the proinflammatory immune response of CD4(+) T cells. These observations suggest that MDSCs play crucial roles in the regulation of autoimmune arthritis, which could be exploited in new cell-based therapies for human rheumatoid arthritis.

The rapid efficacy of abatacept in a patient with rheumatoid vasculitis.

We report a case of rheumatoid vasculitis (RV) that responded well to abatacept, a cytotoxic T lymphocyte-associated antigen 4 (CTLA4)-immunoglobulin fusion protein. A 38-year-old woman developed RV despite treatment with methotrexate and tumor necrosis factor (TNF) inhibitors. The effects of steroid therapy, immunoabsorption plasmapheresis, and interleukin-6 inhibitor were insufficient, however, administration of abatacept rapidly improved her clinical symptoms with almost normalization of the immunological findings. This is the first published case report of the successful treatment of RV with abatacept.

[A case of Goodpasture's syndrome complicated with cytomegalovirus pneumonia during treatment with plasma exchange and immunosuppressive therapy].

A 63-year-old man presented to another hospital with hematuria and an elevated serum creatinine level. He was admitted, and hemodialysis was initiated. One month after admission, hemoptysis developed and the patient was referred to our hospital for further evaluation. Goodpasture's syndrome was diagnosed on the basis of elevated anti-glomerular basement membrane (anti-GBM) antibody levels. Due to massive alveolar hemorrhage, the patient was treated with plasma exchanges, pulse methylprednisolone and pulse cyclophosphamide followed by oral prednisolone, which lowered his anti-GBM antibody level. However, a chest radiograph obtained on the 56th hospital day showed bilateral ground glass opacities, and his condition deteriorated. Since his blood was positive for the cytomegalovirus pp65 antigen, ganciclovir was started. This improved his condition, with improvement in his chest radiograph and a decrease in the number of antigen-positive cells. It is important to consider the development of cytomegalovirus infection during the treatment of Goodpasture's syndrome with immunosuppressive therapy.

Upregulation of sphingosine-1-phosphate receptor 3 on fibroblast-like synoviocytes is associated with the development of collagen-induced arthritis via increased interleukin-6 production.

BACKGROUND: Sphingosine-1-phosphate receptor 3 (S1P3) is one of five receptors for sphingosine-1-phosphate (S1P). S1P/S1P3 signaling is involved in numerous physiological and pathological processes including bone metabolism, sepsis, cancer, and immunity. In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLSs) are activated by several factors and promote abundant proinflammatory cytokine production and bone destruction. The aim of this study was to investigate whether S1P3 is associated with the development of autoimmune arthritis and the pathogenic function of FLSs. METHODS: Wild-type (WT) and S1P3 knockout (S1P3-KO) collagen-induced arthritis (CIA) mice were evaluated with respect to clinical and histological disease severity, along with the levels of anti-collagen antibodies and expression of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6). S1P3 expression in the synovium was analyzed by real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence staining. FLSs isolated from CIA mice were activated with TNFα and S1P3 expression was analyzed by real-time RT-PCR. The role of S1P/S1P3 signaling in activated and non-activated FLSs was investigated by measuring cell proliferation and cyto/chemokine production by real-time RT-PCR and/or enzyme-linked immunosorbent assay. RESULTS: Clinical and histological scores, and synovial IL-6 expression were significantly lower in S1P3-KO mice with CIA than in WT mice. Arthritic synovia had higher S1P3 expression than intact synovia and FLSs in arthritic joints expressed S1P3 in vivo. Primary cultured FLSs produced IL-6 in a time-dependent manner in response to S1P stimulation and exhibited higher levels of S1P3 expression after activation with TNFα. S1P3-induced production of IL-6 and MMP-3 was increased in FLSs pre-activated with TNFα. CONCLUSION: In this study, we demonstrated that S1P3 expression is associated with the development of autoimmune arthritis via inflammation-induced increases in S1P/S1P3 signaling that increase production of IL-6 in FLSs. Inhibition of S1P/S1P3 signaling could open the door to the development of new therapies for RA.

Efficacy of abatacept in patients with rheumatoid arthritis, as assessed by magnetic resonance imaging of bilateral hands.

AIM: To examine the efficacy of abatacept in patients with rheumatoid arthritis (RA) using magnetic resonance imaging (MRI) of bilateral hands. METHOD: This prospective study included 35 RA patients. MRI of bilateral hands was performed at baseline and after 12 months of treatment with intravenous abatacept. MRI images were scored for synovitis, osteitis, erosion and joint space narrowing (JSN) according to the RA MRI Scoring System (RAMRIS). The primary endpoint was the change in RAMRIS score from baseline. Repair of erosion was defined as a negative change in the erosion score that was greater than the smallest detectable changes (SDCs). RESULTS: Thirty-one patients completed the study. Median synovitis and osteitis scores showed statistically significant reductions at Month 12 when compared to baseline (synovitis score, -5.5 [P < 0.0001]; osteitis score, -0.5 [P = 0.03]). However, median erosion and JSN scores did not significantly change. At Month 12, 83% of patients showed no progression of erosion scores and repair of erosion was observed in 11% of patients. All patients with repair of erosion achieved functional remission (Health Assessment Questionnaire-Disability Index ≤ 0.5). The Simplified Disease Activity Index response rate at Month 1 was identified as an independent factor predicting changes in the erosion scores at Month 12. CONCLUSION: Abatacept treatment reduced synovitis and osteitis scores and did not worsen erosion and JSN scores at Month 12. Over 10% of patients experienced repair of erosion.

Allograft inflammatory factor-1 in the pathogenesis of bleomycin-induced acute lung injury.

Allograft inflammatory factor-1 (AIF-1) is a protein expressed by macrophages infiltrating the area around the coronary arteries of rats with an ectopic cardiac allograft. Some studies have shown that expression of AIF-1 increased in a mouse model of trinitrobenzene sulfonic acid-induced acute colitis and in acute cellular rejection of human cardiac allografts. These results suggest that AIF-1 is related to acute inflammation. The current study used bleomycin-induced acute lung injury to analyze the expression of AIF-1 and to examine its function in acute lung injury. Results showed that AIF-1 was significantly expressed in lung macrophages and increased in bronchoalveolar lavage fluid from mice with bleomycin-induced acute lung injury in comparison to control mice. Recombinant AIF-1 increased the production of IL-6 and TNF-α from RAW264.7 (a mouse macrophage cell line) and primary lung fibroblasts, and it also increased the production of KC (CXCL1) from lung fibroblasts. These results suggest that AIF-1 plays an important role in the mechanism underlying acute lung injury.

Once-weekly teriparatide improves glucocorticoid-induced osteoporosis in patients with inadequate response to bisphosphonates.

BACKGROUND: Patients with glucocorticoid-induced osteoporosis (GIOP) are at very high risk of fracture, and patients with severe GIOP often experience fractures during treatment with bisphosphonates. Teriparatide (TPTD) is the only currently available anabolic agent expected to be effective for GIOP. Once-weekly TPTD decreased bone resorption marker with primary osteoporosis different from daily TPTD, but it has not yet been tested with GIOP. OBJECTIVES: To evaluate the efficacy of once-weekly TPTD for patients with GIOP and inadequate response to bisphosphonates. METHODS: Patients with GIOP and collagen diseases treated with prednisolone for at least 6 months with inadequate responses to bisphosphonates were administered once-weekly TPTD. Bone density of the lumbar spine and femoral neck, measured as percent young adult mean (YAM); serum concentrations of cross-linked N-terminal telopeptides of type I collagen (NTx), bone alkaline phosphatase (BAP), and calcium; and FRAX were measured at baseline and 6, 12 and 18 months after starting TPTD. RESULTS: Of the 12 GIOP patients with collagen diseases enrolled, nine (seven females, two males; mean age 57.4 ± 11.1 years) completed treatment, including six with systemic lupus erythematosus, two with rheumatoid arthritis, and one with adult onset still disease. Only one new fracture event, a lumbar compression fracture, occurred during the study period, although seven patients experienced eight fracture events within 18 months before starting TPTD (p = 0.04). Lumbar spine YAM significantly improved at 18 months (p = 0.04), whereas femoral neck YAM did not (p = 0.477). Serum NTx, BAP, Ca, and FRAX were not significantly affected by TPTD treatment. CONCLUSIONS: Once-weekly TPTD reduces fracture events and increases bone density of the lumbar spine of GIOP patients with inadequate response to bisphosphonates.

Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

OBJECTIVE: Synovial fluid pH is decreased in patients with rheumatoid arthritis (RA); however, the underlying mechanisms are unclear. We undertook this study to examine the mechanism by which synovial fluid pH is regulated and to explore the possibility of a therapeutic strategy by manipulating this mechanism. METHODS: We determined the pH and lactate concentration in synovial fluid from 16 RA patients. Cultured synovial fibroblasts (SFs) from the inflamed joints of 9 RA patients (RASFs) were examined for the expression of ion transporters that regulate intracellular and extracellular pH. The ion transporter up-regulated in RASF lines was then suppressed in RASFs by small interfering RNA (siRNA), and the effect of transfection on viability and proliferation was investigated. Finally, we examined the therapeutic effect of electrotransfer of monocarboxylate transporter 4 (MCT4)-specific siRNA into the articular synovium of mice with collagen-induced arthritis (CIA). RESULTS: Synovial fluid pH correlated inversely with both the Disease Activity Score in 28 joints using the C-reactive protein level and the synovial fluid lactate levels. RASFs exhibited up-regulated transcription of MCT4 messenger RNA. MCT4 exported intracellular lactate into the extracellular space. RASFs had significantly higher MCT4 protein levels than did SFs from patients with osteoarthritis. Knockdown of MCT4 induced intrinsic apoptosis of RASFs, thereby inhibiting their proliferation. Moreover, electrotransfer of MCT4-specific siRNA into the articular synovium of mice with CIA significantly reduced the severity of arthritis. CONCLUSION: RA activity correlated with decreased synovial fluid pH. This may be due to increased MCT4 expression in RASFs. Silencing MCT4 induced apoptosis in RASFs and reduced the severity of CIA, suggesting that MCT4 is a potential therapeutic target for inflammatory arthritis.

Knock out of S1P3 receptor signaling attenuates inflammation and fibrosis in bleomycin-induced lung injury mice model.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in many critical cellular processes, including proliferation, migration, and angiogenesis, through interaction with a family of five G protein-coupled receptors (S1P1-5). Some reports have implicated S1P as an important inflammatory mediator of the pathogenesis of airway inflammation, but the role of S1P3 in the pathogenesis of lung diseases is not completely understood. We used S1P3-deficient (knockout (KO)) mice to clarify the role of S1P3 receptor signaling in the pathogenesis of pulmonary inflammation and fibrosis using a bleomycin-induced model of lung injury. On the seventh day after bleomycin administration, S1P3 KO mice exhibited significantly less body weight loss and pulmonary inflammation than wild-type (WT) mice. On the 28th day, there was less pulmonary fibrosis in S1P3 KO mice than in WT mice. S1P3 KO mice demonstrated a 56% reduction in total cell count in bronchoalveolar lavage fluid (BALF) collected on the seventh day compared with WT mice; however, the differential white blood cell profiles were similar. BALF analysis on the seventh day showed that connective tissue growth factor (CTGF) levels were significantly decreased in S1P3 KO mice compared with WT mice, although no differences were observed in monocyte chemotactic protein-1 (MCP-1) or transforming growth factor ß1 (TGF-ß1) levels. Finally, S1P levels in BALF collected on the 7th day after treatment were not significantly different between WT and S1P3 KO mice. Our results indicate that S1P3 receptor signaling plays an important role in pulmonary inflammation and fibrosis and that this signaling occurs via CTGF expression. This suggests that this pathway might be a therapeutic target for pulmonary fibrosis.