The Follistatin-like Protein 1 Pathway Is Important for Maintaining Healthy Articular Cartilage.

OBJECTIVE: We sought to determine whether follistatin-like protein 1 (FSTL1), a protein produced by articular chondrocytes, promotes healthy articular cartilage and prevents chondrocytes from undergoing terminal differentiation to hypertrophic cells. METHODS: In vitro experiments were performed with immortalized human articular chondrocytes. The cells were transduced with a lentivirus encoding human FSTL1 small hairpin RNA or with an adenovirus encoding FSTL1. A quantitative polymerase chain reaction was used for gene expression analysis. Protein expression was assessed by Western blotting. Co-immunoprecipitation was used to identify interacting partners of FSTL1. FSTL1 expression in human articular cartilage was analyzed using confocal microscopy. RESULTS: Downregulation of FSTL1 expression in transforming growth factor ß (TGFß)-stimulated chondrocyte pellet cultures led to chondrocyte terminal differentiation characterized by poor production of cartilage extracellular matrix and altered expression of genes and proteins involved in cartilage homeostasis, including MMP13, COL10A1, RUNX2, COL2A1, ACAN, Sox9, and phospho-Smad3. We also showed that FSTL1 interacts with TGFß receptor proteins, Alk1 and endoglin, suggesting a potential mechanism for its effects on chondrocytes. Transduction of chondrocytes with an FSTL1 transgene increased COL2A1 expression, whereas it did not affect MMP13 expression. FSTL1 protein expression was decreased in human osteoarthritic cartilage in situ. CONCLUSION: Our data suggest that FSTL1 plays an important role in maintaining healthy articular cartilage and the FSTL1 pathway may represent a therapeutic target for degenerative diseases of cartilage.

FSTL-1 Attenuation Causes Spontaneous Smoke-Resistant Pulmonary Emphysema.

Rationale: The role of FSTL-1 (follistatin-like 1) in lung homeostasis is unknown.Objectives: We aimed to define the impact of FSTL-1 attenuation on lung structure and function and to identify FSTL-1-regulated transcriptional pathways in the lung. Further, we aimed to analyze the association of FSTL-1 SNPs with lung disease.Methods: FSTL-1 hypomorphic (FSTL-1 Hypo) mice underwent lung morphometry, pulmonary function testing, and micro-computed tomography. Fstl1 expression was determined in wild-type lung cell populations from three independent research groups. RNA sequencing of wild-type and FSTL-1 Hypo mice identified FSTL-1-regulated gene expression, followed by validation and mechanistic in vitro examination. FSTL1 SNP analysis was performed in the COPDGene (Genetic Epidemiology of Chronic Obstructive Pulmonary Disease) cohort.Measurements and Main Results: FSTL-1 Hypo mice developed spontaneous emphysema, independent of smoke exposure. Fstl1 is highly expressed in the lung by mesenchymal and endothelial cells but not immune cells. RNA sequencing of whole lung identified 33 FSTL-1-regulated genes, including Nr4a1, an orphan nuclear hormone receptor that negatively regulates NF-κB (nuclear factor-κB) signaling. In vitro, recombinant FSTL-1 treatment of macrophages attenuated NF-κB p65 phosphorylation in an Nr4a1-dependent manner. Within the COPDGene cohort, several SNPs in the FSTL1 region corresponded to chronic obstructive pulmonary disease and lung function.Conclusions: This work identifies a novel role for FSTL-1 protecting against emphysema development independent of smoke exposure. This FSTL-1-deficient emphysema implicates regulation of immune tolerance in lung macrophages through Nr4a1. Further study of the mechanisms involving FSTL-1 in lung homeostasis, immune regulation, and NF-κB signaling may provide additional insight into the pathophysiology of emphysema and inflammatory lung diseases.

Follistatin-like protein 1 modulates IL-17 signaling via IL-17RC regulation in stromal cells.

Follistatin-like protein 1 (FSTL-1) possesses several newly identified roles in mammalian biology, including interleukin (IL)-17-driven inflammation, though the mechanism underlying FSTL-1 influence on IL-17-mediated cytokine production is unknown. Using parallel in vitro bone marrow stromal cell models of FSTL-1 suppression, we employed unbiased microarray analysis to identify FSTL-1-regulated genes and pathways that could influence IL-17-dependent production of IL-6 and granulocyte colony-stimulating factor. We discovered that FSTL-1 modulates Il17rc gene expression. Specifically, FSTL-1 was necessary for Il17rc gene transcription, IL-17RC surface protein expression and IL-17-dependent cytokine production. This work identifies a mechanism by which FSTL-1 influences IL-17-driven inflammatory signaling in vitro and reveals a novel function for FSTL-1, as a modulator of gene expression. Thus enhanced understanding of the interplay between FSTL-1 and IL-17-mediated inflammation may provide insight into potential therapeutic targets of IL-17-mediated diseases and warrants ongoing study of in vivo models and clinical scenarios of FSTL-1-influenced diseases.

Suppression of arthritis-induced bone erosion by a CRAC channel antagonist.

OBJECTIVE: We have shown in vitro and in vivo that osteoclast maturation requires calcium-release activated calcium (CRAC) channels. In inflammatory arthritis, osteoclasts mediate severe and debilitating bone erosion. In the current study, we assess the value of CRAC channels as a therapeutic target to suppress bone erosion in acute inflammatory arthritis. METHODS: Collagen-induced arthritis (CIA) was induced in mice. The CRAC channel inhibitor 3,4-dichloropropionaniline (DCPA) and a placebo was administered 1 day prior to collagen II booster to induce arthritis. Effects on swelling, inflammatory cell invasion in joints, serum cytokines and bone erosion were measured. RESULTS: Assays, by blinded observers, of arthritis severity showed that DCPA, 21 mg/kg/day, suppressed arthritis development over 3 weeks. Bone and cartilage damage in sections of animal feet was reduced approximately 50%; overall swelling of joints was reduced by a similar amount. Effects on bone density by µCT showed clear separation in DCPA-treated CIA animals from CIA without treatment, while differences between controls without CIA and CIA treated with DCPA differed by small amounts and in most cases were not statistically different. Response was not related to anticollagen titres. There were no adverse effects in the treated group on animal weight or activity, consistent with low toxicity. The effect was maximal 12-17 days after collagen booster, during the rapid appearance of arthritis in untreated CIA. At 20 days after treatment (day 40), differences in arthritis score were reduced and tumour necrosis factor α, interleukin (IL)-1, or IL-6 in the serum of the animals were similar in treated and untreated animals. CONCLUSIONS: DCPA, a novel inhibitor of CRAC channels, suppresses bone erosion associated with acute arthritis in mice and might represent a new treatment modality for acute arthrits.

Follistatin-like protein 1 regulates chondrocyte proliferation and chondrogenic differentiation of mesenchymal stem cells.

OBJECTIVES: Chondrocytes, the only cells in the articular cartilage, play a pivotal role in osteoarthritis (OA) because they are responsible for maintenance of the extracellular matrix (ECM). Follistatin-like protein 1 (FSTL1) is a secreted protein found in mesenchymal stem cells (MSCs) and cartilage but whose function is unclear. FSTL1 has been shown to modify cell growth and survival. In this work, we sought to determine whether FSTL1 could regulate chondrogenesis and chondrogenic differentiation of MSCs. METHODS: To study the role of FSTL1 in chondrogenesis, we used FSTL1 knockout (KO) mice generated in our laboratory. Proliferative capacity of MSCs, obtained from skulls of E18.5 embryos, was analysed by flow cytometry. Chondrogenic differentiation of MSCs was carried out in a pellet culture system. Gene expression differences were assessed by microarray analysis and real-time PCR. Phosphorylation of Smad3, p38 MAPK and Akt was analysed by western blotting. RESULTS: The homozygous FSTL1 KO embryos showed extensive skeletal defects and decreased cellularity in the vertebral cartilage. Cell proliferation of FSTL1-deficient MSCs was reduced. Gene expression analysis in FSTL1 KO MSCs revealed dysregulation of multiple genes important for chondrogenesis. Production of ECM proteoglycans and collagen II expression were decreased in FSTL1-deficient MSCs differentiated into chondrocytes. Transforming growth factor ß signalling in FSTL1 KO cells was significantly suppressed. CONCLUSIONS: FSTL1 is a potent regulator of chondrocyte proliferation, differentiation and expression of ECM molecules. Our findings may lead to the development of novel strategies for cartilage repair and provide new disease-modifying treatments for OA.

Follistatin-like protein 1 enhances NLRP3 inflammasome-mediated IL-1ß secretion from monocytes and macrophages.

Follistatin-like protein 1 (FSTL-1) is overexpressed in a number of inflammatory conditions characterized by elevated IL-1ß. Here, we found that FSTL-1 serum concentration was increased threefold in patients with bacterial sepsis and fourfold following administration of LPS to mice. To test the contribution of FSTL-1 to IL-1ß secretion, WT and FSTL-1-deficient mice were injected with LPS. While LPS induced IL-1ß in the sera of WT mice, it was low or undetectable in FSTL-1-deficient mice. Monocytes/macrophages, a key source of IL-1ß, do not normally express FSTL-1. However, FSTL-1 was found in tissue macrophages after injection of LPS into mouse footpads, demonstrating that macrophages are capable of taking up FSTL-1 at sites of inflammation. In vitro, intracellular FSTL-1 localized to the mitochondria. FSTL-1 activated the mitochondrial electron transport chain, increased the production of ATP (a key activator of the nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome) and IL-1ß secretion. FSTL-1 also enhanced transcription of the NLRP3 and procaspase 1 genes, two components of the NLRP3 inflammasome. Adenovirus-mediated overexpression of FSTL-1 in mouse paws led to activation of the inflammasome complex and local secretion of IL-1ß and IL-1ß-related proinflammatory cytokines. These results suggest that FSTL-1 may act on the NLRP3 inflammasome to promote IL-1ß secretion from monocytes/macrophages.

FSTL1 promotes arthritis in mice by enhancing inflammatory cytokine/chemokine expression.

OBJECTIVE: FSTL1 is a secreted glycoprotein that exacerbates murine arthritis and is overexpressed in human arthritis. The aim of this study was to determine the mechanism by which FSTL1 promotes arthritis. METHODS: Collagen-induced arthritis was induced in mice hypomorphic for FSTL1, generated with a gene trap-targeted mutant embryonic stem cell line. Arthritis was assessed by measuring paw swelling and using a qualitative arthritis index. Bone marrow-derived mesenchymal stromal cells from hypomorphic mice, as well as mouse stromal ST2 cells transduced with short hairpin RNA to suppress FSTL1 expression, were stimulated with interleukin-1ß (IL-1ß), tumor necrosis factor α, and IL-17. The monocyte cell line U937, which does not express FSTL1, was transfected with a plasmid encoding FSTL1 and stimulated with phorbol myristate acetate and lipopolysaccharide. Cell supernatants were assayed for IL-6, IL-8, monocyte chemotactic protein 1 (MCP-1), and FSTL1 by enzyme-linked immunosorbent assay. RESULTS: FSTL1 hypomorphic mice had reduced levels of FSTL1 compared to littermate controls. Following induction of arthritis, a significant correlation was observed between serum FSTL1 levels and both paw swelling and the arthritis index. Similar correlations were observed between the amount of FSTL1 produced by mesenchymal stromal cells, stromal ST2 cells, and monocytes and the secretion of IL-6, IL-8, and MCP-1. CONCLUSION: These findings demonstrate that FSTL1 up-regulates proinflammatory mediators important in the pathology of arthritis, and that serum levels of FSTL1 correlate with severity of arthritis. The latter supports the possibility that FSTL1 might be a target for treatment of certain forms of arthritis.

Akt fine-tunes NF-κB-dependent gene expression during T cell activation.

Activation of the NF-κB signaling pathway is critical for leukocyte activation and development. Although previous studies suggested a role for the Akt kinase in coupling the T cell antigen receptor and CD28 to NF-κB activation in T cells, the nature of the role of Akt in this pathway is still unclear. Using a targeted gene profiling approach, we found that a subset of NF-κB-dependent genes required Akt for optimal up-regulation during T cell activation. The selective effects of Akt were manifest at the level of mRNA transcription and p65/RelA binding to upstream promoters and appear to be due to altered formation of the Carma1-Bcl10 complex. The proinflammatory cytokine TNF-α was found to be particularly sensitive to Akt inhibition or knockdown, including in primary human blood T cells and a murine model of rheumatoid arthritis. Our findings are consistent with a hierarchy in the expression of NF-κB-dependent genes, controlled by the strength and/or duration of NF-κB signaling. More broadly, our results suggest that defining the more graded effects of signaling, such as those demonstrated here for Akt and the NF-κB pathway, is important to understanding how cells can fine-tune signaling responses for optimal sensitivity and specificity.

Follistatin-like protein 1 is a mesenchyme-derived inflammatory protein and may represent a biomarker for systemic-onset juvenile rheumatoid arthritis.

OBJECTIVE: To examine both the source of follistatin-like protein 1 (FSTL-1) and the factors that induce its expression in arthritis, and to determine whether juvenile rheumatoid arthritis (JRA) is characterized by overexpression of FSTL-1. METHODS: FSTL-1 expression patterns were analyzed by immunohistochemical staining of joint tissue derived from mice with collagen-induced arthritis. Induction of FSTL-1 secretion was assessed in osteoblasts, adipocytes, and human fibroblast-like synoviocytes in response to transforming growth factor beta (TGFbeta), interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and IL-6. In addition, sera and synovial fluid from children with oligoarticular, polyarticular, or systemic-onset JRA were assayed for FSTL-1 using a custom enzyme-linked immunosorbent assay. FSTL-1 concentrations in these patients were assessed for correlations with the erythrocyte sedimentation rate (ESR) and platelet count. RESULTS: Immunohistochemical staining of murine joint sections demonstrated expression of FSTL-1 in all cell types of the mesenchymal lineage, including osteocytes, chondrocytes, adipocytes, and fibroblasts. FSTL-1 could be induced in osteoblasts, adipocytes, and human fibroblast-like synoviocytes by TGFbeta, IL-1beta, TNFalpha, and IL-6. The IL-1beta response was significantly greater than the TNFalpha response (P < 0.05). In human serum and synovial fluid, only those samples from children with the systemic-onset JRA subtype had elevated concentrations of FSTL-1. The synovial fluid concentrations of FSTL-1 were 2-3-fold higher than the serum concentrations. The elevation in serum FSTL-1 concentrations seen in children with systemic-onset JRA correlated closely with elevations in the ESR and platelet count. CONCLUSION: These findings demonstrate that the arthritic joint matrix is a major source of FSTL-1 and that IL-1beta is a central mediator of FSTL-1 secretion. Furthermore, FSTL-1 may represent a useful biomarker of disease activity in systemic-onset JRA.

Pediatric pigmented villonodular synovitis mimicking a septic hip.

We describe a child with pigmented villonodular synovitis initially treated for a presumed hip infection. The correct diagnosis was not made until 2(1/2) years later on a second admission. This is a rare disease with vague presenting symptoms that requires a high index of suspicion. Magnetic resonance imaging and tissue biopsy are usually needed for a definitive diagnosis. Surgery is the primary treatment option; however, the patient described was unusual in that she did well to date with conservative measures.

Follistatin-like protein-1 is a novel proinflammatory molecule.

While analyzing gene expression in collagen-induced arthritis, we discovered that a poorly characterized gene, follistatin-like protein 1 (FSTL-1), is highly overexpressed in mouse paws during early arthritis, especially at the interface of synovial pannus and eroding bone. In this study, we show that FSTL-1 is a novel proinflammatory molecule with a previously unrecognized role in inflammation. Transfection of FSTL-1 into macrophages and fibroblasts leads to up-regulation of proinflammatory cytokines, including IL-1beta, TNF-alpha, and IL-6. Overexpression of FSTL-1 in mouse paws by gene transfer results in severe paw swelling and arthritis.

Inflammatory cytokine regulation of transgene expression in human fibroblast-like synoviocytes infected with adeno-associated virus.

OBJECTIVE: An ideal gene transfer vector for chronic inflammatory diseases such as rheumatoid arthritis (RA) would provide local transgene expression only when the disease is active. To determine whether adeno-associated virus (AAV) possesses this ability, the effects of inflammatory cytokines on transgene expression were evaluated in human RA fibroblast-like synoviocytes (FLS). METHODS: Human FLS were infected with AAV in the presence or absence of inflammatory cytokines or synovial fluid obtained from patients with RA. Transgene expression was monitored by either enzyme-linked immunosorbent assay or flow cytometry. Transgene messenger RNA (mRNA) was measured by real-time quantitative reverse transcription-polymerase chain reaction. RESULTS: Inflammatory cytokines increased transgene expression in FLS by up to 60-fold. Synovial fluid from patients with RA, but not from patients without arthritis, was also able to increase expression in synoviocytes. Protein expression correlated with transgene mRNA levels. The enhanced expression required the continued presence of cytokines because, upon removal, transgene expression returned to baseline levels. Expression could be repeatedly reinduced by reexposure to cytokines. The effect was not promoter specific and was demonstrated to be phosphatidylinositol 3-kinase-dependent. CONCLUSION: These results suggest that expression of a therapeutic transgene can be controlled by the presence of inflammation following AAV gene transfer, making it an attractive vector for chronic inflammatory diseases such as RA.

NOD.c3c4 congenic mice develop autoimmune biliary disease that serologically and pathogenetically models human primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune disease with a strong genetic component characterized by biliary ductular inflammation with eventual liver cirrhosis. The serologic hallmark of PBC is antimitochondrial antibodies that react with the pyruvate dehydrogenase complex, targeting the inner lipoyl domain of the E2 subunit (anti-PDC-E2). Herein we demonstrate that NOD.c3c4 mice congenically derived from the nonobese diabetic strain develop an autoimmune biliary disease (ABD) that models human PBC. NOD.c3c4 (at 9-10 wk, before significant biliary pathology) develop antibodies to PDC-E2 that are specific for the inner lipoyl domain. Affected areas of biliary epithelium are infiltrated with CD3+, CD4+, and CD8+ T cells, and treatment of NOD.c3c4 mice with monoclonal antibody to CD3 protects from ABD. Furthermore, NOD.c3c4-scid mice develop disease after adoptive transfer of splenocytes or CD4+ T cells, demonstrating a central role for T cells in pathogenesis. Histological analysis reveals destructive cholangitis, granuloma formation, and eosinophilic infiltration as seen in PBC, although, unlike PBC, the extrahepatic biliary ducts are also affected. Using a congenic mapping approach, we define the first ABD (Abd) locus, Abd1. These results identify the NOD.c3c4 mouse as the first spontaneous mouse model of PBC.

A soluble divalent class I MHC/IgG1 fusion protein activates CD8+ T cells in vivo.

CD8+ T lymphocytes recognize tumor and viral antigens bound to class I major histocompatibility complexes (MHC). Tumors and viruses may evade detection by preventing antigen presentation. The present study was designed to determine whether a soluble divalent fusion protein, containing the extracellular domains of a class I MHC molecule fused to beta2-microglobulin and the constant domains of IgG1, could induce an immune response in vivo. Administration to mice of the fusion protein loaded with a tumor peptide induced peptide-specific T cell activation and retarded tumor growth. Administration of the fusion protein loaded with a glycoprotein B (gB) peptide derived from herpes simplex virus type 1 (HSV-1) induced gB-specific cytotoxic T lymphocytes and protected mice from a lethal HSV-1 challenge. These data suggest that antigen-loaded MHC/IgG fusion proteins may enhance T cell immunity in conditions where antigen presentation is altered.

Proteasome inhibition enhances AAV-mediated transgene expression in human synoviocytes in vitro and in vivo.

To explore the potential applicability of recombinant adeno-associated virus (rAAV) vectors in the treatment of rheumatoid arthritis (RA), primary human fibroblast-like synoviocytes (FLS) derived from patients with RA were infected with rAAV encoding mouse IL-10 under the control of the CMV promoter. Addition of the proteasome inhibitor carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (zLLL) to the cultures dramatically enhanced expression of the IL-10 transgene, in a dose-dependent manner. The increased expression was transient, peaking at 3 days and returning to near baseline by 7 days. The enhancement was observed even when zLLL was added 13 days after infection with rAAV. The effect of zLLL was not specific to either the mIL-10 transgene or the CMV promoter, as similar findings were observed using an rAAV construct encoding alpha1-anti-trypsin under the control of the chick beta-actin promoter or GFP, driven by the CMV promoter. Transgene expression could be repeatedly induced by reexposure to zLLL. Transgene mRNA levels increased in parallel with protein levels. Transgene expression could also be repeatedly induced in vivo by administering zLLL to SCID mice previously injected with rAAV-infected FLS. These data demonstrate that proteasome inhibition can dramatically enhance transgene expression in human RA FLS following infection with rAAV and suggest a possible approach to regulating synovial transgene expression in vivo.

Recombinant adeno-associated virus preferentially transduces human, compared to mouse, synovium: implications for arthritis therapy.

Despite a number of published reports, including from our own laboratory, suggesting that adeno-associated virus (AAV) transduces mouse synovium, a careful analysis demonstrated transduction predominantly of the subsynovial muscle tissue, while the synovial lining is poorly transduced. To investigate the potential of AAV to transduce human synovium, three human rheumatoid arthritis (RA) and two murine collagen-induced arthritis (CIA) synovial cell lines were infected with recombinant AAV (rAAV) vectors encoding either mouse IL-10 or IL-4. Low-level transgene expression was observed. However, either Gamma-irradiation or the addition of a low-titer E1-, E3-deleted recombinant adenovirus resulted in up to a 100-fold increase in transgene product in the human, but not the mouse, cell lines. RA synovial tissues implanted subcutaneously in severe combined immunodeficiency (SCID) mice, which were subsequently infected with rAAV, showed marked increases in transgene expression when co-infected with adenovirus. To our knowledge, this is the first study to show that intact human synovial tissues can be transduced by rAAV, and it suggests that murine arthritis may not be an optimal model to study rAAV as a gene transfer vector. Further studies to elucidate the mechanisms limiting gene transduction in human synovium may allow optimization of this vector for the treatment of arthritis.