Increased WISP1 expression in human osteoarthritic articular cartilage is epigenetically regulated and decreases cartilage matrix production.

OBJECTIVES: Previously, we have shown the involvement of Wnt-activated protein Wnt-1-induced signaling protein 1 (WISP1) in the development of OA in mice. Here, we aimed to characterize the relation between WISP1 expression and human OA and its regulatory epigenetic determinants. METHODS: Preserved and lesioned articular cartilage from end-stage OA patients and non-OA-diagnosed individuals was collected. WISP1 expression was determined using immunohistochemistry and damage was classified using Mankin scoring. RNA expression and DNA methylation were assessed in silico from genome-wide datasets (microarray analysis and RNA sequencing, and 450 k-methylationarrays, respectively). Effects of WISP1 were tested in pellet cultures of primary human chondrocytes. RESULTS: WISP1 expression in cartilage of OA patients was increased compared with non-OA-diagnosed controls and, within OA patients, WISP1 was even higher in lesioned compared with preserved regions, with expression strongly correlating with Mankin score. In early symptomatic OA patients with disease progression, higher synovial WISP1 expression was observed as compared with non-progressors. Notably, increased WISP1 expression was inversely correlated with methylation levels of a positional CpG-dinucleotide (cg10191240), with lesioned areas showing strong hypomethylation for this CpG as compared with preserved cartilage. Additionally, we observed that methylation levels were allele-dependent for an intronic single-nucleotide polymorphism nearby cg10191240. Finally, addition of recombinant WISP1 to pellets of primary chondrocytes strongly inhibited deposition of extracellular matrix as reflected by decreased pellet circumference, proteoglycan content and decreased expression of matrix components. CONCLUSION: Increased WISP1 expression is found in lesioned human articular cartilage, and appears epigenetically regulated via DNA methylation. In vitro assays suggest that increased WISP1 is detrimental for cartilage integrity.

The level of synovial AXL expression determines the outcome of inflammatory arthritis, possibly depending on the upstream role of TGF-ß1.

OBJECTIVE: To investigate the role of AXL, a member of the anti-inflammatory TYRO3, AXL MER (TAM) receptor family, in arthritis. METHODS: KRN serum transfer arthritis was induced in Axl-/- and wild-type mice. Knee and ankle joints were scored macro- and microscopically. Synovial gene and protein expression of Axl was determined in naïve and TGF-ß1-overexpressing joints. AXL expression was determined in M1-like or M2-like macrophages and RA synovium. Human macrophages, fibroblasts and synovial micromasses were stimulated with TGF-ß1 or the AXL inhibitor R428. RESULTS: Ankle joints of Axl-/- mice showed exacerbated arthritis pathology, whereas no effect of Axl gene deletion was observed on gonarthritis pathology. To explain this spatial difference, we examined the synovium of naïve mice. In contrast to the knee, the ankle synovial cells prominently expressed AXL. Moreover, the M2-like macrophage phenotype was the dominant cell type in the naïve ankle joint. Human M2-like macrophages expressed higher levels of AXL and blocking AXL increased their inflammatory response. In the murine ankle synovium, gene expression of Tgfb1 was increased and Tgb1 correlated with Axl. Moreover, TGFB1 and AXL expression also correlated in human RA synovium. In human macrophages and synovial micromasses, TGF-ß1 enhanced AXL expression. Moreover, TGF-ß1 overexpression in naïve murine knee joints induced synovial AXL expression. CONCLUSION: Differences in synovial AXL expression are in accordance with the observation that AXL dampens arthritis in ankle, but not in knee joints. We provide evidence that the local differences in AXL expression could be due to TGF-ß1, and suggest similar pathways operate in RA synovium.

Treatment of collagenase-induced osteoarthritis with a viral vector encoding TSG-6 results in ectopic bone formation.

OBJECTIVE: Tumor necrosis factor-inducible gene 6 (TSG-6) has anti-inflammatory and chondroprotective effects in mouse models of inflammatory arthritis. Because cartilage damage and inflammation are also observed in osteoarthritis (OA), we determined the effect of viral overexpression of TSG-6 in experimental osteoarthritis. METHODS: Bone marrow-derived cells were differentiated to multinucleated osteoclasts in the presence of recombinant TSG-6 or after transduction with a lentiviral TSG-6 expression vector. Multi-nucleated osteoclasts were analyzed after tartrate resistant acid phosphatase staining and resorption activity was determined on dentin slices. Collagenase-induced osteoarthritis (CIOA) was induced in C57BL/6 mice after intra-articular injection of an adenoviral TSG-6 or control luciferase expression vector. Inflammation-related protease activity was measured using bioluminescent Prosense probes. After a second adenovirus injection, cartilage damage was assessed in histological sections stained with Safranin-O. Ectopic bone formation was scored in X-ray images of the affected knees. RESULTS: TSG-6 did not inhibit the formation of multi-nucleated osteoclasts, but caused a significant reduction in the resorption activity on dentin slices. Adenoviral TSG-6 gene therapy in CIOA could not reduce the cartilage damage compared to the luciferase control virus and no significant difference in inflammation-related protease activity was noted between the TSG-6 and control treated group. Instead, X-ray analysis and histological analysis revealed the presence of ectopic bone formation in the TSG-6 treated group. CONCLUSION: Gene therapy based on the expression of TSG-6 could not provide cartilage protection in experimental osteoarthritis, but instead resulted in increased ectopic bone formation.

Protective Role of the MER Tyrosine Kinase via Efferocytosis in Rheumatoid Arthritis Models.

Objective: Rheumatoid arthritis (RA) is a chronic and progressive joint disease. It appears that anti-inflammatory feedback mechanisms that could restrain joint inflammation and restore homeostasis are insufficient to perform this control. In this study, we investigated the contribution of the MER tyrosine kinase-mediated anti-inflammatory response on arthritis and whether targeting MER could be a valid approach to treat RA. Methods: KRN serum transfer arthritis (KRN STA) was induced in either Mertk-deficient mice or in mice that adenovirally overexpressed Pros1. Human synovial micromasses were treated with MER-specific antibodies or PROS1. Collagen-induced arthritis (CIA) mice were treated with MER-specific agonistic antibodies or by viral overexpression of Pros1. Results: Mertk-/- mice showed exacerbated arthritis pathology, whereas Pros1 overexpression diminished joint pathology in KRN STA. Human synovial micromasses challenged with MER-specific antibodies enhanced the secretion of inflammatory cytokines, whereas stimulating MER with PROS1 reduced the secretion of these cytokines, confirming the protective role of MER. Next, we treated CIA mice with MER-specific agonistic antibodies, and this unexpectedly resulted in exacerbated arthritis pathology. This was associated with increased numbers of apoptotic cells in their knee joints and higher serum levels of interleukin (IL)-16C, a cytokine released by secondary necrotic neutrophils. Apoptotic cell numbers and IL-16C levels were enhanced during arthritis in Mertk-/- mice and reduced in Pros1-overexpressing mice. Conclusion: MER plays a protective role during joint inflammation and activating MER by its ligand PROS1 ameliorates disease. Treatment of mice with MER receptor agonistic antibodies is deleterious due to its counterproductive effect of blocking efferocytosis in the arthritic joint.

Imaging fibroblast activation protein to monitor therapeutic effects of neutralizing interleukin-22 in collagen-induced arthritis.

Objectives: RA is a chronic autoimmune disease leading to progressive destruction of cartilage and bone. RA patients show elevated IL-22 levels and the amount of IL-22-producing Th cells positively correlates with the extent of erosive disease, suggesting a role for this cytokine in RA pathogenesis. The purpose of this study was to determine the feasibility of SPECT/CT imaging with 111In-labelled anti-fibroblast activation protein antibody (28H1) to monitor the therapeutic effect of neutralizing IL-22 in experimental arthritis. Methods: Mice (six mice/group) with CIA received anti-IL-22 or isotype control antibodies. To monitor therapeutic effects after treatment, SPECT/CT images were acquired 24 h after injection of 111In-28H1. Imaging results were compared with macroscopic, histologic and radiographic arthritis scores. Results: Neutralizing IL-22 before CIA onset effectively prevented arthritis development, reaching a disease incidence of only 50%, vs 100% in the control group. SPECT imaging showed significantly lower joint tracer uptake in mice treated early with anti-IL-22 antibodies compared with the control-treated group. Reduction of disease activity in those mice was confirmed by macroscopic, histological and radiographic pathology scores. However, when treatment was initiated in a later phase of CIA, progression of joint pathology could not be prevented. Conclusion: These findings suggest that IL-22 plays an important role in CIA development, and neutralizing this cytokine seems an attractive new strategy in RA treatment. Most importantly, SPECT/CT imaging with 111In-28H1 can be used to specifically monitor therapy responses, and is potentially more sensitive in disease monitoring than the gold standard method of macroscopic arthritis scoring.

Alteration of the intestinal microbiome characterizes preclinical inflammatory arthritis in mice and its modulation attenuates established arthritis.

Perturbations of the intestinal microbiome have been observed in patients with new-onset and chronic autoimmune inflammatory arthritis. However, it is currently unknown whether these alterations precede the development of arthritis or are rather a consequence of disease. Modulation of intestinal microbiota by oral antibiotics or germ-free condition can prevent arthritis in mice. Yet, the therapeutic potential of modulation of the microbiota after the onset of arthritis is not well characterized. We here show that the intestinal microbial community undergoes marked changes in the preclinical phase of collagen induced arthritis (CIA). The abundance of the phylum Bacteroidetes, specifically families S24-7 and Bacteroidaceae was reduced, whereas Firmicutes and Proteobacteria, such as Ruminococcaceae, Lachnospiraceae and Desulfovibrinocaceae, were expanded during the immune-priming phase of arthritis. In addition, we found that the abundance of lamina propria Th17, but not Th1, cells is highly correlated with the severity of arthritis. Elimination of the intestinal microbiota during established arthritis specifically reduced intestinal Th17 cells and attenuated arthritis. These effects were associated with reduced serum amyloid A expression in ileum and synovial tissue. Our observations suggest that intestinal microbiota perturbations precede arthritis, and that modulation of the intestinal microbiota after the onset of arthritis may offer therapeutic opportunities.

Brief Report: Induction of Matrix Metalloproteinase Expression by Synovial Wnt Signaling and Association With Disease Progression in Early Symptomatic Osteoarthritis.

OBJECTIVE: Increased Wnt signaling in chondrocytes is associated with development of osteoarthritis (OA). However, OA is considered a disease of the entire joint, where the synovium has been attributed an important role in disease pathogenesis and progression. This study was undertaken to determine whether Wnt signaling in synovial tissue could contribute to pathologic development of OA through the production of matrix metalloproteinases (MMPs), and to assess the relationship of synovial expression of Frizzled (FZD) receptors and the Wnt inhibitor FRZB to MMP expression and disease progression in patients with early OA in the Dutch Cohort Hip and Cohort Knee (CHECK) study cohort. METHODS: In mouse knee joints, human WNT8A and mouse Wnt16 were overexpressed using adenoviral vectors, and expression of messenger RNA (mRNA) for MMPs in the synovium was determined by reverse transcription-polymerase chain reaction or Luminex assay. In human synovial tissue from a subgroup of patients with early OA with knee pain enrolled in the CHECK cohort, levels of Wnt family members were assessed for linkage to MMP expression and disease progression. In addition, MMP production in human synovium from patients with end-stage OA was determined after stimulation of Wnt signaling with WNT3A or inhibition with FRZB or DKK1 in the synovium. RESULTS: Overexpression of WNT8A and Wnt16 in mouse knee joints induced MMP expression in vivo. Expression of MMPs relevant to human OA in the synovium from CHECK study participants significantly correlated with expression of FZD1, FZD10, and FRZB mRNA. Moreover, increased FZD1 mRNA expression and decreased FRZB mRNA expression were observed in CHECK study patients who experienced disease progression compared to those who were nonprogressors. Stimulation of human OA synovium with WNT3A induced the production of various MMPs, whereas inhibition of Wnt signaling with FRZB or DKK1 reduced the production of MMPs. CONCLUSION: Wnt signaling in the synovium may potently induce progression of OA via increased production of MMPs.

Aberrant intestinal microbiota due to IL-1 receptor antagonist deficiency promotes IL-17- and TLR4-dependent arthritis.

BACKGROUND: Perturbation of commensal intestinal microbiota has been associated with several autoimmune diseases. Mice deficient in interleukin-1 receptor antagonist (Il1rn -/- mice) spontaneously develop autoimmune arthritis and are susceptible to other autoimmune diseases such as psoriasis, diabetes, and encephalomyelitis; however, the mechanisms of increased susceptibility to these autoimmune phenotypes are poorly understood. We investigated the role of interleukin-1 receptor antagonist (IL-1Ra) in regulation of commensal intestinal microbiota, and assessed the involvement of microbiota subsets and innate and adaptive mucosal immune responses that underlie the development of spontaneous arthritis in Il1rn -/- mice. RESULTS: Using high-throughput 16S rRNA gene sequencing, we show that IL-1Ra critically maintains the diversity and regulates the composition of intestinal microbiota in mice. IL-1Ra deficiency reduced the intestinal microbial diversity and richness, and caused specific taxonomic alterations characterized by overrepresented Helicobacter and underrepresented Ruminococcus and Prevotella. Notably, the aberrant intestinal microbiota in IL1rn -/- mice specifically potentiated IL-17 production by intestinal lamina propria (LP) lymphocytes and skewed the LP T cell balance in favor of T helper 17 (Th17) cells, an effect transferable to WT mice by fecal microbiota. Importantly, LP Th17 cell expansion and the development of spontaneous autoimmune arthritis in IL1rn -/- mice were attenuated under germ-free condition. Selective antibiotic treatment revealed that tobramycin-induced alterations of commensal intestinal microbiota, i.e., reduced Helicobacter, Flexispira, Clostridium, and Dehalobacterium, suppressed arthritis in IL1rn -/- mice. The arthritis phenotype in IL1rn -/- mice was previously shown to depend on Toll-like receptor 4 (TLR4). Using the ablation of both IL-1Ra and TLR4, we here show that the aberrations in the IL1rn -/- microbiota are partly TLR4-dependent. We further identify a role for TLR4 activation in the intestinal lamina propria production of IL-17 and cytokines involved in Th17 differentiation preceding the onset of arthritis. CONCLUSIONS: These findings identify a critical role for IL1Ra in maintaining the natural diversity and composition of intestinal microbiota, and suggest a role for TLR4 in mucosal Th17 cell induction associated with the development of autoimmune disease in mice.

Higher efficacy of anti-IL-6/IL-21 combination therapy compared to monotherapy in the induction phase of Th17-driven experimental arthritis.

Th17 cells and their cytokines are linked to the pathogenesis of rheumatoid arthritis, a chronic autoimmune disease characterized by joint inflammation. Th17 development is initiated by combined signaling of TGF-ß and IL-6 or IL-21, and can be reduced in the absence of either IL-6 or IL-21. The aim of this study was to assess whether combinatorial IL-6/IL-21 blockade would more potently inhibit Th17 development, and be more efficacious in treating arthritis than targeting either cytokine. We assessed in vitro Th17 differentiation efficacy in the absence of IL-6 and/or IL-21. To investigate in vivo effects of IL-6/IL-21 blockade on Th17 and arthritis development, antigen-induced arthritis (AIA) was induced in IL-6-/- x IL-21R-/- mice. The therapeutic potential of this combined blocking strategy was assessed by treating mice with collagen-induced arthritis (CIA) with anti-IL-6R antibodies and soluble (s)IL-21R.Fc. We demonstrated that combined IL-6/IL-21 blocking synergistically reduced in vitro Th17 differentiation. In mice with AIA, absence of IL-6 and IL-21 signaling more strongly reduced Th17 levels and resulted in stronger suppression of arthritis than the absence of either cytokine. Additionally, anti-IL-6/anti-IL-21 treatment of CIA mice during the arthritis induction phase reduced disease development more potent than IL-6 or IL-21 inhibition alone, as effective as anti-TNF treatment. Collectively, these results suggest dual IL-6/IL-21 inhibition may be a more efficacious therapeutic strategy compared to single cytokine blockade to suppress arthritis development.

A Dual Role of Upper Zone of Growth Plate and Cartilage Matrix-Associated Protein in Human and Mouse Osteoarthritic Cartilage: Inhibition of Aggrecanases and Promotion of Bone Turnover.

OBJECTIVE: Cartilage damage and subchondral bone changes are closely connected in osteoarthritis. Nevertheless, how these processes are interlinked is, to date, incompletely understood. This study was undertaken to investigate the mechanistic role of a cartilage-derived protein, upper zone of growth plate and cartilage matrix-associated protein (UCMA), in osteoarthritis-related cartilage and bone changes. METHODS: UCMA expression was assessed in healthy and osteoarthritic human and mouse cartilage. For analysis of cartilage and bone changes, osteoarthritis was induced by destabilization of the medial meniscus (DMM) in wild-type (WT) and Ucma-deficient mice. UCMA-collagen interactions, the effect of UCMA on aggrecanase activity, and the impact of recombinant UCMA on osteoclast differentiation were studied in vitro. RESULTS: UCMA was found to be overexpressed in human and mouse osteoarthritic cartilage. DMM-triggered cartilage changes, including increased structural damage, proteoglycan loss, and chondrocyte cell death, were aggravated in Ucma-deficient mice compared to WT littermates, thereby demonstrating the potential chondroprotective effects of UCMA. Moreover, UCMA inhibited ADAMTS-dependent aggrecanase activity and directly interacted with cartilage-specific collagen types. In contrast, osteoarthritis-related bone changes were significantly reduced in Ucma-deficient mice, showing less pronounced osteophyte formation and subchondral bone sclerosis. Mechanistically, UCMA directly promoted osteoclast differentiation in vitro. CONCLUSION: UCMA appears to link cartilage with bone changes in osteoarthritis by supporting cartilage integrity as an endogenous inhibitor of aggrecanases while also promoting osteoclastogenesis and subchondral bone turnover. Thus, UCMA represents an important link between cartilage and bone in osteoarthritis.

Milk-Derived Nanoparticle Fraction Promotes the Formation of Small Osteoclasts But Reduces Bone Resorption.

The general consensus is that milk promotes bone growth and density because is a source of calcium and contains components that enhance intestinal calcium uptake or directly affect bone metabolism. In this study, we investigated the effect of bovine-derived milk 100,000 g pellet (P100), which contains nanoparticles (<220 nm) including extracellular vesicles, on osteoclast differentiation and bone resorption. Bone marrow-derived osteoclast precursor cells were differentiated into osteoclasts by M-CSF and RANKL (control) and in the presence of milk P100. Milk P100 treatment until day 4 increased the number of TRAP-positive mononuclear cells and small (≤5 nuclei) osteoclasts. The number of large (≥6 nuclei) osteoclasts remained the same. These alterations were associated with increased expression of TRAP, NFATc1, and c-Fos. Cells seeded in a calcium-phosphate coated plate or bone slices showed reduced resorption area when exposed to milk P100 during the differentiation phase and even after osteoclast formation. Interestingly, milk P100 treatment enhanced Cathepsin K expression but reduced Carbonic Anhydrase 2 gene expression. Moreover, intracellular acid production was also decreased by milk P100 treatment. Oral delivery of milk P100 to female DBA1/J mice for 7 weeks did not alter bone area; however, increased osteoclast number and area in tibia without changes in serum RANKL and CTX-I levels. We showed for the first time the effect of milk P100 on osteoclast differentiation both in vitro and in vivo and found that milk P100 increased the formation of small osteoclasts but this does not lead to more bone resorption probably due to reduced acid secretion. J. Cell. Physiol. 232: 225-233, 2017. © 2016 Wiley Periodicals, Inc.

Alarmin S100A9 Induces Proinflammatory and Catabolic Effects Predominantly in the M1 Macrophages of Human Osteoarthritic Synovium.

OBJECTIVE: The alarmins S100A8 and S100A9 have been shown to regulate synovial activation, cartilage damage, and osteophyte formation in osteoarthritis (OA). Here we investigated the effect of S100A9 on the production of proinflammatory cytokines and matrix metalloprotease (MMP) in OA synovium, granulocyte macrophage colony-stimulating factor (GM-CSF)-differentiated/macrophage colony-stimulating factor (M-CSF)-differentiated macrophages, and OA fibroblasts. METHODS: We determined which cell types in the synovium produced S100A8 and S100A9. Further, the production of proinflammatory cytokines and MMP, and the activation of canonical Wnt signaling, was determined in human OA synovium, OA fibroblasts, and monocyte-derived macrophages following stimulation with S100A9. RESULTS: We observed that S100A8 and S100A9 were mainly produced by GM-CSF-differentiated macrophages present in the synovium, and to a lesser extent by M-CSF-differentiated macrophages, but not by fibroblasts. S100A9 stimulation of OA synovial tissue increased the production of the proinflammatory cytokines interleukin (IL) 1ß, IL-6, IL-8, and tumor necrosis factor-α. Additionally, various MMP were upregulated after S100A9 stimulation. Experiments to determine which cell type was responsible for these effects revealed that mainly stimulation of GM-CSF-differentiated macrophages and to a lesser extent M-CSF-differentiated macrophages with S100A9 increased the expression of these proinflammatory cytokines and MMP. In contrast, stimulation of fibroblasts with S100A9 did not affect their expression. Finally, stimulation of GM-CSF-differentiated, but not M-CSF-differentiated macrophages with S100A9 activated canonical Wnt signaling, whereas incubation of OA synovium with the S100A9 inhibitor paquinimod reduced the activation of canonical Wnt signaling. CONCLUSION: Predominantly mediated by M1-like macrophages, the alarmin S100A9 stimulates the production of proinflammatory and catabolic mediators and activates canonical Wnt signaling in OA synovium.

Suppression of the inflammatory response by disease-inducible interleukin-10 gene therapy in a three-dimensional micromass model of the human synovial membrane.

BACKGROUND: Gene therapy has the potential to provide long-term production of therapeutic proteins in the joints of osteoarthritis (OA) patients. The objective of this study was to analyse the therapeutic potential of disease-inducible expression of anti-inflammatory interleukin-10 (IL-10) in the three-dimensional micromass model of the human synovial membrane. METHODS: Synovial tissue samples from OA patients were digested and the cells were mixed with Matrigel to obtain 3D micromasses. The CXCL10 promoter combined with the firefly luciferase reporter in a lentiviral vector was used to determine the response of the CXCL10 promoter to tumour necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß) and lipopolysaccharide (LPS). The effects of recombinant IL-10 on gene expression were determined by quantitative PCR. The production of IL-10 from the CXCL10p-IL10 vector and the effects on pro-inflammatory cytokine production were assessed by multiplex ELISA. RESULTS: Micromasses made from whole synovial membrane cell suspensions form a distinct surface composition containing macrophage and fibroblast-like synoviocytes thus mimicking the synovial lining. This lining can be transduced by lentiviruses and allow CXCL-10 promoter-regulated transgene expression. Adequate amounts of IL-10 transgene were produced after stimulation with pro-inflammatory factors able to reduce the production of synovial IL-1ß and IL-6. CONCLUSIONS: Synovial micromasses are a suitable model to test disease-regulated gene therapy approaches and the CXCL10p-IL10 vector might be a good candidate to decrease the inflammatory response implicated in the pathogenesis of OA.

Secukinumab for rheumatology: development and its potential place in therapy.

Rheumatic disease is not a single disorder, but a group of more than 100 diseases that affect joints, connective tissues, and/or internal organs. Although rheumatic diseases like rheumatoid arthritis (RA), psoriatic arthritis, and ankylosing spondylitis (AS) differ in their pathogenesis and clinical presentation, the treatment of these inflammatory disorders overlaps. Non-steroid anti-inflammatory drugs are used to reduce pain and inflammation. Additional disease-modifying anti-rheumatic drugs are prescribed to slowdown disease progression, and is in RA more frequently and effectively applied than in AS. Biologicals are a relatively new class of treatments that specifically target cytokines or cells of the immune system, like tumor necrosis factor alpha inhibitors or B-cell blockers. A new kid on the block is the interleukin-17 (IL-17) inhibitor secukinumab, which has been recently approved by the US Food and Drug Administration for moderate-to-severe plaque psoriasis, psoriatic arthritis, and AS. IL-17 is a proinflammatory cytokine that has an important role in host defense, but its proinflammatory and destructive effects have also been linked to pathogenic processes in autoimmune diseases like RA and psoriasis. Animal models have greatly contributed to further insights in the potential of IL-17 blockade in autoimmune and autoinflammatory diseases, and have resulted in the development of various potential drugs targeting the IL-17 pathway. Secukinumab (AIN457) is a fully human monoclonal antibody that selectively binds to IL-17A and recently entered the market under the brand name Cosentyx(®). By binding to IL-17A, secukinumab prevents it from binding to its receptor and inhibits its ability to trigger inflammatory responses that play a role in the development of various autoimmune diseases. With secukinumab being the first in class to receive Food and Drug Administration approval, this article will further focus on this new biologic agent and review the milestones in its development and marketing.

Low heme oxygenase-1 levels in patients with systemic sclerosis are associated with an altered Toll-like receptor response: another role for CXCL4?

OBJECTIVE: SSc is a disease characterized by inflammation and fibrosis. Heme Oxygenase-1 (HO-1) is a haem-degrading enzyme that mediates resolution of inflammation and is induced upon mediators abundantly present in SSc. We aimed to assess whether HO-1 expression/function is disturbed in SSc patients and could therefore be contributing to the ongoing inflammation. METHODS: In total, 92 SSc patients and 48 healthy controls were included. By measuring total bilirubin in plasma in vivo, HO-activity was assessed. HO-1 expression levels were determined with western blot in monocytes before and after induction of HO-1 with cobalt protoporphyrin (CoPP) with or without CXCL4. Monocyte-derived dendritic cells (DCs) were stimulated with several Toll-like receptor (TLR) ligands with or without pre-stimulation with CoPP for 24 h. Cytokine levels were measured in the supernatants using the Luminex Bead Array. RESULTS: SSc patients have lower plasma levels of bilirubin, suggestive of an aberrant HO-1 function. We demonstrated low HO-1 expression in immune cells from SSc patients, whereas induction with CoPP was able to restore HO-1 levels in DCs from SSc patients, almost normalizing the increased TLR response observed in SSc. Co-exposure to CXCL4 completely abrogated CoPP-induced HO-1 expression, suggesting that the high CXCL4 levels present in SSc patients block the normal induction of HO-1 and its function. CONCLUSION: We demonstrate that HO activity in SSc patients is decreased and show its functional consequences. Since CXCL4 blocks the induction of HO-1 expression, neutralization of CXCL4 in SSc patients could have clinical benefits by diminishing overactivation of immune cells and other anti-inflammatory effects of HO-1.

Sclerostin inhibition promotes TNF-dependent inflammatory joint destruction.

Sclerostin, an inhibitor of the Wnt/ß-catenin pathway, has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. Mutations in the human sclerostin gene (SOST) lead to sclerosteosis with progressive skeletal overgrowth, whereas sclerostin-deficient (Sost(-/-)) mice exhibit increased bone mass and strength. Therefore, antibody-mediated inhibition of sclerostin is currently being clinically evaluated for the treatment of postmenopausal osteoporosis in humans. We report that in chronic TNFα (tumor necrosis factor α)-dependent arthritis, fibroblast-like synoviocytes constitute a major source of sclerostin and that either the lack of sclerostin or its antibody-mediated inhibition leads to an acceleration of rheumatoid arthritis (RA)-like disease in human TNFα transgenic (hTNFtg) mice with enhanced pannus formation and joint destruction. Inhibition of sclerostin also failed to improve clinical signs and joint destruction in the partially TNFα-dependent glucose-6-phosphate isomerase-induced arthritis mouse model, but ameliorated disease severity in K/BxN serum transfer-induced arthritis mouse model, which is independent of TNF receptor signaling, thus suggesting a specific role for sclerostin in TNFα signaling. Sclerostin effectively blocked TNFα- but not interleukin-1-induced activation of p38, a key step in arthritis development, pointing to a previously unrealized protective role of sclerostin in TNF-mediated chronic inflammation. The possibility of anti-sclerostin antibody treatment worsening clinical RA outcome under chronic TNFα-dependent inflammatory conditions in mice means that caution should be taken both when considering such treatment for inflammatory bone loss in RA and when using anti-sclerostin antibodies in patients with TNFα-dependent comorbidities.

Chronic skin inflammation leads to bone loss by IL-17-mediated inhibition of Wnt signaling in osteoblasts.

Inflammation has important roles in tissue regeneration, autoimmunity, and cancer. Different inflammatory stimuli can lead to bone loss by mechanisms that are not well understood. We show that skin inflammation induces bone loss in mice and humans. In psoriasis, one of the prototypic IL-17A-mediated inflammatory human skin diseases, low bone formation and bone loss correlated with increased serum IL-17A levels. Similarly, in two mouse models with chronic IL-17A-mediated skin inflammation,K14-IL17A(ind)andJunB(Δep), strong inhibition of bone formation was observed, different from classical inflammatory bone loss where osteoclast activation leads to bone degradation. We show that under inflammatory conditions, skin-resident cells such as keratinocytes, γδ T cells, and innate lymphoid cells were able to express IL-17A, which acted systemically to inhibit osteoblast and osteocyte function by a mechanism involving Wnt signaling. IL-17A led to decreased Wnt signaling in vitro, and importantly, pharmacological blockade of IL-17A rescued Wnt target gene expression and bone formation in vivo. These data provide a mechanism where IL-17A affects bone formation by regulating Wnt signaling in osteoblasts and osteocytes. This study suggests that using IL-17A blocking agents in psoriasis could be beneficial against bone loss in these patients.

Milk extracellular vesicles accelerate osteoblastogenesis but impair bone matrix formation.

The claimed beneficial effect of milk on bone is still a matter for debate. Recently extracellular vesicles (EVs) that contain proteins and RNA were discovered in milk, but their effect on bone formation has not yet been determined. We demonstrated previously that bovine milk-derived EVs (BMEVs) have immunoregulatory properties. Our aim was to evaluate the effect of BMEVs on osteogenesis by mice and human mesenchymal stem cells (hMSCs). Oral delivery of two concentrations of BMEVs to female DBA/1J mice during 7weeks did not alter the tibia trabecular bone area; however, the osteocytes number increased. In addition, the highest dose of BMEVs markedly increased the woven bone tissue, which is more brittle. The exposure of hMSCs to BMEVs during 21days resulted in less mineralization but higher cell proliferation. Interestingly BMEVs reduced the collagen production, but enhanced the expression of genes characteristic for immature osteoblasts. A kinetic study showed that BMEVs up-regulated many osteogenic genes within the first 4days. However, the production of type I collagen and expression of its genes (COL1A1 and COL1A2) were markedly reduced at days 21 and 28. At day 28, BMEVs again lead to higher proliferation, but mineralization was significantly increased. This was associated with increased expression of sclerostin, a marker for osteocytes, and reduced osteonectin, which is associated to bone matrix formation. Our study adds BMEVs to the list of milk components that can affect bone formation and may shed new light on the contradictory claims of milk on bone formation.

The interferon type I signature is present in systemic sclerosis before overt fibrosis and might contribute to its pathogenesis through high BAFF gene expression and high collagen synthesis.

BACKGROUND: Interferon (IFN) signature has been reported in definite systemic sclerosis (SSc) but it has not been characterised in early SSc (EaSSc). We aim at characterising IFN type I signature in SSc before overt skin fibrosis develops. METHODS: The expression of 11 IFN type I inducible genes was tested in whole-blood samples from 30 healthy controls (HCs), 12 subjects with primary Raynaud's phenomenon (RP), 19 patients with EaSSc, 7 patients with definite SSc without cutaneous fibrosis, 21 limited cutaneous SSc and 10 diffuse cutaneous SSc subjects. The correlation between IFN activity in monocytes, B cell activating factor (BAFF) mRNA expression and type III procollagen N-terminal propeptide (PIIINP) serum levels was tested. RESULTS: In all the SSc groups, higher IFN scores were observed compared with HC. An IFN score ≥7.09 discriminated HCs from patients with SSc (sensitivity=0.7, specificity=0.88, area under receiving operating characteristic (AUROC)=0.82); the prevalence of an elevated IFN score was: HC=3.3%; RP=33.3%, EaSSc=78.9%, definite SSc=100%, limited cutaneous SSc=42.9%, diffuse cutaneous SSc=70.0%. In monocytes an IFN score ≥4.12 distinguished HCs from patients with fibrotic SSc (sensitivity=0.62, specificity=0.85, AUROC=0.76). Compared with IFN-negative subjects, IFN-positive subjects had higher monocyte BAFF mRNA levels (19.7±5.2 vs 15.20±4.0, p=2.1×10(-5)) and serum PIIINP levels (median=6.0 (IQR 5.4-8.9) vs median=3.9 (IQR 3.3-4.7), p=0.0004). CONCLUSIONS: An IFN type I signature is observed in patients with SSc from the earliest phases of the disease, even before overt skin fibrosis. The presence of IFN type I signature in monocytes is correlated with BAFF mRNA expression and serum PIIINP levels, supporting a contribution in the pathogenesis and progression of SSc.

Impaired Porphyromonas gingivalis-Induced Tumor Necrosis Factor Production by Dendritic Cells Typifies Patients With Rheumatoid Arthritis.

OBJECTIVE: The prevalence of periodontitis is increased in patients with rheumatoid arthritis (RA), and the severity of periodontitis can affect the level of arthritis. Porphyromonas gingivalis is one of the main bacteria involved in periodontitis. Our aim was to determine if there are differences in the innate immune response against P gingivalis between healthy controls and RA patients. METHODS: Monocyte-derived dendritic cells (DCs) from healthy controls, RA patients, and patients with psoriatic arthritis (PsA) were stimulated with P gingivalis, a range of other bacteria, and Toll-like receptor agonists. Cytokine production was determined, and blocking studies were performed to determine which receptors were involved in differential recognition of P gingivalis. Effects on T cell cytokines were also determined in cultures of peripheral blood mononuclear cells (PBMCs). RESULTS: Upon stimulation with P gingivalis, RA patient DCs produced less tumor necrosis factor as compared to healthy control DCs, which was not observed in PsA patients or upon stimulation with other bacteria. In addition, P gingivalis-mediated activation of RA patient PBMCs showed a clear reduction of interferon-γ production. Among the various possible underlying mechanisms investigated, only blockade of CR3 abolished the difference between RA patients and healthy controls, suggesting the involvement of CR3 in this process. CONCLUSION: Immune cells from RA patients display a reduced response to P gingivalis, which has functional consequences for the immune response. This may result in prolonged survival of P gingivalis, possibly driving autoantibody formation and a self-perpetuating loop of chronic inflammation. The possible role of CR3 in this process warrants further investigation.