Alarmins (also known as danger signals) are endogenous molecules that are released to the extracellular milieu after infection or tissue damage. Extracellular alarmins interact with specific receptors expressed by cells that are engaged in host defence to stimulate signalling pathways that result in initiation of innate and adaptive immune responses, triggering inflammation or tissue repair. Alarmins are considered to be markers of destructive processes that occur in degenerative joint diseases (primarily osteoarthritis (OA)) and chronic inflammatory joint diseases (such as rheumatoid arthritis, psoriatic arthritis and spondylarthropathy). In OA, high mobility group protein B1 (HMGB1) and S100 proteins, along with many other alarmins, are abundantly secreted by joint cells, promoting cartilage matrix catabolism, osteophyte formation, angiogenesis and hypertrophic differentiation. The involvement of alarmins in chronic inflammatory arthritides is suggested by their presence in serum at high levels in these conditions, and their expression within inflamed synovia and synovial fluid. S100 proteins, HMGB1, IL-33 and other endogenous molecules have deleterious effects on joints, and can recruit immune cells such as dendritic cells to inflamed synovia, initiating the adaptive immune response and perpetuating disease. Improving our understanding of the pathological mechanisms associated with these danger signals is important to enable the targeting of new therapeutic approaches for arthritis.

Osteoarthritis is a whole-joint disease characterized by the progressive destruction of articular cartilage involving abnormal communication between subchondral bone and cartilage. Our team previously identified 14-3-3ε protein as a subchondral bone soluble mediator altering cartilage homeostasis. The aim of this study was to investigate the involvement of CD13 (also known as aminopeptidase N, APN) in the chondrocyte response to 14-3-3ε. After identifying CD13 in chondrocytes, we knocked down CD13 with small interfering RNA (siRNA) and blocking antibodies in articular chondrocytes. 14-3-3ε-induced MMP-3 and MMP-13 was significantly reduced with CD13 knockdown, which suggests that it has a crucial role in 14-3-3ε signal transduction. Aminopeptidase N activity was identified in chondrocytes, but the activity was unchanged after stimulation with 14-3-3ε. Direct interaction between CD13 and 14-3-3ε was then demonstrated by surface plasmon resonance. Using labeled 14-3-3ε, we also found that 14-3-3ε binds to the surface of chondrocytes in a manner that is dependent on CD13. Taken together, these results suggest that 14-3-3ε might directly bind to CD13, which transmits its signal in chondrocytes to induce a catabolic phenotype similar to that observed in osteoarthritis. The 14-3-3ε–CD13 interaction could be a new therapeutic target in osteoarthritis.

Objective: The innate immune system plays a central role in osteoarthritis (OA). We identified 14-3-3ε as a novel mediator that guides chondrocytes toward an inflammatory phenotype. 14-3-3ε shares common characteristics with alarmins. These endogenous molecules, released into extracellular media, are increasingly incriminated in sustaining OA inflammation. Alarmins bind mainly to TLR2 and TLR4 receptors and polarize macrophages in the synovium. We investigated the effects of 14-3-3ε in joint cells and tissues and its interactions with TLRs to define it as a new alarmin involved in OA.Design: Chondrocyte, synoviocyte and macrophage cultures from murine or OA human samples were treated with 14-3-3ε. To inhibit TLR2/4 in chondrocytes, blocking antibodies were used. Moreover, chondrocytes and bone marrow macrophage (BMM) cultures from KO TLRs mice were stimulated with 14-3-3ε. Gene expression and release of inflammatory mediators (IL-6, MCP-1, TNFα) were evaluated via RT-qPCR and ELISA.Results: In vitro, 14-3-3ε induced gene expression and release of IL6 and MCP1 in the treated cells. The inflammatory effects of 14-3-3ε were significantly reduced following TLRs inhibition or in TLRs KO chondrocytes and BMM.Conclusions: 14-3-3ε is able to induce an inflammatory phenotype in synoviocytes, macrophages and chondrocytes in addition to polarizing macrophages. These effects seem to involve TLR2 or TLR4 to trigger innate immunity. Our results designate 14-3-3ε as a novel alarmin in OA and as a new target either for therapeutic and/or prognostic purposes.

Salt Inducible Kinases (SIKs), of which there are 3 isoforms, are established to play roles in innate immunity, metabolic control and neuronal function, but their role in adaptive immunity is unknown. To address this gap, we used a combination of SIK knockout and kinase-inactive knock-in mice. The combined loss of SIK1 and SIK2 activity did not block T cell development. Conditional knockout of SIK3 in haemopoietic cells, driven by a Vav-iCre transgene, resulted in a moderate reduction in the numbers of peripheral T cells, but normal B cell numbers. Constitutive knockout of SIK2 combined with conditional knockout of SIK3 in the haemopoietic cells resulted in a severe reduction in peripheral T cells without reducing B cell number. A similar effect was seen when SIK3 deletion was driven via CD4-Cre transgene to delete at the DP stage of T cell development. Analysis of the SIK2/3 Vav-iCre mice showed that thymocyte number was greatly reduced, but development was not blocked completely as indicated by the presence of low numbers CD4 and CD8 single positive cells. SIK2 and SIK3 were not required for rearrangement of the TCRβ locus, or for low level cell surface expression of the TCR complex on the surface of CD4/CD8 double positive thymocytes. In the absence of both SIK2 and SIK3, progression to mature single positive cells was greatly reduced, suggesting a defect in negative and/or positive selection in the thymus. In agreement with an effect on negative selection, increased apoptosis was seen in thymic TCRbeta high/CD5 positive cells from SIK2/3 knockout mice. Together, these results show an important role for SIK2 and SIK3 in thymic T cell development.

Fibroblasts, derived from the embryonic mesenchyme, are a diverse array of cells with roles in development, homeostasis, repair, and disease across tissues. In doing so, fibroblasts maintain micro-environmental homeostasis and create tissue niches by producing a complex extracellular matrix (ECM) including various structural proteins. Although long considered phenotypically homogenous and functionally identical, the emergence of novel technologies such as single cell transcriptomics has allowed the identification of different phenotypic and cellular states to be attributed to fibroblasts, highlighting their role in tissue regulation and inflammation. Therefore, fibroblasts are now recognised as central actors in many diseases, increasing the need to discover new therapies targeting those cells. Herein, we review the phenotypic heterogeneity and functionality of these cells and their roles in health and disease.

IntroductionThe synovial membrane is the main site of inflammation in rheumatoid arthritis (RA). Here several subsets of fibroblasts and macrophages, with distinct effector functions, have been recently identified. The RA synovium is hypoxic and acidic, with increased levels of lactate as a result of inflammation. We investigated how lactate regulates fibroblast and macrophage movement, IL-6 secretion and metabolism via specific lactate transporters.MethodsSynovial tissues were taken from patients undergoing joint replacement surgery and fulfilling the 2010 ACR/EULAR RA criteria. Patients with no evidence of degenerative or inflammatory disease were used as control. Expression of the lactate transporters SLC16A1 and SLC16A3 on fibroblasts and macrophages was assessed by immunofluorescence staining and confocal microscopy. To test the effect of lactate in vitro we used RA synovial fibroblasts and monocyte-derived macrophages. Migration was assessed via scratch test assays or using trans-well inserts. Metabolic pathways were analysed by Seahorse analyser. IL-6 secretion was determined by ELISA. Bioinformatic analysis was performed on publicly available single cell and bulk RNA sequencing datasets.ResultsWe show that: i) SLC16A1 and SLC16A3 which regulate lactate intake and export respectively, are both expressed in RA synovial tissue and are upregulated upon inflammation. SLC16A3 is more highly expressed by macrophages, while SLC16A1 was expressed by both cell types. ii) This expression is maintained in distinct synovial compartments at mRNA and protein level. iii) Lactate, at the concentration found in RA joints (10 mM), has opposite effects on the effector functions of these two cell types. In fibroblasts, lactate promotes cell migration, IL-6 production and increases glycolysis. In contrast macrophages respond to increases in lactate by reducing glycolysis, migration, and IL-6 secretion.DiscussionIn this study, we provide the first evidence of distinct functions of fibroblasts and macrophages in presence of high lactate levels, opening new insights in understanding the pathogenesis of RA and offering novel potential therapeutic targets.

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Purpose: Synovial inflammation associated with production of proinflammatory mediators and active immune cells proliferation, plays an important role in the onset and progression of osteoarthritis (OA). Regenerative therapies based on mesenchymal stem/stromal cells (MSCs) hold great promise for the disease modulation of OA, however the precise therapeutic mechanisms and interactions with the inflammatory environment in the OA joint still needs to be elucidated. Our previous studies identified elevated production of pre-and antiinflammatory cytokines in the synovial washouts from OA joints in a murine model. The aim of the present study was to investigate the in vitroimmunomodulatory effects of cytokine licensed MSCs on resting and activated bone-marrow derived macrophages (BMDMs). The proposed modulation strategy could provide a better understanding of the interaction between MSCs and immune cells, which gives valuable insight on the development of new therapies for the treatment of OA. Methods: MSCs isolated from bone marrow of C57/BL6 mice, were licensed singularly and simultaneously with IL -6 (50ng/ml) and Monocyte Chemoattractant Protein-1 (MCP-1) (50ng/ml) for 72h. BMDMs of C57BL/6 mice were plated and differentiated for 6 days in macrophage media. Polarization was induced by stimulation with LPS (10ng/ml for 4h) and IFN-gamma (100ng/ml for 24 hours), to generate classical BMDMs. Alternate activated macrophages were obtained following an incubation with 10ng/ml IL-4 and IL-13 for 24h. Licensed MSCs were directly co-cultured respectively with resting (M0), classical activated (M1) and alternate activated (M2) macrophages for 72 hours, in ratio 2:1 (5x10 4 MSCs: 2.5 x 10 4 BMDMs). Co-cultured BMDMs were collected for flow cytometry analysis (FACS Canto cytometer). The release of pro-and anti-inflammatory cytokines in cell supernatants was measured with ELISA, and Griess assay was performed to quantify the production of nitrite. Results: Flow cytometric characterization of differentiated macrophages showed the expression of CD11b, a pan-macrophage marker. The successful generation of activated macrophages was confirmed with the positive expression of MHC-II in M1 and CD206 in M2 macrophages. Flow cytometrical analysis and ELISA showed a modulating effect of licensed MSCs on resting, classical and alternate activated macrophages. Co-cultured resting macrophages had an increased expression of MHC-II and CD206 expression compared to control BMDMs, associated with a higher production of PGE2 and MCP-1. Similarly, licensed MSCs increased the expression of CD206, while maintaining a high MHC-II expression on classical activated M1 macrophages, characterized with elevated production of PGE2 and MCP-1. M2 macrophages were characterized with a high expression of CD206 and MHC-II expression, and increased PGE2 and MCP-1 secretion. Griess assay showed that co-cultured M0, M1 and M2 macrophages had a higher production of NO, compared to control BMDMs. Conclusions: In summary, the present work demonstrates the effect of IL...