K Dalwigk

Quantitative and qualitative deficiencies of regulatory T cells in patients with systemic lupus erythematosus (SLE)

The objective of the study was that the regulatory T cells (Treg) that specialize in the suppression of immune responses might be critically involved in the pathogenesis of autoimmune disease. As for systemic lupus erythematosus (SLE), however, published data concerning Treg phenotype and function are partly conflicting. We therefore performed quantitative and qualitative analyses of naturally occurring CD4(+)CD25(+) Treg from SLE patients as compared with healthy controls (HC) in order to further elucidate the role of Treg in this systemic autoimmune disease. The phenotype of peripheral blood CD4(+)CD25(+) Treg was determined by flow cytometry (FACS) in SLE patients and HC. Treg were isolated from SLE patients and HC and their functional capacity was analyzed in suppression assays. Phenotypic and functional data were correlated with clinical data. Decreased proportions of CD4(+) Treg with high-level expression of CD25 (CD4(+)CD25(hi)) were observed in active and inactive SLE patients (0.96 +/- 0.08 and 1.17 +/- 0.08%, respectively) as compared with HC (2 +/- 0.1%). In contrast to HC, Treg from SLE patients displayed an activated phenotype as determined by the expression of CD69, CD71 and HLA-DR. The suppressive capacity of isolated Treg from SLE patients, however, was significantly reduced as compared with HC. Proportions of CD4(+)CD25(hi) T cells and the suppressive capacity of Treg were inversely correlated with the clinical disease activity in SLE patients. Our data describe quantitative and qualitative defects of Treg in SLE patients. These deficiencies might contribute to the breakdown of self-tolerance and the development of the autoimmune response in SLE patients.

Activation of the interferon‐γ signaling pathway in systemic lupus erythematosus peripheral blood mononuclear cells

OBJECTIVE To investigate interferon-gamma (IFNgamma) signaling in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) by analyzing IFNgamma receptor (IFNgammaR) expression, STAT-1 expression and phosphorylation, and the regulation of IFNgamma-inducible genes. METHODS Fluorocytometry was used to investigate expression of STAT-1, pSTAT-1, CD95, HLA-DR, class I major histocompatibility complex (MHC), IFNgamma-inducible 10-kd protein (IP-10), monokine induced by IFNgamma (Mig), and IFNgammaR in PBMCs from SLE patients and healthy individuals. STAT-1 phosphorylation was determined by fluorocytometry and Western blotting after stimulation with IFNalpha or IFNgamma. Quantitative polymerase chain reaction was used to assess messenger RNA (mRNA) expression of the IFNgamma-inducible genes IP-10 and Mig shortly after preparation or after stimulation with IFNgamma in monocytes. RESULTS STAT-1 expression was increased in PBMCs from SLE patients and correlated significantly with disease activity and with the IFN-inducible expression of CD95 and HLA-DR. STAT-1 expression also showed a trend toward association with class I MHC expression. In addition, the expression of other IFNgamma-inducible genes, such as IP-10 or Mig, was increased in SLE monocytes. While STAT-1 phosphorylation in SLE PBMCs and PBMCs from healthy individuals was similar after IFNalpha stimulation, incubation with IFNgamma induced STAT-1 phosphorylation only in SLE lymphocytes. Moreover, SLE monocytes showed a considerably higher increase in pSTAT-1 expression upon IFNgamma stimulation than monocytes from healthy individuals. Increased responsiveness of SLE monocytes to IFNgamma was also confirmed on the mRNA level, where expression of the IFN-inducible, STAT-1-dependent genes IP-10 and Mig was more efficiently increased in SLE cells. However, IFNgammaR was similarly expressed on SLE lymphocytes and monocytes and those from healthy individuals. CONCLUSION In addition to supporting the role of IFNs in SLE immunopathogenesis in general, the findings of the present study support a role of IFNgamma in this disease.

IFN-gamma promotes fibroblast-like synoviocytes motility

Rheumatoid arthritis (RA) is the most common chronic inflammatory joint disease. In RA, a systemic autoimmune response translates into an inflammatory attack on the synovium that yields the formation of an aggressive cell mass called pannus which attaches to, encroaches over and destroys the articular cartilage. Cartilage destruction is mediated by fibroblast-like synoviocytes (FLS) which are the prevailing cell type of the …

AB0075 MTOR: An Unexpected Role on the TNF-Regulated MRNA Transcriptome in Rheumatoid Fibroblast-Like Synoviocytes

Background Accumulating evidence supports the concept that resident mesenchymal cells, namely fibroblast-like synoviocytes (FLS) actively participate in the destructive, inflammatory process of rheumatoid arthritis (RA). FLS maintain a synovial microenvironment that helps to recruit, retain, and activate immune cells, resulting in chronic inflammation with attendant joint destruction. Here, we provide evidence that the mechanistic target of rapamycin (mTOR), which has evolved as a major factor for the maintainance of tissue homeostasis and function, is a critical component of the inflammatory process in synovitis. Results Immunohistochemical studies revealed that mTOR is activated in rheumatoid synovitis, particularly in FLS of the hyperplastic synovial lining. By using western blot or advanced cell biological methods, such as a recently described synovial organ culture system, we demonstrate that the proinflammatory cytokine TNF activates the mTOR pathway in FLS. To further define the biological significance of these findings, we made use of genome-wide expression profiling. Remarkably, this analysis revealed a so far unknown modulatory role for mTOR in the gene expression programs induced by TNF. Specific inhibition of mTOR by Torin-1 or PP242 increased the TNF-induced expression of genes, known to be regulated by the transcription complex NFkB (e.g. PTGS2, IL8, CCL20). By contrast, FLS treatment with Torin-1 or PP242 diminished the TNF-mediated induction of interferon regulated genes (IRGs), including TNFSF10, CXCL11 and TNFSF13B (BAFF). Conclusions In conclusion, these studies provide insight into determinants of the synovial tissue response to inflammation and suggest a multifaceted regulatory role for mTOR in synovial inflammatory processes. Disclosure of Interest None declared

Targeted inhibition of Janus kinases abates interfon gamma-induced invasive behaviour of fibroblast-like synoviocytes

Objectives The aim was to explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodelling in RA and to determine whether IFNγ signalling controls the invasive potential of fibroblast-like synoviocytes (FLS). Methods To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analysed in migration and invasion assays. Signalling events relevant to cellular motility were defined by western blots. Baricitinib and small interfering RNA pools were used to suppress Janus kinase (JAK) functions. Results Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration upon IFNγ stimulation. Given that cell shape and cell motility are under the control of the focal adhesion kinase (FAK), we next analysed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. Small interfering RNA knockdown of JAK2, but not JAK1, substantially abrogated FAK activation by IFNγ. Correspondingly, IFNγ-induced FAK activation and invasion of FLS was abrogated by the JAK inhibitor, baricitinib. Conclusion Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA.

mTOR Senses Environmental Cues to Shape the Fibroblast-like Synoviocyte Response to Inflammation

Summary Accumulating evidence suggests that metabolic master regulators, including mTOR, regulate adaptive and innate immune responses. Resident mesenchymal tissue components are increasingly recognized as key effector cells in inflammation. Whether mTOR also controls the inflammatory response in fibroblasts is insufficiently studied. Here, we show that TNF signaling co-opts the mTOR pathway to shift synovial fibroblast (FLS) inflammation toward an IFN response. mTOR pathway activation is associated with decreased NF-κB-mediated gene expression (e.g., PTGS2, IL-6, and IL-8) but increased STAT1-dependent gene expression (e.g., CXCL11 and TNFSF13B). We further demonstrate how metabolic inputs, such as amino acids, impinge on TNF-mTORC1 signaling to differentially regulate pro-inflammatory signaling circuits. Our results define a critical role for mTOR in the regulation of the pro-inflammatory response in FLSs and unfold its pathogenic involvement in TNF-driven diseases, such as rheumatoid arthritis (RA).

02.36 Lasp-1 regulates the migratory behaviour of synovial fibroblasts in rheumatoid arthritis

Background In rheumatoid arthritis (RA) the attachment of synovial fibroblasts to articular cartilage is an important prerequisite in the process of cartilage degradation. The actin-associated protein Lasp-1 is involved in processes of actin organisation and polymerization and focal adhesion turnover, respectively. Therefore, we investigated its role in regulating cell-cell contacts and ECM interactions of synovial fibroblasts in RA. Methods Lasp-1 expression was analysed in human RA tissue and in arthritic mouse models such as the hTNFtg and G6PI mouse model by using WB and immunohistochemistry. Lasp-1 expression and its subcellular distribution was investigated in isolated synovial fibroblasts by immunofluorescence in all genotypes. Furthermore, the formation of cell-cell and -ECM contacts was investigated in an electrical cell/substrate impedance sensing assay (ECIS) as well as by immunofluorescence. Additionally, we performed an in vitro three-dimensional organ culture system to identify genotype-specific differences of cellular distribution and formation of the artificial synovial lining layer. Results An increased Lasp-1 expression was observed in synovial tissue from RA patients as well as in RASF. Isolated SF from hTNFtg mice also produced higher levels of Lasp-1 compared to wild type (wt) SF. Interestingly, this was completely reproducible in the G6PI model. Lasp-1 deficient mice that had been crossed into the hTNFtg background exhibited less cartilage degradation (−4% vs. hTNFtg) and less attachment of synovial tissue to the cartilage (−30% vs. hTNFtg) compared to hTNFtg mice. Functional analyses indicated that Lasp-1-/- hTNFtg SF form closer (+20% vs hTNFtg SF) and more stable cell-cell contacts in comparison to hTNFtg SF. Furthermore, we detected Lasp-1 to be located at structures of adherens junction complexes. The loss of Lasp-1 led to alterations of these structures. Histological analysis of 3D matrices showed that Lasp-1 deletion in the hTNFtg background resulted in an organised cellular lining layer at the interface between the matrix and fluid phases comparable with wild type SF. In contrast, hTNFtg SF formed unorganised cellular condensations with no synovial architecture evident. Conclusions Lasp-1 regulates the migratory behaviour of synovial fibroblasts and their invasion into cartilage matrix in rheumatoid arthritis by controlling the dynamics of cell-cell contacts.

FRI0018 Targeted inhibition of janus kinases abates IFN-GAMMA-INDUCED invasive behavior of fibroblast-like synoviocytes

Background Emerging evidence suggests that fibroblast-like synoviocytes (FLS) are key effector cells in rheumatoid arthritis (RA) and research into the mechanisms defining FLS activity in RA indicate that cytokines secreted by leukocytes play a crucial role. Nevertheless, the contribution of IFNγ, which is increased in rheumatoid synovitis, to the inflammatory synovial tissue reaction is not known. Objectives To explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodeling in RA, and to determine whether IFNγ-signaling controls the invasive potential of FLS. Methods To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analyzed in migration-, spreading- and invasion assays. Signaling events relevant to cellular motility were defined by western blots. Baricitinb and siRNA pools were used to suppress Janus Kinase (JAK) functions. Results Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration, pronounced actin and focal adhesion (FA) re-organization upon IFNγ stimulation. Since actin and FA dynamics and, thus, cell motility are integrated by the focal adhesion kinase (FAK), we next analyzed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. siRNA knockdown of JAK2, but not JAK1, abrogated FAK activation by IFNγ. Correspondingly, IFNγ-inudced FAK activation and invasion of FLS was abrogated by the JAK-inhibitor baricitinib. Conclusions Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA. Disclosure of Interest None declared

04.05 The metabolic checkpoint kinase mtor regulates the rheumatoid mesenchymal tissue response to inflammation

Background By producing cytokines, chemokines and other inflammatory factors fibroblast-like synoviocytes (FLS) frame a microenvironemt that significantly contributes to persistent synovial inflammation in rheumatoid arthritis (RA). The molecular mechanisms and pathways that regulate these responses within FLS are, however, insufficiently explored. Since mTOR is emerging as a regulator of tissue homeostasis that coordinates the cellular reponse to environmental stress, such as inflammation, we hypothesised that this kinase might also determine the FLS response to inflammation. Material and methods mTOR activation was assessed by immunhistochemistry and western blots. For functional in-vitro studies RA-FLS were stimulated with TNF and specific mTOR inhibitors (Torin-1, PP242 and rapamycin) were applied. Affymetrix microarrays were used for gene expression profiling. NF-κB and STAT1 pathway activation was assessed by western-blots and EMSA. Results Immunohistochemistry revealed increased mTOR activity in RA synovitis when compared to osteoarthritis. TNF stimulation of FLS ex vivo promoted the phosphorylation of mTOR in an AKT-dependent manner indicating that the pro-inflammatory synovial milieu may drive mTOR activation in RA. Gene expression profiling revealed a so far unknown role for mTOR in the regulation of TNF-induced gene programs: mTOR activation limits the TNF-induced expression of NF-κB-regulated genes (eg, IL6, IL8, PTGS2) by promoting the re-appearence of the NF-κB-inhibitor IκB-α. Contrary, mTOR activation augments the TNF-mediated induction of interferon regulated genes (IRGs; eg, CXCL11, TNFSF13B) by increasing the activity of the transcription factor STAT1. Conclusions The metabolic checkpoint kinase mTOR fine-tunes the induced gene expression program in FLS. Thus, mTOR emerges as crucial factor for the development of the RA-typical synovial, inflammatory microenvironment.

A7.05 Baricitinib abrogates IFNγ-induced focal adhesion kinase (FAK) activation in fibroblast-like synoviocytes

While evidence implicates both the adaptive and innate immune system in rheumatoid arthritis (RA) pathogenesis, accumulating data indicate that the synovial tissue itself actively participates in the destructive inflammatory process. Specifically, resident fibroblast-like synoviocytes (FLS), together with macrophages, re-organise to form an aggressive cell mass, called pannus, which destroys the articular cartilage and the subchondral bone. The exact molecular mechanisms of synovial pannus formation, FLS expansion and invasion into adjacent tissues are not yet known. Our data strongly suggest that the T-cell derived cytokine IFNγ is involved in FLS-mediated joint destruction. Migration and invasion assays revealed increased migratory activity for IFNγ-stimulated FLS, when compared to unstimulated FLS. Further, biochemical studies showed that IFNγ promotes the migratory and invasive activity of FLS via Janus kinase 2 (JAK2) and the focal adhesion kinase (FAK), a kinase known to integrate focal adhesion turnover and thus, regulates cell migration. In detail, IFNγ stimulation of FLS distinctly resulted in the phosphorylation of FAK-Y925, a phospho-site that has recently been demonstrated to be required for FAK-mediated cell migration. siRNA knockdown of JAK2, but not JAK1, abrogated the IFNγ-induced activation of FAK. Correspondingly, baricitinib, a JAK inhibitor that is currently successfully probed in RA clinical trials, abrogated IFNγ-stimulated activation of FAK. In conclusion, our studies contribute insight into the synovial response to IFNγ and reveal JAK2 and FAK as potential targets for synoviocyte-mediated joint destruction in arthritis, especially in RA.