Victoria Saferding

Macrophage PTEN Regulates Expression and Secretion of Arginase I Modulating Innate and Adaptive Immune Responses

The activation of innate immune cells triggers numerous intracellular signaling pathways, which require tight control to mount an adequate immune response. The PI3K signaling pathway is intricately involved in innate immunity, and its activation dampens the expression and release of proinflammatory cytokines in myeloid cells. These signaling processes are strictly regulated by the PI3K antagonist, the lipid phosphatase, PTEN, a known tumor suppressor. Importantly, PTEN is responsible for the elevated production of cytokines such as IL-6 in response to TLR agonists, and deletion of PTEN results in diminished inflammatory responses. However, the mechanisms by which PI3K negatively regulates TLR signaling are only partially resolved. We observed that Arginase I expression and secretion were markedly induced by PTEN deletion, suggesting PTEN−/− macrophages were alternatively activated. This was mediated by increased expression and activation of the transcription factors C/EBPβ and STAT3. Genetic and pharmacologic experimental approaches in vitro, as well as in vivo autoimmunity models, provide convincing evidence that PI3K/PTEN-regulated extracellular Arginase I acts as a paracrine regulator of inflammation and immunity.

Loss of phosphatase and tensin homolog (PTEN) in myeloid cells controls inflammatory bone destruction by regulating the osteoclastogenic potential of myeloid cells

Objective Local bone destruction in rheumatic diseases, which often leads to disability and severely reduced quality of life, is almost exclusively mediated by osteoclasts. Therefore, it is important to understand pathways regulating the generation of osteoclasts. Here, we analysed the impact of the Phosphoinositide-3-Kinase (PI3K)/Phosphatase and tensin homolog (PTEN) axis on osteoclast generation and bone biology under basal and inflammatory conditions. Methods We analysed osteoclastogenesis of wildtype (wt) and PTEN−/− cells in vitro and in vivo, pit resorption and qPCR of osteoclasts in vitro. Mice with a myeloid cell-specific deletion of PTEN and wt littermate mice were investigated by bone histomorphometry and clinical and histological assessment in the human tumour necrosis factor (TNF)-transgenic (hTNFtg) arthritis model. Results We show that myeloid-specific PTEN−/− mice display increased osteoclastogenesis in vitro and in vivo compared to wt mice. Loss of PTEN did not affect the generation or survival of osteoclast precursor cells. However, PTEN deficiency greatly enhanced receptor activator of nuclear factor κ-B ligand (RANKL)-induced expression of the master transcription factor of osteoclastogenesis, nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), resulting in markedly increased terminal differentiation of osteoclasts in vitro. We also observed increased osteoclastogenesis under inflammatory conditions in the hTNFtg mouse model of arthritis, where hTNFtg/myeloid-specific PTEN−/− mice displayed enhanced local bone destruction as well as osteoclast formation in the inflamed joints. The extent of synovial inflammation, however, as well as recruitment of osteoclast precursor cells was not different between wt and myeloid-specific PTEN−/− mice. Conclusions These data demonstrate that loss of PTEN and, therefore, sustained PI3-Kinase signalling in myeloid cells especially, elevates the osteoclastogenic potential of myeloid cells, leading to enhanced inflammatory local bone destruction. Therefore, although our study allows no direct translational conclusion since we used a conditional knockout approach, the therapeutic targeting of the PI3-Kinase pathway may be of benefit in preventing structural joint damage.

Phosphatase and tensin homolog (PTEN) in antigen-presenting cells controls Th17-mediated autoimmune arthritis

IntroductionAutoreactive T cells are a central element in many systemic autoimmune diseases. The generation of these pathogenic T cells is instructed by antigen-presenting cells (APCs). However, signaling pathways in APCs that drive autoimmune diseases, such as rheumatoid arthritis, are not understood.MethodsWe measured phenotypic maturation, cytokine production and induction of T cell proliferation of APCs derived from wt mice and mice with a myeloid-specific deletion of PTEN (myeloid PTEN-/-) in vitro and in vivo. We induced collagen-induced arthritis (CIA) and K/BxN serum transfer arthritis in wt and myeloid-specific PTEN-/- mice. We measured the cellular composition of lymph nodes by flow cytometry and cytokines in serum and after ex vivo stimulation of T cells.ResultsWe show that myeloid-specific PTEN-/- mice are almost protected from CIA. Myeloid-specific deletion of PTEN leads to a significant reduction of cytokine expression pivotal for the induction of systemic autoimmunity such as interleukin (IL)-23 and IL-6, leading to a significant reduction of a Th17 type of immune response characterized by reduced production of IL-17 and IL-22. In contrast, myeloid-specific PTEN deficiency did not affect K/BxN serum transfer arthritis, which is independent of the adaptive immune system and solely depends on innate effector functions.ConclusionsThese data demonstrate that the presence of PTEN in myeloid cells is required for the development of CIA. Deletion of PTEN in myeloid cells inhibits the development of autoimmune arthritis by preventing the generation of a pathogenic Th17 type of immune response.

Loss of Phosphatase and Tensin Homolog in APCs Impedes Th17-Mediated Autoimmune Encephalomyelitis

The PI3K signaling cascade in APCs has been recognized as an essential pathway to initiate, maintain, and resolve immune responses. In this study, we demonstrate that a cell type–specific loss of the PI3K antagonist phosphatase and tensin homolog (PTEN) in myeloid cells renders APCs toward a regulatory phenotype. APCs deficient for PTEN exhibit reduced activation of p38 MAPK and reduced expression of T cell–polarizing cytokines. Furthermore, PTEN deficiency leads to upregulation of markers for alternative activation, such as Arginase 1, with concomitant downregulation of inducible NO synthase in APCs in vitro and in vivo. As a result, T cell polarization was dysfunctional in PTEN−/− APCs, in particular affecting the Th17 cell subset. Intriguingly, mice with cell type–specific deletions of PTEN-targeting APCs were protected from experimental autoimmune encephalomyelitis, which was accompanied by a pronounced reduction of IL-17– and IL-22–producing autoreactive T cells and reduced CNS influx of classically activated monocytes/macrophages. These observations support the notion that activation of the PI3K signaling cascade promotes regulatory APC properties and suppresses pathogenic T cell polarization, thereby reducing the clinical symptoms and pathology of experimental autoimmune encephalomyelitis.

Therapeutic Modulation of Plasmacytoid Dendritic Cells in Experimental Arthritis

The role of plasmacytoid dendritic cells (PDCs) and type I interferons (IFNs) in rheumatoid arthritis (RA) remains a subject of controversy. This study was undertaken to explore the contribution of PDCs and type I IFNs to RA pathogenesis using various animal models of PDC depletion and to monitor the effect of localized PDC recruitment and activation on joint inflammation and bone damage.

A1.02 Important role of microrna-146A in inflammatory arthritis by controlling local bone destruction

Background MicroRNA (MiR-) 146a is a key regulator of the innate immune response and has also been shown to suppress cancer development in myeloid cells., Although in late stages of arthritis elevated expression of miR-146a in synovial tissue of rheumatoid arthritis patients was detected, the level of this miRNA was found to be down regulated in early disease, but its role in the development of inflammatory arthritis is yet unknown. Materials and Methods We induced K/BxN serum transfer arthritis in wild type and miR-146a-/- mice. As a second inflammatory arthritis disease model we crossed miR-146adeficient into hTNFtg mice. Disease severity was assessed clinically and histologically in both arthritis models. Blood of arthritis animals was analysed by flow cytometry. Serum cytokine levels were measured by Elisa. RNA expression levels were measured by qPCR. Results Absence of miR-146a leads to increased clinical signs of the induced serum transfer arthritis. In line, higher serum levels of the proinflammatory cytokines IL-12 and IL-6 were measured in miR146a deficient compared to wt mice. When we crossed miR-146a-/- into hTNFtg mice, histological examination revealed a significant increase in synovial inflammation and even more striking a more than twofold increase in local bone destruction due to increased generation of osteoclasts in the tarsal joints in miR-146a-/-/hTNFtg mice compared to hTNFtg mice. Interestingly, systemic bone loss was comparable in hTNFtg compared to miR-146a-/-/hTNFtg mice, suggesting an important local role of miR-146a. Indeed, we detected increased levels of IL-1β and RANKL and decreased expression of OPG locally in the paws of miR-146a-/-/hTNFtg compared to hTNFtg mice. Analysing the content of myeloid cells in the blood of arthritis diseased mice, revealed significantly increased numbers of circulating CD11b+ as well as CD11c+ cells in mice lacking miR-146a. Bone marrow transplants demonstrated a pivotal role for miR-146a in mesenchymal cells in controlling local osteoclast generation and bone destruction. Conclusion These data demonstrate an important mitigating role of miR-146a in inflammatory arthritis, most importantly in local bone destruction, by controlling mesenchymal expression of osteoclastogenic factors. This shows a crucial anti-inflammatory role of miR-146a, which might possibly be exploited for therapeutic purposes.

4.8 Role of Toll-like receptor 9 in the pathogenesis of inflammatory autoimmune arthritis and osteoclast activation

Background and Objectives There is some evidence that release and insufficient removal of endogenous nucleic acids may be involved in triggering harmful autoimmune reactions that might be important in the initiation of RA. Nucleic acid-sensing molecules, such as the endosomal Toll-like receptors (TLRs) 7 and 9, have been linked to pathogenic autoimmune processes, particularly in SLE, but their role in RA is less clear. Data recently obtained in rats with pristane-induced arthritis (PIA) suggested involvement of TLR9, because antagonizing this receptor in the initiation phase of the disease led to an improved clinical outcome of arthritis. To gain more insight into the role of TLR9 in autoimmune arthritis we have extended our studies to the K/BxN serum-transfer arthritis model which is reflecting the effector phase of erosive autoimmune arthritis. Materials and Methods Arthritis was induced in C57BL/6 mice by two subsequent injections of arthritogenic serum. Antagonists for TLR7 and TLR9 or an agonist for TLR9, respectively, a non-inhibitory control sequence or PBS as placebo was applied every third day. The first treatment was given one day before disease induction. To further investigate TLR9 involvement, arthritis was also induced in TLR9 knock-out mice. Furthermore, the effect of TLR9 on osteoclast differentiation and activation was investigated in an in vitro osteoclast formation assay. Results Neither inhibitor affected arthritis onset and severity in the serum transfer model, which is independent of the adaptive immune system, in contrast to PIA. Furthermore, arthritis severity was not changed in mice lacking a functional tlr9 gene in comparison to wild type animals. In contrast, treatment with a TLR9 agonist led to an improved clinical outcome. Remarkably, in vitro formation of osteoclasts appeared to be slightly enhanced in cells isolated from TLR9-/- mice compared to those from wild type animals. Moreover, osteoclastogenesis was reduced by ~60% in cells from wild type animals grown in the presence of the TLR9 agonist whereas, as expected, no effect was seen in cells from TLR9-/- animals. Conclusions These results suggest that TLR9 might play a beneficial regulatory role in the effector phase of erosive autoimmune arthritis by negatively affecting differentiation and activation of bone resorbing osteoclasts. Nevertheless, the precise role of TLR9 in the different stages of arthritis needs to be further elucidated.

A1.82 Limited role of microrna 155 innate immunity dependent arthritis

Background MicroRNA 155 (miR155) has been demonstrated to be essential for the development of collagen induced arthritis by controlling the generation of autoreactive T and B cells. However, the contribution of miR155 in innate immune cells is not known. Materials and Methods We analysed activation and cytokine production of macrophages and dendritic cells (DCs) in vitro and in vivo. We analysed T-cells stimulatory capacity of DCs. We crossed miR155 deficient mice into hTNFtg mice and analysed arthritis development clinically as well as histologically. Results MiR155 deficiency did not alter the differentiation of BMDC from bone marrow cells or the expression of costimulatory molecules or MHCII expression after stimulation of macrophages and DCs in vitro and in vivo. Proinflammatory cytokine production (IL-12p40, IL-6) after LPS challenge in vivo was not different in serum of wt mice and miR155-/-mice. We also Facs-sorted DCs after stimulation with LPS in vivo and determined the production of proinflammatory cytokines such as IL-23, IL-6 as well as TNF. We did not detect differences between wt and miR155-/- mice. In addition, the T cell stimulatory capacity of wt and miR155-/-was identical in vitro and in vivo. When we analysed miR155-/-mice compared to wt mice in the hTNFtg model of inflammatory arthritis, which is independent of the adaptive immune system, we did not detect differences in the clinical signs and symptoms of arthritis. Histologically, we even found slightly increased synovial inflammation in hTNFtg/ miR155-/-mice compared to wt mice. Conclusion In contrast to the pivotal role of miR155 in autoimmunity requiring the adaptive immune system, the role of miR155 in innate immunity seems to be limited. This is emphasised by the fact that miR155 hardly influences the course of TNF-driven arthritis, which is mainly dependent on components of the innate immune system.

Non-classical monocytes as mediators of tissue destruction in arthritis

Objectives Bone destruction in rheumatoid arthritis is mediated by osteoclasts (OC), which are derived from precursor cells of the myeloid lineage. The role of the two monocyte subsets, classical monocytes (expressing CD115, Ly6C and CCR2) and non-classical monocytes (which are CD115 positive, but low in Ly6C and CCR2), in serving as precursors for OC in arthritis is still elusive. Methods We investigated CCR2−/− mice, which lack circulating classical monocytes, crossed into hTNFtg mice for the extent of joint damage. We analysed monocyte subsets in hTNFtg and K/BxN serum transfer arthritis by flow cytometry. We sorted monocyte subsets and analysed their potential to differentiate into OC and their transcriptional response in response to RANKL by RNA sequencing. With these data, we performed a gene ontology enrichment analysis and gene set enrichment analysis. Results We show that in hTNFtg arthritis local bone erosion and OC generation are even enhanced in the absence of CCR2. We further show the numbers of non-classical monocytes in blood are elevated and are significantly correlated with histological signs of joint destruction. Sorted non-classical monocytes display an increased capacity to differentiate into OCs. This is associated with an increased expression of signal transduction components of RANK, most importantly TRAF6, leading to an increased responsiveness to RANKL. Conclusion Therefore, non-classical monocytes are pivotal cells in arthritis tissue damage and a possible target for therapeutically intervention for the prevention of inflammatory joint damage.

FRI0024 MICRORNA-146A controls local bone destruction by regulating fibroblast induced osteoclastogenesis in inflammatory arthritis

Background MicroRNA (MiR-) 146a plays an important role in the regulation of the innate immune response and has also been shown to suppress cancer development in myeloid cells. Although in late stages of arthritis elevated expression of miR-146a in synovial tissue of rheumatoid arthritis patients was detected, the level of this miRNA was found to be down regulated in early disease, but its role in the development of inflammatory arthritis is still elusive. Objectives The objective of this study is to analyse the role of miR-146a in arthritis by the use of a chronic arthritis disease model. We aim to investigate the regulatory function of this miRNA in the pathogenic stroma, therefore in fibroblasts but also in immune cells. Methods To induce arthritis we used the chronic inflammatory hTNFtg disease model, therefore we crossed miR-146a deficient into hTNFtg mice. Disease severity was assessed clinically and histologically. Blood of arthritis animals was analysed by flow cytometry. Serum cytokine levels were measured by Elisa. Synovial fibroblasts were isolated from metatarsal bones and their proliferation was analysed histologically and by 3[H]thymidine incorporation. RNA expression levels were measured by qPCR. Results When we crossed miR-146a-/- into hTNFtg mice, histological examination revealed a significant increase in synovial inflammation and even more striking a more than twofold increase in local bone destruction, due to increased generation of osteoclasts in the tarsal joints of miR-146a-/-/hTNFtg mice compared to hTNFtg mice. Interestingly, systemic bone loss was comparable in hTNFtg compared to miR-146a-/-/hTNFtg mice, suggesting an important local role of miR-146a. Indeed, we detected increased levels of IL-1β, TRAF6, a major target of miR-146a and RANKL, in addition the expression level of OPG was decreased locally in the paws of miR-146a-/-/hTNFtg compared to hTNFtg mice. By performing bone marrow transplants we could indeed show a pivotal role for miR-146a in mesenchymal cells in controlling local osteoclast generation and bone destruction. Analysis of important mesenchymal cells in arthritis, the synovial fibroblasts exhibited enhanced proliferation if miR-146a is missing, in vitro and in vivo. Moreover stimulation of these cells with IL-1β, a prominent cytokine in arthritis which was also shown to be negatively regulated by miR-146a, led to increased expression of RANKL and TRAF6 in miR-146a deficient synovial fibroblasts. Conclusions These data demonstrate an important mitigating role of the miR-146a in inflammatory arthritis, most importantly in local bone destruction, by controlling mesenchymal expression of osteoclastogenic factors. This shows an important anti-inflammatory role of miR-146a, which might possibly be exploited for therapeutic purposes. Disclosure of Interest None declared