Immunomodulatory Nanoparticles for Modulating Arthritis Flares.

Disease-modifying drugs have improved the treatment for autoimmune joint disorders, such as rheumatoid arthritis, but inflammatory flares are a common experience. This work reports the development and application of flare-modulating poly(lactic-co-glycolic acid)-poly(ethylene glycol)-maleimide (PLGA-PEG-MAL)-based nanoparticles conjugated with joint-relevant peptide antigens, aggrecan70-84 and type 2 bovine collagen256-270. Peptide-conjugated PLGA-PEG-MAL nanoparticles encapsulated calcitriol, which acted as an immunoregulatory agent, and were termed calcitriol-loaded nanoparticles (CLNP). CLNP had a ∼200 nm hydrodynamic diameter with a low polydispersity index. In vitro, CLNP induced phenotypic changes in bone marrow derived dendritic cells (DC), reducing the expression of costimulatory and major histocompatibility complex class II molecules, and proinflammatory cytokines. Bulk RNA sequencing of DC showed that CLNP enhanced expression of Ctla4, a gene associated with downregulation of immune responses. In vivo, CLNP accumulated in the proximal lymph nodes after intramuscular injection. Administration of CLNP was not associated with changes in peripheral blood cell numbers or cytokine levels. In the collagen-induced arthritis and SKG mouse models of autoimmune joint disorders, CLNP reduced clinical scores, prevented bone erosion, and preserved cartilage proteoglycan, as assessed by high-resolution microcomputed tomography and histomorphometry analysis. The disease protective effects were associated with increased CTLA-4 expression in joint-localized DC and CD4+ T cells but without generalized suppression of T cell-dependent immune response. The results support the potential of CLNP as modulators of disease flares in autoimmune arthropathies.

Immunomodulatory Microparticles Epigenetically Modulate T Cells and Systemically Ameliorate Autoimmune Arthritis.

Disease modifying antirheumatic drugs (DMARDs) have improved the prognosis of autoimmune inflammatory arthritides but a large fraction of patients display partial or nonresponsiveness to front-line DMARDs. Here, an immunoregulatory approach based on sustained joint-localized release of all-trans retinoic acid (ATRA), which modulates local immune activation and enhances disease-protective T cells and leads to systemic disease control is reported. ATRA imprints a unique chromatin landscape in T cells, which is associated with an enhancement in the differentiation of naïve T cells into anti-inflammatory regulatory T cells (Treg ) and suppression of Treg destabilization. Sustained release poly-(lactic-co-glycolic) acid (PLGA)-based biodegradable microparticles encapsulating ATRA (PLGA-ATRA MP) are retained in arthritic mouse joints after intra-articular (IA) injection. IA PLGA-ATRA MP enhance migratory Treg which in turn reduce inflammation and modify disease in injected and uninjected joints, a phenotype that is also reproduced by IA injection of Treg . PLGA-ATRA MP reduce proteoglycan loss and bone erosions in the SKG and collagen-induced arthritis mouse models of autoimmune arthritis. Strikingly, systemic disease modulation by PLGA-ATRA MP is not associated with generalized immune suppression. PLGA-ATRA MP have the potential to be developed as a disease modifying agent for autoimmune arthritis.

Systems-biology analysis of rheumatoid arthritis fibroblast-like synoviocytes implicates cell line-specific transcription factor function.

Rheumatoid arthritis (RA) is an immune-mediated disease affecting diarthrodial joints that remains an unmet medical need despite improved therapy. This limitation likely reflects the diversity of pathogenic pathways in RA, with individual patients demonstrating variable responses to targeted therapies. Better understanding of RA pathogenesis would be aided by a more complete characterization of the disease. To tackle this challenge, we develop and apply a systems biology approach to identify important transcription factors (TFs) in individual RA fibroblast-like synoviocyte (FLS) cell lines by integrating transcriptomic and epigenomic information. Based on the relative importance of the identified TFs, we stratify the RA FLS cell lines into two subtypes with distinct phenotypes and predicted active pathways. We biologically validate these predictions for the top subtype-specific TF RARα and demonstrate differential regulation of TGFß signaling in the two subtypes. This study characterizes clusters of RA cell lines with distinctive TF biology by integrating transcriptomic and epigenomic data, which could pave the way towards a greater understanding of disease heterogeneity.

Activated low-density granulocytes in peripheral and intervillous blood and neutrophil inflammation in placentas from SLE pregnancies.

OBJECTIVE: Women with SLE face an increased risk of adverse pregnancy outcomes compared with healthy women, but the underlying immunological mechanisms are unknown. Given the recognised association of neutrophil activation with SLE pathogenesis, we examined whether there is increased neutrophil activation and inflammation in blood and placenta in SLE relative to healthy pregnancy. METHODS: At delivery, peripheral blood, maternal-derived intervillous blood and placentas were collected from 12 SLE and 10 healthy control pregnancies. The proportion of low-density granulocytes (LDGs) and the activation status of LDG and normal-density granulocytes were examined with flow cytometry. The chemokines CXCL8 and CXCL1 were quantified with a cytometric bead-based assay and interferon alpha (IFNα) protein levels with a Simoa method. IFNα-stimulated maternal-derived decidual stromal cells were examined for CXCL8 gene expression with qPCR. A pathologist, blinded to the patient background, examined all placentas. RESULTS: Women with SLE had significantly higher proportions of LDG in peripheral blood compared with controls (p=0.02), and LDG in both peripheral and intervillous blood were more activated in SLE relative to healthy pregnancies (peripheral blood: p=0.002 and intervillous blood: p=0.05). There were higher levels of CXCL8 and CXCL1 in intervillous compared with peripheral blood in women with SLE (p=0.004 and p=<0.0001, respectively) but not in controls. In SLE pregnancy, IFNα was detectable in 6 out of 10 intervillous blood samples but only in one control. Stimulation with IFNα upregulated CXCL8 gene expression in decidual stromal cells from both SLE and healthy pregnancy. Histological chorioamnionitis was present in 6 out of 12 placentas from women with SLE and in 1 out of 10 controls. CONCLUSIONS: In women with SLE, locally produced chemokines in the placenta are increased and may attract and activate neutrophils. This in turn could contribute to placental inflammation and dysfunction and increased risk of placenta-related pregnancy complications.

Synoviocyte-targeted therapy synergizes with TNF inhibition in arthritis reversal.

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.

PTPN22 phosphorylation acts as a molecular rheostat for the inhibition of TCR signaling.

The hematopoietic-specific protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity risk gene. PTPN22 inhibits T cell activation by dephosphorylating substrates involved in proximal T cell receptor (TCR) signaling. Here, we found by mass spectrometry that PTPN22 was phosphorylated at Ser751 by PKCα in Jurkat and primary human T cells activated with phorbol ester/ionomycin or antibodies against CD3/CD28. The phosphorylation of PTPN22 at Ser751 prolonged its half-life by inhibiting K48-linked ubiquitination and impairing recruitment of the phosphatase to the plasma membrane, which is necessary to inhibit proximal TCR signaling. Additionally, the phosphorylation of PTPN22 at Ser751 enhanced the interaction of PTPN22 with the carboxyl-terminal Src kinase (CSK), an interaction that is impaired by the PTPN22 R620W variant associated with autoimmune disease. The phosphorylation of Ser751 did not affect the recruitment of PTPN22 R620W to the plasma membrane but protected this mutant from degradation. Together, out data indicate that phosphorylation at Ser751 mediates a reciprocal regulation of PTPN22 stability versus translocation to TCR signaling complexes by CSK-dependent and CSK-independent mechanisms.

RPTPα phosphatase activity is allosterically regulated by the membrane-distal catalytic domain.

Receptor-type protein tyrosine phosphatase α (RPTPα) is an important positive regulator of SRC kinase activation and a known promoter of cancer growth, fibrosis, and arthritis. The domain structure of RPTPs comprises an extracellular region, a transmembrane helix, and two tandem intracellular catalytic domains referred to as D1 and D2. The D2 domain of RPTPs is believed to mostly play a regulatory function; however, no regulatory model has been established for RPTPα-D2 or other RPTP-D2 domains. Here, we solved the 1.8 Å resolution crystal structure of the cytoplasmic region of RPTPα, encompassing D1 and D2, trapped in a conformation that revealed a possible mechanism through which D2 can allosterically inhibit D1 activity. Using a D2-truncation RPTPα variant and mutational analysis of the D1/D2 interfaces, we show that D2 inhibits RPTPα phosphatase activity and identified a 405PFTP408 motif in D1 that mediates the inhibitory effect of D2. Expression of the gain-of-function F406A/T407A RPTPα variant in HEK293T cells enhanced SRC activation, supporting the relevance of our proposed D2-mediated regulation mechanism in cell signaling. There is emerging interest in the development of allosteric inhibitors of RPTPs but a scarcity of validated allosteric sites for RPTPs. The results of our study not only shed light on the regulatory role of RPTP-D2 domains, but also provide a potentially useful tool for the discovery of chemical probes targeting RPTPα and other RPTPs.

Altered thymic differentiation and modulation of arthritis by invariant NKT cells expressing mutant ZAP70.

Various subsets of invariant natural killer T (iNKT) cells with different cytokine productions develop in the mouse thymus, but the factors driving their differentiation remain unclear. Here we show that hypomorphic alleles of Zap70 or chemical inhibition of Zap70 catalysis leads to an increase of IFN-γ-producing iNKT cells (NKT1 cells), suggesting that NKT1 cells may require a lower TCR signal threshold. Zap70 mutant mice develop IL-17-dependent arthritis. In a mouse experimental arthritis model, NKT17 cells are increased as the disease progresses, while NKT1 numbers negatively correlates with disease severity, with this protective effect of NKT1 linked to their IFN-γ expression. NKT1 cells are also present in the synovial fluid of arthritis patients. Our data therefore suggest that TCR signal strength during thymic differentiation may influence not only IFN-γ production, but also the protective function of iNKT cells in arthritis.

IGF-1R signalling contributes to IL-6 production and T cell dependent inflammation in rheumatoid arthritis.

BACKGROUND: Signalling through insulin-like growth factor 1 receptor (IGF-1R) is essential for cell survival, but may turn pathogenic in uncontrolled tissue growth in tumours. In rheumatoid arthritis (RA), the IGF-1R signalling is activated and supports expansion of the inflamed synovia. AIM: In the present study, we assess if disruption of IGF-1R signalling resolves arthritis. MATERIAL AND METHODS: Clinical associations of IGF-1R expression in leukocytes of the peripheral blood were studied in 84 RA patients. Consequences of the IGF-1R signalling inhibition for arthritis were studied in mBSA immunised Balb/c mice treated with NT157 compound promoting degradation of insulin receptor substrates. RESULTS: In RA patients, high expression of IGF-1R in leukocytes was associated with systemic inflammation as verified by higher expression of NF-kB, serum levels of IL6 and erythrocyte sedimentation rate, and higher pain perception. Additionally, phosphorylated IGF-1R and STAT3 enriched T cells infiltrate in RA synovia. Treatment with NT157 inhibited the phosphorylation of IGF-1R and STAT3 in synovia, and alleviated arthritis and joint damage in mice. It also reduced expression of IGF-1R and despaired ERK and Akt signalling in spleen T cells. This limited IL-6 production, changed RoRgt/FoxP3 balance and IL17 levels. CONCLUSION: IGF-1R signalling contributes to T cell dependent inflammation in arthritis. Inhibition of IGF-1R on the level of insulin receptor substrates alleviates arthritis by restricting IL6-dependent formation of Th17 cells and may open for new treatment strategies in RA.

RAGE Deficiency Impairs Bacterial Clearance in Murine Staphylococcal Sepsis, but Has No Significant Impact on Staphylococcal Septic Arthritis.

BACKGROUND: Septic arthritis is a serious joint disease often caused by Staphylococcus aureus (S. aureus). Receptor for Advanced Glycation End products (RAGE) has an important role in several infections. We sought to investigate the role of RAGE in staphylococcal septic arthritis and sepsis in mice. METHODS: Wild-type (WT) and RAGE deficient (RAGE-/-) mice were intra-articularly or intravenously inoculated with an arthritic or septic dose of S. aureus LS-1 strain. Clinical arthritis, weight development and mortality were monitored for 14 days. Serum levels of cytokines, kidney bacterial loads as well as micro-CT and histopathology of the joints were assessed. RESULTS: RAGE-/- mice with septic arthritis had significantly lower IL-17A and higher bone mineral density (BMD) compared to the control group. However, no significant differences between the groups were observed regarding the weight loss, the severity and frequency of arthritis, and bacterial loads in the kidneys. In mice with sepsis, the overall mortality rate was similar in RAGE-/- (39%) and in WT mice (45%). However, RAGE-/- mice with sepsis had significantly higher bacterial load in their kidneys compared to the WT controls. In line with data from hematogenous S. aureus arthritis, RAGE deficiency had no impact on arthritis severity in local joint infection. CONCLUSIONS: Our results indicate that lack of RAGE has no significant impact on septic arthritis. However, RAGE-/- mice had significantly higher BMD compared to WT mice, which coincided with lower IL-17A in RAGE-/- mice. In sepsis, RAGE deficiency impairs bacterial kidney clearance.

Receptor Protein Tyrosine Phosphatase α-Mediated Enhancement of Rheumatoid Synovial Fibroblast Signaling and Promotion of Arthritis in Mice.

OBJECTIVE: During rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the extracellular matrix of the joint. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to the anomalous behavior of RA FLS. The receptor protein tyrosine phosphatase α (RPTPα), which is encoded by the PTPRA gene, is a key promoter of FAK signaling. The aim of this study was to investigate whether RPTPα mediates FLS aggressiveness and RA pathogenesis. METHODS: Through RPTPα knockdown, we assessed FLS gene expression by quantitative polymerase chain reaction analysis and enzyme-linked immunosorbent assay, invasion and migration by Transwell assays, survival by annexin V and propidium iodide staining, adhesion and spreading by immunofluorescence microscopy, and activation of signaling pathways by Western blotting of FLS lysates. Arthritis development was examined in RPTPα-knockout (KO) mice using the K/BxN serum-transfer model. The contribution of radiosensitive and radioresistant cells to disease was evaluated by reciprocal bone marrow transplantation. RESULTS: RPTPα was enriched in the RA synovial lining. RPTPα knockdown impaired RA FLS survival, spreading, migration, invasiveness, and responsiveness to platelet-derived growth factor, tumor necrosis factor, and interleukin-1 stimulation. These phenotypes correlated with increased phosphorylation of Src on inhibitory Y(527) and decreased phosphorylation of FAK on stimulatory Y(397) . Treatment of RA FLS with an inhibitor of FAK phenocopied the knockdown of RPTPα. RPTPα-KO mice were protected from arthritis development, which was due to radioresistant cells. CONCLUSION: By regulating the phosphorylation of Src and FAK, RPTPα mediates proinflammatory and proinvasive signaling in RA FLS, correlating with the promotion of disease in an FLS-dependent model of RA.

Impaired signaling through the Fms-like tyrosine kinase 3 receptor increases osteoclast formation and bone damage in arthritis.

Osteoclasts are bone-resorbing cells that accumulate in the joints of patients with rheumatoid arthritis causing severe bone damage. Fms-like tyrosine kinase 3 ligand is enriched in the synovial fluid of patients with rheumatoid arthritis, and local exposure to Fms-like tyrosine kinase 3 ligand aggravates arthritis in mice. Because Fms-like tyrosine kinase 3 ligand has been suggested to facilitate osteoclast differentiation, we asked whether Fms-like tyrosine kinase 3 ligand affects bone remodeling in arthritis. The effect of Fms-like tyrosine kinase 3 signaling on osteoclast development was studied by immunohistochemistry in methylated bovine serum albumin-induced arthritis using mice that lack the gene for Flt3l (Flt3L(-/-)) and by an in vitro assay. Bone and joint changes were studied morphologically and by microcomputer tomography. We found that Flt3L(-/-) mice had increased accumulations of osteoclasts in the periarticular area of the arthritic joint. This triggered bone destruction and trabecular bone loss. The increased number of osteoclasts in Flt3L(-/-) mice may be a consequence of insufficient expression of interferon regulatory factor 8. Treatment of Flt3L(-/-) mice with Fms-like tyrosine kinase 3 ligand increased expression of interferon regulatory factor 8, reduced the number of osteoclasts in arthritic mice, and promoted trabecular bone formation. Finally, the reduced number of regulatory T cells in the bone marrow of Flt3L(-/-) mice could further contribute to the increased osteoclastogenesis by reducing the ratio of regulatory T cells to T helper 17 cells. This study shows that Fms-like tyrosine kinase 3 ligand may serve as a negative regulator of osteoclast development by promoting transcription of interferon regulatory factor 8 and sustaining a balance between protective regulatory T cells and pathogenic T helper 17 cells in the pathogenesis of arthritis.

Murine germinal center B cells require functional Fms-like tyrosine kinase 3 signaling for IgG1 class-switch recombination.

Switched antibody classes are important for efficient immune responses. Aberrant antibody production to otherwise harmless antigens may result in autoimmunity. The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development, but the role of Flt3 in peripheral B cells has not been assessed before. Herein we describe a previously unappreciated role for Flt3 in IgG1 class-switch recombination (CSR) and production. We show that Flt3 is reexpressed on B-cell lymphoma 6(+) germinal center B cells in vivo and following LPS activation of peripheral B cells in vitro. Absence of Flt3 signaling in Flt3 ligand-deficient mice results in impaired IgG1 CSR and accumulation of IgM-secreting plasma cells. On activated B cells, Flt3 is coexpressed and functions in synergy with the common-gamma chain receptor family. B cells from Flt3 ligand-deficient mice have impaired IL-4R signaling, with reduced phosphorylation of signal transducer and activator of transcription (Stat) 6, and demonstrate a failure to initiate CSR to IgG1 with low expression of γ1 germ-line transcripts, resulting in impaired IgG1 production. Thus, functional synergy between Flt3 and IL-4R signaling is critical for Stat-mediated regulation of sterile γ1 germ-line transcripts and CSR to IgG1.

Survivin co-ordinates formation of follicular T-cells acting in synergy with Bcl-6.

Follicular T helper (Tfh) cells are recognized by the expression of CXCR5 and the transcriptional regulator Bcl-6. Tfh cells control B cell maturation and antibody production, and if deregulated, may lead to autoimmunity. Here, we study the role of the proto-oncogene survivin in the formation of Tfh cells. We show that blood Tfh cells of patients with the autoimmune condition rheumatoid arthritis, have intracellular expression of survivin. Survivin was co-localized with Bcl-6 in the nuclei of CXCR5+CD4 lymphocytes and was immunoprecipitated with the Bcl-6 responsive element of the target genes. Inhibition of survivin in arthritic mice led to the reduction of CXCR5+ Tfh cells and to low production of autoantibodies. Exposure to survivin activated STAT3 and induced enrichment of PD-1+Bcl-6+ subset within Tfh cells. Collectively, our study demonstrates that survivin belongs to the Tfh cell phenotype and ensures their optimal function by regulating transcriptional activity of Bcl-6.

Trabecular bone loss in collagen antibody-induced arthritis.

INTRODUCTION: Postmenopausal women with rheumatoid arthritis (RA) have increased risk of developing osteoporosis due to chronic inflammation and estrogen deprivation. Collagen antibody-induced arthritis (CAIA), an experimental polyarthritis model representing the effector phase of arthritis, is mainly mediated by the innate immune system. Compared to the widely used collagen-induced arthritis model, CAIA is conveniently short and can be used in C57BL/6 mice, enabling studies with knock-out mice. However, the impact on bone of the CAIA model in C57BL/6 mice has not previously been studied. Therefore, the aim of this study was to determine if CAIA can be used to study postmenopausal arthritis-induced osteoporosis. METHODS: CAIA was induced by administration of collagen-type II antibodies and lipopolysaccharide to ovariectomized female C57BL/6J mice. Control mice received lipopolysaccharide, but no antibodies. Nine days later, femurs were collected for high-resolution micro-CT and histomorphometry. Serum was used to assess cartilage breakdown and levels of complement. Frequencies of immune cell subsets from bone marrow and lymph nodes were analyzed by flow cytometery. RESULTS: Trabecular bone mass was decreased and associated with increased number of osteoclasts per bone surface in the CAIA model. Also, the frequency of interleukin-17(+) cells in lymph nodes was increased in CAIA. CONCLUSION: The present study show that CAIA, a short reproducible arthritis model that is compatible with C57BL/6 mice, is associated with increased number of osteoclasts and trabecular bone loss.

IL-1 Receptor Antagonist Treatment Aggravates Staphylococcal Septic Arthritis and Sepsis in Mice.

BACKGROUND: Interleukin-1 receptor antagonist (IL-1Ra) is the primary therapy against autoinflammatory syndromes with robust efficacy in reducing systemic inflammation and associated organ injury. However, patients receiving IL-1Ra might be at increased risk of acquiring serious infections. AIMS: To study whether IL-1Ra treatment deteriorates Staphylococcus aureus (S. aureus) septic arthritis and sepsis in mice. METHOD: NMRI mice were treated with anakinra (IL-1Ra) daily for 7 days before intravenous inoculation with S. aureus strain Newman in both arthritogenic and lethal doses. The clinical course of septic arthritis, histopathological and radiological changes of the joints, as well as the mortality were compared between IL-1Ra treated and control groups. RESULTS: IL-1Ra treated mice developed more frequent and severe clinical septic arthritis. Also, the frequency of polyarthritis was significantly higher in the mice receiving IL-1Ra therapy. In line with the data from clinical arthritis, both histological and radiological signs of septic arthritis were more pronounced in IL-1Ra treated group compared to controls. Importantly, the mortality of IL-1Ra treated mice was significantly higher than PBS treated controls. CONCLUSION: IL-1Ra treatment significantly aggravated S. aureus induced septic arthritis and increased the mortality in these mice.

Targeting phosphatase-dependent proteoglycan switch for rheumatoid arthritis therapy.

Despite the availability of several therapies for rheumatoid arthritis (RA) that target the immune system, a large number of RA patients fail to achieve remission. Joint-lining cells, called fibroblast-like synoviocytes (FLS), become activated during RA and mediate joint inflammation and destruction of cartilage and bone. We identify RPTPσ, a transmembrane tyrosine phosphatase, as a therapeutic target for FLS-directed therapy. RPTPσ is reciprocally regulated by interactions with chondroitin sulfate or heparan sulfate containing extracellular proteoglycans in a mechanism called the proteoglycan switch. We show that the proteoglycan switch regulates FLS function. Incubation of FLS with a proteoglycan-binding RPTPσ decoy protein inhibited cell invasiveness and attachment to cartilage by disrupting a constitutive interaction between RPTPσ and the heparan sulfate proteoglycan syndecan-4. RPTPσ mediated the effect of proteoglycans on FLS signaling by regulating the phosphorylation and cytoskeletal localization of ezrin. Furthermore, administration of the RPTPσ decoy protein ameliorated in vivo human FLS invasiveness and arthritis severity in the K/BxN serum transfer model of RA. Our data demonstrate that FLS are regulated by an RPTPσ-dependent proteoglycan switch in vivo, which can be targeted for RA therapy. We envision that therapies targeting the proteoglycan switch or its intracellular pathway in FLS could be effective as a monotherapy or in combination with currently available immune-targeted agents to improve control of disease activity in RA patients.

CTLA4 Immunoglobulin but Not Anti-Tumor Necrosis Factor Therapy Promotes Staphylococcal Septic Arthritis in Mice.

BACKGROUND: The development of biologics has greatly increased the quality of life and the life expectancy of many patients with rheumatoid arthritis. However, a large number of these patients have an increased risk of developing serious infections. The aim of this study was to examine differential effects of anti-tumor necrosis factor (TNF) treatment and CTLA4 immunoglobulin (Ig) treatment on both immunological response and host defense in a murine model of septic arthritis. METHODS: Abatacept (CTLA4-Ig), etanercept (anti-TNF), or phosphate-buffered saline were given to NMRI mice intravenously inoculated with Staphylococcus aureus. The clinical course of septic arthritis and histopathological and radiological changes of joints were compared among the groups. RESULTS: Mice receiving CTLA4-Ig treatment had more-severe septic arthritis, compared with controls and mice receiving anti-TNF treatment. Anti-TNF treatment led to more-severe weight loss and kidney abscesses, as well as a higher bacterial burden in the kidneys. Mice receiving CTLA4-Ig therapy had lower serum levels of interleukin 4, whereas mice receiving anti-TNF therapy had higher levels of TNF-α. Both iNOS and arginase-1 expression were reduced in peritoneal macrophages from mice receiving CTLA4-Ig, compared with expression in the anti-TNF group. CONCLUSIONS: CTLA4-Ig therapy significantly increased the susceptibility to S. aureus septic arthritis in mice, whereas anti-TNF therapy deteriorated host bacterial clearance, resulting in more-severe weight loss and kidney abscesses.

S100A4 regulates the Src-tyrosine kinase dependent differentiation of Th17 cells in rheumatoid arthritis.

OBJECTIVES: To evaluate the role of S100A4, a calcium-binding regulator of nonmuscle myosin assembly, for T-cell responses in rheumatoid arthritis. METHODS: Arthritis was induced in the methylated bovine serum albumin (mBSA)-immunized mice lacking the entire S100A4 protein (S100A4KO) and in wild-type counterparts treated with short hairpin ribonucleic acid (shRNA)-lentiviral constructs targeting S100A4 (S100A4-shRNA). The severity of arthritis was evaluated morphologically. T-cell subsets were characterized by the expression of master transcription factors, and functionally by proliferation activity and cytokine production. The activity of the Scr-kinases Fyn and Lck was assessed by the autophosphorylation of C-terminal thyrosine and by the phosphorylation of the CD5 cytodomain. The interaction between S100A4 and the CD5 cytodomain was analysed by nuclear magnetic resonance spectrophotometry. RESULTS: S100A4-deficient mice (S100A4KO and S100A4-shRNA) had significantly alleviated morphological signs of arthritis and joint damage. Leukocyte infiltrates in the arthritic joints of S100A4-deficient mice accumulated Foxp3(+) Treg cells, while the number of RORγt(+) and (pTyr705)STAT3(+) cells was reduced. S100A4-deficient mice had a limited formation of Th17-cells with low retinoic acid orphan receptor gamma t (RORγt) mRNA and IL17 production in T-cell cultures. S100A4-deficient mice had a low expression and activity of T-cell receptor (TCR) inhibitor CD5 and low (pTyr705)STAT3 (signal transducer and activator of transcription 3), which led to increased (pTyr352)ZAP-70 (theta-chain associated protein kinase of 70kDa), lymphocyte proliferation and production of IL2. In vitro experiments showed that S100A4 directly binds Lck and Fyn and reciprocally regulates their kinase activity towards the CD5 cytodomain. Spectrometry demonstrates an interaction between the CD5 cytodomain and EF2-binding sites of S100A4. CONCLUSION: The present study demonstrates that S100A4 plays an important part in the pathogenesis of arthritis. It controls CD5-dependent differentiation of Th17 cells by regulating the activity of the Src-family kinases Lck and Fyn.

Fms-like tyrosine kinase 3 ligand controls formation of regulatory T cells in autoimmune arthritis.

Fms-like tyrosine kinase 3 ligand (Flt3L) is known as the primary differentiation and survival factor for dendritic cells (DCs). Furthermore, Flt3L is involved in the homeostatic feedback loop between DCs and regulatory T cell (Treg). We have previously shown that Flt3L accumulates in the synovial fluid in rheumatoid arthritis (RA) and that local exposure to Flt3L aggravates arthritis in mice, suggesting a possible involvement in RA pathogenesis. In the present study we investigated the role of Flt3L on DC populations, Tregs as well as inflammatory responses in experimental antigen-induced arthritis. Arthritis was induced in mBSA-immunized mice by local knee injection of mBSA and Flt3L was provided by daily intraperitoneal injections. Flow cytometry analysis of spleen and lymph nodes revealed an increased formation of DCs and subsequently Tregs in mice treated with Flt3L. Flt3L-treatment was also associated with a reduced production of mBSA specific antibodies and reduced levels of the pro-inflammatory cytokines IL-6 and TNF-α. Morphological evaluation of mBSA injected joints revealed reduced joint destruction in Flt3L treated mice. The role of DCs in mBSA arthritis was further challenged in an adoptive transfer experiment. Transfer of DCs in combination with T-cells from mBSA immunized mice, predisposed naïve recipients for arthritis and production of mBSA specific antibodies. We provide experimental evidence that Flt3L has potent immunoregulatory properties. Flt3L facilitates formation of Treg cells and by this mechanism reduces severity of antigen-induced arthritis in mice. We suggest that high systemic levels of Flt3L have potential to modulate autoreactivity and autoimmunity.