Proteoglycan-induced Arthritis Research Articles

Autoantigen-Dexamethasone Conjugate-Loaded Liposomes Halt Arthritis Development in Mice.

There is no curative treatment for chronic auto-inflammatory diseases including rheumatoid arthritis, and current treatments can induce off-target side effects due to systemic immune suppression. This work has previously shown that dexamethasone-pulsed tolerogenic dendritic cells loaded with the arthritis-specific antigen human proteoglycan can suppress arthritis development in a proteoglycan-induced arthritis mouse model. To circumvent ex vivo dendritic cell culture, and enhance antigen-specific effects, drug delivery vehicles, such as liposomes, provide an interesting approach. Here, this work uses anionic 1,2-distearoyl-sn-glycero-3-phosphoglycerol liposomes with enhanced loading of human proteoglycan-dexamethasone conjugates by cationic lysine tetramer addition. Antigen-pulsed tolerogenic dendritic cells induced by liposomal dexamethasone in vitro enhanced antigen-specific regulatory Tcells to a similar extent as dexamethasone-induced tolerogenic dendritic cells. In an inflammatory adoptive transfer model, mice injected with antigen-dexamethasone liposomes have significantly higher antigen-specific type 1 regulatory Tcells than mice injected with antigen only. The liposomes significantly inhibit the progression of arthritis compared to controls in preventative and therapeutic proteoglycan-induced arthritis mouse models. This coincides with systemic tolerance induction and an increase in IL10 expression in the paws of mice. In conclusion,a single administration of autoantigen and dexamethasone-loaded liposomes seems to be a promising antigen-specific treatment strategy for arthritis in mice.

Regulation of autoimmune arthritis by the SHP-1 tyrosine phosphatase

BackgroundThe Src homology region 2 domain-containing phosphatase-1 (SHP-1) is known to exert negative regulatory effects on immune cell signaling. Mice with mutations in the Shp1 gene develop inflammatory skin disease and autoimmunity, but no arthritis.We sought to explore the role of SHP-1 in arthritis using an autoimmune mouse model of rheumatoid arthritis. We generated Shp1 transgenic (Shp1-Tg) mice to study the impact of SHP-1 overexpression on arthritis susceptibility and adaptive immune responses.MethodsSHP-1 gene and protein expression as well as tyrosine phosphatase activity were evaluated in spleen cells of transgenic and wild type (WT) mice. WT and Shp1-Tg (homozygous or heterozygous for the transgene) mice were immunized with human cartilage proteoglycan (PG) in adjuvant, and arthritis symptoms were monitored. Protein tyrosine phosphorylation level, net cytokine secretion, and serum anti-human PG antibody titers were measured in immune cells from WT and Shp1-Tg mice. WT mice were treated with regorafenib orally to activate SHP-1 either before PG-induced arthritis (PGIA) symptoms developed (preventive treatment) or starting at an early stage of disease (therapeutic treatment). Data were statistically analyzed and graphs created using GraphPad Prism 8.0.2 software.ResultsSHP-1 expression and tyrosine phosphatase activity were elevated in both transgenic lines compared to WT mice. While all WT mice developed arthritis after immunization, none of the homozygous Shp1-Tg mice developed the disease. Heterozygous transgenic mice, which showed intermediate PGIA incidence, were selected for further investigation. We observed differences in interleukin-4 and interleukin-10 production in vitro, but serum anti-PG antibody levels were not different between the genotypes. We also found decreased tyrosine phosphorylation of several proteins of the JAK/STAT pathway in T cells from PG-immunized Shp1-Tg mice. Regorafenib administration to WT mice prevented the development of severe PGIA or reduced disease severity when started after disease onset.ConclusionsResistance to arthritis in the presence of SHP-1 overexpression likely results from the impairment of tyrosine phosphorylation (deactivation) of key immune cell signaling proteins in the JAK/STAT pathway, due to the overwhelming tyrosine phosphatase activity of the enzyme in Shp1-Tg mice. Our study is the first to investigate the role of SHP-1 in autoimmune arthritis using animals overexpressing this phosphatase. Pharmacological activation of SHP-1 might be considered as a new approach to the treatment of autoimmune arthritis.

Positive transcriptional response on inflammation and joint remodelling influenced by physical exercise in proteoglycan-induced arthritis: An animal study.

AimsTo assess the effect of physical exercise (PE) on the histological and transcriptional characteristics of proteoglycan-induced arthritis (PGIA) in BALB/c mice.MethodsFollowing PGIA, mice were subjected to treadmill PE for ten weeks. The tarsal joints were used for histological and genetic analysis through microarray technology. The genes differentially expressed by PE in the arthritic mice were obtained from the microarray experiments. Bioinformatic analysis in the DAVID, STRING, and Cytoscape bioinformatic resources allowed the association of these genes in biological processes and signalling pathways.ResultsArthritic mice improved their physical fitness by 42.5% after PE intervention; it induced the differential expression of 2,554 genes. The bioinformatic analysis showed that the downregulated genes (n = 1,371) were significantly associated with cellular processes that mediate the inflammation, including Janus kinase-signal transducer and activator of transcription proteins (JAK-STAT), Notch, and cytokine receptor interaction signalling pathways. Moreover, the protein interaction network showed that the downregulated inflammatory mediators interleukin (IL) 4, IL5, IL2 receptor alpha (IL2rα), IL2 receptor beta (IL2rβ), chemokine ligand (CXCL) 9, and CXCL12 were interacting in several pathways associated with the pathogenesis of arthritis. The upregulated genes (n = 1,183) were associated with processes involved in the remodelling of the extracellular matrix and bone mineralization, as well as with the processes of aerobic metabolism. At the histological level, PE attenuated joint inflammatory infiltrate and cartilage erosion.ConclusionPhysical exercise influences parameters intimately linked to inflammatory arthropathies. Research on the effect of PE on the pathogenesis process of arthritis is still necessary for animal and human models.Cite this article: Bone Joint Res. 2020;9(1):36–48.

MiR-21 may Act as a Potential Mediator Between Inflammation and Abnormal Bone Formation in Ankylosing Spondylitis Based on TNF-α Concentration-Dependent Manner Through the JAK2/STAT3 Pathway.

Objective:To explore the role of microRNA (miR-21) in new bone formation in ankylosing spondylitis (AS) as mediated by different concentration of tumor necrosis factor-α (TNF-α).Methods:Fibroblasts isolated from the hips of patients with AS were induced to osteogenesis. These cells were then stimulated with varying concentrations of TNF-α. MicroRNA-21 expressions were evaluated using reverse transcription–polymerase chain reaction (RT-PCR) and osteogenesis was detected via Alizarin Red S (ARS) staining and measurement of alkaline phosphatase (ALP) activity. Relative expressions of p-STAT3, Nuclear STAT3, cytoplasm STAT3, Runx2, BMP2, osteopontin, osteocalcin, and LC3B in AS fibroblasts were measured after exposure to different concentrations of TNF-α. The STAT3-inhibiting small interfering RNA allowed further exploration on its impact on miR-21 and primary miR-21 expressions. A proteoglycan-induced arthritis (PGIA) Balb/c mouse model was established in order to monitor sacroiliac joint (SIJ) inflammation and subsequent damage through magnetic resonance image. Serum miR-21 and TNF-α expressions were evaluated using RT-PCR and enzyme-linked immunosorbent assay. At week 16, mice models were transfected intravenously with miR-21 overexpressing agomir and miR-21 inhibiting antagomir for 7 successive days. The rate of abnormal bone formation at SIJ was evaluated using microcomputed tomography and hematoxylin and eosin staining at week 24. Western blot analysis enabled quantification of STAT-3, JAK-2, and interleukin (IL)-17A expressions present in the SIJ.Results:The in vitro miR-21 expression and osteogenesis activity were noted to be augmented in the setting of low TNF-α concentrations (0.01-0.1 ng/mL) while they were depressed in settings with higher TNF-α concentrations (1-10 ng/mL). Samples with the most distinct ARS manifestation and ALP activity as well as the highest miR-21 expressions were those who received 0.1 ng/mL of TNF-α. Primary miR-21 was found to be notable raised by Si-STAT3, while the converse effect was seen in mature miR-21 expressions. Intravenous injection of exogenous miR-21 contributed to new bone formation and significantly elevated expressions of STAT3, JAK2, and IL-17 in PGIA mice.Conclusions:The results revealed that miR-21 may act as a potential mediator between new bone formation and inflammation in AS.

A Novel Targeted Approach to Achieve Immune System Reset: CD45-Targeted Antibody Drug Conjugates Enable Autologous HSCT, Ameliorate Disease in Autoimmune Models, Potently Kill Human Immune Cells from Normal Donors and MS Patients, and Achieve Immune Depletion in Non-Human Primates (NHP)

Resetting the immune system through autologous hematopoietic stem cell transplant (autoHSCT) is a highly effective treatment in selected patients with autoimmune diseases. AutoHSCT can induce long-term remission (up to 15 years) with 70-80% progression free survival in patients with relapsed refractory and secondary progressive multiple sclerosis (Muraro 2017) that is superior to standard of care agents in a randomized study (Burt 2019). Likewise, use of autoHSCT in scleroderma patients achieved superior outcomes in two randomized studies (Tyndall 2014, Sullivan 2018). These impressive results are achieved by a combination of the eradication of autoreactive immune effector cells and re-establishment of a self-tolerant immune system, i.e., immune system reset. However, only a small fraction of eligible patients undergo autoHSCT, in part due to toxicity associated with current conditioning protocols. To address these issues, we are developing antibody drug conjugates (ADCs) that selectively target CD45 to eradicate autoimmune cells and enable autoHSCT as a potential one-time curative treatment for patients with autoimmune disease. To model this approach in mice, we generated an anti-mouse CD45 ADC that was evaluated for the ability to condition recipients in a murine congenic transplant model following a single myeloablative dose. This ADC was further evaluated for its ability to eliminate pathogenic host-reactive cells and enable immune reset in recipients in multiple murine models of autoimmune disease, including MOG-induced experimental autoimmune encephalitis (EAE), proteoglycan-induced arthritis (PGIA), and sclerodermatous graft-vs-host disease (scGVHD). A single-dose of tool anti-mouse CD45-ADC at 3 mg/kg achieved full myeloablation in recipient mice (>99% depletion of LT-HSCs (Lin-Kit+Sca-1+CD150+CD48-). Transplanted mice achieved full engraftment with congenic BMT (>90% chimerism at 16 weeks). In EAE, conditioning with a non-myeloablative dose of the CD45-ADC followed by congenic transplant prior to disease onset significantly delayed disease onset and reduced disease severity (onset at 21 days, peak disease score 2.1 for with 1 mg/kg CD45-ADC; onset at 42 days, peak disease score 0.75 for 3 mg/kg CD45-ADC + BMT; onset at 11 days with peak disease score 3.1 for vehicle-treated)[Figure 1]. In active EAE, treatment with 3 mg/kg of CD45-ADC on day 10 or 13 followed by congenic transplant halted progression of disease activity (no increase from disease score at time of treatment; peak disease scores of 0.75 and 2.3, respectively). The effect observed with CD45-ADC treatment at day 13 with congenic transplant was comparable to that achieved by treatment with a clinically validated standard of care, FTY-720 (approved S1P1 antagonist equivalent to Gilenya) at day 13 which also halted disease at a peak score of 2.3. Disease control in this study compared favorably to a prior study where mice were treated with 9 Gy TBI + congenic BMT at day 9. These data show that CD45-ADC conditioning followed by congenic transplant is effective at immune reset and shows comparable efficacy to clinically validated therapies. Evaluation of this ADC in additional autoimmune models of arthritis and scleroderma are ongoing and will be presented. To translate these encouraging pre-clinical data, we generated novel anti-human CD45 ADCs that cross react with nonhuman primates (NHP) and evaluated these for the ability to deplete hematopoietic and immune cells in vitro and in vivo in humanized NSG (hNSG) mice. The CD45-ADC showed efficient killing of human BM CD34+ (EC50 2.44 x 10-9 M) and peripheral CD3+ cells from normal donor (EC50 7.6 x10-10 M) and MS patients (EC50 1.5 x 10-10 M). In vivo in hNSG, single doses of the CD45-ADCs were well-tolerated and led to substantial (>95%) depletion of human cells. In NHPs, single doses of CD45-ADCs were well tolerated and achieved >90% peripheral lymphocyte depletion and >80% depletion of HSCs. Dose escalation studies are continuing and will be reported. These results suggest that targeted immune depletion with a single treatment of CD45-ADC may be sufficient for auto-HSCT and allow immune reset and re-establishment of immune tolerance. Targeted CD45-ADCs may represent a safer and better tolerated approach for conditioning patients prior to immune reset through autoHSCT and may significantly reduce the side effects associated with current conditioning. Disclosures Gillard: Magenta Therapeutics: Employment, Equity Ownership. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Lamothe:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Mikse:Magenta Therapeutics: Employment, Equity Ownership. McDonough:Magenta Therapeutics: Employment, Equity Ownership. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership. Bhat:Magenta Therapeutics: Employment, Equity Ownership. Sarma:Magenta Therapeutics: Employment, Equity Ownership. Bhattarai:Magenta Therapeutics: Employment, Equity Ownership. Sawant:Magenta Therapeutics: Employment, Equity Ownership. Pearse:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mcdonough:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Matured Tolerogenic Dendritic Cells Effectively Inhibit Autoantigen Specific CD4+ T Cells in a Murine Arthritis Model.

Tolerogenic dendritic cells (tolDCs) are a promising treatment modality for diseases caused by a breach in immune tolerance, such as rheumatoid arthritis. Current medication for these diseases is directed toward symptom suppression but no real cure is available yet. TolDC-based therapy aims to restore immune tolerance in an antigen-specific manner. Here we used a mouse model to address two major questions: (i) is a maturation stimulus needed for tolDC function in vitro and in vivo and is maturation required for functioning in experimental arthritis and (ii) can tolDCs modulate CD4+ T cell responses? To answer these questions, we compared matured and immature dexamethasone/vitamin D3-generated tolDCs in vitro. Subsequently, we co-transferred these tolDCs with naïve or effector CD4+ T cells to study the characteristics of transferred T cells after 3 days with flow cytometry and Luminex multiplex assays. In addition, we tested the suppressive capabilities of tolDCs in an experimental arthritis model. We found that tolDCs cannot only modulate naïve CD4+ T cell responses as shown by fewer proliferated and activated CD4+ T cells in vivo, but also effector CD4+ T cells. In addition, Treg (CD4+CD25+FoxP3+) expansions were seen in the proliferating cell population in the presence of tolDCs. Furthermore, we show that administered tolDCs are capable to inhibit arthritis in the proteoglycan-induced arthritis model. However, a maturation stimulus is needed for tolDCs to manifest this tolerizing function in an inflammatory environment. Our data will be instrumental for optimization of future tolDC therapies for autoimmune diseases.

Amelioration of Autoimmune Arthritis in Mice Treated With the DNA Methyltransferase Inhibitor 5'-Azacytidine.

Disease-associated, differentially hypermethylated regions have been reported in rheumatoid arthritis (RA), but no DNA methyltransferase inhibitors have been evaluated in either RA or any animal models of RA. The present study was conducted to evaluate the therapeutic potential of 5'-azacytidine (5'-azaC), a DNA methyltransferase inhibitor, and explore the cellular and gene regulatory networks involved in the context of autoimmune arthritis. A disease-associated genome-wide DNA methylation profile was explored by methylated CpG island recovery assay-chromatin immunoprecipitation (ChIP) in arthritic B cells. Mice with proteoglycan-induced arthritis (PGIA) were treated with 5'-azaC. The effect of 5'-azaC on the pathogenesis of PGIA was explored by measuring serum IgM and IgG1 antibody levels using enzyme-linked immunosorbent assay, investigating the efficiency of class-switch recombination (CSR) and Aicda gene expression using real-time quantitative polymerase chain reaction, monitoring germinal center (GC) formation by immunohistochemistry, and determining alterations in B cell subpopulations by flow cytometry. The 5'-azaC-induced regulation of the Aicda gene was explored using RNA interference, ChIP, and luciferase assays. We explored arthritis-associated hypermethylated regions in mouse B cells and demonstrated that DNA demethylation had a beneficial effect on autoimmune arthritis. The 5'-azaC-mediated demethylation of the epigenetically inactivated Ahr gene resulted in suppressed expression of the Aicda gene, reduced CSR, and compromised GC formation. Ultimately, this process led to diminished IgG1 antibody production and amelioration of autoimmune arthritis in mice. DNA hypermethylation plays a leading role in the pathogenesis of autoimmune arthritis and its targeted inhibition has therapeutic potential in arthritis management.

Regulatory role of capsaicin-sensitive peptidergic sensory nerves in the proteoglycan-induced autoimmune arthritis model of the mouse

ObjectiveThe regulatory role of capsaicin-sensitive peptidergic sensory nerves has been shown in acute inflammation, but little is known about their involvement in T/B-cell driven autoimmune arthritis. This study integratively characterized the function of these nerve endings in the proteoglycan-induced chronic arthritis (PGIA), a translational model of rheumatoid arthritis.MethodsPeptidergic afferents were defunctionalized by resiniferatoxin (RTX) pretreatment in BALB/c mice, PGIA was induced by repeated antigen challenges. Hind paw volume, arthritis severity, grasping ability and the mechanonociceptive threshold were monitored during the 17-week experiment. Myeloperoxidase activity, vascular leakage and bone turnover were evaluated by in vivo optical imaging. Bone morphology was assessed using micro-CT, the intertarsal small joints were processed for histopathological analysis.ResultsFollowing desensitization of the capsaicin-sensitive afferents, ankle edema, arthritis severity and mechanical hyperalgesia were markedly diminished. Myeloperoxidase activity was lower in the early, but increased in the late phase, whilst plasma leakage and bone turnover were not altered. Desensitized mice displayed similar bone spurs and erosions, but increased trabecular thickness of the tibia and bony ankylosis of the spine. Intertarsal cartilage thickness was not altered in the model, but desensitization increased this parameter in both the non-arthritic and arthritic groups.ConclusionThis is the first integrative in vivo functional and morphological characterization of the PGIA mouse model, wherein peptidergic afferents have an important regulatory function. Their overall effect is proinflammatory by increasing acute inflammation, immune cell activity and pain. Meanwhile, their activation decreases spinal ankylosis, arthritis-induced altered trabecularity, and cartilage thickness in small joints.

An epitope-specific DerG-PG70 LEAPS vaccine modulates T cell responses and suppresses arthritis progression in two related murine models of rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune joint disease maintained by aberrant immune responses involving CD4+ T helper (Th)1 and Th17 cells. In this study, we tested the therapeutic efficacy of Ligand Epitope Antigen Presentation System (LEAPS™) vaccines in two Th1 cell-driven mouse models of RA, cartilage proteoglycan (PG)-induced arthritis (PGIA) and PG G1-domain-induced arthritis (GIA). The immunodominant PG peptide PG70 was attached to a DerG or J immune cell binding peptide, and the DerG-PG70 and J-PG70 LEAPS vaccines were administered to the mice after the onset of PGIA or GIA symptoms. As indicated by significant decreases in visual and histopathological scores of arthritis, the DerG-PG70 vaccine inhibited disease progression in both PGIA and GIA, while the J-PG70 vaccine was ineffective. Splenic CD4+ cells from DerG-PG70-treated mice were diminished in Th1 and Th17 populations but enriched in Th2 and regulatory T (Treg) cells. In vitro spleen cell-secreted and serum cytokines from DerG-PG70-treated mice demonstrated a shift from a pro-inflammatory to an anti-inflammatory/regulatory profile. DerG-PG70 peptide tetramers preferentially bound to CD4+ T-cells of GIA spleen cells. We conclude that the DerG-PG70 vaccine (now designated CEL-4000) exerts its therapeutic effect by interacting with CD4+ cells, which results in an antigen-specific down-modulation of pathogenic T-cell responses in both the PGIA and GIA models of RA. Future studies will need to determine the potential of LEAPS vaccination to provide disease suppression in patients with RA.

Key serum cytokine markers for evaluating the efficacy of vaccine therapy in autoimmune models of rheumatoid arthritis

Abstract Background Pro-inflammatory T helper (Th)17 or Th1 cell responses (IL17, IFNγ, TNFα) drive autoimmune pathology in rheumatoid arthritis (RA) at the expense of anti-inflammatory/regulatory (IL4, IL10, TGFβ) T cell responses. Ligand Epitope Antigen Presenting System (LEAPS) vaccines, composed of cell-binding and immunodominant peptides, can down-modulate Th17 or Th1 activity and thus have the potential to treat ongoing RA. Choice of appropriate treatment requires knowledge of key cytokines driving disease as well as responses to therapy. Experimental design Serum cytokines were assayed in mouse models of RA including proteoglycan-induced arthritis (PGIA) recombinant proteoglycan G1 domain-induced arthritis (GIA) and collagen-induced arthritis (CIA) after arthritic mice had been treated with LEAPS vaccines. Serum cytokine levels and ratios of anti-inflammatory to pro-inflammatory cytokines were correlated with visual and histopathologic scores of disease. Results Serum levels of pro-inflammatory cytokines were elevated in control mice with either PGIA, GIA, or CIA. Reduced disease progression in mice treated with LEAPS vaccines was accompanied by decreased pro-inflammatory cytokine levels and increased ratios of anti-inflammmatory/regulatory to pro-inflammatory cytokines. Conclusion Serum concentrations and ratios of key disease-related cytokines can predict therapeutic efficacy in different animal models of RA. Successful vaccine therapy is associated with ratios shifted in favor of anti-inflammatory/regulatory cytokines. The ability to predict cytokine responses to therapy will allow better design or choice of appropriate immunomodulatory LEAPS vaccines and other therapies in RA.

Immune Recognition of Citrullinated Proteoglycan Aggrecan Epitopes in Mice with Proteoglycan-Induced Arthritis and in Patients with Rheumatoid Arthritis.

BackgroundRheumatoid arthritis (RA) is an autoimmune inflammatory disease affecting the joints. Anti-citrullinated protein antibodies (ACPA) are frequently found in RA. Previous studies identified a citrullinated epitope in cartilage proteoglycan (PG) aggrecan that elicited pro-inflammatory cytokine production by RA T cells. We recently reported the presence of ACPA-reactive (citrullinated) PG in RA cartilage. Herein, we sought to identify additional citrullinated epitopes in human PG that are recognized by T cells or antibodies from RA patients.MethodsWe used mice with PG-induced arthritis (PGIA) as a screening tool to select citrulline (Cit)-containing PG peptides that were more immunogenic than the arginine (R)-containing counterparts. The selected peptide pairs were tested for induction of pro-inflammatory T-cell cytokine production in RA and healthy control peripheral blood mononuclear cell (PBMC) cultures using ELISA and flow cytometry. Anti-Cit and anti-R peptide antibodies were detected by ELISA.ResultsSplenocytes from mice with PGIA exhibited greater T-cell cytokine secretion in response to the Cit than the R version of PG peptide 49 (P49) and anti-P49 antibodies were found in PGIA serum. PBMC from ACPA+ and ACPA- RA patients, but not from healthy controls, responded to Cit49 with robust cytokine production. High levels of anti-Cit49 antibodies were found in the plasma of a subset of ACPA+ RA patients. Another PG peptide (Cit13) similar to the previously described T-cell epitope induced greater cytokine responses than R13 by control (but not RA) PBMC, however, anti-Cit13 antibodies were rarely detected in human plasma.ConclusionsWe identified a novel citrullinated PG epitope (Cit49) that is highly immunogenic in mice with PGIA and in RA patients. We also describe T-cell and antibody reactivity with Cit49 in ACPA+ RA. As citrullinated PG might be present in RA articular cartilage, Cit PG epitope-induced T-cell activation or antibody deposition may occur in the joints of RA patients.

An Arthritis-Suppressive and Treg Cell-Inducing CD4+ T Cell Epitope Is Functional in the Context of HLA-Restricted T Cell Responses.

We previously showed that mycobacterial Hsp70-derived peptide B29 induced B29-specific Treg cells that suppressed experimental arthritis in mice via cross-recognition of their mammalian Hsp70 homologs. The aim of the current study was to characterize B29 binding and specific CD4+ T cell responses in the context of human major histocompatibility complex (MHC) molecules. Competitive binding assays were performed to examine binding of peptide B29 and its mammalian homologs to HLA molecules. The effect of B29 immunization in HLA-DQ8-transgenic mice with proteoglycan-induced arthritis was assessed, followed by ex vivo restimulation with B29 to examine the T cell response. Human peripheral blood mononuclear cells were used to investigate the presence of B29-specific T cells with immunoregulatory potential. The binding affinity of the B29 peptide was high to moderate for multiple HLA-DR and HLA-DQ molecules, including those highly associated with rheumatoid arthritis. This binding was considered to be functional, because B29 immunization resulted in the suppression of arthritis and T cell responses in HLA-DQ8-transgenic mice. In humans, we demonstrated the presence and expansion of B29-specific CD4+ T cells, which were cross-reactive with the mammalian homologs. Using HLA-DR4+ tetramers specific for B29 or the mammalian homolog mB29b, we showed expansion of cross-reactive T cells, especially the human FoxP3+ CD4+CD25+ T cell population, after in vitro stimulation with B29. These results demonstrated a conserved fine specificity and functionality of B29-induced Treg cell responses in the context of the human MHC. Based on these findings, a path for translation of the experimental findings for B29 into a clinical immunomodulatory therapeutic approach is within reach.

Autologous stem cell transplantation aids autoimmune patients by functional renewal and TCR diversification of regulatory T cells

AbstractAutologous hematopoietic stem cell transplantation (HSCT) is increasingly considered for patients with severe autoimmune diseases whose prognosis is poor with standard treatments. Regulatory T cells (Tregs) are thought to be important for disease remission after HSCT. However, eliciting the role of donor and host Tregs in autologous HSCT is not possible in humans due to the autologous nature of the intervention. Therefore, we investigated their role during immune reconstitution and re-establishment of immune tolerance and their therapeutic potential following congenic bone marrow transplantation (BMT) in a proteoglycan-induced arthritis (PGIA) mouse model. In addition, we determined Treg T-cell receptor (TCR) CDR3 diversity before and after HSCT in patients with juvenile idiopathic arthritis and juvenile dermatomyositis. In the PGIA BMT model, after an initial predominance of host Tregs, graft-derived Tregs started dominating and displayed a more stable phenotype with better suppressive capacity. Patient samples revealed a striking lack of diversity of the Treg repertoire before HSCT. This ameliorated after HSCT, confirming reset of the Treg compartment following HSCT. In the mouse model, a therapeutic approach was initiated by infusing extra Foxp3GFP+ Tregs during BMT. Infusion of Foxp3GFP+ Tregs did not elicit additional clinical improvement but conversely delayed reconstitution of the graft-derived T-cell compartment. These data indicate that HSCT-mediated amelioration of autoimmune disease involves renewal of the Treg pool. In addition, infusion of extra Tregs during BMT results in a delayed reconstitution of T-cell compartments. Therefore, Treg therapy may hamper development of long-term tolerance and should be approached with caution in the clinical autologous setting.

Unconventional T-cell recognition of an arthritogenic epitope of proteoglycan aggrecan released from degrading cartilage.

It has been proposed that peptide epitopes bind to MHC class II molecules to form distinct structural conformers of the same MHC II-peptide complex termed type A and type B, and that the two conformers of the same peptide-MHC II complex are recognized by distinct CD4 T cells, termed type A and type B T cells. Both types recognize short synthetic peptides but only type A recognize endosomally processed intact antigen. Type B T cells that recognize self peptides from exogenously degraded proteins have been shown to escape negative selection during thymic development and so have the potential to contribute to the pathogenesis of autoimmunity. We generated and characterized mouse CD4 T cells specific for an arthritogenic epitope of the candidate joint autoantigen proteoglycan aggrecan. Cloned T-cell hybridomas specific for a synthetic peptide containing the aggrecan epitope showed two distinct response patterns based on whether they could recognize processed intact aggrecan. Fine mapping demonstrated that both types of T-cell recognized the same core epitope. The results are consistent with the generation of aggrecan-specific type A and type B T cells. Type B T cells were activated by supernatants released from degrading cartilage, indicating the presence of antigenic extracellular peptides or fragments of aggrecan. Type B T cells could play a role in the pathogenesis of proteoglycan-induced arthritis in mice, a model for rheumatoid arthritis, by recognizing extracellular peptides or protein fragments of joint autoantigens released by inflamed cartilage.

DEC205+ Dendritic Cell-Targeted Tolerogenic Vaccination Promotes Immune Tolerance in Experimental Autoimmune Arthritis.

Previous studies in mouse models of autoimmune diabetes and encephalomyelitis have indicated that the selective delivery of self-antigen to the endocytic receptor DEC205 on steady-state dendritic cells (DCs) may represent a suitable approach to induce Ag-specific immune tolerance. In this study, we aimed to examine whether DEC205(+) DC targeting of a single immunodominant peptide derived from human cartilage proteoglycan (PG) can promote immune tolerance in PG-induced arthritis (PGIA). Besides disease induction by immunization with whole PG protein with a high degree of antigenic complexity, PGIA substantially differs from previously studied autoimmune models not only in the target tissue of autoimmune destruction but also in the nature of pathogenic immune effector cells. Our results show that DEC205(+) DC targeting of the PG peptide 70-84 is sufficient to efficiently protect against PGIA development. Complementary mechanistic studies support a model in which DEC205(+) DC targeting leads to insufficient germinal center B cell support by PG-specific follicular helper T cells. Consequently, impaired germinal center formation results in lower Ab titers, severely compromising the development of PGIA. Overall, this study further corroborates the potential of prospective tolerogenic DEC205(+) DC vaccination to interfere with autoimmune diseases, such as rheumatoid arthritis.

B cells expressing IFN-γ suppress Treg-cell differentiation and promote autoimmune experimental arthritis.

Clinical efficacy in the treatment of rheumatoid arthritis with anti-CD20 (Rituximab)-mediated B-cell depletion has garnered interest in the mechanisms by which B cells contribute to autoimmunity. We have reported that B-cell depletion in a murine model of proteoglycan-induced arthritis (PGIA) leads to an increase in Treg cells that correlate with decreased autoreactivity. Here, we demonstrate that the increase in Treg cells after B-cell depletion is due to an increase in the differentiation of naïve CD4(+) Tcells into Treg cells. Since the development of PGIA is dependent on IFN-γ and B cells are reported to produce IFN-γ, we hypothesized that B-cell-specific IFN-γ plays a role in the development of PGIA. Accordingly, mice with B-cell-specific IFN-γ deficiency were as resistant to the induction of PGIA as mice that were completely IFN-γ deficient. Importantly, despite a normal frequency of IFN-γ-producing CD4(+) Tcells, B-cell-specific IFN-γ-deficient mice exhibited a higher percentage of Treg cells compared with that in WT mice. These data indicate that B-cell IFN-γ production inhibits Treg-cell differentiation and exacerbates arthritis. Thus, we have established that IFN-γ, specifically derived from B cells, uniquely contributes to the pathogenesis of autoimmunity through prevention of immunoregulatory mechanisms.

Suppression of Development of Ankylosing Spondylitis Through Soluble Flt-1

Background/Aims: Circulating monocytes/macrophages are origins of osteoclasts that mediate the development of ankylosing spondylitis (AS). Moreover, infiltrated macrophages facilitate the AS progression through production and secretion of pro-inflammatory cytokines. Thus, suppression of the recruitment of circulating monocytes/macrophages may be an effective AS treatment, which is, however, not available so far in clinic. Soluble fms-like tyrosine kinase-1 (sFlt-1) is a decoy receptor for vascular endothelial growth factor (VEGF) to compete with VEGF receptor (VEGFR2) for VEGF binding in endothelial cells, while its application in treating AS and effects on the recruitment of circulating monocytes/macrophages has not been reported before. Methods: We used a proteoglycan-induced arthritis (PGIA) mouse model for human AS. We injected sFlt-1 into the articular cavity and evaluated its effects on PGIA by incidence of arthritis, and clinical and pathological arthritis severity. We isolated and analyzed macrophages and endothelial cells in the articular cavity before and after treatment. Results: Injection of sFlt-1 significantly decreased the incidence and severity of PGIA in mice, and significantly reduced the number of infiltrated macrophages, possibly through reduction of vessel permeability, in a VEGFR2-dependent manner. Conclusion: Our data suggest that sFlt-1 may have a therapeutic effect on AS, resulting from suppression of VEGF signaling-mediated recruitment of circulating monocytes/macrophages.

IL-4 Modulates Macrophage Polarization in Ankylosing Spondylitis

Background/Aims: Osteoclasts (OC) originate from monocytes/macrophages and play a critical role in the development of ankylosing spondylitis (AS). Receptor activator of NF-kB ligand (RANKL) is critical for the differentiation and maturation of OC from monocytes/macrophages, the underlying mechanisms of which processes have not been completely elucidated. Interleukin 4 (IL-4) attenuates the pathogenesis of AS via ill-defined mechanisms. Methods: We used a proteoglycan-induced arthritis (PGIA) model in Balb/c mice to study AS in humans. We injected IL-4 into the articular cavity and evaluated its effects on PGIA by incidence of arthritis, clinical and pathological arthritis severity and PET tracer uptake. We isolated and analyzed the number and polarization of macrophages in the articular cavity before and after IL-4 treatment. We analyzed RANKL levels in macrophage subtypes. We then isolated bone-marrow derived macrophages and treated them with IL-4 in vitro, with or without histone deacetylase inhibitors trichostatin A (TSA), and then analyzed the polarization of cultured macrophages before and after IL-4 treatment and RANKL levels in macrophage subtypes. Results: IL-4 treatment decreased the incidence and severity of arthritis in a mouse AS model, and polarized macrophages from a classical M1 subtype to an M2 subtype in vivo and in vitro. RANKL was predominantly produced by M1, but not by M2 macrophages. IL-4-mediated inhibition of RANKL in macrophages was abolished by TSA. Conclusion: Our data suggest that the therapeutic effects of IL-4 on AS may result from a M1-to-M2 macrophage polarization and its inhibition of RANKL expression on macrophages, possibly through enhanced histone deacetylation.

Proteoglycan-induced arthritis model in mice: research progress

Proteoglycan-induced arthritis(PGIA) is a unique systemic autoimmune disease model of spondyloarthritis(SpA)induced by intraperitoneal immunization of either BALB / cor certain C3H colonies with cartilage proteoglycan.This model is currently the only systemic autoimmune mice model with axial skeleton manifestation,which has become a focus of study in Sp A modeling.Here in this review we summarize the methods of animal model building,phenotype identification,immunological characteristics and pathways involved in bone remodeling of PGIA based on previous studies,hoping to provide references for study on SpA animal model.

Suppression of proteoglycan-induced autoimmune arthritis by myeloid-derived suppressor cells generated in vitro from murine bone marrow.

BackgroundMyeloid-derived suppressor cells (MDSCs) are innate immune cells capable of suppressing T-cell responses. We previously reported the presence of MDSCs with a granulocytic phenotype in the synovial fluid (SF) of mice with proteoglycan (PG)-induced arthritis (PGIA), a T cell-dependent autoimmune model of rheumatoid arthritis (RA). However, the limited amount of SF-MDSCs precluded investigations into their therapeutic potential. The goals of this study were to develop an in vitro method for generating MDSCs similar to those found in SF and to reveal the therapeutic effect of such cells in PGIA.MethodsMurine bone marrow (BM) cells were cultured for 3 days in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). The phenotype of cultured cells was analyzed using flow cytometry, microscopy, and biochemical methods. The suppressor activity of BM-MDSCs was tested upon co-culture with activated T cells. To investigate the therapeutic potential of BM-MDSCs, the cells were injected into SCID mice at the early stage of adoptively transferred PGIA, and their effects on the clinical course of arthritis and PG-specific immune responses were determined.ResultsBM cells cultured in the presence of GM-CSF, IL-6, and G-CSF became enriched in MDSC-like cells that showed greater phenotypic heterogeneity than MDSCs present in SF. BM-MDSCs profoundly inhibited both antigen-specific and polyclonal T-cell proliferation primarily via production of nitric oxide. Injection of BM-MDSCs into mice with PGIA ameliorated arthritis and reduced PG-specific T-cell responses and serum antibody levels.ConclusionsOur in vitro enrichment strategy provides a SF-like, but controlled microenvironment for converting BM myeloid precursors into MDSCs that potently suppress both T-cell responses and the progression of arthritis in a mouse model of RA. Our results also suggest that enrichment of BM in MDSCs could improve the therapeutic efficacy of BM transplantation in RA.