Treatment Of Autoimmune Arthritis Research Articles

Metabolomic Profiling of Red Blood Cells to Identify Molecular Markers of Methotrexate Response in the Collagen Induced Arthritis Mouse Model

Although methotrexate (MTX) is the first line disease-modifying therapy used in the treatment of autoimmune arthritis, it is limited by its unpredictable and variable response profile and lack of therapeutic biomarkers to predict or monitor therapeutic response. The purpose of this work is to evaluate the utility of red blood cell (RBC) metabolite profiles to screen for molecular biomarkers associated with MTX response. Methods: Utilizing the collagen-induced arthritis mouse model, DBA/1J mice were treated with subcutaneous MTX (20 mg/kg/week) and RBC samples were collected and analyzed by semi-targeted global metabolomic profiling and analyzed by univariate analysis. Results: MTX treatment normalized the following RBC metabolite levels that were found to be altered by disease induction: N-methylisoleucine, nudifloramide, phenylacetylglycine, 1-methyl-L-histidine, PC 42:1, PE 36:4e, PC 42:3, PE 36:4e (16:0e/20:4), and SM d34:0. Changes in the RBC metabolome weakly but significantly correlated with changes in the plasma metabolome following MTX treatment (ρ = 0.24, p = 1.1 × 10−13). The RBC metabolome resulted in the detection of nine significant discriminatory biomarkers, whereas the plasma metabolome resulted in two. Overall, the RBC metabolome yielded more highly sensitive and specific biomarkers of MTX response compared to the plasma metabolome. N-methylisoleucine was found to be highly discriminatory in both plasma and RBCs. Conclusions: Our results suggest that RBCs represent a promising biological matrix for metabolomics and future studies should consider the RBC metabolome in their biomarker discovery strategy.

Methotrexate Disposition, Anti-Folate Activity, and Metabolomic Profiling to Identify Molecular Markers of Disease Activity and Drug Response in the Collagen-Induced Arthritis Mouse Model.

Methotrexate (MTX) is widely used in the treatment of autoimmune arthritis but is limited by its unpredictable and variable response profile. Currently, no biomarkers exist to predict or monitor early therapeutic responses to MTX. Using a collagen-induced arthritis (CIA) mouse model, this study aimed to identify biochemical pathways and biomarkers associated with MTX efficacy in autoimmune arthritis. Following arthritis disease induction, DBA/1J mice were treated with subcutaneous MTX (20 mg/kg/week) and disease activity was assessed based on disease activity scores (DAS) and paw volume (PV) measurements. Red blood cell (RBC) and plasma samples were collected at the end of the study and were assessed for folate and MTX content. Plasma samples were analyzed by semitargeted global metabolomic profiling and analyzed by univariate and multivariate analysis. Treatment with MTX was associated with significant reductions in disease activity based on both DAS (p = 0.0006) and PV (p = 0.0006). MTX therapy resulted in significant reductions in 5-methyltetrahydrofolate (5mTHF) levels in plasma (p = 0.02) and RBCs (p = 0.001). Reductions in both RBC and plasma 5mTHF were associated with lower DAS (p = 0.0007, p = 0.01, respectively) and PV (p = 0.001, p = 0.005, respectively). Increases in RBC MTX were associated with lower DAS (p = 0.003) but not PV (p = 0.23). Metabolomic analysis identified N-methylisoleucine (NMI) and quinolone as metabolites significantly altered in disease mice, which were corrected towards healthy control levels in mice treated with MTX. Reductions in plasma NMI were associated with lower DAS (p = 0.0002) and PV (p = 9.5 × 10−6). Increases in plasma quinolone were associated with lower DAS (p = 0.02) and PV (p = 0.01). Receiver-operating characteristic curve analysis identified plasma NMI (AUC = 1.00, p = 2.4 × 10−8), RBC 5mTHF (AUC = 0.99, p = 2.4 × 10−5), and plasma quinolone (AUC = 0.89, p = 0.01) as top discriminating metabolites of MTX treatment. Our data support a relationship between MTX efficacy and its effect on circulating folates and identified 5mTHF, NMI, and quinolone as potential therapeutic biomarkers of disease activity and MTX response in the CIA mouse model of autoimmune arthritis.

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.

Metabolomic Profiling to Identify Molecular Biomarkers of Cellular Response to Methotrexate In Vitro.

Variation in methotrexate (MTX) efficacy represents a significant barrier to early and effective disease control in the treatment of autoimmune arthritis. We hypothesize that the utilization of metabolomic techniques will allow for an improved understanding of the biochemical basis for the pharmacological activity of MTX, and can promote the identification and evaluation of novel molecular biomarkers of MTX response. In this work, erythroblastoid cells were exposed to MTX at the physiologic concentration of 1,000 nM and analyzed using three metabolomic platforms to give a broad spectrum of cellular metabolites. MTX pharmacological activity, defined as cellular growth inhibition, was associated with an altered cellular metabolomic profile based on the analysis of 724 identified metabolites. By discriminant analysis, MTX treatment was associated with increases in ketoisovaleric acid, fructose, galactose, and 2‐deoxycytidine, and corresponding reductions in 2‐deoxyuridine, phosphatidylinositol 32:0, orotic acid, and inosine monophosphate. Inclusion of data from analysis of folate metabolism in combination with chemometric and metabolic network analysis demonstrated that MTX treatment is associated with dysregulated folate metabolism and nucleotide biosynthesis, which is in line with its known mechanism of action. However, MTX treatment was also associated with alterations in a diversity of metabolites, including intermediates of amino acid, carbohydrate, and lipid metabolism. Collectively, these findings support a robust metabolic response following exposure to physiologic concentrations of MTX. They also identify various metabolic intermediates that are associated with the pharmacological activity of MTX, and are, therefore, potential molecular biomarker candidates in future preclinical and clinical studies of MTX efficacy in autoimmune arthritis.

Myeloid-derived suppressor cells regulate T cell and B cell responses during autoimmune disease.

MDSCs are a heterogeneous group of myeloid cells that suppress T cell activity in cancer and autoimmune disease. The effect of MDSCs on B cell function is not clear. Using the CIA model of autoimmune disease, we found an increase in M-MDSCs in the periphery of WT mice with CIA compared with naïve mice. These MDSCs were absent from the periphery of CCR2(-/-) mice that developed exacerbated disease. M-MDSCs, isolated from immunized mice, inhibited autologous CD4(+) T cell proliferation. The M-MDSC-mediated suppression of T cell proliferation was NO and IFN-γ dependent but IL-17 independent. Furthermore, we demonstrated for the first time that M-MDSCs from CIA mice also inhibited autologous B cell proliferation and antibody production. The suppression of B cells by M-MDSCs was dependent on the production of NO and PGE2 and required cell-cell contact. Administration of M-MDSCs rescued CCR2(-/-) mice from the exacerbated CIA phenotype and ameliorated disease in WT mice. Furthermore, adoptive transfer of M-MDSCs reduced autoantibody production by CCR2(-/-) and WT mice. In summary, M-MDSCs inhibit T cell and B cell function in CIA and may serve as a therapeutic approach in the treatment of autoimmune arthritis.

A cytokine-centric view of the pathogenesis and treatment of autoimmune arthritis.

Cytokines are immune mediators that play an important role in the pathogenesis of rheumatoid arthritis (RA), an autoimmune disease that targets the synovial joints. The cytokine environment in the peripheral lymphoid tissues and the target organ (the joint) has a strong influence on the outcome of the initial events that trigger autoimmune inflammation. In susceptible individuals, these events drive inflammation and tissue damage in the joints. However, in resistant individuals, the inflammatory events are controlled effectively with minimal or no overt signs of arthritis. Animal models of human RA have permitted comprehensive investigations into the role of cytokines in the initiation, progression, and recovery phases of autoimmune arthritis. The discovery of interleukin-17 (IL-17) and its association with inflammation and autoimmune pathology has reshaped our viewpoint regarding the pathogenesis of arthritis, which previously was based on a simplistic T helper 1 (Th1)-Th2 paradigm. This review discusses the role of the newer cytokines, particularly those associated with the IL-17/IL-23 axis in arthritis. Also presented herein is the emerging information on IL-32, IL-33, and IL-35. Ongoing studies examining the role of the newer cytokines in the disease process would improve understanding of RA as well as the development of novel cytokine inhibitors that might be more efficacious than the currently available options.

Prevention and Treatment of Autoimmune Arthritis Using IL-12 Gene-Silenced Dendritic Cells Friday, June 8 10:45 am−11:05 am

Prevention and Treatment of Autoimmune Arthritis Using IL-12 Gene-Silenced Dendritic Cells Friday, June 8 10:45 am−11:05 am

STAT3 and NF-κB Signal Pathway Is Required for IL-23-Mediated IL-17 Production in Spontaneous Arthritis Animal Model IL-1 Receptor Antagonist-Deficient Mice

IL-23 is a heterodimeric cytokine composed of a p19 subunit and the p40 subunit of IL-12. IL-23 has proinflammatory activity, inducing IL-17 secretion from activated CD4(+) T cells and stimulating the proliferation of memory CD4(+) T cells. We investigated the pathogenic role of IL-23 in CD4(+) T cells in mice lacking the IL-1R antagonist (IL-1Ra(-/-)), an animal model of spontaneous arthritis. IL-23 was strongly expressed in the inflamed joints of IL-1Ra(-/-) mice. Recombinant adenovirus expressing mouse IL-23 (rAd/mIL-23) significantly accelerated this joint inflammation and joint destruction. IL-1beta further increased the production of IL-23, which induced IL-17 production and OX40 expression in splenic CD4(+) T cells of IL-1Ra(-/-) mice. Blocking IL-23 with anti-p19 Ab abolished the IL-17 production induced by IL-1 in splenocyte cultures. The process of IL-23-induced IL-17 production in CD4(+) T cells was mediated via the activation of Jak2, PI3K/Akt, STAT3, and NF-kappaB, whereas p38 MAPK and AP-1 did not participate in the process. Our data suggest that IL-23 is a link between IL-1 and IL-17. IL-23 seems to be a central proinflammatory cytokine in the pathogenesis of this IL-1Ra(-/-) model of spontaneous arthritis. Its intracellular signaling pathway could be useful therapeutic targets in the treatment of autoimmune arthritis.

CXCL13 neutralization reduces the severity of collagen‐induced arthritis

To investigate the role of CXCL13 in the development and pathogenesis of collagen-induced arthritis (CIA), and to determine the mechanisms involved in the modulation of arthritogenic response by CXCL13 neutralization. Mice were immunized with type II collagen (CII) and treated with anti-CXCL13 or control antibodies during boosting. Mice were monitored for the development and severity of arthritis. The effects of CXCL13 neutralization on immune response to CII were evaluated by cytokine production by CII-specific T cells and CII-specific antibody production. Follicular response in the spleen and in synovial tissue was determined by in situ immunohistology. Mice receiving neutralizing antibodies to CXCL13 developed significantly less severe arthritis compared with mice injected with phosphate buffered saline or control antibodies. Follicular response both in the spleen and in synovial tissue was inhibited by anti-CXCL13 treatment. Injection with anti-CXCL13 antibodies did not significantly affect antigen-specific recall lymphocyte proliferation or type 1 cytokine production in vitro. Antibody response specific to CII was not inhibited by anti-CXCL13 treatment. However, anti-CXCL13 treatment induced significantly higher levels of interleukin-10 production after in vitro CII stimulation. Neutralization of CXCL13 inhibits the development of CIA and reduces follicular response in both lymphoid and nonlymphoid tissues. These findings may have important implications regarding the pathogenesis and treatment of autoimmune arthritis.

Treatment of autoimmune arthritis by protective antibodies against a heat shock protein surface epitope that induce IL-10 secretion

Treatment of autoimmune arthritis by protective antibodies against a heat shock protein surface epitope that induce IL-10 secretion

Treatment of autoimmune arthritis by protective antibodies against a heat shock protein surface epitope that induce IL-10 secretion

Treatment of autoimmune arthritis by protective antibodies against a heat shock protein surface epitope that induce IL-10 secretion