Helminth-induced impairment of humoral immunity differently contribute to their anti-arthritic effects in mice: Comparison of Schistosoma mansoni and Trichinella spiralis.

AIMS: We have previously reported reduction of anti-type II collagen (IIC) IgG levels in collagen-induced arthritis (CIA) by Schistosoma mansoni (Sm) and Trichinella spiralis (Ts). To clarify the contribution of the impairment of humoral immunity to their anti-arthritic activities, we herein investigated the relationship between anti-IIC IgG levels and arthritic swelling in Sm- or Ts-infected mice. METHODS AND RESULTS: Male DBA/1J mice were infected with Sm cercariae or Ts muscle larvae prior to the IIC immunization. In the Sm-infected mice, paw swelling and anti-IIC IgG levels were continuously lower than those of non-infected control group. In contrast, arthritic swelling in the Ts-infected mice only decreased in the early phase of CIA progression, despite the continued impairment of anti-IIC IgG production throughout the experimental period. Correlation coefficients between residual paw swelling and anti-IIC IgG titers were similar or higher in the Sm group than in the control group, but were similar or lower in the Ts group than in the control group. CONCLUSION: The down-modulations of anti-IIC IgG levels by the two parasitic infections and the correlation analyses suggest that the anti-arthritic activity of Sm was primarily attributed to the modulation of IgG-independent arthritogenic mechanisms and secondarily to the impairment of anti-IIC IgG production. In contrast, Ts could alleviate CIA mainly via the impairment of antibody production.

In vitro elimination of autoreactive B cells from rheumatoid arthritis patients by universal chimeric antigen receptor T cells.

OBJECTIVES: Autoreactive B cells play a crucial role in the pathogenesis of rheumatoid arthritis (RA), and B cell-depleting therapies using an antibodies, such as rituximab, have been suggested to be effective in RA treatment. However, transient B cell depletion with rituximab is associated with significant safety challenges related to global suppression of the immune system and thus increases the risks of infection and cancer development. To address selective and persistent issues associated with RA therapy, we developed a customised therapeutic strategy employing universal antifluorescein isothiocyanate (FITC) chimeric antigen receptor T cells (CAR-T cells) combined with FITC-labelled antigenic peptide epitopes to eliminate autoreactive B cell subsets recognising these antigens in RA. METHODS: For a proof-of-concept study, four citrullinated peptide epitopes derived from citrullinated autoantigens, namely, citrullinated vimentin, citrullinated type II collagen, citrullinated fibrinogen and tenascin-C, and a cyclocitrulline peptide-1 were selected as ligands for targeting autoreactive B cells; Engineered T cells expressing a fixed anti-FITC CAR were constructed and applied as a universal CAR-T cell system to specifically eliminate these protein-specific autoreactive B cells via recognition of the aforementioned FITC-labelled autoantigenic peptide epitopes. RESULTS: We demonstrated that anti-FITC CAR-T cells could be specifically redirected and kill hybridoma cells generated by immunisation with antigenic peptides, and autoreactive B cell subsets from RA patients via recognition of corresponding FITC-labelled citrullinated peptide epitopes. Additionally, the cytotoxicity of the CAR-T cells was dependent on the presence of the peptides and occurred in a dose-dependent manner. CONCLUSIONS: The approach described here provides a direction for precise, customised approaches to treat RA and can likely be applied to other systemic autoimmune diseases.

Kinase activity profiling reveals contribution of G-protein signaling modulator 2 deficiency to impaired regulatory T cell migration in rheumatoid arthritis.

The ability of regulatory T (Treg) cells to migrate into inflammatory sites is reduced in autoimmune diseases, including rheumatoid arthritis (RA). The reasons for impaired Treg cell migration remain largely unknown. We performed multiplex human kinase activity arrays to explore possible differences in the post-translational phosphorylation status of kinase related proteins that could account for altered Treg cell migration in RA. Results were verified by migration assays and Western blot analysis of CD4+ T cells from RA patients and from mice with collagen type II induced arthritis. Kinome profiling of CD4+ T cells from RA patients revealed significantly altered post-translational phosphorylation of kinase related proteins, including G-protein-signaling modulator 2 (GPSM2), protein tyrosine kinase 6 (PTK6) and vitronectin precursor (VTNC). These proteins have not been associated with RA until now. We found that GPSM2 expression is reduced in CD4+ T cells from RA patients and is significantly downregulated in experimental autoimmune arthritis following immunization of mice with collagen type II. Interestingly, GPSM2 acts as a promoter of Treg cell migration in healthy individuals. Treatment of RA patients with interleukin-6 receptor (IL-6R) blocking antibodies restores GPSM2 expression, thereby improving Treg cell migration. Our study highlights the potential of multiplex kinase activity arrays as a tool for the identification of RA-related proteins which could serve as targets for novel treatments.

Deficiency of ß-arrestin2 exacerbates inflammatory arthritis by facilitating plasma cell formation.

ß-arrestin2 (ß-arr2) is, a key protein that mediates desensitization and internalization of G protein-coupled receptors and participates in inflammatory and immune responses. Deficiency of ß-arr2 has been found to exacerbate collagen antibody-induced arthritis (CAIA) through unclear mechanisms. In this study we tried to elucidate the molecular mechanisms underlying ß-arr2 depletion-induced exacerbation of CAIA. CAIA was induced in ß-arr2-/- and wild-type (WT) mice by injection of collagen antibodies and LPS. The mice were sacrificed on d 13 after the injection, spleen, thymus and left ankle joints were collected for analysis. Arthritis index (AI) was evaluated every day or every 2 days. We showed that ß-arr2-/- mice with CAIA had a further increase in the percentage of plasma cells in spleen as compared with WT mice with CAIA, which was in accordance with elevated serum IgG1 and IgG2A expression and aggravating clinical performances, pathologic changes in joints and spleen, joint effusion, and joint blood flow. Both LPS stimulation of isolated B lymphocytes in vitro and TNP-LPS challenge in vivo led to significantly higher plasma cell formation and antibodies production in ß-arr2-/- mice as compared with WT mice. LPS treatment induced membrane distribution of toll-like receptor 4 (TLR4) on B lymphocytes, accordingly promoted the nuclear translocation of NF-κB and the transcription of Blimp1. Immunofluorescence analysis confirmed that more TLR4 colocalized with ß-arr2 in B lymphocytes in response to LPS stimulation. Depletion of ß-arr2 restrained TLR4 on B lymphocyte membrane after LPS treatment and further enhanced downstream NF-κB signaling leading to additional increment in plasma cell formation. In summary, ß-arr2 depletion exacerbates CAIA and further increases plasma cell differentiation and antibody production through inhibiting TLR4 endocytosis and aggravating NF-κB signaling.

The impact of Clonorchis sinensis infection on immune response in mice with type II collagen-induced arthritis.

BACKGROUND: Clonorchis sinensis infection could trigger strong immune responses in mice and humans. However, whether the C.sinensis infection has an impact on arthritis is unknown. Here we investigated the effect of C.sinensis infection on type II collagen-induced arthritis in BALB/c mice. RESULTS: The mice were firstly infected with 45 C.sinensis metacercariae by oral gavage. Four weeks later, arthritis in mice was induced by type II collagen. Joint inflammation with severe redness and swelling in hind paws was observed in type II collagen-induced arthritis (CIA) mice. Besides, the physical activity was significantly reduced, but the respiratory exchange ratio was increased in CIA mice. Compared with CIA mice, C.sinensis infection could increase the severity of arthritis in CIA mice, based on the results of disease score and pathological changes. Compared to CIA mice, increased neutrophils and Ly6Chi monocytes, decreased B cells and CD4+T cells, were found in C.sinensis infected CIA mice. Besides these, C.sinensis infected mice also displayed significantly higher levels of serum IL-4 and IL-17 than those in CIA mice. CONCLUSIONS: Taken together, our data suggest that C.sinensis infection have a bad effect on arthritis, and could induce the abnormality of the immune response in mice with CIA.

Immunoglobulin A antibodies to oxidized collagen type II as a potential biomarker for the stratification of spondyloarthritis from rheumatoid arthritis.

OBJECTIVES: The discovery of diseased tissue-specific neoantigens offers the opportunity to develop important disease tissue-specific biomarkers that can help in the prediction, diagnosis, and stratification of diseases. This opportunity is specifically significant for autoimmune diseases where diagnostic biomarkers are not available. Inflammatory autoimmune diseases are commonly associated with local generation of large amounts of reactive oxidants. We have previously identified oxidative post-translationally modified (oxPTM) tissue-specific neoantigens in rheumatoid arthritis (RA) and type 1 diabetes that elicit an immune response. In the current study, we studied the presence and clinical significance of antibodies to oxPTM collagen type II (CII) in patients with spondyloarthritis (SpA). METHOD: Levels of antibodies specific to native CII and oxPTM-CII were assessed by enzyme-linked immunosorbent assay. RESULTS: Immunoglobulin G (IgG) binding to oxPTM-CII was observed in 52%, 83%, and 28% of serum samples from patients with axial spondyloarthritis (axSpA), RA, and psoriatic arthritis (PsA), respectively. Importantly, while strong IgA anti-oxPTM-CII responses were detected in axSpA and PsA patients, with 47% and 84% respective binders, no IgA anti-oxPTM-CII was detected in RA patients. IgA anti-oxPTM-CII reactivity in axSpA patients treated with biologics was higher and more frequent, with 85% binders compared to 9% binders in patients treated with synthetic disease-modifying anti-rheumatic drugs. CONCLUSION: Our data imply that SpA and PsA are associated with the presence of antibodies to oxPTM-CII, suggesting that there may be a humoral component that may distinguish patients with SpA from RA. Our approach could be adapted to other diseases, particularly to inflammatory autoimmune diseases.

STAT6 and IL-10 are required for the anti-arthritic effects of Schistosoma mansoni via different mechanisms.

To investigate possible roles of T helper type 2 (Th2) cytokines in the anti-arthritic effects of a blood fluke, Schistosoma mansoni (Sm), for mouse collagen-induced arthritis (CIA), wild-type (WT), signal transducer and activator of transcription 6 (STAT6) knock-out (KO) and interleukin (IL)-10 KO mice were infected with Sm. Three weeks after infection, the mice were immunized with bovine type II collagen (IIC). Arthritis severity was monitored by scoring, measurement of paw thickness and the presence of ankylosis. Serum anti-IIC IgG levels, splenic cytokine production and cytokine gene expression in the popliteal lymph nodes (PLNs) were measured and compared among WT and gene-KO mice. Consistent with our previous findings, Sm infection reduced the arthritis severity in WT mice. Splenic production of IL-17A and tumor necrosis factor (TNF)-α was reduced by the infection. In contrast, Sm infection markedly exacerbated CIA in STAT6 KO mice. In the KO mice, IL-17A production was increased by the infection. Conversely, Sm infection did not affect the exacerbated arthritis in IL-10 KO mice, although IL-17A production was reduced by the helminth. Our results suggest that signaling via STAT6 (presumably IL-4 and/or IL-13) and IL-10 is required for the suppression of CIA by Sm infection, but through different mechanisms. STAT6 was essential for helminth-induced reduction of IL-17A, whereas regulation of the basal arthritis severity by IL-10 was needed in order for it to be sufficiently suppressed by the helminth.

The autoantibody response to cyclic citrullinated collagen type II peptides in rheumatoid arthritis.

OBJECTIVES: The detection of anti-citrullinated peptide antibodies (ACPAs) is a serological hallmark of RA. Autoantibodies reactive with collagen type II (CII) are present in RA sera and synovial fluid and are potentially pathogenic. Here, we investigate the prevalence and specificity of the autoantibody responses to defined citrullinated cyclic peptides derived from CII in a China RA cohort. METHODS: Using bead-based multiplex assay, we examined the presence of autoantibodies binding to 54 cyclic 17-mer citrullinated CII peptides, encompassing all citrullinate epitopes in CII, and the corresponding unmodified peptides in 415 RA patients, in addition to 304 patients with OA. Furthermore, the autoantibody responses to a selected set of 10 cyclic citrullinated peptides were also examined in 203 healthy individuals. RESULTS: Autoantibody responses to cyclic citrullinated CII peptides were higher in RA patients as compared with OA patients or healthy individuals, whereas little or negligible antibody responses to cyclic unmodified CII peptides were observed. Interestingly, several novel citrullinated CII epitopes were identified. Antibodies to these novel citrullinated CII epitopes showed not only substantial overlapping reactivities but also had unique specificities. CONCLUSION: We found a high prevalence of autoantibodies against cyclic citrullinated CII in the sera of patients in a China RA cohort. The present study revealed heterogeneous binding patterns against novel citrullinated CII epitopes, which may help to stratify RA patients into different subgroups.

Evaluation of Targeting Efficiency of Joints with Anticollagen II Antibodies.

Diseases of the joints affect over 10% of the world's population, resulting in significant morbidity. There is an unmet need in strategies for specific delivery of therapeutics to the joints. Collagen type II is synthesized by chondrocytes and is mainly restricted to the cartilage and tendons. Arthrogen-CIA is a commercially available anticollagen II antibody cocktail that reacts with 5 different epitopes on human, bovine, and mouse collagen II. Arthrogen has been used for induction of experimental rheumatoid arthritis (RA) in mice because of high complement activation on the cartilage surface. Native collagen II might serve as a useful target for potential delivery of therapeutics to the joint. To evaluate the efficiency and specificity of targeting collagen II, Arthrogen was labeled with near-infrared (NIR) dye IRDye 800 or IRDye 680. Using ex vivo NIR imaging, we demonstrate that Arthrogen efficiently and specifically accumulated in the limb joints regardless of the label dye or injection route (intravenous and subcutaneous). After subcutaneous injection, the mean fluorescence of the hind limb joints was 19 times higher than that of the heart, 8.7 times higher than that of the liver, and 3.7 times higher than that of the kidney. Control mouse IgG did not show appreciable accumulation. Microscopically, the antibody accumulated on the cartilage surface of joints and on endosteal surfaces. A monoclonal antibody against a single epitope of collagen II showed similar binding affinity and elimination half-life, but about three times lower targeting efficiency than Arthrogen in vitro and ex vivo, and about two times lower targeting efficiency in vivo. We suggest that an antibody against multiple epitopes of collagen II could be developed into a highly effective and specific targeting strategy for diseases of the joints or spine.

Vamorolone, a dissociative steroidal compound, reduces collagen antibody-induced joint damage and inflammation when administered after disease onset.

OBJECTIVE AND DESIGN: The objective of this study was to assess the effect of vamorolone, a first-in-class dissociative steroidal compound, to inhibit inflammation when administered after disease onset in the murine collagen antibody-induced arthritis model of arthritis. ANIMALS: 84 DBA1/J mice were used in this study (n = 12 per treatment group). TREATMENT: Vamorolone or prednisolone was administered orally after disease onset for a duration of 7 days. METHODS: Disease score and bone erosion were assessed using previously described scoring systems. Cytokines were measured in joints via immunoassay, and joint cathepsin B activity (marker of inflammation) was assessed using optical imaging of joints on live mice. RESULTS: We found that vamorolone treatment led to a reduction of several disease parameters including disease score, joint inflammation, and the presence of pro-inflammatory mediators to a degree similar of that observed with prednisolone treatment. More importantly, histopathological analysis of affected joints showed that vamorolone treatment significantly reduced the degree of bone erosion while this bone-sparing property was not observed with prednisolone treatment at any of the tested doses. CONCLUSIONS: While many intervention regimens in other studies are administered prior to disease onset in animal models, the current study involves delivery of the potential therapeutic after disease onset. Based on the findings, vamorolone may offer an efficacious, yet safer alternative to conventional steroidal compounds in the treatment of rheumatoid arthritis and other inflammatory diseases.

The Role of Leukocyte-Associated Ig-like Receptor-1 in Suppressing Collagen-Induced Arthritis.

Several observations implicate a critical role for T cell dysregulation as a central problem in rheumatoid arthritis. We investigated a mechanism for suppressing T cell activation by stimulating a natural inhibitory receptor called leukocyte-associated Ig-like receptor-1 (LAIR-1). The collagen-induced arthritis (CIA) model and DR-1 transgenic mice were used to study the importance of LAIR-1 in autoimmune arthritis. Splenocytes from wild-type or LAIR-1-/- mice were stimulated with soluble anti-CD3 Ab in the presence or absence of α1(II) and supernatants were collected for cytokine analysis. B6.DR1 mice were immunized with type II collagen/CFA to induce arthritis and were treated with either the stimulatory mAb to LAIR-1 or a hamster IgG control. Finally, B6.DR1/LAIR-1-/- and B6.DR1/LAIR-1+/+ mice were challenged for CIA and mean severity scores were recorded thrice weekly. Using splenocytes or purified CD4+ cells that were sufficient in LAIR-1, CD3-induced cytokine secretion was significantly suppressed in the presence of collagen, whereas LAIR-1-deficient splenocytes had no attenuation. Treatment with a stimulatory mAb to LAIR-1 also significantly attenuated CIA in the LAIR+/+ mice. When B6.DR1/LAIR-1-/- mice were immunized with type II collagen they developed more severe arthritis and had a greater percentage of affected limbs than the wild-type mice. These data demonstrate that collagen can suppress the T cell cytokine response through the action of LAIR-1. Treatment with stimulating LAIR-1 Abs suppresses CIA whereas B6.DR1/LAIR-1-/- mice develop more severe arthritis than wild-type controls. These data suggest that LAIR-1 may be a potential therapeutic target for suppressing rheumatoid arthritis.

Dynamic profiles, biodistribution and integration evaluation after intramuscular/intravenous delivery of a novel therapeutic DNA vaccine encoding chicken type II collagen for rheumatoid arthritis in vaccinated normal rodent.

BACKGROUND: The persistence, biodistribution, and risk of integration into the host genome of any new therapeutic DNA vaccine must be established in preclinical studies. We previously developed the DNA vaccine pcDNA-CCOL2A1 encoding chicken type II collagen (CCII) for the treatment of rheumatoid arthritis (RA). In the present study, we characterized its dynamic profile, biodistribution, and potential for genomic DNA integration in normal vaccinated rodent. RESULTS: A real-time quantitative PCR analysis (RT-qPCR) of animals administered a single dose of pcDNA-CCOL2A1 (300 µg/kg by intramuscular injection) showed that CCOL2A1 mRNA level in the blood peaked between 2 and 6 h post-immunization and then rapidly declined, and was undetectable between day 1-42. CCOL2A1 transcript was detected at the muscle injection site on days 3-14 post-immunization. Starting from day 14, the transcript was detected in the heart, liver, lung, and kidney but not in the spleen or thymus, and was expressed only in the lung on day 28. There was no CCOL2A1 mRNA present in the testes or ovaries at any time point. Non-invasive in vivo fluorescence imaging revealed CCII protein expression from 2 h up to day 10 and from 2 h up to day 35 after administration of pcDNA-CCOL2A1 via the intravenous and intramuscular routes, respectively; the protein had disappeared by day 42. Importantly, CCOL2A1 was not integrated into the host genome. CONCLUSIONS: These results indicate that pcDNA-CCOL2A1 vaccine is rapidly cleared within a short period of time and is therefore safe, and merits further development as a therapeutic vaccine for RA treatment.

The role of Syk in peripheral T cells.

The aim of this study was to understand how Syk affects peripheral T cell function. T cells from Syk-/- chimeric mice and DR1 Sykfl/fl CD4cre conditional mice gave strong CD3-induced Th1, Th2, and Th17 cytokine responses. However, an altered peptide ligand (APL) of human CII (256-276) with two substitutions (F263N, E266D), also called A12, elicited only Th2 cytokine responses from Sykfl/fl T cells but not Sykfl/fl-CD4cre T cells. Western blots revealed a marked increase in the phosphorylation of Syk, JNK and p38 upon A12/DR1 activation in WT or Sykfl/fl T cells but not in Sykfl/flCD4-cre cells. We demonstrate that Syk is required for the APL- induction of suppressive cytokines. Chemical Syk inhibitors blocked activation of GATA-3 by peptide A12/DR1. In conclusion, this study provides novel insights into the role that Syk plays in directing T cell activity, and may shape therapeutic approaches for autoimmune diseases.

CD11b regulates the Treg/Th17 balance in murine arthritis via IL-6.

Th17 cells are often associated with autoimmunity and been shown to be increased in CD11b-/- mice. Here, we examined the role of CD11b in murine collagen-induced arthritis (CIA). C57BL/6 and CD11b-/- resistant mice were immunized with type II collagen. CD11b-/- mice developed arthritis with early onset, high incidence, and sustained severity compared with C57BL/6 mice. We observed a marked leukocyte infiltration, and histological examinations of the arthritic paws from CD11b-/- mice revealed that the cartilage was destroyed in association with strong lymphocytic infiltration. The CD11b deficiency led to enhanced Th17-cell differentiation. CD11b-/- dendritic cells (DCs) induced much stronger IL-6 production and hence Th17-cell differentiation than wild-type DCs. Treatment of CD11b-/- mice after establishment of the Treg/Th17 balance with an anti-IL-6 receptor mAb significantly suppressed the induction of Th17 cells and reduced arthritis severity. Finally, the severe phenotype of arthritis in CD11b-/- mice was rescued by adoptive transfer of CD11b+ DCs. Taken together, our results indicate that the resistance to CIA in C57BL/6 mice is regulated by CD11b via suppression of IL-6 production leading to reduced Th17-cell differentiation. Therefore, CD11b may represent a susceptibility factor for autoimmunity and could be a target for future therapy.

Identification of antibodies against extracellular matrix proteins in human osteoarthritis.

We investigated the presence of autoantibodies against the extracellular matrix proteins thrombospondin-4 (TSP-4), cartilage oligomeric matrix protein (COMP), C-type lectin domain family 3 member A (CLEC3A), collagen II, collagen VI, matrilin-3, and fibrillin-2 in the serum of osteoarthritis (OA) patients. We compared those results with the presence of such antibodies in rheumatoid arthritis (RA) patients and in healthy donors (HD). Our study examines whether antibodies against extracellular proteins can be used as potential biomarkers to support the clinical diagnosis of OA. 10 OA, 10 RA patients and 10 HD were enrolled in this explorative cross-sectional study. SDS-PAGE and immunoblot were used to investigate the presence of antibodies against extracellular matrix proteins. The serum of 5/10 OA patients but 0/10 HD exhibited TSP-4 IgG isotype antibodies (P = 0.033). The serum of 8/10 OA patients but only 1/10 HD exhibited IgG isotype antibodies against TSP-4 or COMP (P = 0.005). The serum of 9/10 OA patients but only 1/10 HD exhibited IgG isotype antibodies against TSP-4, COMP or CLEC3A (P = 0.005). We found strong evidence for the presence of IgG isotype autoantibodies against the cartilage extracellular matrix proteins TSP-4, COMP and CLEC3A in OA. The detection of IgG isotype autoantibodies against TSP-4, COMP and CLEC3A may support the clinical diagnosis of OA. OA with autoantibodies against cartilage extracellular matrix proteins defines a new OA subgroup suggesting that patients with high concentrations of autoantibodies may benefit from an immune suppressive therapy.

Acacetin regulated the reciprocal differentiation of Th17 cells and Treg cells and mitigated the symptoms of collagen-induced arthritis in mice.

BACKGROUND AND AIM: The immune-regulative effect of acacetin on the development of autoimmune arthritis remains unexplored. This study aims to investigate the potential effect of acacetin on the treatment of collagen-induced arthritis (CIA) in mice and clarify its underlying mechanism. METHODS AND RESULTS: In a type II collagen (CII)-induced autoimmune arthritis model, acacetin significantly repressed the incidence of CIA, prevented the pathological alteration, and reduced CII-specific IgG and IgG1 levels. Flow cytometry assay suggested that the recipients of acacetin showed the expansion of Treg cells and the decreasing Th17 cells in spleen and inguinal lymph nodes after the initiation of CIA. In vitro experiment suggested that in addition to altering the pro-inflammatory production in dendritic cells, engagement of acacetin relieved the generation of Th17 cells and maintained the ratio of Treg cells under Th17-polarized condition. The addition of acacetin inhibited the T cell proliferation, as well as the expression of the transcriptional coactivator TAZ, which regulated the balance of Treg/Th17 immunity, in a dose-dependent manner. CONCLUSION: Our data demonstrated that acacetin mitigated the development of CIA and might be a potential agent for the treatment of autoimmune arthritis.

Characterization of the Syk-Dependent T Cell Signaling Response to an Altered Peptide.

Rheumatoid arthritis is an autoimmune disorder characterized by T cell dysregulation. We have shown that an altered peptide ligand (A9) activates T cells to use an alternate signaling pathway that is dependent on FcRγ and spleen tyrosine kinase, resulting in downregulation of inflammation. In the experiments described in this study, we have attempted to determine the molecular basis of this paradox. Three major Src family kinases found in T cells (Lck, Fyn, and Lyn) were tested for activation following stimulation by A9/I-Aq Unexpectedly we found they are not required for T cell functions induced by A9/I-Aq, nor are they required for APL stimulation of cytokines. On the other hand, the induction of the second messenger inositol trisphosphate and the mobilization of calcium are clearly triggered by the APL A9/I-Aq stimulation and are required for cytokine production, albeit the cytokines induced are different from those produced after activation of the canonical pathway. DBA/1 mice doubly deficient in IL-4 and IL-10 were used to confirm that these two cytokines are important for the APL-induced attenuation of arthritis. These studies provide a basis for exploring the effectiveness of analog peptides and the inhibitory T cells they induce as therapeutic tools for autoimmune arthritis.

Treatment mechanism of tolerogenic dendritic cells on rheumatoid arthritis.

This study aims to explore the possible mechanism of treatment of collagen-induced rheumatoid arthritis (RA) by tolerogenic dendritic cells (tDCs). Different methods were used to induce and cultivate tDCs, and suitable conditions for tDC cultivation were explored. The experimental RA induced by collagen in mouse was treated by the obtained tDCs, and the possible mechanism was explored. The serum concentration of TNF-α, IFN-ß, IL-4 and anti-type II collagen antibody were detected by ELISA. The anti-type II collagen antibody of mice without treatment was higher than that without disease onset, while the Blank-DC group, VIP-DC group and Bay-D had no statistically significant differences (P>0.05). Compared to the group without disease onset, the TNF-α level of those without treatment was significantly higher, while INF-γ, IL-1ß and IL-4 concentration showed no significant difference (P>0.05). Compared to the untreated group, the TNF-α and IL-1ß concentration after VIP-DC treatment were significantly decreased, while IL-4 was increased (P less than0.05). In summary, VIP-DC and Bay-DC alleviate joint inflammation, synovitis and bone erosion by reducing the production of anti-type II collagen antibody, inhibiting proinflammatory factors and increasing inflammation inhibitors.

A Novel High Sensitivity Type II Collagen Blood-Based Biomarker, PRO-C2, for Assessment of Cartilage Formation.

N-terminal propeptide of type II collagen (PIINP) is a biomarker reflecting cartilage formation. PIINP exists in two main splice variants termed as type IIA and type IIB collagen NH2-propeptide (PIIANP, PIIBNP). PIIANP has been widely recognized as a cartilage formation biomarker. However, the utility of PIIBNP as a marker in preclinical and clinical settings has not been fully investigated yet. In this study, we aimed to characterize an antibody targeting human PIIBNP and to develop an immunoassay assessing type II collagen synthesis in human blood samples. A high sensitivity electrochemiluminescence immunoassay, hsPRO-C2, was developed using a well-characterized antibody against human PIIBNP. Human cartilage explants from replaced osteoarthritis knees were cultured for ten weeks in the presence of growth factors, insulin-like growth factor 1 (IGF-1) or recombinant human fibroblast growth factor 18 (rhFGF-18). The culture medium was changed every seven days, and levels of PIIBNP, PIIANP, and matrix metalloproteinase 9-mediated degradation of type II collagen (C2M) were analyzed herein. Serum samples from a cross-sectional knee osteoarthritis cohort, as well as pediatric and rheumatoid arthritis samples, were assayed for PIIBNP and PIIANP. Western blot showed that the antibody recognized PIIBNP either as a free fragment or attached to the main molecule. Immunohistochemistry demonstrated that PIIBNP was predominately located in the extracellular matrix of the superficial and deep zones and chondrocytes in both normal and osteoarthritic articular cartilage. In addition, the hsPRO-C2 immunoassay exhibits acceptable technical performances. In the human cartilage explants model, levels of PIIBNP, but not PIIANP and C2M, were increased (2 to 7-fold) time-dependently in response to IGF-1. Moreover, there was no significant correlation between PIIBNP and PIIANP levels when measured in knee osteoarthritis, rheumatoid arthritis, and pediatric serum samples. Serum PIIBNP was significantly higher in controls (KL0/1) compared to OA groups (KL2/3/4, p = 0.012). The hsPRO-C2 assay shows completely different biological and clinical patterns than PIIANP ELISA, suggesting that it may be a promising biomarker of cartilage formation.

Targeting IgG in Arthritis: Disease Pathways and Therapeutic Avenues.

Rheumatoid arthritis (RA) is a polygenic and multifactorial syndrome. Many complex immunological and genetic interactions are involved in the final outcome of the clinical disease. Autoantibodies (rheumatoid factors, anti-citrullinated peptide/protein antibodies) are present in RA patients' sera for a long time before the onset of clinical disease. Prior to arthritis onset, in the autoantibody response, epitope spreading, avidity maturation, and changes towards a pro-inflammatory Fc glycosylation phenotype occurs. Genetic association of epitope specific autoantibody responses and the induction of inflammation dependent and independent changes in the cartilage by pathogenic autoantibodies emphasize the crucial contribution of antibody-initiated inflammation in RA development. Targeting IgG by glyco-engineering, bacterial enzymes to specifically cleave IgG/alter N-linked Fc-glycans at Asn 297 or blocking the downstream effector pathways offers new avenues to develop novel therapeutics for arthritis treatment.