Severity Of Arthritis In Mice Research Articles

NAT10 promotes synovial aggression by increasing the stability and translation of N4-acetylated PTX3 mRNA in rheumatoid arthritis

ObjectiveRecent studies indicate that N-acetyltransferase 10 (NAT10)-mediated ac4C modification plays unique roles in tumour metastasis and immune infiltration. This study aimed to uncover the role of NAT10-mediated ac4C in fibroblast-like...

The acute phase reactant orosomucoid-2 directly promotes rheumatoid inflammation

Acute phase proteins involved in chronic inflammatory diseases have not been systematically analyzed. Here, global proteome profiling of serum and urine revealed that orosomucoid-2 (ORM2), an acute phase reactant, was differentially expressed in rheumatoid arthritis (RA) patients and showed the highest fold change. Therefore, we questioned the extent to which ORM2, which is produced mainly in the liver, actively participates in rheumatoid inflammation. Surprisingly, ORM2 expression was upregulated in the synovial fluids and synovial membranes of RA patients. The major cell types producing ORM2 were synovial macrophages and fibroblast-like synoviocytes (FLSs) from RA patients. Recombinant ORM2 robustly increased IL-6, TNF-α, CXCL8 (IL-8), and CCL2 production by RA macrophages and FLSs via the NF-κB and p38 MAPK pathways. Interestingly, glycophorin C, a membrane protein for determining erythrocyte shape, was the receptor for ORM2. Intra-articular injection of ORM2 increased the severity of arthritis in mice and accelerated the infiltration of macrophages into the affected joints. Moreover, circulating ORM2 levels correlated with RA activity and radiographic progression. In conclusion, the acute phase protein ORM2 can directly increase the production of proinflammatory mediators and promote chronic arthritis in mice, suggesting that ORM2 could be a new therapeutic target for RA.

TDO2-overexpressed Dendritic Cells Possess Tolerogenicity and Ameliorate Collagen-induced Arthritis by Modulating the Th17/Regulatory T Cell Balance.

Tolerogenic dendritic cells are promising for restoring immune homeostasis and may be an alternative therapy for autoimmune diseases such as rheumatoid arthritis. The kynurenine pathway is a vital mechanism that induces tolerance in dendritic cells (DCs). Tryptophan 2,3-dioxygenase (TDO2) is an important rate-limiting enzyme in the kynurenine pathway and participates in immune regulation. However, the role of TDO2 in shaping the tolerogenic phenotypes of DCs remains unclear. In this study, we investigated the effects and mechanisms of TDO2-overexpressed DCs in regulating the T cell balance both invivo and invitro. TDO2-overexpressed DC2.4 and TDO2-/- mouse bone marrow-derived DCs (BMDCs) were generated to verify the role of TDO2 in DC maturation and functionality. TDO2 overexpression in BMDCs via PGE2 treatment exhibited an immature phenotype and tolerogenic state, whereas TDO2-/- BMDCs exhibited a mature phenotype and a proinflammatory state. Furthermore, transplant of TDO2-overexpressed BMDCs alleviated collagen-induced arthritis severity in mice, which was correlated with a reduction in Th17 populations and an increase in regulatory T cells. Collectively, these results indicate that TDO2 plays an important role in the tolerogenic phenotype and may be a promising target for the generation tolerogenic DCs for rheumatoid arthritis treatment.

Alteration of Gut Microbiota in Individuals at High-Risk for Rheumatoid Arthritis Associated With Disturbed Metabolome and the Initiation of Arthritis Through the Triggering of Mucosal Immunity Imbalance.

In this study, we aimed to decipher the gut microbiome (GM) and serum metabolic characteristic of individuals at high risk for rheumatoid arthritis (RA) and to investigate the causative effect of GM on the mucosal immune system and its involvement in the pathogenesis of arthritis. Fecal samples were collected from 38 healthy individuals and 53 high-risk RA individuals with anti-citrullinated protein antibody (ACPA) positivity (Pre-RA), 12 of 53 Pre-RA individuals developed RA within 5 years of follow-up. The differences in intestinal microbial composition between the healthy controls and Pre-RA individuals or among Pre-RA subgroups were identified by 16S ribosomal RNA sequencing. The serum metabolite profile and its correlation with GM were also explored. Moreover, antibiotic-pretreated mice that received GM from the healthy control or Pre-RA groups were then evaluated for intestinal permeability, inflammatory cytokines, and immune cell populations. Collagen-induced arthritis (CIA) was also applied to test the effect of fecal microbiota transplantation (FMT) from Pre-RA individuals on arthritis severity in mice. Stool microbial diversity was lower in Pre-RA individuals than in healthy controls. The bacterial community structure and function significantly differed between healthy controls and Pre-RA individuals. Although there were differences to some extent in the bacterial abundance among the Pre-RA subgroups, no robust functional differences were observed. The metabolites in the serum of the Pre-RA group were dramatically different from those in the healthy controls group, with KEGG pathway enrichment of amino acid and lipid metabolism. Moreover, intestinal bacteria from the Pre-RA group increased intestinal permeability in FMT mice and zonula occludens-1 expression in the small intestine and Caco-2 cells. Moreover, Th17 cells in the mesenteric lymph nodes and Peyer's patches were also increased in mice receiving Pre-RA feces compared to healthy controls. The changes in intestinal permeability and Th17-cell activation prior to arthritis induction enhanced CIA severity in PreRA-FMT mice compared with HC-FMT mice. Gut microbial dysbiosis and metabolome alterations already occur in individuals at high risk for RA. FMT from preclinical individuals triggers intestinal barrier dysfunction and changes mucosal immunity, further contributing to the development of arthritis.

Latent gammaherpesvirus exacerbates arthritis through modification of age-associated B cells.

Epstein-Barr virus (EBV) infection is associated with rheumatoid arthritis (RA) in adults, though the nature of the relationship remains unknown. Herein, we have examined the contribution of viral infection to the severity of arthritis in mice. We have provided the first evidence that latent gammaherpesvirus infection enhances clinical arthritis, modeling EBV's role in RA. Mice latently infected with a murine analog of EBV, gammaherpesvirus 68 (γHV68), develop more severe collagen-induced arthritis and a Th1-skewed immune profile reminiscent of human disease. We demonstrate that disease enhancement requires viral latency and is not due to active virus stimulation of the immune response. Age-associated B cells (ABCs) are associated with several human autoimmune diseases, including arthritis, though their contribution to disease is not well understood. Using ABC knockout mice, we have provided the first evidence that ABCs are mechanistically required for viral enhancement of disease, thereby establishing that ABCs are impacted by latent gammaherpesvirus infection and provoke arthritis.

IFNβ is a critical regulator of Lyme arthritis severity in mice and is regulated by the tumor suppressor gene Cdkn2a on murine Chr4

Abstract Lyme disease is caused by infection with the tick-borne spirochete Borrelia burgdorferi, and results in a spectrum of clinical disorders. Infected C3H mice develop severe Lyme arthritis while B6 mice display mild disease, allowing analysis of host genetic contribution to disease. We used forward genetics to identify a region on Chr4, Bbaa1, that includes the Type I IFN cluster and regulates arthritis severity. The development of congenic mice, B6.C3-Bbaa1, isolating the C3H allele of Bbaa1 onto the background of B6 mice demonstrated that Bbaa1 regulates arthritis severity by modulating Type I IFN expression. IFNβ was identified as the key effector for Lyme arthritis severity, however, paradoxically there are no polymorphisms between C3H and B6 in this gene. To identify regulators of IFNβ in the Bbaa1 locus, candidate genes were subjected to siRNA silencing. A single gene, Cdkn2a, was identified as the modulator of IFNβ expression, and its encoded protein ARF was found to regulate the magnitude of IFNβ expression. Reconstitution of Arf−/− mice with hematopoietic cells expressing the C3H allele of Arf developed severe arthritis, whereas mice reconstituted with cells expressing the B6 Arf allele developed mild arthritis. ARF was found to regulate B. burgdorferi induced IFNβ through p53 and BCL6 in myeloid cells. Indeed, targeted blocking of BCL6 enhanced IFNβ activation in the joint tissue of B6 mice and resulted in increased severity of Lyme arthritis. Results were similar in Rag-1-deficient mice, indicating dependence on myeloid cells expression of BCL6. Thus, we have identified novel pathways modulating IFNβ expression in Lyme arthritis development which could lead to therapeutic discovery.

Potential role of myeloid-derived suppressor cells in transition from reaction to repair phase of bone healing process.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with immunosuppressive functions; these cells play a key role in infection, immunization, chronic inflammation, and cancer. Recent studies have reported that immunosuppression plays an important role in the healing process of tissues and that Treg play an important role in fracture healing. MDSCs suppress active T cell proliferation and reduce the severity of arthritis in mice and humans. Together, these findings suggest that MDSCs play a role in bone biotransformation. In the present study, we examined the role of MDSCs in the bone healing process by creating a bone injury at the tibial epiphysis in mice. MDSCs were identified by CD11b and GR1 immunohistochemistry and their role in new bone formation was observed by detection of Runx2 and osteocalcin expression. Significant numbers of MDSCs were observed in transitional areas from the reactionary to repair stages. Interestingly, MDSCs exhibited Runx2 and osteocalcin expression in the transitional area but not in the reactionary area. And at the same area, cllagene-1 and ALP expression level increased in osteoblast progenitor cells. These data is suggesting that MDSCs emerge to suppress inflammation and support new bone formation. Here, we report, for the first time (to our knowledge), the role of MDSCs in the initiation of bone formation. MDSC appeared at the transition from inflammation to bone making and regulates bone healing by suppressing inflammation.

Increased SUMO-activating enzyme SAE1/UBA2 promotes glycolysis and pathogenic behavior of rheumatoid fibroblast-like synoviocytes.

Fibroblast-like synoviocytes (FLSs) are critical to joint inflammation and destruction in rheumatoid arthritis (RA). Increased glycolysis in RA FLSs contributes to persistent joint damage. SUMOylation, a posttranslational modification of proteins, plays an important role in initiation and development of many diseases. However, the role of small ubiquitin-like modifier–activating (SUMO-activating) enzyme 1 (SAE1)/ubiquitin like modifier activating enzyme 2 (UBA2) in regulating the pathogenic FLS behaviors is unknown. Here, we found an increased expression of SAE1 and UBA2 in FLSs and synovial tissues from patients with RA. SAE1 or UBA2 knockdown by siRNA and treatment with GA, an inhibitor of SAE1/UBA2-mediated SUMOylation, resulted in reduced glycolysis, aggressive phenotype, and inflammation. SAE1/UBA2-mediated SUMOylation of pyruvate kinase M2 (PKM2) promoted its phosphorylation and nuclear translocation and decreased PK activity. Moreover, inhibition of PKM2 phosphorylation increased PK activity and suppressed glycolysis, aggressive phenotype, and inflammation. We further demonstrated that STAT5A mediated SUMOylated PKM2-induced glycolysis and biological behaviors. Interestingly, GA treatment attenuated the severity of arthritis in mice with collagen-induced arthritis and human TNF-α transgenic mice. These findings suggest that an increase in synovial SAE1/UBA2 may contribute to synovial glycolysis and joint inflammation in RA and that targeting SAE1/UBA2 may have therapeutic potential in patients with RA.

Genetic mechanisms contributing to IFNβ regulation of Lyme Arthritis: involvement of the Borrelia burgdorferi arthritis associated locus 1 (Bbaa1)

Abstract Lyme disease shows a spectrum of clinical disorders in patients that are infected with the tick borne spirochete Borrelia burgdorferi. Studies have shown that C3H mice develop severe arthritis while C57BL/6 (B6) mice display mild arthritis when infected with the same isolate of B. burgdorferi. In order to study the role of the host genetics in regulating the severity of Lyme arthritis development, we used forward genetics and identified a genetic region, Bbaa1, that includes the Type I IFN cluster on Chr4. We further developed congenic mice isolating the C3H allele of Bbaa1 onto the background of B6 mice and determined that Bbaa1 regulates arthritis severity by upregulating Type I IFN. Antibody blocking revealed IFNβ, but not IFNα, as the key effector that drives severe Lyme arthritis. Because there are no SNPs between the C3H and B6 IFNβ genes, we hypothesized that other genetic factors in the Bbaa1 locus regulate the differential expression of IFNβ and Lyme arthritis severity. RNA silencing was used to identify candidate genes within Bbaa1 that regulate IFNβ expression in myeloid cells. Cdkn2a was identified as a strong regulator of IFNβ expression. Although Cdkn2a encodes two proteins, p16 and ARF, only the silencing of Arf resulted in the suppression of IFNβ expression. Additionally, the overexpression of ARF led to enhancement of IFNβ. We propose that ARF regulates IFNβ by two pathways involving the proteins p53 and BCL6. Indeed, blocking of BCL6 by a small molecule inhibitor induced IFNβ activation in the joint tissue of B6 mice and enhanced the severity of Lyme arthritis. Future work will focus on the impact of the two pathways on IFNβ expression and arthritis severity in mice.

Synergetic effect of an autoantigen CII and a mycoplasma superantigen on the onset and development of autoimmune arthritis in mice

Abstract An infection can amplify an autoimmune disease by either exacerbating an ongoing disorder, including a relapse, or by leading to chronic progressive disease. We have been studying a mouse autoimmune arthritis induced by M. arthriditis. M. arthritidis produces a potent superantigen (SAg) MAM, which is shown to be partially responsible for the chronic autoimmune arthritis. Although the MAM clearly plays a role in disease induced by live M. arthritidis, MAM alone does not induce overt disease. It does alter the immune system rendering mice more susceptible to disease that caused by other autoimmune agents such as type II collagen (CII). A key question however is how do CII and MAM act together in the host to initiate inflammation and then an autoimmune arthritis? MAM was injected into mice convalescing from CIA or mice suboptimally immunized with CII. In contrast to controls, MAM caused an exacerbation of arthritis in mice that were recovering from CIA. MAM also triggered arthritis onset in mice that had been immunized with CII up to 71 days previously. Injection of MAM during the onset phase of CIA also triggered and enhanced the severity of arthritis in mice given low doses of CII. We also tested the CII-specific immune responses in mice injected with CII alone and also to the mixtures of the CII and MAM. The T cell profile analysis demonstrated that MAM significantly promoted IL-17 and IFNg following exposure to CII in vivo. Of particular interest was that MAM-enhanced IL-17 can stimulate synoviocytes to release a spectrum of chemokines, CXCL1, CXCL2, CXCL12, CCL2 and CCL5, and attracted inflammatory leukocytes. Thus, the results suggest MAM can serve as an enhancer and a trigger by activating inflammatory responses to other bioactive molecules, i.e. CII.

Kaempferol inhibits the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes by blocking activation of the MAPK pathway

In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLSs) play an essential role in cartilage destruction. Aggressive migration and invasion by FLSs significantly affect RA pathology. Kaempferol has been shown to inhibit cancer cell migration and invasion. However, the effects of kaempferol on RA FLSs have not been investigated. Our study aimed to determine the effects of kaempferol on RA both in vitro and in vivo. In vitro, cell migration and invasion were measured using scratch assays and the Boyden chamber method, respectively. The cytoskeletal reorganization of RA FLSs was evaluated by immunofluorescence staining. Matrix metalloproteinase (MMP) levels were measured by real-time PCR, and protein expression levels were measured by western blotting. In vivo, the effects of kaempferol were evaluated in mice with CIA. The results showed that kaempferol reduced migration, invasion and MMP expression in RA FLSs. In addition, we demonstrated that kaempferol inhibited reorganization of the actin cytoskeleton during cell migration. Moreover, kaempferol dramatically suppressed tumor necrosis factor (TNF)-α-induced MAPK activation without affecting the expression of TNF-α receptors. We also demonstrated that kaempferol attenuated the severity of arthritis in mice with CIA. Taken together, these results suggested that kaempferol inhibits the migration and invasion of FLSs in RA by blocking MAPK pathway activation without affecting the expression of TNF-α receptors.

Experimental colitis delays and reduces the severity of collagen-induced arthritis in mice.

Amongst extraintestinal manifestations (EIM) occurring in IBD patients, rheumatologic manifestations are the most frequent. Understanding the relationships between arthritis and colitis is a prerequisite to improving the management of these patients. Microbiota of patients with IBD or rheumatologic diseases, like spondyloarthritis (SpA) is modified compared to healthy individual. Thus, we have evaluated the impact of colitis in the development of arthritis in mice and we have analyzed microbiota changes. Collagen-induced arthritis (CIA) was induced at day 0 in DBA1 mice exposed or not to Dextran Sodium Sulfate (DSS) to induce colitis between day 14 and day 21. Animals were monitored regularly for arthritis and colitis severity (clinical score, hindpaw edema). Fecal microbiota was studied by 16S rRNA deep sequencing at critical time points (D14, D14, D21 & D41). At day 41, histological scoring of the intestines and ankles were performed at the end of experiment. Induction of colitis slightly delayed arthritis onset (2 ± 1 days of delay) and reduced its severity (5.75 ± 1.62 in arthritis only group vs 4.00 ± 1.48 in arthritis + colitis group (p = 0.02 at day 28) macroscopically and histologically. In contrast, colitis severity was not influenced by arthritis development. Induction of colitis promoted a modification of microbiota composition and a decrease of α-diversity. Fecal microbiota composition was different between “colitis” and “arthritis+colitis” groups during colitis development. Interestingly a milder decrease of bacterial diversity in the “arthritis+colitis” group was observed. Concomitant experimental colitis protects mice against collagen-induced arthritis and this is associated with changes in gut microbiome composition.

Functional characterization of the collagen-binding protein DIP2093 and its influence on host-pathogen interaction and arthritogenic potential of Corynebacterium diphtheriae.

Corynebacterium diphtheriae is typically recognized as the a etiological agent of diphtheria, a toxaemic infection of the respiratory tract; however, both non-toxigenic and toxigenic strains are increasingly isolated from cases of invasive infections. The molecular mechanisms responsible for bacterial colonization and dissemination to host tissues remain only partially understood. In this report, we investigated the role of DIP2093, described as a putative adhesin of the serine-aspartate repeat (Sdr) protein family in host-pathogen interactions of C. diphtheriae wild-type strain NCTC13129. Compared to the parental strain, a DIP2093 mutant RN generated in this study was attenuated in its ability to bind to type I collagen, to adhere to and invade epithelial cells, as well as to survive within macrophages. Furthermore, DIP2093 mutant strain RN had a less detrimental impact on the viability of Caenorhabditis elegans as well as in the clinical severity of arthritis in mice. In conclusion, DIP2093 functions as a microbial surface component recognizing adhesive matrix molecules, and may be included among the factors that contribute to the pathogenicity of C. diphtheriae strains, independently of toxin production.

Neutralization of MMP-2 and TNFR1 Regulates the Severity of S. aureus-Induced Septic Arthritis by Differential Alteration of Local and Systemic Proinflammatory Cytokines in Mice.

Despite advancement in the field of antibiotics septic arthritis remains a serious concern till date. Staphylococcus aureus is the most common bacterium that causes septic arthritis. Severity of this disease is directly correlated with chronic inflammation induced by proinflammatory cytokines like TNF-α, interleukin (IL)-1β, IL-6, and induction of matrix metalloproteinases (MMPs) including MMP-2. The objective of our study was to evaluate the role of MMP-2 and tumor necrosis factor receptor 1 (TNFR1) in the pathogenesis of S. aureus infection-induced septic arthritis. Mice were infected with live S. aureus (5×106 cells/ml) followed by administration of MMP-2 inhibitor and TNFR1 antibody. Arthritis index showed highest reduction in severity of arthritis in mice treated with both MMP-2 inhibitor and TNFR1 antibody after infection. Combined neutralization of MMP-2 and TNFR1 led to marked diminution in bacterial count in the combined group. Lowest levels of pro inflammatory cytokines like TNF-α, IL-1β, IL-6, and IFN-γ were observed in both serum and synovial tissues indicating maximum protection in S. aureus arthritis during combination treatment. Increment in the level of IL-10 in the combination group could be positively correlated with the recovery of arthritis. Similarly, expressions of COX-2 and iNOS, markers of acute inflammation were also significantly reduced in the combination group due to resolution of inflammation. Levels of O2.- and NO also showed a significant fall in case of the group treated with MMP-2 inhibitor and TNFR1 antibody both. Neutralization of both MMP-2 and TNFR1 caused rapid decline in recruitment of neutrophil and macrophages in the synovial tissues as evident from reduced MPO and MCP-1 levels, respectively, compared to other groups. Overall, it can be suggested that administration of MMP-2 inhibitor and TNFR1 antibody in combination is protective against the severity of inflammation and cartilage destruction associated with S. aureus infection-induced septic arthritis by altering the levels of cytokines.

Modulation of autoimmune arthritis severity in mice by apolipoprotein E (ApoE) and cholesterol.

Apolipoprotein E (ApoE) deficiency promoted an exacerbation of autoimmune arthritis in mice by inducing proinflammatory immune responses. In this study we analysed the contribution of hypercholesterolaemia and/or the absence of ApoE anti-inflammatory properties, unrelated to its function in the control of cholesterol metabolism, towards the acceleration of arthritis in these mutant animals. The induction and severity of collagen type II-induced arthritis (CIA) were compared for B10.RIII wild-type (WT), B10.RIII.ApoE+/- , B10.RIII.ApoE-/- and B10.RIII.low-density lipoprotein receptor (LDLR-/- ) mice with different concentrations of circulating ApoE and cholesterol. A 50-70% reduction in serum levels of ApoE was observed in heterozygous B10.RIII.ApoE+/- mice in comparison to B10.RIII.WT, although both strains of mice exhibited similar circulating lipid profiles. This ApoE reduction was associated with an increased CIA severity that remained lower than in homozygous B10.RIII.ApoE-/- mice. An important rise in circulating ApoE concentration was observed in hypercholesterolaemic B10.RIII.LDLR-/- mice fed with a normal chow diet, and both parameters increased further with an atherogenic hypercholesterolaemic diet. However, the severity of CIA in B10.RIII.LDLR-/- mice was similar to that of B10.RIII.WT controls. In conclusion, by comparing the evolution of CIA between several strains of mutant mice with different levels of serum ApoE and cholesterol, our results demonstrate that both hypercholesterolaemia and ApoE regulate the intensity of in-vivo systemic autoimmune responses.

Efficient and Non-Toxic Biological Response Carrier Delivering TNF-α shRNA for Gene Silencing in a Murine Model of Rheumatoid Arthritis.

Small interfering RNA (siRNA) is an effective and specific method for silencing genes. However, an efficient and non-toxic carrier is needed to deliver the siRNA into the target cells. Tumor necrosis factor α (TNF-α) plays a central role in the occurrence and progression of rheumatoid arthritis (RA). In this study, we pre-synthetized a degradable cationic polymer (PDAPEI) from 2,6-pyridinedicarboxaldehyde and low-molecular-weight polyethyleneimine (PEI, Mw = 1.8 kDa) as a gene vector for the delivery of TNF-α shRNA. The PDAPEI/pDNA complex showed a suitable particle size and stable zeta potential for transfection. In vitro study of the PDAPEI/pDNA complex revealed a lower cytotoxicity and higher transfection efficiency when transfecting TNF-α shRNA to macrophages by significantly down-regulating the expression of TNF-α. Moreover, the complex was extremely efficient in decreasing the severity of arthritis in mice with collagen-induced arthritis. PDAPEI delivered TNF-α shRNA has great potential in the treatment of RA.

Collagen triple helix repeat containing 1 is a new promigratory marker of arthritic pannus.

BackgroundThe formation of destructive hypercellular pannus is critical to joint damage in rheumatoid arthritis (RA). The collagen triple helix repeat containing 1 (CTHRC1) protein expressed by activated stromal cells of diverse origin has previously been implicated in tissue remodeling and carcinogenesis. We recently discovered that the synovial Cthrc1 mRNA directly correlates with arthritis severity in mice. This study characterizes the role of CTHRC1 in arthritic pannus formation.MethodsSynovial joints of mice with collagen antibody-induced arthritis (CAIA) and human RA-fibroblast-like synoviocytes (FLS) were immunostained for CTHRC1, FLS and macrophage-specific markers. CTHRC1 levels in plasma from patients with RA were measured using sandwich ELISA. The migratory response of fibroblasts was studied with a transwell migration assay and time-lapse microscopy. Velocity and directness of cell migration was analyzed by recording the trajectories of cells treated with rhCTHRC1.ResultsImmunohistochemical analysis of normal and inflamed synovium revealed highly inducible expression of CTHRC1 in arthritis (10.9-fold). At the tissue level, CTHRC1-expressing cells occupied the same niche as large fibroblast-like cells positive for α-smooth muscle actin (α-SMA) and cadherin 11 (CDH11). CTHRC1 was produced by activated FLS predominantly located at the synovial intimal lining and at the bone-pannus interface. Cultured RA-FLS expressed CDH11, α-SMA, and CTHRC1. Upon treatment with exogenous rhCTHRC1, embryonic fibroblasts and RA-FLS significantly increased migration velocity, directness, and cell length along the front-tail axis (1.4-fold, p < 0.01).ConclusionCTHRC1 was established as a novel marker of activated synoviocytes in murine experimental arthritis and RA. The pro-migratory effect of CTHRC1 on synoviocytes is considered one of the mechanisms promoting hypercellularity of the arthritic pannus.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1067-1) contains supplementary material, which is available to authorized users.

Abnormal PTPN11 enhancer methylation promotes rheumatoid arthritis fibroblast-like synoviocyte aggressiveness and joint inflammation.

The PTPN11 gene, encoding the tyrosine phosphatase SHP-2, is overexpressed in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) compared with osteoarthritis (OA) FLS and promotes RA FLS invasiveness. Here, we explored the molecular basis for PTPN11 overexpression in RA FLS and the role of SHP-2 in RA pathogenesis. Using computational methods, we identified a putative enhancer in PTPN11 intron 1, which contained a glucocorticoid receptor- binding (GR-binding) motif. This region displayed enhancer function in RA FLS and contained 2 hypermethylation sites in RA compared with OA FLS. RA FLS stimulation with the glucocorticoid dexamethasone induced GR binding to the enhancer and PTPN11 expression. Glucocorticoid responsiveness of PTPN11 was significantly higher in RA FLS than OA FLS and required the differentially methylated CpGs for full enhancer function. SHP-2 expression was enriched in the RA synovial lining, and heterozygous Ptpn11 deletion in radioresistant or innate immune cells attenuated K/BxN serum transfer arthritis in mice. Treatment with SHP-2 inhibitor 11a-1 reduced RA FLS migration and responsiveness to TNF and IL-1β stimulation and reduced arthritis severity in mice. Our findings demonstrate how abnormal epigenetic regulation of a pathogenic gene determines FLS behavior and demonstrate that targeting SHP-2 or the SHP-2 pathway could be a therapeutic strategy for RA.

Role of the neutrophil chemorepellent soluble dipeptidyl peptidase IV in decreasing inflammation in a murine model of arthritis.

To determine whether an intraarticular injection of the neutrophil chemorepellent dipeptidyl peptidase IV (DPPIV; CD26) can attenuate inflammation and decrease the severity of arthritis in a murine model. DBA/1 mice were immunized with type II collagen/Freund's complete adjuvant to produce collagen-induced arthritis (CIA). On day 25 postimmunization, recombinant human DPPIV (rhDPPIV) or phosphate buffered saline was injected intraarticularly, and arthritis severity scores were recorded 3 times per week. The hind legs of mice in both groups were fixed, decalcified, paraffin embedded, and sectioned. Pathologic scores for inflammation and neutrophil infiltration were recorded on a scale of 1-8, and the number of neutrophils was determined by morphometric cell counts. In addition, Mac-2-positive macrophages and articular damage were assessed using anti-Mac-2 antibodies and histologic staining, respectively. Injection of rhDPPIV reduced the mean score of arthritis severity in mice with CIA. DPPIV treatment reduced the overall extent of inflammation and articular damage around the arthritic joint and periarticular tissue, and also decreased neutrophil and macrophage infiltration. A localized injection of the neutrophil chemorepellent DPPIV reduces inflammation and the severity of the disease in a murine model of arthritis.

Regulatory effect of nicotine on collagen-induced arthritis and on the induction and function of in vitro-cultured Th17 cells

Objectives. To determine the effect of nicotine stimulation on collagen-induced arthritis (CIA), especially on Th17 cells, and the influence of activated acetylcholine receptor signaling on the induction and function of in vitro-cultured Th17 cells.Methods. Mice were divided into control and experimental (nicotine) group, and PBS or nicotine-PBS was orally administered from Day 21 to Day 28. Phenotypic changes in spleen CD4+ cells were measured by flow cytometry. α7nAChR expression in Th17 cells was detected using flow cytometry, western blotting and real-time PCR. Purified Th17 cells were further stimulated with nicotine. The cytometric bead array (CBA) assay was employed to measure TNF-α levels in mice serum and IL-17A levels in the supernatants of nicotine-treated cell cultures.Results. Compared with their counterparts, mice receiving oral nicotine showed a delayed progress of arthritis and more attenuated signs of histological changes. Moreover, serum TNFα levels were lower in the nicotine-treated group. Spleen IL-17 level of nicotine-treated mice was lower than that of the control group, and the mRNA expression of pro-inflammatory cytokines (IL-17A and IL-6) in splenocytes were also lower than that of the control group. α7nAChR expression was detected on in vitro-cultured IL-17A+ cells. Cells treated with 10 − 6 M nicotine expressed lower IL-17A levels. Similarly, supernatants from nicotine-treated cell cultures also showed lower IL-17A levels.Conclusions. Nicotine stimulation attenuated signs and severity of arthritis in mice. Activation of nicotine acetylcholine receptors on in vitro-cultured Th17 cells decreased their pro-inflammatory function, which may play a potential role in alleviating arthritis in mice.