Resolving the genetic basis of complex diseases like rheumatoid arthritis will require knowledge of the corresponding diseases in experimental animals to enable translational functional studies. Mapping of quantitative trait loci in mouse models of arthritis, such as collagen-induced arthritis (CIA), using F(2) crosses has been successful, but can resolve loci only to large chromosomal regions. Using an inbred-outbred cross design, we identified and fine-mapped CIA loci on a genome-wide scale. Heterogeneous stock mice were first intercrossed with an inbred strain, B10.Q, to introduce an arthritis permitting MHCII haplotype. Homozygous H2(q) mice were then selected to set up an F(3) generation with fixed major histocompatibility complex that was used for arthritis experiments. We identified 26 loci, 18 of which are novel, controlling arthritis traits such as incidence of disease, severity and time of onset and fine-mapped a number of previously mapped loci. © The Author 2011. Published by Oxford University Press. All rights reserved.

OBJECTIVE: Some monoclonal antibodies (mAb) to type II collagen (CII) are arthritogenic upon passive transfer to mice. We undertook this study to investigate whether such mAb are pathogenic in the absence of mediators of inflammation. METHODS: The arthritogenic mAb CIIC1 and M2139, and the nonarthritogenic mAb CIIF4, each reactive with a distinct and well-defined conformational epitope on CII, were compared with control mAb GAD6. Bovine chondrocytes were cultured with one of the mAb, and on days 3, 6, and 9, antibody binding by chondrocytes and newly synthesized extracellular matrix (ECM) was examined by immunofluorescence, morphologic effects were studied by electron microscopy, and synthesis of matrix components was determined by metabolic labeling with (3)H-proline for collagen and (35)S-sulfate for proteoglycans. RESULTS: All 3 mAb to CII bound to the matrix. CIIC1 and M2139 adversely affected the cultures, whereas CIIF4 did not. CIIC1 caused disorganization of CII fibrils in the ECM without affecting chondrocyte morphology, and increased matrix synthesis. M2139 caused thickening and aggregation of CII fibrils in the ECM and abnormal chondrocyte morphology but matrix synthesis was unaffected. CONCLUSION: The unique arthritogenic capacity of particular anti-CII mAb upon passive transfer could be explained by their adverse, albeit differing, effects in primary cultures of chondrocytes. Such effects occur independent of inflammation mediators and are related to the epitope specificity of the mAb. Interference with the structural integrity of CII could precede, and even initiate, the inflammatory expression of disease.

INTRODUCTION: Collagen-induced arthritis (CIA) has traditionally been performed in MHC class II A(q)-expressing mice, whereas most genetically modified mice are on the C57BL/6 background (expressing the b haplotype of the major histocompatibility complex (MHC) class II region). However, C57BL/6 mice develop arthritis after immunisation with chicken-derived collagen type II (CII), but arthritis susceptibility has been variable, and the immune specificity has not been clarified. OBJECTIVE: To establish a CIA model on the C57BL/6 background with a more predictable and defined immune response to CII. RESULTS: Both chicken and rat CII were arthritogenic in C57BL/6 mice provided they were introduced with high doses of Mycobacterium tuberculosis adjuvant. However, contaminating pepsin was strongly immunogenic and was essential for arthritis development. H-2(b)-restricted T cell epitopes on chicken or rat CII could not be identified, but expression of A(q) on the C57BL/6 background induced T cell response to the CII260-270 epitope, and also prolonged the arthritis to be more chronic. CONCLUSIONS: The putative (auto)antigen and its arthritogenic determinants in C57BL/6 mice remains undisclosed, questioning the value of the model for addressing T cell-driven pathological pathways in arthritis. To circumvent this impediment, we recommend MHC class II congenic C57BL/6N.Q mice, expressing A(q), with which T cell determinants have been thoroughly characterised.

T cell recognition of the type II collagen (CII) 260-270 peptide is a bottleneck for the development of collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis. We have earlier made C3H.Q mice expressing CII with glutamic acid instead of aspartic acid at position 266 (the MMC-C3H.Q mouse), similar to the rat and human CII epitope, which increases binding to MHC class II and leads to effective presentation of the peptide in vivo. These mice show T cell tolerance to CII, but also develop severe arthritis. The present investigation shows that non-MHC genes play a decisive role in determining tolerance and arthritis susceptibility. We bred MMC into B10.Q mice, which display similar susceptibility to CIA induced with rat CII as the C3H.Q mice. In contrast to MMC-C3H.Q mice, MMC-B10.Q mice were completely resistant to arthritis. Nontransgenic (B10.Q x C3H.Q)F(1) mice were more susceptible to CIA than either of the parental strains, but introduction of the MMC transgene leads to CIA resistance, showing that the protection is dominantly inherited from B10.Q. In an attempt to break the B10-mediated CIA protection in MMC-transgenic mice, we introduced a transgenic, CII-specific, TCR beta-chain specific for the CII(260-270) glycopeptide, in the highly CIA-susceptible (B10.Q x DBA/1)F(1) mice. The magnification of the autoreactive CII-specific T cell repertoire led to increased CIA susceptibility, but the disease was less severe than in mice lacking the MMC transgene. This finding is important for understanding CIA and perhaps also rheumatoid arthritis, as in both diseases MHC class II-restricted T cell recognition of the glycosylated CII peptide occurs.

Antibodies against type II collagen (CII) are important in the development of collagen-induced arthritis (CIA) and possibly also in rheumatoid arthritis. We have determined the fine specificity and arthritogenicity of the antibody response to CII in chronic relapsing variants of CIA. Immunization with rat CII in B10.Q or B10.Q(BALB/cxB10.Q)F2 mice induces a chronic relapsing CIA. The antibody response to CII was determined by using triple-helical peptides of the major B cell epitopes. Each individual mouse had a unique epitope-specific response and this epitope predominance shifted distinctly during the course of the disease. In the B10.Q mice the antibodies specific for C1 and U1, and in the B10.Q(BALB/cxB10.Q)F2 mice the antibodies specific for C1, U1 and J1, correlated with the development of chronic arthritis. Injection of monoclonal antibodies against these epitopes induced relapses in chronic arthritic mice. The development of chronic relapsing arthritis, initially induced by CII immunization, is associated with an arthritogenic antibody response to certain CII epitopes.

Objective

Pain is one of the most debilitating symptoms reported by rheumatoid arthritis (RA) patients. While the collagen antibody–induced arthritis (CAIA) model is used for studying the effector phase of RA pathologic progression, it has not been evaluated as a model for studies of pain. Thus, this study was undertaken to examine pain-like behavior induced by anticollagen antibodies and to assess the effect of currently prescribed analgesics for RA. In addition, the involvement of spinal glia in antibody-induced pain was explored.

Methods

CAIA was induced in mice by intravenous injection of a collagen antibody cocktail, followed by intraperitoneal injection of lipopolysaccharide. Disease severity was assessed by visual and histologic examination. Pain-like behavior and the antinociceptive effect of diclofenac, buprenorphine, gabapentin, pentoxifylline, and JNK-interacting protein 1 were examined in mechanical stimulation experiments. Spinal astrocyte and microglia reactivity were investigated by real-time polymerase chain reaction and immunohistochemistry.

Results

Following the induction of CAIA, mice developed transient joint inflammation. In contrast, pain-like behavior was observed prior to, and outlasted, the visual signs of arthritis. Whereas gabapentin and buprenorphine attenuated mechanical hypersensitivity during both the inflammatory and postinflammatory phases of arthritis, diclofenac was antinociceptive only during the inflammatory phase. Spinal astrocytes and microglia displayed time-dependent signs of activation, and inhibition of glial activity reversed CAIA-induced mechanical hypersensitivity.

Conclusion

CAIA represents a multifaceted model for studies exploring the mechanisms of pain induced by inflammation in the articular joint. Our findings of a time-dependent prostaglandin and spinal glial contribution to antibody-induced pain highlight the importance of using appropriate disease models to assess joint-related pain.

Rheumatoid arthritis-associated joint pain is frequently observed independent of disease activity, suggesting unidentified pain mechanisms. We demonstrate that antibodies binding to cartilage, specific for collagen type II (CII) or cartilage oligomeric matrix protein (COMP), elicit mechanical hypersensitivity in mice, uncoupled from visual, histological and molecular indications of inflammation. Cartilage antibody-induced pain-like behavior does not depend on complement activation or joint inflammation, but instead on tissue antigen recognition and local immune complex (IC) formation. smFISH and IHC suggest that neuronal Fcgr1 and Fcgr2b mRNA are transported to peripheral ends of primary afferents. CII-ICs directly activate cultured WT but not FcRγ chain-deficient DRG neurons. In line with this observation, CII-IC does not induce mechanical hypersensitivity in FcRγ chain-deficient mice. Furthermore, injection of CII antibodies does not generate pain-like behavior in FcRγ chain-deficient mice or mice lacking activating FcγRs in neurons. In summary, this study defines functional coupling between autoantibodies and pain transmission that may facilitate the development of new disease-relevant pain therapeutics.

OBJECTIVES: To assess the human complement inhibitor C4b-binding protein (C4BP) for treatment of arthritis. METHODS: We have used two mouse models of rheumatoid arthritis (RA) to assess the therapeutic effect of C4BP on different phases of arthritis, the collagen antibody-induced arthritis (CAIA), an acute antibody-induced disease and the collagen-induced arthritis (CIA), which carries the full complexity of arthritis. RESULTS: Purified human C4BP injected intraperitoneally alleviated CAIA significantly in a manner similar to cobra venom factor that depletes complement due to massive activation. Furthermore, C4BP was injected before and after the disease development into CIA mice. In the former case, the disease onset was delayed and in the latter, the severity of the disease was reduced in animals treated with C4BP. However, C4BP did not affect the anti-CII antibody synthesis. C4BP present in mouse sera decreased activity of the classical but not the alternative pathway of the complement system when these were assessed in a fluid phase. However, C4BP was efficiently inhibiting the alternative pathway when present on the activating surface. Taken together, the disease ameliorating effect of C4BP appears to be related to inhibition of both pathways of complement. CONCLUSIONS: Although human C4BP was cleared relatively fast from the circulation and was only moderately affecting complement activity, its effect on the disease severity was substantial, suggesting that minor alterations in complement activity can have significant therapeutic value in RA.

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.

OBJECTIVE: To analyze the fine specificity of IgG autoantibodies in sera from rheumatoid arthritis (RA) patients for type II collagen (CII) epitopes that are arthritogenic in collagen-induced arthritis (CIA), a relevant murine model of RA. METHODS: For enzyme-linked immunosorbent assay (ELISA) analysis of conformation-dependent autoantibody binding, recombinant chimeric collagens that harbor the respective CII epitopes as an insertion within the frame of a constant type X collagen triple helix were constructed. In addition, synthetic peptides mimicking the native collagen structures were applied for the first time in the ELISA assessment of humoral CII autoimmunity. RESULTS: The pathogenicity of IgG responses to certain CII determinants in CIA was demonstrated by arthritis development in BALB/c mice upon the combined transfer of 2 mouse monoclonal antibodies specific for precisely mapped conformational CII epitopes (amino acid residues 359-369 [C1(III)] and 551-564 [J1]), whereas antibodies to another epitope (F4) were not arthritogenic. To test whether human autoimmune responses are similarly directed to these conserved CII determinants, serum IgG was analyzed. The prevalence of sera with increased IgG binding to the C1(III) epitope was significantly higher in RA compared with sera from healthy donors or from patients with other rheumatic conditions, e.g., osteoarthritis (OA), systemic lupus erythematosus (SLE), or relapsing polychondritis (RP), whereas levels of antibodies specific for the nonarthritogenic F4 epitope were associated with OA rather than RA. CONCLUSION: Autoimmunity to CII, although detectable in different rheumatic conditions, differs in fine specificity between distinct disease entities. In RA, in contrast to degenerative joint disease, RP, and SLE, autoantibody responses are directed to an evolutionary conserved CII structure that is also targeted by pathogenic autoimmune responses in murine models of arthritis.

INTRODUCTION: Collagen-induced arthritis (CIA) is a mouse model for rheumatoid arthritis (RA) and is induced after immunization with type II collagen (CII). CIA, like RA, is an autoimmune disease leading to destruction of cartilage and joints, and both the priming and inflammatory phases have been suggested to be dependent on proteases. In particular, the cysteine proteases have been proposed to be detrimental to the arthritic process and even immunomodulatory. A natural inhibitor of cysteine proteases is cystatin C. METHODS: Cystatin C-deficient, sufficient and heterozygous mice were tested for onset, incidence and severity of CIA. The effect of cystatin C-deficiency was further dissected by testing the inflammatory effector phase of CIA; that is, collagen antibody-induced arthritis model and priming phase, that is, T cell response both in vivo and in vitro. In addition, in order to determine the importance of T cells and antigen-presenting cells (APCs), these cell populations were separated and in vitro T cell responses determined in a mixed co-culture system. Finally, flow cytometry was used in order to further characterize cell populations in cystatin C-deficient mice. RESULTS: Here, we show that mice lacking cystatin C, develop arthritis at a higher incidence and an earlier onset than wild-type controls. Interestingly, when the inflammatory phase of CIA was examined independently from immune priming then cystatin C-deficiency did not enhance the arthritis profile. However, in line with the enhanced CIA, there was an increased T cell and B cell response as delayed-type hypersensitivity reaction and anti-CII antibody titers were elevated in the cystatin C-deficient mice after immunization. In addition, the ex vivo naive APCs from cystatin C-deficient mice had a greater capacity to stimulate T cells. Interestingly, dendritic cells had a more activated phenotype in naive cystatin C-deficient mice. CONCLUSIONS: The lack of cystatin C enhances CIA and primarily affects in vivo priming of the immune system. Although the mechanism of this is still unknown, we show evidence for a more activated APC compartment, which would elevate the autoimmune response towards CII, thus resulting in an enhanced development of chronic arthritis.

We have addressed the importance of B cell tolerance to collagen type II, a matrix protein, which is a target in rheumatoid arthritis (RA) and its mouse models. We generated a germline-encoded anti-collagen type II (CII) IgH replacement anti-C1 B cell mouse strain (ACB) to investigate how B cell tolerance to CII, a matrix protein, is subverted and to further understand pathogenesis of RA. Phenotypic analysis revealed that CII-specific B cells were surprisingly neither deleted nor anergized. Instead, they were readily detected in all lymphoid organs. Spontaneously produced autoantibodies could bind directly to cartilage surface without detectable pathology. However, exaggerated arthritis was seen after injection of anti-CII Abs specific for other epitopes. In addition, Abs from CII-specific hybridomas generated from ACB mice induced arthritis. Interestingly, IgH/L chain sequence data in B cell hybridomas revealed a lack of somatic mutations in autoreactive B cells. The ACB model provides the first possibility, to our knowledge, to study B cell tolerance to a matrix protein, and the observations made in the study could not be predicted from previous models. B cell-reactive epitopes on CII are largely shared between human RA and rodent CII-induced arthritis; this study, therefore, has important implications for further understanding of pathological processes in autoimmune diseases like RA.

OBJECTIVE: To develop a new mouse model for arthritis using cartilage oligomeric matrix protein (COMP) and to study the role of major histocompatibility complex (MHC) and Ncf1 genes in COMP-induced arthritis (COMPIA). METHODS: Native (pentameric) and denatured (monomeric) COMP purified from a rat chondrosarcoma was injected into mice with Freund's adjuvant to induce arthritis. C3H.NB, C3H.Q, B10.P, B10.Q, (B10.Q x DBA/1)F1, (BALB/c x B10.Q)F1, Ncf1 mutated, H-2Aq, H-2Ap, and human DR4+-transgenic mice were used. Anti-COMP antibodies and COMP levels in the immune sera were analyzed, and passive transfer of arthritis with purified immune sera was tested. RESULTS: Immunization with rat COMP induced a severe, chronic, relapsing arthritis, with a female preponderance, in the mice. The disease developed in C3H.NB mice, but not in B10.P mice, although they share the same MHC haplotype. Both H-2q and H-2p MHC haplotypes allowed the initiation of COMPIA. Using H-2Aq-transgenic and H-2Ap-transgenic mice, we demonstrated a role of both the Aq and Ep class II molecules in this model. Interestingly, the introduction of a mutation in the Ncf1 gene, which is responsible for the reduced oxidative burst phenotype, into the COMPIA-resistant B10.Q mouse strain rendered them highly susceptible to arthritis. In addition, the transfer of anti-COMP serum was found to induce arthritis in naive mice. Mice transgenic for the rheumatoid arthritis (RA)-associated DR4 molecule were found to be highly susceptible to COMPIA. CONCLUSION: Using rat COMP, we have developed a new and unique mouse model of chronic arthritis that resembles RA. This model will be useful as an appropriate and alternative model for studying the pathogenesis of RA.

Joint cartilage is attacked in both autoimmune inflammatory and osteoarthritic processes. Type IX collagen (CIX) is a protein of importance for cartilage integrity and stability. In this study we have backcrossed a transgenic disruption of the col9a1 gene, which leads to an absence of CIX, into two different inbred mouse strains, DBA/1 and B10.Q. None of the CIX-deficient mice developed observable clinical or microscopic osteoarthritis, but DBA/1 male mice had more pronounced enthesopathic arthritis, the so-called stress-induced arthritis. Both DBA/1 and B10.Q strains are susceptible to the induction of collagen-induced arthritis, and CIX deficiency in both strains led to the development of a more severe arthritis than in the controls. Induction of arthritis with monoclonal antibodies against type II collagen (CII) led to an earlier arthritis in the paws that also involved the knee joints. The antibodies used, which were specific for the J1 and the C1I epitopes of CII, initiate their arthritogenic attack by binding to cartilage. The C1I-specific antibodies bound to cartilage better in CIX-deficient mice than in wild-type animals, demonstrating that the lack of CIX in cartilage leads to an increased accessibility of structures for antibody binding and thus making the joints more vulnerable to inflammatory attack. These findings accentuate the importance of cartilage stability; cartilage disrupted as a result of genetic disorders could be more accessible and vulnerable to an autoimmune attack by pathogenic antibodies.

Toll-like receptor 3 (TLR3), as an important pattern recognition receptor (PRR), dominates the innate and adaptive immunity regulating many acute and chronic inflammatory diseases. Atherosclerosis is proved as an inflammatory disease, and inflammatory events involved in the entire process of initiation and deterioration. However, the contribution of TLR3 to atherosclerosis remains unclear. Herein, we identified the clinical relevance of TLR3 upregulation and disease processes in human atherosclerosis. Besides, activation of TLR3 also directly led to significant expression of atherogenic chemokines and adhesion molecules. Conversely, silencing TLR3 inhibited the uptake of oxLDL by macrophages and significantly reduced foam cell formation. Given the aberrance in TLR3 functions on atherosclerosis progression, we hypothesized that TLR3 could serve as novel target for clinical atherosclerosis therapy. Therefore, we developed the novel ellipticine derivative SMU-CX24, which specifically inhibited TLR3 (IC50 = 18.87 ± 2.21 nmol/L). In vivo, atherosclerotic burden was alleviated in Western diet fed ApoE-/- mice in response to SMU-CX24 treatment, accompanying notable reductions in TLR3 expression and inflammation infiltration within atherosclerotic lesion. Thus, for the first time, we revealed that pharmacological downregulation of TLR3 with specific inhibitor regenerated inflammatory environment to counteract atherosclerosis progression, thereby proposing a new strategy and probe for atherosclerosis therapy.

Toll-like receptor 2 (TLR2) expressed on antigen presenting cells evokes a series of critical cytokines, which favor the development of tumor-specific cytotoxic T lymphocytes (CTLs). Therefore, TLR2 represents an attractive cancer immunotherapeutic target. Here, a synthetic library of 14 000 compounds together with a series of newly developed compounds for NF-κB activation using HEK-Blue hTLR2 cells is initially screened. Following further screening in a variety of cells including HEK-Blue hTLRs reporter cells, murine, and human macrophage cell lines, a potent small molecule agonist 23 (SMU-Z1) is identified, which specifically activates TLR2 through its association with TLR1, with a EC₅₀ of 4.88 ± 0.79 × 10^-9 m. Toxicology studies, proinflammatory cytokines (e.g., TNF-α, IL-1β, IL-6, and nitric oxide) and target-protein based biophysical assays demonstrate the pharmacologically relevant characteristics of SMU-Z1. In addition, SMU-Z1 promotes murine splenocyte proliferation and upregulates the expression of CD8⁺ T cells, NK cells and DCs, which results in a significant antitumor effect in a murine leukemia model. Finally, the induced tumors in three out of seven mice disappear after administration of SMU-Z1. Our studies thus identify a novel and potent TLR1/2 small molecule agonist, which displays promising immune adjuvant properties and antitumor immunity.

PURPOSE: Inflamm-aging is a novel-concept in rheumatoid arthritis (RA) with accelerating aging process. We try to find a correlation between serum albumin/globulin (A/G) ratio and clinical biochemical parameters, incidence of aging-related diseases (ARDs) as well as inflammaging-related molecules.

PATIENTS AND METHODS: Healthy controls (HC) and RA patients were compared with their clinical biochemical parameters including albumin and globulin levels, A/G ratio, and levels of serum lipids. Incidence of ARDs in RA was compared with A/G ratio, having a cut off value of 1.2. Expression levels of leptin and Trf2 genes in PBMCs, and inflammatory factors like IL-1β, IL-6, IL-8 and TNF-ɑ between HC and RA patients were compared, and correlated with the A/G ratio.

RESULTS: Compared to HC, RA patients had decreased levels of albumin, while globulin levels were found to be increased, which led to a significantly lower A/G ratio in RA patients. A/G ratio rather than ESR and CRP had significant correlation with dyslipidemia in RA patients. Patients with A/G <1.2 had a higher risk of ARDs than patients with A/G >1.2. The RR was 2.48 (95% CI: 1.79 to 3.64, p <0.0001). In addition, A/G ratio has positively correlated to leptin and Trf2 expression, while an inverse correlation was observed with the levels of inflamm-aging related cytokines like IL-6, IL-8 and TNF-ɑ.

CONCLUSION: A decreased A/G ratio in RA patients has significantly correlated with dyslipidemia and ARDs, as well as inflammaging- related adipokine and pro-inflammatory cytokines. Thus, A/G ratio could be a reliable marker for evaluating the inflammaging process during clinical management in ARDs.

AMP-activated protein kinase (AMPK) is essential for maintaining energy balance and has a crucial role in various inflammatory pathways. In this study, AMPK levels positively correlated with many inflammatory indexes in rheumatoid arthritis (RA) patients, especially in the affected synovium. In RA sera, a positive correlation between phosphorylated (p-)AMPK-α1 levels and DAS28 (disease activity score 28) activity (r = 0.270, p < 0.0001) was found. Similarly, a positive correlation was observed between AMPK-α1 and tumor necrosis factor α (TNF-α) levels (r = 0.460, p = 0.0002). Differentially expressed genes between osteoarthritis (OA) and RA synovium from NCBI GEO profiles and our RNA sequencing data suggested activation of metabolic pathways specific to RA-fibroblast-like synoviocytes (FLSs). AMPK-α1 was highly expressed in the synovium of RA but not OA patients. An AMPK activator, metformin, inhibited FLS proliferation at higher but not lower concentrations, whereas the inhibitor dorsomorphin promoted the proliferation of RA-FLSs. Interestingly, both metformin and dorsomorphin inhibited the migration of RA-FLSs. After metformin treatment, expression of interleukin 6 (IL-6), TNF-α, and IL-1β were significantly downregulated in RA-FLSs; however, increased expression of p-AMPK-α1, protein kinase A (PKA)-α1, and HAPLN1 (hyaluronan and proteoglycan link protein 1) was observed. Increased levels of HAPLN1 in RA-FLSs by an AMPK activator could potentially be beneficial in protecting the joints. Hence, our results demonstrate the potential of an AMPK activator as a therapeutic for RA.

HAPLN1 maintains aggregation and the binding activity of extracellular matrix (ECM) molecules (such as hyaluronic acid and proteoglycan) to stabilize the macromolecular structure of the ECM. An increase in HAPLN1 expression is observed in a few types of musculoskeletal diseases including rheumatoid arthritis (RA); however, its functions are obscure. This study examined the role of HAPLN1 in determining the viability, proliferation, mobility, and pro-inflammatory phenotype of RA- fibroblast-like synoviocytes (RA-FLSs) by using small interfering RNA (siHAPLN1), over-expression vector (HAPLN1OE), and a recombinant HAPLN1 (rHAPLN1) protein. HAPLN1 was found to promote proliferation but inhibit RA-FLS migration. Metformin, an AMPK activator, was previously found by us to be able to inhibit FLS activation but promote HAPLN1 secretion. In this study, we confirmed the up-regulation of HAPLN1 in RA patients, and found the positive relationship between HAPLN1 expression and the AMPK level. Treatment with either si-HAPLN1 or HAPLN1OE down-regulated the expression of AMPK-ɑ gene, although up-regulation of the level of p-AMPK-ɑ was observed in RA-FLSs. si-HAPLN1 down-regulated the expression of proinflammatory factors like TNF-ɑ, MMPs, and IL-6, while HAPLN1OE up-regulated their levels. qPCR assay indicated that the levels of TGF-β, ACAN, fibronectin, collagen II, and Ki-67 were down-regulated upon si-HAPLN1 treatment, while HAPLN1OE treatment led to up-regulation of ACAN and Ki-67 and down-regulation of cyclin-D1. Proteomics of si-HAPLN1, rHAPLN1, and mRNA-Seq analysis of rHAPLN1 confirmed the functions of HAPLN1 in the activation of inflammation, proliferation, cell adhesion, and strengthening of ECM functions. Our results for the first time demonstrate the function of HAPLN1 in promoting the proliferation and pro-inflammatory phenotype of RA-FLSs, thereby contributing to RA pathogenesis. Future in-depth studies are required for better understanding the role of HAPLN1 in RA.

The previous virtual screening of ten million compounds yielded two novel nonlipopeptide-like chemotypes as TLR2 agonists. Herein, we present the chemical optimization of our initial hit, 1-phenyl-3-(thiophen-2-yl) urea, which resulted in the identification of SMU-C80 (EC₅₀ = 31.02 ± 1.01 nM) as a TLR2-specific agonist with a 370-fold improvement in bioactivity. Mechanistic studies revealed that SMU-C80, through TLR1/2, recruits the adaptor protein MyD88 and triggers the NF-κB pathway to release cytokines such as TNF-α and IL-1β from human, but not murine, cells. To the best of our knowledge, it is the first species-specific TLR1/2 agonist reported until now. Moreover, SMU-C80 increased the percentage of T, B, and NK cells ex vivo and activated the immune cells, which suppressed cancer cell growth in vitro. In summary, we obtained a highly efficient and specific human TLR1/2 agonist that acts through the MyD88 and NF-κB pathway, facilitating cytokine release and the simultaneous activation of immune cells that in turn affects the apoptosis of cancer cells. © 2021 American Chemical Society

Approximately 25% of mature T cells possess two distinct cytoplasmic T cell receptor (TCR) alpha-chains, due to productive gene rearrangements of both alleles. Expression of two different alpha-chains at the cell surface is a potential risk factor for development of autoimmunity. However, it has been difficult to determine the frequency of peripheral T cells with two different alpha-chains at the surface. Our new approach is based on comparing by flow cytometry the percentage of cells that express a given Valpha-chain between wild-type mice and mice that are hemizygous for a disrupted Tcra locus (Tcra+/-) and consequently unable to express two rearranged Tcra genes. We consistently found that approximately 8% of total peripheral T cells express two surface alpha-chains. The importance of dual alpha-T cells in autoimmunity was examined in a mouse model for rheumatoid arthritis, namely collagen-induced arthritis (CIA). No significant difference was observed between Tcra+/- mice and wild-type littermates, considering arthritis incidence, day of disease onset, and maximum arthritic score. We therefore conclude that there is incomplete phenotypic allelic exclusion in TCRalpha, and that the presence of a significant number of potentially multireactive T cells does not increase the susceptibility to develop autoimmune arthritis. © 2001 Academic Press.

Certain monoclonal antibodies (mAbs) to type II collagen (CII) induce arthritis in vivo after passive transfer and have adverse effects on chondrocyte cultures and inhibit self assembly of collagen fibrils in vitro. We have examined whether such mAbs have detrimental effects on pre-existing cartilage. Bovine cartilage explants were cultured over 21 days in the presence of two arthritogenic mAbs to CII (CIIC1 or M2139), a non-arthritogenic mAb to CII (CIIF4) or a control mAb (GAD6). Penetration of cartilage by mAb was determined by immunofluorescence on frozen sections and correlated with changes to the extracellular matrix and chondrocytes by morphometric analysis of sections stained with toluidine blue. The effects of mAbs on matrix components were examined by Fourier transform infrared microspectroscopy (FTIRM). A possible role of Fc-binding was investigated using F(ab)2 from CIIC1. All three mAbs to CII penetrated the cartilage explants and CIIC1 and M2139, but not CIIF4, had adverse effects that included proteoglycan loss correlating with mAb penetration, the later development in cultures of an abnormal superficial cellular layer, and an increased proportion of empty chondrons. FTIRM showed depletion and denaturation of CII at the explant surface in the presence of CIIC1 or M2139, which paralleled proteoglycan loss. The effects of F(ab)2 were greater than those of intact CIIC1. Our results indicate that mAbs to CII can adversely affect preformed cartilage, and that the specific epitope on CII recognised by the mAb determines both arthritogenicity in vivo and adverse effects in vitro. We conclude that antibodies to CII can have pathogenic effects that are independent of inflammatory mediators or Fc-binding.

OBJECTIVE: The type II collagen (CII)-specific monoclonal antibodies (mAb) M2139 and CIIC1 induce arthritis in vivo and degrade bovine cartilage explants in vitro, whereas mAb CIIF4 is nonarthritogenic and prevents arthritis development when given in combination with M2139 and CIIC1. To determine the nature of the protective capacity of CIIF4 antibody, we examined the effects of adding CIIF4 to cartilage explants cultured in vitro with M2139 and CIIC1. METHODS: Bovine cartilage explants were cultured in the presence of M2139 and CIIC1, with or without CIIF4. Histologic changes were examined, and chemical changes to collagens and proteoglycans were assessed by Fourier transform infrared microspectroscopy (FTIRM). Fresh cartilage and cartilage that had been freeze-thawed to kill chondrocytes cultured with or without the addition of GM6001, a broad-spectrum inhibitor of matrix metalloproteinases (MMPs), were compared using FTIRM analysis. RESULTS: M2139 and CIIC1 caused progressive degradation of the cartilage surface and loss of CII, even in the absence of viable chondrocytes. CIIF4 did not cause cartilage damage, and when given with the arthritogenic mAb, it prevented their damage and permitted matrix regeneration, a process that required viable chondrocytes. Inhibition of MMP activity reduced cartilage damage but did not mimic the effects of CIIF4. CONCLUSION: CII-reactive antibodies can cause cartilage damage or can be protective in vivo and in vitro, depending on their epitope specificity. Since CII antibodies of similar specificity also occur in rheumatoid arthritis in humans, more detailed studies should unravel the regulatory mechanisms operating at the effector level of arthritis pathogenesis. © 2010 by the American College of Rheumatology.

Collagen antibody-induced arthritis develops in mice following passive transfer of monoclonal antibodies (mAbs) to type II collagen (CII) and is attributed to effects of proinflammatory immune complexes, but transferred mAbs may react directly and damagingly with CII. To determine whether such mAbs cause cartilage damage in vivo in the absence of inflammation, mice lacking complement factor 5 that do not develop joint inflammation were injected intravenously with two arthritogenic mAbs to CII, M2139 and CIIC1. Paws were collected at day 3, decalcified, paraffin embedded, and 5-mum sections were examined using standard histology and synchrotron Fourier-transform infrared microspectroscopy (FTIRM). None of the mice injected with mAb showed visual or histological evidence of inflammation but there were histological changes in the articular cartilage including loss of proteoglycan and altered chondrocyte morphology. Findings using FTIRM at high lateral resolution revealed loss of collagen and the appearance of a new peak at 1635 cm(-1) at the surface of the cartilage interpreted as cellular activation. Thus, we demonstrate the utility of synchrotron FTIRM for examining chemical changes in diseased cartilage at the microscopic level and establish that arthritogenic mAbs to CII do cause cartilage damage in vivo in the absence of inflammation. © 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).

OBJECTIVE: Murine collagen antibody-induced arthritis (CAIA), like collagen-induced arthritis, has clinical and immunopathologic features that parallel those in human rheumatoid arthritis (RA). This study was undertaken to examine the effects of autoantibodies to type II collagen (CII) on articular cartilage in the paws of mice, under conditions in which other factors that may influence joint pathology could be excluded. METHODS: Mice of 2 different strains, B10.QC5delta and the parental strain B10.Q, were injected intravenously with either saline or arthritogenic monoclonal antibodies (mAb) to CII. B10.QC5delta mice lack complement factor C5 and do not develop CAIA when injected with arthritogenic mAb, whereas B10.Q mice have C5 and develop CAIA when administered the mAb and a subsequent injection of lipopolysaccharide. Three days after injection the paws of the mice were examined by standard histologic methods to assess morphologic appearance and proteoglycan loss, and by synchrotron-enhanced Fourier transform infrared microspectroscopy to assess chemical evidence of structural change. RESULTS: No macroscopic or microscopic signs of inflammation were evident in the mice. However, in contrast to the saline-injected controls, all mAb-injected mice exhibited cartilage damage in all joints, with loss of proteoglycans and collagen, chondrocyte hyperplasia and/or loss, and surface damage in the interphalangeal joints. CONCLUSION: The implication of these findings is that an autoimmune response to CII can disrupt articular cartilage, particularly that of the small joints, and the subsequent integrity of the cartilage depends on a balance between breakdown and repair. This has relevance with regard to RA, in which such autoantibodies occur but the inflammatory response may dominate clinically and mask underlying features of the autoimmune response. © 2013 by the American College of Rheumatology

OBJECTIVE: Rheumatoid arthritis (RA) is considered to be a prototypical autoimmune disorder. Several mechanisms have been proposed for the known pathologic function of B cells in RA, including antigen presentation, cytokine secretion, and humoral immunity. The aim of this study was to address the function of B lymphocytes in experimental arthritis.

METHODS: We mapped the adaptive immune response following collagen-induced arthritis (CIA). We subsequently monitored these responses and disease outcomes in genetically modified mouse strains that lack mature B cell or germinal center (GC) functionality in a B cell-intrinsic manner.

RESULTS: Following primary immunization, the draining lymph nodes broadly reacted against type II collagen (CII) with the formation of GCs and T cell activation. Mice that lacked mature B cell function were fully protected against CIA and had a severely attenuated ability to mount isotype-switched humoral immune responses against CII. Almost identical results were observed in mice that were selectively deficient in GC responses. Importantly, GC-deficient mice were fully susceptible to collagen antibody-induced arthritis.

CONCLUSION: We identified GC formation and anticollagen antibody production as the key pathogenic functions of B cells in CIA. The role of B cells in RA is likely to be more complex. However, targeting the GC reaction could allow for therapeutic interventions that are more refined than general B cell depletion.

OBJECTIVE: Rheumatoid arthritis (RA) is considered to be a prototypical autoimmune disorder. Several mechanisms have been proposed for the known pathologic function of B cells in RA, including antigen presentation, cytokine secretion, and humoral immunity. The aim of this study was to address the function of B lymphocytes in experimental arthritis. METHODS: We mapped the adaptive immune response following collagen-induced arthritis (CIA). We subsequently monitored these responses and disease outcomes in genetically modified mouse strains that lack mature B cell or germinal center (GC) functionality in a B cell-intrinsic manner. RESULTS: Following primary immunization, the draining lymph nodes broadly reacted against type II collagen (CII) with the formation of GCs and T cell activation. Mice that lacked mature B cell function were fully protected against CIA and had a severely attenuated ability to mount isotype-switched humoral immune responses against CII. Almost identical results were observed in mice that were selectively deficient in GC responses. Importantly, GC-deficient mice were fully susceptible to collagen antibody-induced arthritis. CONCLUSION: We identified GC formation and anticollagen antibody production as the key pathogenic functions of B cells in CIA. The role of B cells in RA is likely to be more complex. However, targeting the GC reaction could allow for therapeutic interventions that are more refined than general B cell depletion. © 2017, American College of Rheumatology

BACKGROUND: Anti-malarial drug artesunate (ART), a semi-synthetic derivative of artemisnin, has immunosuppressive effects on several autoimmune diseases, including Systemic lupus erythematosus (SLE), Rheumatoid arthritis (RA), and Colitis. However, molecular mechanisms of ART, especially on follicular helper T cells (Tfh), central players in SLE pathology, are far from clear.

PURPOSE: The object for this work is to investigate the therapeutic effect of ART on lupus-prone MRL/lpr mice and its regulatory function on Tfh cells.

STUDY DESIGN AND METHODS: MRL/lpr mice were used to explore therapeutic effects of ART on lupus-prone MRL/lpr mice and its regulatory functions on Tfh cells. Then, experiments of renal function were accomplished using the biochemical kits. Effects of ART on histopathology of kidneys, inflammatory factors and autoantibodies were examined using H&E staining, ELISA and real-time PCR. Flow cytometry and western blot analysis were used to examine effects of ART on Tfh differentiation and Jak2-Stat3 signaling pathway.

RESULTS: Upon oral administration, ART significantly prolonged the survival of MRL/lpr mice, ameliorated the lupus nephritis symptoms, decreased the levels of anti-dsDNA antibodies deposited in the kidney, and the levels of pathogenic cytokines (IL-6, IFN-γ and IL-21). After ART treatment, T-cell compartment in the spleen of MRL/lpr mice was restored in terms of reduction in the number of Tfh cells and in the maintenance of the ratio of Tfr to follicular regulatory T cells (Tfh). In addition, ART has significantly inhibited the phosphorylation levels of Jak2 and Stat3 in the MRL/lpr mice.

CONCLUSION: ART showed therapeutic effects on lupus-prone MRL/lpr mice by inhibiting the differentiation of Tfh cells as well as altering the activation status of Jak2-Stat3 signaling cascade. Copyright © 2019 Elsevier GmbH

OBJECTIVE: In rheumatoid arthritis, joint inflammation and cartilage destruction are mediated by autoantibodies directed to various self antigens. Type II collagen (CII)-specific antibodies are likely to play a role in this process and have been shown to induce experimental arthritis in susceptible animals. The purpose of this study was to reveal how arthritogenic autoantibodies recognize native CII in its triple-helical conformation. METHODS: Site-directed mutagenesis and crystallographic studies were performed to reveal crucial contact points between the CII antibody and the triple-helical CII peptide. RESULTS: The crystal structure of a pathogenic autoantibody bound to a major triple-helical epitope present on CII was determined, allowing a first and detailed description of the interactions within an arthritogenic complex that is frequently occurring in both mice and humans with autoimmune arthritis. The crystal structure emphasizes the role of arginine residues located in a commonly recognized motif on CII and reveals that germline-encoded elements are involved in the interaction with the epitope. CONCLUSION: The crystal structure of an arthritogenic antibody binding a triple-helical epitope on CII indicates a crucial role of germline-encoded and arginine residues as the target structures.

Based on previous studies, we know that estrogen can protect the joints from arthritis development by increasing IgG glycosylation and inhibiting osteoclast activation. Phytoestrogens, especially genistein and daidzein, are structurally similar to estradiol that can bind to estrogen receptors (ERs). However, how phytoestrogens affect IgG glycosylation and osteoclast activation in vivo are not investigated so far. In this study, we used 20 mg/kg genistein or daidzein to gavage the female DBA1/J mice in collagen induced arthritis (CIA). We assessed arthritis and bone erosion by clinical scores, histopathology, and micro-CT analysis. Inflammatory cells such as neutrophils, B cells, macrophages and T cells in the peripheral blood were analyzed by flow cytometry. Phagocytic function of peritoneal macrophages was assessed by using FITC-labeled Escherichia coli. New monoclonal antibodies against CII were produced, purified and analyzed. Glycosylation levels of polyclonal and monoclonal IgG were detected by lectin-ELISA. Quantitative PCR was used to analyze the genes related to IgG glycosylation (B4galt1, St6gal1) and osteoclasts (TRAP, NFATC1, c-Fos). Expression of NF-κB and Akt signaling pathways as well as downstream transcription factors NFATc1 and c-Fos was studied by Western blot. Our results show that phytoestrogens protect mice from CIA by increasing IgG glycosylation leading to amelioration of inflammation and inhibiting the NF-κB pathway and NFATc1/c-Fos to decrease the activity of osteoclasts. In conclusion, phytoestrogens can protect bone and joints in CIA mice by increasing IgG glycosylation and inhibiting osteoclast activity. © 2020 Elsevier B.V.

In both collagen-induced arthritis (CIA) and rheumatoid arthritis, T cells recognize a galactosylated peptide from type II collagen (CII). In this study, we demonstrate that the CII259-273 peptide, galactosylated at lysine 264, in complex with Aq molecules prevented development of CIA in mice and ameliorated chronic relapsing disease. In contrast, nonglycosylated CII259-273/Aq complexes had no such effect. CIA dependent on other MHC class II molecules (Ar/Er) was also down-regulated, indicating a bystander vaccination effect. T cells could transfer the amelioration of CIA, showing that the protection is an active process. Thus, a complex between MHC class II molecules and a posttranslationally modified peptide offers a new possibility for treatment of chronically active autoimmune inflammation such as rheumatoid arthritis. © 2006 by The American Association of Immunologists, Inc.

OBJECTIVE: 5-alpha reductase inhibitors (5ARIs) decrease the androgen levels in vivo and are currently used for the treatment of benign prostatic hyperplasia (BPH) in men. However, these inhibitors can also increase the risk of gynecomastia, breast tenderness, and breast cancer. Hence, we did a systematic review and meta-analysis to evaluate the rate of breast-related diseases in men treated with 5ARIs.

MATERIALS AND METHODS: PubMed, Embase, Cochrane, and CNKI databases were searched for randomized controlled trials using 5ARIs in patients with BPH. Data were analyzed by using Cochrane Collaboration review manager program and Stata 12.0 software.

RESULTS: In total, 14 studies were included in the meta-analysis. Gynecomastia was significantly more common with 5ARIs treatment when compared with placebo (3.30% vs. 1.84%; P < .00001) or alpha blockers (ABs) monotherapy (2.33% vs. 1.00%; P = .0009). Both dutasteride (2.03% vs. 0.90%; P < .00001) and finasteride (4.08% vs. 2.43%; P < .00001) are associated with significantly higher risk of gynecomastia than placebo. Risk for breast tenderness was elevated in 5ARIs users (0.83% vs. 0.25%; P = .01) or in users having combination therapy with ABs (2.48% vs. 0.58%; P < .0001). Finasteride is associated with significantly higher risk of breast tenderness than placebo (0.80% vs. 0.25%; P = .02).

CONCLUSION: In male patients with BPH, 5ARIs have significantly increased the risk of gynecomastia and breast tenderness but may be not to the breast cancer. In addition, combination therapy is significantly associated with higher risk of breast tenderness compared to single ABs monotherapy. © 2019 Elsevier Inc. All rights reserved.

We identified abnormally methylated, differentially expressed genes (DEGs) and pathogenic mechanisms in different immune cells of RA and SLE by comprehensive bioinformatics analysis. Six microarray data sets of each immune cell (CD19⁺ B cells, CD4⁺ T cells and CD14⁺ monocytes) were integrated to screen DEGs and differentially methylated genes by using R package "limma." Gene ontology annotations and KEGG analysis of aberrant methylome of DEGs were done using DAVID online database. Protein-protein interaction (PPI) network was generated to detect the hub genes and their methylation levels were compared using DiseaseMeth 2.0 database. Aberrantly methylated DEGs in CD19⁺ B cells (173 and 180), CD4⁺ T cells (184 and 417) and CD14⁺ monocytes (193 and 392) of RA and SLE patients were identified. We detected 30 hub genes in different immune cells of RA and SLE and confirmed their expression using FACS sorted immune cells by qPCR. Among them, 12 genes (BPTF, PHC2, JUN, KRAS, PTEN, FGFR2, ALB, SERB-1, SKP2, TUBA1A, IMP3, and SMAD4) of RA and 12 genes (OAS1, RSAD2, OASL, IFIT3, OAS2, IFIH1, CENPE, TOP2A, PBK, KIF11, IFIT1, and ISG15) of SLE are proposed as potential biomarker genes based on receiver operating curve analysis. Our study suggests that MAPK signaling pathway could potentially differentiate the mechanisms affecting T- and B- cells in RA, whereas PI3K pathway may be used for exploring common disease pathways between RA and SLE. Compared to individual data analyses, more dependable and precise filtering of results can be achieved by integrating several relevant data sets.

Rheumatoid arthritis is a systemic, polygenic, and multifactorial syndrome characterized by erosive polyarthritis, damage to joint architecture, and presence of autoantibodies against several self-structures in the serum and synovial fluid. These autoantibodies (anticitrullinated protein/peptide antibodies (ACPAs), rheumatoid factors (RF), anticollagen type II antibodies, antiglucose-6 phosphate isomerase antibodies, anticarbamylated protein antibodies, and antiacetylated protein antibodies) have different characteristics, diagnostic/prognostic value, and pathological significance in RA patients. Some of these antibodies are present in the patients' serum several years before the onset of clinical disease. Various genetic and environmental factors are associated with autoantibody production against different autoantigenic targets. Both the activating and inhibitory FcγRs and the activation of different complement cascades contribute to the downstream effector functions in the antibody-mediated disease pathology. Interplay between several molecules (cytokines, chemokines, proteases, and inflammatory mediators) culminates in causing damage to the articular cartilage and bones. In addition, autoantibodies are proven to be useful disease markers for RA, and different diagnostic tools are being developed for early diagnosis of the clinical disease. Recently, a direct link was proposed between the presence of autoantibodies and bone erosion as well as in the induction of pain. In this review, the diagnostic value of autoantibodies, their synthesis and function as a mediator of joint inflammation, and the significance of IgG-Fc glycosylation are discussed.

Rheumatoid arthritis is a chronic autoimmune syndrome associated with several genetic, epigenetic, and environmental factors affecting the articular joints contributing to cartilage and bone damage. Although etiology of this disease is not clear, several immune pathways, involving immune (T cells, B cells, dendritic cells, macrophages, and neutrophils) and nonimmune (fibroblasts and chondrocytes) cells, participate in the secretion of many proinflammatory cytokines, chemokines, proteases (MMPs, ADAMTS), and other matrix lysing enzymes that could disturb the immune balance leading to cartilage and bone damage. The presence of autoantibodies preceding the clinical onset of arthritis and the induction of bone erosion early in the disease course clearly suggest that initiation events damaging the cartilage and bone start very early during the autoimmune phase of the arthritis development. During this process, several signaling molecules (RANKL-RANK, NF-κB, MAPK, NFATc1, and Src kinase) are activated in the osteoclasts, cells responsible for bone resorption. Hence, comprehensive knowledge on pathogenesis is a prerequisite for prevention and development of targeted clinical treatment for RA patients that can restore the immune balance improving clinical therapy.

Complex autoimmune diseases are sexually dimorphic. An interplay between predisposing genetics and sex-related factors probably controls the sex discrepancy in the immune response, but the underlying mechanisms are unclear. Here we positionally identify a polymorphic estrogen receptor binding site that regulates Cd2 expression, leading to female-specific differences in T cell-dependent mouse models of autoimmunity. Female mice with reduced Cd2 expression have impaired autoreactive T cell responses. T cells lacking Cd2 costimulation upregulate inhibitory Lag-3. These findings help explain sexual dimorphism in human autoimmunity, as we find that CD2 polymorphisms are associated with rheumatoid arthritis and 17-β-estradiol-regulation of CD2 is conserved in human T cells. Hormonal regulation of CD2 might have implications for CD2-targeted therapy, as anti-Cd2 treatment more potently affects T cells in female mice. These results demonstrate the relevance of sex-genotype interactions, providing strong evidence for CD2 as a sex-sensitive predisposing factor in autoimmunity.

It has proven difficult to identify the underlying genes in complex autoimmune diseases. Here, we use forward genetics to identify polymorphisms in the vitamin D receptor gene (Vdr) promoter, controlling Vdr expression and T cell activation. We isolated these polymorphisms in a congenic mouse line, allowing us to study the immunomodulatory properties of VDR in a physiological context. Congenic mice overexpressed VDR selectively in T cells, and thus did not suffer from calcemic effects. VDR overexpression resulted in an enhanced antigen-specific T cell response and more severe autoimmune phenotypes. In contrast, vitamin D3-deficiency inhibited T cell responses and protected mice from developing autoimmune arthritis. Our observations are likely translatable to humans, as Vdr is overexpressed in rheumatic joints. Genetic control of VDR availability codetermines the proinflammatory behavior of T cells, suggesting that increased presence of VDR at the site of inflammation might limit the antiinflammatory properties of its ligand.

OBJECTIVE: To identify genetic factors driving pathogenic autoantibody formation in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA), in order to better understand the etiology of RA and identify possible new avenues for therapeutic intervention. METHODS: We performed a genome-wide analysis of quantitative trait loci controlling autoantibody to type II collagen (anti-CII), anti-citrullinated protein antibody (ACPA), and rheumatoid factor (RF). To identify loci controlling autoantibody production, we induced CIA in a heterogeneous stock-derived mouse cohort, with contribution of 8 inbred mouse strains backcrossed to C57BL/10.Q. Serum samples were collected from 1,640 mice before arthritis onset and at the peak of the disease. Antibody concentrations were measured by standard enzyme-linked immunosorbent assay, and linkage analysis was performed using a linear regression-based method. RESULTS: We identified loci controlling formation of anti-CII of different IgG isotypes (IgG1, IgG3), antibodies to major CII epitopes (C1, J1, U1), antibodies to a citrullinated CII peptide (citC1), and RF. The anti-CII, ACPA, and RF responses were all found to be controlled by distinct genes, one of the most important loci being the immunoglobulin heavy chain locus. CONCLUSION: This comprehensive genetic analysis of autoantibody formation in CIA demonstrates an association not only of anti-CII, but interestingly also of ACPA and RF, with arthritis development in mice. These results underscore the importance of non-major histocompatibility complex genes in controlling the formation of clinically relevant autoantibodies.

By their interaction with IgG immune complexes, FcgammaR and complement link innate and adaptive immunity, showing functional redundancy. In complement-deficient mice, IgG downstream effector functions are often impaired, as well as adaptive immunity. Based on a variety of model systems using FcgammaR-knockout mice, it has been concluded that FcgammaRs are also key regulators of innate and adaptive immunity; however, several of the model systems underpinning these conclusions suffer from flawed experimental design. To address this issue, we generated a novel mouse model deficient for all FcgammaRs (FcgammaRI/II/III/IV(-/-) mice). These mice displayed normal development and lymphoid and myeloid ontogeny. Although IgG effector pathways were impaired, adaptive immune responses to a variety of challenges, including bacterial infection and IgG immune complexes, were not. Like FcgammaRIIb-deficient mice, FcgammaRI/II/III/IV(-/-) mice developed higher Ab titers but no autoantibodies. These observations indicate a redundant role for activating FcgammaRs in the modulation of the adaptive immune response in vivo. We conclude that FcgammaRs are downstream IgG effector molecules with a restricted role in the ontogeny and maintenance of the immune system, as well as the regulation of adaptive immunity.

By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immunity, showing functional redundancy. In complement-deficient mice, IgG downstream effector functions are often impaired, as well as adaptive immunity. Based on a variety of model systems using FcγR-knockout mice, it has been concluded that FcγRs are also key regulators of innate and adaptive immunity; however, several of the model systems underpinning these conclusions suffer from flawed experimental design. To address this issue, we generated a novel mouse model deficient for all FcγRs (FcγRI/II/III/IV^-/- mice). These mice displayed normal development and lymphoid and myeloid ontogeny. Although IgG effector pathways were impaired, adaptive immune responses to a variety of challenges, including bacterial infection and IgG immune complexes, were not. Like FcγRIIb-deficient mice, FcγRI/II/III/IV^-/- mice developed higher Ab titers but no autoantibodies. These observations indicate a redundant role for activating FcγRs in the modulation of the adaptive immune response in vivo. We conclude that FcγRs are downstream IgG effector molecules with a restricted role in the ontogeny and maintenance of the immune system, as well as the regulation of adaptive immunity.

Reduced capacity to produce ROS increases the severity of T cell-dependent arthritis in both mice and rats with polymorphisms in neutrophil cytosolic factor 1 (Ncf1) (p47phox). Since T cells cannot exert oxidative burst, we hypothesized that T cell responsiveness is downregulated by ROS produced by APCs. Macrophages have the highest burst capacity among APCs, so to study the effect of macrophage ROS on T cell activation, we developed transgenic mice expressing functional Ncf1 restricted to macrophages. Macrophage-restricted expression of functional Ncf1 restored arthritis resistance to the level of that of wild-type mice in a collagen-induced arthritis model but not in a T cell-independent anti-collagen antibody-induced arthritis model. T cell activation was downregulated and skewed toward Th2 in transgenic mice. In vitro, IL-2 production and T cell proliferation were suppressed by macrophage ROS, irrespective of T cell origin. IFN-gamma production, however, was independent of macrophage ROS but dependent on T cell origin. These effects were antigen dependent but not restricted to collagen type II. In conclusion, macrophage-derived ROS play a role in T cell selection, maturation, and differentiation, and also a suppressive role in T cell activation, and thereby mediate protection against autoimmune diseases like arthritis.

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is a major noncollagenous component of cartilage and is used as a biomarker in rheumatoid arthritis and experimental arthritis. Injection of COMP leads to severe inflammatory joint disease, and antibodies play a critical role in mediating arthritis. The arthritogenicity of COMP might be due to the lack of self tolerance. This study was undertaken to determine the status of COMP-specific B cell tolerance using COMP-deficient mice. METHODS: Arthritis development and antibody responses were compared between COMP-sufficient and COMP-deficient littermates after immunization with rat COMP. Serum anti-COMP antibody levels were measured using a panel of recombinant mouse COMP proteins, and antibody-secreting cells were enumerated by enzyme-linked immunospot assays. A novel sandwich enzyme-linked immunosorbent assay was developed to assess COMP molecules in serum. RESULTS: COMP-sufficient mice, but not COMP-deficient mice, developed severe arthritis following immunization with rat COMP. However, anti-COMP antibody titers to native COMP and recombinant protein domains covering the entire mouse COMP sequence, except the less immunodominant type 3 repeat domains, were decreased in COMP-sufficient mice compared to COMP-deficient mice. In addition, COMP-sufficient mice had fewer B cells secreting COMP-reactive antibodies. Detectable levels of full-length COMP in arthritic COMP-sufficient B10.Q NCF-1(*/*) and healthy mice suggested systemic availability of COMP to the immune system. CONCLUSION: The lack of arthritis, together with high levels of COMP-specific antibodies, in COMP-deficient mice indicates that susceptibility to arthritis is COMP specific and that endogenous expression of COMP in wild-type mice tolerizes B cells in vivo.

INTRODUCTION: Cartilage oligomeric matrix protein (COMP) is a homopentameric protein in cartilage. The development of arthritis, like collagen-induced arthritis (CIA), involves cartilage as a target tissue. We have investigated the development of CIA in COMP-deficient mice. METHODS: COMP-deficient mice in the 129/Sv background were backcrossed for 10 generations against B10.Q mice, which are susceptible to chronic CIA. COMP-deficient and wild-type mice were tested for onset, incidence, and severity of arthritis in both the collagen and collagen antibody-induced arthritis models. Serum anti-collagen II and anti-COMP antibodies as well as serum COMP levels in arthritic and wild-type mice were measured by enzyme-linked immunosorbent assay. RESULTS: COMP-deficient mice showed a significant early onset and increase in the severity of CIA in the chronic phase, whereas collagen II-antibody titers were similar in COMP-deficient and wild-type controls. COMP antibodies were not found in wild-type mice. Finally, COMP-deficient and wild-type mice responded similarly to collagen antibody-induced arthritis, indicating no difference in how collagen II antibodies interact with COMP-deficient cartilage during the initial stages of arthritis. CONCLUSIONS: COMP deficiency enhances the early onset and development of chronic arthritis but does not affect collagen II autoimmunity. These findings accentuate the importance of COMP in cartilage stability. © 2008 Geng et al.; licensee BioMed Central Ltd.

INTRODUCTION: Cartilage oligomeric matrix protein (COMP) is a major non-collagenous component of cartilage. Earlier, we developed a new mouse model for rheumatoid arthritis using COMP. This study was undertaken to investigate the epitope specificity and immunopathogenicity of COMP-specific monoclonal antibodies (mAbs). METHODS: B cell immunodominant regions on the COMP molecule were measured with a novel enzyme-linked immunosorbent assay using mammalian expressed full-length mouse COMP as well as a panel of recombinant mouse COMP fragments. 18 mAbs specific to COMP were generated and the pathogenicity of mAbs was investigated by passive transfer experiments. RESULTS: B cell immunodominant epitopes were localized within 4 antigenic domains of the COMP but with preferential response to the epidermal growth factor (EGF)-like domain. Some of our anti-COMP mAbs showed interactions with the native form of COMP, which is present in cartilage and synovium. Passive transfer of COMP-specific mAbs enhanced arthritis when co-administrated with a sub-arthritogenic dose of a mAb specific to collagen type II. Interestingly, we found that a combination of 5 COMP mAbs was capable of inducing arthritis in naive mice. CONCLUSIONS: We have identified the specificities of mAbs to COMP and their contribution to the development of arthritis. These findings will further improve our understanding of the autoantibody mediated immunopathologies occurring widely in rheumatoid arthritis (RA), as well as in other autoimmune disorders.

The gut microbiome is recognized as a key modulator of sepsis development. However, the contribution of the gut mycobiome to sepsis development is still not fully understood. Here, we demonstrated that the level of Candida albicans was markedly decreased in patients with bacterial sepsis, and the supernatant of Candida albicans culture significantly decreased the bacterial load and improved sepsis symptoms in both cecum ligation and puncture (CLP)-challenged mice and Escherichia coli-challenged pigs. Integrative metabolomics and the genetic engineering of fungi revealed that Candida albicans-derived phenylpyruvate (PPA) enhanced the bactericidal activity of macrophages and reduced organ damage during sepsis. Mechanistically, PPA directly binds to sirtuin 2 (SIRT2) and increases reactive oxygen species (ROS) production for eventual bacterial clearance. Importantly, PPA enhanced the bacterial clearance capacity of macrophages in sepsis patients and was inversely correlated with the severity of sepsis in patients. Our findings highlight the crucial contribution of commensal fungi to bacterial disease modulation and expand our understanding of the host-mycobiome interaction during sepsis development. © 2023, The Author(s), under exclusive licence to CSI and USTC.

OBJECTIVE: To develop a new chronic rheumatoid arthritis model that is driven by the innate immune system. METHODS: Injection of a cocktail of 4 monoclonal antibodies against type II collagen, followed on days 5 and 60 by intraperitoneal injections of mannan (from Saccharomyces cerevisiae), was used to induce development of chronic arthritis in B10.Q mice. The role of the innate immune system as compared to the adaptive immune system in this arthritis model was investigated using genetically modified mouse strains. RESULTS: A new model of chronic relapsing arthritis was characterized in B10.Q mice, in which a persistently active, chronic disease was found. This relapsing disease was driven by macrophages lacking the ability to mount a reactive oxygen species response against pathogens, and was associated with the classical/alternative pathway, but not the lectin pathway, of complement activation. The disease was independent of Fcgamma receptor type III, and also independent of the activity of adaptive immune cells (B and T cells), indicating that the innate immune system, involving complement activation, could be the sole driver of chronicity. CONCLUSION: Chronic active arthritis can be driven innately by macrophages without the involvement of T and B cells in the adaptive immune system.

OBJECTIVE: To develop a new chronic rheumatoid arthritis model that is driven by the innate immune system.

METHODS: Injection of a cocktail of 4 monoclonal antibodies against type II collagen, followed on days 5 and 60 by intraperitoneal injections of mannan (from Saccharomyces cerevisiae), was used to induce development of chronic arthritis in B10.Q mice. The role of the innate immune system as compared to the adaptive immune system in this arthritis model was investigated using genetically modified mouse strains.

RESULTS: A new model of chronic relapsing arthritis was characterized in B10.Q mice, in which a persistently active, chronic disease was found. This relapsing disease was driven by macrophages lacking the ability to mount a reactive oxygen species response against pathogens, and was associated with the classical/alternative pathway, but not the lectin pathway, of complement activation. The disease was independent of Fcγ receptor type III, and also independent of the activity of adaptive immune cells (B and T cells), indicating that the innate immune system, involving complement activation, could be the sole driver of chronicity.

CONCLUSION: Chronic active arthritis can be driven innately by macrophages without the involvement of T and B cells in the adaptive immune system. © 2018, American College of Rheumatology.

Autoimmune diseases are characterized by the presence of serum autoantibodies of various specificities but the significance of these autoantibodies in the development of symptoms is unclear. Most studies have been carried out using polyclonal sera. However, antibodies’ effects depend on Fab-mediated diversity in epitope specificity, and also on Fc-mediated effects dependent on immunoglobulin class and subclass, immune complex-induced activation of complement, and the milieu in which the reaction occurs. Monoclonal autoantibodies have rarely been studied, but increasingly such mAb are becoming available. These include human mAb to GAD65 from patients with newly diagnosed type 1 diabetes, and mouse mAb to type II collagen that have the capacity to induce collagen antibody induced arthritis (CAIA). In both systems, there is clear evidence that the epitope specificity of the antibodies is related to the expression of the disease, and protective antibodies may occur. © 2004 - 2023 Nova Science Publishers

Relapsing polychondritis is an autoimmune disease that affects cartilage in the ear, nose, and respiratory tract. A pathogenic immune response has been proposed and antibodies to several cartilage proteins are detected in sera from these patients. To investigate the role of the humoral immune response in relapsing polychondritis, we used the matrilin-1-induced relapsing polychondritis model. Mice deficient of B cells (muMT) and mice congenic at the complement factor 5, were immunized with matrilin-1, a cartilage-specific protein mainly detected in the tracheal cartilage. To investigate the binding properties and tissue selection of matrilin-1-specific antibodies we produced matrilin-1-specific B-cell hybridomas. Although 83% of the micro MT heterozygous mice developed respiratory distress and erosive chondritis in the respiratory tract, none of the B-cell-deficient mice were susceptible to disease. In addition, we show that complement factor 5 is important for the induction of matrilin-1-induced relapsing polychondritis. Monoclonal matrilin-1-specific antibodies injected into neonatal mice bound specifically to cartilage of the respiratory tract and adult B-cell-deficient mice injected with the same antibodies developed erosive chondritis in the respiratory tract. We conclude that relapsing polychondritis can be mediated by a pathway involving tissue-specific antibodies and complement activation.

Animal models for inflammatory arthritides such as rheumatoid arthritis (RA) and psoriatic arthritis are widely accepted and frequently used to identify pathological mechanisms and validate novel therapeutic strategies. Unfortunately, many publications reporting on these animal studies lack detailed description and appropriate assessment of the distinct histopathological features of arthritis: joint inflammation, cartilage damage and bone erosion. Therefore, the European consortium BeTheCure, consisting of 38 academic and industrial partners from 15 countries, set as goal to standardise the histological evaluation of joint sections from animal models of inflammatory arthritis. The consensual approach of a task force including 16 academic and industrial scientists as well as laboratory technicians has resulted in the development of the Standardised Microscopic Arthritis Scoring of Histological sections ('SMASH') recommendations for a standardised processing and microscopic scoring of the characteristic histopathological features of arthritis, exemplified by four different rodent models for arthritis: murine collagen-induced arthritis, collagen-antibody-induced arthritis, human tumour necrosis factor transgenic Tg197 mice and rat pristane-induced arthritis, applicable to any other inflammatory arthritis model. Through standardisation, the SMASH recommendations are designed to improve and maximise the information derived from in vivo arthritis experiments and to promote reproducibility and transparent reporting on such studies. In this manuscript, we will discuss and provide recommendations for analysis of histological joint sections: identification of the regions of interest, sample preparation, staining procedures and quantitative scoring methods. In conclusion, awareness of the different features of the arthritis pathology in animal models of inflammatory arthritis is of utmost importance for reliable research outcome, and the standardised histological processing and scoring methods in these SMASH recommendations will help increase uniformity and reproducibility in preclinical research on inflammatory arthritis.