When IL-1 receptor antagonist (IL-1rn) is knocked out, mice have shown strain background dependent and major QTL regulated susceptibility to spontaneously inflammatory arthritis disease (SAD). The impact on bone properties resulting from the interactions of IL-1rn, genomic background strains, and the QTL locus, is unknown. Bone properties in the four specifically bred mouse strains with mutation of IL-1rn and variations in genomic components were investigated with high-resolution MicroCT and genomic analytical tools. Two congenic mouse strains were also measured to evaluate the influence on bone properties by a QTL in the region in chromosome 1. Our results reveal that several bone phenotypes, including bone mineral density (BMD), bone volume, tibial length, and cortical thickness of the tibia are different between wild type and IL-1rn knockout mice in both Balb/c and DBA/1 backgrounds, but IL-1rn knockout affects BMD differently between the two mouse strains. The absence of IL-1rn decreases BMD in Balb/c mice but increases BMD in DBA/1-/- mice compared to their respective wild type counterparts. A QTL transferred from the Balb/c genetic background which affects arthritis in congenic strains appears to also regulate BMD. While several genes, including Ctsg and Prg2, may affect BMD, Ifi202b is the most favored candidate gene for regulating BMD as well as SAD. In conclusion, the previously mentioned bone phenotypes are each influenced in different ways by the loss of IL-1ra when considered in mice from varying genomic backgrounds.

Sex difference has shown in the arthritis diseases in human population and animal models. We investigate how the sex and symmetry vary among mouse models with different genomic backgrounds. Disease data of sex and limbs accumulated in the past more than two decades from four unique populations of murine arthritis models were analyzed. They are (1) interleukin-1 receptor antagonist (IL-1ra) deficient mice under Balb/c background (Balb/c KO); (2) Mice with collagen II induced arthritis under DBA/1 background; (3) Mice with collagen II induced arthritis under C57BL/6 (B6) background and (4) A F2 generation population created by Balb/c KO X DBA/1 KO. Our data shows that there is a great variation in sexual dimorphism for arthritis incidence and severity of arthritis in mice harboring specific genetic modifications. For a F2 population, the incidence of arthritis was 57.1% in female mice and 75.6% in male mice. There was a difference in severity related to sex in two populations: B6.DR1/ B6.DR4 ( P < 0.001) and F2 ( P = 0.023) There was no difference Balb/c parental strain or in collagen-induced arthritis (CIA) in DBA/1 mice. Among these populations, the right hindlimbs are significantly higher than the scores for the left hindlimbs in males ( P < 0.05). However, when examining disease expression using the collagen induced arthritis model with DBA/1 mice, sex-dimorphism did not reach statistical significance, while left hindlimbs showed a tendency toward greater disease expression over the right. Sexual dimorphism in disease expression in mouse models is strain and genomic background dependent. It sets an alarm that potential variation in sexual dimorphism among different racial and ethnic groups in human populations may exist. It is important to not only include both sexes and but also pay attention to possible variations caused by disease expression and response to treatment in all the studies of arthritis in animal models and human populations.

Citrullination of proteins plays an important role in protein function and it has recently become clear that citrullinated proteins play a role in immune responses. In this study we examined how citrullinated collagen, an extracellular matrix protein, affects T-cell function during the development of autoimmune arthritis. Using an HLA-DR1 transgenic mouse model of rheumatoid arthritis, mice were treated intraperitoneally with either native type I collagen (CI), citrullinated CI (cit-CI), or phosphate buffered saline (PBS) prior to induction of autoimmune arthritis. While the mice given native CI had significantly less severe arthritis than controls administered PBS, mice receiving cit-CI had no decrease in the severity of autoimmune arthritis. Using Jurkat cells expressing the inhibitory receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), Western blot analysis indicated that while CI and cit-CI bound to LAIR-1 with similar affinity, only CI induced phosphorylation of the LAIR ITIM tyrosines; cit-CI was ineffective. These data suggest that cit-CI acts as an antagonist of LAIR-1 signaling, and that the severity of autoimmune arthritis can effectively be altered by targeting T cells with citrullinated collagen.

Vitamin D plays a crucial role in regulation of the immune response. However, treatment of autoimmune diseases with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] doses sufficient to be effective is prohibitive due to its calcemic and toxic effects. We use the collagen-induced arthritis (CIA) model to analyze the efficacy of the noncalcemic analog of vitamin D, 20S-hydroxyvitamin D3 [20S(OH)D3], as well as 1,25(OH)2D3, to attenuate arthritis and explore a potential mechanism of action. Mice fed a diet deficient in vitamin D developed a more severe arthritis characterized by enhanced secretion of T cell inflammatory cytokines, compared to mice fed a normal diet. The T cell inflammatory cytokines were effectively suppressed, however, by culture of the cells with 20S(OH)D3. Interestingly, one of the consequences of culture with 1,25(OH)2D3 or 20S(OH)D3, was upregulation of the natural inhibitory receptor leukocyte associated immunoglobulin-like receptor-1 (LAIR-1 or CD305). Polyclonal antibodies which activate LAIR-1 were also capable of attenuating arthritis. Moreover, oral therapy with active forms of vitamin D suppressed arthritis in LAIR-1 sufficient DR1 mice, but were ineffective in LAIR-1−/− deficient mice. Taken together, these data show that the effect of vitamin D on inflammation is at least, in part, mediated by LAIR-1 and that non-calcemic 20S(OH)D3 may be a promising therapeutic agent for the treatment of autoimmune diseases such as Rheumatoid Arthritis.

Multiple observations implicate T-cell dysregulation as a central event in the pathogenesis of rheumatoid arthritis. Here, we investigated mechanisms for suppressing T-cell activation via the inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1). To determine how LAIR-1 affects T-cell receptor (TCR) signaling, we compared 1) T cells from LAIR-1?sufficient and ?deficient mice, 2) Jurkat cells expressing either LAIR-1 mutants or C-terminal Src kinase (CSK) mutants, and 3) T cells from mice that contain a CSK transgene susceptible to chemical inhibition. Our results indicated that LAIR-1 engagement by collagen or by complement C1q (C1Q, which contains a collagen-like domain) inhibits TCR signaling by decreasing the phosphorylation of key components in the canonical T-cell signaling pathway, including LCK proto-oncogene SRC family tyrosine kinase (LCK), LYN proto-oncogene SRC family tyrosine kinase (LYN), ? chain of T-cell receptor?associated protein kinase 70 (ZAP-70), and three mitogen-activated protein kinases (extracellular signal?regulated kinase, c-Jun N-terminal kinase 1/2, and p38). The intracellular region of LAIR-1 contains two immunoreceptor tyrosine-based inhibition motifs that are both phosphorylated by LAIR-1 activation, and immunoprecipitation experiments revealed that Tyr-251 in LAIR-1 binds CSK. Using CRISPR/Cas9-mediated genome editing, we demonstrate that CSK is essential for the LAIR-1?induced inhibition of the human TCR signal transduction. T cells from mice that expressed a PP1 analog?sensitive form of CSK (CskAS) corroborated these findings, and we also found that Tyr-251 is critical for LAIR-1's inhibitory function. We propose that LAIR-1 activation may be a strategy for controlling inflammation and may offer a potential therapeutic approach for managing autoimmune diseases.

The bioavailability of a drug at the target site is vital to repair the degenerated cartilage following trauma or osteoarthritis (OA). Previously, we developed targeted nanosomes with anti-type II collagen monoclonal antibody (MabCII) on their surface that can bind to the damaged cartilage. The efficiency of nanosomes is highly dependent on their physicochemical nature. Therefore, in this study, we presented a rigorous method for examining the physicochemical characteristics and biological efficacy of nanosomes. Nanosomes were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), and thin-layer chromatography (TLC). Specificity of nanosomes for type II collagen was evaluated by enzyme-linked immunosorbent assay (ELISA). Release kinetics of nanosomes was determined by dialysis method using fluorescein isothiocyanate (FITC) dye. The biological efficacy of targeted nanosomes encapsulating TGF-β3 was determined in porcine chondrocytes (pChon). Moreover, the binding specificity of targeted nanosomes to the damaged cartilage was confirmed onto the cartilage explants and in a mouse model of spontaneous osteoarthritis (OA). The synthetic targeted nanosomes were unilamellar with a mean diameter of 200 nm. Retention factor (Rf) values for all the lipids were in accordance with the standards with a mean 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) concentration of 3.22 nM. It was found that nanosomes release approximately 50% the encapsulated product at 37 °C within 24 h. TGF-β3-targeted nanosomes found to reduce the expression of inflammatory marker matrix metalloproteinases (MMP-1) in chondrocytes stimulated with TNFα. In brief, in this study, we present a comprehensive approach to characterize the physicochemical and biological characteristics of nanosomes. Furthermore, this approach can be utilized to deliver the drug or molecule of interest to the diseased or damaged tissues.

IL-1 is an important cytokine that is secreted by various cells, including synovial cells, and can induce the production of proinflammatory mediators. Due to the excessive and uncontrolled IL-1 signaling, IL-1R antagonist knockout mice (IL-1rnKO) develop spontaneous polyarthritis that mimics some pathogenic features of human rheumatoid arthritis (RA). MyD88 is a key signaling adaptor molecule for toll-like receptors (TLRs) and IL-1R. We have previously identified protein kinase D1 (PKD1) to play an indispensable role in the MyD88-dependent proinflammatory response. MyD88 and PKD1 may therefore confer regulatory role on arthritis development in IL- 1rnKO mice. To investigate this, we examined the synovial cell, T cell, and B cell responses, and the incidence and severity of arthritis in IL-1rnKO mice that are deficient in MyD88 or PKD1. We found that MyD88 deficiency and PKD1 deficiency inhibited the development of polyarthritis as well as reduced the levels of synovial macrophages in IL-1rnKO. T cells isolated from lymph nodes expressed significantly reduced CCR6, CXCR3, IFNγ, and IL-17 in MyD88-deficient IL-1rnKO mice. BAFF-R and TACI levels in splenic marginal zone precursor cells (MZP) and maturation of MZP to marginal zone B cells were significantly reduced in MyD88-deficient IL- 1rnKO mice. We are currently investigating whether PKD1 also play a regulatory role in T cell and B cell responses during the pathogenic course of arthritis in IL-1rnKO mice. Taking together, our results suggest that MyD88 and PKD1 play a critical role in the pathogenesis of arthritis in IL-1rnKO. Further insights into PKD1 and its role in arthritis development will contribute to better understanding innate signaling in the development of RA.

Although the etiology of Rheumatoid arthritis (RA) is unknown, it is thought that TLR signaling may be involved in the initiation and progression of the disease. Disruption of the TLR signaling at early stages of RA could be a possible therapeutic strategy. We have previously identified protein kinase D1 (PKD1) as an indispensable component in the MyD88-dependent TLR signaling pathway that plays a critical role in acute and chronic inflammation. Although it is currently not known whether PKD1 plays a pathogenic role in RA in humans, we have previously found that daily treatments of a PKC/PKD inhibitor Gö6976 significantly ameliorated arthritic symptoms in collagen-induced arthritis (CIA). Here, we further investigated the role of PKD1 in pathogenesis of CIA in humanized HLA-DR1 transgenic mice using a tamoxifen-inducible PKD1-gene deletion system. We have found that PKD1 deletion resulted in reduced incidence and severity of arthritis in HLA-DR1 mice immunized with type II collagen. In addition, Gr-1+ myeloid lineage cell infiltration into the joint space was significantly reduced and joint destruction (assessed by joint histopathology study and mCT scanning) was ameliorated in the PKD1-deleted mice compared to wild-type. Our results provide evidences that PKD1 might play an important pathogenic role in inflammatory arthritis and that PKD1 could be a new therapeutic molecular target for arthritis.

Aims: To examine whether the increased expression level of interferon-activatable protein (Ifi) genes is associated with the decreased resistance to SAD in a congenic mouse strain DBA.B-1(-/-) which is IL1rn-deficient and contains the QTL genomic fragment associated with susceptibility to SAD, from a BALB/c(-/-) on the DBA/1(-/-) background. Methods: We produced whole genome expression profiles from the DBA.B-1(-/-) and four parental strains, the wild type BALB/c, DBA/1 and the IL1rn-deficient DBA/1(-/-) and BALB/c(-/-). We analyzed the differential expression levels of genes in DBA.B-1(-/-) in comparison to other strains, compared these genes with that of a previous congenic strain, BALB. D1-1(-/-) , which is IL1rn-deficient and contains the QTL genomic fragment associated with susceptibility to SAD, from a DBA/1(-/-) on the BALB/c(-/-) background and examined the candidacy of genes within the Ifi family. Results: Although there are a considerable number of differentially expressed genes between DBA.B-1(-/-) and the four parental strains, the differences are in the opposite direction to that in previous comparisons between the BALB. D1-1(-/-) and other strains. There were a fewer number of up-regulated genes in BALB. D1-1(-/-) in comparison to DBA/1. Instead of down-regulated Ifi genes in BALB. D1-1(-/-) in comparison to its parental strain BALB/c(-/-), the expression levels of a few Ifi genes in the DBA.B-1(-/-) strain were higher than that of its parental strain DBA/1(-/-). These Ifi genes are also differentially expressed between DBA.B-1(-/-) and DBA/1 strains. Their expression levels in the DBA.B-1(-/-) are similar to that in BALB/c and BALB/c(-/-) strains. Among these genes, only Ifi204 expressed at a significant, high level in these mouse strains. Conclusion: Ifi204 is the most favored candidate gene that regulates susceptibility to spontaneous arthritis in mice deficient in IL-1ra. Both Htra1 and Dpt may be involved in the Ifi204 molecular pathway.

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

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

Toll-like receptor (TLR) signaling can contribute to the pathogenesis of arthritis. Disruption of TLR signaling at early stages of arthritis might thereby provide an opportunity to halt the disease progression and ameliorate outcomes. We previously found that Gö6976 inhibits TLR-mediated cytokine production in human and mouse macrophages by inhibiting TLR-dependent activation of protein kinase D1 (PKD1), and that PKD1 is essential for proinflammatory responses mediated by MyD88-dependent TLRs. In this study, we investigated whether PKD1 contributes to TLR-mediated proinflammatory responses in human synovial cells, and whether Gö6976 treatment can suppress the development and progression of type II collagen (CII)-induced arthritis (CIA) in mouse. We found that TLR/IL-1R ligands induced activation of PKD1 in human fibroblast-like synoviocytes (HFLS). TLR/IL-1R-induced expression of cytokines/chemokines was substantially inhibited in Gö6976-treated HFLS and PKD1-knockdown HFLS. In addition, serum levels of anti-CII IgG antibodies, and the incidence and severity of arthritis after CII immunization were significantly reduced in mice treated daily with Gö6976. Synergistic effects of T-cell receptor and TLR, as well as TLR alone, on spleen cell proliferation and cytokine production were significantly inhibited in the presence of Gö6976. Our results suggest a possibility that ameliorating effects of Gö6976 on CIA may be due to its ability to inhibit TLR/IL-1R-activated PKD1, which might play an important role in proinflammatory responses in arthritis, and that PKD1 could be a therapeutic target for inflammatory arthritis.

BACKGROUND:The mouse strain BALB/c deficient in IL-1 receptor antagonist protein (Il-1ra) develops spontaneous arthritis disease (SAD) while the strain DBA/1 IL1rn (-/-) with the same deficiency does not. Previously, we mapped a QTL on chromosome 1 for SAD and then developed a congenic mouse strain BALB.D1-1(-/-) that contains the QTL genomic fragment associated with resistance from DBA/1(-/-) on a BALB/c(-/-) background. The congenic strain was relatively resistant to spontaneous arthritis and had delayed onset and reduced severity of disease. We obtained whole genome expression profiles from the spleen of the congenic strain BALB.D1-1(-/-) and four other strains, the wild type BALB/c, DBA/1 and the deficient DBA/1 IL1rn (-/-) and the BALB/c IL1rn (-/-). We then compared the similarities and differences between the congenic strain and the four parental strains. Here we report the selected potential causal genes based on differential expression levels as well as function of genes.RESULTS:There is a considerable number of genes that are differentially expressed between the congenic strain and the three parental strains, BALB/c, DBA/1, and DBA/1(-/-). However there only a few differentially expressed genes were identified by comparing the congenic strain and the BALB/c(-/-)strain. These differentially expressed genes are mainly from T-cell receptor beta chain (Tcrb) and interferon-activatable protein (Ifi) genes. These genes are also differentially expressed between congenic strain and BALB/c strains. However, their expression levels in the congenic strain are similar to that in DBA/1 and DBA/1(-/-). The expression level of Tcrb-j gene is positively associated with two genes of Ifi gene 200 cluster.CONCLUSIONS:Decreased expression levels of Ifi genes is associated to the increased resistance to spontaneous arthritis disease and with down regulation of expressions of Tcrb genes in the mouse congenic strain. Ifi genes may play an important role in the susceptibility to SAD in mice.

Detection and intervention at an early stage is a critical factor to impede arthritis progress. Here we present a non-invasive method to detect inflammatory changes in joints of arthritic mice. Inflammation was monitored by dual fluorescence optical imaging for near-infrared fluorescent (750F) matrix-metalloproteinase activatable agent and allophycocyanin-conjugated anti-mouse CD11b. Increased intensity of allophycocyanin (indication of macrophage accumulation) and 750F (indication of matrix-metalloproteinase activity) showed a biological relationship with the arthritis severity score and the histopathology score of arthritic joints. Our results demonstrate that this method can be used to detect early stages of arthritis with minimum intervention in small animal models.

IL-1 receptor antagonist-deficient (IL-1rn−/−) mice develop spontaneous polyarthritis that mimics human rheumatoid arthritis. We investigated whether a Toll-like receptor (TLR)/IL-1R signaling adaptor MyD88 and its downstream protein kinase D1 (PKD1) play a role in the development and progression of arthritis in IL-1rn−/− mice. Serum cytokine levels, and macrophage accumulation, matrix metalloproteinase activities and cartilage damage in joints were significantly lowered in MyD88−/−IL-1rn−/− mice compared to MyD88+/+ IL-1rn−/− mice. Development of arthritis was completely inhibited in MyD88−/− IL-1rn−/− mice, indicating an absolute requirement for MyD88 in the development of arthritis in IL-1rn−/− mice. Because PKD1 is essential for proinflammatory responses mediated by MyD88-dependent TLRs, we further investigated the contribution of PKD1 to the development of arthritis in IL-1rn−/− mice. Suppression of PKD1 expression in vitro resulted in inhibition of TLR-mediated cytokine production in IL-1rn−/− macrophages. Daily treatment with a PKD inhibitor substantially reduced the incidence and severity of arthritis in IL-1rn−/− mice. In addition, deletion of the PKD1 gene in IL-1rn−/− mice using a tamoxifen-inducible system significantly delayed development of arthritis and reduced the incidence and severity of arthritis in IL-1rn−/− mice. Our findings demonstrated that MyD88 and PKD1 are necessary for development of arthritis in IL-1rn−/− mice. This also implies that PKD1 might be one of the key factors that modulate proinflammatory responses in rheumatoid arthritis, and may serve as a therapeutic target.

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

Rheumatoid arthritis (RA) may be initiated in part by signaling through Toll-like receptors (TLRs). Disruption of TLR signaling may provide an opportunity to halt the RA process in the earliest stages. We investigated whether protein kinase D1 (PKD1), which is essential for proinflammatory responses mediated by MyD88-dependent TLRs, plays a role in the development of experimental arthritis. The presence of TLR ligands in joint cavities induces development of reactive arthritis that is characterized by accumulation of activated inflammatory cells in the joints. Deletion of PKD1 in myeloid lineage cells prevented TLR-mediated accumulation of inflammatory cells in joints, thus protecting mice from developing reactive arthritis. Arthritis spontaneously occurring in IL-1R antagonist-deficient (IL-1rn-/-) mice was dependent on MyD88, and suppression of PKD1 expression resulted in inhibition of TLR-mediated cytokine production in IL-1rn-/- macrophages. Daily treatment with a PKD inhibitor substantially reduced the incidence and severity of arthritis in IL-1rn-/- mice. In addition, a PKD inhibitor inhibited the synergistic effect of T cell receptor and TLR2 on IL-6 production in splenocytes, and significantly reduced the incidence and severity of collagen-induced arthritis that closely mimics the pathophysiologic process of human RA. This implies that PKD1 might be one of the key factors that modulate proinflammatory responses in RA, and can be a therapeutic target for RA.