Jared Velasco

β-Glucan triggers spondylarthritis and Crohn's disease-like ileitis in SKG mice

OBJECTIVE The spondylarthritides (SpA), including ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, and arthritis associated with inflammatory bowel disease, cause chronic inflammation of the large peripheral and axial joints, eyes, skin, ileum, and colon. Genetic studies reveal common candidate genes for AS, PsA, and Crohns disease, including IL23R, IL12B, STAT3, and CARD9, all of which are associated with interleukin-23 (IL-23) signaling downstream of the dectin 1 β-glucan receptor. In autoimmune-prone SKG mice with mutated ZAP-70, which attenuates T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire, IL-17-dependent inflammatory arthritis developed after dectin 1-mediated fungal infection. This study was undertaken to determine whether SKG mice injected with 1,3-β-glucan (curdlan) develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process. METHODS SKG mice and control BALB/c mice were injected once with curdlan or mannan. Arthritis was scored weekly, and organs were assessed for pathologic features. Anti-IL-23 monoclonal antibodies were injected into curdlan-treated SKG mice. CD4+ T cells were transferred from curdlan-treated mice to SCID mice, and sera were analyzed for autoantibodies. RESULTS After systemic injection of curdlan, SKG mice developed enthesitis, wrist, ankle, and sacroiliac joint arthritis, dactylitis, plantar fasciitis, vertebral inflammation, ileitis resembling Crohns disease, and unilateral uveitis. Mannan triggered spondylitis and arthritis. Arthritis and spondylitis were T cell- and IL-23-dependent and were transferable to SCID recipients with CD4+ T cells. SpA was associated with collagen- and proteoglycan-specific autoantibodies. CONCLUSION Our findings indicate that the SKG ZAP-70W163C mutation predisposes BALB/c mice to SpA, resulting from innate and adaptive autoimmunity, after systemic β-glucan or mannan exposure.

Interleukin-23 Mediates the Intestinal Response to Microbial β-1,3-Glucan and the Development of Spondyloarthritis Pathology in SKG Mice

Spondyloarthritides (SpA) occur in 1% of the population and include ankylosing spondylitis (AS) and arthropathy of inflammatory bowel disease (IBD), with characteristic spondylitis, arthritis, enthesitis, and IBD. Genetic studies implicate interleukin‐23 (IL‐23) receptor signaling in the development of SpA and IBD, and IL‐23 overexpression in mice is sufficient for enthesitis, driven by entheseal‐resident T cells. However, in genetically prone individuals, it is not clear where IL‐23 is produced and how it drives the SpA syndrome, including IBD or subclinical gut inflammation of AS. Moreover, it is unclear why specific tissue involvement varies between patients with SpA. We undertook this study to determine the location of IL‐23 production and its role in SpA pathogenesis in BALB/c ZAP‐70W163C–mutant (SKG) mice injected intraperitoneally with β‐1,3‐glucan (curdlan).

ZAP-70 Genotype Disrupts the Relationship Between Microbiota and Host, Leading to Spondyloarthritis and Ileitis in SKG Mice

The spondyloarthritides share genetic susceptibility, interleukin‐23 (IL‐23) dependence, and the involvement of microbiota. The aim of the current study was to elucidate how host genetics influence gut microbiota and the relationship between microbiota and organ inflammation in spondyloarthritides.

High Chlamydia Burden Promotes Tumor Necrosis Factor–Dependent Reactive Arthritis in SKG Mice

Chlamydia trachomatis is a sexually transmitted obligate intracellular pathogen that causes inflammatory reactive arthritis, spondylitis, psoriasiform dermatitis, and conjunctivitis in some individuals after genital infection. The immunologic basis for this inflammatory response in susceptible hosts is poorly understood. As ZAP‐70W163C–mutant BALB/c (SKG) mice are susceptible to spondylo‐arthritis after systemic exposure to microbial β‐glucan, we undertook the present study to compare responses to infection with Chlamydia muridarum in SKG mice and BALB/c mice.

Host Genetic Background Disrupts The Relationship Between Microbiota and Gut Mucosal Tolerance Leading To Spondyloarthritis and Ileitis After a Dectin-1 Trigger.

SUPPLEMENTSex Bias In Autoimmune Diseases : Increased Risk Of 47,XXX In Systemic Lupus Erythematosus (SLE) and Sjogrens Syndrome (SS) Supports The Gene Dose HypothesisBackground/Purpose: Human FoxP3+ Th-cells are heterogeneous in function and include not only suppressive cells (TRegs) but also nonsuppressive cells that abundantly secrete proinflammatory cytokines. We have previously shown that FoxP3+ Th-cells were increased in GPA-patients during remission as compared to healthy controls (HCs). In this group of patients, however, we observed a defective suppressor function of TRegs, and an increase in the percentage of Th-17 cells. These observations make it tempting to investigate whether increased FoxP3+ Th-cells in GPA-patients are attributed to an increase in the cytokine-secreting non-suppressive FoxP3+Th-cells. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 46 GPA-patients in remission and from 22 age- and sex-matched HCs. Expression of CD4, CD45RO, and FoxP3 were determined by flow cytometric analysis. The expression levels of FoxP3 and CD45RO were used for distinction between activated suppressor TRegs (FoxP3HighCD45RO+; ASTReg), resting suppressor TRegs (FoxP3LowCD45RO-; RSTReg), and cytokine-secreting non-suppressor TRegs (FoxP3LowCD45RO+; NONTReg) cells. Intracellular expression of IFNg, IL-17, and IL-21 were determined in the various FoxP3+ Th-cell subsets after in vitro activation of PBMCs by PMA and Ca-Ionophore. Results: A significant increase in the frequency of NONTReg cells was observed in GPA-patients as compared with HCs, whereas no differences were detected in RSTReg- and ASTReg cells between GPA-patients and HCs. The distribution of RSTReg- and NONTReg cells did not differ between ANCA-negative and ANCA-positive patients, whereas lower percentages of ASTReg cells were observed in ANCA-positive patients as compared to ANCA-negative patients and HCs. Importantly, a significant increase in the percentage of IL-17+ and IL-21+ cells was seen within the NONTRegcells from ANCA-positive patients (n= 9) when compared to ANCA-negative (n= 10) and HCs (n= 12), whereas no differences were found between ANCA-negative and HCs. Conclusion: Increased FoxP3 expression in Th-cells from GPA-patients is related to an increase in a subset of non-suppressive Th-cells. Increased production of IL-17 and IL-21 cytokines, in NONTReg cells from ANCApositive patients points towards FoxP3+ effector cells and decrease in suppressive TReg cells in relation to ANCA production.Complex Functional Effects Within The HLA Contribute To Sjogrens Syndrome Pathogenesis and May Influence Both Transcriptional Regulation and Peptide Binding

High Chlamydia burden promotes TNF-dependent reactive arthritis in SKG mice

Chlamydia trachomatis is a sexually transmitted obligate intracellular pathogen that causes inflammatory reactive arthritis, spondylitis, psoriasiform dermatitis, and conjunctivitis in some individuals after genital infection. The immunologic basis for this inflammatory response in susceptible hosts is poorly understood. As ZAP‐70W163C–mutant BALB/c (SKG) mice are susceptible to spondylo‐arthritis after systemic exposure to microbial β‐glucan, we undertook the present study to compare responses to infection with Chlamydia muridarum in SKG mice and BALB/c mice.

HighChlamydiaBurden Promotes Tumor Necrosis Factor-Dependent Reactive Arthritis in SKG Mice: TNF-DEPENDENT REACTIVE ARTHRITIS INCHLAMYDIA-INFECTED SKG MICE

Chlamydia trachomatis is a sexually transmitted obligate intracellular pathogen that causes inflammatory reactive arthritis, spondylitis, psoriasiform dermatitis, and conjunctivitis in some individuals after genital infection. The immunologic basis for this inflammatory response in susceptible hosts is poorly understood. As ZAP‐70W163C–mutant BALB/c (SKG) mice are susceptible to spondylo‐arthritis after systemic exposure to microbial β‐glucan, we undertook the present study to compare responses to infection with Chlamydia muridarum in SKG mice and BALB/c mice.

Impaired Bacterial Clearance and An Exuberant Inflammatory Response Promote Chlamydia-Induced Reactive Arthritis In SKG Mice

SUPPLEMENTSex Bias In Autoimmune Diseases : Increased Risk Of 47,XXX In Systemic Lupus Erythematosus (SLE) and Sjogrens Syndrome (SS) Supports The Gene Dose HypothesisBackground/Purpose: Human FoxP3+ Th-cells are heterogeneous in function and include not only suppressive cells (TRegs) but also nonsuppressive cells that abundantly secrete proinflammatory cytokines. We have previously shown that FoxP3+ Th-cells were increased in GPA-patients during remission as compared to healthy controls (HCs). In this group of patients, however, we observed a defective suppressor function of TRegs, and an increase in the percentage of Th-17 cells. These observations make it tempting to investigate whether increased FoxP3+ Th-cells in GPA-patients are attributed to an increase in the cytokine-secreting non-suppressive FoxP3+Th-cells. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 46 GPA-patients in remission and from 22 age- and sex-matched HCs. Expression of CD4, CD45RO, and FoxP3 were determined by flow cytometric analysis. The expression levels of FoxP3 and CD45RO were used for distinction between activated suppressor TRegs (FoxP3HighCD45RO+; ASTReg), resting suppressor TRegs (FoxP3LowCD45RO-; RSTReg), and cytokine-secreting non-suppressor TRegs (FoxP3LowCD45RO+; NONTReg) cells. Intracellular expression of IFNg, IL-17, and IL-21 were determined in the various FoxP3+ Th-cell subsets after in vitro activation of PBMCs by PMA and Ca-Ionophore. Results: A significant increase in the frequency of NONTReg cells was observed in GPA-patients as compared with HCs, whereas no differences were detected in RSTReg- and ASTReg cells between GPA-patients and HCs. The distribution of RSTReg- and NONTReg cells did not differ between ANCA-negative and ANCA-positive patients, whereas lower percentages of ASTReg cells were observed in ANCA-positive patients as compared to ANCA-negative patients and HCs. Importantly, a significant increase in the percentage of IL-17+ and IL-21+ cells was seen within the NONTRegcells from ANCA-positive patients (n= 9) when compared to ANCA-negative (n= 10) and HCs (n= 12), whereas no differences were found between ANCA-negative and HCs. Conclusion: Increased FoxP3 expression in Th-cells from GPA-patients is related to an increase in a subset of non-suppressive Th-cells. Increased production of IL-17 and IL-21 cytokines, in NONTReg cells from ANCApositive patients points towards FoxP3+ effector cells and decrease in suppressive TReg cells in relation to ANCA production.Complex Functional Effects Within The HLA Contribute To Sjogrens Syndrome Pathogenesis and May Influence Both Transcriptional Regulation and Peptide Binding

Incidence and severity of spondyloarthritis and Crohn's Ileitis are determined by interaction between the microbiota and genetic susceptibility in beta-glucan-treated SKG mice

Background/Purpose: MicroRNAs (miRs) are a novel class of posttranscriptional regulators. A single miR can have profound effects on cell activation due to its ability to modulate multiple pathways at once. We have previously shown that miR-155 is upregulated in rheumatoid arthritis (RA) synovial macrophages and promotes the development of autoimmunity and joint inflammation. Pre-clinical arthritis may be associated with lung changes e.g. bronchial wall thickening, thus the aim of this study was to investigate the contribution of miR-155 regulated pathways to lung homeostasis. Methods: Normal human lung tissue was tested by in situ hybridisation with miR-155 and control probes. To model the fibrotic response, WT and miR-155 / mice were given bleomycin (0.06 unit/mouse) intranasally. Intervention included intraperitoneal injections of the Liver X Receptor (LXR) agonist (GW3965 daily; 40 mg/kg). End-points included bronchial lavage (BAL) cytology, lung tissue histology, evaluation of the expression of inflammatory and fibrotic genes by qPCR and concentrations of soluble mediators in serum and BAL fluid by multiplex assays. The validation of miR-155 binding to LXR, and the LXR response element in collagen gene promoters were performed with reporter assays. Results: In situ hybridisation showed an abundant expression of miR-155 in the normal human lung suggesting that this miR may contribute to normal lung homeostasis. miR-155 / mice developed more severe bleomycininduced lung fibrosis compared to WT mice, as seen by increased collagen 1a/3a mRNA expression and protein deposition in the lungs, as well as accumulation of macrophages and lymphocytes in BAL. Gene expression analysis of lung extracts revealed an increase in the M2 pro-fibrotic macrophage markers Arginase 2, IL-13R and Ym1. In addition, the levels of pro-fibrotic cytokines such as VEGF and bFGF were significantly higher in BAL and serum of miR-155 / mice. Primary lung fibroblast lines derived from miR-155 / mice showed higher proliferation rates and motility compared to WT cells in wound healing assays. Computational analysis followed by functional luciferase assays revealed that the transcription activator LXR alpha is a direct target of miR-155 in the lungs. Expression of LXR alpha was significantly upregulated in the lungs of naive miR-155 / mice and was further increased in mice given bleomycin compared to similarly treated WT controls. Injection of the LXR agonist to WT mice increased LXR expression and mirrored the same phenotypic response to bleomycin as the miR-155 deficient mice; shown by increased collagen deposition and M2 macrophage and fibroblast activation. Promoter analysis revealed that LXRs could directly induce collagen production by binding to col1a and col3a promoters. / Conclusion: miR-155 appears important for lung homeostasis, likely by fine tuning levels of LXR thereby protecting from excessive remodelling. Given this and the emerging contribution of miR-155 to development of autoimmunity, this miR may act as a master-switch determining the duration of inflammation and the initiation of remodelling, as well as the balance between the immune and auto-immune responses.Background/Purpose: High mobility group box 1 (HMGB1) is a non-histone DNA binding protein that is passively released by dying cells or actively secreted by immunocompetent cells and the receptor for advanced glycation end-products (RAGE) is one of its receptors. Higher levels of HMGB1 have been found in patients with granulomatosis with polyangiitis (GPA) with active disease whereas higher HMGB1 and lower soluble (sRAGE) levels have been found in patients with acute atherosclerotic events suggesting sRAGE acts as a decoy receptor. This study aims to evaluate HMGB1 levels in relation to subclinical carotid atherosclerosis in GPA, and the impact of therapy on HMGB1 levels. Methods: A cross-sectional study was performed on 23 GPA patients during a quiescent phase of the disease in comparison to 20 matched controls. All study participants underwent carotid ultrasound to assess atherosclerotic plaques and intima-media thickness (IMT) and were tested for traditional risk factors for atherosclerosis, serum HMGB1 levels (ELISA-Shino Test, Kanagawa, Japan), and sRAGE levels (ELISA RD P = 0.978), HDLcholesterol (1.41 ± 0.37 vs. 1.51±0.33 mmol/L; P = 0.359), LDLcholesterol (3.01±0.79 vs. 3.29±0.82 mmol/L; P = 0.267), and a similar frequency of smoking (8.7% vs. 5.0%; P = 0.635), family history of premature coronary artery disease (CAD) (39.1% vs. 40.0%; P = 0.954), and obesity (4.3% vs. 10.0%; P = 0.446). Hypertension was only found in GPA patients (39.1% vs. 0.0%; P = 0.002) while no study participants had diabetes. Overt cardiovascular disease was found only in 13.0% of GPA patients. Statins were prescribed for 21.7% of GPA patients and 5.0% of controls (P = 0.127). Among GPA patients, prednisolone was being used by 34.8% with a median daily dose of 5.0mg (2.5-15.0) and azathioprine by 34.8%. Only two GPA patients used statins and prednisolone concomitantly. Carotid plaques were found in 30.4% of GPA patients and in 15.0% of controls (P = 0.203) and the overall IMT was similar in GPA patients and in controls (0.833±0.256 vs. 0.765±0.133mm; P = 0.861). Median serum HMGB1 levels were similar between GPA patients and controls [2.13ng/mL (1.11-7.22) vs. 2.42ng/mL (0.38-6.75); P = 0.827] as well as mean sRAGE levels (1256.1±559.6 vs. 1483.3±399.8pg/mL; P = 0.155). No correlations were found between HMGB1 and sRAGE ( = 0.068; P = 0.681) and between HMGB1 and maximum IMT in carotid arteries ( = -0.067; P = 0.720). GPA patients on prednisolone (1.77±0.76 vs. 3.53±2.06ng/ mL; P = 0.017) and statins (1.39±0.28 vs. 3.34±1.94ng/mL; P = 0.001) presented significantly lower serum HMGB1 levels whereas no difference in mean HMGB1 levels was found regarding azathioprine use (2.89±2.28 vs. 2.93±1.75; P = 0.970). Conclusion: No association was found between subclinical atherosclerosis in carotid arteries and HMGB1 levels in GPA patients. Furthermore, the use of either prednisone or statins was associated with lower HMGB1 levels in GPA patients. These findings suggest that the anti-inflammatory properties of statins include effects on serum HMGB1 levels in GPA.