Additional file 1 of STING mediates experimental osteoarthritis and mechanical allodynia in mouse

Additional file 1: Supplementary Table 1. Characteristics of individuals with OA from whom cartilage samples were taken. Supplementary Table 2. List of primary and secondary antibodies. Supplementary Table 3. PCR primers and conditions. Supplementary Fig. 1. Characterization of Sting1-/- mice. Supplementary Fig. 2. Experimental design and number of mice assigned to each group. Supplementary Fig. 3. Genetic ablation of Sting1 mitigates mechanical sensitivity in mouse. Supplementary Fig. 4. Stimulation of the STING in mouse knee joints exacerbates OA-associated mechanical allodynia. Supplementary Fig. 5. Expression of pain-sensitizing molecules in joint tissues of DMM-operated WT and Sting1-/- mice. Supplementary Fig. 6. Expression of pain-sensitizing molecules in periosteum of DMM-operated WT and Sting1-/- mice. Supplementary Fig. 7. Expression of pain-sensitizing molecules in joint tissue of sham-operated WT and Sting1-/- (KO) mice

Hypoxia-Inducible Factor-2α Is an Essential Catabolic Regulator of Inflammatory Rheumatoid Arthritis

<div><p>Rheumatoid arthritis (RA) is a systemic autoimmune disorder that manifests as chronic inflammation and joint tissue destruction. However, the etiology and pathogenesis of RA have not been fully elucidated. Here, we explored the role of the hypoxia-inducible factors (HIFs), HIF-1α (encoded by <i>HIF1A</i>) and HIF-2α (encoded by <i>EPAS1</i>). HIF-2α was markedly up-regulated in the intimal lining of RA synovium, whereas HIF-1α was detected in a few cells in the sublining and deep layer of RA synovium. Overexpression of HIF-2α in joint tissues caused an RA-like phenotype, whereas HIF-1α did not affect joint architecture. Moreover, a HIF-2α deficiency in mice blunted the development of experimental RA. HIF-2α was expressed mainly in fibroblast-like synoviocytes (FLS) of RA synovium and regulated their proliferation, expression of RANKL (receptor activator of nuclear factor–κB ligand) and various catabolic factors, and osteoclastogenic potential. Moreover, HIF-2α–dependent up-regulation of interleukin (IL)-6 in FLS stimulated differentiation of T_H17 cells—crucial effectors of RA pathogenesis. Additionally, in the absence of IL-6 (<i>Il6</i>^−/− mice), overexpression of HIF-2α in joint tissues did not cause an RA phenotype. Thus, our results collectively suggest that HIF-2α plays a pivotal role in the pathogenesis of RA by regulating FLS functions, independent of HIF-1α.</p></div

Local deletion of <i>Epas1</i> in joint tissues inhibits CIA.

<p><i>Epas1</i>^fl/fl mice were IA-injected with Ad-C or Ad-<i>Cre</i> (1×10⁹ PFU), immunized with type II collagen (CIA) or NI, and maintained for 3 wk. (A) HIF-2α in joint tissues was detected by immunostaining (<i>n</i> = 10). (B) Synovitis, cartilage destruction, pannus formation, and angiogenesis were detected by H&E staining, safranin-O staining, safranin-O/hematoxylin staining, and CD31 immunostaining, respectively. Representative images were obtained from more than 10 independent experiments. P, pannus. (C) Quantification of synovitis, pannus formation, blood vessels in the synovium, and Mankin score (<i>n</i>>10). Values are means ± SEM (*<i>p</i><0.0005). Scale bar, 50 µm.</p

Normal immune system development and effector function of CD4⁺ T cells in <i>Epas1</i>^+/− mice.

<p>(A) Representative flow cytometric analysis of CD4⁺ and CD8⁺ T-cell populations in the lymph nodes of WT and <i>Epas1</i>^+/− DBA/1J mice. (B) Populations of T_H1, T_H2, and T_H17 cells differentiated from uncommitted CD4⁺ T cells of WT and <i>Epas1</i>^+/− DBA/1J mice. (C) IL17A-producing cells identified by flow cytometry (left), and levels of secreted IL17A determined by ELISA (right), from lymphocytes (LN) and splenocytes (SP) of WT and <i>Epas1</i>^+/− DBA/1J mice (<i>n</i> = 8 mice per group) under CIA conditions. (D) mRNA levels of the indicated cytokines in total knee synovial cells isolated from <i>Epas1</i>^+/− DBA/1J mice under CIA conditions or in Ad-<i>Epas1</i>–injected mice (<i>n</i> = 10). The NI condition and Ad-C injection were used as controls. Values are means ± SEM (*<i>p</i><0.01, **<i>p</i><0.005, ***<i>p</i><0.0005).</p

Overexpression of HIF-2α, but not HIF-1α, in joint tissues causes an RA-like phenotype in mice.

<p>DBA/1J mice were IA-injected with 1×10⁹ PFU of empty virus (Ad-C), Ad-<i>Epas1</i>, or Ad-<i>Hif1a</i>. After 3 wk, mice were sacrificed for further analysis. (A and B) Representative images of HIF-1α (A) and HIF-2α (B) immunostaining in knee joint tissues. (C and D) Scoring of synovial inflammation (<i>n</i> = 20) (C) and representative images of H&E staining (D). (E) Safranin-O staining and scoring of cartilage destruction (<i>n</i> = 20), safranin-O/hematoxylin staining and quantitation of pannus formation (<i>n</i> = 15), and CD31 staining and quantitation of blood vessels (<i>n</i> = 15) in Ad-injected knee joints. Ca, cartilage; P, pannus. Values are means ± SEM (*<i>p</i><0.001, **<i>p</i><0.0002). NS, not significant. Scale bars, 50 µm.</p

HIF-1α and HIF-2α are differentially up-regulated in RA synovium.

<p>(A) Representative images of human RA synovium immunostained for HIF-1α, HIF-2α, IL6, MMP3, and MMP13 (<i>n</i> = 10). (B) Representative images of human RA and mouse CIA synovial sections (<i>n</i> = 8) immunostained for HIF-2α and a RA synovium marker (MMP3, MMP13, or IL6) and counterstained with DAPI (triple stained). Insets are enlarged images of double-stained cells. (C) Representative images of HIF-1α and HIF-2α immunostaining in the knee synovia of CIA and NI control DBA/1J mice (<i>n</i> = 10). (D) Relative expression levels of HIF-1α and HIF-2α in synovial cells (left) (<i>n</i> = 10). HIF-2α–positive cells were counted in the indicated compartments of RA synovium (right) (<i>n</i> = 5). Values are means ± SEM (*<i>p</i><0.0005). Scale bar, 50 µm.</p

Local deletion of <i>Epas1</i> in joint tissues inhibits CIA.

<p><i>Epas1</i>^fl/fl mice were IA-injected with Ad-C or Ad-<i>Cre</i> (1×10⁹ PFU), immunized with type II collagen (CIA) or NI, and maintained for 3 wk. (A) HIF-2α in joint tissues was detected by immunostaining (<i>n</i> = 10). (B) Synovitis, cartilage destruction, pannus formation, and angiogenesis were detected by H&E staining, safranin-O staining, safranin-O/hematoxylin staining, and CD31 immunostaining, respectively. Representative images were obtained from more than 10 independent experiments. P, pannus. (C) Quantification of synovitis, pannus formation, blood vessels in the synovium, and Mankin score (<i>n</i>>10). Values are means ± SEM (*<i>p</i><0.0005). Scale bar, 50 µm.</p

HIF-2α is up-regulated by pro-inflammatory cytokines in FLS of RA synovium.

<p>(A and B) Typical immunofluorescence microscopy images of DAPI, HIF-2α, and FLS markers (vimentin and CD55) or the macrophage marker CD68 in human RA synovium (A) and mouse CIA synovium (B). (C, Left) Primary culture of total synovial cells isolated from DBA/1J mice. DC, dendritic cells; MΦ, macrophages. (Center) Typical immunofluorescence microscopy images of HIF-2α, DAPI, and vimentin or CD68. (Right) The percentage of FLS and macrophages positive for HIF-2α staining was determined from six microscopic fields (<i>n</i> = 4). (D) Raw264.7 cells were treated with LPS (50 ng/ml) or TNFα (50 ng/ml) for 24 h. mRNA levels were detected by RT-PCR analysis (<i>n</i> = 6). (E) Primary cultured FLS were treated with IL1β (1 ng/ml), IL6 (100 ng/ml), IL17 (10 ng/ml), or TNFα (100 ng/ml) for 24 h. mRNA levels of HIF-1α and HIF-2α were quantified by qRT-PCR (<i>n</i> = 6). (F) FLS were treated with PD98059 (PD; 20 µM) to inhibit ERK, SB203580 (SB; 20 µM) to inhibit p38 MAP kinase, SP600125 (SP; 20 µM) to inhibit JNK, or the indicated concentration (µM) of BAY 11-7085 (BAY) to inhibit NF-κB. The cells were exposed to IL1β or TNFα for 24 h, and HIF-2α mRNA levels were quantified (<i>n</i> = 6). (G) Mouse CIA synovium was stained for the hypoxia marker pimonidazole (upper). Primary cultured FLS were maintained under hypoxic conditions or were infected with Ad-<i>Epas1</i> at an MOI of 800 for 24 h. HIF-1α and HIF-2α proteins were detected by Western blotting (lower). Values are presented as means ± SEM (*<i>p</i><0.01, **<i>p</i><0.005, ***<i>p</i><0.001). Scale bar, 50 µm.</p

HIF-2α up-regulates the expression of cytokines, chemokines, and catabolic enzymes in FLS.

<p>(A) qRT-PCR analysis (<i>n</i>≥8) of catabolic enzymes and chemokines in FLS infected with Ad-C (800 MOI) or with Ad-<i>Epas1</i> at the indicated MOI for 24 h. (B) qRT-PCR analysis (<i>n</i>≥8) of mRNA levels of HIF-2α, IL6, and TNFα (Left), and ELISA of secreted IL6 and TNFα proteins (Right) in FLS infected with Ad-C (800 MOI) or with Ad-<i>Epas1</i> at the indicated MOI for 24 h. (C) FLS were infected with Ad-C, Ad-<i>Epsa1</i>, or Ad-<i>Hif1a</i> (800 MOI) for 24 h. The indicated proteins were detected by Western blotting (<i>n</i> = 5). Values are means ± SEM (*<i>p</i><0.01, **<i>p</i><0.001).</p

HIF-2α regulates FLS proliferation, RANKL expression in FLS, osteoclastogenesis, and pannus formation.

<p>(A) BrdU-incorporation assays in FLS infected with Ad-C or Ad-<i>Epas1</i> (MOI 800) (left), and FLS from WT or <i>Epas1</i>^+/− mice treated with 1 ng/ml of IL1β (right) (<i>n</i> = 6). (B) Ki67 staining in synovial sections from WT and <i>Epas1</i>^+/− mice without (NI) or with CIA, or from WT mice injected with Ad-C or Ad-<i>Epas1</i> (MOI 800) (<i>n</i> = 8). (C) Double-immunofluorescence staining for HIF-2α and Ki67 in mouse CIA synovium. Ki67-positive cells among HIF-2α–overexpressing cells were counted (<i>n</i> = 8). (D) RANKL mRNA levels were quantified in FLS infected with Ad-C or Ad-<i>Epas1</i> (MOI 800) or treated with 1 ng/ml of IL1β (<i>n</i> = 10). (E) Representative images of RANKL immunostaining in the knee synovium of WT or <i>Epas1</i>^+/− mice without (NI) or with CIA (Left). Typical immunofluorescence microscopy image of triple-stained CIA synovium (Right). (F) TRAP staining and counting of TRAP-positive multinucleated cells (<i>n</i> = 10) in the pannus of the bone–cartilage interface in WT and <i>Epas1</i>^+/− mice without (NI) or with CIA, or following injection with 1×10⁹ PFU of Ad-C or Ad-<i>Epas1</i>. (G) TRAP staining during <i>in vitro</i> osteoclastogenesis of precursor cells isolated from WT and <i>Epas1</i>^+/− mice or WT precursor cells infected with Ad-C or Ad-<i>Epas1</i> (800 MOI) (Left). Osteoclastogenesis was quantified by measuring the osteoclast area (<i>n</i> = 10) (Right). Values are means ± SEM (*<i>p</i><0.01, **<i>p</i><0.001). Scale bar, 50 µm.</p