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

Cytl1 induces EndMT.

HCAECs were treated with TGF-β2 (10 ng/ml) or Ad-Cytl1 (50 moi) for 48 h. In selected experiments, the cells were pretreated with the TGF-β receptor 1 antagonist SB431542 (10 μM). (A) The cells were immunostained with antibodies against the endothelial marker CD31 and the MFB marker α-SMA. (B) Lysates of HCAECs infected with Ad-Cytl1 were analyzed by western blotting. CD31 and VE-cadherin served as endothelial cell markers, and vimentin served as the MFB marker. (C) Activation of the TGF-β signaling pathway was investigated by western blotting. GAPDH served as the loading control. (D) Quantification of the mRNA levels of several fibrotic markers (TGF-β2, Cytl1, collagen 1 and TNF-α) by qRT-PCR. n = 3–5 for each experimental group. *p p < 0.01.</p

Cytl1 functions independently of CCR2.

<p>Adult primary cardiac FBs were treated with CCL2 (20 ng/ml) or Ad-Cytl1 (50 moi) for 48 h. In selected experiments, the cells were pretreated with the CCR2 antagonist CAS445679-97-0 (6 nM). (A) The cells were immunostained with an antibody against α-SMA. (B) Quantification of the mRNA levels of TGF-β2, Cytl1, collagen 1 and α-SMA by qRT-PCR. n = 3–5 for each experimental group. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

Cytl1 induces transdifferentiation of FBs to MFBs.

<p>Adult primary cardiac FBs were treated with TGF-β2 (10 ng/ml) or Ad-Cytl1 (50 moi) for 48 h. In selected experiments, the cells were pretreated with SB431542 (10 μM). (A) The cells were immunostained with an antibody against the MFB marker α-SMA. (B) Activation of the TGF-β signaling pathway in primary cardiac FBs infected with Ad-Cytl was investigated by western blotting. GAPDH served as the loading control. (C) Quantification of the mRNA levels of TGF-β2, Cytl1, the MFB marker collagen 1 and α-SMA by qRT-PCR. n = 3–5 for each experimental group. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

AAV-mediated overexpression of Cytl1 induces CF.

<p>Control virus or AAV-Cytl1 (5 × 10¹⁰ viral genome) was injected into the tail vein of WT mice, and the phenotype of the heart was examined after 8 wks. (A) Picrosirius staining of heart cross-sections from WT mice injected with control virus or AAV-Cytl. Fibrotic areas in the interstitial and perivascular areas were quantified using MetaMorph software (right panels). (B) Quantification of the mRNA levels of several fibrotic markers (TGF-β2, collagen 1 and TNF-α) by qRT-PCR. (C) Activation of the TGF-β signaling pathway was investigated by western blotting. GAPDH served as the loading control. n = 3–5 for each experimental group. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

CF is attenuated in <i>cytl1</i> KO mice.

<p>WT and <i>cytl1</i> KO mice were subjected to TAC for 6 wks and the extent of fibrosis in the heart was analyzed. (A) Picrosirius staining of heart cross-sections from WT and <i>cytl1</i> KO mice subjected to TAC. Fibrotic areas in the interstitial and perivascular areas were quantified using MetaMorph software (right panels). (B) Quantification of the mRNA levels of several fibrotic markers (TGF-β2, collagen 1 and TNF-α) by qRT-PCR. (C) Activation of the TGF-β signaling pathway was investigated by western blotting. GAPDH served as the loading control. n = 3–5 for each experimental group. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

Cytokine-Like 1 Regulates Cardiac Fibrosis via Modulation of TGF-β Signaling

<div><p>Cytokine-like 1 (Cytl1) is a secreted protein that is involved in diverse biological processes. A comparative modeling study indicated that Cytl1 is structurally and functionally similar to monocyte chemoattractant protein 1 (MCP-1). As MCP-1 plays an important role in cardiac fibrosis (CF) and heart failure (HF), we investigated the role of Cytl1 in a mouse model of CF and HF. Cytl1 was upregulated in the failing mouse heart. Pressure overload-induced CF was significantly attenuated in <i>cytl1</i> knock-out (KO) mice compared to that from wild-type (WT) mice. By contrast, adeno-associated virus (AAV)-mediated overexpression of <i>cytl1</i> alone led to the development of CF <i>in vivo</i>. The endothelial-mesenchymal transition (EndMT) and the transdifferentiation of fibroblasts (FBs) to myofibroblasts (MFBs) have been suggested to contribute considerably to CF. Adenovirus-mediated overexpression of <i>cytl1</i> was sufficient to induce these two critical CF-related processes <i>in vitro</i>, which were completely abrogated by co-treatment with SB-431542, an antagonist of TGF-β receptor 1. Cytl1 induced the expression of TGF-β2 both <i>in vivo</i> and <i>in vitro</i>. Antagonizing the receptor for MCP-1, C-C chemokine receptor type 2 (CCR2), with CAS 445479-97-0 did not block the pro-fibrotic activity of Cytl1 <i>in vitro</i>. Collectively, our data suggest that Cytl1 plays an essential role in CF likely through activating the TGF-β-SMAD signaling pathway. Although the receptor for Cyt1l remains to be identified, Cytl1 provides a novel platform for the development of anti-CF therapies.</p></div

Cytl1 is upregulated under pathological conditions.

<p>Three groups of WT mice were subjected to TAC for 6 wks (TAC), ligation of coronary artery for 4 wks (MI), or ligation of coronary artery for 30 min followed by reperfusion for 24 hrs (I/R). Hearts were harvested and qRT-PCR was performed to determine the transcript levels of Cytl1. n = 3 for sham, n = 3 for TAC, n = 4 for MI, n = 4 for I/R. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

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

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