SOCS2 Suppresses Inflammation and Apoptosis during NASH Progression through Limiting NF-κB Activation in Macrophages

Abstract

Background: Inflammation and apoptosis play a crucial role in the progression of nonalcoholic steatohepatitis (NASH). Suppressor of cytokine signaling 2 (SOCS2) is one of classic negative regulators of cytokine signaling, which has recently been described as anti-inflammatory mediators. However, the role of SOCS2 in macrophages during NASH progression and the relationship among SOCS2, inflammation, apoptosis and NASH is largely unknown. Herein, we aimed to study the function of SOCS2 in NASH progression. Methods: We detected SOCS2 expression in macrophages in human subjects without steatosis, with simple steatosis and with NASH to confirm the relationship between SOCS2 and NASH. Free fatty acids was used to establish stress environment in RAW 264.7 cell lines stably overexpressing or knockdown SOCS2. In vitro and vivo assays also performed to study the molecular function of SOCS2 in NASH progression. Findings: Our human samples illustrated that SOCS2 was decreased in macrophages during NASH progression and was negatively correlated to NASH level. Meanwhile, In vitro assays showed SOCS2 overexpression in macrophages suppressed inflammation and apoptosis via inhibiting NF-κB signaling pathway, while SOCS2 knock-down in macrophages caused an increased activation of NF-κB, which could be blocked by ammonium 1-pyrrolidinedithiocarbamate (PDTC). In addition, SOCS2 in macrophages also suppressed inflammation via limiting the activation of inflammasomes. Consistent with these, our BMT model also confirmed the SOCS2 function in macrophages during NASH. Interpretation: Our data strongly indicate that SOCS2 plays a role in inhibiting inflammation and apoptosis via NF-κB and inflammasome signaling pathway in macrophages during NASH. Further studies are required to explore the potential preventive and therapeutic strategies of SOCS2 for this common liver disease.

Keywords: NF-κB; Nonalcoholic steatohepatitis; Suppressor of cytokine signaling 2; apoptosis; inflammation.

Figure 1

SOCS2 in macrophages was decreased in macorphages during NASH progression and negative related to NASH level. A. The heatmap from RNA-Seq of human liver macrophages with NASH (n=5) or Normal (n=5). B. The related markers expression were detect by RT-qPCR in human liver macrophages with NASH (n=6) or Normal (n=6). C. The related mRNA level of SOCS2 was detected by RT-qPCR in liver tissue macrophage samples of human subjects without steatosis, with simple steatosis and with NASH. D. Immunoblotting of SOCS2 and β-actin (loading control throughout) in liver tissue macrophage samples of human subjects without steatosis (n=3), with simple steatosis (n=3) and with NASH (n=3). E. Pearson comparison analyses of the correlation between SOCS2 mRNA level and NASH (r = -0.9471), BMI (r = -0.8542), serum TG content (r = -0.8908), serum γ-GT concentrations (r = -0.8406), serum AST concentrations (r = -0.9207) and serum ALT concentrations (r = -0.9112) (n = 153). P < 0.0001 for all of these correlations by Spearman′s rank correlation coefficient analysis.

Figure 2

SOCS2 suppresses inflammation via limiting the activation of NF-κB signaling pathway. A. The related protein and mRNA level of SOCS2 was detected by Western blot and RT-PCR in RAW cell lines overexpreesing SOCS2 (RAW-Ctrl/ SOCS2⁺). B. The related protein and mRNA level of SOCS2 was detected by Western blot and RT-PCR in RAW cell lines knockdown SOCS2 (RAW-Ctrl/ shSOCS2). C. The related mRNA level of TNFα, IL-1β, IL-6, IL-8, IL-17, IL-18 was detected by RT-PCR in RAW-Ctrl or -SOCS2⁺ or -shSOCS2 treated with FFAs for 24 hr. D. The level of TNFα, IL-1β, IL-6, IL-8, IL-17, IL-18 was detected by ELISA in RAW-Ctrl or -SOCS2⁺ or -shSOCS2 treated with FFAs for 24 hr. E. Immunoblotting of phospho-IKKα, IKKα, phospho-IKKβ, IKKβ, phospho-P65, P65, SOCS2, and β-actin (loading control) in RAW-Ctrl or -SOCS2⁺ or -shSOCS2 treated with FFAs for 24 hr. F. The levels of IL-1β, TNFα, IL-6, IL-8, IL-17, and IL-18 in cell culture supernatants from RAW-Ctrl and RAW-shSOCS2 cells treated with or without FFAs and/or PDTC for 24 hr were detected by ELISA.

Figure 3

SOCS2 in macrophages suppresses inflammation via limiting the activation of inflammasomes signaling pathway. A. Immunoblotting of NLRP3, Pro-Caspase-1, Caspase-1 p20/p22 and β-actin (loading control) in RAW-Ctrl or -SOCS2⁺ or -shSOCS2 treated with FFAs for 24 hr. B. The levels of IL-1β, TNFα, IL-6, IL-8, IL-17, and IL-18 in cell culture supernatants from RAW-Ctrl and RAW-shSOCS2 cells treated with or without FFAs and/or MCC950 for 24 hr were detected by ELISA. C. The related mRNA level of TNFα, IL-1β, IL-6, IL-8, IL-17, IL-18 was detected by RT-PCR in RAW-Ctrl and RAW-shSOCS2 cells treated with or without FFAs and/or MCC950 for 24 hr were detected by ELISA.

Figure 4

SOCS2 in macrophages suppresses apoptosis via limiting the activation of NF-κB signaling pathway. A. Immunoblotting of BAX, BCL-2, SOCS2 and β-actin (loading control) in RAW-Ctrl or -SOCS2⁺ or -shSOCS2 treated with FFAs for 24 hr. B. The level of apoptosis of RAW-Ctrl / SOCS2⁺ treated with FFAs or PDTC for 24 hr, detected by Flow cytometry.

Figure 5

Overexpression of SOCS2 in macrophages restrains the development of NASH in vivo. A. The Western diet-induced NASH model in BMT mice (n = 6 per group). B. The liver from the mice described in Figure 5A. Liver sections were stained with Hematoxylin and Eosin (H&E), Oil-Red O, Masson and IHC (anti-F4/80), Original magnification, ×20. Bar = 200 μm. C. Quantitative analysis for Figure 5B of staining was shown. D. The level of serum TNFα, IL-1β, IL-6, IL-8, IL-17, IL-18 were determined in the mice described in Figure 5B by ELISA assay (n = 6 per group). E. Immunoblotting of phospho-IKKα, IKKα, phospho-IKKβ, IKKβ, phospho-P65, P65, SOCS2, and β-actin (loading control) in macrophage isolated from mice induced NASH (n = 6 per group).