sNASP Mutation Aggravates to the TLR4-Mediated Inflammation in SLE by TAK1 Pathway

Abstract

Genetic factors play an important role in the pathogenesis of systemic lupus erythematosus (SLE), and abnormal Toll-like receptor (TLR) signaling pathways are closely related to the onset of SLE. In previous studies, we found that the mutant somatic nuclear autoantigenic sperm protein (sNASP) gene in the mouse lupus susceptibility locus Sle2 can promote the development of lupus model mice, but the mechanism is still unclear. Here, we stimulated mouse peritoneal macrophages with different concentrations of lipopolysaccharide. The results showed that sNASP gene mutations can promote the response of the TLR4-TAK1 signaling pathway but have no significant effect on the TLR4-TBK1 signaling pathway. sNASP mutations enhanced TLR4-mediated nuclear factor-κ-gene binding and mitogen-activated protein kinase activation and IL-6, tumor necrosis factor secretion in murine peritoneal macrophages. Collectively, our study revealed the impact of sNASP gene mutation on the sensitivity of TLR4 receptors in mouse peritoneal macrophages and shed light on potential mechanisms underlying inflammation in autoimmune diseases.

Figure 1

sNASP mutation promotes LPS-induced proinflammatory cytokine production in B6.lpr mice. The expression of mRNA ((a)–(c) and (g)–(i)) and protein ((d)–(f) and (j)–(k)) of IL-6 and TNF-α were measured by qPCR and ELISA in peritoneal macrophages, compared between B6.lpr and B6.∆sNASP.lpr mice. Following 0/0.5/1/2 hr stimulation with ((a), (d), (g), (j)) LPS 1 ng/ml, ((b), (e), (h), (k)) LPS 10 ng/ml, and ((c), (f), (i), (l)) LPS 100 ng/ml. Data were shown as means ± SEM (n = 3) of one representative experiment.  ^∗P < 0.05,  ^∗∗P < 0.01.

Figure 2

sNASP mutation augments LPS-induced the cytokine IL-6 and TNF-α expression in B6.WT mice. The peritoneal macrophages were treated with LPS for 0.5, 1, and 2 hr that IL-6 and TNF-α levels were analyzed by qPCR ((a)–(c), (g)–(i)) and ELISA ((d)–(f), (j)–(l)), compared between B6.WT and B6.∆sNASP mice. Following stimulation with ((a), (d), (g), (j)) LPS 1 ng/ml, ((b), (e), (h), (k)) LPS 10 ng/ml, and ((c), (f), (i), (l)) LPS 100 ng/ml. Data were shown as means ± SEM (n = 3) of one representative experiment.  ^∗P < 0.05,  ^∗∗P < 0.01.

Figure 3

sNASP mutation downregulates the protein of TRAF6 expression. The expression of TRAF6 was measured by western blot in peritoneal macrophages and compared among B6.lpr, B6.∆sNASP.lpr, B6.WT, and B6.∆sNASP mice, and mouse β-actin was used as control. Following 0/0.5/1/2 hr stimulation with ((a) and (d)) LPS (1 ng/ml), ((b) and (e)) LPS (10 ng/ml), ((c) and (f)) LPS (100 ng/ml). Data were shown as means ± SEM.  ^∗P < 0.05,  ^∗∗P < 0.01; independent sample t-test. Results shown are representative of 2–3 independent experiments.

Figure 4

sNASP mutation promotes TLR4-induced NF-κB and MAPK activation of the pathways in B6.lpr mice. The expression of phosphorylated and total proteins was measured by western blotting in peritoneal macrophages and compared between B6.lpr and B6.∆sNASP.lpr mice, and mouse β-actin was used as control. Following 0/0.5/1/2 hr stimulation with (a) LPS (1 ng/ml), (b) LPS (10 ng/ml), (c) LPS (100 ng/ml). Data were shown as means ± SEM.  ^∗P < 0.05,  ^∗∗P < 0.01; independent sample t-test. Results shown are representative of 2–3 independent experiments.

Figure 5

sNASP mutation accelerates the signaling of TLR4–TAK1 pathway in B6.WT mice. The phosphorylated and total protein levels were determined by immunoblotting in peritoneal macrophages following 0/0.5/1/2 hr stimulation with (a) LPS (1 ng/ml), (b) LPS (10 ng/ml), (c) LPS (100 ng/ml). Mouse β-actin was detected as control. Data were shown as means ± SEM.  ^∗P < 0.05,  ^∗∗P < 0.01; independent sample t-test. Results shown are representative of 2–3 independent experiments.

Figure 6

sNASP mutation has no influence on TLR4-induced IRF3 activation of the pathway. The expression of phosphorylated and total proteins were measured by western blotting in peritoneal macrophages and compared between ((a)–(c)) B6.lpr and B6.∆sNASP.lpr mice, ((d)–(f)) B6.WT and B6.∆sNASP mice, and mouse β-actin was used as an internal control. Following 0/0.5/1/2 hr stimulation with ((a) and (d)) LPS (1 ng/ml), ((b) and (e)) LPS (10 ng/ml), ((c) and (f)) LPS (100 ng/ml). Data were shown as means ± SEM.  ^∗P < 0.05,  ^∗∗P < 0.01; independent sample t-test. Results shown are representative of 2–3 independent experiments.

Figure 7

Model of TLR4 signaling pathway regulated by sNASP. sNASP mutation promotes LPS-induced TLR4–TAK1 activation in peritoneal macrophages and has no influence on TLR4–TBK1, resulting in the activation of NF-κB and MAPK signaling pathways and the expression of proinflammatory cytokines.