TEC regulates macrophage activation dependent on PPARγ
Previous studies have shown that TEC is a partial peroxisome proliferator-activated receptor-γ (PPARγ) agonist with an IC50 value of 13.3 µM[31]. PPARγ activation inhibits the production of TNF-α and IL-1β in monocytes and macrophages, and promotes alternative macrophage activation[32].
To confirm the regulation of TEC in KCs, we used primary KCs isolated from C57BL/6J mice. In vitro, TEC treatment inhibited LPS-induced activation of KCs, which proved that TEC markedly increased the M2 markers (IL-10, Arg-1, Retnla, CD163 and CD80) and decreased the M1 markers (TNF-α, INF-γ, CCL2, CXCL1 and iNOS) (Figure 4A). Then, GW9662 (PPARγ inhibitor) was used to confirm that TEC regulation of KCs polarity was dependent on PPARγ. Our results showed that TEC treatment did not further influence macrophage activation (TNF-α, CXCL1, iNOS and CCL2) and macrophage alternative activation (IL-10, Arg-1, Retnla, CD206) when treated with GW9662 in the presence of LPS (Figure 4B). Analogical results were obtained by cotreatment with GW1929 (PPARγ agonist) and TEC (Figure S1A and S1B). Importantly, PPARγ expression deficiency dramatically suppressed TEC-mediated inhibition of macrophage activation in the presence of LPS (Figure 4C and 4D).
It is worth noting that BMDMs infiltrated the liver under CLD conditions. Thus, BMDMs were isolated from mice, and treated with TEC and/or ILPS. As expected, TEC increased the ratio of CD206/CD80, decreased M1 marker genes (TNF-α, iNOS, IL-1β and CCL2) and upregulated the expression of M2 marker genes (IL-10, Arg-1, Retnla and CD163) (Figure S2A). BMDMs treated with shPPARγ and TEC supported our findings that TEC did not inhibit LPS-induced macrophage activation when PPARγ was knocked out (Figure S2B).
Accumulated evidence suggests that PPARγ attenuates inflammation by suppressing NFκb activity. Mechanically, PPARγ is an E3 ligase that induces the degradation of NFκb-p65 to terminate NFκB signaling pathway-elicited inflammation[33]. In light of this, we further determined whether TEC-regulated macrophage activation was dependent on the PPARγ/NFκb pathway. BAY 11-7085 (NFκb inhibitor) and/or TEC were used to treat KCs in the presence of LPS (Figure S1B). Our results showed that inhibition of NFκb activity abolished the TEC-mediated regulation of KCs activation.
Taken together, these results indicate that TEC notably inhibited LPS-induced activation of macrophages. Moreover, TEC regulated macrophage activation which was dependent on activating PPARγ.