The qPCR results indicate that Klf3, Klf4, Klf6, and Klf13 exhibited a minor or no increase, whereas Klf10 and Klf11 significantly

decreased (Fig. 1B). In addition, KLF expression and response to LPS were Ibrutinib solubility dmso investigated in GM-BMMs, and the result was similar to that in M-BMMs (Supporting Information Fig. 1). The decline in Klf10 expression in M-BMMs was further verified by western blot analysis (Fig. 1C). This Klf10 downregulation can be induced by LPS even with a concentration as low as 10 ng/mL (Fig. 1D). LPS is a ligand for TLR4, which localizes on the cell surface. Klf10 expression also decreased when TLR3 and TLR9, located in intracellular vesicles [31], were activated by poly I:C and CpG (Supporting Information Deforolimus Fig. 2). TLR stimulation can result in NF-κB activation, and our observation reveals that Klf10 can respond sensitively to these TLRs. Klf10 is an NF-κB-targeted gene [32]. Thus, we further demonstrate that Klf10 was downregulated in an NF-κB-dependent manner. We pretreated M-BMMs with BAY11–7082, an IκB-α inhibitor, to repress the NF-κB pathway

and found that the decrease in Klf10 after LPS challenge can be abrogated (Fig. 1E). Meanwhile, the upregulation of inflammatory cytokines, such as IL-12p40 and IL-6, was abolished (Fig. 1E). These results indicate that klf10 may participate in TLRs and may control the production of inflammatory factors in M-BMMs. Klf10 was overexpressed in M-BMMs to investigate whether it is involved in the regulation of inflammatory cytokines triggered by TLR4 signaling. The result shows that LPS-induced IL-12p40 was significantly inhibited at both the mRNA and protein levels, which also resulted in a decrease in IL-12p70. However, BCKDHB IL-12p35, the other subunit of IL-12p70, was unaffected (Fig. 2A). Other proinflammatory mediators, such as IL-6 and TNF-α, were slightly affected or unaffected by Klf10 (Fig. 2A and B). IL-10 is a key antiinflammatory factor that

can suppress IL-12 and IL-6 expressions in M-BMMs. Thus, we found Klf10 had no effect on IL-10 (Fig. 2B), indicating that the suppression of IL-12p40 and IL-6 was not mediate by IL-10. These observations indicated that Klf10 overexpression inhibited the production of IL-12p40 induced by TLR4 signaling in M-BMMs. We further performed the loss of function assay with Klf10-deficient mice to verify the aforementioned observation. Surface markers of M-BMMs from WT and Klf10-deficient mice were first characterized by flow cytometry. The result reveal that the proportion of F4/80+CD11b+ mature M-BMMs did not differ between these two markers, indicating that Klf10 was not involved in the differentiation of M-BMMs (Supporting Information Fig. 3A). Moreover, we investigated the markers on M-BMMs such as costimulatory molecules CD80, CD86, TLR4 receptor, and MHC class II, and found that these markers were expressed normally (Supporting Information Fig. 3B).