Inhibition of SIK2 and SIK3 during differentiation enhances the anti-inflammatory phenotype of macrophages

Abstract

The salt-inducible kinases (SIKs) control a novel molecular switch regulating macrophage polarization. Pharmacological inhibition of the SIKs induces a macrophage phenotype characterized by the secretion of high levels of anti-inflammatory cytokines, including interleukin (IL)-10, and the secretion of very low levels of pro-inflammatory cytokines, such as tumour necrosis factor α. The SIKs, therefore, represent attractive new drug targets for the treatment of macrophage-driven diseases, but which of the three isoforms, SIK1, SIK2 or SIK3, would be appropriate to target remains unknown. To address this question, we developed knock-in (KI) mice for SIK1, SIK2 and SIK3, in which we introduced a mutation that renders the enzymes catalytically inactive. Characterization of primary macrophages from the single and double KI mice established that all three SIK isoforms, and in particular SIK2 and SIK3, contribute to macrophage polarization. Moreover, we discovered that inhibition of SIK2 and SIK3 during macrophage differentiation greatly enhanced the production of IL-10 compared with their inhibition in mature macrophages. Interestingly, macrophages differentiated in the presence of SIK inhibitors, MRT199665 and HG-9-91-01, still produced very large amounts of IL-10, but very low levels of pro-inflammatory cytokines, even after the SIKs had been reactivated by removal of the drugs. Our data highlight an integral role for SIK2 and SIK3 in innate immunity by preventing the differentiation of macrophages into a potent and stable anti-inflammatory phenotype.

A soy-based probiotic drink modulates the microbiota and reduces body weight gain in diet-induced obese mice

“…The E. faecium CRL 183 strain had already been found to maintain the adipocyte morphology in rats induced to hyperlipidemia (Manzoni et al, 2005), confirming its effect on the control of obesity. Resident macrophages in adipose tissue are classified into two subtypes: M1 (classically activated), characterized by production of high levels of pro-inflammatory cytokines, and M2 (alternatively activated), which produce anti-inflammatory mediators and are dominant in lean adipose tissue (Darling et al, 2017;Lumeng, Bodzin, & Saltiel, 2007;Xu, 2013). In adipose tissue, obesity causes a change in the macrophage profile resulting in the prevalence of the M1 phenotype and accordingly, the production of inflammatory cytokines (TNF, IL-1, IL-6).…”

“…The primers used for gene expression quantification of pro-and antiinflammatory cytokines were: IL-6: Foward (F): 5′-TTCCATCCAGTTG CCTTCTTG-3′ and Reverse (R): 5′-AGGTCTGTTGGGAGTGGTATC-3′ (McGuire et al, 2016); IL-1β: F: 5′-CATCCAGCTTCAAATCTCGCAG-3′ and R: 5′-CACACACCAGCA GGTTATCATC-3′ (Liu et al, 2017); IL-10: F: 5′-CCCTTTGCTAT GGTGTCCTTTC-3′ and R: 5′-GATCTCCCTGGTTTCT CTTCCC-3′ (Darling, Toth, Arthur, & Clark, 2017); TGF-β: F: 5′-TGAC GTCACTGGAGTTGTACGG-3′ and R: 5′-GGTTC ATGTCATGGATGG TGC-3′ (Xu et al, 2017); TNF-α: F: 5′-CATCTTCTCAAAAT TCGAGTGA CAA-3′ and R: 5′-TGGGAGTAGACAAGGTACAACCC-3′ (Nikolaidis et al, 2010); GAPDH: F: 5′-AACTTTGGCATTGTGGAAGG-3′ and R: 5′-ACACATTGGGGGTAGGAACA-3′ (Liu, Guan & Ma, 2007). The conditions of the reaction were 50°C for two minutes, 95°C for 10 min and 40 repetitions of 95°C for 15 s and 60°C for one minute.…”

Salt-inducible kinases (SIKs) regulate TGFβ-mediated transcriptional and apoptotic responses

“…The catalytic activity of SIK isoforms can be ablated via mutation of the activation loop threonine to alanine 39 , which abolishes LKB1-mediated phosphorylation. Indeed, in mouse embryonic fibroblasts (MEFs) derived from embryos harbouring homozygous SIK2 T175A and SIK3 T163A genotypes 39 , the phosphorylation of CRTC3 at S370 is substantially reduced compared with WT control MEFs ( Fig. 4d).…”

“…SIK2 T175A /SIK3 T163A homozygous kinase dead knock-in (KI) mice were bred from SIK2 tm1.1Arte and SIK3 tm1.1Arte mice maintained on a C57BL/6NJ genetic background as described previously 39 . Primary mouse embryonic fibroblasts (MEFs) were generated from SIK2 T175A / SIK3 T163A or SIK3 WT embryos at E11.5-13.5 as detailed previously 62 .…”

Salt-inducible kinases (SIKs) regulate TGFβ-mediated transcriptional and apoptotic responses

“…The catalytic activity of SIK isoforms can be ablated via mutation of the activation loop threonine to alanine 39 , which abolishes LKB1-mediated phosphorylation. Indeed, in mouse embryonic fibroblasts (MEFs) derived from embryos harbouring homozygous SIK2 T175A and SIK3 T163A genotypes 39 , the phosphorylation of CRTC3 at S370 is substantially reduced compared to WT control MEFs (Figure 4D). A time-course treatment WT MEFs with TGFβ 1 resulted in robust SMAD3 phosphorylation and increase in PAI-1 protein levels at 6 hours (Figure 4E).…”