70 Chapter 3 have been deposited on the PASSEL server with the data identifier PASS04816. Data access is available upon request. RESULTS KDAC inhibition inhibits alveolar macrophage activation upon LPS stimulation Previous work from Leus et al. showed that class I KDACs (KDAC 1, 2, 3 and 8), and in particular KDAC3, play a key role in pro-inflammatory gene expression in monocyte-derived RAW 264.7 macrophages and precision-cut lung slices (20,21). To study their influence on the pro- and anti-inflammatory responses in alveolar-like macrophages, we used MS275 (KDAC1,2, and 3 inhibitor) and RGFP966 (KDAC3 inhibitor) (34) in combination with LPS exposure. We first investigated the effects of these treatments on the excretion of the cytokines TNF-α and IL-10 in the culture supernatant. LPS treatment resulted in a significantly more excretion of the pro-inflammatory cytokine TNFα compared to vehicle-treated controls, which was inhibited by pretreatment with KDAC3 inhibitor RGFP966 but not by pretreatment with KDAC1,2 and 3 inhibitor MS275 (Figure 2A). Similar findings were found for IL-6 (supplementary data, Supplementary Figure 1). Conversely, LPS treatment resulted in lower excretion of the antiinflammatory cytokine IL-10 compared to vehicle-treated control (Figure 2B). Again, only KDAC3 inhibitor RGFP966 inhibited this effect of LPS significantly, while MS275 had a minor effect that was not statistically significant. This was specific for the combination of LPS and RGFP966 as treatment of unstimulated macrophages with either KDAC inhibitor alone did not induce IL-10 excretion (supplementary data Supplementary Figure 1) We subsequently investigated the effect of KDAC inhibition on the expression of additional anti-inflammatory factors, (i.e. SOCS3 and TGFβ) (35–37) by qPCR to confirm an anti-inflammatory phenotypic shift in alveolar-like macrophages after treatment with KDAC inhibitors upon LPS treatment. SOCS3 is an important negative regulator of innate immune responses and is induced together with pro-inflammatory cytokines during LPS stimulation in macrophages to control inflammation and eventually induce resolution (38–40). LPS treatment indeed induced the expression of SOCS3 in alveolar-like macrophages and this induction was enhanced when macrophages were pretreated with KDAC1/2/3 inhibitor MS275, but not with KDAC3 inhibitor RGFP966 (Figure 3A). TGFβ, produced in an autocrine manner by adult alveolar macrophages, plays a critical role in alveolar macrophage development (41) and is also an important anti-inflammatory
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