Background

Sirtuins, NAD⁺-dependent deacetylases, have been implicated in many biological processes, including inflammation. In the context of the inflammatory response, SIRT6 in particular has attracted attention due to its apparently coexisting and opposite pro- and anti-inflammatory activities (Kawahara et al., 2009; Xiao et al., 2012; Jiang et al., 2013; 2016). Initially, and similarly to other sirtuins, SIRT6 was proposed to inhibit inflammation by silencing NFκB and c-Jun–dependent transcription. Through its deacetylating enzymatic activity on Ac-H3K9, SIRT6 indirectly regulates cytokine expression (Kawahara et al., 2009; Bauer et al., 2012; Xiao et al., 2012). Recently, it has been reported that SIRT6 exerts pro-inflammatory functions through its ability to remove fatty acid groups from proteins in the cytoplasmic compartment. In particular, SIRT6 promotes tumor necrosis factor (TNFα) secretion by the demyristoylation of pro-TNFα in fibroblasts and macrophages during lipopolysaccharide(LPS)-mediated acute inflammatory response (Jiang et al., 2013; Jiang et al., 2016; Bresque et al., 2022). In addition, SIRT6 is actively regulated during acute inflammation in vivo, and its inhibition dampens TNFα secretion, reducing LPS-induced septic shock, obesity-induced systemic inflammation, and progression of experimental autoimmune encephalomyelitis (EAE) (Sociali et al., 2017; Ferrara et al., 2020; Bresque et al., 2022).

Most studies focusing on the role of SIRT6 in inflammation have been performed in vitro and have not measured changes in its localization and expression during the inflammatory response. Recently, studies regarding conditions for SIRT6 ablation have been developed to assess SIRT6 relevance in chronic inflammation and its consequences on immune cell populations in vivo (Lee et al., 2017; Ferrara et al., 2020). The peritoneal cavity provides a large number of differentiated macrophages and is a useful tool for studying in vivo immune responses. Recently, we demonstrated, by using flow cytometry and immunofluorescence, that there was an increase in both SIRT6-positive cells and SIRT6 fluorescence intensity in CD11b+F4/80^hi peritoneal macrophages stimulated by LPS. Our work is the first to apply flow cytometry to quantify SIRT6 levels in vivo in macrophages isolated from LPS-stimulated mice peritoneum (Bresque et al., 2022). Thus, this protocol sheds light on this issue, providing a new tool to study SIRT6 expression in immune and potentially other cells. This flow cytometry protocol, which employs a commercially available anti-SIRT6 antibody, offers a robust method to quantify the levels of an intracellular protein and may be applied to a wide variety of samples, including macrophages from adipose tissue.