Since the discovery of sirtuins as longevity determination genes in yeast over a decade ago, much effort has been devoted to elucidating the molecular mechanisms and physiological functions of their mammalian homologs. Recently, evidence has emerged pointing toward an association between SIRT1 and the NF-κB transcription factor, which makes targeting this signaling pathway an intriguing possibility for the treatment of chronic inflammatory diseases (29
). In our current study, we used a Mac-SIRT1 KO mouse model to explore SIRT1's role in the regulation of the NF-κB-mediated inflammatory response. We demonstrated that SIRT1 plays an important role in controlling cytokine production in the wake of environmental stimuli, such as TNF-α, bacterial endotoxin, and dietary lipids. Importantly, our study used a novel mouse model to further verify that SIRT1 regulates this in vivo
anti-inflammatory effect by modulating NF-κB gene transcription in immune cells, namely, macrophages.
Our findings suggest that SIRT1 plays a protective role against chronic inflammation and thus development of metabolic syndrome, an observation supported by several independent lines of evidence. First, Mac-SIRT1 KO BMDMs display elevated levels of hyperacetylated nuclear NF-κB. Second, hyperactive NF-κB signaling in macrophages contributed to elevated transcriptional activity and proinflammatory cytokine production. Third, knockdown of NF-κB in Mac-SIRT1 KO and control macrophages results in similar cytokine production in the cell groups, demonstrating that SIRT1 functions primarily through NF-κB. Fourth, loss of SIRT1 leads to compensatory SIRT6 deacetylase activity on histone H3 lysine 9. Finally, the chronic inflammatory phenotype observed in Mac-SIRT1 KO mice is independent of metabolic conditions. However, HFD-induced obesity exacerbates metabolic abnormalities in Mac-SIRT1 KO mice and leads to systemic abrogated insulin signaling.
It is widely reported that NF-κB is subject to regulation by acetylation and to deacetylation by HDACs, including SIRT1 (8
). We note that the promiscuous nature of SIRT1 deacetylase activity at both histone and nonhistone substrates most likely reflects a transient enzyme-substrate interaction. In this context, it appears SIRT1 association at NF-κB target gene promoter sites is an important regulatory mechanism involved in the modulation of this transcription factor. In support of these notions, TSA treatment abrogates deacetylation of NF-κB in Mac-SIRT1 KO macrophages, further highlighting the dynamic nature of this process (Fig. ). Unlike SIRT1, SIRT6 exerts deacetylase activity on histone H3K9 (Fig. ), demonstrating that SIRT1 and SIRT6 act upon NF-κB via distinct molecular mechanisms. These findings imply that the combinatory actions of both sirtuins modulate NF-κB transcriptional activity and thus regulate the immune signaling pathway (Fig. ).
Additional experiments with both primary macrophages and Mac-SIRT1 KO mice revealed that the proinflammatory phenotype associated with myeloid deletion of SIRT1 was independent of metabolic conditions. For example, Mac-SIRT1-deficient mice displayed hyperactive immune signaling in response to both aerosol and systemic LPS injections on a standard diet, where no metabolic defects were observed (Fig. ). These same chow-fed mice showed no signs of obesity or metabolic derangements, while displaying a heightened state of inflammatory response. Additionally, primary bone marrow-derived macrophages from Mac-SIRT1 KO mice expressed higher basal levels of proinflammatory cytokines (Fig. ). These findings indicate that SIRT1 deficiency induces an intrinsic proinflammatory status in macrophages. Additional data from our group (not shown) indicate that SIRT1-deficient macrophages display disruptions in fatty acid oxidative metabolism. It is likely that the intrinsic chronic inflammation in Mac-SIRT1 KO mice primes diet-induced obesity, elevated resting leptin levels, and insulin resistance (Fig. ). Consistent with this notion, excess proinflammatory cytokines have been shown to trigger insulin resistance in animals (9
). This chronic activation may be the cause of insulin resistance. Since NF-κB is a transcription factor involved in a vast array of disease states, understanding its interactions with sirtuins may provide therapeutic benefits.
Excess proinflammatory cytokines have been shown to trigger insulin resistance in animals (10
). In vitro
cell culture studies have shown that TNF-α can render cells insulin resistant through downregulation of the synthesis of the glucose transporter, as well as through interference with insulin signaling (34
). Studies have also shown that mice lacking the TNF-α receptor have improved capacity to facilitate glucose uptake in metabolic tissues (35
). Interestingly, it has been shown that caloric restriction attenuates the age-related upregulation of nuclear factor NF-κB, which is known to induce transcription of TNF-α in adipose tissue and the production of inflammatory cytokines in immune cells (20
). It is possible that upregulation of SIRT1 may contribute to increases in insulin sensitivity and reduction in inflammation.
In summary, we have demonstrated an important role for SIRT1 in immune signaling. In the absence of SIRT1, macrophages display hyperactive NF-κB, leading to increased transcription of proinflammatory genes. Our findings identify SIRT1 as an important link between environmental stress and immune system activation. It will be interesting to investigate how pharmacological activators of sirtuins affect diseases associated with chronic inflammation.