SIRT5 is important for bacterial infection by regulating insulin secretion and glucose homeostasis

Dear Editor, Recent studies indicate that cellular metabolism plays a key role in supporting immune cell development, maintenance and function (Norata et al., 2015). For example, sensing of microbial ligands by macrophages triggers increased glucose metabolism, which delivers energy to support antimicrobial inflammation and the production of cytokines. This effect further enhances the generation of mitochondrial reactive oxygen species (ROS) to fight infections. Tucey et al. show that maintaining host glucose homeostasis is important to prevent life-threatening fungal infection (Tucey et al., 2018). This phenomenon emerges as a bedrock of the emerging concept of how metabolism and immunity are integrated based on bioenergy requirements. Sirtuins contribute to dynamic shifts in immunity, metabolism, and bioenergetics during inflammation (Vachharajani et al., 2016). Therefore, targeting Sirtuins is likely to deliver new opportunities for manipulating immunometabolism as an anti-inflammatory strategy. However, better understanding of Sirtuin biology and its role in regulation of inflammation is in its infancy. SIRT1 acts as a key control point for regulating metabolism and inflammatory outputs from T cells (Liu et al., 2012). In our previous work, we demonstrated that SIRT5 plays an important role in preventing dextran sulfate sodium (DSS)-induced colitis by regulating glycolysis in macrophages (Wang et al., 2017). Inflammation in DSS-induced colitis is not of infectious origin. We would like to know the function of SIRT5 in infection-induced inflammation and host defense. To study the role of SIRT5 in infection response, we treated littermate wild-type (WT) and Sirt5-deficient (Sirt5) mice with lipopolysaccharide (LPS). When Sirt5 mice were treated with LPS for 3 h, the level of blood glucose decreased and the level of serum insulin increased significantly (Fig. 1A and 1B), accompanied with increased concentration of circulating IL-1β (Fig. 1C). Peritoneal macrophages (PMs) from Sirt5 mice treated with LPS also expressed higher level of Il-1β mRNA and secreted more IL1β (Fig. S1A and S1B). These results indicated that SIRT5 has a potential role in regulating glucose homeostasis and Sirt5 deficiency boosts IL-1β production in inflammatory response. Then, we wanted to know the source of the elevated IL1β. The gating strategies of flow cytometry for immune cells have been shown in Fig. S1C. We found that the F4/80/ CD11b macrophages accounted for the largest proportion of the immune cells isolated from peritoneal cavity of Sirt5 mice treated with LPS (Fig. S1D), which is consistent with a previous interesting report (Dror et al., 2017). Depletion of the peritoneal macrophages by intraperitoneal injection of clodronate liposomes (Fig. S1E) eliminated the effect induced by LPS treatment in Sirt5 mice. The blood glucose, circulating insulin and IL-1β level in LPS-treated Sirt5 mice showed no significant difference with that in WT mice (Fig. 1D–F). Therefore, LPS induced elevation of IL-1β in Sirt5 mice is mainly from the intraperitoneal macrophages. The NLRP3 inflammasome was shown to be a pivotal player in sepsis and the activation of the NLRP3 inflammasome cause IL-1β and IL-18 production (Ting et al., 2008). So, we assumed that the inflammasome activation might be enhanced by LPS treatment in Sirt5 macrophages. In Sirt5 bone marrow derived macrophages (BMDMs) cells, treatment with LPS did not change the level of SIRT5 protein, while significantly upregulated mRNAs level of NRLP3 and IL-1β, compared to the WT counterparts (Fig. S2A–C). With LPS priming, ATP promoted the cleavage of caspase 1 and the maturation of IL-1β. The result showed both signal 1 and signal 2 of the NLRP3 inflammasome were activated (Fig. 1G) (Hao et al., 2017). The IL-1β released into BMDMs supernatant was confirmed by ELISA (Fig. 1I). While the caspase-1 inhibitor zYVAD abolished IL-1β release in both WT and Sirt5 BMDMs (Fig. 1G and 1H). Furthermore, we investigated the mechanisms involved in inflammasome activation. ROS are pivotal for inflammasome activation (Schroder and Tschopp, 2010). Treatment with LPS and ATP increased ROS production in Sirt5 macrophages when compared to WT macrophages, and this was inhibited by ROS inhibitor N-acetyl-cysteine (NAC) and NAPDH oxidase inhibitor diphenyleneiodonium (DPI) (Fig. 1I). Inhibition of ROS production also suppressed IL-1β release as assessed by ELISA (Fig. 1J). Together, these findings reveal that LPS-

macrophages with one multiplicities of infection (MOI) of the S. typhimurium in low glucose (5mM glucose) or high glucose medium, non-phagocytosed bacterial were removed by washing three time with PBS; the cells were stained with 0.6 µM DRAQ7 (US Everbright Inc) in the corresponding medium and observed under a Leica AF6000 LX epifluorescence microscope for 24 h. The cell death data were analyzed and quantified using ImageJ.

RNA isolation and quantitative real-time PCR
Total RNA was isolated by Trizol according to manufacturer instructions. RNA was reverse-transcribed with oligo-dT primers and preceded to real-time PCR with genespecific primers in the presence of SYBR Premix Ex Taq (Takara). Target gene expression was normalized to the housekeeping gene and relative quantitation values were calculated using the ΔΔ-CT method.

Supernatant or serum was collected and ELISA performed according to manufacturers'
instructions. For testing insulin levels, add 75 μl of Conjugate to each well, add 5 μl of each standard, control, and sample into their respective wells, then incubate for 2 h at room temperature with shaking at 700-900 rpm. After washing the microplate 6 times, add 100 μl of TMB Substrate into each well and incubate for 15 min at room temperature with shaking at 700-900 rpm. Finally, add 100 μl of Stop Solution and read at 450 nm within 30 min. For testing IL-1β levels, wash microwell strips twice with Wash Buffer. Add 100 μl of each standard, blank and sample into their respective wells, add 50 μl Biotin-Conjugate to all wells, then incubate for 2 h at room temperature. After washing the microplate 4 times, add 100 μl Streptavidin-HRP to all wells and incubate 60 min at room temperature. After washing the microplate 4 times again, add 100 μl of TMB Substrate Solution into all wells and incubate for 10 min at room temperature.
Finally, add 100 μl of Stop Solution and read at 450 nm within 30 min.

Western blotting
Protein concentration was quantified by the BCA kit (Thermo Fisher Scientific) and then subjected to SDS-PAGE and membrane transfer. The transferred membrane was blocked with 5% no-fat milk for 1 h at room temperature, and then primary antibody was incubated at 4°C overnight. Secondary antibody was incubated at room temperature for 1 h, and finally enhanced chemi luminescence (ECL) auto-development was performed.

Islet preparation and insulin secretion assay
Pancreatic islets were isolated from 8-to 12-week-old wild-type and Sirt5 -/mice by collagenase digestion and density-gradient centrifugation. Isolated islets were cultured with indicated reagents in RPMI 1640 medium (0.25% bovine serum albumin).
For insulin secretion assay, freshly isolated and incubated islets were washed twice with glucose-free phosphate buffer and were pre-incubated in Krebs-Ringer Buffer (KRB) containing 3.3mM glucose for 30 min. Then, ten islets per assay in triplicate were incubated with KRB buffer containing either 3.3mM glucose, 16.7mM glucose, or other reagents as indicated (LPS, 100ng/ml or IL-1β, 2.5ng/ml) for 1 h at 37°C. Supernatants containing insulin were removed and stored at − 20 °C until analysis. Insulin content was extracted with acid-ethanol. Insulin levels of all samples were measured by ELISA kit.