Sirtuin 3 deficiency does not alter host defenses against bacterial and fungal infections

Sirtuin 3 (SIRT3) is the main mitochondrial deacetylase. SIRT3 regulates cell metabolism and redox homeostasis, and protects from aging and age-associated pathologies. SIRT3 may drive both oncogenic and tumor-suppressive effects. SIRT3 deficiency has been reported to promote chronic inflammation-related disorders, but whether SIRT3 impacts on innate immune responses and host defenses against infections remains essentially unknown. This aspect is of primary importance considering the great interest in developing SIRT3-targeted therapies. Using SIRT3 knockout mice, we show that SIRT3 deficiency does not affect immune cell development and microbial ligand-induced proliferation and cytokine production by splenocytes, macrophages and dendritic cells. Going well along with these observations, SIRT3 deficiency has no major impact on cytokine production, bacterial burden and survival of mice subjected to endotoxemia, Escherichia coli peritonitis, Klebsiella pneumoniae pneumonia, listeriosis and candidiasis of diverse severity. These data suggest that SIRT3 is not critical to fight infections and support the safety of SIRT3-directed therapies based on SIRT3 activators or inhibitors for treating metabolic, oncologic and neurodegenerative diseases without putting patients at risk of infection.

The innate immune system provides the first line of defense against microbial infections. Innate immune cells such as macrophages and dendritic cells (DCs) detect invading microorganisms through pattern recognition receptors (PRRs). The best-characterized family of PRRs is constituted by Toll-like receptors (TLRs), which mediate the sensing of a broad range of microbial structures 1 . The interaction between PRRs and microbial ligands activates intracellular signaling pathways that coordinate the expression of immune-regulatory genes among which cytokines/chemokines, and the development of humoral and cellular responses required to neutralize or eliminate pathogens and restore homeostasis.
Sirtuins (SIRT1-7) belong to the NAD + -dependent class III subfamily of histone deacetylases (HDACs) 2 . Besides histones, sirtuins target thousands of non-histone proteins, among which chromatin modifiers, transcription regulators, signal transduction molecules, metabolic enzymes and structural cell components 3 . SIRT1-7 localize in the cytosol, nucleus and/or mitochondria, which dictates their accessibility to substrates and effector functions.
In the present study, we used SIRT3 knockout mice to investigate whether SIRT3 deficiency altered the response of immune cells to microbial ligands in vitro. We then analyzed the impact of SIRT3 deficiency in a panel of severe and non-severe models of endotoxemia, peritonitis, pneumonia, listeriosis and candidiasis. Overall, our results suggest that SIRT3 deficiency has no major impact on host defenses against infections, supporting the safety of SIRT3-oriented therapies currently under development.

Materials and Methods
Ethics statement. Animal
Flow cytometry. Single cell suspensions from thymus and spleen were incubated with 2.4G2 monoclonal antibody (mAb) and stained using mAbs listed in Supplementary Table S1 as described previously 55 . Data were acquired using a LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo Version 8.5.3 software (FlowJo LLC, Ashland OR).
Proliferation assay. Splenocytes were cultured in 96-well plates for 48 hours with different stimuli and proliferation quantified by measuring 3 H-thymidine incorporation over 18 hours 56 .
Scientific RepoRts | 7: 3853 | DOI:10.1038/s41598-017-04263-x Statistical analyses. Comparisons between groups were performed using the ANOVA F-test followed by two-tailed unpaired Student's t-test. Survival curves were built using the Kaplan-Meier method and differences were analyzed by the log-rank sum test. All analyses were performed using PRISM (GraphPad, San Diego, CA). P values were two-sided and significance level was set at 0.05.

SIRT3 deficiency does not affect the course of endotoxemia and bacterial and fungal infections.
To address the relevance of our in vitro findings, we developed a model of endotoxemia by challenging mice i.p. with 20 mg/kg LPS (Fig. 3A and B). TNF and IL-12p40 concentrations in blood collected from SIRT3 +/+ and SIRT3 −/− mice 1 hour (TNF) and 6 hours (IL-6) post-challenge were comparable (Fig. 3A). In line with these results, the overall survival of SIRT3 +/+ and SIRT3 −/− mice was similar (75% vs 89%, P = 0.4, Fig. 3B).

Discussion
We report that SIRT3 deficiency has no major impact on immune cell development and host defenses against bacterial and fungal infections. These observations are particularly topical considering the promises of SIRT3-targeting strategies to treat age-related disorders. SIRT3 deficiency did not alter the composition of the main lymphoid and DC subsets in thymus and spleen, in line with a previous study showing normal thymic, splenic and lymph node CD4 + and CD8 + T-cell subpopulations in SIRT3 −/− mice, including CD4 + Foxp3 + T regulatory cells (Tregs) 29 . Immune cells exposed to microbial products or cytokines undergo metabolic reprogramming characterized by a switch from oxidative Figure 2. SIRT3 does not affect TNF and IL-6 production by BMDMs and BMDCs exposed to microbial stimuli. SIRT3 +/+ and SIRT3 −/− BMDMs (A,B) and BMDCs (C) were exposed to LPS (10 ng/ml), Pam 3 CSK 4 (10 ng/ml), CpG (2 µg/ml), E. coli (10 6 CFU/ml) and GBS (2.5 × 10 6 CFU/ml). (A) Expression levels of phosphorylated (p) and total ERK1/2, JNK and p38 were analyzed by Western blotting and quantified by imaging. Data are means ± SD obtained with 3 mice. Full-length blots are presented in Supplementary Figure     phosphorylation to glycolysis associated with the development of inflammatory and antimicrobial functions 59 . Considering that SIRT3 impacts on mitochondrial biogenesis and functions 60 , we expected SIRT3 deficiency to enhance immune cell response to microbial stimulation. MAPK phosphorylation was reduced to some extend early on after LPS stimulation in SIRT3 −/− BMDMs. However, proliferation and cytokine production by splenocytes, macrophages and DCs were largely unaffected by SIRT3 deficiency. In contrast, shRNA-mediated SIRT3 silencing increased baseline TNF mRNA levels in RAW 264.7 macrophages 61 , and adenovirus-mediated SIRT3 overexpression inhibited MAPK phosphorylation in phenylephrine-treated myocytes and palmitate-stimulated pancreatic beta-cells 62,63 . Thus, SIRT3 seems to have cell and possibly context-dependent effects. Supporting this assumption, SIRT3 deficiency impaired in vitro the suppressive function of Tregs, which are particularly dependent on SIRT3-mediated mitochondrial activity and oxidative phosphorylation to develop optimal functions. However, SIRT3 −/− Tregs retained their suppressive functions in an adoptive transfer model of cardiac allograft rejection 29 . SIRT3 deficiency also affected endothelial function in mice fed with a high cholesterol diet but not a normal diet 64 , and organ-specific SIRT3 deficiency increased mitochondrial protein acetylation but did not induce mitochondrial dysfunction and did not impact on overall metabolic homeostasis as observed in germline SIRT3 knockouts 43,65 .
To assess the safety of SIRT3-targeting therapies, it was most important to analyze the contribution of SIRT3 in preclinical models of infection. As a first approach, we tested a model of endotoxemia, which revealed that cytokine response and survival rates were not different in SIRT3 +/+ and SIRT3 −/− mice. Although SIRT3 −/− mice survived slightly better than SIRT3 +/+ mice, whether this was a genuine effect would require large groups of animals (>40 mice per genotype) according to power calculation. Albeit very unlikely, we also cannot totally rule out that some differences in the genetic background of SIRT3 +/+ and SIRT3 −/− mice play a role. To address that question, SIRT3 −/− mice with additional backcrosses should be tested. SIRT3 +/+ mice had a minor yet significant survival advantage (10% vs 0% survival in SIRT3 +/+ vs SIRT3 −/− mice) in a highly stringent model of endotoxemia 38 , suggesting that SIRT3 may provide benefits in sterile, deep inflammatory, processes. Unfortunately, the cytokine response was not reported. Endotoxemia does not recapitulate the complexity to host defense mechanisms generated to fight against living microorganisms. Moreover, immunomodulatory compounds may interfere with innate immune responses and compromise host defenses, as well documented for anti-TNF and anti-IL-1 agents 66,67 . Thus, we elected to test models of sepsis induced by E. coli and K. pneumoniae, two of the most frequent etiologic agents of human sepsis. SIRT3 deficiency did not impact on the development of sub-acute pneumoniae and acute peritonitis, going well along with normal in vitro responses to bacterial stimulation of immune cells. In line with our observations, the survival rates of SIRT3 +/+ and SIRT3 −/− mice were not significantly different following cecal ligation and puncture sepsis 22 . Additionally, SIRT3 +/+ and SIRT3 −/− mice behaved roughly identically following systemic infection with L. monocytogenes and C. albicans given to produce chronic/mild and acute candidiasis. Interestingly, Listeria loads were slightly increased in the blood of SIRT3 −/− mice, which might feature transient alteration of mitochondrial dynamics during L. monocytogenes infection 68 . However, bacteremia was very low when compared to liver and spleen bacterial burdens, which were not different between SIRT3 +/+ and SIRT3 −/− mice. The absence of patent phenotype in a panel of sepsis models suggests that infection-induced phagocyte recruitment and/or activity was not impaired in SIRT3 −/− mice. Indeed, SIRT3 deficiency did not affect endothelial activation, plaque macrophage and T cell infiltration and atherosclerosis in low-density lipoprotein receptor knockout mice 64 . Our observations are somehow reminiscent of that obtained analyzing SIRT1. While SIRT1 was globally shown to inhibit inflammation 69 , it had little impact on macrophage and neutrophil antimicrobial functions, and myeloid deficiency in SIRT1 did not influence the outcome of endotoxemia and Gram-positive sepsis 70 .
SIRT3 activity is strongly associated with metabolism, and there is a tight relationship between metabolism and immune functions 59 . Thus, work will be required to address whether SIRT3 impacts on host defenses under metabolic stress. It is also possible that sirtuins have complementary or redundant effects, as suggested by protein interaction studies 5 . Therefore, future studies should analyze the impact of targeting multiple sirtuins on innate immune responses. Supporting this strategy, dual inhibitors of SIRT1/2 and pan-classical HDAC inhibitors affected host defenses against infections 46,51,56,71 . Considering that SIRT3 has been associated with age-related dysfunctions and that immune functions are decreased in elderly 72 , one should analyze the impact of SIRT3 in populations of different ages. Finally, a limitation of this study is that preclinical mouse models were performed with female mice. In a preliminary experiment using a limited number of males (seven animals), the survival of SIRT3 +/+ and SIRT3 −/− mice to Klebsiella-induced pneumonia was not different. However, larger groups of mice and additional models should be tested to settle whether there is or not a sex-dependent impact of SIRT3 deficiency on susceptibility to infection.
Overall, our data support the assumption that SIRT3 has no major impact on innate immune functions and host defenses against bacterial and fungal infections, at least in healthy immunocompetent hosts. The present data largely support the safety of SIRT3-oriented therapies, in terms of susceptibility to infections, for treating metabolic, oncologic and neurodegenerative diseases.