Impact of the Dual Deletion of the Mitochondrial Sirtuins SIRT3 and SIRT5 on Anti-microbial Host Defenses

The sirtuins SIRT3 and SIRT5 are the main mitochondrial lysine deacetylase and desuccinylase, respectively. SIRT3 and SIRT5 regulate metabolism and redox homeostasis and have been involved in age-associated metabolic, neurologic and oncologic diseases. We have previously shown that single deficiency in either SIRT3 or SIRT5 had no impact on host defenses in a large panel of preclinical models of sepsis. However, SIRT3 and SIRT5 may compensate each other considering that they share subcellular location and targets. Here, we generated a SIRT3/5 double knockout mouse line. SIRT3/5 deficient mice multiplied and developed without abnormalities. Hematopoiesis and immune cell development were largely unaffected in SIRT3/5 deficient mice. Whole blood, macrophages and neutrophils from SIRT3/5 deficient mice displayed enhanced inflammatory and bactericidal responses. In agreement, SIRT3/5 deficient mice showed somewhat improved resistance to Listeria monocytogenes infection. Overall, the double deficiency in SIRT3 and SIRT5 has rather subtle impacts on immune cell development and anti-microbial host defenses unseen in single deficient mice, indicating a certain degree of overlap between SIRT3 and SIRT5. These data support the assumption that therapies directed against mitochondrial sirtuins, at least SIRT3 and SIRT5, should not impair antibacterial host defenses.


INTRODUCTION
The innate immune system plays a central role in host defenses. Innate immune cells among which monocytes/macrophages, granulocytes and dendritic cells (DCs) sense microbial and danger associated molecular patterns (MAMPs/DAMPs) through pattern recognition receptors (PRRs) expressed at the cell surface, in the cytoplasm and in endosomes. The best characterized PRRs belong to the families of Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and cytosolic DNA sensors (1,2). The binding of MAMPs/DAMPs to PRRs activates intracellular signaling cascades that induce the production of effector molecules involved in inflammation and host defense mechanisms, as well as the resolution of inflammation and tissue repair (3,4). Immune cells are plastic and adapt their metabolism and responsiveness to their environment to execute their biological functions (5,6).
Sirtuins belong to the family of so-called histone deacetylases (HDACs) that target lysine posttranscriptional modifications. Classical HDACs (HDAC1-11) are Zn 2+ -dependent, while sirtuins are NAD + -dependent lysine deacetylases. Sirtuins are homologs to yeast Sir2 that gained tremendous attention when it was shown to be activated upon caloric restriction and to increase lifespan (7). The mammalian genome encodes for seven sirtuins that target proteins by removing acetyl functional groups, but also acyl, glutaryl, malonyl, and succinyl groups as demonstrated lately (8). The list of targets of sirtuins has increased dramatically over the years, and high throughput proteomics analyses pinpointed to thousands of substrates for sirtuins. Accordingly, sirtuins are involved in the regulation of many biological and pathological processes and in the development of metabolic, neurodegenerative, cardiovascular, and oncologic diseases (9,10).
SIRT3 and SIRT5 are mainly localized in the mitochondrial matrix, where SIRT3 is the main deacetylase (11) and SIRT5 is the main desuccinylase (12,13). Of note, SIRT5 also catalyzes lysine demalonylation and deglutarylation (13,14). SIRT3 promotes glucose and fatty acid metabolism, urea cycle and the activity of the electron transport chain. During caloric restriction, SIRT3 regulates mitochondrial acetylome and multiple metabolic pathways in the liver (15,16). SIRT3 protects from oxidative stress by activating the reactive oxygen species (ROS) detoxifying enzyme superoxide dismutase 2 (SOD2) and the redox controlling enzyme isocitrate dehydrogenase 2 (IDH2) (17,18). Similar to SIRT3, SIRT5 activates enzymes involved in ROS detoxification (i.e., SOD1, IDH1, and IDH2), promotes mitochondrial functions and integrity and regulates the urea cycle and other metabolic pathways (14,(19)(20)(21)(22)(23)(24)(25). The genetic ablation of SIRT3 or SIRT5 in mice has been associated with increased susceptibility to age-associated diseases including insulin resistance, obesity, neurodegeneration, cardiac dysfunction and fibrosis, while contrasting context-dependent effects have been reported for tumorigenesis (10,(26)(27)(28)(29)(30). Deficiencies in SIRT3 or SIRT5 have also been reported to promote colitis, acute lung injury and ischemia reperfusion injury (10,(31)(32)(33)(34)(35)(36). Overall, targeting the activity of sirtuins and particularly mitochondrial sirtuins is viewed as an attractive therapeutic strategy to tackle the development of age-related disorders (10,(28)(29)(30). Considering that inflammation is an essential component of innate immune defenses, we analyzed the impact of SIRT3 and SIRT5 deficiencies on the response of mice subjected to a broad panel of preclinical models of bacterial and fungal sepsis (37,38). Neither SIRT3 nor SIRT5 was critical to fight against infections. Additionally, SIRT3 −/− mice were not particularly susceptible to cecal ligation and puncture (CLP), a stringent model of sepsis (39,40). Hence, SIRT3 and SIRT5 appear to have a more prominent influence on chronic metabolic and inflammation-related disorders than on infectious diseases characterized by acute inflammatory reactions.
SIRT3 and SIRT5 share subcellular location and targets, so they might compensate each other in single knockout mice. To bypass this hurdle, we generated a SIRT3/5 deficient mouse line. SIRT3/5 −/− mice were fertile and developed without apparent abnormalities. In vitro and in vivo investigations revealed somewhat enhanced inflammatory and bactericidal responses of whole blood, macrophages, and neutrophils and a moderate improved resistance to Listeria monocytogenes in the double knockouts. Altogether SIRT3 and SIRT5 have subtle, redundant roles during antimicrobial host defenses. Overall, therapies directed against mitochondrial sirtuins should not dramatically impact on antimicrobial host defenses.

Key Resources
See Supplementary Information.

Western Blot Analyses
Total and nuclear proteins were extracted, submitted to PAGE and transferred onto membranes as described (47,48). Membranes were incubated with primary and secondary HRP-coupled antibodies and revealed by chemiluminescence

Flow Cytometry
Single cell suspensions from thymus, spleen and BM were incubated with 2.4 G2 to block Fc receptors and stained with antibodies described in Supplementary Information (50).

ROS Measurement
BMDMs were plated in half-area black 96-well plates in RPMI without phenol red (Invitrogen). Cells were incubated for 10 min at 37 • C with 5 µM MitoSOX (Thermofisher). Stimuli were added and fluorescence (Ex 510 , Em 580 ) recorded using a Synergy plate reader (BioTek, Winooski, VT). Neutrophils in HBSS without calcium and magnesium (ThermoFisher) were incubated for 1 h with 100 nM PMA (Enzo Life Sciences, Farmingdale, NY) and 5 µM MitoSOX during the last 10 min of incubation. ROS were measured by flow cytometry.

Metabolic Activity
The metabolic activity of BMDMs was measured using the XF Cell Mito Stress, Glycolysis Stress and Mito Fuel Flex Test Kits on a 96-well format Seahorse XFe R system (Agilent Technologies, Santa Clara, CA) (46).

Neutrophil Killing and NETosis Assays
Neutrophils were incubated with live L. monocytogenes for 1 h in RPMI medium. Serial dilutions of reaction mixtures were plated on blood agar plates (BD Biosciences). Twenty-four hours later, colonies were enumerated. To measure NETosis, neutrophils were incubated for 3 h with 100 nM PMA and 5 µM of the cell impermeable dye Sytox green. Fluorescence (Em 504 , Ex 523 ) was recorded using the Synergy plate reader.

In vivo Models
Listeriosis was induced by challenging intravenously (i.v.) age and sex-matched mice with a low (7.3 × 10 3 cfu) or a high (0.9-1 × 10 5 cfu) inoculum of L. monocytogenes. Blood and organs were collected 1-3 days post-infection to quantify bacteria and cytokines and analyze cell populations. A model of endotoxemia was developed by challenging mice intraperitoneally (i.p.) with 10 mg/kg LPS. Body weight loss, severity score and survival were registered at least twice daily (52, 53) by 2-3 operators. The severity score was graded from 0 to 4 based on the mobility, the posture, the appearance and the weight loss of mice (detailed criteria were approved by the Service des Affaires Vétérinaires, DGAV, and are available upon request). Mice were sacrificed when they met a severity score of 4. A mice found dead was assigned a score of 5.

Statistical Analyses
Groups were compared by variance analysis followed by two-tailed unpaired Student's t-test or a Mann-Whitney test when appropriate. Survival was analyzed using the Kaplan-Meier method. P < 0.05 was used to indicate statistical significance. Analyses were performed using PRISM 8.0.1 (GraphPad Software, San Diego, CA).

SIRT3/5 Deficiency Has No Dramatic Impact on Mouse Development and Macrophage Metabolism
We generated a SIRT3/5 double knockout mouse line (SIRT3/5 −/− , see Materials and Methods) to study the interaction between SIRT3 and SIRT5. Genomic-DNA based PCR genotyping ( Figure 1A) and Western blotting analyses ( Figure 1B) confirmed the truncation of the Sirt3 and Sirt5 genes and the absence of SIRT3 and SIRT5 protein expression in SIRT3/5 −/− mice. Fecundity and development were normal. The size ( Figure 1C) and the female/male sex ratio ( Figure 1D) of the litters as well as the weight of adult female and male mice ( Figure 1E) were like those of SIRT3/5 +/+ , SIRT3 −/− , and SIRT5 −/− mouse lines. Autopsy did not reveal gross abnormalities in SIRT3/5 −/− mice.

DISCUSSION
This is the first report about the impact of the dual deficiency of SIRT3 and SIRT5 on immune cell development and antimicrobial host defenses. Double knockout mice developed normally and showed subtle, minor alterations of immune cell subpopulations and host responses to infection. Together with the fact that SIRT3 −/− and SIRT5 −/− mice are susceptible to bacterial sepsis like wild-type mice (37)(38)(39)(40), these observations strengthen the development of pharmacological modulators of the activity of mitochondrial sirtuins for clinical purposes.
Notwithstanding that SIRT3 and SIRT5 orchestrate metabolism and oxidative stress responses, SIRT3 and SIRT5 whole body knockout mice have no macroscopic abnormalities (41,42). The SIRT3/5 −/− mouse line we generated here developed normally. No problem of fertility, sex distribution and growth were noticed. Surprisingly, the metabolism of SIRT3/5 −/− BMDMs was similar to that of SIRT3/5 +/+ BMDMs. Yet, the impact of SIRT3 and SIRT5 on metabolism was mainly demonstrated in cells or tissues such as the liver and the heart that are rich in mitochondrial sirtuins when compared to macrophages (38,41,60). Another SIRT3/5 −/− mouse line has been recently generated. In line with our observations, no developmental defects were reported. Moreover, these SIRT3/5 −/− mice were susceptible to streptozotocin-induced hyperglycemia like controls, while showing only a modest inner retinal dysfunction (61,62).
Studies on the role of sirtuins in hematopoiesis and immune cell development are scarce. SIRT3/5 −/− mice had a normal pool of HSCs and MPPs and a slightly increased number of CMP (and GMP as a trend) in their bone marrow. The primary and secondary immune organs of SIRT3/5 −/− mice were largely unaffected, according to absolute numbers and proportions of immune cell subpopulations. There was only a slight reduction of thymus size, which did not impact the proportion of thymocyte subpopulations. This reminds the phenotype of SDHD-ESR mice with deletion of the succinate dehydrogenase, subunit D gene encoding for one of the subunits of the mitochondrial complex II (63). SDHD mice have a thymic atrophy without perturbation of thymocyte development. Overall, deficiencies in SIRT3, SIRT5, and SIRT3/5 do not seem to have a dramatic impact on immune cell development and/or functions [(37, 38) and present study]. Nonetheless, a role for these enzymes might come to light under stress or stimulatory conditions, or in aged mice. For example, SIRT1 shaped the T helper (Th) and T regulatory (Treg) responses of naïve T cells (64)(65)(66)(67)(68). Furthermore, in SIRT1 −/− mice, the percentages of CD4 + , CD8 + , and CD4 + CD8 + T cell subpopulations were normal, but thymocytes were at increased sensitivity to ionizing radiation induced DNA damaging (69). Finally, SIRT3 −/− mice of 18-24 months had a lower frequency of bone marrow hematopoietic progenitors than mice of 12 weeks (70). SIRT3/5 −/− macrophages exposed to TLR agonists produced more inflammatory cytokines and less IL-10 than SIRT3/5 +/+  macrophages, contrary to SIRT3 −/− and SIRT5 −/− macrophages that behaved like wild-type cells (37,38). Accordingly, NF-κB and MAPK signaling pathways were increased in resting and/or LPS-stimulated SIRT3/5 −/− macrophages. These data somehow support the possibility that SIRT3 and SIRT5 compensate each other in single knockout animals. Sirtuins are generally considered to drive anti-inflammatory responses. However, as nicely reviewed recently for SIRT1 (68), sirtuins may promote proinflammatory and anti-inflammatory effects depending on the context and whether myeloid or lymphoid cells are considered. For example, SIRT5 deficiency was associated with both increased and decreased innate inflammatory response in vivo (33,71). More generally, contrasting observations have been reported for most sirtuins (SIRT1-3, SIRT5-6). Methodological differences may explain these differences when studying monocytes/macrophages (37,38,46): the origin/fate of the cells (BMDMs vs. peritoneum macrophages vs. established macrophage cell lines, growth factors used for differentiation and maturation state of macrophages), strategies to delete or overexpress sirtuins or to modulate sirtuin activity (siRNA, shRNA, expression vectors, full or cell-specific knockouts, pharmacological activators, and inhibitors), readouts, and subtle variations in NAD + concentrations and circadian rhythm known to affect sirtuin activity or expression.
The expression levels of SIRT3 and SIRT5 decreased gradually from CMP to GMP and from GMP to granulocytes, which mirror the decline of mitochondrial mass and mitochondrial DNA during hematopoietic differentiation (72). Granulopoiesis relies on the expression of CCAAT/enhancer binding protein (C/EBP). The expression of SIRT1, which deacetylates C/EBPε and represses neutrophil terminal differentiation (73), declines during granulopoiesis (727 ± 13, 643 ± 20, and 307 ± 61 mRNA arbitrary units in CMP, GMP, and granulocytes, respectively). Thus, the downregulation of sirtuins seems to be a general feature associated with neutrophil development. The fact that neutrophil counts were normal and not increased in SIRT3/5 −/− mice suggests either the implementation of compensatory mechanisms, possibly through SIRT1, or that SIRT3 and SIRT5 have a modest influence on granulopoiesis.
Neutrophils produce cytotoxic compounds that target pathogenic bacteria and fungi but are harmful for host tissues (74). Contrary to SIRT3/5 +/+ neutrophils, and better than SIRT3 −/− and SIRT5 −/− neutrophils, SIRT3/5 −/− neutrophils killed L. monocytogenes, which was associated with an augmented production of ROS but not of NETs. In comparison, neutrophils deficient in SIRT3 had a mild increase of intracellular ROS but performed either normal or increased NETosis (56,57). SIRT1 deficiency did not impact on neutrophil functions, and the role of the remaining sirtuins has not been reported. Whereas, SIRT3 −/− mice had increased neutrophil infiltration in lungs during sterile injury (36,75) and mycobacterial infection impairing the survival of mice (76), SIRT5 −/− had reduced inflammation and ischemia/reperfusion brain injury (77). Finally, SIRT3 −/− and SIRT5 −/− mice behaved like wild-type mice in models of sepsis requiring neutrophils to fight the infectious agents (37)(38)(39)(40). Interestingly, SIRT3/5 −/− mice resisted better than SIRT3/5 +/+ mice to acute listeriosis, showing decreased signs of morbidity, reduced blood bacterial loads and significant albeit modest delayed mortality. SIRT3/5 −/− mice expressed higher concentrations of cytokines but normal counts of leukocytes in blood, suggesting that the reactivity rather than the number of leukocytes protected SIRT3/5 −/− mice from listeriosis. Interestingly, SIRT3/5 −/− mice were not more resistant to mild listeriosis than their wild type counterparts, and behaved like wild type mice in a model of endotoxemia. These observations support the assumption that drugs targeting SIRT3 and SIRT5 should not have a deletary impact on host defenses, which would contrast with drugs targeting classical HDACs that strongly impaired innate immune defenses against infections in preclinical models and clinical settings (78)(79)(80)(81)(82)(83)(84).
Overall, the double deficiency in SIRT3 and SIRT5 had rather modest and subtle impacts on immune cell development and anti-microbial host defenses. It might be that SIRT4, the remaining mitochondrial sirtuin in SIRT3/5 −/− mice, compensated for SIRT3 and SIRT5 absence. Unfortunately, whether SIRT4 affects immune responses has not been reported. Considering the link between sirtuins, metabolism and ageassociated pathologies, it is possible that phenotypes will emerge in aged mice or in mice submitted to metabolic stress. SIRT3/5 −/− mice should be tested in other preclinical models of sepsis. Nonetheless, putting together the data from the present study together with the fact that single deficiencies in SIRT3 and SIRT5 had no impact in a large panel of experimental sepsis (37)(38)(39)(40), one may foresee that therapies directed against mitochondrial sirtuins or concomitant targeting of SIRT3 and SIRT5 activity should have no deep impact on antibacterial host defenses.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the manuscript/Supplementary Files.

AUTHOR CONTRIBUTIONS
TH and EC performed in vitro experiments. TH, EC, and DLR performed in vivo experiments. TR and TH conceived the project, designed the experiments and wrote the paper. All the authors revised the paper. Supplementary Figure S3 | (A) Extracellular acidification rate (ECAR) by SIRT3/5 +/+ and SIRT3/5 −/− BMDMs exposed to LPS (10 ng/ml) measured using the Seahorse technology. (B) Oxygen consumption rate (OCR) and ECAR by SIRT3/5 +/+ and SIRT3 −/− BMDMs measured using the Seahorse technology. Data are means ± SD from three mice aged 10-12 weeks analyzed in quadruplicate.