Sirtuin 3 is required for pro-inflammatory and pro-regenerative aspects of senescent cells

doi:10.21203/rs.3.rs-4656269/v1

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Sirtuin 3 is required for pro-inflammatory and pro-regenerative aspects of senescent cells

https://doi.org/10.21203/rs.3.rs-4656269/v1

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Cellular senescence is a multifaceted stress response marked by stable proliferative arrest and the secretion of diverse biologically active factors, collectively known as the senescence-associated secretory phenotype (SASP). The senescent phenotype is remarkably variable and subject to various regulatory influences. We previously demonstrated that mitochondrial dysfunction induced by diverse stimuli, including the loss of sirtuin 3 (SIRT3), leads to the hyperactivation of AMPK and p53, culminating in senescence while concurrently suppressing much of the proinflammatory SASP. Here, we extend our findings by revealing that the absence of SIRT3 can suppress segments of the SASP, even in the absence of p53. Intriguingly, SIRT3 deficiency renders cells resistant to stimulation by exogenous cytokines, such as interleukin-1. Fibroblasts derived from Sirt3 knockout mice exhibit a diminished SASP, including reduced levels of Pdgfa, and these mice display impaired wound healing and a more expansive granulation area. Furthermore, Sirt3 knockout mice accumulate more senescent cells than their age-matched counterparts and increases in many segments of the SASP, yet specifically exhibit lower levels of key proinflammatory SASP factors. Collectively, these data underscore a key role for SIRT3 in orchestrating cellular senescence phenotypes, shedding light on its regulatory influence beyond the p53-dependent pathway.

Cellular senescence is a stress or damage response characterized by an essentially irreversible proliferative arrest accompanied by the secretion of various bioactive molecules including, but not limited to, cytokines, chemokines, growth factors, proteases and signaling lipids, collectively known as the senescence-associated secretory phenotype (SASP) [1, 2]. Senescent cells accumulate with age and have been associated with age-related pathologies such as neurodegenerative diseases, atherosclerosis, and cancer [3-7]. Clearance of senescent cells in mice has been linked to both health span and lifespan extension [3, 8].

Sirtuins (SIRTs) are a family of nicotinamide adenine dinucleotide (NAD⁺)-dependent enzymes comprised of seven homologs, three of which (SIRT3, 4, and 5) are localized to the mitochondria [9]. Sirtuin 3 (SIRT3), the primary mitochondrial protein deacetylase, has a complicated relationship with senescence and aging. For example, disease-accelerating conditions, such as metabolic disease [10], neuro-inflammation [11], and pulmonary arterial hypertension [12] are exacerbated in Sirt3 knockout (Sirt3-KO) mice. However, lifespans of Sirt3-KO mice are comparable to wild type mice and are extended upon caloric restriction [13]. Adding to this complexity, a separate study reported that Sirt3-KO mice under X conditions were shorter lived than their WT counterparts, citing cardiac abnormalities as the major pathological difference between the two [14]. Thus, the role of Sirt3 in murine aging remains unclear, as are the mechanisms that mediate its effects.

Loss of Sirt3 activity in human or murine fibroblasts or human mesenchymal stem cells results in adoption of cellular senescence, but these cells lack many of the proinflammatory features of the SASP that are found in response to other inducers of senescence [15], so it is unclear if the net result of Sirt3 loss is beneficial or detrimental. Additionally, Sirt3-KO mice are viable, and MEFs isolated from these animals do not show increased levels of senescence [15], suggesting that germline transmission is sufficient to spare Sirt3-KO mice from accumulation of senescent cells during early life. Other drivers of mitochondrial dysfunction, including loss of HSPA9, mitochondrial DNA depletion, and electron transport chain inhibition also result in this mitochondrial dysfunction-associated senescence (MiDAS). MiDAS is distinct from other inducers of senescence, as these cells show a loss of many proinflammatory SASP factors at least in part due to activation of an AMPK-p53 signaling cascade. Despite showing a weaker metabolic phenotype than other forms of MiDAS, depletion of SIRT3 resulted in stronger SASP suppression than other forms of MiDAS [15], but the mechanism behind this remains unexplored.

We show here that loss of SIRT3 is required for many additional aspects of the SASP, independent of p53 activity or cell cycle arrest. Rather, SIRT3 is required for robust activation of the IL-1 signaling pathway, rendering SIRT3-depleted cells resistant to a major autocrine and paracrine driver of the proinflammatory SASP. Sirt3 knockout also lowers Pdgfa levels in senescent cells, and Sirt3-KO mice display commensurate alterations in wound healing kinetics. Finally, aged Sirt3-KO mice have increased numbers of senescent cells relative to age-matched controls, but also an altered SASP. Together, our data help explain some of the complexity observed in phenotypes associated with Sirt3 depletion and age-related conditions via disrupted regulation of the senescent phenotype.

Loss of Sirt3 suppresses proinflammatory segments of the SASP in a p53-independent manner.

We previously reported that mitochondrial dysfunction can both drive senescence and suppress proinflammatory aspects of the SASP via AMPK-dependent activation of p53 [15]. However, we observed a notable difference between other inducers of mitochondrial dysfunction and loss of SIRT3. For example, while depletion of p53 allows SIRT3-depleted cells to continue to divide [15], we did not observe a commensurate restoration of IL-6 secretion (Fig. 1A) or RNA levels of proinflammatory cytokines IL1A, IL1B, or IL6 (Fig. 1B) following loss of both SIRT3 and p53, including in response to ionizing radiation – a robust inducer of senescence and proinflammatory aspects of the SASP - suggesting that SIRT3 has effects that extend beyond the AMPK-p53 axis.

NF-κB is a major mediator of the proinflammatory arm of the SASP [16, 17]. We therefore measured NF-κB activity by luciferase reporter assay. NF-κB activity was elevated in control cells when induced to senesce by IR, contrasted by a strongly reduced NF-κB activity in SIRT3 knockdown cells, regardless of IR treatment (Fig. 1C). Furthermore, western blots revealed a decrease in the phosphorylated forms of three major MAPK signaling proteins: p38MAPK, ERK1/2, and JNK [18, 19] (Fig. 1D). Thus, SIRT3 is required for key stress and inflammatory signaling pathways in senescent cells, and this is independent of p53 status.

Sirt3 is required for activation of the IL-1 signaling pathway

We next sought to determine if SIRT3 was required for cell non-autonomous promotion of the SASP. We therefore co-cultured either mCherry-labeled scramble or shSIRT3 cells with IR-induced senescent cells for 3 days and stained them for interleukin 6 (IL-6) by immunofluorescence. IR-induced senescent cells promoted IL-6 expression in scramble control cells, similar to previous studies [20]. However, shSIRT3 cells did not elevate IL-6 levels in response to co-culture (Figs. 2A-2B), suggesting that SIRT3 is required for cell non-autonomous activation of the SASP.

Since interleukin-1 (IL-1) signaling can drive the SASP by autocrine, juxtacrine, and paracrine mechanisms [20, 21], we treated scramble or shSIRT3 cells with recombinant IL-1α or IL-1β (rIL-1A or rIL-1B; 100/pg//mL) and analyzed conditioned media for release of IL-6, a SASP factor induced by the IL-1 signaling pathway. Depletion of SIRT3 significantly lowered IL-6 secretion in response to IL-1 treatment (Fig. 2C).

We also sought to determine if blunted responses are a common feature of multiple forms of MiDAS. We therefore induced MiDAS by rotenone (ROT, 250 nM) or by depleting mitochondrial DNA in the absence of pyruvate (Rho0) and treated cells with rIL-1A (Fig. 2D). Mitochondrial DNA depletion had no effect on IL-6 secretion in response to IL-1, while ROT elevated IL-6 secretion relative to controls, indicating that the observed effects were SIRT3-specific and not a common feature of MiDAS.

The toll-like receptor 4 (TLR4) signaling pathway uses a similar signal transduction pathway to the IL-1 receptor. We therefore measured IL-6 secretion following treatment with lipopolysaccharides (LPS), a TLR4 agonist (Fig. 2E). LPS induced IL-6 secretion in both shSIRT3 and scramble controls, with shSIRT3 responding even more strongly than scramble controls, similar to previous reports [22].

IL-1 signaling can occur from both secreted forms of IL-1α and IL1β, and also from jutacrine signaling from membrane-bound IL-1α [21]. Notably, IR-induced senescent cells failed to induce IL-6 expression in co-cultured shSIRT3 cells, and shIL1A prevented IR-induced cells from promoting IL-6 positivity, regardless of shSIRT3 status (Fig. 2F-2G). To further test whether loss of SIRT3 disrupts the IL-1 signaling pathway, we treated scramble or shSIRT3 cells with rIL-1A plus calyculin A for 0, 30, or 60 minutes – followed by protein extraction and analysis by western blot (Fig. 2H). Treatment with rIL-1A resulted in increased phosphorylation of IRAK4, IRAK1, TAK1, and MEKK1, along with lowered total IRAK1 and IkBα - all consistent with activation of IL-1 signaling [23], and each of these markers was blunted by loss of SIRT3. Together, these data indicate that SIRT3 is required activation of the IL-1 signaling pathway.

Sirt3 is required for wound healing-promoting features of senescent cells

We previously showed that cells from germline Sirt3-KO mice proliferate normally in culture, whereas depletion of Sirt3 in WT cells resulted in senescence [15]. However, we did not measure the SASP in these cells at the time. We therefore irradiated MEFs from either WT or Sirt3-KO mice and measured expression of multiple SASP factors for qPCR. RNA levels of multiple inflammatory SASP factors including Il1a, Il6, Mmp3, and Ccl2 were diminished in Sirt3-KO mice (Fig. 3A). Additionally, cells from Sirt3-KO mice also showed reduced RNA levels of Pdgfa (Fig. 3B), and accumulation of senescent cells at the site of a wound is known to be beneficial in healing, at least partially due to the secretion of platelet-derived growth factor AA (PDGF-AA) [24]. We therefore conducted wound healing assays following punch biopsy and measured wound closure over time. Sirt3-KO mice displayed reduced wound healing kinetics (Fig. 3C), in agreement with previous studies [25, 26]. These early kinetics also correlated with the time frame in which elimination of p16-positive cells slows wound healing [24]. Furthermore, Sirt3-KO mice also displayed larger granulation thickness and area compared to WT mice 14 days after wounding (Figs. 3D-3F). These data suggest that Sirt3 is required for features of the SASP that promote regeneration.

Aged Sirt3-KO mice have increased numbers of senescent cells and an altered SASP

Since Sirt3 both promotes senescence and antagonizes elements of the SASP, we sought to determine if it plays a role in the development of senescence or the SASP during chronological aging. We therefore crossed Sirt3-KO mice with p16-3MR mice, which allow detection of p16-positive cells by luminescence [24]. Using these mice, we observed that whole body luminescence was increased at 18 months of age in Sirt3-KO mice relative to WT mice (Fig. 4A), suggesting that more senescent cells were present. Furthermore, gonadal adipose tissue from aged Sirt3-KO mice also had significantly higher levels of senescence-associated beta-galactosidase (SA-Bgal) staining relative to WT mice (Figs. 4B and 4C). Despite increases in whole body luminescence, p16 levels were not elevated in adipose tissue from Sirt3-KO mice (Fig. 4D), but adipose tissue from Sirt3-KO mice instead showed higher levels of the senescence markers p21 (Fig. 4E) and p15 (Fig. 4F). In addition, much like in culture and wound healing assays, aged Sirt3-KO mice had greatly altered SASP profiles compared to age-matched WT mice. In Sirt3-KO mice, levels of SASP factors and oxylipin synthases that are common to multiple inducers, such as Gdf15, Serpine1, and Alox15 [27–29] were significantly elevated, but inflammation-associated SASP factors such as Il6, Tnf, and Il1a were significantly decreased (Fig. 4G). Thus, Sirt3 deficient animals, much like cultured cells, have increased senescent cells but display an altered SASP during aging, though it remains unclear what effects this altered senescence phenotype might have on longevity and healthspan. Together, our data elucidate a complex relationship between SIRT3, cellular senescence, and outcomes generated by senescent cells.

Deficiency in SIRT3, a mitochondrially localized sirtuin, has been linked to senescence and to regulation of age-related phenotypes, but our understanding of the mechanisms behind this regulation remains incomplete. Here, we describe new mechanistic insights on how these relationships might occur – including both loss of proinflammatory IL-1 signaling and other secretory elements and increases in age-related senescent cell accumulation.

While we show that SIRT3 is required for IL-1 signaling during senescence, it remains unclear how loss of SIRT3 blunts IL-1 signaling. We initially considered the possibility that the IL-1 signaling pathway connects with the mitochondrion through the ECSIT adapter protein [30, 31]. However, ECSIT is also required for TLR4 signaling, which was enhanced – rather than blunted – by loss of SIRT3 (Fig. 2E). Additionally, ECSIT is an intermediate protein in the IL-1 signaling pathway [30], and therefore would not be expected to lower IRAK4 and IRAK1 phosphorylation, which are immediately downstream of IL-1 receptor activation and upstream of ECSIT in the IL-1 signaling pathway [23]. Rather, it seems likely that SIRT3 is required for IL-1 signaling at or near the plasma membrane by an as-yet unidentified mechanism. Since TLR4 signaling was enhanced by loss of SIRT3 and uses many of the same proteins to drive the signaling response [32], it is possible that SIRT3 plays an unknown role in favoring myddosome assembly at TLR4 at the expense of IL-1R, but this remains speculative.

Sirt3-KO mice have previously been shown to heal wounds more slowly [25, 26], but links to senescent cells have not been explored. Notably, Sirt3 is decreased in macrophages during diabetes, and this slows wound healing. Similarly, Sirt3 deficiency is linked to increased necroptosis during skin wound healing – a process we and others have shown can be driven by senescent cells during premature and chronological aging [33, 34]. Thus, it is tempting to speculate that these phenotypes are driven either directly or indirectly by aberrant senescence responses in Sirt3-KO mice.

Our findings in aged Sirt3-KO mice suggest a perturbation in age-related senescence responses that are not clearly pro- or anti-geronic. For example, most senescence markers assayed, including cyclin-dependent kinase inhibitors and multiple SASP factors, were increased in aged Sirt3-KO mice relative to their wild type littermates. Conversely, many of the best-studied inflammatory SASP factors are decreased by loss of Sirt3 both in culture and in vivo. Thus, it appears that the role of Sirt3 in age-related senescence under ad libitum conditions is more complex than directional. Reports that Sirt3-KO mice have roughly equivalent lifespans to wild type mice would seem to support this idea [13]. However, Sirt3 is also a key player in lifespan and disease following metabolic challenges, as calorically restricted Sirt3-KO mice live longer than their wild type counterparts [13], and disease-promoting features of high fat diets and diabetes are exacerbated in these mice [10, 35, 36]. Thus, it may be that Sirt3 is more of a key integrator of metabolic responses during aging, and these in turn might be mediated at least in part through control of cellular senescence.

Cell culture. IMR-90 human fibroblasts were mycoplasma-free, cultured in cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin at 3% O₂ and 5% CO₂ 3% O2 as described previously [15], and used under 40 population doublings. Cells were mock irradiated or irradiated with 10 Gy X-rays, as described in [37], or treated with 100 nM rotenone. To deplete mitochondrial DNA for rho⁰ cells, cells were cultured in 100 ng/ml ethidium bromide, 100 mM sodium pyruvate, and 50 mg/ml uridine for 2 months, followed by pyruvate removal to induce MiDAS.

Signaling ligands. Recombinant IL-1α or IL-1β (R&D Systems, 100 pg/mL), or LPS (1 µg/mL) were administered to cells in culture media for 24 hours, followed by wash out and replacement with serum free media for 24 h and analysis by IL-6 ELISA.

Co-culture experiments. “Donor” cells were transduced with shRNAs to GFP or IL1A, were selected using puromycin, and 4 days later received 10 Gy of ionizing radiation. Donor cells were used 7 days later. “Acceptor” cells were transduced with shSIRT3 or the empty pSICOR vector, selected using puromycin, and cells were confirmed to be > 90% mCherry positive before use. Acceptor cells were then plated 1:3 with donor cells and co-cultured for 2 days before immunofluorescence and analysis.

Lentiviral transduction. IMR-90 fibroblasts were transduced with lentiviral vectors to p53 [37]

or SIRT3 [15] in polybrene as previously described. 24 hours after transduction, cells were selected using puromycin (2 µg/mL) for 48 hours.

IL-6 ELISA. 3x10⁴ cells in 12-well plates were treated as indicated and cultured in 0.5 ml serum-free DMEM for 24 hr. CM were collected and clarified at 2,000 x g for 10 min. Supernatants were transferred to a tube; cells were trypsinized and counted. Conditioned media (2.5 ml) were analyzed by bead-based ELISAs (AlphaLISA, Perkin-Elmer) as instructed by the manufacturer and normalized to cell number.

Quantitative real-time RT-PCR. RNA was isolated from cells using a kit (Bioline), and from IAT using a Lipid Tissue RNA Isolation Kit (Qiagen), according to the manufacturer instructions. RNA (250 ng/ml) was used to synthesize cDNA, which was analyzed by qPCR using the universal probe library (Roche) or using TaqMan assays (ThermoFisher). mRNA levels were normalized to beta actin, unless noted otherwise. Primer sequences and probes were described previously [15].

NF-κB reporter assays. IMR-90 fibroblasts were transduced with Cignal NF-κB Reporter Assay and Cignal renilla control lentiviral particles (Qiagen), followed by selection and the indicated treatment regimens. Cells were then lysed and analyzed via a dual luciferase reporter assays kit (Promega).

Western blots. Cells were lysed in 5% SDS in 10 mM Tris, pH 7.4, with protease and phosphatase inhibitors. After BCA assay quantitation, 20 mg protein was electrophoresed and

transferred to PVDF membranes. Membranes were blocked in TBST + 5% BSA, incubated overnight with primary antibody, washed thrice in TBST, incubated with HRP-conjugated secondary antibody for 30 min and visualized by chemiluminescence. Antibodies were from Cell Signaling Technology (p38 - #9212, phospho-JNK – #4668, phospho-ERK1/2 - #4370, phospho-IRAK4 - #7652, phospho-TAK1 - # 4531, phosphor-IRAK1 - # 4365), PhosphoSolutions (phospho-p38 - p190-1802), Santa Cruz (IRAK1 - sc-5288, phospho-MEKK1 - sc-130202, MEKK1 - sc-252, IkB-a - sc-371, SIRT3 - sc-49744), and Abcam (beta actin - ab8226, alpha-tubulin - ab4074).

Immunofluorescence. Immunofluorescence was performed as described previously [1]. Briefly, cells were treated as described, formalin fixed for 10 minutes, washed, and permeabilized in triton-X-100 for 30 minutes. Cells were washed again and incubated overnight in IL6 antibody (R&D Systems AF-206-NA), washed, and incubated in donkey-anti-Goat secondary antibody conjugated to ALEXA-488. Cells were counterstained with DAPI and photographed using fluorescence microscopy.

Animal models. All mice were maintained in the AALAC-accredited Buck Institute for Research on Aging animal facility. All procedures were approved by the Institutional Animal Care and Use Committee. All mice were bred in house and fed an ad libitum diet. Sirt3-KO mice in the 129S1 and C57Bl/6 backgrounds were provided by the Eric Verdin lab. P16-3MR were crossed to Sirt3-KO mice in the C57Bl/6 background and F2 hybrids were genotyped for homozygosity. WT and Sirt3-KO x p16-3MR were aged for 18 months before analysis.

Wound healing assays. Mice were used at 3 months of age. Two equal-sized wounds were created on dorsal skin by a 6 mm punch, and wounds were measured every 2 days by caliper measurement and averaged across both wounds for each animal. At 14 days all animals were euthanized and dorsal skin surrounding the wound site was collected.

In vivo luminescence. Homozygous WT and SIRT3-KO mice in the p16-3MR C57Bl/6 background were intraperitoneally injected with 15 µg of Xenolight RediJect Coelentarazine and quantified 25 minutes after injection on an IVIS Lumina S5 In Vivo Imaging system.

Senescence-associated beta galactosidase. Staining for senescence-associated beta-galactosidase was accomplished as previously describes [38] using a commercial kit (Biovision-Abcam). Briefly, 50 µg gonadal adipose tissue samples were fixed in 200 µl of fixation solution for 15 minutes, followed by 3 washes using ice cold PBS. Tissues were then placed in 200 µl of staining solution and incubated at 37°C with observation every 3 hours until color (blue) developed. Stained tissues were photographed together and analyzed for beta-galactosidase intensity using the blue channel in ImageJ.

Author contributions.

CW – experimental and conceptual design and execution, wrote manuscript. BM and STJ – execution of experiments. MCV – wound healing assays.

Acknowledgements.

This work was supported by NIH Grant AG051729 (PI: Campisi) and USDA-ARS 58-8050-9-004. S.T.J. is supported by NIDDK T32 DK124170 (PI: Greenberg). B.A.M. was supported by a fellowship from the SENS Research Foundation.

Data availability.

All data are presented in the manuscript.

Competing Interests.

CW holds patents related to the detection, modification, and elimination of senescent cells and declares no non-financial interests. BM, SJ, and MV declare no competing financial or non-financial interests.

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There is a conflict of interest CW holds patents related to the detection, modification, and elimination of senescent cells and declares no non-financial interests.

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Sirtuin 3 is required for pro-inflammatory and pro-regenerative aspects of senescent cells

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Abstract

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Introduction

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Sirt3 is required for activation of the IL-1 signaling pathway

Sirt3 is required for wound healing-promoting features of senescent cells

Aged Sirt3-KO mice have increased numbers of senescent cells and an altered SASP

Discussion

Methods

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