Senolytics reduce coronavirus-related mortality in old mice

SnCs have an altered response to SARS-CoV-2 spike protein

Viral entry through cell surface receptors and dampening of host antiviral gene expression are critical steps in successful infections and virus propagation (18). The spike 1 (S1) glycoprotein of SARS-CoV-2, antibodies against which are currently being tested in clinical trials (NCT04425629), mediates entry into host cells through binding to angiotensin-converting enzyme 2 (ACE-2), resulting in elevated nuclear factor κB (NF-κB) signaling and inflammatory cytokine production (19, 20). Endothelial cells can be infected directly by SARS-CoV-2, leading to amplification of inflammation with subustantial changes in endothelial morphology and disruption of intercellular junctions (21).

To address specifically how the SARS-CoV-2 PAMPs affect SnCs, senescent human kidney endothelial cells (fig. S4) were treated with pyrogen-free recombinant S1 protein. Exposing endothelial SnCs to S1 for 24 hours significantly increased secretion of the majority of endothelial SASP factors measured in the conditioned media (composite score P < 0.0089 comparing SnCs to non-SnCs) (Fig. 2A and table S2). Similar albeit less distinctive results were obtained by using kidney endothelial cells in which senescence was induced through replication rather than radiation (fig. S5 and table S3). In addition, treatment of human subcutaneous adipocyte progenitor SnCs with S1 increased expression of the key preadipocyte SASP factors, IL1α and IL1β, at the mRNA level (fig. S6). Consistent with the LPS data, these data suggest that S1 is a PAMP that can trigger a hyperinflammatory state in SnCs, possibly through stimulation of a Toll-like receptor (TLR) (22, 23), with the inflammatory profile differing among types of SnCs.

Inflammatory SASP factors contribute to clearing pathogens. However, certain inflammatory/SASP factors released by senescent human lung cell types—including IL-1α, IL-1β, IL-6, MCP-1, TNFα, and MMP-1—are central to the pathological cytokine storm seen in some COVID-19 patients (4, 5, 24–33). Initially, to determine whether the SASP affects the response of human endothelial cells to pathogen exposure, nonsenescent primary kidney endothelial cells were exposed to conditioned media (CM) from SnCs or non-SnCs (Fig. 2B). The CM from SnC endothelial cells significantly reduced expression of the key viral defense genes IFITM2 and IFITM3 (Fig. 2C). IL-1α is a natural pyrogen as well as a master up-stream regulator of the senescence-associated IL-6/IL-8 cytokine network (34). It is increased in COVID-19 patients (35), increased in SnCs treated with S1 (fig. S6), and increased in LPS-treated mice (Fig. 1B and figs. S2 and S3). Directly treating nonsenescent primary human endothelial cells with IL-1α significantly reduced expression of IFITM2 and IFITM3 (Fig. 2D). Suppressing the SASP factors IL-18, PAI-1, and IL-1α by pretreating the CM from SnCs with neutralizing antibodies against these proteins partially restored IFITM2 and IFITM3 expression (Fig. 2C). These data support the conclusion that the SASP from preexisting SnCs could exacerbate SARS-CoV-2 infection of nonsenescent human endothelial cells.

Next, we examined the impact of the SASP on human lung epithelial cells, another target cell type in COVID-19. Treating nonsenescent primary human lung epithelial cells with CM from senescent human preadipocytes, kidney endothelial cells, or human umbilical vein endothelial cells (HUVECs) significantly increased expression of the SARS-CoV-2 viral entry genes ACE2 and TMPRSS2 (Fig. 2E). Similarly, treating nonsenescent human primary kidney endothelial cells with CM from SnCs induced expression of TMPRSS2 (Fig. 2E). Adding neutralizing antibodies against IL-1α to the CM from SnC kidney endothelial cells reduced expression of TMPRSS2, whereas antibodies against IL-18 did not (Fig. 2F). Treating nonsenescent human primary endothelial cells directly with IL-1α increased TMPRSS2 expression fivefold (Fig. 2F), and IL-1α treatment of nonsenescent human lung epithelial cells increased both ACE2 and TMPRSS2 expression twofold (fig. S7A). Treating nonsenescent human kidney endothelial cells with IL-1α also significantly increased expression of IL6, IL8, IP10, and MCP1 (fig. S7B). In addition, although ACE2 and TMPRSS2 were not up-regulated in senescent human preadipocytes (fig. S7C) in which these genes are not normally expressed, TMPRSS2 was up-regulated in senescent human endothelial cells (fig. S7D). Consistent with these in vitro results, in healthy human lung tissue resected from five elderly patients for clinical indications of focal, noninfectious causes, there were more TMPRSS2⁺ cells adjacent to p16^INK4a+ cells as detected with immunofluorescence, with the abundance of p16^INK4a+ cells correlating with TMPRSS2⁺ cell abundance (Fig. 2, G and H). Collectively, these data further support the conclusion that SnCs could promote SARS-CoV-2 pathogenesis by decreasing viral defenses and increasing expression of viral entry proteins in neighboring non-SnCs through amplified secretion of SASP factors.

Old mice are hypersensitive to pathogen exposure, including β-coronavirus infection

To investigate the role of SnCs in driving adverse outcomes upon infection in vivo, we exploited an experimental paradigm developed to study the response of laboratory [specified-pathogen free (SPF)] mice to infection with common mouse microbes, creating what is termed a “normal microbial experience” (NME) (36–38). Experimental mice are exposed to pathogens through cohousing with pet-store mice or through exposure to their dirty bedding. NME exposure for many months rarely compromises the viability of young mice (89% survival across all experiments) (Fig. 3A) (36–38). By contrast, exposing old mice (20+ months of age) to the same NME rapidly caused nearly 100% lethality in <2 weeks and in both sexes (Fig. 3A and fig. S8A). In mice euthanized on day 6 or 7 after NME exposure, expression of senescence markers (p21^Cip1 and p16^Ink4a) and SASP factors (Il6, Mcp1, and Tnfα) in liver, kidney, and to a lesser extent in lung were increased in old NME mice compared with old SPF, young SPF, or young NME mice (Fig. 3B). In addition, there was an increase in infiltration of CD45⁺ cells into the liver by day 6 or 7 after NME exposure in both young and aged mice (fig. S8B). The percent of infiltrating immune cells was significantly higher in aged mice than in young animals. These results are consistent with spread of senescence and inflammation after pathogen exposure. In addition, there was a significant increase in SASP-related inflammatory cytokines (IL-6, IL-10, EOTAXIN/CCL11, and TNFα) in the serum of old NME mice compared with young NME mice (Fig. 3C), which is consistent with preexisting SnCs creating an environment that contributes to hyperinflammation upon infection.

Several viruses were detected in saliva and fecal pellets from the NME mice a week after exposure to pet store mice, including the β-coronavirus mouse hepatitis virus (MHV), a virus in the same family as SARS-CoV-1 and -2 (table S4). However, by day 11, when the majority of old mice had succumbed to infection, NME mice were serologically positive for MHV but not the other pathogens carried by pet store mice (Fig. 3D). Histopathology indicated that old but not young mice had evidence of active MHV infection, manifested as multifocal necrotizing hepatitis and the presence of MHV-specific syncytial cells within areas of necrosis (Fig. 3E). In addition, MHV-induced syncytial cells were observed among epithelial cells in the small and large intestines of aged mice (fig. S8C). These findings are consistent with active infection in aged animals, in contrast to rapid clearance in the young animals.

To determine whether MHV infection contributes to NME-mediated mortality in old mice, young and old mice were directly infected with a sublethal dose of MHV (strain A59) before NME exposure (Fig. 3F). Old mice challenged with MHV generated a reduced antibody response compared with young mice (Fig. 3F). However, MHV immunization prevented death of the old mice after NME exposure, although the animals were infected with multiple other viruses (table S5), whereas naïve, old mice succumbed (Fig. 3G). This provides compelling evidence that the β-coronavirus MHV is the primary driver of mortality in old mice in the NME paradigm.

Senolytics reduce senescence, inflammation, and mortality after pathogen exposure

To determine whether drugs that induce apoptosis specifically of SnCs, termed senolytics, reduce the mortality of old mice acutely infected with pathogens, we tested fisetin, a natural flavonoid found in many fruits and vegetables (39, 40) that we established as senolytic (14, 41). fisetin improves tissue homeostasis, reverses age-related tissue damage, and extends median life span of mice, even when administered late in life, with no observable adverse effects (14, 41).

Old mice were exposed to NME for 1 week starting on day 0 and were then treated with 20 mg/kg fisetin by means of oral gavage on days 3 to 5, 10 to 12, and 17 to 19 after pathogen exposure (Fig. 4A), with no evidence of adverse effects. In between fisetin dosing, the mice were on a maintenance dose of fisetin [500 parts per million (ppm) Fisetin in chow ad libitum]. Consistent with our previous results (Fig. 3A), 100% of the old mice in the vehicle control groups died within 2 weeks (Fig. 4B, sexes combined, and fig. S9A, graphed by sex). However, 64% of the fisetin-treated male mice and 22% of the female mice survived long-term with a significant extension of overall life span for both sexes. Whether there is a true sex difference in the effect of fisetin on survival needs to be explored further because the ages of the old male and female mice were not identical.

On day 11 after NME, relative levels of antibodies against MHV were dramatically lower in the old than young mice (Fig. 4C), which is consistent with the premature death of the old mice. However, in old mice treated with fisetin, antibodies against MHV were increased to youthful levels by day 16. All mice exposed to NME were confirmed MHV-positive by means of reverse transcription polymerase chain reaction (RT-PCR) at 8 days after exposure (Fig. 4D). The old mice had significantly more viral mRNA than that of young mice (Fig. 4D), which is consistent with impaired immune responses in aged organisms (Fig. 3F) and impaired viral defenses because of SnCs (Fig. 2C). However, a short duration of fisetin treatment initiated 3 days after NME exposure tended to reduce the viral mRNA burden in old mice (P = 0.09) (Fig. 4D).

To evaluate how fisetin mediates its protective effects on NME-induced mortality in aged mice, we measured senescence and SASP markers before death. Cellular senescence markers (p16^Ink4a or p21^Cip1) were reduced in the liver, kidney, lung, and spleen of the old fisetin-treated NME mice compared with old mice receiving vehicle only (Fig. 4E). Furthermore, expression of multiple SASP inflammatory factors—including Ifnγ, Il1α, Il1β, Il6, Il17, Tnfα, Cxcl1, Cxcl2, Cxcl10, Mcp1, Mip1, Pai1, Pai2, Il2, and Il7—was reduced to varying extents in the same tissues (Fig. 4F and fig. S9B). Similarly, the levels of circulating IL-1β, IL-6, MCP-1, and TNFα were reduced after fisetin treatment (Fig. 4G). Thus, although the old mice were MHV-infected, fisetin reduced senescence, the SASP, and inflammation after infection and prolonged survival, enabling an improved antibody response to the virus.

Senolysis contributes to improved outcomes in old mice exposed to pathogens

To determine whether the mechanism of action of fisetin in suppressing adverse outcomes upon viral infection includes senolysis, two approaches were taken. First, INK-ATTAC mice were studied under NME conditions to enable genetic ablation of p16^Ink4a-expressing SnCs (42). INK-ATTAC mice express a caspase 8-FKBP fusion protein, ATTAC (43), from the p16^Ink4a promoter. Old INK-ATTAC mice (>24 months) were treated with AP20187 to drive dimerization of FKBP, activation of caspase-8, and apoptosis of p16^Ink4a-expressing cells (3 days per week × 2 weeks), before exposure to NME and then weekly after NME (Fig. 5A). Both control and AP20187-treated mice were positive for MHV RNA at day 8 after NME exposure (Fig. 5B). AP20187 treatment reduced the expression of the SnC markers p16^Ink4a and p21^Cip-1 and of enhanced green fluorescent protein (eGFP), which is also driven by the p16^Ink4a promoter in INK-ATTAC mice after NME exposure (Fig. 5C), as well as certain inflammatory/SASP genes in kidney, liver, brain, pancreas, and/or colon (fig. S10). AP20187 treatment significantly delayed NME-induced mortality in both male and female aged mice (Fig. 5D and fig. S10A), providing evidence that senolysis improves outcomes in aged organisms acutely exposed to pathogens. The level of MHV RNA also trended down after AP20187 treatment (Fig. 5B), which is consistent with the results with fisetin treatment.

Second, we tested a different well-established senolytic cocktail, Dasatinib plus Quercetin (D+Q) (12, 13), and directly compared it with fisetin in the same survival experiment. D+Q or fisetin was administered to aged female mice at days 3 or 4 then 11 or 12, respectively, after initiation of NME (Fig. 5E). As expected, whereas 100% of the old, vehicle-treated mice succumbed to infection, ~50% of the old mice treated with D+Q or fisetin survived (Fig. 5E). The similarity in survival curves between the two treatment groups is notable. This combination of genetic and pharmacologic studies provides strong support for the conclusion that clearing SnCs in old organisms contributes to improved outcomes upon acute exposure to viral pathogens.

Last, to determine whether pretreating old mice with fisetin after infection could prevent adverse outcomes, old WT mice were treated with a single round of high-dose fisetin (20 mg/kg/day for 2 consecutive days beginning 3 days before NME exposure), followed by low-dose fisetin after infection (fig. S11A). This suppressed mortality in both male and female mice by 40% (fig. S11, A and B). Additionally, antibodies against MHV were detected in fisetin-treated mice on days 16 and 21 (fig. S11C), a time by which all vehicle-treated old mice had died (fig. S11A). To evaluate whether a shorter regimen of senolytic therapy could improve outcomes in old NME mice, animals were given after NME exposure two doses of fisetin once (days 3 and 4) (Fig. 5F) or twice (days 3 and 4 then days 10 and 11) (Fig. 5G). These short-course treatments, in the absence of continuous exposure to fisetin through chow, were sufficient to delay mortality significantly (Fig. 5, F and G). Because Fisetin has an elimination half-life of less than 5 hours (44), these data are consistent with a “hit-and-run” mechanism, in which fisetin is acting as a senolytic, reducing overall SnC burden, rather than being required to be present constantly to engage with a molecular target to confer benefit. The data also reveal that fisetin can be administered in a pulsatile fashion before or after viral infection to reduce mortality of old organisms.