·       Aged, non-proliferating (senescent) cell burden in old mice confers the reduced lifespan observed following exposure to new viral pathogens.
·       Both pharmacological and genetic clearance of senescent cells yielded significant delay or reduction in mortality in both old male and female mice.

The COVID-19 pandemic has revealed the pronounced vulnerability of the elderly and chronically ill to SARS-CoV-2-induced morbidity and mortality. Cellular senescence in aged people contributes to inflammation, multiple chronic diseases, and age-related dysfunction, but its effects on responses to viral infection are unclear.

Here, researchers from the University of Minnesota and the Mayo Clinic demonstrate that targeting senescent cells using senolytic drugs before or after exposure to SARS-CoV-2-related mouse β-coronavirus significantly reduced mortality, cellular senescence, and inflammatory markers and increased antiviral antibodies. This study, published in Science, demonstrates that reducing the senescent cell burden in diseased or aged individuals should enhance resilience and reduce mortality following viral infection, including SARS-CoV-2.

“Even though vaccine use is growing, senolytics could still be helpful to those who cannot receive the vaccine, and especially to older individuals in nursing homes with comorbidities or immunity issues,” said James Kirkland, M.D., Ph.D., director of the Kogod Center on Aging and, together with Tamar Tchkonia, Ph.D., senior author for the Mayo Clinic on the study.

Senolytics reduce senescence, inflammation, and mortality following pathogen exposure

Although there are now vaccines for SARS-CoV-2 being distributed, it will take a long time for a significant percentage of the world’s population to be vaccinated. Even if the 95% effectiveness rate of the vaccines in healthy populations is borne out in elderly nursing home residents, still at least 1 out of 20 vaccinated elderly residents is anticipated to become infected by COVID-19 and will need treatment, potentially with senolytics and antivirals.

“We wanted to determine if therapeutically targeting fundamental aging mechanisms, such as cellular senescence, could reduce morbidity and mortality following viral infection,” said Christina Camell, Ph.D., an assistant professor in the Department of Biochemistry, Molecular Biology and Biophysics, and a first author of the study.

To find out if drugs that induce cell death specifically in senescent cells, termed senolytics, reduce the mortality of old mice acutely infected with pathogens, Camell and colleagues tested Fisetin, a natural flavonoid found in many fruits and vegetables that is established as a senolytic. Fisetin is known to reverse age-related tissue damage and extend the median lifespan of mice, even when administered late in life, with no observable adverse effects.

To test Fistein’s effects, Camell and colleagues exposed young and old mice to a normal microbial environment (NME) that included a mouse β-coronavirus related to SARS-CoV-2. Old mice were exposed to NME for 1 week starting on day 0 and were then treated with 20 mg/kg Fisetin in water (oral gavage) on days 3, 4, and 5 and 10, 11, and 12 following pathogen exposure, with no evidence of adverse effects. In between Fisetin dosing, the mice were on a maintenance dose of Fisetin (500 ppm fisetin in chow ad libitum).

All of the old mice in the vehicle control groups died within 2 weeks. However, 64% of the Fisetin-treated male mice and 22% of the female mice survived long-term with a significant extension of overall lifespan for both sexes. Whether there is a true sex difference in the effect of Fisetin on survival needs to be explored further since the ages of the old male and female mice were not identical.

(Camell et al., 2021 | Science) The senolytic Fisetin decreases mortality in old mice exposed to a SARS-CoV-2 related mouse β-coronavirus. The left image is a schematic of the experiment. Young (6-7 months) and old (20-24 months) mice were exposed to a normal microbial environment (NME) bedding containing mouse β-coronavirus MHV for 7 days. Mice were treated with 20 mg/kg/day Fisetin or vehicle only by oral gavage daily for 3 consecutive days starting on day 3 post-initiation of NME. The 3 days of treatment were repeated (3 days on, 4 days off) for 3 weeks. Animals were also fed standard chow with Fisetin added (500 ppm) ad libitum after initiation of treatment. The right plot shows the survival of these animals, which was measured for 36 days following initiation of NME. While all old untreated mice (black) died within 16 days of the experiment, very few of the Fisetin-treated old mice (blue) died in this same time frame. Over the course of 36 days, roughly half of the Fisetin-treated mice survived.

Notably, on day 11 after exposure to the NME, the relative levels of antibodies related to the virus were dramatically lower in the old than young mice, consistent with the premature death of the old mice. However, in old mice treated with Fisetin, antibodies against the virus were increased to youthful levels by day 16.

To evaluate whether a shorter regimen of senolytic therapy could improve outcomes in old NME mice, animals were given two doses of Fisetin after NME exposure once (days 3 and 4) or twice (days 3,4 and 10,11). These short-course treatments, in the absence of continuous exposure to Fisetin via chow, were sufficient to delay mortality significantly.

(Camell et al., 2021 | Science) Fisetin decreases mortality in old mice exposed to a normal microbial environment (NME) that includes a SARS-CoV-2 related mouse β-coronavirus. The left plot shows survival curves for 20-month-old untreated (WT) female mice treated with 20 mg/kg Fisetin or vehicle orally on days 3 and 4 following initiation of NME exposure. The right plot shows Survival of 22-month-old WT female mice treated with 20 mg/kg Fisetin or vehicle only by oral gavage at days 3, 4, 10, and 11 post-NME exposure monitored out to 60 days post.

To evaluate how Fisetin mediates its protective effects on pathogen-induced mortality in aged mice, Camell and colleagues measured senescence and the milieu of discharged molecules associated with senescence, called the senescence-associated secretory phenotype (SASP), before death. Senescent cell markers were reduced in the liver, kidney, lung, and spleen of the old Fisetin-treated mice exposed to an NME that included a mouse β-coronavirus related to SARS-CoV-2 compared to old mice receiving the vehicle only. Furthermore, the levels of multiple SASP inflammatory factors were reduced to varying extents in the same tissues. Thus, although the old mice were infected with a coronavirus, Fisetin reduced senescence, the SASP, and inflammation post-infection and prolonged survival, enabling an improved antibody response to the virus.

“We have been working on a new approach to help the elderly remain healthy, which is to find therapeutics to treat aging rather than treating each disease associated with old age. The fact that senolytics worked to protect old organisms from a viral infection proves that approach is accurate,” said Paul Robbins, Ph.D., co-director of the Institute on the Biology of Aging and Metabolism at the University of Minnesota Medical School.

“By getting rid of a piece of aging biology, senescent cells, with senolytics, the older mice were able to withstand the stress of infection. This suggests that reducing the burden of senescent cells in ill or elderly individuals could improve their resilience and reduce their risk of dying from COVID-19.”

Inflammation “Amplifier/Rheostat” Hypothesis

This study demonstrates that senescent cells are primed to respond to pathogen-associated molecular patterns (PAMPs) by expressing and secreting even higher levels of inflammatory SASP factors. These PAMPs include the SARS-CoV-2 spike protein-1, which exacerbates the SASP of human senescent cells and, in turn, reduces innate viral defenses and increases expression of SARS-CoV-2 viral entry proteins in non-senescent human lung cells and tissue.

Based on these observations, Camell and colleagues formulated the “Amplifier/Rheostat” Hypothesis, whereby PAMPs, such as SARS-CoV-2 S1 viral antigen, cause a shift in SASP to a more highly inflammatory, tissue damaging state. The amplified SASP factors include inflammatory signaling molecules called cytokines and chemokines that exacerbate systemic inflammation and drive senescence in non-senescent cells. These secondary senescent cells can then: 1) further exacerbate and prolong inflammation; 2) reduce viral defenses in non-senescent cells; 3) facilitate viral entry in non-senescent cells; 4) attenuate or delay recovery; 5) contribute to persistent frailty; 6) cause tissue scarring (fibrosis); and 7) contribute to hyper-inflammation and multi-organ failure.

(Camell et al., 2021 | Science) SASP Amplifier/Rheostat Hypothesis. Schematic of the hypothesis generated from these data and tested herein. Senescent cells amplified the response to pathogen-associated molecular patterns (PAMPs), resulting in increased production of pro-inflammatory cytokines and chemokines. This could exacerbate acute systemic inflammatory responses and cytokine release by innate immune cells and amplify the spread of senescence. This model could explain the increased risk of cytokine storm during COVID-19 or other infections and adverse outcomes observed in the elderly or those with chronic conditions associated with an increased burden of senescent cells (obesity, diabetes, chronic lung or kidney disease, and cardiovascular disease).

Importantly, the SASP Amplifier Hypothesis, supported by data presented here, led to the initiation of a clinical trial (NCT04476953) to test whether Fisetin prevents disease progression in hospitalized older COVID-19 patients. A similar, but larger multi-site trial to test Fisetin in elderly COVID-19 patients in nursing homes (NCT04537299) also has been initiated.