key: cord-0729914-joqkwes2
authors: Justice, Jamie N.; Gubbi, Sriram; Kulkarni, Ameya S.; Bartley, Jenna M.; Kuchel, George A.; Barzilai, Nir
title: A geroscience perspective on immune resilience and infectious diseases: a potential case for metformin
date: 2020-09-09
journal: GeroScience
DOI: 10.1007/s11357-020-00261-6
sha: 8d6dbd2d0b41d52d138fc173f397cff620631753
doc_id: 729914
cord_uid: joqkwes2

We are in the midst of the global pandemic. Though acute respiratory coronavirus (SARS-COV2) that leads to COVID-19 infects people of all ages, severe symptoms and mortality occur disproportionately in older adults. Geroscience interventions that target biological aging could decrease risk across multiple age-related diseases and improve outcomes in response to infectious disease. This offers hope for a new host-directed therapeutic approach that could (i) improve outcomes following exposure or shorten treatment regimens; (ii) reduce the chronic pathology associated with the infectious disease and subsequent comorbidity, frailty, and disability; and (iii) promote development of immunological memory that protects against relapse or improves response to vaccination. We review the possibility of this approach by examining available evidence in metformin: a generic drug with a proven safety record that will be used in a large-scale multicenter clinical trial. Though rigorous translational research and clinical trials are needed to test this empirically, metformin may improve host immune defenses and confer protection against long-term health consequences of infectious disease, age-related chronic diseases, and geriatric syndromes.

Abstract We are in the midst of the global pandemic. Though acute respiratory coronavirus (SARS-COV2) that leads to COVID-19 infects people of all ages, severe symptoms and mortality occur disproportionately in older adults. Geroscience interventions that target biological aging could decrease risk across multiple age-related diseases and improve outcomes in response to infectious disease. This offers hope for a new hostdirected therapeutic approach that could (i) improve outcomes following exposure or shorten treatment regimens; (ii) reduce the chronic pathology associated with the infectious disease and subsequent comorbidity, frailty, and disability; and (iii) promote development of immunological memory that protects against relapse or improves response to vaccination. We review the possibility of this approach by examining available evidence in metformin: a generic drug with a proven safety record that will be used in a large-scale multicenter clinical trial. Though rigorous translational research and clinical trials are needed to test this empirically, metformin may improve host immune defenses and confer protection against long-term health consequences of infectious disease, age-related chronic diseases, and geriatric syndromes.

Keywords Metformin . Aging . Geroscience . Immunity . COVID-19

The promise of geroscience is that interventions that target biological aging could fortify organismal resilience and delay the onset or lessen the severity of multiple age-related diseases en masse. Never has this promise been more critical than now, in the midst of the global pandemic. Though acute respiratory coronavirus (SARS-COV2) infects people of all ages, severe symptoms and mortality occur disproportionately in older adults. In the USA, 8 out of 10 deaths occurred in persons over the age of 65 years [1] , and in Britain, persons above age 80 years were~180 fold more likely to die that those in their 40s [2] . Severity and complications also increase nonlinearly with age. In China, the odds of hospitalization at ages 40-49 years were 4.3% but nearly doubled to 8.2%, for 50-59-year-olds, and continued to climb for each decade of advancing age up to 18.4% in persons over the age of 80 years [3, 4] .

This trend is not unique to COVID-19. It is well known that older persons generally have greater susceptibility to infections than younger adults due to agerelated changes in cell-mediated immunity, underlying chronic disease, and geriatric syndromes [5, 6] . Moreover, the pattern of exponential increase in mortality rate with age in COVID-19 exhibits a doubling time estimated between 6.6 to 9 years (USA, China, Italy); this is mirrored by the exponential increase in age-related risk of mortality from all causes (doubling time of~9 years, USA), and cause specific which ranges from 4.7 years (chronic obstructive pulmonary disease, COPD) to 7.1 years (diabetes mellitus, DM) [7] . This suggests contributions of age-related declines in immune and inflammatory responses in harmony with systemic age-related physiologic dysregulation and comorbidity [3, 8] . Chronically elevated inflammation and dysregulated cell-cell communication constitute examples of biological hallmarks of aging and represent both contributors to and consequences of varied chronic conditions that are independently associated with increased COVID-19 vulnerability. For example, the presence of multiple chronic conditions or comorbidities such as diabetes, chronic kidney disease, and heart failure are associated with proinflammatory cytokines and other biomarkers of biological hallmarks of aging [9] [10] [11] , and have been shown to be independently associated with severe COVID-19 illness and death [12] [13] [14] [15, 16] . Therefore, while virus-specific treatments and vaccines are critically needed, other complementary strategies are also needed to address increased vulnerability to severe COVID-19 during this window of vulnerability as vaccines and antiviral agents are being developed. To that end, treatment of the biological underpinnings which render older adults more vulnerable may not only improve outcomes during this COVID-19 pandemic, but could also offer benefits in terms of future pandemics involving other pathogens, while also impacting positively the onset and progression of varied acute and chronic diseases [17] . Geroscience interventions that target biological aging could decrease risk across multiple age-related diseases and improve outcomes in response to infectious disease. This offers hope for a new host-directed therapeutic approach that could (i) improve outcomes following exposure or shorten treatment regimens; (ii) reduce the chronic pathology associated with the infectious disease and subsequent comorbidity, frailty, and disability; and (iii) promote development of immunological memory that protects against relapse or improves response to vaccination [18, 19] (Fig. 1) .

Preclinical interventions testing programs show that biological aging pathways can be therapeutically targeted. Perhaps, the most robust intervention is caloric restriction, which has been shown to prolong the lifespan of experimental animal models such as nematodes, flies, and mice [20, 21] and may improve immune response [22] . In addition, promising pharmacological and nutraceutical candidates have been identified. In most cases, median lifespan extension is accompanied by improved health and ability of preclinical models to withstand both chronic and acute challenges [23, 24] . A select subset of these have intriguing effects in humans on overall health and immune function, including acarbose [25] , and mTORC1 inhibitors rapamycin, everolimus, and RTB101 [26] . For example, mice treated with 6 weeks of rapamycin mounted a greater response to influenza vaccine than age-matched control mice [27] , and two phase II studies of mTORC inhibitors demonstrated improved antibody responses to an influenza vaccine and decreased rates of upper respiratory tract infections by varied pathogens in healthy community-dwelling older adults [26, 28] . Phase III trial of mTORC1 inhibitor RTB101 did not meet its primary endpoint for prevention of respiratory illness. However, two new phase I-II trials with mTOR inhibitor sirolimus are now in progress in hospitalized patients with COVID-19 pneumonia (NCT04341675 and NCT04371640) and those with RTB101 in the nursing home setting (NCT04409327).

A critical obstacle for larger clinical trials and widespread clinical application of any drug targeting aging is safety, tolerability, and expense. So far, only a select couple of drugs can overcome these translational barriers. One such drug is metformin. Though metformin offers modest improvements in lifespan in preclinical interventions testing [29] , it targets molecular and cellular drivers of aging which may exert positive effects more broadly on health span. Moreover, metformin (i) is a generic and relatively inexpensive medication, (ii) has an excellent safety record for over 5 decades, and (iii) can be generally well tolerated with long-term use in a large population of aging adults. Metformin will be tested in a first-of-its-kind trial designed to create a regulatory pathway for aging as a target for drug development, the phase III multicenter randomized doubleblind placebo-controlled trial Targeting Aging with MEtformin (TAME) [30, 31] . With recruitment start coinciding with what is anticipated to be the waning of the COVID-19 global pandemic, TAME provides an excellent platform to probe preventive effects on agerelated chronic disease and long-term consequences of infectious disease and vaccine response in older persons. In light of the recent global pandemic, we provide a discussion of metformin in immune aging and infectious disease focusing on clinical evidence.

Metformin is a biguanide and is currently the most widely prescribed oral hypoglycemic medication for type 2 diabetes treatment or prevention prescribed worldwide. However, its history dates back to the seventeenth century, when metformin-like guanidine compounds found in traditional herbal medicine Galega officinalis were used to treat conditions like plague, fever, and snake bites. Metformin was rediscovered in the 1940s, in a search for antimalarial agents, and in 1949, metformin was found to be helpful in treating a local influenza outbreak in the Philippines [32, 33] . Its immune effects were accompanied by the beneficial side effect of lowered blood glucose in some of the influenza patients. In the 1950s, the physician Dr. Jean Stern translated this glucose-lowering potential into diabetes treatments, though metformin was not finally approved by the Food and Drug Administration (FDA) as an antidiabetic until 1995.

Interest in metformin's clinical effects beyond glucose regulation originated from epidemiologic studies, which observed a reduction in cardiovascular risk and all-cause mortality in individuals with diabetes treated with metformin in the Diabetes Prevention Program (DPP) and UK Prospective Diabetes Study (UKPDS) [34, 35] . Since then, there is growing interest in metformin's clinical benefits beyond diabetes (reviewed in [30] ) in diseases ranging from gestational diabetes, polycystic ovary syndrome (PCOS), and human immunodeficiency virus (HIV)-associated lipodystrophy, to dementia-type time, and improved return to baseline health compared to nonuse (black line). Greater long-term health effects could be observed as improved response to vaccine and prevention or delay of age-related diseases and geriatric syndromes neurodegenerative disorders and certain cancers in individuals with and without type 2 diabetes.

Metformin has emerged as a leading candidate in clinical trials in aging and age-related acute and chronic diseases given its cellular effects which are distinct from its effects on glucose metabolism in diabetics. Kulkarni et al. [36] recently reviewed metformin's effects on the biological hallmarks of aging, with supporting evidence from C. elegans, drosophila, and rodents. Metformin acts through a few widely accepted cellular and molecular mechanisms that are highly interdependent and implicated metformin's downstream effects on immune response, chronic diseases, and geriatric syndromes ( Table 1) . Chief among these cellular mechanisms are metformin's actions on the mitochondrion as a weak inhibitor of complex I of the mitochondrial electron transport chain, activation of the energy sensor AMPactivated protein kinase, inhibition of mTORC1, and regulation of inflammatory cytokine signaling [30, 36] . Converging evidence also implicates lysosomes [37] , gut microbiome [38] , and epigenetic modulation as mediators of metformin's actions on biological aging [39, 40] . Importantly, metformin is not proposed to exert action by any one single or distinct mechanism but through multiple mechanisms. Moreover, it is precisely this plurality of pleiotropic cellular effects that makes metformin a valuable tool to achieve downstream effects on immunoprotection, and multiple age-related diseases and geriatric conditions.

Metformin also exerts actions in immune modulation and inflammatory response, lending resilience to infectious diseases and acute respiratory illnesses (Fig. 2 , Table 2 ). Metformin's interference with key immunomodulatory mechanisms are both dependent and independent of its effects on improving in metabolic parameters. Several mechanisms may underlie metformin's effects on immune resilience and responses to infectious disease; however, it is important to note that the effects of metformin on the immune system are dependent on the pathological context (reviewed in [41] ). In the context of some cancers, metformin has been shown to be immunostimulatory with a shift favoring M1 m a c r o p h a g e p o l a r i z a t i o n o v e r t h e m o r e antiinflammatory M2 [42, 43] and increased antigenspecific T cell responses coupled with decreased immunosuppressive T regulatory cells [44, 45] . However, this shift towards an immunostimulatory phenotype seems specific to pathological immunosuppressive conditions such as cancer. Conversely, in most other situations, metformin is able to quell aberrant inflammation and promote antiinflammatory pathways. Indeed, in the context of wound healing, metformin induces M2 polarization [46] . Additionally, during autoimmune diseases [47] , graft-versus-host disease [48] and others more proinflammatory conditions, metformin is able to increase T regulatory cells and decrease T helper (Th)17 cells [47] [48] [49] , an effect likely partially mediated by AMPK activation [50] . Moreover, metformin treatment in uninfected mice increases in vitro T cell IFN-γ responses as well, indicating that metformin treatment has the ability to modulate immune cells independent of current infections and potentially improve responses to future pathogens [51] . For coronaviruses, the timing of type I IFN production is critical to infection outcome: an early burst of type I IFN leads to protection, but a delay in IFN production causes an inability to control viral replication or pathogen spread, leading to cellular damage of airway epithelia and the lung parenchyma and an eventual lethal inflammatory cytokine storm [52] . Thus, metformin has the potential to improve the initial type I IFN response that is crucial for coronavirus immune responses and prevent or reduce cellular damage from exaggerated, prolonged inflammatory responses.

Indeed, metformin's effect on immune responses to chronic pathogen exposures or challenges, as well as general states with elevated inflammation, tends to be overall immunosuppressive and antiinflammatory ( Fig.  2 ). This dysregulated immune signaling is particularly relevant to SARS-CoV-2, which causes exaggerated and aberrant host immune responses that are uniquely challenging in persons of advanced age [53]. This 'cytokine storm' of perpetuating proinflammatory signaling loops is thought to lead to acute respiratory distress syndrome and poorer outcomes, especially in older adults [54, 55] . Metformin improves the immune response and reduces inflammation by promoting the formation of M2 macrophages and T regulatory and CD8 memory T cells [56] , and may prevent the differentiation of monocytes to macrophages via an AMPKdependent inhibition of STAT3 [57] . In the face of chronic infection or disease, metformin also reduces genes encoding for cytokines associated with inflammation response by suppression of tumor necrosis factor-α (TNF-α) dependent NF-κB signaling and expression of interleukin (IL)-6 (IL-6), IL-β (IL-1β), MCP-1 (CCL2 chemokine ligand 2), CXCL5, and IP-10 (CXCL10) [51, 58, 59] . Collectively, this results in lower levels of circulating proinflammatory cytokines, and in a recent report in older persons with T2DM, the reduction in proinflammatory signaling was associated 5-year allcause mortality risk [60] . Furthermore, metformin treatment diminished LPS-induced acute lung injury [61] , suggesting that metformin may be able to reduce the cytokine storm-like response and prevent exaggerated lung damage due to SARS-CoV-2 as well. In addition, metformin can induce autophagy and facilitate phagosome-lysosome fusion and induces phagocytosis of neutrophils, which points to a role for metformin in killing or containing pathogens, controlling inflammation, and activating innate and adaptive immune response in the host [62] .

Metformin may also reduce inflammation through gut microbiota [38, 62] . Multiple studies have shown that the glucose lowering ability of metformin is partly mediated through the gut microbiota changes [63] [64] [65] .

Others have further shown that the antiinflammatory effects of metformin may also be partly mediated through the gut microbiota, even in nondiabetic conditions where the pattern of gut microbe changes with metformin are negatively correlated with a variety of inflammatory diseases [66] . While the mechanism of effect on the microbiota is not known, it seems that metformin may reduce dysfunctional gut permeability ("leaky gut") by increasing goblet cell mass and mucin production [67] , and may alleviate gut dysbiosis by Table 1 Summary of metformin's mechanisms in attenuating hallmarks of aging. Metformin has been postulated to exert gerotherapeutic effects through several molecular pathways related to biological hallmarks of aging [36] Aging hallmarks Metformin's effects on key targets and pathways

• Antiinflammatory response and immunomodulation ↓ pro-inflammatory cytokines (IL-6, IL-1β, CXCL1/2) [59] ↓ NF-κB and IKKα/β signaling [111] • Regulation of the gut microbiota by altering microbial folate Anti-inflammatory response via ↑ Akkermansia, ↑ Bacteroides, ↑ Butyricimonas and ↑ Parabacteroides genera [112, 113] Methionine restriction [114] Deregulated nutrient sensing • Direct targeting of key energy sensors and modulation of nutrient sensing pathways ↑ AMPK [37, 115, 116] , ↑ SIRT1 [117, 118] ↓ mTORC1 (via ↓ Rad-GTPase, ↑ TSC2, ↑ REDD1) [119] [120] [121] [122] ↓ Insulin and IGF-1 signaling [123] Genomic instability • Genome protective effects with ↑ DNA-damage-like response and ↑ DNA repair and regulation of ATM-protein kinases [124] [125] [126] Loss of proteostasis • Increased autophagy and rescue of protein misfolding ↑ LAMP-1 and ↑ Beclin-1 [127, 128] ↑ CEBPD-mediated autophagy [129] Mitochondrial dysfunction • Inhibition of mitochondrial complex I of the electron transport chain • Lowering mitochondria-induced oxidative stress [130] [131] [132] ↓ Endogenous production of ROS [130] [131] [132] [133] • Improved mitochondrial biogenesis via ↑ PGC-1α [134] Stem cell exhaustion • Improved stem cell rejuvenation capacity [135] [136] [137] • Delayed stem cell aging [138] Epigenetic alterations • Transcriptional regulation via histone modifications, DNA methylation and miRNAs [40] Phosphorylation of Histone Acetyl Transferases (HATs) ↓ Class II Histone Deacetylases (HDACs) ↑ DICER1 [139] Telomere attrition • Reduction of telomere shortening and ↑ telomeric repeat containing RNA (TERRA) [140] Cellular senescence • Suppression of senescence-associated secretome ↓ p16, ↓ p21 and RNA-levels of SASP hallmarks [111, 139, 141] ↓ β-gal activity and ↓proinflammatory senescence [142] increasing Akkermansia muciniphila and short-chain fatty acid-producing bacteria, such as Bacteroides and Butyricimonas [68] . Of particular interest, these alterations were still observed in aged obese mice despite the presence of preexisting age-related microbiota changes and were correlated with lower IL-6 and Il1β [67] [68] . This suggests that metformin may also be able to mitigate exaggerated inflammatory responses via changes in gut microbiota.

Metformin and resilience to severe infectious disease

The origin and pathogenesis of the acute respiratory disease caused by the novel coronavirus SARS-CoV2 is increasingly well understood [54] . SARS-CoV2 is an enveloped nonsegmented positive-sense RNA virus. The virus gains entrance to host cells through angiotensin-converting enzyme 2 (ACE2) receptors, and upon entry the virion releases its RNA in the cytoplasm. Translation and replication occur and new virions are then released from the host cell [69, 70] . However, key to COVID-19 pathogenesis is the attendant explosive and deleterious host immune response and immunopathogenesis [71] . This cytokine storm, or cytokine release syndrome, may occur approximately 5-8 days after symptom onset and is associated with dismal outcomes including ICU care, mechanical ventilation, prolonged hospital care, and in some cases, death. Inflammatory factors elevated during this storm include IL-6, IFNγ, TNFα, IL-1β, IL-8, MCP-1, (CXCL10), and lower T cell counts (CD8+ and CD4+ T lymphocytes) in older adults with severe COVID-19 cases [71] [72] [73] [74] [75] [76] . Importantly, elevated levels of the proinflammatory factors are associated severity of COVID-19 and unfavorable outcomes like acute respiratory distress syndrome, multiple-organ failure [73, 76] . Interestingly, the list proinflammatory cytokines implicated in the cytokine storm are nearly identical to those suppressed with metformin administration. Intriguing epidemiologic evidence indicates a potential benefit of metformin on COVID-19 health outcomes (Table 3 ). In a retrospective analysis of 283 T2DM patients from Wuhan, China, with confirmed COVID-19, investigators found no difference in the length of stay in hospital, but persons taking metformin had significantly lower in-hospital mortality (3 of 104, 2.9%) than those not taking metformin (22 of 179, 12.3%) [77] . Upon hospital admission, both groups were under strict glucose control and all received antiviral and supportive treatments including antibacterial and anticoagulants, with no significant differences in treatment. This report is further supported by an in-review analysis of de-identified claims data from UnitedHealth of 6256 persons hospitalized with COVID-19 [78] . Women who had at least 90 days of metformin claims in the 12 months before hospitalization were found to havẽ 21% lower odds of mortality compared with persons with T2DM but without metformin. This association was not observed for men. Though difficult to quantify, metformin's immunoprotective effects in the context of COVID-19 may be masked if fewer persons taking metformin are being admitted to hospital.

Importantly, these findings are relevant only in the context of long-term treatment with metformin prior to infection. Therefore, these epidemiologic studies do not generalize to potential actions of metformin administered in hospitalized patients with active infection as a treatment adjuvant. While metformin use is generally safe, side effects and gastrointestinal discomfort do occur in a proportion of the population, and it is unknown what the additional risk of initiating metformin in hospital for those with comorbid or critical conditions upon admission would be. Basic research coupled with carefully controlled clinical trials would be required prior to translation to the clinic. Moreover, the protective effects of metformin on health outcomes due to acute infectious disease must be replicated and confirmed with rigorous prospective studies with and without overt T2DM. Finally, emerging evidence supports close investigation of both potential sexual dimorphism and mediating effects of metformin on proinflammatory components of the cytokine storm also require close investigation.

Evidence of metformin as a therapeutic strategy in other infectious diseases and respiratory illnesses is emerging from a range of experimental designs, model systems, and pathogens or exposures ( Table 2 ). In a screen of 13 FDA-approved autophagy activators and AMPK modulators, metformin (i) displayed direct antimycobacterial effects; (ii) controlled the growth of drug-resistant bacterial strains, (iii) increased production of mitochondrial reactive oxygen species (mROS), and (iv) facilitated phagosome-lysosome fusion [51, 79] . In mice, use of metformin monotherapy enhanced ameliorated lung pathology and enhanced host immune responses, and in combination therapy, metformin enhanced the specific immune response and the efficacy of conventional antimycobacterial drugs [51] . Collectively, this confers host protection and lessens burden of active infection. For example, retrospective analyses of tuberculosis patients who were on metformin had fewer pulmonary cavities, less advanced disease, and lower mortality rate than those not on metformin [51, 80] . Moreover, in 2416 persons being treated for tuberculosis in Taiwan, coexisting metformin use was significantly associated with decreased mortality during treatment; strikingly, metformin users had similar mortality as patients without T2DM [80] .

Laboratory studies using cells or model organisms suggest metformin could be effective against numerous other pathogens, including Trypanosoma cruzi, Trichinella spiralis, Staphylococcus aureus ( S. aureus), and Pseudomonas aeruginosa (P. aeruginosa) [81, 82] . Of relevance to COVID-19, bacterial infections of the respiratory system by S. aureus and P. aeruginosa are associated with local (vs. systemic) hyperglycemia, which may be associated with respiratory tract infections in conditions like chronic obstructive pulmonary disease [83] . Interestingly, metformin's effects on airway epithelial tight junctions [84] and bacterial load are independent of effects on blood glucose but use an AMPK-and PKCζ-mediated pathway [84, 85] . Moreover, in a murine Legionella pneumophila pneumonia model, metformin treatment improved survival of mice, which was associated with a significant reduction in bacterial number in the lung, which was mediated by mitochondrial ROS production and AMPK signaling and enhanced macrophage bactericidal activity [86] . Epidemiologic evidence are conflicting (Table 3) ; some studies show a net benefit of metformin use on outcomes like 30-day mortality in US veterans hospitalized with pneumonia [87] , whereas a population-based Taiwanese case-control study failed to demonstrate a preventive effect of metformin on hospitalization for pneumonia in persons with T2DM and COPD [88] . Clinical trials are needed to bridge the promising preclinical evidence and epidemiologic evidence. A planned small randomized crossover placebocontrolled trial will explore effects on airway glucose and bacterial growth in nondiabetic persons with COPD (NCT03651895); however, trials with follow-up time sufficient to capture a broad range of respiratoryrelated outcomes may be necessary.

Insights can also be drawn from metformin use in the context of chronic viruses, such as liver targeting hepatitis B virus (HBV) and hepatitis C virus (HCV), and human immunodeficiency virus (HIV). HBV and HCV are among the leading causes of liver disease. In HBV, approximately 90% of infections are acute, whereas 10% progress to chronic infection. Metformin-mediated decreases in hepatitis B surface antigen (HBsAg) levels in culture supernatants and in cell lysates and may work synergistically with other antivirals [89] . This will be tested in a randomized placebo-controlled trial in nondiabetic persons with chronic HBV: an investigative team in China will examine the addition of 24-week metformin to ongoing standard HBV treatment entecavir on cumulative rate of HBsAg loss (NCT04182321). Trials on long-term disease outcomes are have not been conducted, but a population-based cohort study of 71,824 HBVinfected patients in Taiwan showed that metformin use was chemoprotective; patients on metformin had a lower incidence of all-cancer, not just HBV-related liver cancer (adjusted hazard ratios, HR 0.75 (95% CI, 0.67-0.84)) [90] . This is interesting because with chronic viral exposure some of metformin's protective effects may extend beyond the direct long-term health consequences of HBV. HCV, like SARS-CoV 2, is a ribonucleic acid (RNA) virus. HCV poses a serious health threat across all ages, but those over the age of 65 years now account for a large portion of all chronic hepatitis C infections in the USA and have the highest rate of hepatitis C-related deaths [91] . In patients with HCV and T2DM, metformin's effects on sustained viral response (SVR) are mixed (reviewed in [81] ). However, overall metformin is associated with benefit on liver injury and reduces the rate of incident hepatocellular carcinoma [92] , possibly by activating type I INF signaling against HCV via activation of AMPK.

Metformin is a first-line medication for management of T2DM and insulin resistance in persons living with HIV, but metformin may have a role in HIV pathogenesis as well [93, 94] . Dysregulated metabolism is implicated in the relationship between viral proteins, immune activation, and inflammation in persons infected with HIV, and is hypothesized to dampen the protective innate and adaptive arms of immunity [95] . In persons living with HIV and T2DM in Botswana, metformin use is associated with improved CD4 lymphocyte count recovery [96] . An independent study in China suggests metformin inhibit NF-κB/p65 phosphorylation to suppress CD54 expression on CD4+ T cells, which is associated with disease progression in persons living with HIV [97] . In a recent 24-week pilot study involving 12 virally suppressed HIV-infected individuals without T2DM who were randomized to metformin or observation, metformin reduced CD4 T cell exhaustion as measured by negative immune checkpoint receptors (NCR) such as programmed cell death protein-1 (PD1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) [98] . Other trials are planned including a pilot trial in nondiabetic HIV-infected persons to evaluate metformin's effect on various virological assays, inflammation, and disease outcomes (NCT02659306) [99, 100] ; follow-up trials will be needed to uncover common mechanisms and best drug combinations [101] . Additionally, an emerging hypothesis posits that metformin may improve gut microbiota composition, which could reducing inflammation and mitigate risk of nonAIDS comorbidities [62, 102, 103] .

In 2014, it was reported that an mTORC1 inhibitor increased antibody titers to flu vaccination in 218 healthy elderly subjects [28] . Whether metformin improves vaccine response in nondiabetic older adults remains an active area of exploration. Recent reports are mixed. For example, a 2017 study measured in vivo and in vitro influenza vaccine responses in newly diagnosed treatment naïve T2DM patients and patients taking metformin for at least 3 years. Ongoing metformin use was associated with recovered B cell function and vaccine response compared with those who were just recently diagnosed with T2DM. Moreover, metformin administered in vitro reduced B cell intrinsic inflammation and increased antibody responses in B cells harvested from the recently diagnosed T2DM patients, resulting in B cell function similar to patients already taking metformin [104] . In contrast, a recent cross-sectional study or 67 older adults showed reduced antibody responses to influenza vaccination quantified by virus neutralizing Ab (VNA) titers and immunoglobulin (Ig) isotypes to H1N1 and H3N2 in the 11 metformin users compared with nonmetformin users [105] . Clinical trials in nondiabetic older adults are needed to resolve these seemingly discrepant results of observational studies in order to evaluate metformin as a potential adjuvant to boost response to vaccine. Two noteworthy studies on metformin and vaccine response in nondiabetic older adults are underway (Table 4) . A pilot investigation is ongoing to determine whether metformin can improve the immune response to the pneumococcal conjugate vaccine (PCV-13) in older adults and if this effect is mediated by the gut microbiota (NCT03713801). An independent ongoing pilot trial will evaluate metformin's effect on both cellular and humoral influenza vaccine responses, in Geroscience: from acute care to long-term health and prevention

Evaluating intervention effects on comorbidity and resilience to acute challenge is even more critical in the aftermath of COVID-19. The lowered overall resilience of older COVID-19 patients is easily observed in the high incidence of fatalities, hospitalizations, and potential for long-term complications in patients with advanced ages. These outcomes are associated with or exacerbated the prevalence of age-related disease, comorbidity, frailty, and loss of endogenous protective and repair mechanisms. Most importantly, once the immediate challenge of treating acute cases of COVID-19 have passed, the need to treat these newly acquired or exacerbated long-term chronic age-related diseases in our population will be greater than ever. Efforts to treat the infectious diseases presenting in our hospitals worldwide are essential. At the same time, we must also delve deeper to understand how molecular pathways inextricably linked to biological aging, such as mTOR signaling, cellular senescence, insulin/IGF-1 signaling, and AMPK activation, are linked to immune response. This focus could provide novel host, as opposed to pathogen-directed therapies to respond to the current pandemic crisis, but more importantly aid our ability to help those affected by COVID-19 and its attendant chronic health consequences. Understanding the interaction between aging biology, viral susceptibility and immune response, and progression to comorbid chronic disease, frailty, and disability remains one of the most important biomedical issues worldwide. Geroscience represents a major strategic advancement and new therapeutic approach to considering long-term options for prevention and mitigating the anticipated swell in long-term health complications that will be left in the wake of COVID-19.

Ample evidence links metformin to lower all-cause mortality and reduced rates of multiple disease of aging, even in nondiabetic populations [106] [107] and suggest a broad geroprotective role of metformin. Clinical trials exploring the effects of metformin in nondiabetic older adults on gerocentric outcomes are underway [108] or planned [30, 31] . For example, a placebo-controlled, double-blinded clinical trial of 2-year metformin treatment for the prevention of frailty in 120 older adults with prediabetes is ongoing at University of Texas Health Science Center at San Antonio [108] . The TAME trial will evaluate metformin's effects on incidence of any new agerelated disease (cardiovascular disease, mild cognitive impairment/dementia, most cancers, or death) in 3000 older adults without diabetes [30, 31] . Both trials include collection of biospecimens and a variety of assessment measures to support ancillary investigations to complement the primary outcome. For example, in addition to an FDA-facing clinical disease composite, primary outcome TAME will include several indices of health span measures including measures of physical and cognitive function and frailty assessments, assays for key biomarkers, and biobanking of biofluids, cells, and feces for discovery and mechanistic investigations. While neither randomized controlled trial is designed to evaluate efficacy of metformin on COVID-19-related outcomes, they nonetheless represent unique and time-sensitive resources to the scientific community. These trials coincide with the pandemic, which provides an opportunity for long-term follow-up and deep phenotyping of older adults with and without exposure to COVID-19 in the course of the study and may include smaller subgroups who are symptomatic vs. asymptomatic. Fresh collection of peripheral blood mononuclear cells and tissues included in these trials provide an unprecedented opportunity for immunophenotyping; investigator-initiated studies could leverage ongoing trials to examine metformin's effects on immune cell activation and inflammatory cytokine production. Similar to other ongoing trials or large cohort studies [109, 110] , TAME will include measures to query history, symptoms, and burden of COVID-19 as exploratory assessments. Moreover, TAME's large sample size could permit exploratory investigations on the effects of metformin on agerelated diseases or functional decline in older adults with or without history of COVID-19, though such explorations would not bear the burden of proof necessary for a virus-related FDA indication. Other ancillary investigations could be envisioned to explore metformin's effects on immune response to vaccines in older adults once a vaccine against SARS-CoV2 is developed. Ultimately, these existing or upcoming clinical trials provides a scaffolding for future investigations on long-term health effects of COVID-19 and metformin's potential to improve resilience following immune challenge.

In conclusion, metformin is an attractive tool or probe for clinical trials targeting aging and to improve host immune defense and resilience in COVID-19 and infectious disease. Its immunoprotective effects are hypothesized to (i) lessen severity of unfavorable health outcomes or death in the event of exposure to infectious disease; (ii) delay or prevent long-term chronic diseases or conditions which can stem from acute challenges or viral infections; and (iii) bolster immune response to vaccine. However, this hypothesis has yet to be rigorously tested. It is time for definitive geroscience trials of not only metformin but other promising geroprotective interventions like caloric restriction, mTOR inhibitors, and senolytics. Trials informed by geroscience will offer opportunity to investigate intervention effects on vaccine response and resilience to age-related chronic diseases and geriatric syndromes and provides a unique opportunity to advance study of host-immune defense with implication for recovery from the current pandemic and unforeseen future challenges.

Authors' contributions JNJ is the primary author responsible for drafting, editing, figure development, and literature reviews; SG aided in literature reviews and vetting of studies listed in Table 3 , and editing all manuscript drafts; ASK provided content expertise and development of Table 1 ; JMB provided content expertise and draft materials related to mechanisms of metformin on immune defenses and vaccine response; GAK and NB provided guidance on development, oversight, and editing throughout.

Funding The authors acknowledge the support by the American Federation of Aging Research (JMB, Irene Diamond AFAR award), the Glenn Center for the Biology of Human Aging (NB), and the National Institutes of Health (NIH): JNJ (K01 AG059837, P30 AG021332); GAK (R33 AG061456, R21 AG063528, R56 AG060746, R35 GM124922, U01 AI124297, R21 AG060018, R01 AG058814, R01 AI142086, UH3 AG056925); NB (P30AG038072).Data availabilityNot applicable.

Conflict of interest ASK is now a Senior Scientist, Computational Genomics at AbbVie; his recent employment in Abbvie did not influence the literature reviewed. JNJ, SG, JMB, GAK, NB have no conflicts to report.

Code availability Not applicable.

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Targeting age-related pathways in heart failure

Presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with COVID-19 in the New York City area

COVID-19 in patients with kidney disease

Clinical features of maintenance hemodialysis patients with 2019 novel coronavirus-infected pneumonia in Wuhan, China

Cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (COVID-19)

Association of cardiac infection with SARS-CoV-2 in confirmed COVID-19 autopsy cases

Geroscience and the coronavirus pandemic: the whack-a-mole approach is not enough

Metformin as a host-directed therapeutic in tuberculosis: is there a promise?

Reducing mortality and morbidity in patients with severe COVID-19 disease by advancing ongoing trials of mesenchymal stromal (stem) cell (MSC) therapy -achieving global consensus and visibility for cellular host-directed therapies

Daily fasting improves health and survival in male mice independent of diet composition and calories

Mechanisms of lifespan regulation by calorie restriction and intermittent fasting in model organisms

Impact of age, caloric restriction, and influenza infection on mouse gut microbiome: an exploratory study of the role of age-related microbiome changes on influenza responses

Fisetin is a senotherapeutic that extends health and lifespan

mTOR is a key modulator of ageing and age-related disease

Acarbose treatment and the risk of cardiovascular disease and hypertension in patients with impaired glucose tolerance: the STOP-NIDDM trial

TORC1 inhibition enhances immune function and reduces infections in the elderly

mTOR regulation and therapeutic rejuvenation of aging hematopoietic stem cells

mTOR inhibition improves immune function in the elderly

Metformin improves healthspan and lifespan in mice

Metformin as a tool to target aging

Development of clinical trials to extend healthy lifespan

Flumamine, a new synthetic analgesic and antiflu drug

Metformin: historical overview

Effect of intensive blood-glucose control with metformin on complications in overweight patients with type 2 diabetes (UKPDS 34). UK Prospective Diabetes Study (UKPDS) Group

Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin

Benefits of metformin in attenuating the hallmarks of aging

Metformin activates AMPK through the lysosomal pathway

The effects of metformin on gut microbiota and the immune system as research frontiers

Metformin as anti-aging therapy: is it for everyone?

Epigenetic effects of metformin: from molecular mechanisms to clinical implications

Context-dependent pharmacological effects of metformin on the immune system

Metformin prevents cancer metastasis by inhibiting M2-like polarization of tumor associated macrophages

Metformin induces CD11b+−cell-mediated growth inhibition of an osteosarcoma: implications for metabolic reprogramming of myeloid cells and anti-tumor effects

Metformin exerts antitumor activity via induction of multiple death pathways in tumor cells and activation of a protective immune response

Immune-mediated antitumor effect by type 2 diabetes drug, metformin

Metformin induces the M2 macrophage polarization to accelerate the wound healing via regulating AMPK/mTOR/NLRP3 inflammasome singling pathway

Metformin ameliorates the development of experimental autoimmune encephalomyelitis by regulating T helper 17 and regulatory T cells in mice

Metformin attenuates graft-versus-host disease via restricting mammalian target of rapamycin/signal transducer and activator of transcription 3 and promoting adenosine monophosphate-activated protein kinase-autophagy for the balance between T helper 17 and Tregs

Metformin suppresses systemic autoimmunity in Roquin(san/san) mice through inhibiting B cell differentiation into plasma cells via regulation of AMPK/mTOR/STAT3

The energy sensor AMPK regulates T cell metabolic adaptation and effector responses in vivo

Metformin as adjunct antituberculosis therapy

SARS-CoV-2 and COVID-19 in older adults: what we may expect regarding pathogenesis, immune responses, and outcomes

The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak -an update on the status

The possible pathophysiology mechanism of cytokine storm in elderly adults with COVID-19 infection: the contribution of "inflame-aging

Metformin, a new era for an old drug in the treatment of immune mediated disease?

Metformin inhibits monocyte-tomacrophage differentiation via AMPK-mediated inhibition of STAT3 activation: potential role in atherosclerosis

Metformin inhibits pro-inflammatory responses via targeting nuclear factor-kappaB in HaCaT cells

Anti-inflammatory effects of metformin irrespective of diabetes status

Metformin monotherapy downregulates diabetes-associated inflammatory status and impacts on mortality

Mitochondrial respiratory complex I regulates neutrophil activation and severity of lung injury

Metformin effect on gut microbiota: insights for HIV-related inflammation

Metformin alters the gut microbiome of individuals with treatment-naive type 2 diabetes, contributing to the therapeutic effects of the drug

Modulation of gut microbiota by berberine and metformin during the treatment of high-fat diet-induced obesity in rats

Disentangling type 2 diabetes and metformin treatment signatures in the human gut microbiota

Metformin alters gut microbiota of healthy mice: implication for its potential role in gut microbiota homeostasis

Metformin reduces aging-related leaky gut and improves cognitive function by beneficially modulating gut microbiome/goblet cell/mucin axis

An increase in the Akkermansia spp. population induced by metformin treatment improves glucose homeostasis in diet-induced obese mice

The COVID-19 epidemic

Emerging coronaviruses: genome structure, replication, and pathogenesis

SARS-CoV-2: a storm is raging

The epidemiology and pathogenesis of coronavirus disease (COVID-19) outbreak

The pathogenesis and treatment of the 'Cytokine Storm' in COVID-19

Imbalanced host response to SARS-CoV-2 drives development of COVID-19

Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients

Cytokine release syndrome in severe COVID-19

Metformin treatment was associated with decreased mortality in COVID-19 patients with diabetes in a retrospective analysis

Observational study of metformin and risk of mortality in patients hospitalized with Covid-19

Metformin as host-directed therapy for TB treatment: scoping review

Metformin use reverses the increased mortality associated with diabetes mellitus during tuberculosis treatment

Is metformin poised for a second career as an antimicrobial?

The brighter (and evolutionarily older) face of the metabolic syndrome: evidence from Trypanosoma cruzi infection in CD-1 mice

Role of airway glucose in bacterial infections in patients with chronic obstructive pulmonary disease

Metformin prevents the effects of Pseudomonas aeruginosa on airway epithelial tight junctions and restricts hyperglycaemia-induced bacterial growth

Metformin attenuates the effect of Staphylococcus aureus on airway tight junctions by increasing PKCzeta-mediated phosphorylation of occludin

Metformin mediates protection against Legionella pneumonia through activation of AMPK and mitochondrial reactive oxygen species

Association of metformin and mortality for patients with diabetes who are hospitalized with pneumonia

Respiratory outcomes of metformin use in patients with type 2 diabetes and chronic obstructive pulmonary disease

Metformin inhibits hepatitis B virus protein production and replication in human hepatoma cells

Cancer risk in HBV patients with statin and metformin use: a population-based cohort study

Estimating prevalence of hepatitis C virus infection in the United States

Safety and efficacy of metformin in patients with type 2 diabetes mellitus and chronic hepatitis C

The efficacy and safety of insulinsensitizing drugs in HIV-associated lipodystrophy syndrome: a meta-analysis of randomized trials

Drug therapies for HIV-related metabolic disorders

Current topics in HIV-1 pathogenesis: the emergence of deregulated immunometabolism in HIV-infected subjects

Risk of hepatocellular carcinoma in diabetic patients and risk reduction associated with anti-diabetic therapy: a population-based cohort study

Elevated CD54 expression renders CD4+ T cells susceptible to natural killer cell-mediated killing

Short communication: metformin reduces CD4 T cell exhaustion in HIV-infected adults on suppressive antiretroviral therapy

Effect of metformin on the size of the HIV reservoir in non-diabetic ART-treated individuals: singlearm non-randomised Lilac pilot study protocol

Improving treatment and liver fibrosis outcomes with metformin in HCV-HIV co-infected and HCV mono-infected patients with insulin resistance: study protocol for a randomized controlled trial

Diabetes and hepatitis C: a two-way association

Low abundance of colonic butyrate-producing bacteria in HIV infection is associated with microbial translocation and immune activation

Alterations in bacterial communities, SCFA and biomarkers in an elderly HIVpositive and HIV-negative population in western Mexico

Metformin improves in vivo and in vitro B cell function in individuals with obesity and type-2 diabetes

Immune response to influenza vaccination in the elderly is altered by chronic medication use

Metformin reduces all-cause mortality and diseases of ageing independent of its effect on diabetes control: a systematic review and meta-analysis

Differential effects of metformin on age related comorbidities in older men with type 2 diabetes

Rationale and study design of a randomized clinical trial of metformin to prevent frailty in older adults with prediabetes

Assessing the impact of the covid-19 pandemic and accompanying mitigation efforts on older adults

Up and about: older adults' wellbeing during the COVID-19 pandemic in a Swedish longitudinal study

Metformin inhibits the senescence-associated secretory phenotype by interfering with IKK/NF-kappaB activation

Metformin alters upper small intestinal microbiota that impact a glucose-SGLT1-sensing glucoregulatory pathway

Effect of metformin on metabolic improvement and gut microbiota

Metformin retards aging in C. elegans by altering microbial folate and methionine metabolism

The antidiabetic drug metformin activates the AMP-activated protein kinase cascade via an adenine nucleotideindependent mechanism

Metformin induces a dietary restriction-like state and the oxidative stress response to extend C. elegans Healthspan via AMPK, LKB1, and SKN-1

Metformin is a direct SIRT1-activating compound: computational modeling and experimental validation

AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity

Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPase-dependent manner

Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells

independent of AMPK, induces mTOR inhibition and cellcycle arrest through REDD1

Metformin inhibits hepatic mTORC1 signaling via dose-dependent mechanisms involving AMPK and the TSC complex

Metformin downregulates the insulin/IGF-I signaling pathway and inhibits different uterine serous carcinoma (USC) cells proliferation and migration in p53-dependent or -independent manners

Mechanism of metformin: inhibition of DNA damage and proliferative activity in Drosophila midgut stem cell

Combined metformin and resveratrol confers protection against UVC-induced DNA damage in A549 lung cancer cells via modulation of cell cycle checkpoints and DNA repair

Metformin activates an ataxia telangiectasia mutated (ATM)/Chk2-regulated DNA damage-like response

Autophagy was involved in the protective effect of metformin on hyperglycemia-induced cardiomyocyte apoptosis and Connexin43 downregulation in H9c2 cells

Metformin induces autophagy and G0/G1 phase cell cycle arrest in myeloma by targeting the AMPK/mTORC1 and mTORC2 pathways

Metformin promotes apoptosis in hepatocellular carcinoma through the CEBPD-induced autophagy pathway

Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I

Metformin promotes lifespan through mitohormesis via the peroxiredoxin PRDX-2

Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis

Metformin reduces endogenous reactive oxygen species and associated DNA damage

Metformin induces PGC-1alpha expression and selectively affects hepatic PGC-1alpha functions

Metformin alleviates human cellular aging by upregulating the endoplasmic reticulum glutathione peroxidase 7

Metformin restores CNS remyelination capacity by rejuvenating aged stem cells

Metformin acts on two different molecular pathways to enhance adult neural precursor proliferation/self-renewal and differentiation

Deficiency of Atg6 impairs beneficial effect of metformin on intestinal stem cell aging in Drosophila

Metformin-mediated increase in DICER1 regulates microRNA expression and cellular senescence

Nuclear respiratory factor 1 and endurance exercise promote human telomere transcription

Antioxidant and anti-senescence effect of metformin on mouse olfactory ensheathing cells (mOECs) may be associated with increased brain-derived neurotrophic factor levels-an ex vivo study

Metformin reduces the senescence of renal tubular epithelial cells in diabetic nephropathy via the MBNL1/miR-130a-3p/STAT3 pathway

Metformin relieves acute respiratory distress syndrome by reducing miR-138 expression

Activation of AMPK attenuates LPS-induced acute lung injury by upregulation of PGC1alpha and SOD1

Impaired efferocytosis and neutrophil extracellular trap clearance by macrophages in ARDS

Metformin reverses established lung fibrosis in a bleomycin model

Metformin alters human host responses to mycobacterium tuberculosis in healthy subjects

Metformin promotes mycobacterium tuberculosis killing and increases the production of human beta-defensins in lung epithelial cells and macrophages

Metformin adjunctive therapy does not improve the sterilizing activity of the firstline antitubercular regimen in mice

Increased airway glucose increases airway bacterial load in hyperglycaemia

Metformin promotes innate immunity through a conserved PMK-1/ p38 MAPK pathway

Fructose transport-deficient Staphylococcus aureus reveals important role of epithelial glucose transporters in limiting sugar-driven bacterial growth in airway surface liquid

Metformin reduces airway glucose permeability and hyperglycaemia-induced Staphylococcus aureus load independently of effects on blood glucose

Host AMPK is a modulator of plasmodium liver infection

Targeting liver stage malaria with metformin

Metformin promotes the protection of mice infected with Plasmodium yoelii independently of gammadelta T cell expansion

Simvastatin and metformin inhibit cell growth in hepatitis C virus infected cells via mTOR increasing PTEN and autophagy

Inhibition of hepatitis C virus replication through adenosine monophosphate-activated protein kinase-dependent andindependent pathways

Treatment of insulin resistance with metformin in naive genotype 1 chronic hepatitis C patients receiving peginterferon alfa-2a plus ribavirin

The effect of metformin on the efficacy of antiviral therapy in patients with genotype 1 chronic hepatitis C and insulin resistance

Efficacy of adding metformin to pegylated interferon and ribavirin in treatment naive patients with chronic hepatitis C: a randomized double-blind controlled trial

Sirolimus and metformin synergistically inhibit hepatocellular carcinoma cell proliferation and improve long-term survival in patients with HCC related to hepatitis B virus induced cirrhosis after liver transplantation

The effect of metformin treatment on CRBP-I level and cancer development in the liver of HBx transgenic mice

Metformin inhibits tumorigenesis in HBVinduced hepatocellular carcinoma by suppressing HULC overexpression caused by HBX

Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner

Peroxisome proliferator-activated receptor and AMP-activated protein kinase agonists protect against lethal influenza virus challenge in mice. Influenza Other Respir Viruses

Metformin associated with lower mortality in diabetic patients with early stage hepatocellular carcinoma after radiofrequency ablation

Impact of metformin on the prognosis of cirrhosis induced by viral hepatitis C in diabetic patients

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