Cellular senescence is a double-edged sword in regulating aged immune responses to influenza.

Clearance of senescent cells has demonstrated therapeutic potential in the context of chronic age-related diseases. Little is known, however, how clearing senescent cells affects the ability to respond to an acute infection and form quality immunological memory. We aimed to probe the effects of clearing senescent cells in aged mice on the immune response to influenza (flu) infection. We utilized a p16 trimodality reporter mouse model (p16-3MR) to allow for identification and selective clearance of p16-expressing cells upon administration of ganciclovir (GCV). While p16-expressing cells may exacerbate dysfunctional responses to a primary infection, our data suggest they may play a role in fostering memory cell generation. We demonstrate that although clearance of p16-expressing cells enhanced viral clearance, this also severely limited antibody production in the lungs of flu-infected aged mice. 30 days later, there were fewer flu-specific CD8 memory T cells and lower levels of flu-specific antibodies in the lungs of GCV-treated mice. Furthermore, GCV-treated mice were unable to mount an optimal memory response and demonstrated increased viral load following heterosubtypic challenge. These results suggest that targeting senescent cells may potentiate primary responses while limiting the ability to form durable and protective immune memory with age.

Unraveling the association between major depressive disorder and senescent biomarkers in immune cells of older adults: a single-cell phenotypic analysis.

Background: Little is known about the prevalence of cellular senescence among immune cells (i.e., immune cells expressing senescence markers, iSCs) nor is there a gold-standard to efficiently measure iSCs. Major depressive disorder (MDD) in older adults has been associated with many hallmarks of senescence in whole blood, leukocytes, and plasma, supporting a strong connection between iSCs and MDD. Here, we investigated the prevalence and phenotype of iSCs in older adults with MDD. Using a single-cell phenotypic approach, circulating immune cells were examined for iSC biomarkers and their relationship to depression and inflammation. Results: PBMCs from older adults with MDD (aged 69.75 ± 5.23 years) and healthy controls (aged 71.25 ± 8.8 years) were examined for immune subset distribution and senescence biomarkers (i.e., lack of proliferation, senescence-associated heterochromatin foci (SAHF), and DNA damage). Dual-expression of SAHF and DNA damage was categorized by low, intermediate, and high expression. A significant increase in the number of high expressing total PBMCs (p = 0.01), monocytes (p = 0.008), a trending increase in the number of high expressing CD4 T cells (p = 0.06) was observed overall in those with MDD. There was also a significantly lower proportion of intermediate expressing cells in monocytes and CD4 T cells in MDD (p = 0.01 and p = 0.05, respectively). Correlation analysis revealed associations between iSCs and mRNA expression of factors related to SASP and immune cell function. Conclusion: MDD is associated with increased senescent cell biomarkers in immune cell populations delineated by distinct levels of SAHF and DNA damage. Inflammatory markers might serve as potent indicators of iSC burden in MDD.

Respiratory viral infection promotes the awakening and outgrowth of dormant metastatic breast cancer cells in lungs.

Breast cancer is the second most common cancer globally. Most deaths from breast cancer are due to metastatic disease which often follows long periods of clinical dormancy1. Understanding the mechanisms that disrupt the quiescence of dormant disseminated cancer cells (DCC) is crucial for addressing metastatic progression. Infection with respiratory viruses (e.g. influenza or SARS-CoV-2) is common and triggers an inflammatory response locally and systemically2,3. Here we show that influenza virus infection leads to loss of the pro-dormancy mesenchymal phenotype in breast DCC in the lung, causing DCC proliferation within days of infection, and a greater than 100-fold expansion of carcinoma cells into metastatic lesions within two weeks. Such DCC phenotypic change and expansion is interleukin-6 (IL-6)-dependent. We further show that CD4 T cells are required for the maintenance of pulmonary metastatic burden post-influenza virus infection, in part through attenuation of CD8 cell responses in the lungs. Single-cell RNA-seq analyses reveal DCC-dependent impairment of T-cell activation in the lungs of infected mice. SARS-CoV-2 infected mice also showed increased breast DCC expansion in lungs post-infection. Expanding our findings to human observational data, we observed that cancer survivors contracting a SARS-CoV-2 infection have substantially increased risks of lung metastatic progression and cancer-related death compared to cancer survivors who did not. These discoveries underscore the significant impact of respiratory viral infections on the resurgence of metastatic cancer, offering novel insights into the interconnection between infectious diseases and cancer metastasis.

Patient-derived organoids recapitulate glioma-intrinsic immune program and progenitor populations of glioblastoma.

Glioblastoma multiforme (GBM) is a highly lethal human cancer thought to originate from a self-renewing and therapeutically-resistant population of glioblastoma stem cells (GSCs). The intrinsic mechanisms enacted by GSCs during 3D tumor formation, however, remain unclear, especially in the stages prior to angiogenic/immunological infiltration. In this study, we performed a deep characterization of the genetic, immune, and metabolic profiles of GBM organoids from several patient-derived GSCs (GBMO). Despite being devoid of immune cells, transcriptomic analysis across GBMO revealed a surprising immune-like molecular program, enriched in cytokine, antigen presentation and processing, T-cell receptor inhibitors, and interferon genes. We find two important cell populations thought to drive GBM progression, Special AT-rich sequence-binding protein 2 (SATB2+) and homeodomain-only protein homeobox (HOPX+) progenitors, contribute to this immune landscape in GBMO and GBM in vivo. These progenitors, but not other cell types in GBMO, are resistant to conventional GBM therapies, temozolomide and irradiation. Our work defines a novel intrinsic immune-like landscape in GBMO driven, in part, by SATB2+ and HOPX+ progenitors and deepens our understanding of the intrinsic mechanisms utilized by GSCs in early GBM formation.

Dysfunctional Bladder Morphology and Functional Impairments Are Identified in the Alzheimer's Disease APPNL-G-F/NL-G-F Murine Model.

BACKGROUND: While symptoms related to lower urinary tract dysfunction (LUTD) are common in individuals with Alzheimer's disease (AD), pathophysiological links between AD and LUTD remain unclear. OBJECTIVE: This study aimed to investigate whether AD neuropathology would cause autonomic dysfunction along the spinal cord-bladder axis, which could result in alterations in bladder muscle kinetics. METHODS: We utilized APPNL-G-F/NL-G-F knock-in (APP KI) and APPwt/wt (wild-type) mice at two different ages, 4- and 10-month-old, to investigate how AD impacts bladder tissue function by immunohistochemistry, western blotting, and pharmacomyography. RESULTS: We showed that the mucosal layer partially separated from the detrusor in 10-month-old APP KI mouse bladders. Although there was no detectable amyloid deposition in the APP KI bladder, we found amyloid plaques in APP KI lumbar spinal cord. Further immunoblot analysis revealed that tyrosine hydroxylase protein levels were significantly reduced in both 4- and 10-month-old bladder tissues, suggesting reduction of norepinephrine synthesis in APP KI mouse bladders. In contrast, the level of ß2 adrenergic receptor was increased in 4-month-old but not 10-month-old APP KI bladders. In bladder strips, the adrenergic agonist isoproterenol induced increased relaxation in 4- but not 10-month-old APP KI bladders. With 10 Hz electrical field stimulation, 10-month-old APP KI bladder strips were more responsive than wild-type controls, with no differences observed in 4-month-old APP KI bladders. CONCLUSIONS: APP KI mice exhibit LUTD, which is likely arising from amyloid pathology in the spinal cord, and results in maturational declines in presynaptic activity combined with compensatory postsynaptic upregulation.

Old drug, new tricks: the utility of metformin in infection and vaccination responses to influenza and SARS-CoV-2 in older adults.

In the face of global pathogens such as influenza (flu) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), strategies beyond standard vaccines and virus-specific treatments are critically needed for older populations who are more susceptible to severe disease and death from these infections due to age-related immune dysregulation. Thus, complimentary therapeutics are needed to address the increased risk of complications and death in older adults. Metformin, an FDA approved diabetes drug, is an attractive therapeutic candidate to improve immune defenses and resilience in older adults facing viral challenge. Metformin is already a candidate anti-aging drug, but its benefits have potential to span beyond this and improve specific immune responses. Metformin can target multiple aging hallmarks as well as directly impact innate and adaptive immune cell subsets. Both retrospective and prospective studies have demonstrated metformin's efficacy in improving outcomes after SARS-CoV-2 or flu infections. Moreover, evidence from clinical trials has also suggested that metformin treatment can improve vaccination responses. In totality, these findings suggest that metformin can improve age-related declines in immunological resilience. Strategies to improve outcomes after infection or improve vaccine-induced protection are invaluable for older adults. Moreover, the ability to repurpose an already FDA approved drug has significant advantages in terms of necessary time and resources. Thus, metformin has great potential as a therapeutic to improve age-related immune dysregulation during flu and SARS-CoV-2 infections and should be further explored to confirm its ability to improve overall immunological resilience in older adults.

Senolytic treatment with dasatinib and quercetin does not improve overall influenza responses in aged mice.

Age is the greatest risk factor for adverse outcomes following influenza (flu) infection. The increased burden of senescent cells with age has been identified as a root cause in many diseases of aging and targeting these cells with drugs termed senolytics has shown promise in alleviating many age-related declines across organ systems. However, there is little known whether targeting these cells will improve age-related deficits in the immune system. Here, we utilized a well characterized senolytic treatment with a combination of dasatinib and quercetin (D + Q) to clear aged (18-20 months) mice of senescent cells prior to a flu infection. We comprehensively profiled immune responses during the primary infection as well as development of immune memory and protection following pathogen reencounter. Senolytic treatment did not improve any aspects of the immune response that were assayed for including: weight loss, viral load, CD8 T-cell infiltration, antibody production, memory T cell development, or recall ability. These results indicate that D + Q may not be an appropriate senolytic to improve aged immune responses to flu infection.

A pilot metabolomic study of drug interaction with the immune response to seasonal influenza vaccination.

Many human diseases, including metabolic diseases, are intertwined with the immune system. The understanding of how the human immune system interacts with pharmaceutical drugs is still limited, and epidemiological studies only start to emerge. As the metabolomics technology matures, both drug metabolites and biological responses can be measured in the same global profiling data. Therefore, a new opportunity presents itself to study the interactions between pharmaceutical drugs and immune system in the high-resolution mass spectrometry data. We report here a double-blinded pilot study of seasonal influenza vaccination, where half of the participants received daily metformin administration. Global metabolomics was measured in the plasma samples at six timepoints. Metformin signatures were successfully identified in the metabolomics data. Statistically significant metabolite features were found both for the vaccination effect and for the drug-vaccine interactions. This study demonstrates the concept of using metabolomics to investigate drug interaction with the immune response in human samples directly at molecular levels.

Metformin for Treatment of Acute COVID-19: Systematic Review of Clinical Trial Data Against SARS-CoV-2.

BACKGROUND: Observational and preclinical data suggest metformin may prevent severe coronavirus disease 2019 (COVID-19) outcomes. PURPOSE: We conducted a systematic review of randomized, placebo-controlled clinical trials of metformin treatment for COVID-19 to determine whether metformin affects clinical or laboratory outcomes in individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and present a structured summary of preclinical data. STUDY SELECTION: Two independent reviewers searched PubMed, Scopus, Cochrane COVID-19 Study Register, and ClinicalTrials.gov on 1 February 2023 with no date restrictions for trials where investigators randomized adults with COVID-19 to metformin versus control and assessed clinical and/or laboratory outcomes of interest. The Cochrane Risk of Bias 2 tool was used to assess bias. DATA EXTRACTION: Two reviewers extracted data pertaining to prespecified outcomes of each interest from each included trial. DATA SYNTHESIS: The synthesis plan was developed a priori and was guided by Synthesis Without Meta-analysis (SWiM) guidelines. Summary tables and narrative synthesis were used (PROSPERO, 2022, CRD42022349896). Three randomized trials met inclusion criteria. In two of the trials investigators found that metformin improved clinical outcomes (prevented need for oxygen and prevented need for acute health care use), and in the third trial a larger portion of adults with diabetes were enrolled but results did show a direction of benefit similar to that of the other trials in the per-protocol group. In the largest trial, subjects were enrolled during the delta and omicron waves and vaccinated individuals were included. The certainty of evidence that metformin prevents health care use due to COVID-19 was moderate per Grading of Recommendations Assessment, Development and Evaluation (GRADE) criteria. Many preclinical studies have shown metformin to be effective against SARS-CoV-2. LIMITATIONS: Limitations include inclusion of only three trials and heterogeneity between trials. CONCLUSIONS: Future trials will help define the role of metformin in COVID-19 treatment guidelines.

Targeting the hallmarks of aging to improve influenza vaccine responses in older adults.

Age-related declines in immune response pose a challenge in combating diseases later in life. Influenza (flu) infection remains a significant burden on older populations and often results in catastrophic disability in those who survive infection. Despite having vaccines designed specifically for older adults, the burden of flu remains high and overall flu vaccine efficacy remains inadequate in this population. Recent geroscience research has highlighted the utility in targeting biological aging to improve multiple age-related declines. Indeed, the response to vaccination is highly coordinated, and diminished responses in older adults are likely not due to a singular deficit, but rather a multitude of age-related declines. In this review we highlight deficits in the aged vaccine responses and potential geroscience guided approaches to overcome these deficits. More specifically, we propose that alternative vaccine platforms and interventions that target the hallmarks of aging, including inflammation, cellular senescence, microbiome disturbances, and mitochondrial dysfunction, may improve vaccine responses and overall immunological resilience in older adults. Elucidating novel interventions and approaches that enhance immunological protection from vaccination is crucial to minimize the disproportionate effect of flu and other infectious diseases on older adults.

The effect of metformin on influenza vaccine responses in nondiabetic older adults: a pilot trial.

BACKGROUND: Aging is associated with progressive declines in immune responses leading to increased risk of severe infection and diminished vaccination responses. Influenza (flu) is a leading killer of older adults despite availability of seasonal vaccines. Geroscience-guided interventions targeting biological aging could offer transformational approaches to reverse broad declines in immune responses with aging. Here, we evaluated effects of metformin, an FDA approved diabetes drug and candidate anti-aging drug, on flu vaccination responses and markers of immunological resilience in a pilot and feasibility double-blinded placebo-controlled study. RESULTS: Healthy older adults (non-diabetic/non-prediabetic, age: 74.4 ± 1.7 years) were randomized to metformin (n = 8, 1500 mg extended release/daily) or placebo (n = 7) treatment for 20 weeks and were vaccinated with high-dose flu vaccine after 10 weeks of treatment. Peripheral blood mononuclear cells (PBMCs), serum, and plasma were collected prior to treatment, immediately prior to vaccination, and 1, 5, and 10 weeks post vaccination. Increased serum antibody titers were observed post vaccination with no significant differences between groups. Metformin treatment led to trending increases in circulating T follicular helper cells post-vaccination. Furthermore, 20 weeks of metformin treatment reduced expression of exhaustion marker CD57 in circulating CD4 T cells. CONCLUSIONS: Pre-vaccination metformin treatment improved some components of flu vaccine responses and reduced some markers of T cell exhaustion without serious adverse events in nondiabetic older adults. Thus, our findings highlight the potential utility of metformin to improve flu vaccine responses and reduce age-related immune exhaustion in older adults, providing improved immunological resilience in nondiabetic older adults.

Cellular Senescence is a Double-Edged Sword in Regulating Aged Immune Responses to Influenza.

Clearance of senescent cells has demonstrated therapeutic potential in the context of chronic age-related diseases. Little is known, however, how clearing senescent cells affects the ability to respond to an acute infection and form quality immunological memory. We aimed to probe the effects of clearing senescent cells in aged mice on the immune response to influenza (flu) infection. We utilized a p16 trimodality reporter mouse model (p16-3MR) to allow for identification and selective deletion of p16-expressing senescent cells upon administration of ganciclovir (GCV). While p16-expressing senescent cells may exacerbate dysfunctional responses to a primary infection, our data suggest they may play a role in fostering memory cell generation. We demonstrate that although deletion of p16-expressing cells enhanced viral clearance, this also severely limited antibody production in the lungs of flu-infected aged mice. 30 days later, there were fewer flu-specific CD8 memory T cells and lower levels of flu-specific antibodies in the lungs of GCV treated mice. GCV treated mice were unable to mount an optimal memory response and demonstrated increased viral load following a heterosubtypic challenge. These results suggest that targeting senescent cells may potentiate primary responses while limiting the ability to form durable and protective immune memory with age.

Loss of resilience contributes to detrusor underactivity in advanced age.

Volume hyposensitivity resulting from impaired sympathetic detrusor relaxation during bladder filling contributes to detrusor underactivity (DU) associated with aging. Detrusor tension regulation provides an adaptive sensory input of bladder volume to the brainstem and is challenged by physiological stressors superimposed upon biological aging. We recently showed that HCN channels have a stabilizing role in detrusor sympathetic relaxation. While mature mice maintain homeostasis in the face of stressors, old mice are not always capable. In old mice, there is a dichotomous phenotype, in which resilient mice adapt and maintain homeostasis, while non-resilient mice fail to maintain physiologic homeostasis. In this DU model, we used cystometry as a stressor to categorize mice as old-responders (old-R, develop a filling/voiding cycle) or old-non-responders (old-NR, fail to develop a filling/voiding cycle; fluctuating high pressures and continuous leaking), while also assessing functional and molecular differences. Lamotrigine (HCN activator)-induced bladder relaxation is diminished in old-NR mice following HCN-blockade. Relaxation responses to NS 1619 were reduced in old-NR mice, with the effect lost following HCN-blockade. However, RNA-sequencing revealed no differences in HCN gene expression and electrophysiology studies showed similar percentage of detrusor myocytes expressing HCN (Ih) current between old-R and old-NR mice. Our murine model of DU further defines a role for HCN, with failure of adaptive recalibration of HCN participation and intensity of HCN-mediated stabilization, while genomic studies show upregulated myofibroblast and fibrosis pathways and downregulated neurotransmitter-degradation pathways in old-NR mice. Thus, the DU phenotype is multifactorial and represents the accumulation of age-associated loss in homeostatic mechanisms.

Altered T cell infiltration and enrichment of leukocyte regulating pathways within aged skeletal muscle are associated impaired muscle function following influenza infection.

Older adults have diminished immune responses that increase susceptibility to infectious diseases, such as influenza (flu). In older adults, flu infection can lead to hospitalization, catastrophic disability, and mortality. We previously demonstrated severe and prolonged muscle degradation and atrophy in aged mice during flu infection. Here, we utilized an unbiased transcriptomic analysis to elucidate mechanisms of flu-induced muscular declines in a mouse model. Our results showed age-related gene expression differences including downregulation of genes associated with muscle regeneration and organization and upregulation of genes associated with pro-inflammatory cytokines and migratory immune pathways in aged mice when compared to young. Pathway analysis revealed significant enrichment of leukocyte migration and T cell activation pathways in the aged muscle during infection. Intramuscular CD4 T cells increased in both young and aged mice during infection, while intramuscular CD8 T cells increased exclusively in aged muscle. CD4 T cells in young muscle were regulatory T cells (Treg), while those in aged were T follicular helper (Tfh) and Th2 cells. Correspondingly, IL-33, an important cytokine for Treg accumulation within tissue, increased only in young flu-infected muscle. Conversely, CXCL10 (IP-10) increased only in aged muscle suggesting a continued recruitment of CD8 T cells into the aged muscle during flu infection. Overall, our findings elucidate a link between flu-induced disability and dysregulated intracellular T cell recruitment into flu-injured muscle with aging. Furthermore, we uncovered potential pathways involved that can be targeted to develop preventative and therapeutic interventions to avert disability and maintain independence following infection.

Increased Levels of Circulating Cell-Free mtDNA in the Plasma of Subjects With Late-Life Depression and Frailty: A Preliminary Study.

OBJECTIVE: To evaluate the circulating cell-free mitochondrial DNA (ccf-mtDNA) levels, a marker of cellular stress and damage, in older adults with late-life depression (LLD) and frailty. We hypothesize that individuals with both frailty and LLD will have higher ccf-mtDNA levels than individuals with either condition in isolation. METHODS: Fifty-three older adults (Never Depressed+Robust (reference group, n = 16), LLD+Robust (n = 9), Never Depressed+Prefrail/Frail (n = 5), and LLD+Prefrail/Frail (n = 23)) were included in the study. DNA was extracted from EDTA plasma samples, and ccf-mtDNA was quantified by RT-PCR. RESULTS: We found a statistically significant difference in the levels of ccf-mtDNA across groups (F(3,49) = 3.07, p = 0.036), with individuals in the LLD+Prefrail/Frail group showing the highest levels of ccf-mtDNA. CONCLUSION: The coexistence of LLD and frailty is associated with increased markers of cellular damage and stress (i.e., ccf-mtDNA). Our results suggest that these conditions may share cellular stress and mitochondrial dysfunction phenomena as a common biological mechanism, offering potential future opportunities for geroscience-guided interventions for these conditions.

Senescence-induced changes in CD4 T cell differentiation can be alleviated by treatment with senolytics.

Aging and senescence impact CD4 T helper cell (Th) subset differentiation during influenza infection. In the lungs of infected aged mice, there were significantly greater percentages of Th cells expressing the transcription factor FoxP3, indicative of regulatory CD4 T cells (Treg), when compared to young. TGF-beta levels, which drive FoxP3 expression, were also higher in the bronchoalveolar lavage of aged mice and blocking TGF-beta reduced the percentage of FoxP3+ Th in aged lungs during influenza infection. Since TGF-beta can be the product of senescent cells, these were targeted by treatment with senolytic drugs. Treatment of aged mice with senolytics prior to influenza infection restored the differentiation of Th cells in those aged mice to a more youthful phenotype with fewer Th cells expressing FoxP3. In addition, treatment with senolytic drugs induced differentiation of aged Th toward a healing Type 2 phenotype, which promotes a return to homeostasis. These results suggest that senescent cells, via production of cytokines such as TGF-beta, have a significant impact on Th differentiation.

Targeting Aging: Lessons Learned From Immunometabolism and Cellular Senescence.

It is well known that aging is associated with dysregulated metabolism. This is seen both in terms of systemic metabolism, as well as at the cellular level with clear mitochondrial dysfunction. More recently, the importance of cellular metabolism in immune cells, or immunometabolism, has been highlighted as a major modifier of immune cell function. Indeed, T cell activation, differentiation, and effector function partly depend on alterations in metabolic pathways with different cell types and functionality favoring either glycolysis or oxidative phosphorylation. While immune system dysfunction with aging is well described, what remains less elucidated is how the integral networks that control immune cell metabolism are specifically affected by age. In recent years, this significant gap has been identified and work has begun to investigate the various ways immunometabolism could be impacted by both chronological age and age-associated symptoms, such as the systemic accumulation of senescent cells. Here, in this mini-review, we will examine immunometabolism with a focus on T cells, aging, and interventions, such as mTOR modulators and senolytics. This review also covers a timely perspective on how immunometabolism may be an ideal target for immunomodulation with aging.