PAST TIME - Repurposing of PPAR modulators: a novel STrategy to awaken T-cell IMmunity in the Elderly

Infections and cancers account for more than 25% of total deaths among elderly subjects worldwide (Global burden of disease 2019), and age-related deficits of the immune system are thought to greatly contribute to this phenomenon. The aim of PAST TIME is to reverse immunosenescence and improve the control of pathogens and malignancies in old subjects by repurposing molecules already in use for metabolic disorders.
Our published and unpublished observations suggest that T cells form elderly subjects are characterized by several dysfunctions, partly due by a dysregulated lipid metabolism. Notably, we demonstrated that the treatment of old naïve CD8+ T cells with two lipid lowering molecules (fenofibrate, a PPAR-alfa agonist, and rosiglitazone, a PPAR-gamma agonist) improves their functionality. In this project we will extend these findings and validate in vivo the use of PPAR agonists to reverse T-cell senescence.
We will assess the activity of the two lead candidates (fenofibrate and rosiglitazone) and of other analogues belonging to the same drug classes (fibrates and thiazolidinediones) on the restoration of functional and metabolic properties of naïve and memory CD4+ and CD8+ T cells from elderly subjects (WP1). The two molecules showing the strongest activity in vitro and with the best pharmacokinetic, pharmacodynamic and safety profile will be selected for further in vivo validation.
The selected molecules will be administered to naïve young and old mice to define immune cell functional, transcriptional and metabolic profiles (WP2). Next, their capability to foster the control of infectious diseases (WP3) and malignancies (WP4) will be assessed in old mice. Infection models will include both viral and bacterial infective agents toward which pathogen-specific immune responses will be measured. Tumor models will include breast and lung cancer, against which the PPAR agonists will be also assessed in combination with currently used therapies.
In parallel, a pharmaco-epidemiological analysis will be conducted, to assess the risk of occurrence of infections and tumors in adult and elderly subjects using fibrates or thiazolidinediones (WP5).
The consortium brings together research groups with complementary expertise and belonging to the Universities of Ferrara (UNIFE, as coordinator), Milan (UNIMI) and Siena (UNISI).
Expected results will provide new strategies to be rapidly transferred into the clinic to correct immune dysfunctions in the elderly. Drug repurposing is considered a faster approach for the introduction of new therapeutic agents and the use, in this project, of drugs already approved for humans will foster the translational potential of the findings.
Economical and societal benefits derived from the development of immune restoration strategies may be relevant considering the constant increase of the aged population.

Risultati attesi:

Introduction:
Advances in medicine and improvements in public health systems have significantly increased life expectancy, leading to a growing elderly population that is more susceptible to age-related diseases. Among the various physiological changes that occur with aging, lipid dysfunctions are frequently observed both at systemic and cellular levels. Notably excessive lipid accumulation within cells is a well-established trigger to apoptosis. However, whether this phenomenon also affect the immune system and determines the loss of immune functionality associated with aging is still unclear. Therefore, interventions that promote lipid catabolism may have potential applications in the elderly to reverse immunosenescence and enhance T cell-mediated immunity against pathogens and tumors. To this end, the use of agonist drugs targeting Peroxisome Proliferator-Activated Receptors (PPARs)—a family of transcription factors involved in the regulation of lipid metabolism—has shown promising preliminary results in restoring both lipid metabolism and lymphocyte functionality.
Objectives:
This study aimed to investigate whether aging is associated with alterations in lipid metabolism, increased susceptibility to apoptosis, and impaired priming capacity in different subsets of CD4⁺ and CD8⁺ T lymphocytes, as well as B lymphocytes. After defining and characterizing the immunological alterations associated with aging, we proceeded to repurpose PPAR-α and PPAR-γ agonist drugs —already approvedfor the treatment of metabolic disorders—as a strategy to target lymphocyte lipid metabolism. Our goals was to restore metabolic balance and thereby improved the impaired immune function typical of elderly individuals.
WP1. IN VITRO SCREENING OF FIBRATES AND THIAZOLIDINEDIONES EFFECTS ON T-CELL METABOLISM AND FUNCTIONS
We analyzed peripheral blood samples collected from healthy donors aged 18 to 90 years. Intracellular staining was performed to assess both the metabolic and functional characteristics of lymphocyte populations in the cohort, highlighting differences between young adults (under 40) and older adults (over 65). Additionally, we investigated the potential of PPAR agonist drugs to reverse key features of immunosenescence in vitro. We observed that aging is associated with an increase in neutral lipid content in various subsets—including naïve and different memory subsets—of CD4⁺ T lymphocytes, CD8⁺ T lymphocytes, and B lymphocytes. Furthermore, we found that this increase in lymphocyte lipid content is strongly correlated with blood triglyceride levels, suggesting a close relationship between systemic and cellular metabolism. We then assessed apoptosis susceptibility in different T lymphocyte subsets (naïve and memory) by measuring active Caspase-3 expression levels. Our results show that with aging, T lymphocyte subsets—particularly within the CD8⁺ compartment—become more prone to apoptosis. These alterations contribute to a reduced CD8⁺ T cell priming capacity in healthy older individuals compared to younger subjects. In vitro treatment with PPAR-α and PPAR-γ agonists reduces intracellular lipid content and apoptosis susceptibility in CD4⁺, CD8⁺, and B lymphocytes. Notably, these treatments also enhance CD8⁺ T cell priming capacity, highlighting their potential to boost immune responses against novel antigens.
WP2. ASSESSMENT OF THE EFFECTS OF PPAR AGONISTS ON T-CELL SUBSETS OF OLD MICE
After the promising in vitro findings, the two most effective drugs were selected for in vivo validation. These drugs will be administered to immunocompetent young (2 months of age) and old (18 months of age) mice to determine their ability to modulate and restore immunometabolic alterations characteristic of immunosenescence. Various samples, including blood and spleen, will be collected and analyzed at different time points to assess changes inT cell compartments during and after treatment. We expect that PPAR agonists will restore both the proportions of circulating immune cells and their metabolic properties in aged mice, potentially leading to improved immune function, comparable to those seen in younger animals.
WP3. ASSESSMENT OF THE IN VIVO CAPABILITY OF PPAR AGONISTS TO CONTROL INFECTIOUS DISEASES IN OLD MICE
The two selected compounds will also be evaluated in a murine model of herpes simplex virus type 1 (HSV-1) infection. We aim to assess whether pharmacological treatment improves pathogen-specific immune responses in aged animals. This assessment will involve the analysis of multiple organs and immune-relevant tissues, with the goal of comprehensively determining the impact of compounds to modulate and restore the metabolic and immunological functions of both innate and adaptive immune compartments. PPAR agonists are anticipated to enhance pathogen-specific immune responses in aged mice by restoring compromised immunometabolic functions to levels comparable to those observed in younger animals.
WP4. ASSESSMENT OF THE IN VIVO CAPABILITY OF PPAR AGONISTS TO CONTROL TUMOR PROGRESSION IN OLD MICE
Based on preliminary in vitro and in vivo evidence and literature data, we expect that pharmacological modulation of lipid metabolism through PPAR agonists will restore the metabolic and functional competence of CD8⁺ T cells in aged murine models, thus improving cancer immune surveillance. In murine models of primary breast cancer and metastatic lung cancer, treatment with selected PPAR-α and PPAR-γ agonists is expected to reduce tumor growth and metastatic spread by enhancing T cell-mediated anti-tumor immunity, particularly in aged mice, validating the relevance of immunometabolic reprogramming in the context of immunosenescence. Furthermore, combining PPAR agonists with immune checkpoint inhibitors—already in clinical use—may synergistically improve their therapeutic efficacy. Overall, the project aims to demonstrate that restoring T cell function via lipid metabolism modulation not only rejuvenates cancer immune surveillance but also enhances the efficacy of existing cancer immunotherapies. These findings may offer a translational strategy for improving cancer outcomes in the aging population.
WP5 EPIDEMIOLOGICAL ANALYSIS ON THE SUSCEPTIBILITY OF ELDERLY SUBJECTS TAKING FIBRATES AND THIAZOLIDINEDIONES TOINFECTIONS AND MALIGNANCIES
The epidemiological analysis on the susceptibility of elderly subjects taking fibrates and thiazolidinediones to infections and malignancies, consists of two parts. The first is a drug utilization study analyzing patterns of use for both fibrates and thiazolidinediones, estimating the number of users and preliminarily describing the occurrence of infections and tumors among elderly patients in Romagna. The second part is a risk assessment analysis basedon two retrospective cohort studies. The first will evaluate the risk of acute infections (HZV, pneumonitis, sepsis), while the second one will assess cancer incidence in userof one of the drug classes. Exposure groups will consist ofincident subjects of fibrates or thiazolidinediones, while comparator groups will include subjects exposed to other second-line drugs used to treat hyperlipidemia or diabetes. The outcome will be considered as a record of infection/tumor occurrence registered in administrative databanks.
Conclusions:
Our results highlight that aging is associated with significant immunometabolic dysfunction, particularly lipid accumulation in lymphocytes, which compromised their functionality. This may partly explain the reduced vaccine efficacy and the increased susceptibility to infections and tumors observed in older adults. The use of PPAR agonist drugs, by targeting cellular lipid metabolism, emerged as a promising strategy to restore the functional capacity of lymphocytes in elderly individuals. This approach holds translational potential for improving immune defense and enhancing therapeutic outcomes in age-related diseases, including cancer.