Mechanistic insights and biomarker discovery in immune cell aging and age-associated diseases

Primary lymphoid organs, including the bone marrow and thymus, play a crucial role in generating diverse immune cells. While all immune cells originate and mature in the bone marrow, T cells require additional maturation in the thymus. These organs possess a highly organized microenvironment comprising precursor immune cells and stromal cells that support immune cell development (32) However, aging leads to thymic involution, bone marrow HSC decline, and lymph node fibrosis, collectively impairing the generation and maintenance of naive immune cells and compromising adaptive immunity.

Thymic involution results in the gradual replacement of functional thymic tissue with adipose deposits, reducing naive T-cell output. In the bone marrow, hematopoietic stem cell (HSC) activity declines, impairing the body's ability to generate new immune cells. Additionally, aging leads to a reduction in the number and size of lymph nodes, which are essential for mounting immune responses (33). In older individuals, diminished lymphadenopathy in response to infections indicates a compromised ability to activate adaptive immunity. A key structural change in aging lymph nodes is the disruption of distinct T-cell and B-cell zones due to excessive collagen deposition. This disorganization interferes with the accessibility of IL-7, a crucial cytokine necessary for T-cell survival and homeostasis. As a result, the maintenance of naive T cells is impaired, further compromising adaptive immunity (34).

Aging is associated with alterations in both the frequency and function of HSCs, with variations observed across individuals based on genetic factors and underlying conditions. Studies in murine models have demonstrated that HSC frequency differs across strains, with DBA/2 mice exhibiting higher HSC counts than C57BL/6 and BALB/C strains (35). Studies have shown that the DBA/2 strain in mice exhibits a higher frequency of HSCs compared to the C57BL/6 and BALB/c strains. However, aging universally compromises HSC self-renewal and differentiation, as demonstrated by experiments transplanting aged HSCs into irradiated mice, which resulted in impaired self-renewal and reduced lymphopoiesis. A hallmark of aging is the shift in HSC differentiation toward myeloid lineages, termed skewed myelopoiesis (36). This shift is reflected in the upregulation of myeloid lineage genes over lymphoid genes, resulting in an age-related increase in myeloid cell production. Concurrently, there is a decrease in B-cell precursors, such as common lymphoid progenitors (CLPs), pro-B cells, and pre-B cells in the bone marrow, leading to diminished lymphopoiesis. Additionally, aging leads to enhanced production of reactive oxygen species (ROS), negatively impacting HSC function and further compromising immune response and hematopoietic homeostasis in the elderly population (37).

Aging is also associated with reduced expression of various surface and endosomal Toll-like receptors (TLRs), which play a crucial role in the innate immune response by detecting pathogen-associated molecular patterns. This diminished expression affects the production of type I interferons (IFNα and IFNβ), key cytokines with potent antiviral effects. Type I IFNs function by inducing the expression of interferon-stimulated genes (ISGs) such as IFIT1, IFITM1, and ISG15, which are critical for inhibiting viral replication and enhancing the host's defense mechanisms. Additionally, type I IFNs are essential in boosting antigen presentation by dendritic cells (DCs), facilitating the processing and presentation of antigens to T cells. This process promotes the expansion and activation of cognate T cells necessary for an effective adaptive immune response. The decline in type I IFN production with age weakens both the antiviral response and the overall capacity of the immune system to respond to infections and malignancies (38).

The immune system relies on the homeostasis and functionality of innate immune cells such as DCs, macrophages, NK cells, and neutrophils. However, aging disrupts these functions, leading to compromised immune responses. This dysfunction manifests as impaired DC antigen presentation, skewed macrophage activation, accumulated yet less effective NK cells, and altered neutrophil activity (39). DCs serve as key antigen-presenting cells (APCs), bridging innate and adaptive immunity. Their primary role is to process and present antigens to T cells, initiating adaptive immune responses. In aging individuals, DC function declines due to reduced expression of costimulatory molecules like CD80 and CD86, which are essential for effective T cell activation. This impairment weakens T cell expansion and adaptive immune responses. Additionally, aging affects DC antigen uptake, processing, and migration to lymphoid tissues, leading to delayed pathogen recognition and a diminished ability to mount robust immune responses (40). Reduced efficiency in major histocompatibility complex (MHC)-mediated antigen presentation further hampers T cell recognition, contributing to increased susceptibility to infections and malignancies in older adults. Furthermore, impaired DC function diminishes vaccine efficacy, as DCs are crucial for antigen presentation and memory cell generation. This highlights the need for age-specific vaccine formulations or adjuvants to enhance DC functionality (4).

Macrophages, critical for clearing pathogens and maintaining tissue balance, undergo functional decline with aging (41). Older macrophages show a diminished ability to engulf and eliminate pathogens and cellular debris, heightening vulnerability to infections (42). Aging also shifts macrophage polarization toward the pro-inflammatory M1 state, marked by elevated secretion of TNF-α, IL-6, and IL-1β. This imbalance fosters chronic low-grade inflammation, a driver of age-associated conditions like cardiovascular diseases, type 2 diabetes, Alzheimer's disease, and cancer. In contrast, M2 macrophages, which support tissue repair and anti-inflammatory processes, become less functional in aging, worsening impaired wound healing and unresolved inflammation (43). Aged macrophages may also develop a senescent phenotype, leading to the secretion of the SASP, which includes pro-inflammatory cytokines, chemokines, and proteases. SASP exacerbates inflammation, accelerates tissue aging, and fosters a cycle of immune dysfunction. This persistent inflammatory environment impairs immune homeostasis, increasing the risk of autoimmunity and chronic disease (44). Neutrophils, the first responders against infections, experience age-related functional impairments, including reduced phagocytosis and diminished ability to form neutrophil extracellular traps (NETs) through NETosis. These deficiencies compromise pathogen elimination, allowing infections to spread more readily in elderly individuals. Additionally, aged neutrophils exhibit prolonged inflammatory responses, contributing to tissue damage and exacerbating age-related inflammation. Chemotactic responsiveness also declines with age, reducing the efficiency of neutrophil migration to infection sites and further delaying immune responses (45). NK cells, crucial for viral defense and tumor surveillance, undergo significant changes with aging. Although their overall numbers increase, there is an accumulation of dysfunctional CD56-dim NK cells with diminished cytotoxic activity. These nonfunctional NK cells are less effective in targeting infected or malignant cells, leading to increased cancer risk and impaired viral immunity in the elderly population. The reduced cytotoxic potential of NK cells further contributes to immune senescence, as the ability to eliminate aberrant cells declines over time (46). Myeloid-derived suppressor cells (MDSCs), which regulate immune responses by inhibiting T cell activation and proliferation, accumulate excessively with age. Under normal conditions, MDSCs prevent excessive inflammation and maintain immune tolerance. However, in aged individuals, their overabundance creates an overly suppressive environment that dampens adaptive immunity. MDSCs interfere with antigen-presenting cells, release immunosuppressive molecules such as arginase, reactive oxygen species (ROS), and nitric oxide (NO), and directly inhibit T cells. This suppression not only weakens the response to infections but also reduces vaccine efficacy, as MDSCs impair T cell-mediated immunity. The excessive presence of MDSCs in older adults contributes to an overall decline in immune competence, increasing susceptibility to infections and malignancies. Targeted therapies aimed at reducing MDSC accumulation or mitigating their suppressive effects could enhance immune function in aging individuals. Recent studies on aged DC showed that Rab8a overexpression, which is accompanied by the upregulation of Rab11, restores the functionality of these aged DCs, whereas knockdown of Rab8a reduces functionality of DCs from young mice (47). Such interventions may improve T-cell responses, bolster vaccine efficacy, and enhance pathogen clearance, ultimately promoting healthier aging (48). Aging profoundly affects innate immune cell function, leading to compromised pathogen defense, chronic inflammation, and weakened vaccine responses. The decline in DC antigen presentation, macrophage phagocytic efficiency, neutrophil chemotaxis, NK cell cytotoxicity, and the excessive accumulation of MDSCs collectively contribute to immunosenescence (49). Understanding these changes is crucial for developing targeted therapeutic strategies to enhance immune resilience in older populations and mitigate age-related disease risks.

As we age, our immune system undergoes significant changes, including alterations in the function and composition of B and T cells. These two primary lymphocyte populations are critical for adaptive immune responses. One notable effect of aging is that T cells experience a decline in proliferation and functionality. In contrast, B cells show impaired antibody production, contributing to an increased susceptibility to infections and a diminished response to vaccinations in older individuals. Adaptive immunity undergoes remodeling during aging, including B cell composition and function changes. A distinct subset of B cells, termed "age-associated B cells," expands in aged mice's spleen and bone marrow. These cells differ from conventional naive and memory B cells (6). Hao et al. identified them as CD43-CD21/CD35-CD23-B cells, while Rubtsov et al. described them as CD11b+CD11c+ B cells. Notably, these age-associated B cells express T-bet (encoded by Tbx21), similar to B cells linked to lupus-like autoimmunity in mice. Although the factors driving their expansion aren't fully understood, evidence suggests that damage-associated molecular patterns, such as debris and chromatin from apoptotic cells, activate age-associated B cells via the TLR7 or TLR9 pathway (50, 51). Once formed, their survival in aged mice depends on IFNγ signaling. Age-associated B cells contribute to "inflammaging" by disrupting immune homeostasis, secreting IL-4 and IL-10 upon activation and presenting antigens to T cells. The recent advancements in ssRNA sequencing indicate new subsets and molecular markers for the aged B cell population like ApoE, although APOE protein is related to lipid metabolism and is expressed in other immune subpopulations. B Cells are very diverse and function in the different compartments of the body. Like splenic age-associated B cells, peritoneal B1-like cells express the transcription factor gene Zbtb32 but form a distinct transcriptional cluster. This cluster is marked by high expression of unique genes, including Zcwpw1 and Ctla4. With age, B1-like cells become more activated, strongly induce cytotoxic CD8+ T cells, and respond to commensal bacteria. Single-cell studies reveal the heterogeneity of age-associated B cells, suggesting their potential antigen specificity and tissue-specific inflammatory roles in older organisms (52). Additionally, regulatory T cells tend to increase in number with age, which may further exacerbate T cell dysfunction and contribute to a more profound immune suppression observed in elderly populations, highlighting the complex interplay between T cell dynamics and aging-related immune decline. Furthermore, this phenomenon of immune suppression due to an increased presence of regulatory T cells may be linked to the chronic inflammatory state often seen in older adults, which complicates the overall immune response and increases the risk of various age-associated morbidities, including autoimmunity and cancer (53). The role of sex hormones in modulating the aging-associated changes to the immune system has also been an area of active research. Emerging evidence suggests that sex hormones can influence the degree of immune senescence, with notable differences observed between males and females as they age, potentially leading to disparities in disease susceptibility and progression among the sexes (54).

Aging significantly impacts the T cell compartment, leading to immunosenescence- a decline in naive T cell numbers that weakens responses to new infections and vaccines. This decline is largely attributed to thymic involution, where the thymus shrinks and loses functional capacity, reducing T cell progenitor output and compromising immune diversity (33). Sex differences in T cell aging suggest that males and females experience distinct immune aging patterns, likely influenced by circulating sex hormones. These differences may contribute to varying susceptibilities to age-related diseases and responses to immunization (55). Regulatory T cells (Tregs), which suppress excessive immune responses and maintain homeostasis, increase in frequency with age. While Tregs help prevent autoimmunity, their accumulation can dampen effector T cell responses, impairing the immune system's ability to combat infections and tumors (56).

Genetic studies in mice show that premature T cell aging, such as through TFAM gene knockout- leading to systemic aging effects, affecting metabolic, musculoskeletal, cardiovascular, and cognitive health. This suggests that aging T cells may accelerate aging across multiple organ systems, making them a key target for interventions (57). Maria et al. proposed eight hallmarks of T cell aging: four primary (thymic involution, mitochondrial dysfunction, genetic/epigenetic changes, and proteostasis loss) and four secondaries (TCR repertoire reduction, naive–memory imbalance, T cell senescence, and loss of effector plasticity). These lead to two integrative hallmarks- immunodeficiency and inflammaging- characterizing aged immune function (58). Aging alters CD8+ T cells, reducing naive populations while increasing memory subsets and cloning. Single-cell RNA and TCR sequencing in mice reveal a decrease in naive CD8+ T cells across multiple tissues. A distinct PD1+TOX+CD8+ T cell subset accumulates with age, which is absent in young mice (59). Additionally, aging contributes to CD4+ T cell exhaustion, though this phenomenon is still under investigation. Using scRNA-seq-derived cell classification with our full lifespan coverage, it has been observed that although both naive T cell subsets (CD8-Naïve-LEF1 and CD4-Naïve-CCR7) exhibited a decreased trend with age (60). Understanding these age-related T cell changes is crucial for developing targeted strategies to enhance vaccine efficacy, and mitigate age-related immune dysfunction.

CD8+ T cells in humans demonstrate notable age-associated alterations in both phenotype and function, most evident in changes to granzyme expression. Granzymes, a family of serine proteases, are critical for cytotoxic and immunomodulatory roles. With advancing age, there is a marked expansion of granzyme K (GZMK)-expressing effector memory CD8+ T cells (TEM), identifying a subset that differs significantly from granzyme B (GZMB)-expressing TEM cells. In murine models, these GZMK+ CD8+ TEM cells, sometimes referred to as Taa (age-associated GZMK+ CD8+ T) cells, exhibit clonal proliferation and possess distinct epigenetic and transcriptional signatures associated with tissue homing, exhaustion, and a pro-inflammatory phenotype (61). Unlike their GZMB+ counterparts, GZMK+ TEM cells show reduced cytolytic activity but drive persistent low-grade inflammation by acting on non-immune cells. The accumulation of these cells is linked to increased cytokine release, recruitment of additional immune cells, and tissue damage, all of which contribute to the impaired immune landscape observed in aging.

From a mechanistic perspective, GZMK+ CD8+ TEM cells are distinguished from GZMB+ TEM cells by their capacity to induce cellular senescence in tissue fibroblasts and to intensify inflammatory niches. These cells express markers characteristic of T cell exhaustion, such as PD-1 and TOX, which closely link them to age-related immune decline. Expansion of the GZMK+ subset has been documented in several chronic conditions, including atherosclerosis, emphasizing their dual role as both indicators and potential therapeutic targets for immune aging (62). Consequently, the gradual accumulation of GZMK+ TEM cells provide crucial mechanistic insight into age-related immune remodeling and represents a promising avenue for biomarker development to track immunosenescence, persistent inflammation, and age-linked diseases (63).

Similarly, CD4+ T cells undergo age-related phenotypic shifts. The proportion of naive CD4+ T cells decline with advancing age, whereas memory and terminally differentiated subsets become more prevalent. A defining feature of senescent CD4+ T cells is the loss of CD28 expression, resulting in the emergence of CD4+CD28^null cells that exhibit diminished proliferative potential and natural killer (NK)-like properties, including expression of NK cell receptors and cytotoxic mediators such as perforin and GZMB (60). Although less extensively characterized, certain CD4+ T cell subsets in older individuals also upregulate GZMK, which may further amplify chronic inflammatory responses and disrupt immune regulation (64). These cytotoxic CD4+ T cells produce elevated levels of pro-inflammatory cytokines, such as IFN-γ and TNF, and preferentially accumulate in aging tissues, linking them to immunosenescence and age-associated diseases (65). Age-driven epigenetic and transcriptional adaptation supports their cytotoxic and senescent profiles, reinforcing their significance in immune aging (3).

Collectively, the expansion of GZMK+ effector memory CD8+ T cells and cytotoxic, granzyme-expressing CD4+ T cell populations positions granzymes as pivotal indicators of age-related immune remodeling. These lymphocyte subsets contribute to the perpetuation of chronic inflammation and functional deterioration, and they represent promising mechanistic biomarkers and therapeutic candidates for age-associated immune dysfunction and disease (66).

Sex hormones play a critical role in modulating age-associated changes in the immune system, contributing to distinct immune function and disease susceptibility between men and women. Estrogens, androgens, and progestins act through receptors on various immune cells, including B cells, T cells, dendritic cells, macrophages, and NK cells. Estrogens typically enhance immune responses, leading to heightened antibody production, improved B and T cell maturation, and stronger immunity in females (67, 68). Androgens, more abundant in males, generally exert immunosuppressive effects, resulting in greater susceptibility to infections but lower rates of autoimmune disorders in men. With aging, both sexes experience declines in sex hormones, but the effects differ (69). Postmenopausal women face a sharp drop in estrogen, increased inflammation (higher IL-1, IL-6, and TNF-α), and reduced immune function, contributing to "inflammaging" and a higher risk of age-related diseases. In men, declining testosterone is linked to increased inflammatory markers and changes in immune cell populations, with their adaptive immune system aging more rapidly than in women. Older women tend to have a more inflammatory immune profile but retain better adaptive immunity, explaining higher late-onset autoimmune disease rates but lower cancer incidence compared to men (70). Conversely, older men, with a more immunosuppressive hormonal environment, face greater infection and cancer susceptibility. Hormone replacement therapy in postmenopausal women can partially restore immune balance by enhancing B and T cell responses and reducing inflammation. In men, testosterone supplementation may help support immune health, whereas testosterone deficiency is linked to immune aging and is commonly treated in older populations.

SASP includes proinflammatory cytokines, chemokines, growth factors, and proteases that promote chronic inflammation and further induce senescence in neighboring cells. This cascade leads to inflammatory cell death, tissue and organ inflammation, and is influenced by alterations in the gut microbiome (71). Collectively, these processes drive age-associated systemic inflammation (inflammaging) and functional decline in aged individuals.

P21 is a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, also referred to as p21 WAF1/Cip1 or CDK inhibitor 1A. Growing evidence suggests that p21^high represents distinct senescent subpopulations found in different tissues. It is predominantly expressed in adipocytes, callus at fracture sites, lung fibroblasts, and trophoblast cells in the placenta. It is upregulated in senescent cells and induced cell cycle arrest. scRNA-seq has identified an increased expression of CDKN1A in various aging tissues, including fibroblasts, endothelial cells, and immune cells. Cdkn1a transcript variant 2 serves as a novel and more sensitive marker of cellular senescence compared to variant 1 or total p21Cip1/Waf1 protein. It is selectively elevated during natural aging in almost all tissues of male and female mice and shows distinct temporal dynamics in response to genotoxic stress, with higher sensitivity to senolytic treatments. p21, regulated by Tet1, plays a key role in embryonic stem cell proliferation by promoting the G1 to S phase transition through the modulation of EZH2 and H3K27 trimethylation at its promoter (72). Its role in controlling cell cycle progression, cellular senescence, and tissue formation underscores its importance in both normal development and in diseases characterized by abnormal proliferation or differentiation. p21 is crucial in tissueogenesis, regulating endothelial cell proliferation during vascular growth, where Ddx21 mutation triggers Vegfc-Flt4-driven proliferation and increased p21 expression, leading to cell cycle arrest and inhibited lymph angiogenesis. p21-dependent cellular senescence is essential for neural development, where loss of Rack1 activates p21 signaling, inducing aging in neural stem cells, while removal of p21 can alleviate microcephaly caused by Rack1 knockout. It has been found that p21 maintains the viability of DNA damage-induced senescent cells by preventing DNA damage and NF-κB-mediated cell death. p21 knockout in mice reduced liver senescent stellate cells, alleviating liver fibrosis and collagen production, revealing a new pathway regulating senescent cell survival and fibrosis have reviewed many diseases such as osteoporosis, osteoarthritis, metabolic disease, chronic obstructive pulmonary disease, pulmonary fibrosis, Parkinson's disease (PD) linked to p21 (73).

Recent works highlight an ongoing debate regarding the reversibility of immune aging via senolytic or metabolic interventions. While the clearance of p16+ cells restore immune profiles in some models, the functional plasticity of aged cells remains limited by persistent epigenetic scars and an altered signaling landscape. P16INK4a is a well-established marker of cellular senescence and is involved in regulating the cell cycle, particularly in inhibiting CDKs. scRNA-seq has revealed that CDKN2A expression is markedly elevated in senescent cells across multiple tissues, including the liver and skin. Its expression is significantly upregulated in senescent cells and during both natural aging and age-related diseases, making p16 a key biomarker for identifying senescent cells and assessing the aging process. P16 acts as a potent inhibitor of cyclin-dependent kinases CDK4 and CDK6, preventing the G1 to S phase transition and inducing cell cycle arrest by maintaining the retinoblastoma protein (pRB) in a hypophosphorylated state (74). This regulation is thought to be part of a feedback loop, where pRB phosphorylation activates E2F, which in turn promotes p16 expression. Factors such as telomere shortening, DNA damage, and UV exposure activate the p16/pRB and p19^ARF-p53^–p21 signaling pathways, leading to an accumulation of senescent cells and impairing the regenerative capacity of tissues, particularly in skin stem cells. Notably, p16 expression during development coincides with the onset of cellular differentiation, a process that requires reduced cell proliferation. Consequently, p16's role in tumor suppression aligns with its function in cell cycle inhibition. In this context, senescence in aging, marked by elevated p16 levels, can be seen as an extreme form of cell cycle arrest (75). In numerous preclinical models, clearance of senescent cells or modulation of the SASP has shown beneficial effects, with early clinical trials using senolytics yielding promising results. However, the impact of p16 ablation on health span is not straightforward, as different mouse models with p16 deletion have yielded conflicting outcomes. A comprehensive comparison of the SASP profiles across these models could provide valuable insights into how targeting specific SASP factors might improve health outcomes in aging populations. Furthermore, the high expression of p16, particularly in endothelial cells during aging, and the potential liver damage caused by the removal of p16^high cells warrant further investigation into the functional consequences of p16 in these cell types. A significant correlation was observed between CDKN2A mRNA expression levels and age. In Alzheimer's disease (AD) patients, CDKN2A mRNA expression levels in blood were notably reduced, while in healthy controls, these levels increased with age. Additionally, CDKN2A mRNA expression levels in AD patients showed a significant positive correlation with DNA methylation rates (76). While CDKN1A/p21 and CDKN2A/p16INK4a are hallmarks of cellular senescence, their induction often results from upstream metabolic and signaling alterations in aged immune cells, linking mitochondrial dysfunction to epigenetic reprogramming and dysfunctional inflammatory cascades.

While not directly measured by scRNA-seq (since it's an enzymatic activity), genes associated with SA-β-gal expression, such as GLB1, are often identified as markers for senescent cells in scRNA-seq studies. This marker is used to detect the accumulation of senescent cells in tissues. Using a second-generation fluorogenic substrate for β-galactosidase and multi-parameter flow cytometry, a study shows that peripheral blood mononuclear cells (PBMCs) from healthy humans exhibit an increasing number of cells with high senescence-associated β-galactosidase (SA-βGal) activity as donor age advances (77). The most pronounced age-related increase was observed in CD8+ T cells, with up to 64% of these cells in donors in their 60s showing high SA-βGal activity. These senescent CD8+ T cells exhibited telomere dysfunction-induced and p16-mediated senescence characteristics, including impaired proliferation, various T-cell differentiation states, and a gene expression profile similar to that seen in senescent human fibroblasts. β-galactosidase is commonly used as a tumor marker, with increased activity linked to malignant potential in cells. Oncogene activation induces oncogene-induced senescence (OIS), a process that halts the cell cycle and limits tumor cell proliferation. Initially observed in somatic non-tumor cells, OIS was later found in atypical cells (78). It leads to DNA damage, activating the p53-p21-p16 pathway, which overexpresses senescence-associated SA-βGal. This mechanism prevents atypical cells from entering mitosis, thereby halting tumor growth due to the accumulation of irreversible DNA damage. Valieva et al., reviewed SA-βGal involvement in various cancers (79).

Senescent cells exhibit distinctive molecular signatures characterized by changes in cell cycle and metabolism-related genes, along with alterations in genes encoding secretory proteins that constitute the SASP. This complex secretory program encompasses various components, including soluble signaling molecules, secreted proteases, and extracellular matrix (ECM) components, which collectively influence the tissue microenvironment (80). Through single-cell RNA sequencing analysis, researchers have identified key SASP components consistently upregulated in senescent cells. These include pro-inflammatory cytokines such as IL-6, IL-8, TNF-α, and matrix metalloproteinases (MMPs). Additionally, chemokines like CXCL1, CCL2, and growth differentiation factor 15 (GDF15) show increased expression, particularly in aging tissues. The SASP proteases are crucial in modifying the microenvironment through multiple mechanisms, including membrane protein shedding, signaling molecule degradation, and ECM modification (81). The SASP components work through interconnected pathways to maintain and amplify the senescent state. Interleukins, particularly IL-6 and IL-1, are central to this process, with IL-6 being closely linked to DNA damage-induced senescence and IL-1 activating inflammatory pathways through receptor signaling. Chemokines, including IL-8 and various MCPs, contribute to a self-reinforcing secretory network that maintains growth arrest. The insulin-like growth factor (IGF) pathway also plays a significant role, with senescent cells expressing elevated levels of IGF-binding proteins that influence senescence and apoptosis in neighboring cells (82).

Matrix metalloproteinases represent another crucial component of the SASP, with specific proteins like stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), and collagenase-1 (MMP-1) being upregulated in senescent fibroblasts. These MMPs actively modify the SASP composition by cleaving various components, including MCP-1, -2, -4, and IL-8. Additionally, serine proteases, such as urokinase- and tissue-type plasminogen activators and their inhibitors, contribute to the senescent phenotype, with PAI-1 particularly important in reinforcing growth arrest (83). Beyond protein factors, senescent cells also produce non-protein molecules, including ROS and nitric oxide, which significantly impact cellular phenotypes and contribute to cancer cell aggressiveness. The influence of senescent cells extends to neighboring cell differentiation, with effects ranging from triggering epithelial-mesenchymal transition in epithelial cells to promoting angiogenesis through proangiogenic SASP components in endothelial cells. Furthermore, senescent cells actively modify the immune landscape by altering immune cell infiltration patterns, particularly in tumors, where they can recruit pro-tumorigenic T cells and macrophages (84).

Many studies have identified CpG sites whose methylation correlates with age; however, these epigenetic clocks are limited in their mechanistic understanding and only capture a small fraction of the genome, missing the broader context of the aging process. The researchers found that CpG sites gaining methylation with age are enriched in Polycomb Repressive Complex 2 (PRC2) targets. This age-dependent methylation gains accounts for about 90% of the increase in DNA methylation across the genome. PRC2 is primarily known for its role in transcriptional repression through the trimethylation of histone H3 at lysine 27 (H3K27me3), a marker of gene silencing. The core components of PRC2 include: (i) Enhancer of Zeste Homolog 2 (EZH2): The catalytic subunit responsible for the methylation of H3K27, which involves transferring methyl groups to the histone tail and promoting gene silencing. (ii)SUZ12: A crucial component for the stability and activity of PRC2. It is necessary to recruit the complex to chromatin and maintain H3K27me3 levels. (iii) Embryonic Ectoderm Development (EED): Binds to the methylated H3K27 mark, helping stabilize the PRC2 complex and enhancing its activity. (iv) Retinoblastoma-Binding Proteins (RBBP4/7): Co-factors that assist in chromatin binding and contribute to the overall stability of the complex. The two critical subunits, EZH2 and EED, defined below, are essential for the function of PRC2, playing a central role in regulating DNA methylation and gene silencing.

Immunosenescence, significantly impacts the susceptibility of older adults to various infections. Increases vulnerability to respiratory infections, urinary tract infections (UTIs), and herpes zoster (shingles) in the elderly population. Respiratory infections, particularly influenza and pneumonia, emerge as major contributors to morbidity and mortality among older adults. The compromised immune system, characterized by diminished innate and adaptive responses, struggles to combat respiratory pathogens effectively. Specifically, the reduction in T cell function and antibody production impairs the body's ability to resist novel strains of viruses and bacteria, leaving the elderly more susceptible to these infections (95). Urinary tract infections also show increased prevalence in the aging population. This heightened occurrence stems from a combination of factors, including weakened immune surveillance, alterations in urinary tract function, and shifts in the microbiome. Notably, recurring UTIs are common among older adults, with a higher incidence observed in elderly women (96). Herpes zoster, commonly known as shingles, represents another significant health concern for older individuals. This painful condition results from the reactivation of the varicella-zoster virus, which causes chickenpox in its initial infection. The re-emergence of this latent virus is primarily attributed to immunosenescence, particularly the decline in T-cell surveillance. Consequently, older populations face an elevated risk of developing shingles (97). Understanding these age-related changes in immune function and their impact on infection susceptibility is crucial for developing targeted interventions and preventive strategies. By addressing the underlying mechanisms of immunosenescence, healthcare providers can work towards improving the quality of life and reducing infection-related complications in the elderly population (98).

Aging is closely linked to the onset and progression of multiple chronic conditions that compromise health and quality of life. Among these, cardiovascular disease, type 2 diabetes, and osteoarthritis represent major contributors to morbidity in older populations. Understanding the interplay between aging-related biological changes and these disorders is crucial for developing targeted interventions and preventive strategies.