Inflammation and aging: signaling pathways and intervention therapies

The aging of bone tissue inevitably affects HSCs. With age, red bone marrow is gradually replaced by fat cells, leading to yellow bone marrow formation that inhibits hematopoietic function.¹⁰⁵ The decreased secretion of nutrient factors by bone marrow stromal cells can result in an enhanced differentiation of HSCs into myeloid cells and a reduced differentiation into lymphocytes. This imbalance in myeloid/lymphoid differentiation is one of the manifestations of HSC aging.^105–107 Importantly, aged HSCs tend to differentiate more towards myeloid cells, while their ability to support lymphoid cell maturation decreases. This leads to a reduction in the number of precursors for T and B cells with increasing age.^108,109 Taken together, with aging, the number of HSCs increases, while their function including self-renewal and clonogenicity, decreases. In addition to the HSC changes mentioned above, aging marrow also has decreased Wnt signaling and the accumulation of senescent cells and inflammatory cytokines.¹¹⁰

The thymus is a central T-lymphatic organ that produces functional initial T-lymphocytes and immune tolerance. In most mammals, aging is accompanied by degeneration of the thymus gland. In humans, thymocyte numbers and hormone secretion levels typically increase during early development and then decrease over time. In addition, the majority of functional cells are substituted with senescent fibroblasts and adipocytes, and stromal cells during thymus aging.^88,111,112 In the aged mouse thymus, elevated levels of phosphorylated histone H2AX and the p53 binding protein suggest heightened oxidative stress and DNA damage, consequently leading to cellular senescence,¹¹³ providing support for the notion that the aging thymus exhibits a greater proportion of senescent cells.

Thymic degeneration results in reduced generation of new T-cells, an accumulation of memory T-cells, and a decline in the diversity of T-cell receptors. As a consequence, this leads to a weakened immune response and decreased overall immunity. It has been observed that apparent aging-associated alteration, especially a progressively reduced population of naive T cells, in murine T cell compartment during thymic involution.¹¹⁴ However, in human, there is a progressive loss of CD8+ naive T cells while, notably, a relatively stable naive CD4+ compartment is efficiently maintained via homeostatic proliferation.^115–117

Consequently, both in mice and humans, age-related variations in the production of naive T cells from the thymus result in qualitative disparities in the overall T cell repertoire.^118–121 In addition, it has been observed that naive T cells from older individuals exhibit reduced responsiveness to the superantigen toxic shock syndrome toxin-1 compared to younger individuals,^122,123 which may be caused by low dual specificity phosphatase 6 levels in naive T cells, resulting in a rise in the threshold of TCR activation.

The spleen acts as a secondary lymphoid organ promoting immune defense and is the main pivotal organ for initiating the activities required for the adaptive immune responses. During the aging process, significant alterations occur in the cellular composition and microarchitecture of the spleen. The clear distinction between T-cell and B-cell areas within the white pulp becomes less defined, and there are noticeable changes in the organization and functionality of marginal zone macrophages, stromal cells, and marginal metallophilic macrophages.^124,125

Furthermore, recent advancements in single-cell RNA sequencing studies have revealed that the proportion of T cells in the spleen decreases with age, while the relative abundance of plasma cells increases.¹²⁶ Impaired migration of B cells and the phagocytic capacity of macrophages in the marginal zone can also be seen in aged spleens.¹²⁷ Also, impaired function of microenvironment-mediated antigen-presenting cells was observed, which may provide an explanation for the observed delayed responses to stimulation even in T cells derived from young HSCs (Fig. 3).^102,128,129

Lymph nodes serve as crucial sites where T cells and B cells reside and where immune responses are initiated, playing a vital role in establishing an effective immune response. However, the number, integrity, and functionality of lymph nodes undergo significant declines with age, as evidenced by previous studies.^102,125 The exact cause of lymph node atrophy remains unknown; however, it can result in the deterioration of the microenvironment where immune cells reside, thereby negatively impacting immune function. Age-related alterations in cellularity and the functionality of different cell types within the lymph nodes have been extensively documented.¹³⁰ Specifically, the number of fibroblastic reticular cells in lymph nodes diminishes, resulting in a compressed and less reticular stromal network.¹³¹ In addition, older individuals, aged 60 years and above, exhibit increased fat deposition and fibrosis in their lymph nodes.^124,125 Moreover, stromal cells within aged lymph nodes exhibit reduced replication potential when stimulated and are unable to maintain a balance of naive T cells.^126,127,131 The accumulation of senescent cells in lymph nodes, along with heightened inflammation, may negatively impact the migration and recruitment of immune cells, thereby serving as detrimental factors (Fig. 3).¹³⁰

The main cause of brain aging appears to be neuroinflammation¹³² via aged brain cells and a weakened immune system. The process of brain aging significantly contributes to the decline of various cognitive functions, encompassing decreased speed of information processing, reduced capacity of working memory, impaired spatial memory, and diminished plasticity (Fig. 3).¹³³

Aging of brain cells including neurons and glial cells (i.e., microglia and astrocytes) leads to the upregulation of inflammatory-related pathways, causing brain function weakness and increased inflammation damage. During the aging process, microglia gradually lose their ability to efficiently clear misfolded proteins that are linked to neurodegeneration. This impairment in protein clearance significantly contributes to the neuroinflammatory response observed in the brain, with microglia playing a central role in this process. Except for the supporting role, Shao et al. found that astrocytes can also be a mastermind of neuroinflammation, depending on the Dopamine D2 receptor (Drd2), normally an important brake on it.¹³⁴ During aging, the level of Drd2 and its ligand dopamine both decline with high neuroinflammation in the brain. Subsequently, activated astrocytes produce SASP factors, such as IL-1β, IL-6, TNF-α, IFN-γ, COX-2, and other inflammatory factors (Table 1). In turn, these factors further promote astrocyte activation. The excessive production of pro-inflammatory mediators disrupts the intricate equilibrium necessary for the induction of long-term potentiation, leading to a decrease in the production of brain plasticity-related molecules such as BDNF and IGF-1, consequently impairing synaptic plasticity.¹³⁵ Remarkably, even older adults without neurological impairments demonstrate a gradual escalation in neuroinflammation, characterized by elevated homeostatic levels of inflammatory cytokines and reduced production of anti-inflammatory molecules (Fig. 3).¹³⁶

Immunosenescence and inflammaging can both contribute to neuroinflammation, resulting in impaired neuronal function and the accumulation of brain tissue damage.^137,138 Consequently, various central nervous system disorders, including Alzheimer’s disease, Parkinson’s disease, and stroke, are characterized by degenerative neurological conditions.¹³⁹

Most cardiac tissue is composed of cardiomyocytes, cardiac fibroblasts, and macrophages. The aging process in the heart is characterized by the gradual occurrence of several hallmarks, including progressive cardiomyocyte hypertrophy, the gradual onset of cardiac fibrosis, and the presence of inflammation (Fig. 3).¹⁴⁰

Hypertrophic cardiomyocytes, characterized by heightened oxygen and energy requirements, create a hypoxic environment of low oxygen levels. This imbalance in oxygen levels leads to the generation of excessive free radicals, which can potentially damage cellular components. In response to hypoxia, cardiomyocytes release pro-inflammatory cytokines and chemokines. These molecules stimulate an immune response and contribute to an increase in the number of macrophages within the left ventricle.¹⁴⁰ In addition, because mature cardiomyocytes have a low rate of proliferation, the injured area in the aging heart is replaced by fibrotic scar tissue, resulting in organ failure.¹⁴¹

The main effector cells in cardiac fibrosis are activated myofibroblasts. Long-term inflammation promotes cardiac and vascular fibrosis. The cardioprotective effects of AMPK and GDF11 on cardiomyocytes have been extensively documented, and the decline in AMPK and GDF11 expression associated with aging is likely a contributing factor to the heightened cardiac fibrosis observed during the aging process.¹⁴²

In the steady-state heart, macrophages play a crucial role in eliminating senescent and dying cells, contributing to the normal homeostatic maintenance of the myocardium and facilitating tissue repair following injury. However, in the aging heart, macrophages recruited to the site of infarction exhibit a pro-inflammatory M1 phenotype initially, but subsequently transition to an anti-inflammatory M2 phenotype after myocardial infarction (MI). This phenotypic switch promotes angiogenesis and scar formation, aiding in the recovery process.¹⁴³

In addition, vascular smooth muscle cells (VSMCs) play a crucial role in coordinating vascular function alongside endothelial cells, regulating blood pressure, vascular tone, and blood flow. However, during the aging process, the dysfunction and decline of VSMCs have a detrimental impact on the structural integrity of the aorta, ultimately leading to the development of transthoracic aortic aneurysms.¹⁴² Classical molecular IGF-1 signaling causes cardiac hypertrophy and heart failure.¹⁴² IGF-1 increases cellular senescence in VSMCs by inducing DNA damage and increasing ROS production via the p53 pathway.

As individuals age, the kidneys undergo various structural impairments, such as fibrosis, and experience functional issues, including mitochondrial dysfunction (Fig. 3).^144–146 Moreover, older individuals become more susceptible to acute kidney injury (AKI) and chronic kidney disease (CKD).^123,147 Age-related alterations in multiple cell types, such as tubular epithelial cells, resident and circulating leukocytes, contribute to kidney injury. Proximal tubular cells depend on autophagy to effectively eliminate defective mitochondria and other organelles under both normal and pathological conditions.^148,149 However, during aging, their diminished proliferative capacity leads to impaired clearance abilities.¹⁵⁰

Aging in the kidneys is associated with various physiological changes, including chronic low-grade inflammation. This inflammatory state, often referred to as inflammaging, has been observed to have detrimental effects on the kidneys. Chronic low-grade inflammation in the kidneys can impair the normal repair mechanisms that occur following injury. This inflammation hampers intrinsic cellular repair mechanisms following injury and promotes immunosenescence and organ damage.^151,152 Elderly individuals exhibit immunological phenotypes characterized by reduced numbers of naive lymphocytes, increased pro-inflammatory T cells, and diminished phagocytic activity in monocyte lineage cells, similar to CKD patients. These alterations in the kidneys form the basis for prevalent pathological conditions that are commonly observed in both elderly individuals and patients with CKD.¹²³

Aging raises the risk of chronic liver disease and liver fibrosis, which is highly related to hepatic stellate cells, hepatocytes, and macrophages. Liver cells initially activate compensatory mechanisms in response to time-dependent damage caused by aging, which can lead to the development of pathologies of the liver if overstimulated.¹⁵³ Activated hepatic stellate cells are the major functional population during liver fibrogenesis.¹⁵⁴ During senescence, their replication, immune-recruiting signals, and clearance are important for the regulation of liver fibrogenesis.¹⁵⁵ An illustration of the contribution of senescent hepatocytes to hepatic stellate cell activation and liver fibrogenesis is evident in p53-deficient mice with nutrition-induced steatohepatitis. It was discovered that these mice displayed reduced levels of hepatocyte p21, as well as decreased activation of hepatic stellate cells and expression of fibrotic markers such as SMA and collagen.¹⁵⁶ This finding supports the involvement of senescent hepatocytes in the activation of hepatic stellate cells and the development of liver fibrosis. Furthermore, M2 macrophages secrete pro-fibrogenic mediators, including TGF-β1, which promote the progression of liver fibrosis.¹⁵⁷ In brief, the recruitment and mobilization of immune cells, the accumulation of inflammation, and the activation of hepatic stellate cells and hepatocytes contributes to the development of liver fibrosis and the aging process (Fig. 3).

During the aging process, the skin accumulates senescent cells that, despite their inability to divide, remain metabolically active. These senescent cells exhibit an altered secretome known as SASP, which significantly disrupts the skin microenvironment.¹⁵⁸ For instance, senescent dermal fibroblasts secrete a higher amount of extracellular vesicles (EV) compared to their non-senescent counterparts. This increased EV secretion hampers the normal differentiation of keratin-forming cells and compromises the skin’s barrier function. In addition, it triggers the elevated production of the pro-inflammatory cytokine IL-6.¹⁵⁹

Furthermore, skin aging can occur due to age-related factors or exposure to environmental stressors like ultraviolet radiation.

The process of skin aging can also have systemic effects on the overall aging process of the body, primarily through the activation of SASP.¹⁶⁰ The presence of p16-positive cells in the skin, which is a marker of cellular senescence, has been found to be associated with markers of CD4+ T-cell senescence and biological age.^161,162 Notably, the microbiome of skin has been found to predict a person’s actual age accurately.^160,163 While the numbers of CD4 T cells remain consistent with age, the levels of CD8 T cells are higher in older skin compared to younger skin.¹⁶⁴ The ratio of cutaneous CD4 T cells to CD8 T cells is greater in aged individuals, but the number of CD4 T cells is not elevated in aged skin.¹⁶⁵ Moreover, aged skin exhibits increased numbers of regulatory T cells (Tregs)¹⁶⁶ and elevated expression of the immunosuppressive receptor PD-1,^165,167 which may contribute to weakened adaptive immunity. These changes could be a response to an inflammatory state exacerbated by impaired epidermal barrier function or fibroblast senescence, further promoting an inflammatory microenvironment (Fig. 3).

The aging process brings about notable transformations in the structure and function of the lungs, including a decline in mucociliary clearance and heightened vulnerability to pulmonary infections.^168,169 These alterations contribute to the onset and progression of various lung diseases like idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD).¹⁶⁹ Several cell types within the lungs undergo modifications during aging, including respiratory epithelial cells, lung progenitor cells, lung immune cells, and lung interstitial cells.¹⁶⁹ Among these cell types, alveolar epithelial type II cells (AT2) are a significant population responsible for regenerating the alveolar parenchyma. However, as these cells age, the airway epithelium experiences quantitative and qualitative defects. The number of basal and spherical cells decreases, while the count of AT2 cells remains unchanged but exhibits impairments in self-renewal and differentiation capacity.^170–172 Moreover, age-related changes in the lung environment, such as alterations in extracellular matrix (ECM) components, tissue and circulating cytokines, SASP, and structural abnormalities, can lead to abnormal intercellular communication mechanisms. This is evident through distorted interactions with microbial pathogens and a shift in innate and adaptive immunity towards increased inflammation, disrupted adaptive immune responses, and impaired immune surveillance (Fig. 3).¹⁷³

The increased susceptibility of elderly individuals to lung diseases can be ascribed to age-related changes in immunity and anti-infection responses. The phagocytic capacity of pulmonary and alveolar macrophages diminishes with age, impairing the clearance of pathogens from the lungs.^51,174,175 Dendritic cells, neutrophils, and NK cells also experience age-related alterations in their numbers and functionality.¹⁶⁹ In addition, the aging process is linked with a decrease in CD4, and CD8 T cell populations. The decline in naive T cell numbers is accompanied by an increase in the number of memory T cells. The CD4 to CD8 lymphocyte ratio in bronchoalveolar lavage fluid tends to rise with age, indicating a reduction in the pool of naive T cells available for conversion into memory cells in response to new antigens. Furthermore, aging is associated with reduced CD4 and CD8 T cell responses, diminished TCR repertoire diversity, impaired Th cell differentiation, and reduced Th cell activity.¹⁷⁶ These age-related changes in T cell number and function can compromise influenza vaccination immunity and cytotoxicity against the virus. The adaptive immune response to antigens also declines with age, which explains why older individuals are more susceptible to environmental stimuli. Notably, immune cell disorganization associated with aging may contribute to the heightened severity of COVID-19 and chronic obstructive pulmonary disease (COPD) in the elderly.^169,177

To recapitulate, age-related changes in T cell-mediated adaptive immune responses enhance vulnerability to infectious agents and result in severe diseases.

Age-related perturbations in the gut microbiome have emerged as crucial factors contributing to age-related pathological conditions, including chronic inflammation,¹⁷⁸ neurodegeneration,¹⁷⁹ cognitive decline,¹⁸⁰ and type 1 and type 2 diabetes.¹⁸¹ The gut microbiota comprises probiotic, commensal, and pathogenic bacteria, and the imbalance between intestinal flora and aging mutually influences and exacerbates each other. Older adults (>65 years) exhibit reduced microbiota diversity compared to adults, along with greater inter-individual variation in microbiota composition.¹⁸² This is characterized by diminished populations of beneficial bacteria such as Bifidobacterium, Bacillus, E. coli, Clostridium XIV, Blautia coccoides-Eubacterium rectal, and Bacteroidetes, and increased presence of Enterobacteriaceae.¹⁸³ However, it’s important to note that data regarding age-related changes in microbiome composition can vary among populations.

Age-related alterations in the intestinal microbiota, particularly due to prolonged immune system stimulation, can contribute to the accumulation of inflammation and a decline in immune system function known as immunosenescence.¹⁸⁴ Interestingly, two previous studies have indicated that changes in the relative abundance of gut microbiota are more likely to be influenced by inflammation rather than age, with TNF playing a significant role.^185,186 This suggests that the changes in gut microbiota are more closely related with inflammatory processes rather than solely being a consequence of aging (Fig. 3).^187,188

Oxidative stress leads to oxidative damage to biomolecules (especially DNA),²⁰¹ causing endogenous damage-associated molecular patterns (DAMPs) production and cytokine release in the organism.^202,203 Cytokines activate downstream signaling pathways of pattern recognition receptors,²⁰³ causing systemic chronic inflammatory responses in the body.²⁰⁴ Consequently, oxidative stress is recognized as a concurrent occurrence within the inflammatory process, amplifying the inflammatory response through oxidation. At the same time, inflammation promotes oxidation through inflammatory mediators.^205,206 Based on the close relationship among oxidative stress, inflammation, and aging, Dela Fuente et al. proposed the theory of aging by oxidation-inflammation (oxi-inflamm-aging)^207,208 and concluded that oxidative stress leads to inflammatory aging. Oxidative stress has been ensured as a crucial factor for cellular senescence through shortening telomere and causing DNA double-strand breaks.^209,210 Moreover, infections,²¹¹ environmental pollution,^212,213 and adverse lifestyle habits²¹⁴ can increase oxidative stress.

Recently, there has been an increasing focus on studying age-specific changes in the intestinal microbiome and its role in regulating inflammation. A healthy gut microbiome is essential for body metabolism, infection resistance, inflammation regulation, prevention of autoimmunity and cancer, and brain-gut axis regulation.²¹⁵ However, with age, there is a decrease in beneficial microorganisms^216,217 and an accumulation of potentially pro-inflammatory microorganisms in the gut,²¹⁸ leading to a change in microbial composition and a decrease in microbial diversity. Moreover, this phenomenon exists simultaneously in species such as Drosophila,²¹⁹ fish,²²⁰ mice,²²¹ rats,²²² and humans.²²³ The detailed gut microbiota changes with aging have been collected and discussed by Du et al.²²⁴ Recent studies have revealed that the transplantation of fecal matter from young donors into the gastrointestinal tract of middle-aged fish can effectively prolong lifespan and delay the onset of behavioral decline,²²⁰ and that fecal transplantation from young mice slows HSC senescence in the bone marrow.²²⁵ Lachnospiraceae and tryptophan-associated metabolites have emerged as key players in important biological processes, but the exact mechanism and involvement of other factors are not yet clear. Nonetheless, it is evident that the senescence-related remodeling of microorganisms mediates the accumulation of chronic inflammation, which is highly correlated with their metabolites and their induced immune responses.

As the body ages, tissues and cells gradually experience damage, leading to a decline in their abilities and functions. Aging cells may be more susceptible to damage from external stimuli, increasing the risk of necrosis. In addition, certain age-related diseases such as cardiovascular diseases and neurodegenerative disorders may be accompanied by cellular necrosis.

Necroptosis (previously named programmed necrosis) is a regulated form of inflammatory necrosis that occurs when apoptosis (the programmed cell death process) fails. Its identification questioned the conventional notion that necrosis is exclusively an inert process induced by overwhelming stress. Necroptosis is distinguished by the early disruption of plasma membrane integrity, release of intracellular contents, and enlargement of organelles. Apoptosis is generally regarded as non-immunogenic since the regulated dismantling of apoptotic cells restricts the liberation of DAMPs. However, necroptosis triggers inflammation through the massive release of DAMPs from the disintegrating cell.²³⁷

The contribution of DAMPs from dying cells in the RIPK1-RIPK3 inflammasome-dependent pathway of cytokine production varies. Upregulation of RIPK3 has been observed in hepatocytes, suggesting that RIPK3-dependent necroptosis may play a role in inflammation and hepatocyte death. Immunostaining with antibodies recognizing phosphorylated mixed lineage kinase domain-like protein (MLKL) serves as a specific marker of necroptosis.^238,239 In addition, immunostaining using antibodies that target phosphorylated MLKL has been identified as a specific marker for necroptosis.²⁴⁰

Focus on liver, its aging has been linked to an increase in necroptosis, and this process has been found to contribute to chronic liver inflammation, which in turn appears to be involved in the development of liver fibrosis.²⁴¹ On the other hand, in the livers of old mice (specifically, those aged 18 months and older), there was a significant upregulation of phosphorylated MLKL and MLKL oligomers, which are markers associated with necroptosis. In addition, the phosphorylation of RIPK3 and RIPK1, two key proteins involved in necroptosis signaling, was also significantly increased in the livers of old mice compared to young mice. In comparison to young mice, hepatocytes and liver macrophages from old mice had higher levels of necroptosis markers and higher expression of pro-inflammatory cytokines M1 macrophage markers, pro-inflammatory cytokines (TNF-α, IL-1, and IL-6), and fibrosis markers. In the livers of old mice, short-term treatment with the necroptosis inhibitor necrostatin-1s (Nec-1s) reduced necroptosis, M1 macrophage markers, cellular senescence, fibrosis, and pro-inflammatory cytokines.²⁴¹ Importantly, nerve injury-induced protein 1 (Ninjurin1/Ninj 1) plays a crucial role in facilitating the ultimate breach of the plasma membrane that takes place in necroptosis, pyroptosis, and secondary necrosis. Secondary necrosis refers to the phenomenon where cells undergoing apoptosis fail to be engulfed by adjacent phagocytes (Fig. 4).²⁴² These findings suggest an age-associated dysregulation of necroptosis signaling, indicating a potential role for necroptosis in aging and related pathologies.

As the body ages, tissues and cells gradually experience damage, leading to a decline in their abilities and functions. Similar to other forms of inflammatory cell death, aging cells may also be more susceptible to damage from external stimuli that can trigger pyroptosis, increasing the risk of pyroptosis occurrence. In addition, age-related diseases such as neurodegenerative disorders and cardiovascular diseases may be accompanied by cellular pyroptosis. Specifically, pyroptosis may play a specific role in aging. Inflammation and cell death have important regulatory roles in aging, and pyroptosis, as an inflammatory form of cell death, may contribute to the inflammatory response and cellular dysregulation in the aging process.²⁴³ Moreover, pyroptosis may be involved in the development and progression of age-related diseases.

In the non-classical pathway, human-derived caspase-4, 5, and murine-derived caspase-11 can be activated upon direct contact with bacterial lipopolysaccharide (LPS). LPS cleaves gasdermin D (GSDMD), which indirectly activates caspase-1, leading to pyroptosis. Alternatively, caspase-1 can be recruited and activated by inflammatory vesicles that detect danger signals. Activated caspase-1 cleaves and activates inflammatory factors, which in turn cleave the N-terminal sequence of GSDMD. This results in the binding of GSDMD to the membrane and the generation of membrane pores, ultimately leading to pyroptosis (Fig. 4). Evidence of pyroptosis, coincided with elevated levels of IL-1 and IL-18, and inflammasome activation, has been illustrated in various conditions such as atherosclerosis, neurodegenerative diseases, cancer, and chimeric antigen receptor (CAR)-T therapy.^244–247

As the body ages, changes in iron levels and iron metabolism may occur. The accumulation of iron in cells during the aging process may be associated with the development of age-related diseases such as neurodegenerative diseases²⁴⁸ and cardiovascular diseases.²⁴⁹ This iron accumulation can lead to increased generation of ROS within cells, thereby triggering iron-dependent cell death known as ferroptosis. The main drivers of ferroptosis are the inactivation of the lipid repair enzyme glutathione peroxidase 4 (GPX4) and the induction of ROS, particularly lipid ROS. GPX4 plays a cytoprotective role by reducing cellular lipid hydroperoxide levels, which are associated with inflammation. In cancer cells, certain inflammatory cytokines such as TNF, PGE2, IL-1, and IL-6 have been shown to directly affect GPX4 levels and activity. Treatment with TNF, for example, downregulates GPX4, leading to ferroptosis.

Ferroptosis is an inflammatory form of cell death that is distinct from apoptosis. It is characterized by iron-dependent lipid peroxidation and can contribute to various pathological processes, including neurodegenerative diseases, inflammatory diseases, autoimmune diseases, and cancer. The inactivation of the lipid repair enzyme glutathione peroxidase 4 (GPX4) and the induction of ROS, particularly lipid ROS, are the main causes of iron death. GPX4 has been shown to have a cytoprotective effect by lowering the levels of cellular lipid hydroperoxides.²⁵⁰ Several pro-inflammatory cytokines, including TNF, PGE2, IL-1, and IL-6, have been demonstrated to exert a direct influence on the levels and function of GPX4 within cancer cells;²⁵¹ for example, TNF treatment causes GPX4 downregulation that can lead to ferroptosis.²⁵² High mobility group box 1 (HMGB1), a DAMP, has been implicated in inflammation and its pathogenesis.^253,254 In the context of ferroptosis, inhibiting HMGB1 release has been shown to limit the inflammatory response during cell death. Anti-HMGB1 antibodies have demonstrated their ability to reduce the inflammatory response in macrophages induced by ferroptotic cells.²⁵² Ferroptosis inhibitors have shown promise in the treatment of certain diseases due to their anti-inflammatory properties. In an oxalate-induced mouse model of AKI, evidence of inflammation was observed, and the ferroptosis inhibitor Ferrostatin-1 successfully inhibited neutrophil infiltration and the expression of pro-inflammatory cytokines such as CXCL-2 and IL-6.^255,256 Conversely, the ferroptosis inducer RSL-3 significantly increased the protein levels of pro-inflammatory cytokines like TNF, IL-1, and IL-6, exacerbating hepatosteatosis, lobular inflammation, and apoptosis (Fig. 4).²⁵⁷ The results indicate a possible interplay between ferroptosis and inflammation.

In addition, certain biological processes and molecular mechanisms associated with aging may be related to ferroptosis. During the aging process, alterations in cellular function and metabolism can increase the sensitivity of cells to external stimuli, including sensitivity to ferroptosis. Recent studies have reported the involvement of ferroptosis as a mechanism that promotes skeletal muscle aging.²⁵⁸ With skeletal muscle aging, there is a decreased expression of Tfr1 and an increased expression of Slc39a14, which is enriched on the cell membrane surface of aging mouse skeletal muscle cells. This increase in Slc39a14 leads to enhanced non-transferrin-bound iron uptake, resulting in the accumulation of free iron ions within skeletal muscle and the occurrence of ferroptosis.²⁵⁸

Lastly, the interaction between ferroptosis and aging may be bidirectional. On one hand, ferroptosis may play a role in the development of certain age-related diseases, accelerating tissue and cellular aging processes. On the other hand, the cellular functional and metabolic changes that occur during the aging process may increase the sensitivity of cells to ferroptosis, further promoting disease progression.

NETosis is a special form of cell death closely associated with inflammation and immune response. It is a cell death program executed by neutrophils and is characterized by the release of net-like structures (neutrophil extracellular traps/ NETs). These structures are composed of DNA, histones, and microbial toxins, among other components, and serve the purpose of capturing and killing microorganisms.²²⁸ NETosis plays a significant role in various pathologies, including COVID-19, Kawasaki syndrome, and rheumatoid arthritis (RA). Excessive NETosis has been implicated in the development of cytokine storms and thrombosis.²⁵⁹ In COVID-19, NETosis can be caused by virus-infected epithelial and endothelial cells, thereby activating inflammatory cytokines and platelets. Excessive NETosis, accompanied by increased circulating free DNA and Neutrophil Extracellular (NE)-DNA complexes, is also found in acute Kawasaki syndrome, a vasculitis occurring in children.²⁶⁰

In RA, the disease pathology is characterized by the accumulation of DNA-MPO complexes and the presence of antibodies targeting guanylated histones (NETosis markers).^261,262 In myocarditis, NETosis probably promotes PMN trafficking via MK and substantially contributes to cardiac inflammation.²⁶³ In systemic lupus erythematosus, NETosis activates the plasmacytoid and induces the production of IFN-α and ROS, which contribute the following further inflammation.^264–267 On the other hand, the anti-microbial effects of NETosis have been observed to slow down the spread of pathogens in infected lesions. NETs in staphylococcal skin infections inhibit the penetration of pathogens into the bloodstream.²⁶⁸ Knockout of the PAD4 gene in mice prevents NET formation and leads to more severe necrotizing fasciitis caused by streptococcus pyogenes. In summary, NETosis could cause inflammation or conversely slow the onset of age-related diseases (Fig. 4).

The ability of neutrophils to undergo NETosis may be affected by aging.²⁶⁹ Senescent neutrophils exhibit several distinct characteristics during NETosis, including a reduced capacity to release NETs, instability in the quality of formed NETs, and decreased activity of DNA degrading enzymes (DNases) within NETs. These age-related changes can result in impaired functionality of aging neutrophils, leading to deficiencies in their ability to effectively combat microbial infections and regulate inflammatory responses.

Moreover, aging is often accompanied by a phenomenon called inflammaging, which refers to a chronic low-grade inflammatory state. Inflammaging can further contribute to the occurrence of NETosis. This persistent inflammatory condition enhances the activity of inflammatory cells, including neutrophils, thereby increasing the likelihood of NETosis. A previous study found that aged mice exhibited an increased propensity for NETosis compared to younger mice. This heightened NETosis activity was associated with the activation of peptidylarginine deiminase 4 (PAD4), an enzyme involved in the formation of NETs. The excessive formation of NETs, in turn, was implicated in the development of age-related organ fibrosis.²⁷⁰

In conclusion, the process of aging can adversely affect the ability of neutrophils to undergo NETosis. Senescent neutrophils may experience limitations in NET release, compromised stability of formed NETs, and reduced DNase activity within NETs. In addition, the presence of inflammaging, the age-associated inflammatory state, can intensify the occurrence of NETosis by stimulating inflammatory cell activity. However, further research is necessary to fully comprehend the intricate mechanisms and interactions between aging and NETosis.

PANoptosis, is a united modality of inflammatory programmed cell death, accompanied by markers of apoptosis, necrosis, and pyroptosis pathways.^271–275 Influenza A virus was first discovered to cause PANoptosis, followed by many other infections, of bacterial, fungal, and viral origin.²⁷⁵

PANoptosis is involved in the occurrence of cytokine storms (CS) characterized by excessive cytokine production.²⁷⁶ The combination of TNF-α and IFN-γ activates the JAK/STAT1/IRF1 signaling pathway, leading to the production of nitric oxide (NO). This NO release triggers PANoptosis through the involvement of GSDME (pyroptotic), CASP8/3/7 (apoptotic), and pMLKL (necroptotic) pathways. In vivo, blocking CS by giving mice anti-TNF-α and anti-IFN-γ antibodies prevents death from SARS-CoV-2 infection, hemophagocytic lymph histiocytosis, and LPS shock (sepsis).²⁷⁶ This highlights the critical role of TNF-α and IFN-γ released from PANoptosis in driving cytokine storms during infections and inflammatory conditions.²⁷⁵ Cytokines or PAMPs trigger the assembly of a multiprotein complex called the PANoptosome. This complex includes various molecules necessary for the activation of downstream programmed cell death (PCD) effectors such as GSDMD, GSDME, CASP3/7, and MLKL (Fig. 4). No direct evidence yet links PANoptosis to aging. However, aging can affect cell responses to inflammation and cell death. Further research is needed to explore the potential connection between aging and PANoptosis, shedding light on its impact on immune and cell death mechanisms.

In summary, DAMPs from senescent, damaged, and dying cells trigger various cell death modalities, including necrosis, pyroptosis, necroptosis, PANoptosis, NETosis, and ferroptosis (Fig. 4). DAMPs bind to specific receptors, initiating inflammation and orchestrating a coordinated response involving immune cells. This response includes the recruitment of neutrophils and monocytes, which play crucial roles in tissue repair and healing processes. When leukocytes fail to clear immunostimulatory molecules, inflammation persists, which further causes cancer and aging. Endogenous DAMPs can activate PRRs and non-PRR transmembrane proteins, resulting in massive inflammation, cellular senescence, diseases of the organs, and aging.^277–279 The shift of understanding in aging mechanisms, from the cellular and organ level to the molecular level, aid in identifying novel targets for anti-inflammatory therapies and effective anti-aging interventions.

Replicative senescence is closely associated with the shortening of telomeres. In the laboratory aging of human diploid fibroblasts (HDFs), as the cells undergo a certain number of population doublings, telomeres become shorter, leading to cell cycle arrest, reduced cell saturation density, and increased cell surface and volume.²⁸⁰ Hydrogen peroxide is commonly used to induce stress-induced premature senescence (SIPS), which shares similarities with replicative senescence. When young HDFs are exposed to prolonged low doses of hydrogen peroxide, they enter irreversible G1 cell cycle arrest and exhibit senescence-associated beta-galactosidase activity. These cellular senescence markers are accompanied by increased expression of p21, gadd45, and enhanced p53 binding activity.²⁸¹ In addition, DNA repair capability decreases, and telomere shortening accelerates. Hydrogen peroxide-induced senescence also triggers inflammation, characterized by the upregulation of pro-inflammatory cytokines such as IL-6, TNF-α, and MCP-1.^282,283

Oncogene-induced senescence (OIS) is observed following the activation of various oncogenes such as B-RAFV600E or H-RAS G12V, as well as the loss of tumor suppressor proteins like PTEN or NF-1, in different cell types.²⁸⁴ OIS is often associated with DNA replication stress and hyper-replication. It is characterized by the upregulation of CDK inhibitors, including p15INK4B, p16INK4A, p21CIP1, and an increased senescence-associated β-galactosidase (SA-β-Gal) activity.^285,286

Kuilman et al. discovered that OIS is specifically associated with the induction of an inflammatory gene expression profile, which includes the expression of various genes such as the pleiotropic cytokines IL-6, IL-1α, IL-1β, and IL-8. In addition, the transcription factor C/EBPbeta collaborates with IL-6 to enhance initiation of the pro-inflammatory cascade, as demonstrated in cells carrying B-RAFV600E and H-RAS G12V mutations.²⁸⁷

In physically induced models, the inhalation of ozone is a frequently employed technique for inducing premature senescence. When male BALB/c mice are exposed to ozone at a concentration of 1.2 mg/m³ for 10 h per day, they exhibit thymic atrophy and an elevated level of oxidative damage. Subsequently, there is a decline in the immune function of the mice, which is closely associated with oxidative stress. This decline is characterized by an increase in IL-6 levels, reduced splenocyte proliferation, decreased production of IL-2, diminished natural killer (NK) cell activity, and a weakened antigen-specific response.²⁹⁸

For chemical induction models, the D-galactose-induced aging model is widely preferred in chemical induction studies due to its convenience, higher survival rate, and minimal side effects. This model effectively mimics aging in vivo by inducing changes in various tissues and organs. When mice are treated with certain concentrations of D-galactose, they exhibit increased levels of ROS and inflammatory cytokines such as NOS-2, IL-1β, IL-6, TNF-α, and NF-κB. These alterations contribute to the aging-like phenotype observed in this model. Moreover, researchers have developed combined methods involving D-galactose to induce premature aging. For instance, the D-galactose and AlCl3 model and the D-galactose and NaNO2 model are commonly used. These combinations enhance the aging effects and provide additional insights into the mechanisms underlying accelerated aging.²⁹⁹

Senescence-accelerated mouse/prone (SAMP) strains, specifically the SAMP1/Yit substrain, have been recognized as valuable models for studying the genetic aspects of aging. In particular, the SAMP1/Yit mice have been used as a model for Crohn’s disease, which is a chronic and recurring inflammatory bowel disease. The mice exhibit both acute and chronic inflammation in the ileum and cecum, displaying a non-continuous pattern of inflammation.³⁰⁰ On the other hand, the SAMP8 mouse strain is considered an excellent model for investigating Alzheimer’s disease (AD), a cognitive decline disorder that predominantly affects the elderly. The SAMP8 mice exhibit reduced expression and lower activity of anti-aging factors including silent information regulator type (sirtuin/Sirt), Forkhead box class O (FoxOs), and Klothos. These factors play crucial roles in the aging process.³⁰¹

Aging models with apparent inflammation phenotypes include Nfkb1 deficient mice (Nfkb1−/−). Nfkb1−/− mice have shortened lifespan, kyphosis, osteoporosis, tissue inflammation, and gliosis of the central nervous system.³⁰² Interestingly, the accumulation of senescent cells with telomere-dysfunction in Nfkb1−/− tissues can be effectively hindered through the implementation of anti-inflammatory or antioxidant treatment in mice.³⁰³ This noteworthy observation underscores the promising utilization of these mice as valuable models for investigating age-related interventions. Details of other in vivo aging models are also shown in Table 2.

Premature aging models can be categorized into two main types: progeroid syndrome models and other models exhibiting premature aging phenotypes. Progeroid syndromes are exceptionally uncommon human disorders characterized by early onset aging and a reduced lifespan. These syndromes include laminopathies, such as Hutchinson-Gilford progeria syndrome (HGPS), which disrupt the balance of the nuclear envelope, as well as conditions that affect telomere length and DNA repair mechanisms, like Werner syndrome and Cockayne syndrome.

The identification of specific mutations causing these syndromes has enabled the development of mouse models that simulate premature aging.³⁰⁴ For instance, mouse models of HGPS have been created by modifying the Lmna gene or its processing enzyme. Among these models, the LmnaG609G mouse model (featuring the mutation 1827C > T; Gly609Gly) closely resembles human phenotypes.³⁰⁵ This includes heightened inflammation markers like IL-6, caspase 1, and Nlrp3, increased oxidative stress, persistent DNA damage, and cell cycle arrest.^306,307 In addition, two independently generated mouse models deficient in the Zmpste24 gene (Zmpste24 − /− mice) also exhibit elevated expression of caspase 1 and Nlrp3, severe growth retardation, dilated cardiomyopathy, muscular dystrophy, lipodystrophy and premature death.^307,308

Many animals naturally have long lifespans. Decoding the underlying mechanisms for provide insights for developing anti-aging strategies. One such remarkable example is the naked mole rat, a socially oriented mammal that dwells in subterranean burrows. This extraordinary creature holds the esteemed distinction of being the longest-living rodent, with a maximum lifespan that surpasses an astonishing 30 years.³⁰⁹ With aging, naked mole rats will not lose their physiological functions, and their mortality will not increase significantly.³¹⁰ As a successful aging specifications, nude mole also achieved resistance to tumor through a variety of methods, such as efficient DNA damage repair, synthesis of unique anti-inflammatory high molecular weight hyaluronan.³¹¹ The special longevity mechanism of naked mole makes it a good animal model for longevity research and provides a blueprint for exploring the strategies of delaying human aging.

Planaria is a type of flatworms. For planarians, small fragments of almost any tissue can be regenerated into an entire individual. This has led it to be considered immortal. The shortening of telomeres during cell division is a major obstacle to infinite cell division, and planarians overcome this problem by upregulating telomerase expression during regeneration.³¹² On the other hand, planarians also achieve resistance to tumors through a variety of means, such as efficient DNA repair mechanisms.³¹³ All of these mechanisms provide enlightenment for us to study human aging.

As an amphibian, the salamander is also very long-lived. Their limbs and many of their organs are capable of regenerating. Axolotls age very slowly, and their phenotypes are less pronounced. Its physiological mechanism to clear senescent cells in time prevents senescent cells from accumulating in its body, which may explain its slow aging.³¹⁴ The salamander’s large genome may have provided lines of defense for potentially harmful mutations, such as regulators. This also laid the foundation for the prevention of tumors.³¹⁵

Turtles are a typical group of long-lived animals. The protection of the carapace keeps them from predators. During diving, turtles are chronically deprived of oxygen, which allows them to upregulate glutathione-related enzymes to clear away ROS.³¹⁶ Inhibition of ROS and unique telomerase and perhaps DNA repair mechanisms make turtles’ longevity possible.³¹⁷