The efferocytosis process in aging: Supporting evidence, mechanisms, and therapeutic prospects for age-related diseases

Graphical abstract

Highlights

• •Efferocytosis, immunological resolution, ageing biomarker, inflammatory secretion, and autophagy functions are all affected by phagocyte ageing.
• •The deficiencies in efferocytosis promote chronic inflammation and ageing, eventually leading to various diseases.
• •Hallmarks of ageing, such as mitochondrial dysfunction, cellular senescence, altered intercellular communication, and chronic inflammation, interact with efferocytosis in aging-related diseases.
• •The concurrent implementation of strategies aimed at augmenting efferocytic mechanisms and anti-ageing treatments can serve as drug targets for extending the duration of a healthy lifespan.
• •Anti-ageing therapies will be combined with other well-established methods for treating age-related diseases.

Abstract

Background

Aging is characterized by an ongoing struggle between the buildup of damage caused by a combination of external and internal factors. Aging has different effects on phagocytes, including impaired efferocytosis. A deficiency in efferocytosis can cause chronic inflammation, aging, and several other clinical disorders.

Aim of review

Our review underscores the possible feasibility and extensive scope of employing dual targets in various age-related diseases to reduce the occurrence and progression of age-related diseases, ultimately fostering healthy aging and increasing lifespan.

Key scientific concepts of review

Hence, the concurrent implementation of strategies aimed at augmenting efferocytic mechanisms and anti-aging treatments has the potential to serve as a potent intervention for extending the duration of a healthy lifespan. In this review, we comprehensively discuss the concept and physiological effects of efferocytosis. Subsequently, we investigated the association between efferocytosis and the hallmarks of aging. Finally, we discuss growing evidence regarding therapeutic interventions for age-related disorders, focusing on the physiological processes of aging and efferocytosis.

Introduction

Over the past 200 years, human life expectancy has increased substantially, leading to a notable increase in the percentage of older people in demographic and increasingly aging societies. It is anticipated that this upward trend in life expectancy will persist, culminating in developed world citizens having a life expectancy of over 85 years by 2030 [1]. The deterioration in physiological conditions correlates with advancing age, which subsequently contributes to an increase in age-related diseases. Therefore, a substantial correlation exists between the extension of life expectancy and the increased incidence of various diseases, including diabetes, metabolic disorders, cardiovascular disorders, cancer, and neurodegenerative disorders [2], [3]. The mitigation of adverse effects associated with advanced age and extension of the human health span have thus emerged as significant objectives in the global field of aging and anti-aging research [4], [5], [6]. On the one hand, researchers are currently gaining insights into the physiological processes that contribute to aging and are striving to identify biomarkers associated with aging. On the other hand, the advancement of modern medicine and material chemistry have significantly increased life expectancy and improved human health [7], [8], [9], [10].

Aging has been described as an ongoing struggle between the accumulation of harm caused by endogenous and exogenous processes [11]. It is characterized by a decline in the function of tissues and cells, as well as a substantial increase in the likelihood of developing a range of diseases associated with aging [12]. In 2023, Lopez-Otin et al. provided a concise overview of recent advancements in the study of aging hallmarks over the past decade based on a review in 2013. Additionally, the author delineates and elaborates on 12 significant attributes of aging. This comprehensive analysis serves as a foundation for investigating the aging process and devising novel approaches to counteract it [11].

Despite the large number of apoptotic cells (ACs) produced regularly, they are rarely observed in vivo. Efferocytosis, the phagocytic clearance of ACs, is associated with cell death [13]. In most tissues, tissue-resident professional phagocytes, such as macrophages and dendritic cells, or neighboring non-professional phagocytes, such as epithelial cells and fibroblasts, undergo efferocytosis. Efferocytosis has the following benefits [14], [15]: the process rapidly eliminates ACs, thereby preventing their transition into a state of secondary necrosis and subsequent release of potentially harmful intracellular contents that can induce inflammation. Additionally, it induces a shift in phagocytes towards a more “pro-resolution” phenotype, characterized by the enhanced release of anti-inflammatory agents and initiation of transcriptional processes linked to tissue restoration. This process removes billions of dead cells without inflammation or immunogenicity. With advancing age, the phagocytosis of ACs decreases. A comparison was made between aged (24-month-old) and younger (2-month-old) mice in a mouse paradigm in which the efferocytosis of intraperitoneally injected apoptotic Jurkat cells was impaired by peritoneal macrophages in vivo [16]. Arnardottir et al. conducted a comparable investigation using in vitro murine bone marrow-derived macrophages co-cultured with human peripheral blood apoptotic neutrophils. Furthermore, the clearance of neutrophils by geriatric (20-month-old) mice was less efficient than that by macrophages from 2-month-old mice. This factor has been hypothesized to play a role in the general deterioration of inflammation resolution with increasing age [17].

In summary, efferocytosis, immunological resolution, aging-related biomarker expression, inflammatory cytokine secretion, and autophagy are all affected by phagocytic aging. Efferocytosis deficiencies can promote chronic inflammation and aging, eventually leading to various diseases. Nevertheless, an in-depth and comprehensive understanding of the recent achievements in this domain is lacking. In this review, we comprehensively discuss the association between efferocytosis and hallmarks of aging. Although this overview does not encompass all aspects of aging, our attention will mostly be on representative factors, including mitochondrial dysfunction, cellular senescence, altered intercellular communication, and chronic inflammation. This review highlights the feasibility and extensive scope of employing dual targets for various age-related diseases. In doing so, we aimed to reduce the occurrence and progression of age-related diseases, ultimately fostering the promotion of healthy aging and an increased lifespan.

The process of efferocytosis

The concept and physiological effect of efferocytosis

Efferocytosis is a process of phagocytosis specifically pertaining to ACs, as it entails the engulfment of extracellular materials. Phagocytosis refers to the biological mechanism by which phagocytes engulf extracellular particles exceeding a diameter of 0.5 µm. Efferocytosis is carried out by professional phagocytes, such as macrophages, dendritic cells, and other nonprofessional cells, to engulf ACs [18]. Maintaining physiological equilibrium throughout the body is of paramount importance.

Efferocytosis encompasses a range of functions, as outlined in previous research [19]. Initially, phagocytes expeditiously and securely eliminate the membrane structure of the deceased cell before its disintegration and subsequent release into adjacent tissue. This process serves to safeguard the surrounding tissue against the deleterious effects induced by toxic enzymes, oxidants, and intracellular components, such as protease antibodies and caspases within ACs. Additionally, efferocytosis can generate a significant number of biological factors, including vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), which are hypothesized to facilitate cellular regeneration within the body. In addition, efferocytosis elicits subsequent intracellular signalling cascades, including anti-inflammatory, anti-protease, and growth-promoting actions. The coordination of many processes is essential for the proper differentiation of ACs from healthy cells and their subsequent elimination through efferocytosis. When these deceased cells remain uninterrupted, they undergo a disruptive process, resulting in harm to the organism, triggering an inflammatory reaction, and perhaps giving rise to a range of ailments. Numerous human diseases, such as atherosclerosis, cancer, systemic lupus erythematosus, diabetes, obesity, rheumatoid arthritis, and aging, have been observed to exhibit associations with deficiencies or alterations in the processes of efferocytosis. Efferocytosis is a multistep physiological process and enhancing any one of these steps can promote efferocytosis while suppressing tissue inflammation [20]. Next, we outline the key signalling molecules and therapeutic strategies for the different steps.

Mechanics of efferocytosis

Efferocytosis is subject to various conditions and involves multiple processes. Among these steps is a preliminary phase referred to as the “smell phase,” in which phagocytes sense the existence of ACs and subsequently find them. Subsequently, the “eating phase” ensues, wherein ligands present on ACs selectively bind to phagocytic receptors located on phagocytes, facilitating the targeted recognition and engulfment of the dying cells. The third phase, commonly referred to as the “digestion phase,” involves the process of engulfing deceased cells and assimilating their internal contents (Fig. 1).

Fig. 1.

The mechanisms of efferocytosis. The smell phase: “find-me” signals are released by apoptotic cells to attract macrophages to the site of death. The eating phase: Phosphatidylserine (PS) is the predominant “eat-me” signal utilized by macrophages to identify apoptotic cells. There are two distinct mechanisms by which this process takes place: direct binding of PS to macrophage surface receptors (e.g., Stabilin2, Tim4, and BAI-1), and indirect binding to PS in the presence of bridging molecules. Additionally, calreticulin functions as an eat-me signal by binding directly to LRP1 on the macrophage surface. The digestion phase: phagocytosis follows the canonical process, which involves the gradual transformation of the early phagosome into the late phagosome, with the latter undergoing fusion with the lysosomal compartment so as to form phagolysosomes. The supplemental pathway comprises the phagocytosis mechanism that is facilitated by light chain 3 (LC3) of microtubule-associated protein 1A/1B. The phagocytosed cell corpses are decomposed by proteases and other enzymes contained within the newly formed phagolysosome; the digested products are either excreted from the body or reused by macrophages. Created with BioRender.com.

Efferocytotic processes must be perfectly coordinated for ACs to be distinguished from healthy cells that function normally and for ACs to be eliminated. “Efferocytosis escape” refers to the accumulation of dead cells within the lesion and the subsequent progression and expansion of the lesion if any of the preceding phases is compromised. In summary, cell fate is determined by the equilibrium between particular “eat me” and “don't eat me” molecules [21]. The molecules involved in the different stages of efferocytosis are summarized in Table 1.

Table 1.

The signals involved in the different phases of efferocytosis.

Phase	Signal	Receptor	Disease
The smell phase
Find me	ATP/UTP	P2Y2[25]	Allergic airway inflammation [229]
	LPC	G2A [230]	Autoimmunity [231],colitis [232]
	S1P	S1PRS[233]	Systemic lupus erythematosus [234]
	CX3CL1	CX3CR1 [24]	Atherosclerosis [235]
	TSP1	CD36 [236]	Lung injury [237], obesity [238]
The eating and digestion phase
Eat me	PS	BAI-1 [33]	Learning and memory [239]
		TIM4 [34]	Ischemia reperfusion injury [240]
		Stabilin1/2 [36]	Glomerular fibrosis [241]
		MerTK [242]	Systemic lupus erythematosus [243], allergic airway inflammation [244]
		CD36 [32]	Lung injury [237], obesity [238]
	Calreticulin	LRP1 [36]	Atherosclerosis [245]
Don’t eat me	CD47	SIPα [20]	Glioblastoma [246]
	CD24	Siglec10 [20]	Non-small cell lung cancer [247]

The smell phase

“Find-me” signals are released by ACs to attract macrophages to the site of death. These signals include nucleotides, such as adenosine triphosphate (ATP)/ UTP, sphingosine 1-phosphate (S1P), and chemokines, such as CX3CL1. These signals possess the ability to attract phagocytes and prime them for engagement in defensive actions, such as augmenting the expression of phagocytic receptors and digesting processes. Notably, find-me signals serve the dual purpose of guiding phagocytes to the precise site of ACs and augmenting their capacity to effectively eliminate these cells, while also eliciting anti-inflammatory responses [22]. Fractalkine is a protein that is localized inside the cellular membrane and is secreted by apoptotic B cells and neurons. Apoptosis is a cellular process characterized by the formation of membrane blebs that are subsequently released as microparticles (MPs). The production of fractalkine-associated MPs from ACs stimulates monocyte chemotaxis towards apoptotic B cells [23], [24]. Research has demonstrated that CX3CR1 receptors present on phagocytes can detect CX3CL1, thereby triggering the migration of phagocytes into ACs. Nevertheless, the tissue distribution of CX3CL1 is constrained by its exclusive expression in a limited number of cell types. Nucleotides, including ATP and UTP, are also used as find-me signals during efferocytosis. Phagocyte migration is induced by the release of ATP via the purine receptor P2Y. Nucleotides can modulate the immune responses of macrophages. For instance, the conversion of ATP or AMP from ACs to adenosine inhibits inflammation via adenosine receptors and increases the expression of pro-resolution and anti-inflammatory genes [25]. The integrity of the lipid membranes in non-ACs is compromised, resulting in the release of inflammatory signals into the extracellular environment. Infected cells generate pathogen-associated molecular patterns, which interact with pattern recognition receptors located on or inside phagocytes. This interaction potentially influences the macrophage activity and immune activation. Furthermore, non-ACs are capable of releasing damage-associated molecular patterns, which have the ability to initiate inflammatory reactions and function as chemokines for macrophages [26].

The eating phase

Following phagocyte migration towards ACs, surface receptors bind to the “Eat me” signal emitted by the ACs to identify ACs. AC surfaces undergo various modifications, including alterations in charge and glycosylation conformation, as well as epitope changes in the intracellular adhesion molecule-1 (ICAM-1) and calreticulin, which are both induced by exposure to phosphatidylserine (PS) to the outer membrane lobules [15], [27]. PS is the most effective and conserved signalling molecule during evolution. One common feature observed in all forms of cell death is the perturbation of phospholipid asymmetry inside the plasma membrane, which exposes PS externally to the cell and facilitates phagocytosis. PS, located in the interior of the cell, undergoes an early transition from the interior to the surface during apoptosis. PS receptors on the surface of phagocytes can recognize PS either directly or indirectly. By binding to PS, PS receptor initiates intracellular signalling via the complex to prepare apoptotic cells for “eating” [28], [29], [30], [31]. This process operates via two distinct mechanisms: direct binding and indirect binding via bridging molecules. The first mechanism involves the direct binding of PS to certain macrophage surface receptors, including brain-specific angiogenesis inhibitor-1 (BAI-1), T cell immunoglobulin mucin 4 (Tim4), and Stabilin2 (Stab2). The second mechanism involves indirect binding of PS, which is facilitated by bridging molecules. Calreticulin serves as an eat-me signal by directly interacting with the LRP1 receptors located on the surface of macrophages. PS directly interacts with phagocytic receptors, including BAI-1, Tim1-4 (Tim family), and Stab2. Soluble bridging molecules, including Gas6 and protein S, facilitate the binding of PS to tyrosine kinase Tyro3/Axl/Mer family receptors, the scavenger receptor SCARF1, integrin receptor family complexes with CD36, and transglutaminase-2 [32]. BAI-1 is responsible for the recruitment and activation of the ELMO1-DOCK180 complex, which initiates intracellular signalling, induces reorganization of the actin cytoskeleton, and facilitates efferocytosis [33] subsequent to its direct binding to PS via the thrombospondin repeat. The TIM receptor family refers to a group of type I cell surface glycoproteins that play a crucial role in facilitating phagocytosis, specifically in ACs [34] by binding to PS via the N-terminal IgV domain. TIM-3 facilitates cross-presentation of antigens associated with ACs [34]. In contrast, TIM-4-mediated phagocytosis can initiate light chain 3 (LC3)-associated phagocytosis (LAP), a distinct non-classical form of autophagy [34], [35]. Notably, while TIM-4 can directly interact with PS, the transduction of cell signals requires its collaboration with integrins or other receptors and cannot be executed in isolation [20]. Stabilin2, a type I surface receptor, enables direct contact with PS and promotes efferocytosis through the adaptor proteins GULP and thymosin β4, which are involved in the regulation of actin polymerization [36], [37]. Additionally, Stab2 can promote the binding of cultured cells to ACs [38].

There will be “don 't eat me” if there is “eat me.” Exposing the “don't eat me” signal on cells helps keep them from being engulfed. Several “don't eat me” molecules have been found, including CD47 and CD24 [39]. Recently, CD24 was reported to release antiphagocytic signals through its interaction with sialic acid–binding immunoglobulin (Ig)-like lectin 10 (Siglec-10), which is located on the surface of macrophages, to form a novel immune checkpoint. This is a novel target for cancer immunotherapy using macrophages in ovarian and breast cancers [40], [41]. Phagocytic cells receive a ”don't eat me“ signal from CD47 via interaction with its receptor signal regulatory protein (SIRPα). As an effective immunotherapy strategy, blocking CD47 has been demonstrated to be effective against a wide range of malignancies [42], [43], [44]. Therefore, an interesting question is whether concurrent inhibition of CD47 and CD24 with a bispecific antibody can lead to potential synergy. A recent study has demonstrated that the function of CD47 and CD24 coordinated the transmission of the “don’t eat me” signal from macrophages to cancer cells. Initially, researchers designed and created a bi-specific antibody fusion protein, PPAB001, to simultaneously target CD47 and CD24. Evidence shows that the concurrent inhibition of CD47/SIRPα and CD24/Siglec-10 signalling by PPAB001 significantly enhanced the ability of macrophages to engulf tumor cells. Utilizing a bispecific approach to target two specific checkpoints has the potential to effectively remodel the tumor microenvironment, leading to an enhanced antitumor response [45].

The digestion phase

The canonical process of phagocytosis entails progressive development of the early phagosome into the late phagosome, ultimately leading to its fusion with the lysosomal compartment. After binding to dying cells, phagocytes activate actin, rearrange their own skeleton, form phagosomes via plasma membrane invagination and local extravasation, then “eat” dying cells via efferocytosis. RabGTPase protein family regulates lipid membrane modifications during efferocytosis. After maturation, the phagosome unites with the lysosome via a Ca2 + -dependent SNARE complex comprising VAMP7 and Syntaxin7. Many proteases, nucleases, and lipases are found in lysosomes and are responsible for digesting ACs in phagosomes. The supplemental pathway encompasses the mechanism of phagocytosis mediated by microtubule-associated protein 1A/1B LC3 [1], [23]. Subsequent to phagocytosis, LAPPI3K complexes are recruited into LAP-associated phagosomes (LAPosomes), which contain ACs, thereby instigating the LC3 binding mechanism. The connectivity of LC3 facilitates the fusion of lysosomes and LAPosomes, thereby enhancing the efficacy of dead cell clearance and preserving immune silencing. The lysosome, with a pH range of 4.5–5.0, comprises a substantial quantity of protease, nuclease, and lipase enzymes, the majority of which are responsible for digestion; these enzymes bind to the phagosome to form the phagolysosome. Some remain viable after the degradation of expiring cells by their acidity and active cathepsin [46]. When multiple corpses are ingested, phagocytes are required to regulate their cellular volume and surface area efficiently. Ingested cargo, which includes membranes, cholesterol, proteins, nucleic acids, and other substances, imposes a metabolic burden on phagocytes. These substances must be metabolized and incorporated into the metabolic cycles of phagocytes or eliminated. Additionally, the ingested cargo may contain harmful components, requiring phagocytes to effectively digest and degrade the apoptotic cargo while transitioning towards an anti-inflammatory and pro-repair state after efferocytosis. This poses a distinct challenge for phagocytes [47], [48], [49]. Most of the time, the number of dead cells in the environment exceeds the capacity of phagocytes to conduct phagocytosis; therefore, phagocytes must eliminate multiple cells simultaneously. The accumulation of metabolically decomposing cell membrane structures, cholesterol, proteins, and nucleic acids poses an enormous burden. Phagocytes must maintain precise surface area regulation amid a dynamic immune and metabolic milieu. Ultimately, neither the path nor the environment is immutable. Regardless of the scale (macro or micro), environmental interaction is perpetually dynamic and non-conserved.

The dynamic tracking of efferocytosis

A routine in vitro efferocytosis study was performed. For example, efferocytosis can be observed under a microscope after immunofluorescence staining of primary human macrophages and apoptotic Jurkat T cells. However, because ACs are easily eliminated, their in vivo detection remains challenging. Raymond et al. developed a gene-encoded fluorescent reporter gene, CharON, that enables live cell tracking of efferocytosis during Drosophila embryonic development [50]. To monitor efferocytosis in vivo, scientists have generated transgenic Drosophila melanogaster specimens, known as CharON. During the intermediate and advanced phases of Drosophila embryogenesis, a pronounced occurrence of programmed cell death, also known as apoptosis, occurs within the developing central nervous system (CNS). CharON offers a comprehensive depiction of efferocytosis in vivo, encompassing many stages, such as apoptosis, recruitment of phagocytes, identification, engulfment, and subsequent digestion. CharON observed that the task of clearing ACs from the CNS of embryonic Drosophila melanogaster was undertaken by phagocytic glial cells and scattered ventral blood cells (macrophages). Furthermore, there was a disparity in the phagosome dimensions between glial cells and macrophages. Additionally, the fluorescence intensity of the ACs within the macrophages was more concentrated.

Researchers have conducted additional investigations into the functional heterogeneity of macrophages and have observed that distinct cells play distinct roles in the course of efferocytosis. Macrophages can undergo phagocytosis and ingest varying quantities of ACs, ranging from to 1–3 to 4–6, or even beyond 7. When macrophages become overwhelmed, an increase in acidification is observed, suggesting the presence of adaptive and regulatory functions at the cellular level within the body. Subsequently, researchers inserted a repo mutant to ascertain macrophage heterogeneity. The absence of phagocytic glial cells in this mutant resulted in an elevated efferocytosis load in the macrophages. These findings indicate that the presence of repo mutants results in a compromised inflammatory response in macrophages, hindering their ability to effectively phagocytose AC debris, even when they are able to reach the site of injury.

In summary, this study verified the feasibility of using apoptotic Jurkat cells and BFP + mouse J774 macrophages or mouse bone marrow-derived macrophages in vitro by constructing a CharON system that can simultaneously monitor apoptosis (pH-CaspGFP) and acidification degradation (pHlorina). The tracer of efferocytosis during Drosophila early embryonic development clearly revealed the entire process of recruitment, acidification, and degradation of ACs in vivo, providing excellent new tools and methods for future research on efferocytosis.

The linkage to hallmarks of aging and efferocytosis

The precise relationship between efferocytosis and the hallmarks of aging is shown in Fig. 2. Here, we focused on representative factors, including mitochondrial dysfunction, cellular senescence, altered intercellular communication, and chronic inflammation. In summary, the precise nature between efferocytosis and the hallmarks of aging is two-way interaction and mutual influence. As the Fig. 2 shows, chronic inflammation has the potential to induce mitochondrial dysfunction and cellular senescence. The impact of senescent cells on macrophage function and polarization is mediated by the release of senescence-associated secretory phenotype (SASP). Inflammation can be induced by mitochondrial dysfunction and impaired efferocytosis. Meanwhile, mitochondrial dysfunction can lead to the occurrence of chronic inflammation and cellular senescence, and then affect the physiological function of phagocytes through SASP. In addition to serving as intercellular communication for SASP, inflammatory chemicals have the capacity to influence the activity and polarization of macrophages, thereby impacting efferocytosis [11], [51], [52], [53], [54], [55], [56].

Fig. 2.

The interconnection between efferocytosis and hallmarks of ageing. Efferocytosis and hallmarks of aging interact with each other. As shown in the figure, aging markers interact with each other. On the one hand, they can directly affect the physiological function and polarization of phagocytes represented by macrophages; on the other hand, they can also indirectly affect phagocytes and efferocytosis through inflammatory factors and SASP. Finally, aging markers and efferocytosis jointly promote the occurrence and development of age-related diseases.

Mitochondrial dysfunction

The preservation of mitochondrial health is critical during aging. A multitude of factors can substantially accelerate mitochondrial dysfunction due to the increased susceptibility of mitochondria to environmental fluctuations, which can ultimately lead to age-related diseases [57]. In recent years, there has been increased recognition of the role of mitochondria in immune cell activation and differentiation. After activation, immune cells exhibit a significant energy demand. Immune cells utilize diverse mechanisms to produce energy. Energy is produced through glycolysis in pro-inflammatory cells, including activated monocytes, T and B cells, regulatory cells, M2 macrophages, and regulatory T cells. Conversely, energy is generated via β-oxidation and mitochondrial function in regulatory cells. Mitochondria provide energy during activation and stimulate immune responses [58]. Macrophages, by virtue of their pro-inflammatory and anti-inflammatory properties, exert both beneficial and deleterious effects and are thus central to a number of diseases [59].

Efferocytosis, which plays a crucial role in macrophage function, prevents post-apoptotic necrosis and mitigates inflammation. To facilitate effective efferocytosis, phagocytes must possess the ability to internalize ACs. The results of this study suggest that the process by which macrophages take up multiple ACs depends on dynamin-related protein 1-mediated mitochondrial fission. Fission is initiated in response to AC uptake. Disabling mitochondrial fission reduces the effects of AC-induced cytosolic calcium increase due to mitochondrial calcium sequestration and hinders the formation of calcium-dependent phagosomes around the subsequently encountered ACs. These abnormalities can be rectified by inhibiting mitochondrial calcium uniporters. Therefore, mitochondrial fission plays a crucial role in allowing macrophages to effectively eliminate numerous ACs and prevent adverse effects associated with impaired efferocytosis in an in vivo setting [60]. Moreover, the process of heart repair following acute myocardial infarction (MI) includes the recruitment and stimulation of immune cells, such as macrophages. During the initial stage following MI, macrophages exhibit a pro-inflammatory phenotype, whereas they transition towards a reparative phenotype in later stages. Despite the observation of metabolic reprogramming during this shift, the molecular connections to macrophage differentiation remain poorly understood. Cai et al. have provided evidence to support the notion that the resolution of inflammation and tissue healing is influenced by mitochondrial function in macrophages. This influence is exerted through the modulation of two key processes: the phagocytic clearance of ACs, often known as efferocytosis, and the activation of myofibroblasts [61].

Recent research has revealed that the release of detrimental inflammatory senescence-associated secretory phenotype (SASP) molecules by senescent cells is attributed to mitochondria inside the cellular structure. Research has revealed that the process of cell senescence involves the permeability of the mitochondrial outer membrane permeabilization (MOMP) permeabilization, which is influenced by the activation of mitochondrial DNA (mtDNA) and the subsequent activation of the cyclic guanosine 5′-monophosphate (GMP)-AMP synthase - stimulator of interferon genes pathway. This activation pathway leads to the secretion of SASP. This discovery established a connection between apoptosis and the aging process, indicating that the permeability of the mitochondrial outer membrane, which is intricately associated with cell death, facilitates inflammatory reactions in aging cells. These findings present novel avenues for further investigation and hold promise for the development of therapeutic interventions targeting the aging process [62].

In summary, among the various mechanisms that influence the aging process, those regulated by the mitochondria exhibit the highest degree of sensitivity to environmental alterations. Consequently, it is imperative to acquire a comprehensive understanding of the involvement of the mitochondria in human well-being and aging process to achieve significant advancements in combating frailty and age-related ailments.

Cellular senescence

Cellular senescence refers to a state of stable cell cycle arrest that can be induced in normal cells by many internal and external stimuli as well as developmental cues. Cellular aging is primarily attributed to the activation of stress responses triggered by various factors that result in DNA damage. These factors include oxidative stress, exposure to UV radiation, and substances that possess the ability to harm DNA. Cellular senescence has a significant impact on tissue repair and regeneration, leading to aging in both organs and individuals. Numerous studies have provided evidence that the elimination of senescent cells can ameliorate age-related tissue malfunctions and prolong the duration of a healthy lifespan [63].

Senescent cells exhibit continued viability during modified metabolic processes and exhibit significant alterations in gene expression. These changes lead to the development of a multifaceted SASP that encompasses various pro-inflammatory factors [e.g., interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8)], and additional growth factors [e.g., HGF, transforming growth factor-β (TGF-β), and granulocyte–macrophage colony-stimulating factor (GM-CSF)]. Chemokines, including CXCL-1/3 and CXCL-10, as well as matrix remodelling enzymes, such as metalloproteinases, exert significant paracrine and autocrine effects on cells and tissues. Additionally, they function as signalling molecules during various phases of efferocytosis [64], [65], [66]. Moreover, chronic senescence can induce pathological inflammation. In their study, Schloesser et al. illustrated how senescent cells utilize “don't eat me” signals that impede macrophages' capacity to ingest them and hinder macrophages from eliminating adjacent corpses, that is efferocytosis; thus, they uncover an additional mechanism by which senescence might play a role in instigating inflammation [67]. The roles and mechanisms of age-related phenotypes and age-related secretory phenotypes, which are linked to the occurrence and progression of numerous diseases, have received increasing attention in recent years. Age-related diseases, such as hypertension, cardiac remodeling and dysfunction, myocardial ischemia, infarction, and heart failure (HF), are associated with cellular senescence, senescent cell accumulation, and the production and release of SASP components, according to clinical and experimental evidence [50], [68], [69]. Nevertheless, the precise function of senescent cells under these circumstances remains ambiguous, and certain instances have documented both detrimental and advantageous consequences. Uncertainty surrounds the relationship between cellular senescence and other significant factors [70], [71], [72], [73]. The inhibitory effect of aging on the growth and spread of cancer cells renders aging a formidable antitumor mechanism. The cellular process of aging may be likened to a dual-edged phenomenon with both advantageous and disadvantageous implications for the regular functioning of the human body. It is capable of preserving tissue homeostasis, facilitating tissue remodelling and repair, and promoting wound healing. Conversely, it has the potential to impede tissue repair and regeneration while accelerating the aging process of both tissues and the entire body [74], [75], [76], [77], [78].

In summary, cellular senescence is an important physiological response to stress and injury. Under normal physiological conditions, this response is followed by clearance of immune cells. However, in the context of aging or chronic injury, immune mechanisms cannot completely eliminate cellular senescence. Consequently, the accumulation of senescent cells leads to disease development, primarily due to the substantial secretion of pro-inflammatory and pro-fibrotic factors. Therapeutic techniques targeting the elimination of senescent cells have been widely investigated in animal models and are currently under clinical trials [79], [80], [81]. Further investigations are warranted to identify additional inducible factors, markers, regulatory elements, and signalling pathways associated with cellular aging; devise safer and more efficient techniques for identifying, eliminating, or regulating senescent cells; employ cellular aging as a therapeutic target or intervention to ameliorate or treat cancers and diseases associated with aging; and enhance the efficacy of regenerative medicine.

Altered intercellular communication

The accumulation of senescent cells and active intercellular communication are serious consequences of aging and age-related diseases. The SASP is a unique means of intercellular communication in senescent cells. Classical SASP is characterized by the secretion of soluble factors, growth factors, and extracellular matrix (ECM) remodeling enzymes by senescent cells. However, newly discovered SASP and other non-classical means of intercellular communication have also been recognized in cellular senescence, including extracellular vesicles (EVs), non-cellular metabolites, and ions. Juxtacrine signalling is a type of intercellular communication that depends on receptor and ligand binding. Senescent cells also “communicate” with neighboring cells through cell-to-cell or paracrine signalling. For example, IL-1A is believed to be a major regulator of soluble factor paracrine aging and also regulates paracrine aging. Senescent cells express membrane-binding IL-1A, which interacts with IL-1R to regulate IL-6 and IL-8 levels. Intercellular communication in senescent cells is much more complex than initially believed and does not entirely rely on these pathways [82]. Therefore, it is very likely that there are other unidentified modes of communication, and it will be necessary to systematically identify and analyze cellular communication during aging and senescence.

Throughout the various stages of efferocytosis, there is a continuous exchange of information between ACs and phagocytes, facilitating the collaborative elimination of ACs. Additionally, this communication between the surrounding cells plays a crucial role in fostering the resolution of inflammation and restoration of tissue integrity. It also has the ability to slow the aging process. For example, spermidine and GMP are released from ACs through caspase-activated pannexin 1 channels and can modify the gene programs of neighboring cells, thereby establishing anti-inflammatory and wound healing responses [83]. It is also possible for ACs to secrete cytokines, such as TGF-β [84] and interleukin-10 [85] in certain circumstances, which can contribute to the formation of an immunosuppressive milieu. Additionally, it is critical to specify that ACs can produce big ApoEVs, also known as apoptotic bodies (ApoBDs), as well as small ApoEVs, which are vesicles that resemble exosomes and microvesicles, during the process of cell death. ApoEVs are responsible for regulating a range of processes that utilize cargo wrapped within or exposed to EVs [86]. It is possible that a certain percentage of ACs or ApoBDs may unavoidably lead to membrane lysis [87], despite the fact that efferocytosis is both quick and effective. This scenario also revealed the presence of intracellular inflammatory components, highlighting that the immunological outcome of efferocytosis is regulated by a multifaceted interplay of several factors, even in the absence of pathogens.

Chronic inflammation

The aging process is characterized by chronic systemic inflammation and cellular and immunosenescence, followed by organ dysfunction and the development of age-related diseases. The SASP, which consists of factors secreted by senescent cells, has been found to aid in the progression of chronic inflammation and induces senescence in non-senescent cells. Simultaneously, persistent inflammation expedites immune cell senescence, leading to compromised immune functionality and an inability to eliminate inflammatory agents and senescent cells. This phenomenon establishes a negative feedback cycle, in which inflammation and senescence are interconnected. As a result, it has been acknowledged that inflammation is an intrinsic aspect of the aging process and that reducing inflammation may represent a viable approach to mitigate the effects of aging [88], [89], [90], [91], [92], [93]. Bidirectional crosstalk occurs when the senescent and immune cells engage in reciprocal interactions. Schloesser et al. described [67] a novel mechanism by which senescent cells can impede the phagocytic activity of immune cells, thereby impeding efferocytosis and the ability of immune cells to efficiently eradicate cellular debris. Collectively, these actions may contribute to the pathogenesis of chronic inflammation.

Recent data have established a direct correlation between wound repair in the myocardium and efferocytosis by inflammatory immune cells. Moreover, the results of this study indicate that phagocytic receptors, which are found on monocytes and macrophages, are essential for establishing a link between the resolution of inflammation and healthy operation of organs. Insufficient elimination of necrotic cells in elderly individuals may lead to an enduring inflammatory reaction [94] and compromised cardiac repair mechanisms, consequently accelerating the progression of HF [95]. Specifically, when there is a defect in efferocytosis, the ACs can not be cleared in time, resulting in secondary necrosis and releasing toxic cell contents to induce inflammation; Meanwhile, due to the functional defect, phagocytes can not increase the secretion of anti-inflammatory mediators and complete the phenotypic transformation of “pro-resolving”, so that the dead cells can not be cleared, thus causing inflammatory reaction. In addition, efferocytosis, another name for macrophages getting rid of dead cells (ACs), is a very important process in fixing tissue damage caused by inflammation; it causes macrophages to cytolyze, activates signalling pathways through receptors, stops AC necrosis, reduces tissue inflammation, and accelerates the healing of tissue damage. Instead, exocytosis does not work well in many long-term inflammatory diseases, such as atherosclerosis, which damages tissues and keeps them inflamed. Through efferocytosis, some metabolic processes inside the cell affect how macrophages repair damage and cause inflammation. ACs are important in macrophages during efferocytosis, which occurs when phagocytic lysosomes break down metabolites. Researchers have found that DNase2a breaks down the DNA of ACs into nucleotides in the phagocytic lysosomes of macrophages during efferocytosis. Subsequently, these nucleotides increase Myc expression via the DNA-PKCS- mammalian target of rapamycin (mTOR)C2/Rictor axis. This makes it easy for non-inflammatory macrophages to grow, which helps stop inflammation and repair harm [96].

In reverse, efferocytosis can generate a significant quantity of growth factors, including TGF-β [97], VEGF, and HGF. These growth factors play crucial roles in preserving cellular stability. Dysfunctional efferocytosis can result in chronic inflammatory responses, autoimmune disorders, and tissue injury. Moreover, it is plausible that the characteristics of inflammation manifest as a result of the failure of macrophages to undergo polarization towards a phenotype that promotes the resolution of inflammation. Macrophage activity, characterized by increased efferocytosis and a heightened release of anti-inflammatory and pro-resolution cytokines, is known as alternative macrophage activation or M2 activation [98]. It is uncertain whether macrophage polarization caused by efferocytosis is the same as that induced by other stimuli, such as IL-4 or specific pro-resolving mediators. If these two polarizations are similar, it is possible that there is relevant data indicating that aging may inhibit polarization.

The diseases are driven by defective efferocytosis and aging

A significant number of human aging-related diseases, such as atherosclerosis, cancer, diabetes, obesity, and rheumatoid arthritis, have been identified to be correlated with efferocytosis defects or mutations. Next, we focused on the pathogenesis of the following diseases (Fig. 3).

Fig. 3.

Efferocytosis, ageing and ageing-related diseases. Efferocytosis and ageing markers are correlated and interact with each other, which includes mitochondrial dysfunction, cellular senescence, altered intercellular communication, and chronic inflammation. This eventually leads to a range of age-related diseases. Created with BioRender.com.

Atherosclerosis

Advanced age is a prominent determinant of the development of atherosclerotic cardiovascular disease. There are two primary ways in which aging contributes to the development of atherosclerosis: endogenous and exogenous mechanisms of the vascular system [99]. Regarding the external mechanism, the process of aging leads to the differentiation of hematopoietic cells in the bone marrow into myeloid cells. Furthermore, the process of aging can also facilitate the development of hematopoietic cell clones. Notably, there are no apparent indications of hematopoietic malignant tumors or other recognized markers of illness. This occurrence is commonly referred to as clonal hematopoiesis of indeterminate potential. Regarding the internal mechanism, aging has been found to adversely affect both mitochondrial function and the removal of damaged mitochondria, known as mitochondrial autophagy. A previous study indicated that the elevation of IL-6 levels in the aorta due to aging is linked to impaired mitochondrial function in blood vessels, which contributes to the development of atherosclerosis [100].

Atherosclerotic plaques are generated because of the accumulation of altered lipoproteins in the subendothelial layer of the arteries. This accumulation triggers an inflammatory response leading to an influx of leukocytes into the arterial wall. A significant proportion of these leukocytes undergo apoptosis, and while they are effectively eliminated during the early stages of lesion development, efferocytosis begins to decline in advanced plaques. Consequently, there is a build-up of secondary necrotic cells within a specific region of the plaque, known as the necrotic core [101], [102], [103]. In humans, large necrotic cores are commonly observed in high-risk plaques that have a heightened propensity to induce MI and stroke [104]. Hence, investigation of the underlying processes involved in impaired efferocytosis represents a significant aim in the field of atherosclerosis research [105]. One potential mechanism underlying impaired efferocytosis in the context of atherosclerosis involves the proteolytic degradation of the efferocytosis receptors, MERTK and LRP1. The significance of MERTK in the process of lesional efferocytosis is evident from the observation that mice lacking Mertk and fed a Western diet exhibit larger lesion sizes, expanded necrotic cores, and diminished synthesis of specialized pro-resolving mediators [106], [107]. In both humans and mice, it has been observed that advanced lesions contain lesional macrophages that exhibit elevated expression of the enzyme calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ). This enzyme plays a role in suppressing the ATF6–LXRα–MERTK pathway (1 3 9). Consequently, it was observed that atherosclerotic lesions in Ldlr−/− mice fed a Western diet and lacking myeloid CaMKIIγ exhibited elevated levels of macrophage MERTK expression, enhanced efferocytosis, and reduced necrotic cores [108]. Apoptosis is induced when macrophages are inundated by excessive accumulation of lipid and lipoprotein deposits. Both inflammation and severe metabolic dysregulation have been found to impede efferocytosis in atherosclerotic plaques, thereby exacerbating disease progression. The promotion of efferocytosis not only alleviates the burden of apoptosis, but also enhances plaque stability.

Type 2 diabetes mellitus (T2DM)

In animal models, one of the principal indications of efferocytosis deficiencies caused by obesity or diabetes is delayed wound repair. However, the precise mechanism by which phagocytes operate at the site of the wound is still under investigation. Macrophages obtained from ob/ob animals lacking leptin or from mice with obesity induced by dietary factors exhibit compromised efferocytosis, as reported in previous studies [106], [109]. In the context of atherosclerosis, it has been observed that Ldlr−/− ob/ob mice have compromised lesional efferocytosis and bigger necrotic cores in comparison to Ldlr−/− animals. When mice with the genetic mutation Ldlr−/− ob/ob are provided with a diet that is abundant in ω − 3 fatty acids, there is an observed enhancement in the ability of their macrophages to perform efferocytosis. Furthermore, it has been observed that human senescent β cells have a response to senolysis, which lays the groundwork for potential translation into practical applications. The aforementioned unique discoveries establish the groundwork for further exploration into the senolysis of β cells as a potential preventive and ameliorative approach for T2DM [110]. Furthermore, it has been observed in animal models that senomorphic medicines, such as resveratrol and metformin not only promote senolysis but also have the potential to mitigate β-cell senescence and enhance metabolic equilibrium [111], [112]. Metformin has been proposed as a potential intervention for enhancing glucose tolerance and insulin sensitivity in individuals with diabetes. This is believed to be achieved by various mechanisms, including reduction of telomere shortening, prevention of inflammation and oxidation, and alteration of the composition of the gut microbiota [113], [114], [115], [116]. However, metformin use in older individuals may lead to deficits in folate-related B vitamins, which have been linked to a decline in cognitive function [117].

Overall, the range of therapeutic choices tailored to individual patients can be broadened by considering their medical histories, which may involve the use of anti-inflammatory metabolites or dietary changes.

Hypertension

Cardiomyocyte apoptosis is abundant in hypertrophic hearts of patients with hypertension. It is induced by various factors, such as pressure overload and angiotensin [118]. The mobilization of macrophages to eliminate and process ACs is crucial for restoration following cardiac damage. Macrophages efficiently discharge ACs in a process called efferocytosis, which prevents post-apoptotic necrosis and decreases inflammation. In contrast, impaired efferocytosis leads to chronic inflammation and dysfunction of the heart muscles [119], [120]. Consequently, improving efferocytosis has been suggested as a therapeutic approach for managing hypertensive cardiac diseases. A study [121] showed that sirtuin-3 (SIRT3) can reduce inflammation in the heart by enhancing efferocytosis and anti-inflammatory functions. The makeup of resident or recruited macrophages was unaffected by SIRT3 ablation, but it worsened the decline in MerTK expression in macrophages, which led to a pro-inflammatory phenotype and the release of cytokines, including IL-6 and tumor necrosis factor (TNF)-α. Notably, there was a decrease in the efferocytosis receptor MerTK index in tandem with the increasing accumulation of ACs. Under hypertensive conditions, SIRT3 overexpression restored impaired efferocytosis and enhanced the anti-inflammatory macrophage phenotype.

Hypertension has been described as a condition of premature vascular aging relative to actual chronological age. Many factors contributing to the deterioration of vascular function with age are accelerated in hypertension [122]. Besides phenotypic recognition, our understanding of senescence during hypertension-associated vascular aging remains incomplete. The majority of literature suggests a pressure-dependent link between accelerated cellular senescence and hypertension [123]. Cellular senescence is a physiological process that occurs with aging and causes cells to stop growing. Nevertheless, there is a lack of research determining whether senescence can facilitate blood pressure elevation. Similarly, it remains uncertain whether elimination of senescent cells using senolytic therapy can effectively reduce blood pressure and enhance arterial function in hypertensive animals. Enhancing our understanding of cellular senescence beyond phenotypic recognition could further refine vascular age determination as a prognostic and diagnostic index of cardiovascular disease risk and offer an alternative therapeutic target for patients with hypertension resistant to all currently available treatments.

MI and HF

Ischemic heart disease is the leading cause of death worldwide, and MI accounts for the largest number of deaths [124]. HF is a common cause of morbidity and mortality after MI. The risk of HF caused by MI is increasing. Therefore, new treatment methods need to be explored. After MI, cardiac-resident macrophages are responsible for the effective removal and degradation of apoptotic cardiomyocytes, a process also known as efferocytosis, which is necessary for inflammation regression and tissue repair. Therefore, strategies based on the removal of dead cardiomyocytes may improve inflammation and prevent the widespread death of cardiomyocytes, thereby slowing the process of HF [61]. Professional phagocytes are often regulated by a variety of signal pathways in “digesting” phagocytic cells. Macrophages have different origins, regeneration mechanisms, and functions among heterogeneous cardiac macrophage populations. A study [61] revealed that cardiac-resident macrophage subsets specifically overexpressed the legumain (Lgmn) gene and then participated in efferocytosis. Researchers have found that Lgmn deficiency leads to a significant deterioration of cardiac function, accompanied by the accumulation of apoptotic cardiomyocytes and weakening of efferocytosis in the boundary region. In addition, the deletion of Lgmn can also led to the downregulation of anti-inflammatory mediators IL-10 and TGF-β and the upregulation of pro-inflammatory mediators IL-1β and TNF-α. Subsequently, a mouse MI model confirmed that macrophage-derived Lgmn was specifically secreted during the clearance and degradation of dead cardiomyocytes. Mice that do not possess MERTK macrophages show an amplified reaction to ischemia–reperfusion injury in the left anterior descending artery. This response is characterized by the accumulation of ACs, increased infarct size, and greater impairment of cardiac function. In contrast, it has been observed that mice that express MERTK with resistance to cleavage exhibit a reduced presence of ACs in the cardiac tissue, hence displaying increased resistance to myocardial injury [119], [120], [125]. A recent study provided evidence that EVs released by a specific group of progenitor cells, referred to as cardiosphere-derived cells, can induce the upregulation of MERTK in macrophages. This in turn leads to enhanced efferocytosis and subsequent repair following MI in both rat and mouse models [126], [127].

Cardiac macrophages play an essential role in preserving cardiac homeostasis by eliminating senescent and damaged heart cells [128], [129], [130], [131]. Nevertheless, as individuals age, cardiac macrophages that dwell inside the heart also experience an upregulated release of substances, such as MMP9. This, in turn, contributes to the development of cardiomyocyte hypertrophy and an imbalance in the regulation of the ECM, which is characterized by the excessive accumulation of collagen and indicates the presence of fibroblast senescence [132]. After experiencing an acute MI, regulatory CD4 + T lymphocytes release cytokines, such as TGF-β1, IL-10, and IL-13, that facilitate the process of wound repair and the proliferation of cardiomyocytes [133]. Nevertheless, T cells potentially contribute to the pathogenesis of cardiac dysfunction in the context of aging. This is supported by the observation that older wild-type mice exhibit more pronounced systolic dysfunction than older animals lacking CD4þ T cells. This disparity may be attributed to elevated levels of inflammatory cytokines, such as tumour necrosis factor (TNF) [134]. Mast cells are responsible for tryptase production of tryptase following MI, which subsequently modulates the activity of protein kinases. One potential strategy for maintaining cardiac function involves preventing the hyperphosphorylation of troponin I and myosin-binding proteins [135]. Nevertheless, in the context of diabetic cardiomyopathy, mast cells exert a detrimental impact through the secretion of TNF-α and IL-6, which subsequently promote the death of cardiomyocytes and the manufacture of collagen via the activation of TGF-β signalling in cardiac fibroblasts [136]. Enhancing our understanding of the mechanisms by which immune cells might leverage these advantageous effects may present potential therapeutic approaches for mitigating the process of cardiac aging.

Liver pathologies

Liver-resident macrophages known as Kupffer cells are primarily responsible for executing the necessary process of efferocytosis. However, other liver cells, such as hepatocytes, can also facilitate efferocytosis [137], [138], [139]. Impaired efferocytosis is associated with several liver disorders, including autoimmune hepatitis, fatty liver disease, and primary biliary cholangitis. Molecules, such as TIM4 and GAS6, play crucial roles in the resolution of hepatic ischemia–reperfusion injury, indicating that clearance of deceased cells is vital for regulating hepatic inflammation [140].

An association between non-alcoholic fatty liver disease (NAFLD) and chronic liver disease due to aging has been reported. The primary distinguishing feature of NAFLD is fat in hepatocytes [141], [142], [143], [144]. NAFLD can be categorized into two distinct groups based on the liver pathology: non-alcoholic fatty liver, also known as simple hepatic steatosis, and non-alcoholic steatohepatitis (NASH). NASH is characterized by the presence of inflammation and fibrosis in the liver, which can subsequently lead to the development of severe cirrhosis and perhaps hepatocellular carcinoma [144]. The contribution of impaired mitochondrial function to NAFLD is a significant factor [145], [146]. Hepatocytes possess an abundance of mitochondria that play a crucial role in energy metabolism. The pathophysiology of NAFLD is associated with a decrease in fatty acid β-oxidation (FAO) caused by impaired carnitine palmitoyl transferase-1 (CPT-1) activity and reduced fatty acid clearance, which can be attributed to mitochondrial malfunction [147], [148]. Furthermore, impaired hepatic FAO is correlated with the advancement of NAFLD in individuals with obesity.

Recent studies have revealed that the protein known as A + T-rich interaction domain protein 3a (Arid3a) plays a role in MERTK-mediated efferocytosis in cholestasis. Consequently, this phenomenon directly affects the prompt elimination of apoptotic bile duct cells, thereby increasing the risk of cholestatic liver injury. The Arid3a-Mertk axis holds promise as an innovative treatment target for cholestatic liver disease [149].

Cancer

Cancer is frequently described as a pathological condition characterized by inflammation, and chronic refractory inflammation is believed to play a significant role in the development of cancer [150], [151], [152]. Conversely, there is growing recognition that the tumor microenvironment functions as an immunosuppressive mechanism, enabling tumor cells to evade immunosurveillance. Macrophages are abundant in the microenvironment of tumors and serve important immunomodulatory functions [153], [154]. An additional association between macrophages and inhibition of tumor immunosurveillance is a poor prognosis for the disease. The efferocytosis apparatus facilitates the transformation of naïve macrophages into “M2”-like cells, a phenotype that is unequivocally associated with a negative prognosis for cancer, owing to its involvement in tissue repair responses, inhibition of antitumor activity, and promotion of angiogenesis [153].

However, the relationship between malignancies and age is complex. Senescence contributes to tumorigenesis in two ways [155], [156], [157], [158], [159]. The impact of senescent cells on tumor promotion or inhibition is contingent on a multitude of factors, including the underlying causes of senescence, the quantity and durability of senescent cells, the nature and stage of the tumor tissue, the body's immune system, and the condition of critical senescent proteins, such as TP53. Recent progress has been made in understanding the biological mechanisms of aging in cancer, but its treatment remains fraught with obstacles. Although there is considerable evidence that senescent cells inhibit tumorigenesis, some studies have shown that senescent cells continuously exhibit tumorigenic properties under certain conditions. Senescent cells initiate both intrinsic and extrinsic mechanisms to impede tumorigenesis. A typical intrinsic antitumor mechanism involves the formation of a natural barrier to tumorigenesis through the induction of sustained growth arrest. Oncogene-induced senescence is characterized by the proliferation of malignant cells due to uncontrolled DNA replication induced by abnormal activation of proto-oncogenes in rat sarcoma (RAS). Subsequently, cells undergo senescence and robust proliferative arrest through the activation of critical cell cycle arrest genes to inhibit malignant proliferation [160]. Senescent cells augment tumor suppression in an extrinsic manner. Using direct cell-to-cell interactions or the release of inflammatory SASP factors, senescent cells can induce senescence or death in adjacent cells. This results in increased generation of reactive oxygen species (ROS) and a sustained DNA damage response; consequently, proliferation of neighboring precancerous or cancerous cells is inhibited [160].

COVID-19

SARS-CoV-2 is a respiratory virus responsible for the onset of COVID-19. Analysis of transcriptome sequencing data obtained from the postmortem brain tissue of individuals affected by COVID-19 revealed a correlation between the cognitive deterioration exhibited by patients severely afflicted with COVID-19 and the molecular characteristics associated with brain aging. Furthermore, individuals infected with SARS-CoV-2 show a notable increase in pulmonary aging [161]. Over the last decade, scientists have discovered several techniques for targeting senescent cells. Senolytics, a 2015 medication combination that selectively kills senescent cells, was licensed by the US FDA for several human clinical trials. These early clinical trials demonstrated that the therapy was safe for human patients [162], [163]. A recent Phase 1 clinical research demonstrated that senolytics are safe, practical, and well-tolerated in patients with Alzheimer’s disease [164]. Surprisingly, recent research has shown that senescent cells accumulate in senescent human brain organoids. SARS-CoV-2 infection causes senescence in human brain organoids. Senolytic treatment has the potential to reduce age-related inflammation while resetting the transcriptome aging clock. Senolytic therapy enhanced clinical symptoms and survival in a mouse model expressing human ACE2 and infected with SARS-CoV-2, reduced intracranial viral load, boosted dopaminergic neuron survival, and reduced viral and pro-inflammatory gene expression. These findings show that cellular senescence plays a significant role in brain aging and SARS-CoV-2-induced neuropathology and that senolytic treatment may be beneficial [165]. Importantly, this study found an abundance of senescent cells in the postmortem brain tissue of patients with COVID-19, demonstrating the direct function of SARS-CoV-2 and highly neurotropic viruses in the senescence of human brain organoid cells. Furthermore, senolytic therapy for SARS-CoV-2-infected brain organoids selectively eliminates senescent cells, reduces SASP-associated inflammation, and SARS-CoV-2 RNA expression, implying that senescent cells may play a role in viral retention [166].

The functional plasticity of macrophages enables them to enhance the elimination of pathogens and induce inflammation, while also suppressing inflammation and facilitating tissue remodeling and repair. Efferocytosis governs the interplay between these distinct processes during infection. Studies have shown that efferocytosis is involved in the pathophysiology of SARS-CoV-2 [167]. Numerous cell receptors facilitate efferocytosis by identifying PS on the surface of dead cells. Macrophages phagocytose numerous ACs to prevent subsequent necrotic cell build-up and tissue damage. The authors discovered that when macrophages phagocytosed virus-carrying ACs, the transcription of PS receptors on their surfaces decreased, resulting in poor efferocytosis. The expression of efferocytosis-associated genes in invading macrophages was strongly decreased in patients with both moderate and severe COVID-19, with the degree of inhibition being greater in severe patients. This observation is consistent with the notion that swallowing infected cells decreases the ability of macrophages to remove dead cells. Immunological failure can exacerbate COVID-19 and antiviral medications and other treatments that reduce viral replication may be ineffective in severely ill patients. The clearance of ACs infected with SARS-CoV-2 via the efferocytotic pathway is critical for maintaining tissue homeostasis and decreasing the consequences of SARS-CoV-2 infection [168].

Opportunities for therapy

The pursuit of healthy longevity, free from age-related ailments, has been a longstanding endeavor among human beings throughout history. The prevention and treatment of disorders associated with aging have considerable potential, yet pose significant challenges. Nevertheless, a multitude of aging mechanisms exist along with variations in the progression of distinct diseases associated with aging. Employing various targets for the treatment of age-related disorders holds significant promise for future research. The pathogenesis of numerous age-related diseases have been attributed to the intricate relationship between efferocytosis and aging. Consequently, we propose a combination of targeted cell burial and anti-aging therapy, which can not only improve the therapeutic efficacy for a single target, but also reduce the amount of treatment for a single therapy, thus reducing side effects and human tolerance. Strategies encompassing the promotion of a healthy lifestyle and the implementation of therapeutic interventions, such as small molecules, gene therapies, and cell transplantation are among the several approaches employed (Fig. 4).

Fig. 4.

Intervention and treatment opportunities for age-related diseases. The strategies encompassing the promotion of a healthy lifestyle and the implementation of therapeutic interventions, such as small molecules, gene therapies, and cell transplantation, are among the several approaches employed. Created with BioRender.com.

Targeting anti-aging

Optimal lifestyle decisions strongly correlate with age. Maintaining good health can occasionally result in an extended lifespan [169], [170]. An increasing body of research has demonstrated that aging can be effectively postponed by adhering to a healthy lifestyle, which includes adequate nutrition [171], [172], [173], [174], moderate exercise [175], [176], [177], and a positive mental state [178], [179], [180], [181], [182]. A nutritionally balanced diet has beneficial effects on the aging process. A considerable number of ingested nutrients, such as minerals and probiotics, are crucial for regulating immunity and mitigating inflammation. In a similar vein, research has demonstrated that the ingestion of polyunsaturated fatty acids substantially diminishes systemic concentrations of inflammatory cytokines [183]. Exercise can effectively postpone the aging process by diminishing the expression of age-related markers. Additionally, maintaining excellent mental health can delay aging. Neuroendocrine function is influenced by psychological stress via the hypothalamus–pituitary–adrenal axis [184], [185], [186]. Ongoing stimulation of this circuit results in sustained elevation of glucocorticoid levels, a process that precipitates hippocampal atrophy, which is intricately linked to aging [178].

Recent research has provided evidence that targeting the pro-inflammatory cytokine network using anti-aging medications, including metformin, aspirin, rapamycin, and ibuprofen, is a viable strategy. Metformin has the potential to ameliorate healthy middle-aged aging and mitigate chronic inflammation through its action on potential targets, including GPX7/NRF2 [187], IKK/NF-κB in patients diagnosed with type 2 diabetes [188], and a recently identified target, PEN2 [189], [190]. Repetitive senescence can be delayed by the mechanism by which aspirin reduces oxidative stress. Since 2015, the development of several senolytics has progressed from identification to clinical trials. Quercetin and dasatinib constitute the initial drug combination that resembled a senolytic drug. In contrast, dasatinib eliminates senescent human adipocyte progenitor cells, whereas quercetin exhibits greater cytotoxicity towards senescent human endothelial cells and mouse bone marrow stem cells [191]. The most effective elimination of senescent cells occur with a combination of these two compounds. Immune cell-mediated senescent cell clearance is recognized as a potentially effective approach for combating various chronic ailments and aging process [192], [193], [194]. Anti-uPAR-CAR-T cells were found to be efficacious in eliminating senescent cells in vitro, as well as precancerous and malignant cells in mouse models of the liver and lung, even when exposed to potentially toxic substances [195], [196], [197], [198], [199], [200]. According to clinical trials, numerous symptoms improve and inflammatory marker levels decline in older debilitated patients who receive stem cell injections [201]. Without adverse effects, patients with ischemic stroke who undergo MSC transplantation for acute stroke experience ameliorated symptoms and enhanced neurological recovery [202]. Organ transplantation, akin to stem cell transplantation, is an exceptionally efficacious anti-aging technique because of its capacity to “repair” the detrimental effects of aging in a straightforward and effective manner [203]. Human disease treatment and anti-aging will advance if animal organ transplantation is successfully accomplished and at a reasonable cost. As we are presently elucidating the mechanisms underlying aging, several compounds are undergoing preclinical evaluation to determine whether or not they are effective at decelerating the aging process. This category includes inhibitors of the mTOR [204], sirtuin activators [204], and mitochondrial inhibitors [205], [206], [207]. The significance of mitochondria in the regulation of aging has been established [208], [209], [210], [211], [212], [213], [214]. Although the principal role of these organelles is to produce ATP, which serves as an energy source for organisms, they also participate in various physiological processes associated with aging. These include apoptosis, autophagy, and ROS. A recent study has shown that pyrroloquinoline quinone can improve the recovery of mitochondrial activity and has the potential to prevent oxidative stress-induced premature aging of auditory cells [215]. Additionally, the transcriptional induction of HLH-30/TFEB has been employed as a more reliable measure for screening natural products that have the capacity to augment ALP. Initial screening conducted in mammalian cell culture revealed that coumarin (MIC) has a noteworthy impact on retarding the aging process and prolonging lifespan [216].

Targeting efferocytic machinery

Given the clear and distinct nature of the stages involved in efferocytosis, the integration of molecular pathway interventions alongside the advancement of pharmacometabolic omics holds significant therapeutic potential. One the one hand, given that efferocytosis is ubiquitous in all organs, when the function of the receptor is impaired, it can lead to a number of age-related diseases, and targeting the appropriate receptors may have therapeutic potential; One the other hand, efferocytosis is a multistage mechanism for eliminating ACs and consists of smell, eating, and digestion phases. Targeting critical signalling molecules and associated signalling pathways at each stage could be advantageous in the development of pharmaceuticals for conditions characterized by aberrant cell interactions.

As stated previously, the reduced expression of TIM-4 in mononuclear phagocytes in older adults has been linked to a disturbance in the p38 MAPK signalling pathway, which consequently impairs efferocytosis and leads to the resolution of inflammation [217]. Losmapimod, a p38 inhibitor used during clinical development, restored TIM-4 expression and efferocytic activity in mononuclear phagocytes generated from aged adults in vitro to therapeutically target this impairment. Moreover, specific types of malignancies [218] and atherosclerotic plaques [219] can upregulate the expression of the DMT-signal CD47. It has been demonstrated that inhibiting CD47 with anti-CD47 antibodies facilitates phagocytosis, which aids in the removal of cancer cells and apoptotic detritus from atherosclerotic plaques [220], [221], [222]. Inflammation is also associated with the development of cancer and cardiovascular diseases [223], [224], [225]. Therefore, anti-CD47 therapy represents an additional therapeutic alternative for enhancing efferocytosis during aging. Recently, additional methodologies for augmenting efferocytosis in vitro and in vivo via genetic and pharmacological methods have been documented. Skin dendritic cells exhibit pharmacological inhibition of the cysteine/glutamate transporter solute carrier family 7 member 11 (Slc7a11), which significantly enhances wound healing and efferocytosis in mice predisposed to diabetes as well as in control mice. Moreover, Slc7a11-deficient rodents exhibited enhanced wound healing after sustaining full-thickness skin injury [226]. An interesting untested hypothesis is that the delivery of such chimeric receptors via virus-like particles or lipid nanoparticles under particular inflammatory conditions could provide an advantage in resolving or dampening inflammation.

Challenges and prospects for the future

The overall aging of the population has led to a substantial increase in human life expectancy; however, this progress has also resulted in an increase in the prevalence of age-related illnesses. Research on aging is currently undergoing tremendous progress. The emergence of novel pharmaceuticals, research methodologies, and clinical interventions presents prospects for elucidating the mechanisms underlying aging and for developing preventive or therapeutic interventions for age-related ailments. The study of aging characteristics is helpful to improve the framework of aging mechanism. In the research of anti-aging drugs, it has been found that a variety of potential drugs and small molecular compounds such as metformin, rapamycin, spermidine, Senolytics drugs, Sirtuin activators and NAD + prodrugs can prolong the healthy life of organisms. So far, more than 2000 life-regulating genes and more than 400 life-prolonging compounds have been studied and discussed, but a large number of model biological studies still need to be supplemented by human trials and clinical observations [227]. It is gratifying that the research and development process of anti-aging drugs has been promoted with the emergence of new technologies, and the development of clinical trials of the first kind of anti-aging drugs has also provided valuable experience for later drug research and development. It is believed that anti-aging drugs and interventions that can effectively improve the healthy life span of human beings will emerge in the future. Putative geroprotectors have demonstrated encouraging outcomes in animal models; however, to authenticate these findings, placebo-controlled multi-center clinical investigations employing biomarkers of human aging and mortality data are necessary. Therefore, obtaining conclusive findings is difficult because of the insufficient data necessary to satisfy the fundamental standards for geroprotectors. In addition, over time, potential advantages may be superseded by detrimental consequences. For example, instances of toxicity and adverse effects resulting from prolonged and high-dose administration of anti-aging medications are increasing. Recent studies have demonstrated that metformin has the potential to significantly worsen acute kidney injury and may even result in fatal side effects. Specifically, its interaction with iron within the body induces neutrophil NETosis, an immune response characterized by the exacerbation of kidney parenchymal cell death in rodents. This serves as a critical foundation for scientific assessment and direction regarding the clinical implementation of metformin [228]. Therefore, it is necessary to address the objective consequences of antiaging therapies. Thus, therapeutic outcomes may be enhanced by combining anti-aging therapies with other well-established approaches to age-related maladies as opposed to relying solely on individual strategies. However, there are many gaps exist between basic research on aging and the implementation of anti-aging interventions in clinical trials, which mainly include the identification of the most effective amalgamation of synergistic treatment protocols for individual patients on the premise of ensuring individual safety. In addition, the development of anti-aging intervention strategies still faces many challenges, including regulatory difficulties, clinical design problems, inadequate verification of biomarkers of human aging and the commercial challenge of bringing new interventions to market. Therefore, future research should focus on the following points: First, large-scale preclinical experiments, primarily using large animal models, particularly non-human primates, can promote molecular targets and drug therapy in clinical trials. Second, establishing larger multi-center cohort studies, including more patients with aging-related diseases and classifying them according to their different types and characteristics, may provide ideas for basic research on aging. Third, focusing on the development of more efficient and sensitive technical equipment, such as a combination of artificial intelligence and clinical examination equipment, can promote data collection and analysis in this field.

Conclusion

Twelve hallmarks of aging were identified [11]. Several hallmarks, including chronic inflammation, mitochondrial dysfunction, cellular senescence, and altered intercellular communication, have been identified in this review as having a direct impact on efferocytosis. Compromised efferocytosis may contribute to the progression of chronic inflammation, which manifests with age. The precise nature of the intricate connection between efferocytosis and the hallmarks of aging is not entirely understood. However, at present, most evidence regarding the relationship between efferocytosis and aging markers only provides a correlation and not a causal relationship. Therefore, in the face of an aging society, more in-depth and extensive studies are needed to clarify the pathogenesis of aging-related diseases, which will be helpful in developing more effective and safe interventions and therapeutic measures. However, many anti-aging therapies are still in the laboratory, even though some have advanced to clinical trials. This indicates a substantial gap between basic research and real-world applications. The emergence of novel pharmaceuticals, research methodologies, and clinical interventions presents prospects for elucidating the mechanisms underlying aging and for developing preventive or therapeutic interventions for age-related ailments. In the future, anti-aging therapies will be combined with other well-established methods for treating age-related diseases rather than implementing these methods alone.

CRediT authorship contribution statement

Meng Zhang: Conceptualization, Writing – original draft, Writing – review & editing, Visualization, Validation. Jin Wei: Conceptualization, Data curation, Writing – review & editing, Visualization. Yu Sun: Conceptualization, Data curation, Writing – review & editing, Visualization. Chang He: Data curation. Shiyin Ma: Data curation. Xudong Pan: Writing – review & editing, Validation, Formal analysis, Methodology, Supervision. Xiaoyan Zhu: Writing – review & editing, Validation, Formal analysis, Methodology, Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Biographies

Pan Xudong, The corresponding author, is the professor and doctoral supervisor of Qindgao University, and the chief physician of Department of Neurology in the Affiliated Hospital of Qingdao University, the visiting scholar in Germany. Dr. Pan has been engaged in the research on the mechanism of atherosclerosis for more than 20 years, and has a certain influence in neuroscience, especially in China and participated in one major national research project. He has been sponsored by many national natural science foundations. So far, he has published more than 100 high-quality papers and written many high-quality works in the field of neurology. Among them, there are more than 70 SCI papers. He has directed several neurologist researchers as corresponding author, 5 of published papers had an IF> 10. At present, his research direction focuses on mitochondria, macrophages and atherosclerosis, and has won a national natural science fund.

Xiaoyan Zhu, The corresponding author, is the professor and doctoral supervisor of Qindgao University, and the chief physician of Department of Critical Care Medicine in the Affiliated Hospital of Qingdao University. Dr. Zhu has been engaged in the research on the mechanism of Neurocritical medicine for more than 10 years. She has been sponsored by many national natural science foundations. So far, she has published more than 50 high-quality papers. Among them, there are more than 30 SCI papers. She has directed several neurologist researchers as corresponding author, 5 of published papers had an IF> 10. At present, her research direction focuses on mitochondria, macrophages and atherosclerosis, and has won a national natural science fund.

Meng Zhang, PHD candiadate, majoring in Neurology, jioned Study of Master and Doctoral Degree in Qingdao University, a doctor in the affiliated Hospital of Qingdao University. She has participated in the research in the field of atherosclerosis for more than 5 years. More than 10 papers have been published. During the COVID-19 epidemic, she wrote a review and a letter of the role of viruses in nervous system diseases, which were widely quoted all over the world. At present, doctoral research topics mainly focus on mitochondria, efferocytosis and atherosclerosis.

Jin Wei, PHD candiadate, majoring in Neurology, a doctor in the affiliated Hospital of Qingdao University. 7 papers have been published, of which 6 are SCI papers. She has participated in the research in the field of atherosclerosis for more than 5 years and participated in the writing of a review related in aging(IF>13). At present, doctoral research topics mainly focus on aging and atherosclerosis.

Yu Sun, PHD candiadate, majoring in Neurology, jioned Study of Master and Doctoral Degree in Qingdao University, a doctor in the affiliated Hospital of Qingdao University. She has participated in the research in the field of atherosclerosis for more than 5 years. The published review related in aging which had highest IF is greater than 9. At present, doctoral research topics mainly focus on mitochondria function, aging and atherosclerosis.

Chang He, Master candiadate, majoring in Critical medicine in Qingdao University, a doctor in the affiliated Hospital of Qingdao University. Responsible for the development and utilization of the team's special database for cerebrovascular diseases. At present, the master's research topics mainly focus on aging markers.

Shiyin Ma, Master candiadate, majoring in Neurology in Qingdao University, a doctor in the affiliated Hospital of Qingdao University. As a researcher in the early stage of the study, she has participated in many studies related to atherosclerosis. At present, the master's research topics mainly focus on aging markers.