Counteracting Immunosenescence—Which Therapeutic Strategies Are Promising?

Monocytes constitute motile precursors of tissue-resident macrophages but exert similar functions, such as pathogen recognition, cytokine secretion, and antigen presentation [34]. However, so far most studies have focused rather on immunosenescent aspects of macrophages than monocytes. Macrophages are specialized phagocytic cells that are involved in recognition, phagocytosis, and degradation of pathogens and cell debris. In combination with neutrophil polymorphonuclear granulocytes (PMNs), macrophages constitute the first line of defense to infections. Moreover, activated macrophages also act as APCs [39,40]. Differentiated macrophages are generally divided into classically activated, pro-inflammatory “M1-like” and alternatively activated, anti-inflammatory “M2-like” macrophages, although the existence of respective subpopulations has been recognized [41].

Hearps and coworkers reported on alterations in the composition of monocytes in the elderly with a decrease in the main population of cluster of differentiation (CD)14⁺CD16⁻ monocytes and concomitantly an increase in so-called intermediate CD14⁺CD16⁺ and non-classical CD14^dimCD16⁺ monocytes [42]. In a geriatric mouse model, increased numbers of macrophages were observed in the bone marrow and in the spleen [43]. However, in the bone marrow of old patients no significant changes were noted [44]. Further, in mice numbers of hepatic macrophages accumulated with age [45].

Several studies reported reduced expression of Toll-like receptors (TLRs) in monocytes [42] and macrophages of the elderly, thereby limiting their responsiveness towards DAMPs [46]. Further, splenic macrophages derived from aged mice showed impaired activation of NF-κB and p38 mitogen-activated protein kinase (MAPK) signaling in response to TLR-4 activation [47]. Similarly, human blood monocytes of the elderly showed attenuated expression of TLR-1 and impaired activation of extracellular signal-regulated kinase (ERK)1/2 when applying a TLR-1/2 agonist [48].

In this line, microarray analysis of lipopolysaccharide (LPS)-stimulated murine M1-like macrophages revealed lower induction of genes encoding pro-inflammatory cytokines/chemokines in the case of cells derived from aged mice [49]. Likewise, macrophages of the elderly were characterized by an attenuated production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β upon S. pneumoniae infection and an impaired capacity to kill the pathogen compared to macrophages of younger adults [50]. Further, activated macrophages of old mice presented with decreased major histocompatibility complex (MHC)II expression leading to impaired antigen presentation to T cells [51]. Consequently, old mice presented with a higher susceptibility to infection [51,52]. Interestingly, aging also resulted in impaired activity of M2-like macrophages in wound healing [53].

The central nervous system (CNS) is also affected by immunosenescence. Microglia, the brain-resident macrophage population, constitutes the main immune cell type of the CNS and accounts for roughly 10% of all cells in the brain [54]. Aside from immune surveillance, the microglia are important for maintaining brain homeostasis [55] and undergo various changes throughout aging, such as elevated secretion of pro-inflammatory cytokines [56] and impaired motility and phagocytosis [57]. These processes drive neuroinflammation, which is a hallmark for many neurodegenerative diseases like Alzheimer’s disease (AD) and Parkinson’s disease (PD) [58].

PMNs are the most abundant leukocyte in the blood and constitute the first line of defense against bacterial, fungal, and yeast pathogens at infected sites [59,60]. Neutrophils have a rather short lifespan under homeostatic conditions but persist much longer upon stimulation, e.g., by pathogens [61].

Aging was not found to alter the numbers of circulating PMNs in healthy and hospitalized patients [62,63]. Also, their adhesion to endothelium at inflamed sites and tissue immigration was not affected by age [64]. However, human PMNs isolated from aged people were characterized by decreased chemotaxis towards granulocyte–macrophage colony-stimulating factor (GM-CSF), N-formylmethionyl-leucyl-phenylalanine (fMLP), and LPS [65], and similar findings were obtained for PMNs derived from aged mice [66]. Further, in aged mice reverse transendothelial migration of PMNs to re-enter the circulation occurred more frequently, which was mediated by increased production of chemokine C-X-C motif ligand (CXCL)1 by tissue-resident mast cells [67]. On a functional level, human PMNs derived from the elderly exerted lower phagocytic activity [68]. Both defects in migration and phagocytosis may be attributed at least in part to dysregulated expression of required effector proteins.

In this regard the β2 integrins CD11a/CD18 (lymphocyte function-associated antigen 1, LFA-1) and CD11b/CD18 (macrophage-1 antigen, MAC-1), which commonly interact with intercellular adhesion molecule 1 to facilitate leukocyte rolling as a prerequisite for transendothelial migration, play an important role [69]. Moreover, MAC-1 recognizes complement C3-opsonized pathogens and immune complexes and mediates their phagocytic uptake [70]. In addition, MAC-1 constitutes a coreceptor of Fc receptors, which confer internalization of antibody-opsonized pathogens and immune complexes [71,72]. Whereas Butcher and coworkers observed no age-dependent alterations of LFA-1 and MAC-1 on PMNs [68], Gasparato et al. reported on attenuated MAC-1 expression in PMNs derived from the elderly [73]. In the same study, PMNs derived from aged people showed increased expression of FcγRIIa (CD32) but lower surface levels of FcγRIII (CD16). The latter was also observed by Butcher and coworkers [68]. Furthermore, CD32 expression was refractory towards stimulation-induced upregulation in PMNs of the elderly, whereas MAC-1 was strongly upregulated [73]. In contrast to humans, in the same study murine PMNs were found to express and upregulate Fcγ receptors to a similar extent, irrespective of mouse age. Further, murine PMNs of old mice expressed MAC-1 to a higher extent under basal conditions but were refractory towards stimulation which resulted in MAC-1 upregulation in PMNs of young mice. This age-dependent defect was not observed in human PMNs. However, in human monocytes [74] and T cells [75] LFA-1 expression increased with age.

Besides attenuated chemotactic and phagocytic activity, murine PMNs of aged mice were also demonstrated to release fewer neutrophil extracellular traps (NETs), comprising chromatin and bactericidal mediators derived from the cytoplasm and granules [76], in response to pathogens, and to produce attenuated amounts of CXCL2 [77]. CXCL2 serves to attract PMNs, thereby promoting a positive feedback loop [78]. The impaired response of PMNs derived from the elderly towards GM-CSF has been attributed to the elevated activity of different inhibitory proteins like Src homology domain-containing protein tyrosine phosphatase-1 (SHP-1) and suppressors of cytokine signaling (SOCSs) [79]. As mentioned above, tissue repair is negatively affected by advanced age [80]. In association with defective tissue regeneration, the abundance of CD11b⁺Ly6C^intLy6G⁺ PMNs significantly decreased with age [81].

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of myeloid cells, which expand in the course of infections and tumor progression [82]. In cancer, MDSCs in combination with regulatory T cells (Treg) contribute to tumor immune evasion by inhibiting the activity of APCs and T effector cells by various mechanisms [83]. In aged mice, elevated numbers of MDSCs have been observed in bone marrow, spleen, lymph nodes, and blood [84]. Also, in elderly humans MDSC numbers were increased [85]. Further, in aged mice MDSCs were shown to exert an elevated T cell inhibitory function, attributed in part to elevated activities of arginase-1 [86,87] and inducible nitric oxide synthase (iNOS) [87]. The latter was considered a consequence of age-dependently impaired PI3K signaling, which resulted in enhanced release of IFN-γ triggering both iNOS expression and activity.

FOXO3 was shown to play a crucial role in HSC homeostasis by enhancing the expression of genes required for repair of oxidative DNA damage [187] and promoting mitochondrial metabolism [188] and autophagy [189]. In the elderly, FOXO3 was demonstrated to be downregulated by age-induced miRNA species (see below). Further, p53-induced phosphatase Wiskott–Aldrich syndrome protein-interacting protein (WIP)1 [190] was reported as required for HSC repopulation efficacy [191]. Consequently, impaired HSC activity in the elderly may be in part a consequence of age-dependent downregulation of both p53 and WIP1 [192].

In human T cells and B cells an age-associated impaired expression of BTB domain and CNC homolog (BACH)2 has been observed [193]. BACH2 was shown to promote the differentiation of CD4⁺ T cells towards Tregs [194] and to delay the differentiation of B cells towards plasma cells to enable class switch recombination and somatic hypermutation of immunoglobulins [195]. BACH2 downregulation was accompanied by an increase in BACH2-repressed PR domain-containing 1, with ZNF domain (PRDM1), demonstrated to promote both T cell and B cell differentiation [196]. The age-dependent shift in expression of both TFs may contribute to the overall attenuated numbers of naïve lymphocytes in the elderly. Autophagy describes the lysosomal degradation of cellular macromolecules and is downregulated in an age-dependent manner [197]. The endogenous polyamine metabolite spermidine, which drives expression of the autophagy-inducing transcription factor EB [198], was found downregulated in T cells [199] and B cells [200] of the elderly. In vitro application of spermidine largely restored functional activity of both adaptive immune cell types.

T cell factor (TCF)1 activity is necessary for T cell development as well as T cell differentiation [201]. TCF1 mRNA expression was reduced in the elderly due to hypermethylation of the gene promoter [202] and attenuated expression of forkhead box protein (FOX)O1 that confers expression of TCF1 [203]. On a posttranscriptional level, TCF1 was inhibited by elevated levels of microRNA (miRNA)-21 [204]. FOXO1 was shown to be downregulated by sustained PI3K and mTOR activity in activated T cell populations of the elderly [205]. Of note, TCF1 also conferred expression of miR-181a, which in turn positively regulates TCR activation by inhibiting the expression of TCR-attenuating phosphatases [206]. Therefore, attenuated TCF1/miR-181a may explain in part the age-dependently reduced T cell activity. In general, exhausted T cells that also arise in the course of chronic infection [7] and cancer [207] are characterized by TCF1 deficiency [208].

Besides TCF1, B cell lymphoma (BCL)-6 expression was also reduced in activated T cells of aging donors [204]. Both TFs are required for the differentiation of activated CD4⁺ T cells towards Tfhs [209]. In addition, FOXO1 is necessary for CD8⁺ T memory cell differentiation [210]. T cells of the elderly were characterized by elevated expression of the TF PRDM1 and of the runt-related TF (RUNX)3 [193,211]. Of these, PRDM1 was reported to promote Tregs [212] and to inhibit Tfh differentiation [213], whereas RUNX3 promoted Th1 in favor of Th2 differentiation [214,215]. Hu and coworkers reported on preferential Th9 polarization of activated CD4⁺ T cells of the elderly due to enhanced responsiveness towards TGF-β via increased expression of TGF-β receptor 3 elevating expression of spleen focus forming virus pro-viral integration oncogene (SPI)1 [216]. SPI1 is required to confer Th9 polarization [217,218]. In the same study the basic leucine zipper TF and ATF-like [219] and IRF4 [220] previously demonstrated to promote Th9 differentiation were also found upregulated in CD4⁺ T cells obtained from the elderly. Most recently, Zhang et al. demonstrated that activated CD4⁺ T cells derived from the elderly displayed attenuated expression of HELIOS as compared to CD4⁺ T cells obtained from younger donors and concomitantly elevated phosphorylation of STAT5 [221]. Activated STAT5 conferred vast epigenetic changes and consequently changes in the gene expression profile, promoting the differentiation towards inflammatory effector cells.

Piskor and coworkers demonstrated that inactivation of Myc-interacting zinc finger protein 1 (Miz-1) mimicked features of B cell immunosenescence in B cells downstream of the pro-B cell state [222]. Further, BCL-6 interacted with Miz-1 to inhibit expression of cyclin-dependent kinase inhibitor, thereby promoting germinal center formation [223], which is strongly affected in the elderly [224]. However, it needs to be shown whether Miz-1 activity is altered in an age-dependent manner. Common lymphoid progenitors of aged mice were found to express lower levels of early B cell factor and paired box (PAX)5 [225]. Forced expression of constitutively active STAT5 restored expression levels of both TFs and rescued age-dependent defects in B cell generation. Expression levels of E47 and its target activation-induced cytidine deaminase, which are both required for Ig class switching and somatic hypermutation of Ig genes [226], were attenuated in B cells of the elderly [227]. B cells derived from adults were reported to contain higher levels of inhibitor of binding or differentiation 2 that by inhibiting its target E47 interferes with immunoglobulin gene rearrangements in B cells [228]. Therefore, B cells in the elderly are less able to optimize antibody affinity to pathogenic proteins [229]. T-box expressed in T cells has been suggested as crucial for the differentiation of murine [230] and human [231] ABCs like those induced by Epstein–Barr virus (EBV) [232] and in systemic lupus erythematosus patients [233]. In the latter study IRF5 was identified as an ABC-promoting factor. However, it is unclear yet whether IRF5 is also involved in age-associated ABC differentiation. Additionally, ABCs obtained from older individuals displayed lower paired box (PAX)5 levels as compared to the ABC population of younger donors [234]. Further, levels of PAX5 were also diminished in ABCs obtained from older individuals that generated only poor antibody amounts 2–4 months after influence vaccination and therefore were considered non-responders as compared to ABCs obtained from elderly vaccination responders.

Early on, the NF-κB family members p52 and p65 were recognized to display elevated activity in immune cells obtained from aged mice [235] and to constitute the main TFs driving inflammaging and SASP in immune cells as induced, for example, by DNA damage [236] and as a consequence of activated MAPK p38 signaling [237]. The activities of both MAPK p38 [238] and NF-κB [239] are negatively regulated by WIP1. Age-dependent downregulation of the WIP1 regulator p53 and WIP1 thereby may contribute to promotion of SASP [192]. Further, in different non-immune senescent cells Janus kinase activity promoted a SASP phenotype [240]. More recently, both innate and adaptive immune cells of mice were demonstrated to display enhanced expression of several activator protein-1 TF family members that may contribute to SASP as exemplified by Jun driving IL-6 production in myeloid cells of aged mice [241]. In senescent cells the histone H3 lysine (K)-specific demethylase 4 (KDM4) was identified as upregulated and to confer expression of SASP-associated factors [242]. In human monocytes KDM4 was identified as an important positive regulator of cytokine responses induced by repetitive stimulation [243]. It remains to be shown whether KDM4 also plays a role in inflammaging in immune cells.

lncRNA (>200 nucleotides) acts on several levels to regulate gene expression, including epigenetic modulators like histone modifiers as exemplified by lncRNA-AW112010↗, which promoted CD4⁺ T cell activation by engaging the histone demethylase lysine demethylase 5A [244]. By this, histone H3K4 methylation of the IL-10 gene locus was reduced and IL-10 production attenuated. Further, several lncRNAs were reported to modulate the activity of DNA methyltransferase, for example, in polarizing Th populations [245]. Concerning the CD8⁺ T cell compartment, the lncRNA-NRON was found downregulated in senescent CMV_pp65CD8⁺ CD28^– T cells of the elderly, whereas its target gene nuclear factor of activated T cells was concomitantly upregulated [246].

lncRNA may also control the activity of TFs, for example, lncRNA-Gm9866, which promoted M2 macrophage polarization by inhibiting NF-κB activity [247], and act on a posttranscriptional level by various mechanisms. For example, in activated B cells lncRNA-XLOC_098131 acted as a decoy substrate for miR-548, resulting in elevated stability of the Fos proto-oncogene, activator protein-1 transcription factor subunit encoding mRNA, and consequently elevated B cell survival and differentiation towards plasma cells [248]. In macrophages, stimulation-induced lncRNA-FIRRE interacted with the RNA-binding protein heterogeneous nuclear ribonucleoproteins U stabilizing vascular cell adhesion molecule 1 and IL-12p40 mRNA [249].

Several lncRNAs have been shown to regulate cellular senescence, for example, lncRNA-SENEBLOC by inhibiting expression of the senescence effector p21 [250]. However, further studies are required to elucidate the contribution of lncRNAs that regulate signaling pathways like NF-κB and of pro-inflammatory cytokines as SASP components for immunosenescence.

In contrast to lncRNA, miRNA (~22 nucleotides) regulates gene expression exclusively on a posttranscriptional level [251]. miRNAs are embedded in the so-called RNA-induced silencing complex and bind sequence-complementary stretches in mRNA [252]. In case of several mismatches, target mRNA translation is inhibited by various mechanisms, including interference with binding of ribosomal factors, while near perfect binding of miRNA affects mRNA half-life. As outlined above, FOXO3 plays an important role in HSC maintenance [253]. Disturbed hematopoiesis in the elderly may be in part a consequence of age-dependently upregulated expression of members of the miR-132/212 cluster in HSCs, which in turn inhibit FOXO3 expression [254]. In macrophages derived from aged mice stimulation elevated expression of induced cyclooxygenase (COX-)2 mRNA to a much higher extent as observed in macrophages obtained from young mice due to impaired expression of the COX-2 mRNA inhibiting miR-26b and miR-101b in the former group [255]. The authors showed that expression of these miRNA species was attenuated in macrophages of old mice on an epigenetic level by histone deacetylation. As another example, the regulation of stimulation-induced miR-146a was found dysregulated in aging mice [256]. Macrophages of old mice displayed about 6-fold higher expression of miR-146a in unstimulated macrophages as compared to the expression level of cells of young mice. Further, whereas LPS stimulation increased expression of miR-146a by 12-fold in macrophages derived from young mice, no upregulation was observed for cells obtained from old mice.

Further, Xu et al. observed an age-correlating decrease in miR-17, miR-92a, and miR-181a expression in human PBMCs [122], which was in line with previous observations [257,258] and correlated with decreased TCR sensitivity [259] and reduced numbers of naïve CD8⁺ T cells [6]. Activated CD4⁺ T cells of elderly donors showed much stronger expression of miR-21 than T cells obtained from younger donors, causing attenuated expression of various TFs (e.g., TCF1 and BCL6) and activation of ERK and mTOR signaling, all contributing to the differentiation towards short-lived effector cells at the expense of memory precursors [260].

Resveratrol has been reported to activate AMPK and thereby limit mTOR [365] and PI3K [366] signaling. Further, resveratrol was demonstrated to induce the autophagy regulators SIRT1 [367] and FOXO1 [368], most probably via AMPK activation [369]. Due to these properties as well as additional diverse anti-tumorigenic modes of action like modulation of glucose metabolism [370] and histone deacetylase activities [364] resveratrol has been tested as an anti-tumor [371] agent, as a therapeutic for other diseases [372], and with regard to anti-aging properties [373]. Of note, resveratrol enhanced the lifespan of aged mice on a high-calorie diet, which was associated with improved insulin sensitivity and elevated AMPK activity [374]. Several studies have also shown a beneficial effect of resveratrol on neurodegenerative diseases, such as AD [375].

Owed to its poor solubility and bioavailability a number of distinct nanoformulations of resveratrol have been assayed for anti-tumor efficacy both in vitro and in vivo as reviewed in [376]. For this, different types of NC systems were used, for example, encapsulating chitosan NCs [377], liposomes [378], and mesoporous silica NCs [379] but also gold NCs that were coated with resveratrol [380].

In contrast to resveratrol the polyamine spermidine was demonstrated to enhance autophagy in an SIRT1-independent manner [381] by inhibiting the acyltransferase E1A-binding protein E 300 [361] and histone acetyltransferases [382], enhancing the expression and stability of autophagy-controlling components like TFEB [198] and microtubule-associated protein 1S [383] and promoting AMPK signaling [384], thereby reducing mTOR signaling [361]. Besides its prominent effects on autophagy and AMPK/mTOR signaling spermidine also increased the activity of age-dependently downregulated DNA methyltransferase 3 [385] which counteracted age-dependent upregulation of the β2 integrin LFA-1 in human T cells [386] and monocytes [74]. Cellular levels of spermidine decline in an age-dependent manner [387]. Administration of spermidine increased the lifespan of mice [383,388], which was associated with improved autophagy and attenuated oxidative stress [382]. In humans, increased uptake of spermidine via food was associated with lower mortality (NCT03378843↗) [389]. Spermidine was also demonstrated to reduce neuroinflammation in a murine AD model [390] and to improve cognitive abilities in older adults suffering from dementia [391].

Sermidine-based NPs have been generated to improve polyamine metabolism in tumor cells [392] and to exploit their strong anti-bacterial activity upon topical application [393]. Further, due to their cationic character spermidine and derived spermin have been employed to condense nucleic acids for improved gene delivery into tumor cells [394,395]. More recently, Huang and coworkers demonstrated that spermidine/DNA-based NCs that contained mTOR small interfering (si)RNA alleviated symptoms in a murine acute lung injury model due to synergistic autophagy-promoting effects of the carrier spermidine and its cargo, resulting in M2 polarization of lung macrophages [396].

Various mTOR inhibitors have been evaluated for the treatment of age-related diseases [397,398]. They are broadly divided into mTORC1- and mTORC2-specific as well as pan-mTORC inhibitors. Rapamycin, a naturally occurring macrolide produced by Streptomyces hygroscopius, inhibits mTORC1 and has been frequently used to evaluate its anti-cancer properties [399]. To overcome limitations in bioavailability and the tolerance-promoting activity of rapamycin upon long-term application [400] the suitability of nanoencapsulated rapamycin has been tested in several preclinical studies. For example, solid lipid NCs loaded with rapamycin and coated with polysorbate for stabilization [401] and PLGA-based NCs containing rapamycin to improve its oral bioavailability [402] performed better than soluble rapamycin in in vitro studies. Nanomicelles composed of a grafted copolymer of polyvinyl caprolactam–polyvinylalcohol–polyethylene glycol (PEG) loaded with the rapamycin derivative everolimus, employed due to its immunosuppressive activity in posterior uveitis, showed better permeation into the cornea than in its soluble form [403]. In preclinical in vivo studies PLGA-based NCs encapsulating rapamcycin and a model antigen conferred antigen-specific tolerance by passively addressing endocytic immune cells [404,405]. Based on the finding that senescent cells overexpress surface CD9 [406], PEGylated liposomes [407] and lactose-coated calcium carbonate NCs [408] were conjugated with CD9-specific antibody, thereby achieving preferential uptake by senescent fibroblasts and, upon loading with rapamycin, they counteracted cell cycle arrest.

Pan-mTORC inhibitors, also known as second-generation mTOR inhibitors, compete with ATP for the active site of the mTOR kinase and are extensively studied as anti-cancer drugs with several drugs currently in clinical trials [409]. However, several rapalogs, i.e., rapamycin derivatives which have a more favorable pharmacokinetic profile, have also been studied with regard to their anti-aging effects [398]. In this regard, Mannick and coworkers reported that in a clinical trial combined application of the dual PI3K/mTOR inhibitor BEZ235 and the mTOR inhibitor everolimus to elderly patients attenuated the rate of observed infections and increased CD4⁺ and CD8+ T cell responses after influenza vaccination, which was accompanied by a decrease in expression of the T cell inhibitory receptor programmed cell death protein (PD)-1 [410].

The first-line anti-diabetic drug metformin [425] has also been demonstrated to inhibit PI3K and mTOR [21,426] signaling at least in part via AMPK activation [427]. Its numerous distinct positive effects on metabolic pathways have been reviewed in [428]. Effects of metformin on the immune compartment have been elucidated primarily in the context of tumor therapy. Here, metformin was shown to exert anti-tumor effects on several levels, including the reprogramming of cells within the tumor microenvironment via activation of AMPK and by inhibition of STAT3 [429] and NF-κB [430]. In agreement, metformin inhibited the SASP phenotype of senescent cells by preventing nuclear translocation of NF-κB and activating NF-κB inhibitor α in an AMPK-independent manner [431]. In addition, metformin inhibited MDSC activity by inhibiting expression of programmed cell death protein ligand (PD-L)1 [432] and of the ATP-degrading ectoenzymes CD39 and CD73 [433] in an AMPK-dependent manner and ROS production via FOXO3 induction [434]. Further, metformin stimulated CTL activation, including stronger IFN-γ and TNF-α production in response to stimulation, via metabolic reprogramming, by stimulating glycolysis and under low-glucose conditions via fatty acid oxidation and autophagy-dependent glutaminolysis [432]. Moreover, metformin inhibited Foxp3-dependent Treg induction by activating AMPK, which in turn inhibited the Foxp3 regulator mTORC1 [435]. So far, the anti-tumor effects of metformin have been assessed in a number of clinical trials, albeit with controversial outcomes [436]. All of the aforementioned effects are also able to counteract immunosenescence. However, the efficacy of metformin as an anti-aging drug is still under debate and further clinical trials are needed to substantiate the preliminary beneficial findings obtained in the course of clinical studies [437]. Several studies have demonstrated a correlation between type 2 diabetes mellitus and AD, indicating a potential overlap in the underlying pathophysiological mechanisms. The potential use of metformin for AD treatment has been reviewed by Khezri et al. [438].

Nanoformulated metformin may overcome limited bioavailability and enable cell targeting. In agreement, in a number of in vivo diabetes models [439,440] but also in vitro tumor cell studies [441,442] and some in vivo mouse tumor studies [443,444] encapsulated metformin exerted stronger effects than in its soluble form. So far, a variety of NCs have been employed for nanoformulation of metformin, quite often polymeric carriers [442], nanoemulsions [440], and liposomes [444]. Again, the suitability of nanoformulated metformin for treatment of immunosenescence has been scarcely addressed so far. Hunt and coworkers reported that in mice quantum dot-conjugated metformin after oral uptake accumulated in the liver and yielded stronger AMPK and SIRT1 activation in hepatocytes and liver sinusoidal endothelial cells by several orders of magnitude than soluble metformin [360]. Further, hepatocytes derived from old mice internalized nanoformulated metformin to a higher extent than when applied in its soluble form. Whereas cellular uptake of soluble metformin is mediated by organic cation transporters [445], nanoformulated metformin was endocytosed in a clathrin-dependent manner [360]. The authors concluded that the alteration of the uptake route compensated in part for the overall reduced endocytic activity of hepatocytes of old mice.

Interestingly, NCs themselves may modulate mTOR activity. Activated mTORC1 localizes to the outside of the membrane of lysosomes, controlling autophagy in the lysosome, and in turn is regulated by various factors, including arginine, within the lysosome [446]. Of note, polystyrene NCs with amino groups that accumulated in lysosomes have been reported to increase the lysosomal pH value and thereby to inhibit mTOR signaling, whereas carboxylated NCs stimulated mTOR activity [447]. These findings underscore that intrinsic properties of NCs need to be tested in the course of designing functionalized derivatives and that such properties may even be exploited.

The dual Abelson interactor (ABL)/sarcoma (SRC) tyrosine kinase inhibitor dasatinib is clinically used for treatment of chronic myeloid leukemia that is characterized by constitutively active breakpoint cluster region–ABL activity [459]. The polyphenol quercetin was reported to exert anti-inflammatory and anti-oxidant activities [460] and has also been reported to show tumor-inhibiting effects in part due to modulation of the PI3K and MAPK signaling axis [461]. A combination of both agents was shown to effectively kill senescent cells [462] and thereby revealed the role of senescent cells for the progression of various diseases in different organs.

For example, in a mouse model of idiopathic pulmonary fibrosis the frequency of senescent fibroblasts was enhanced, and their depletion by combined administration of dasatinib and quercetin improved pulmonary health [463]. Aging constitutes a risk factor for non-alcoholic fatty liver disease [464]. Ogodnik and colleagues showed that disease progression was correlated with an increase in senescent hepatocytes, which was counteracted by application of dasatinib/quercetin [465]. In aged mice, depletion of senescent cells by treatment with dasatinib/quercetin reduced levels of pro-inflammatory mediators in the intestine and altered the composition of the gut microbiome [466]. Further, infection of old mice with a mouse β-coronavirus that resulted in an expansion of senescent cells and was lethal in most cases could be counteracted by treatment with dasatinib/quercetin [467]. Furthermore, Xu and colleagues demonstrated that injection of preadipocytes that were irradiated beforehand to induce senescence exerted deleterious effects on physical fitness [468]. Combined treatment with dasatinib and quercetin improved these parameters and prolonged the lifespan of young mice preinjected with senescent cells as well as of aged mice.

To date, several agents have been described to exert senolytic activity and many of these act by inhibiting members of the BCL-2 family that exert anti-apoptotic functions. In this regard, navitoclax, which inhibits BCL-2 and BCL-xL [469], was reported to deplete various types of senescent cells, including senescent HSCs, in aged mice as well as in mice irradiated to promote cellular senescence, thereby rejuvenating the HSC compartment [470]. Senolytic activity has also been demonstrated for the flavone fisetion, known to target the same BCL-2 family members as navitoclax [471] and two BCL-xL-specific inhibitors (A1331852, A1155463) that were considered favorable due to low toxicity [472].

The curcumin analog EF24 has been shown to inhibit BCL-2 and other anti-apoptotic proteins like X-linked inhibitor of apoptosis and baculoviral IAP repeat-containing (Birc)2 and Birc7, but also NF-κB [473], and to modulate miRNA expression and proteasomal activity [474]. EF24 exerted senolytic activity, which was synergistically enhanced upon coadministration of navitoclax [475]. Senolytic activity has also been shown for the histone deacetylase (HDAC) inhibitor panobinostat [476] and the dual HDAC/PI3K inhibitor CUDC907 [477] shown to decrease BXL-xL (panobinostat) and Bcl-2 homology domain 3 (CUDC-907), respectively, and that have been applied for treatment of multiple myeloma [478,479]. The members of the bromodomain and exterminal domain family (BET) regulate expression of inflammation-associated genes with acetylated H3 and H3 histones by recruiting various transcription factors [480]. Hence, BET activity has been linked to SASP induction [481]. Wakita et al. reported that BET protein degrader exerted senolytic activity but identified attenuated DNA repair and enhanced expression of autophagy-related genes as causative [482]. Additionally, inhibitors of heat shock protein 90, in part via upregulation of autophagy [483] and the polyphenol procyanidin C1 in a ROS-dependent manner [484], deleted senescent cells and prolonged survival in a mouse model of progeroid syndrome and of aged mice, respectively. The efficacy of senolytics to improve treatment of various diseases has prompted a number of clinical trials (phases 1–2) employing most often dasatinib/quercetin and fisetin, respectively, as recently reviewed in [485]. Most of these studies are still ongoing.

A number of studies have reported on NCs functionalized with senolytics that killed senescent cells in vitro, but due to their non-selective targeting properties their therapeutic usage may be limited [486]. Pronounced lysosomal β-galactosidase activity is a hallmark of senescent cells [487]. Encapsulation of drugs into galactose-coated NCs [488] and the administration of galactose-modified prodrugs [489,490] has been shown to enable targeting of senescent cells and to improve specific killing of such cells, thereby minimizing adverse effects. Xu and coworkers demonstrated that NCs targeting β-2-microglobulin (B2M), found overexpressed by senescent cells [491], preferentially engaged senescent cells in vitro and in vivo in aged mice [492]. NCs encapsulating dasatinib specifically killed senescent cells, whereas no cytotoxic activity was observed when applying dasatinib to the cell culture at an equimolar concentration.