Cooperation between inhibitory immune checkpoints of senescent cells with immunosuppressive network to promote immunosenescence and the aging process

Ruggieri E., Domenico E.D., Locatelli A.G., Isopo F., Damanti S., Lorenzo R.D., Milan E., Musco G., Rovere-Querini P., Cenci S., Vénéreau E.

Hou J., Chen K., He C., Li X., Huang M., Jiang Y., Jiao Y., Xiao Q., He W., Liu L., Zou N., Huang M., Wei J., Xiao Y., Yang M., et. al.

The accumulation and systemic propagation of senescent cells contributes to physiological aging and age-related pathology. However, which cell types are most susceptible to the aged milieu and could be responsible for the propagation of senescence has remained unclear. Here we found that physiologically aged bone marrow monocytes/macrophages (BMMs) propagate senescence to multiple tissues, through extracellular vesicles (EVs), and drive age-associated dysfunction in mice. We identified peroxisome proliferator-activated receptor α (PPARα) as a target of microRNAs within aged BMM-EVs that regulates downstream effects on senescence and age-related dysfunction. Demonstrating therapeutic potential, we report that treatment with the PPARα agonist fenofibrate effectively restores tissue homeostasis in aged mice. Suggesting conservation to humans, in a cohort study of 7,986 participants, we found that fenofibrate use is associated with a reduced risk of age-related chronic disease and higher life expectancy. Together, our findings establish that BMMs can propagate senescence to distant tissues and cause age-related dysfunction, and they provide supportive evidence for fenofibrate to extend healthy lifespan. Hou, Chen et al. show that aged bone marrow macrophages propagate senescence to multiple tissues in vivo, through extracellular vesicles containing PPARα-targeted microRNAs. They demonstrate the therapeutic potential of intervening in this process using the PPARα agonist fenofibrate.

Majewska J., Agrawal A., Mayo A., Roitman L., Chatterjee R., Sekeresova Kralova J., Landsberger T., Katzenelenbogen Y., Meir-Salame T., Hagai E., Sopher I., Perez-Correa J., Wagner W., Maimon A., Amit I., et. al.

AbstractThe accumulation of senescent cells promotes ageing and age-related diseases, but molecular mechanisms that senescent cells use to evade immune clearance and accumulate in tissues remain to be elucidated. Here we report that p16-positive senescent cells upregulate the immune checkpoint protein programmed death-ligand 1 (PD-L1) to accumulate in ageing and chronic inflammation. We show that p16-mediated inhibition of cell cycle kinases CDK4/6 induces PD-L1 stability in senescent cells via downregulation of its ubiquitin-dependent degradation. p16-expressing senescent alveolar macrophages elevate PD-L1 to promote an immunosuppressive environment that can contribute to an increased burden of senescent cells. Treatment with activating anti-PD-L1 antibodies engaging Fcγ receptors on effector cells leads to the elimination of PD-L1 and p16-positive cells. Our study uncovers a molecular mechanism of p16-dependent regulation of PD-L1 protein stability in senescent cells and reveals the potential of targeting PD-L1 to improve immunosurveillance of senescent cells and ameliorate senescence-associated inflammation.

Salminen A.

AbstractThe accumulation of pro-inflammatory senescent cells within tissues is a common hallmark of the aging process and many age-related diseases. This modification has been called the senescence-associated secretory phenotype (SASP) and observed in cultured cells and in cells isolated from aged tissues. Currently, there is a debate whether the accumulation of senescent cells within tissues should be attributed to increased generation of senescent cells or to a defect in their elimination from aging tissues. Emerging studies have revealed that senescent cells display an increased expression of several inhibitory immune checkpoint ligands, especially those of the programmed cell death protein-1 (PD-1) ligand-1 (PD-L1) proteins. It is known that the PD-L1 ligands, especially those of cancer cells, target the PD-1 receptor of cytotoxic CD8+ T and natural killer (NK) cells disturbing their functions, e.g., evoking a decline in their cytotoxic activity and promoting their exhaustion and even apoptosis. An increase in the level of the PD-L1 protein in senescent cells was able to suppress their immune surveillance and inhibit their elimination by cytotoxic CD8+ T and NK cells. Senescent cells are known to express ligands for several inhibitory immune checkpoint receptors, i.e., PD-1, LILRB4, NKG2A, TIM-3, and SIRPα receptors. Here, I will briefly describe those pathways and examine whether these inhibitory checkpoints could be involved in the immune evasion of senescent cells with aging and age-related diseases. It seems plausible that an enhanced inhibitory checkpoint signaling can prevent the elimination of senescent cells from tissues and thus promote the aging process.

Zeng X., Wang T., Yamaguchi K., Hatakeyama S., Yamazaki S., Shimizu E., Imoto S., Furukawa Y., Johmura Y., Nakanishi M.

Adipose tissue is an endocrine and energy storage organ composed of several different cell types, including mature adipocytes, stromal cells, endothelial cells, and a variety of immune cells. Adipose tissue aging contributes to the pathogenesis of metabolic dysfunction and is likely induced by crosstalk between adipose progenitor cells (APCs) and immune cells, but the underlying molecular mechanisms remain largely unknown. In this study, we revealed the biological role of p16high senescent APCs, and investigated the crosstalk between each cell type in the aged white adipose tissue. We performed the single-cell RNA sequencing (scRNA-seq) analysis on the p16high adipose cells sorted from aged p16-CreERT2/Rosa26-LSL-tdTomato mice. We also performed the time serial analysis on the age-dependent bulk RNA-seq datasets of human and mouse white adipose tissues to infer the transcriptome alteration of adipogenic potential within aging. We show that M2 macrophage-derived TGF-β induces APCs senescence which impairs adipogenesis in vivo. p16high senescent APCs increase with age and show loss of adipogenic potential. The ligand-receptor interaction analysis reveals that M2 macrophages are the donors for TGF-β and the senescent APCs are the recipients. Indeed, treatment of APCs with TGF-β1 induces senescent phenotypes through mitochondrial ROS-mediated DNA damage in vitro. TGF-β1 injection into gonadal white adipose tissue (gWAT) suppresses adipogenic potential and induces fibrotic genes as well as p16 in APCs. A gWAT atrophy is observed in cancer cachexia by APCs senescence, whose induction appeared to be independent of TGF-β induction. Our results suggest that M2 macrophage-derived TGF-β induces age-related lipodystrophy by APCs senescence. The TGF-β treatment induced DNA damage, mitochondrial ROS, and finally cellular senescence in APCs.

Lin X., Kang K., Chen P., Zeng Z., Li G., Xiong W., Yi M., Xiang B.

AbstractImmune evasion contributes to cancer growth and progression. Cancer cells have the ability to activate different immune checkpoint pathways that harbor immunosuppressive functions. The programmed death protein 1 (PD-1) and programmed cell death ligands (PD-Ls) are considered to be the major immune checkpoint molecules. The interaction of PD-1 and PD-L1 negatively regulates adaptive immune response mainly by inhibiting the activity of effector T cells while enhancing the function of immunosuppressive regulatory T cells (Tregs), largely contributing to the maintenance of immune homeostasis that prevents dysregulated immunity and harmful immune responses. However, cancer cells exploit the PD-1/PD-L1 axis to cause immune escape in cancer development and progression. Blockade of PD-1/PD-L1 by neutralizing antibodies restores T cells activity and enhances anti-tumor immunity, achieving remarkable success in cancer therapy. Therefore, the regulatory mechanisms of PD-1/PD-L1 in cancers have attracted an increasing attention. This article aims to provide a comprehensive review of the roles of the PD-1/PD-L1 signaling in human autoimmune diseases and cancers. We summarize all aspects of regulatory mechanisms underlying the expression and activity of PD-1 and PD-L1 in cancers, including genetic, epigenetic, post-transcriptional and post-translational regulatory mechanisms. In addition, we further summarize the progress in clinical research on the antitumor effects of targeting PD-1/PD-L1 antibodies alone and in combination with other therapeutic approaches, providing new strategies for finding new tumor markers and developing combined therapeutic approaches.

Lu S., Li R., Deng Y., Bai J., Ji B., Chu Y., Xu Y., Qu H., Guo X., Li P., Meng M.

AbstractGrowth differentiation factor 15 (GDF15) as a stress response cytokine is involved in the development and progression of several diseases associated with metabolic disorders. However, the regulatory role and the underlying mechanisms of GDF15 in sepsis remain poorly defined. Our study analyzed the levels of GDF15 and its correlations with the clinical prognosis of patients with sepsis. In vivo and in vitro models of sepsis were applied to elucidate the role and mechanisms of GDF15 in sepsis-associated lung injury. We observed strong correlations of plasma GDF15 levels with the levels of C-reactive protein (CRP), procalcitonin (PCT), lactate dehydrogenase (LDH), and lactate as well as Sequential Organ Failure Assessment (SOFA) scores in patients with sepsis. In the mouse model of lipopolysaccharide-induced sepsis, recombinant GDF15 inhibited the proinflammatory responses and alleviated lung tissue injury. In addition, GDF15 decreased the levels of cytokines produced by alveolar macrophages (AMs). The anti-inflammatory effect of glycolysis inhibitor 2-DG on AMs during sepsis was mediated by GDF15 via inducing the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) and the expression of activating transcription factor 4 (ATF4). Furthermore, we explored the mechanism underlying the beneficial effects of GDF15 and found that GDF15 inhibited glycolysis and mitogen-activated protein kinases (MAPK)/nuclear factor-κB (NF-κB) signaling via promoting AMPK phosphorylation. This study demonstrated that GDF15 inhibited glycolysis and NF-κB/MAPKs signaling via activating AMP-activated protein kinase (AMPK), thereby alleviating the inflammatory responses of AMs and sepsis-associated lung injury. Our findings provided new insights into novel therapeutic strategies for treating sepsis.

Tao W., Yu Z., Han J.J.

Cellular senescence underlies many aging-related pathologies, but its heterogeneity poses challenges for studying and targeting senescent cells. We present here a machine learning program senescent cell identification (SenCID), which accurately identifies senescent cells in both bulk and single-cell transcriptome. Trained on 602 samples from 52 senescence transcriptome datasets spanning 30 cell types, SenCID identifies six major senescence identities (SIDs). Different SIDs exhibit different senescence baselines, stemness, gene functions, and responses to senolytics. SenCID enables the reconstruction of senescent trajectories under normal aging, chronic diseases, and COVID-19. Additionally, when applied to single-cell Perturb-seq data, SenCID helps reveal a hierarchy of senescence modulators. Overall, SenCID is an essential tool for precise single-cell analysis of cellular senescence, enabling targeted interventions against senescent cells.

Salminen A.

Abstract The accumulation of senescent cells within tissues is a hallmark of the aging process. Senescent cells are also commonly present in many age-related diseases and in the cancer microenvironment. The escape of abnormal cells from immune surveillance indicates that there is some defect in the function of cytotoxic immune cells, e.g., CD8+ T cells and natural killer (NK) cells. Recent studies have revealed that the expression of programmed death-ligand 1 (PD-L1) protein is abundantly increased in senescent cells. An increase in the amount of PD-L1 protein protects senescent cells from clearance by the PD-1 checkpoint receptor in cytotoxic immune cells. In fact, the activation of the PD-1 receptor suppresses the cytotoxic properties of CD8+ T and NK cells, promoting a state of immunosenescence. The inhibitory PD-1/PD-L1 checkpoint pathway acts in cooperation with immunosuppressive cells; for example, activation of PD-1 receptor can enhance the differentiation of regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC), and M2 macrophages, whereas the cytokines secreted by immunosuppressive cells stimulate the expression of the immunosuppressive PD-L1 protein. Interestingly, many signaling pathways known to promote cellular senescence and the aging process are crucial stimulators of the expression of PD-L1 protein, e.g., epigenetic regulation, inflammatory mediators, mTOR-related signaling, cGAS-STING pathway, and AhR signaling. It seems that the inhibitory PD-1/PD-L1 immune checkpoint axis has a crucial role in the accumulation of senescent cells and thus it promotes the aging process in tissues. Thus, the blockade of the PD-1/PD-L1 checkpoint signaling might be a potential anti-aging senolytic therapy. Key messages Senescent cells accumulate within tissues during aging and age-related diseases. Senescent cells are able to escape immune surveillance by cytotoxic immune cells. Expression of programmed death-ligand 1 (PD-L1) markedly increases in senescent cells. Age-related signaling stimulates the expression of PD-L1 protein in senescent cells. Inhibitory PD-1/PD-L1 checkpoint pathway suppresses clearance of senescent cells.

Isenberg J.S., Montero E.

AbstractCluster of differentiation 47 (CD47) occupies the outer membrane of human cells, where it binds to soluble and cell surface receptors on the same and other cells, sculpting their topography and resulting in a pleiotropic receptor‐multiligand interaction network. It is a focus of drug development to temper and accentuate CD47‐driven immune cell liaisons, although consideration of on‐target CD47 effects remain neglected. And yet, a late clinical trial of a CD47‐blocking antibody was discontinued, existent trials were restrained, and development of CD47‐targeting agents halted by some pharmaceutical companies. At this point, if CD47 can be exploited for clinical advantage remains to be determined. Herein an airing is made of the seemingly conflicting actions of CD47 that reflect its position as a junction connecting receptors and signalling pathways that impact numerous human cell types. Prospects of CD47 boosting and blocking are considered along with potential therapeutic implications for autoimmune diseases and cancer.

Tarallo D., Martínez J., Leyva A., Mónaco A., Perroni C., Tassano M., Gambini J.P., Cappetta M., Durán R., Moreno M., Quijano C.

AbstractCellular senescence is a therapy endpoint in melanoma, and the senescence-associated secretory phenotype (SASP) can affect tumor growth and microenvironment, influencing treatment outcomes. Metabolic interventions can modulate the SASP, and mitochondrial energy metabolism supports resistance to therapy in melanoma. In a previous report we showed that senescence, induced by the DNA methylating agent temozolomide, increased the level of fusion proteins mitofusin 1 and 2 in melanoma, and silencing Mfn1 or Mfn2 expression reduced interleukin-6 secretion by senescent cells. Here we expanded these observations evaluating the secretome of senescent melanoma cells using shotgun proteomics, and explored the impact of silencing Mfn1 on the SASP. A significant increase in proteins reported to reduce the immune response towards the tumor was found in the media of senescent cells. The secretion of several of these immunomodulatory proteins was affected by Mfn1 silencing, among them was galectin-9. In agreement, tumors lacking mitofusin 1 responded better to treatment with the methylating agent dacarbazine, tumor size was reduced and a higher immune cell infiltration was detected in the tumor. Our results highlight mitochondrial dynamic proteins as potential pharmacological targets to modulate the SASP in the context of melanoma treatment.

Dyachkova U., Vigovskiy M., Basalova N., Efimenko A., Grigorieva O.

Fibrosis and the associated decline in organ functionality lead to an almost 50% mortality rate in developed countries. Multipotent mesenchymal stromal cells (MSC) were shown to suppress the development and progression of fibrosis through secreted factors including specific non-coding RNAs transferred within extracellular vesicles (EV). However, age-associated chronic inflammation can provoke MSC senescence and change secretome composition, thereby affecting their antifibrotic properties. Alternatively activated macrophages (M2-type) are key players in chronic inflammation that may interact with MSC through paracrine mechanisms and decrease their antifibrotic functions. To confirm this hypothesis, we evaluated the M2-macrophage conditioned medium (CM-M2) effect on human adipose-tissue-derived MSC senescence in vitro. We found that CM-M2, as well as a pro-senescence agent, hydrogen peroxide (H2O2), increased p21+–MSC number and secretion of IL-6 and MCP-1, which are considered main senescence-associated secretory phenotype (SASP) components. Thus, both exposures led to the senescent phenotype acquisition of MSC. EV from both CM-M2 and H2O2-exposed MSC, which showed a decreased effect on the suppression of TGFβ-induced fibroblast-to-myofibroblast differentiation compared to EV from control MSC according to αSMA level and the αSMA+–stress fiber reduction. After two weeks of subsequent cultivation under standard conditions, MSC demonstrated a decrease in senescence hallmarks and fibroblast differentiation suppression via EV. These results suggest that M2-macrophage-induced chronic inflammation can reversibly induce MSC senescence, which reduces the MSC’s ability to inhibit fibroblast-to-myofibroblast differentiation.

Evans D.S., Young D., Tanaka T., Basisty N., Bandinelli S., Ferrucci L., Campisi J., Schilling B.

Abstract Cellular senescence, a hallmark of aging, results in a senescence-associated secretory phenotype (SASP) with an increased production of proinflammatory cytokines, growth factors, and proteases. Evidence from non-human models demonstrates that SASP contributes to tissue dysfunction and pathological effects of aging. However, there are relatively few human studies on the relationship between SASP and aging-related health outcomes. Proteins from the SASP Atlas were measured in plasma using aptamer-based proteomics (SomaLogic). Regression models were used to identify SASP protein associations with aging-related traits representing multiple aspects of physiology in 1,201 participants from two human cohort studies (BLSA/GESTALT and InCHIANTI). Traits examined were fasting glucose, C-reactive protein, interleukin-6, alkaline phosphatase, blood urea nitrogen, albumin, red blood cell distribution width, waist circumference, systolic and diastolic blood pressure, gait speed and grip strength. Study results were combined with fixed-effect inverse-variance weighted meta-analysis. In the meta-analysis, 28 of 77 SASP proteins were significantly associated with age. Of the 28 age-associated SASP proteins, 18 were significantly associated with one or more clinical traits, and seven SASP proteins were significantly associated with three or more traits. Growth/differentiation factor 15 (GDF-15), Insulin-like growth factor-binding protein 2 (IGFBP-2), and Cystatin-C showed significant associations with inflammatory markers and measures of physical function (grip strength or gait speed). These results support the relevance of SASP proteins to human aging, identify specific traits that are potentially impacted by SASP, and prioritize specific SASP proteins for their utility as biomarkers of human aging.

Nawas A.F., Solmonson A., Gao B., DeBerardinis R.J., Minna J.D., Conacci-Sorrell M., Mendelson C.R.

Abstract Introduction Inflammation plays a significant role in various cancers, including lung cancer, where the inflammatory cytokine IL-1β is often elevated in the tumor microenvironment. Patients with lung adenocarcinoma show higher levels of serum IL-1β compared to healthy individual. Moreover, IL-1β blockade reduces the incidence and mortality of lung cancer. Our prior studies revealed that alveolar type-II cells, the precursors for lung adenocarcinoma, display an induction in the expression of the enzyme tryptophan 2,3-dioxygenase (TDO2) during normal lung development. This induction of TDO2 coincides with an increase in IL-1β levels and is likely caused by IL-1β. Given that cancer cells can co-opt developmentally regulated pathways, we hypothesized that IL-1β may exert its pro-tumoral function by stimulating TDO2 and indoleamine 2, 3-dioxygenase-1 (IDO1), parallel enzymes involved in the conversion of tryptophan (Trp) into the immune-suppressive oncometabolite kynurenine (Kyn). Our goal was to determine whether IL-1β is a common upstream regulator of immune checkpoint regulators. Methods To determine whether IL-1β regulates IDO1, TDO2, PD-L1, and PD-L2, we measured mRNA and protein levels in lung adenocarcinoma cells lines (A549, H1792, H1838, H2347, H2228, HCC364 and HCC827) grown in 2D or 3D and in immortalized normal lung epithelial cells (HBEC3-KT and HSAEC1-KT). To determine the importance of the NFκB pathway in mediating IL-1β -regulated cellular effects, we used siRNA to knockdown RelA/p65 in IL-1β treated cells. The levels of Trp and Kyn in the IL-1β-treated cells and media were measured by mass spectrometry. Results Upon IL-1β stimulation, lung adenocarcinoma cells exhibited significant increases in IDO1 mRNA and protein levels, a response that depended on the NFκB pathway. Interestingly, this induction was more pronounced in 3D spheroid cultures compared to monolayer cultures and was not observed in normal immortalized lung epithelial cells. Furthermore, the conversion of Trp to Kyn increased in cells exposed to IL-1β, aligning with the heightened IDO1 expression. Remarkably, IL-1β also upregulated the expression of programmed death ligand-1 (PD-L1) and PD-L2 in multiple cell lines, indicating that IL-1β triggers parallel immune-suppressive mechanisms in lung adenocarcinoma cells. Conclusions Our studies demonstrate that lung adenocarcinoma cells, but not normal immortalized lung epithelial cells, respond to IL-1β signaling by inducing the expression of parallel immune checkpoint proteins that have the potential to promote immune evasion.

Redondo-García S., Barritt C., Papagregoriou C., Yeboah M., Frendeus B., Cragg M.S., Roghanian A.

Human leukocyte immunoglobulin (Ig)-like receptors (LILR) are a family of 11 innate immunomodulatory receptors, primarily expressed on lymphoid and myeloid cells. LILRs are either activating (LILRA) or inhibitory (LILRB) depending on their associated signalling domains (D). With the exception of the soluble LILRA3, LILRAs mediate immune activation, while LILRB1-5 primarily inhibit immune responses and mediate tolerance. Abnormal expression and function of LILRs is associated with a range of pathologies, including immune insufficiency (infection and malignancy) and overt immune responses (autoimmunity and alloresponses), suggesting LILRs may be excellent candidates for targeted immunotherapies. This review will discuss the biology and clinical relevance of this extensive family of immune receptors and will summarise the recent developments in targeting LILRs in disease settings, such as cancer, with an update on the clinical trials investigating the therapeutic targeting of these receptors.