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ScienceDirect Cellular signaling in the aging immune system Tamas Fulop1, Aure´lie Le Page1, Carl Fortin1, Jacek M Witkowski2, Gilles Dupuis3 and Anis Larbi4 Causes for immunosenescence and inflamm-aging have to be established. Efficient function of the immune system requires homeostatic regulation from receptor recognition of antigenic challenge to cell responses and adaptation to its changing environment. It is reasonable to assume that one of the most important molecular causes of immunosenescence is alteration in the regulation of signaling pathways. Indeed, alterations in feed-forward and negative feedback (inhibitory) signaling have been highlighted in all cells involved in the immune response including short-lived (neutrophils) and long-lived (T lymphocytes) cells. These dysregulations tip the balance in favor of altered (less efficient) function of the immune system. In this review, we summarize our knowledge on signal transduction changes in the aging immune system and propose a unifying mechanism as one of the causes of immunosenescence. Modulation of these pathways with aging represents a major challenge to restore the immune response to functional levels. Addresses 1 Research Center on Aging, Department of Medicine, Graduate Program in Immunology, Faculty of Medicine and Health Sciences, Universite´ de Sherbrooke, Sherbrooke, QC, Canada 2 Department of Pathophysiology, Medical University of Gdan´sk, Gdan´sk, Poland 3 Department of Biochemistry, Graduate Program in Immunology, Faculty of Medicine and Health Sciences, Universite´ de Sherbrooke, Sherbrooke, QC, Canada 4 Singapore Immunology Network (SIgN), Immunos Building at Biopolis, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Singapore 138648, Singapore Corresponding author: Fulop, Tamas ([email protected])
Current Opinion in Immunology 2014, 29:105–111 This review comes from a themed issue on Immune senescence Edited by Tamas Fu¨lo¨p and Ruth Montgomery
http://dx.doi.org/10.1016/j.coi.2014.05.007 0952-7915/# 2014 Elsevier Ltd. All rights reserved.
defective early signaling events, inter/intra-cellular communication, in adaptive and innate cells [5,6, 7–9,10,11]. In this review, we summarize our knowledge on signal transduction changes in the aging immune system and propose a unifying mechanism as one of the causes of immunosenescence. We will provide examples illustrating changes in neutrophils following GM-CSFR and Toll like receptors (TLR) stimulation and, in T cells activated by occupation of T cell receptor (TCR)/CD28. We will also argue for a unique general signaling alteration underlying immunosenescence and inflamm-aging. Alterations in neutrophil signaling with aging
Neutrophils are the first cells to arrive at the site of inflammation [2,9]. Neutrophils use an impressive array of mechanisms to phagocyte and kill intruders [12]. Neutrophils phagocytosis, chemotaxis, free radical production and apoptosis are altered with aging [2,9,13,14]. These functions are elicited by receptor–ligand interactions, suggesting that reduced functionality of neutrophils with aging results from alterations in signaling pathways downstream of receptors. For example, functions mediated by formylmethionylleucylproline (fMLP), Fcg and the C3b receptors are altered with aging which clearly resulted from changes in MAP kinases, Jak/STAT and PI3K-Akt signaling pathways [15–17]. These changes are not due to variations in the number of receptors but to alterations in signalosome formation resulting in impaired cell responses [13]. Additionally, neutrophils of aged individuals display alterations in TLR signaling through MyD88 and IRAKs [18,19]. The number of TLRs is not affected with aging but there is an alteration in the trafficking of pathway-associated signaling molecules in the plasma membrane [9,19]. The inflammasome, an important component of the immune response, is activated in response to an aggression by activating inflammatory caspase-1 and caspase-5 which results in the production of a wide range of cytokines [20–22]. There is however no data on inflammasomes in aging neutrophils and how this translates into inflamm-aging and vice versa, but it is reasonable to assume that neutrophil is a target population.
Immune cell signaling in aging Of the nine conditions associated with aging [1], altered intercellular communication was emphasized as the integration of declining immune responsiveness (immunosenescence) with low-grade inflammation (inflamm-aging) [2–4]. Altered immune functions are associated with www.sciencedirect.com
T cell signaling through TCR/CD28 with aging Feedforward signaling
T cell activation depends on recognition of antigenic epitopes presented by professional antigen-presenting cells (APC) within the context of major histocompatibility Current Opinion in Immunology 2014, 29:105–111
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complex (MHC) molecules (signal 1) at the immunological synapse (IS) where assembly of the T cell signaling processes occur within lipid rafts (LR) [23–26]. The first events of TCR signaling are activation of Lck, phosphorylation of ITAM motifs of the CD3 complex and recruitment/activation of ZAP70/LAT/SLP76 [27,28,29]. Further participation/phosphorylation of accessory proteins assembles the signalosome [30]. Signal 2 provided by costimulation (CD28) leads to full and efficient T cell activation [31] through the NF-kB pathway and prevent anergy [32]. Activation of CD28 results in decreased levels of reduced glutathione (GSH) and production of reactive oxygen species (ROS) [33,34] that may result in increased levels of TNFa [7]. The persistence of low amounts of proinflammatory cytokines and increased production of ROS, which are both features of inflamm-aging converge to hamper T cell responses [5,35–39] via altered signaling. Overall, these observations suggest that the reduced efficiency of the immune response with aging may be due to the dysregulated proximal events of TCR signaling [6,27,39]. In fact, nearly all the proximal events of TCR signaling or IL-2 responses are altered with aging [5,7,40], including impairment of protein tyrosine kinase (PTK) phosphorylation, decreased Ca2+ mobilization and lowered PKC activation that lead to sub-optimal NF-kB and NF-AT activities [36,32]. Feedback regulation of signaling
The activity of a key component of TCR signaling, Lck, is finely tuned by opposite effects of plasma membraneembedded CD45 protein tyrosine phosphatase (PTPase) and cytoplasmic Csk PK bound to the scaffold protein PAG [28,41] and, autophosphorylation. Csk phosphorylation of Lck C-terminal Y505 favors a conformationrelated inactivation whereas CD45-dependent dephosphorylation of Y505 and autophosphorylation of Y394 result in Lck activation [42,43]. Recent data have also implicated FAK as a negative regulator of Lck [44]. Our group has shown that dysregulation of the Csk/PAG/ CD45 loop in T cells of elderly increases the levels of inactive Lck [6], providing a molecular clue to altered T cell responses in aging. In addition, PTPases CD45, SHP1 and Lyp (PTPN22) play a key role in regulating Lck activity [41,45]. Although PTPases have overlapping substrate specificities, there are differences between their actions based on subcellular location, substrate binding, cellular maturation (e.g. T cell subsets) and strength of recruitment in response to TCR stimulation [45–47]. SHP-1 activity is crucial to maintain peripheral T cells in the resting state and to prevent uncontrolled T cell activation due to the fact that Lck is one of the targets of SHP-1 [48,49]. Under strong stimulation, SHP-1 activity is reduced [50,51], tipping the balance toward T cell activation. Since abrogation of SHP-1 enhances the quality of effector T cells functions, it can be suggested that targeting SHP-1 may prove useful as an enhancer of the CD8+ T cell polyfunctionality in immunotherapy [52]. Current Opinion in Immunology 2014, 29:105–111
We have reported that the Csk/PAG loop in activated T cells of healthy elderly individuals was dysregulated and that tipped the balance in favor of the inactive form of pLck-Y505 [6]. Feedback inhibitory events were also compromised in these subjects. In fact, SHP-1 activity was higher in elderly subjects than in young individuals, consistent with decreased T cell responses. Pharmacological inhibition of SHP-1 resulted in recovery of TCR/ CD28-dependent lymphocyte proliferation and IL-2 production to levels similar to young adults. These studies provide a lead for a strategy aimed at modulation of the inhibitory feedback loop of T cell activation by targeting SHP-1 and PTPases. This is supported by recent data showing that inhibition of protein phosphatase activities (DUSP4/6) resulted in increased T cell signaling and function [53,54] and that modulation of inhibitory co-receptors such as PD-1, signaling also via SHP-1, successfully increased the functions of CD8+ T cells [55]. These approaches can serve as a basis to improve immune functions in aged populations at risk (e.g. frail category or patients with impaired immunity). Oxidative status and mTOR regulation of TCR signaling
PTPases are regulated by phosphorylation and reversible oxidation of active site-located cysteine residues [7,37,51,56,57]. Mitochondrial respiration and NADPH oxidase are the major generators of ROS in T cells [58]. It is reasonable to suggest that altered redox potential may cause signaling abnormalities in aging T cells. This is of importance since healthy aging is characterized by increased levels of ROS (O2 , peroxidized lipids) and decreased content of antioxidants (e.g. GSH) [59,60]. Redox-associated stimulation of TCR signaling alters the balance between PTK and PTPase activities [67] favoring the activation. In aging where there is a sustained level of increased ROS [59,61], SHP-1 regulation may be affected and its negative regulatory role would be favored. Inhibition or decreased ROS production should rescue PTPase activity [30,62]. In this situation, the effect of ROS would be blunted and SHP-1 would remain active. However, upon TCR and CD28 stimulation, increased ROS levels which result in oxidation/inhibition of SHP-1 would favor activation of Lck [63]. An alteration in this finely regulated system in aging would lead to aberrant signaling, thus contributing to immunosenescence, inflamm-aging and their adverse effects [7,64]. Recent data have provided evidence that mTOR serine threonine kinase activity may also play a role in T cell activation and differentiation, especially of naı¨ve CD4+ T cells, toward the Th1 or Th17 phenotypes [65]. mTOR signaling pathway activation is under the control of TCR/ CD28 stimulation [65,66]. There are only very few studies on T cell mTOR alterations with aging and most of them come from murine models. Perkey et al. [67] showed that TORC2 signaling was increased in murine CD4+ T cells in aging and its overexpression in CD4+ T www.sciencedirect.com
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cells of young mice reproduced age-related CD4+ T cell functional changes. Our data (unpublished) have suggested differential phosphorylation status (CREB, Akt, S6, ElF4E, 4EBP1) of memory T cells as compared to naı¨ve T cells. Recently, Arnold et al. have reported that TCR stimulation induced autophagy in human CD8+ T cells, whereas autophagy was decreased in CD8+CD28 subset, which largely seems to compromise their survival under antigen stimulation [66]. These emerging data emphasize the importance of mTOR-related metabolic data intertwined with TCR feed-forward and negative feedback and its potential relationship to immunosenescence. Further investigations of the mutual influence of these signaling pathways are fundamental to understand and improve functions in T cells with aging.
Immunosenescence, signaling and inflammaging Immunosenescence can be viewed as the consequence of the immune system trying to adapt and maintain homeostasis in face of persistent intrinsic and extrinsic challenging inputs and outputs during life-time [68]. These include chronic disease states such as diabetes, obesity
and atherosclerosis, nutritional status, genetic background and environmental conditions [69]. Highly differentiated memory T cells accumulate in aged humans [3,4,70,71], likely due to persistent antigenic stimulation, pro-inflammatory environment associated with increased levels of TNFa derived from senescent cells and/or activation of the NLRP3 inflammasome and the NFkB pro-inflammatory pathways [55,37]. Recently the Nlrp3 was shown to control Inflamm-aging in rodents, independently of noncanonical caspase-11 inflammasome. Deletion of Nlrp3 leads to reduction of brain inflammation, suggesting that modulating Nlrp-3-dependent pathways may slow down chronic conditions in the elderly [72]. Thus, the unbalanced changes in adaptive versus innate immunity may contribute to the low-grade inflammatory process especially in syndromes such as frailty [73]. Pro-inflammatory cytokines and acute-phase proteins (IL-6, IL-8, TNFa, C-reactive protein) are two characteristics of inflamm-aging that negatively modulate TCR signaling pathways [74]. This is also true for ROS [27]. Thus inflamm-aging and increased oxidative stress converge to impair T cell activation and functions in aging [75]. For instance, high
Figure 1
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Changes in lipid rafts composition and function with aging in immune cell membranes including all the susceptible early signaling molecules independently of the immune cells. www.sciencedirect.com
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levels of phosphorylated p38 have been recorded in CD27 CD45RA+ CD4+ T cell phenotype [71]. As p38 activation involves T cell production of pro-inflammatory cytokines (including TNFa) and anti-TNF treatment in rheumatoid arthritis patients depletes CD8+ EMRA T cells [76], it suggests a role for TNFa in the mechanisms generating CD8+ EMRA T cells in vivo. The relationship between cytokine secretion switch and p38 signalingmediated T cell senescence remains to be further defined [71].
A unifying mechanistic explanation of signaling in immunosenescence Lipid rafts (LR), T cells and aging
LR are dynamic cholesterol-rich plasma membrane microdomains that function as organizing centers for
assembly of signaling molecules, protein trafficking and regulation of neurotransmission [77] (Figure 1). In T cells, LR move freely across the plane of the plasma membrane lipid bilayer as discrete entities to assemble the signalosome [78] before or concomitantly with formation of the immune synapse. Whereas some signaling components are constitutive constituents of LR (Lck, Cbp/PAG), others are recruited during activation (TCR, CD28, IL-2R, LAT, PI3K). Our group initially reported alterations in LR in aged humans [39]. Whereas LR were distributed homogeneously in T cells of young subjects, this was not the case in quiescent T cells of older subjects. LR coalescence was altered with aging in TCR CD28 activated T cells. LR poorly coalesced in CD4+ T cells of older subjects but this was less pronounced in CD8+ T cells. Furthermore, recruitment
Figure 2
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Schematic representation of the unified concept of signaling by various receptors on neutrophils (a) and T cells (b) with aging. The signaling molecules shown are only those demonstrated to be altered in aging. Besides the changes in the lipid rafts composition and function which induce alterations in the proximal signaling pathways such as Src kinases and PI3K (feedforward pathways), there are alterations in the feedback regulation pathways mainly mediated by protein tyrosine phosphatases (e.g. SHP-1). These intrinsic changes are influenced and potentiated by the extrinsic milieu of aging such as the reactive oxygen species (ROS) production and inflamm-aging. Current Opinion in Immunology 2014, 29:105–111
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and activation of Lck, LAT and SHP-1 in LR of aged donors was altered [39,79]. Of significance, CD28 and IL2R were poorly recruited in LR of CD4+ T cells of older subjects. By contrast, these proteins were already located in LR in CD8+ T cells from older subjects before stimulation. These differences may be related to intrinsic regulation of cholesterol metabolism in older subjects [39]. These observations suggested that assembly of the signaling machinery in CD4+ T cells relied largely on recruitment in LR, whereas a preassembly of the signalosome may be present in CD8+ T lymphocytes, as proposed by us and others [8,39,80]. The differential function of LR may be a key factor influencing T cell differentiation. Lipid rafts (LR), neutrophils and aging.
LR are required for neutrophils (PMN) to efficiently fulfill their aggressive protection against intruders (Figure 1). We investigated the role of SHP-1 in aged donors in response to GM-CSF stimulation. Down-regulation of SHP-1 activity was significantly altered with aging. In young donors, SHP-1 was displaced from lipid rafts, but not in the elderly [81]. We observed unbalance of tyrosine/serine phosphorylation of LR-located SHP-1 in PMN of elderly subjects, suggesting that colony-stimulating factor 2 (CSF2) was unable to inhibit serinedependent SHP-1 activity. CSF2 triggered Lyn activation in neutrophils but its activation/translocation into LR was affected with aging. We identified a permanent association of SHP-1 with Lyn in neutrophils of elderly donors that was not reversed by CSF2. Our results suggested alteration of SHP-1 regulation by CSF2 in LR of PMN with aging, explaining the decreased capacity of CSF2 to rescue PMN from apoptosis in the elderly.
Conclusions Increased human longevity represents major social and medical challenges as the incidence of age-related diseases, many of which are influenced by the dysregulation of the immune system, will increase [82]. A large body of data suggests that the immune response is dysregulated in aging. These functional changes may result in serious clinical consequences such as increased infections, cancers and autoimmune diseases. Evidence for alterations in T cell and PMN signaling is widely accepted as an underlying cause of this dysregulation. One unifying cause for these alterations could involve the signaling machinery which combines key signaling and regulatory molecules as well as composition of the plasma membrane (Figures 1 and 2) at least in T cells and PMN as almost no data are available for the other immune cells, however it can be suggested that similar changes may also occur in these cells with aging. Data have provided a basis to pursue studies to understand the molecular mechanisms of T cell signaling deficiencies associated with aging, the involvement of LR, the formation of the immune synapse and the assembly of the signaling machinery. www.sciencedirect.com
Moreover, investigating cholesterol metabolism will help to understand the regulation of cholesterol content in the membrane of T cell during key processes: maturation, activation, differentiation and apoptosis. The role of free radicals in the regulation of membrane integrity, qualitatively and quantitatively for cholesterol content, is of particular importance. We suggest that combined approaches that consist of maintaining adequate early feed-forward activation while down-regulating feedback inhibition would be beneficial in restoring several T cell functions in aging. Current strategies of blocking receptors such as PD-1 are efficiently used in a cancer setting however it is uncertain whether this strategy can be applied in the context of boosting immunity in healthy elderly. Early intervention should reduce the influence of factors that contribute to the increased feedback inhibition and will rescue T cells from exhaustion.
Acknowledgements This work was supported by grants from the Canadian Institutes of Health Research (CIHR) (No. 106634 and No. 106701), the Universite´ de Sherbrooke, and the Research Center on Aging.
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66. Arnold CR, Theresa Pritz T, Brunner S, Knabb C, Salvenmoser W, Holzwarth B, Thedieck K, Grubeck-Loebenstein B: T cell receptor-mediated activation is a potent inducer of macroautophagy in human CD8+CD28+ T cells but not in CD8+CD28S T cells. Exp Gerontol 2014. doi.org/10.1016/ j.exger.2014.01.018. This article describes for the first time the induction of autophagy in human CD8+ T cells following TCR stimulation and the decreased ability of the CD8+CD28 T cell subsets autophagy which suggests that they cannot suvive when metabolic requirements are not fulfilled. 67. Perkey E, Fingar D, Miller RA, Garcia GG: Increased mammalian target of rapamycin complex 2 signalling promotes agerelated decline in CD4 T cell signaling and function. J Immunol 2013, 191:4648-4655. This article shows that the increased TORC2 signaling in naı¨ve CD4+ T cells may lead to CD4 functional defects in old mice. 68. Mu¨ller L, Fu¨lo¨p T, Pawelec G: Immunosenescence in vertebrates and invertebrates. Immun Aging 2013, 10:12. 69. Fu¨lo¨p T, Larbi A, Witkowski JM, McElhaney J, Loeb M, Mitnitski A, Pawelec G: Aging, frailty and age-related diseases. Biogerontology 2010, 11:547-563. 70. Fu¨lo¨p T, Le Page A, Garneau H, Azimi N, Baehl S, Dupuis G, Pawelec G, Larbi A: Aging, immunosenescence and membrane rafts: the lipid connection. Longevity Healthspan 2012, 1:6. 71. Di Mitri D, Azevedo RI, Henson SM, Libri V, Riddell NE, Macaulay R, Kipling D, Soares MV, Battistini L, Akbar AN: Reversible senescence in human CD4+CD45RA+CD27memory T cells. J Immunol 2011, 187:2093-2100. 72. Youm YH, Grant RW, McCabe LR, Albarado DC, Nguyen KY, Ravussin A, Pistell P, Newman S, Carter R, Laque A et al.: Canonical Nlrp3 inflammasome links systemic low-grade inflammation to functional decline in aging. Cell Metab 2013, 18:519-532. 73. Franceschi C, Bonafe` M, Valensin S, Olivieri F, De Luca M, Ottaviani E, De Benedictis G: Inflamm-aging. An evolutionary perspective on immunosenescence. Ann N Y Acad Sci 2000, 908:244-254. 74. Larbi A, Dupuis G, Douziech N, Khalil A, Fu¨lo¨p T Jr: Low-grade inflammation with aging has consequences for T-lymphocyte signaling. Ann N Y Acad Sci 2004, 1030:125-133. 75. Larbi A, Cabreiro F, Zelba H, Marthandan S, Combet E, Friguet B, Petropoulos I, Barnett Y, Pawelec G: Reduced oxygen tension results in reduced human T cell proliferation and increased intracellular oxidative damage and susceptibility to apoptosis upon activation. Free Radic Biol Med 2010, 48: 26-34. 76. Macauulay R, Akbar AN, Henson SM: The role of the T cell in agerelated inflammation. Age 2013, 35:563-572. 77. Pizzo P, Viola A: Lipid rafts in lymphocyte activation. Microbes Infect 2004, 6:686-692. 78. Taner SB, Onfelt B, Pirinen NJ, McCann FE, Magee AI, Davis DM: Control of immune responses by trafficking cell surface proteins, vesicles and lipid rafts to and from the immunological synapse. Traffic 2004, 5:651-661. 79. Ohno-Iwashita Y, Shimada Y, Hayashi M, Inomata M: Plasma membrane microdomains in aging and disease. Geriatr Gerontol Int 2010, 10(Suppl. 1):S41-S52. 80. Larbi A, Douziech N, Dupuis G, Khalil A, Pelletier H, Guerard KP, Fu¨lo¨p T: Age-associated alterations in the recruitment of signal-transduction proteins to lipid rafts in human T lymphocytes. J Leukoc Biol 2004, 75:373-381. 81. Fortin CF, Larbi A, Lesur O, Douziech N, Fulop T Jr: Impairment of SHP-1 down regulation in the lipid rafts of human neutrophils under GM-CSF stimulation contributes to their age-related, altered functions. J Leukoc Biol 2006, 79:1061-1072. 82. Montecino-Rodrigez E, Berent-Maoz B, Dorschkind K: Causes, consequences and reversal of immune system aging. J Clin Invest 2013, 123:958-965.
Current Opinion in Immunology 2014, 29:105–111
ScienceDirect Cellular signaling in the aging immune system Tamas Fulop1, Aure´lie Le Page1, Carl Fortin1, Jacek M Witkowski2, Gilles Dupuis3 and Anis Larbi4 Causes for immunosenescence and inflamm-aging have to be established. Efficient function of the immune system requires homeostatic regulation from receptor recognition of antigenic challenge to cell responses and adaptation to its changing environment. It is reasonable to assume that one of the most important molecular causes of immunosenescence is alteration in the regulation of signaling pathways. Indeed, alterations in feed-forward and negative feedback (inhibitory) signaling have been highlighted in all cells involved in the immune response including short-lived (neutrophils) and long-lived (T lymphocytes) cells. These dysregulations tip the balance in favor of altered (less efficient) function of the immune system. In this review, we summarize our knowledge on signal transduction changes in the aging immune system and propose a unifying mechanism as one of the causes of immunosenescence. Modulation of these pathways with aging represents a major challenge to restore the immune response to functional levels. Addresses 1 Research Center on Aging, Department of Medicine, Graduate Program in Immunology, Faculty of Medicine and Health Sciences, Universite´ de Sherbrooke, Sherbrooke, QC, Canada 2 Department of Pathophysiology, Medical University of Gdan´sk, Gdan´sk, Poland 3 Department of Biochemistry, Graduate Program in Immunology, Faculty of Medicine and Health Sciences, Universite´ de Sherbrooke, Sherbrooke, QC, Canada 4 Singapore Immunology Network (SIgN), Immunos Building at Biopolis, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Singapore 138648, Singapore Corresponding author: Fulop, Tamas ([email protected])
Current Opinion in Immunology 2014, 29:105–111 This review comes from a themed issue on Immune senescence Edited by Tamas Fu¨lo¨p and Ruth Montgomery
http://dx.doi.org/10.1016/j.coi.2014.05.007 0952-7915/# 2014 Elsevier Ltd. All rights reserved.
defective early signaling events, inter/intra-cellular communication, in adaptive and innate cells [5,6, 7–9,10,11]. In this review, we summarize our knowledge on signal transduction changes in the aging immune system and propose a unifying mechanism as one of the causes of immunosenescence. We will provide examples illustrating changes in neutrophils following GM-CSFR and Toll like receptors (TLR) stimulation and, in T cells activated by occupation of T cell receptor (TCR)/CD28. We will also argue for a unique general signaling alteration underlying immunosenescence and inflamm-aging. Alterations in neutrophil signaling with aging
Neutrophils are the first cells to arrive at the site of inflammation [2,9]. Neutrophils use an impressive array of mechanisms to phagocyte and kill intruders [12]. Neutrophils phagocytosis, chemotaxis, free radical production and apoptosis are altered with aging [2,9,13,14]. These functions are elicited by receptor–ligand interactions, suggesting that reduced functionality of neutrophils with aging results from alterations in signaling pathways downstream of receptors. For example, functions mediated by formylmethionylleucylproline (fMLP), Fcg and the C3b receptors are altered with aging which clearly resulted from changes in MAP kinases, Jak/STAT and PI3K-Akt signaling pathways [15–17]. These changes are not due to variations in the number of receptors but to alterations in signalosome formation resulting in impaired cell responses [13]. Additionally, neutrophils of aged individuals display alterations in TLR signaling through MyD88 and IRAKs [18,19]. The number of TLRs is not affected with aging but there is an alteration in the trafficking of pathway-associated signaling molecules in the plasma membrane [9,19]. The inflammasome, an important component of the immune response, is activated in response to an aggression by activating inflammatory caspase-1 and caspase-5 which results in the production of a wide range of cytokines [20–22]. There is however no data on inflammasomes in aging neutrophils and how this translates into inflamm-aging and vice versa, but it is reasonable to assume that neutrophil is a target population.
Immune cell signaling in aging Of the nine conditions associated with aging [1], altered intercellular communication was emphasized as the integration of declining immune responsiveness (immunosenescence) with low-grade inflammation (inflamm-aging) [2–4]. Altered immune functions are associated with www.sciencedirect.com
T cell signaling through TCR/CD28 with aging Feedforward signaling
T cell activation depends on recognition of antigenic epitopes presented by professional antigen-presenting cells (APC) within the context of major histocompatibility Current Opinion in Immunology 2014, 29:105–111
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complex (MHC) molecules (signal 1) at the immunological synapse (IS) where assembly of the T cell signaling processes occur within lipid rafts (LR) [23–26]. The first events of TCR signaling are activation of Lck, phosphorylation of ITAM motifs of the CD3 complex and recruitment/activation of ZAP70/LAT/SLP76 [27,28,29]. Further participation/phosphorylation of accessory proteins assembles the signalosome [30]. Signal 2 provided by costimulation (CD28) leads to full and efficient T cell activation [31] through the NF-kB pathway and prevent anergy [32]. Activation of CD28 results in decreased levels of reduced glutathione (GSH) and production of reactive oxygen species (ROS) [33,34] that may result in increased levels of TNFa [7]. The persistence of low amounts of proinflammatory cytokines and increased production of ROS, which are both features of inflamm-aging converge to hamper T cell responses [5,35–39] via altered signaling. Overall, these observations suggest that the reduced efficiency of the immune response with aging may be due to the dysregulated proximal events of TCR signaling [6,27,39]. In fact, nearly all the proximal events of TCR signaling or IL-2 responses are altered with aging [5,7,40], including impairment of protein tyrosine kinase (PTK) phosphorylation, decreased Ca2+ mobilization and lowered PKC activation that lead to sub-optimal NF-kB and NF-AT activities [36,32]. Feedback regulation of signaling
The activity of a key component of TCR signaling, Lck, is finely tuned by opposite effects of plasma membraneembedded CD45 protein tyrosine phosphatase (PTPase) and cytoplasmic Csk PK bound to the scaffold protein PAG [28,41] and, autophosphorylation. Csk phosphorylation of Lck C-terminal Y505 favors a conformationrelated inactivation whereas CD45-dependent dephosphorylation of Y505 and autophosphorylation of Y394 result in Lck activation [42,43]. Recent data have also implicated FAK as a negative regulator of Lck [44]. Our group has shown that dysregulation of the Csk/PAG/ CD45 loop in T cells of elderly increases the levels of inactive Lck [6], providing a molecular clue to altered T cell responses in aging. In addition, PTPases CD45, SHP1 and Lyp (PTPN22) play a key role in regulating Lck activity [41,45]. Although PTPases have overlapping substrate specificities, there are differences between their actions based on subcellular location, substrate binding, cellular maturation (e.g. T cell subsets) and strength of recruitment in response to TCR stimulation [45–47]. SHP-1 activity is crucial to maintain peripheral T cells in the resting state and to prevent uncontrolled T cell activation due to the fact that Lck is one of the targets of SHP-1 [48,49]. Under strong stimulation, SHP-1 activity is reduced [50,51], tipping the balance toward T cell activation. Since abrogation of SHP-1 enhances the quality of effector T cells functions, it can be suggested that targeting SHP-1 may prove useful as an enhancer of the CD8+ T cell polyfunctionality in immunotherapy [52]. Current Opinion in Immunology 2014, 29:105–111
We have reported that the Csk/PAG loop in activated T cells of healthy elderly individuals was dysregulated and that tipped the balance in favor of the inactive form of pLck-Y505 [6]. Feedback inhibitory events were also compromised in these subjects. In fact, SHP-1 activity was higher in elderly subjects than in young individuals, consistent with decreased T cell responses. Pharmacological inhibition of SHP-1 resulted in recovery of TCR/ CD28-dependent lymphocyte proliferation and IL-2 production to levels similar to young adults. These studies provide a lead for a strategy aimed at modulation of the inhibitory feedback loop of T cell activation by targeting SHP-1 and PTPases. This is supported by recent data showing that inhibition of protein phosphatase activities (DUSP4/6) resulted in increased T cell signaling and function [53,54] and that modulation of inhibitory co-receptors such as PD-1, signaling also via SHP-1, successfully increased the functions of CD8+ T cells [55]. These approaches can serve as a basis to improve immune functions in aged populations at risk (e.g. frail category or patients with impaired immunity). Oxidative status and mTOR regulation of TCR signaling
PTPases are regulated by phosphorylation and reversible oxidation of active site-located cysteine residues [7,37,51,56,57]. Mitochondrial respiration and NADPH oxidase are the major generators of ROS in T cells [58]. It is reasonable to suggest that altered redox potential may cause signaling abnormalities in aging T cells. This is of importance since healthy aging is characterized by increased levels of ROS (O2 , peroxidized lipids) and decreased content of antioxidants (e.g. GSH) [59,60]. Redox-associated stimulation of TCR signaling alters the balance between PTK and PTPase activities [67] favoring the activation. In aging where there is a sustained level of increased ROS [59,61], SHP-1 regulation may be affected and its negative regulatory role would be favored. Inhibition or decreased ROS production should rescue PTPase activity [30,62]. In this situation, the effect of ROS would be blunted and SHP-1 would remain active. However, upon TCR and CD28 stimulation, increased ROS levels which result in oxidation/inhibition of SHP-1 would favor activation of Lck [63]. An alteration in this finely regulated system in aging would lead to aberrant signaling, thus contributing to immunosenescence, inflamm-aging and their adverse effects [7,64]. Recent data have provided evidence that mTOR serine threonine kinase activity may also play a role in T cell activation and differentiation, especially of naı¨ve CD4+ T cells, toward the Th1 or Th17 phenotypes [65]. mTOR signaling pathway activation is under the control of TCR/ CD28 stimulation [65,66]. There are only very few studies on T cell mTOR alterations with aging and most of them come from murine models. Perkey et al. [67] showed that TORC2 signaling was increased in murine CD4+ T cells in aging and its overexpression in CD4+ T www.sciencedirect.com
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cells of young mice reproduced age-related CD4+ T cell functional changes. Our data (unpublished) have suggested differential phosphorylation status (CREB, Akt, S6, ElF4E, 4EBP1) of memory T cells as compared to naı¨ve T cells. Recently, Arnold et al. have reported that TCR stimulation induced autophagy in human CD8+ T cells, whereas autophagy was decreased in CD8+CD28 subset, which largely seems to compromise their survival under antigen stimulation [66]. These emerging data emphasize the importance of mTOR-related metabolic data intertwined with TCR feed-forward and negative feedback and its potential relationship to immunosenescence. Further investigations of the mutual influence of these signaling pathways are fundamental to understand and improve functions in T cells with aging.
Immunosenescence, signaling and inflammaging Immunosenescence can be viewed as the consequence of the immune system trying to adapt and maintain homeostasis in face of persistent intrinsic and extrinsic challenging inputs and outputs during life-time [68]. These include chronic disease states such as diabetes, obesity
and atherosclerosis, nutritional status, genetic background and environmental conditions [69]. Highly differentiated memory T cells accumulate in aged humans [3,4,70,71], likely due to persistent antigenic stimulation, pro-inflammatory environment associated with increased levels of TNFa derived from senescent cells and/or activation of the NLRP3 inflammasome and the NFkB pro-inflammatory pathways [55,37]. Recently the Nlrp3 was shown to control Inflamm-aging in rodents, independently of noncanonical caspase-11 inflammasome. Deletion of Nlrp3 leads to reduction of brain inflammation, suggesting that modulating Nlrp-3-dependent pathways may slow down chronic conditions in the elderly [72]. Thus, the unbalanced changes in adaptive versus innate immunity may contribute to the low-grade inflammatory process especially in syndromes such as frailty [73]. Pro-inflammatory cytokines and acute-phase proteins (IL-6, IL-8, TNFa, C-reactive protein) are two characteristics of inflamm-aging that negatively modulate TCR signaling pathways [74]. This is also true for ROS [27]. Thus inflamm-aging and increased oxidative stress converge to impair T cell activation and functions in aging [75]. For instance, high
Figure 1
cholesterol excess cholesterol
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Changes in lipid rafts composition and function with aging in immune cell membranes including all the susceptible early signaling molecules independently of the immune cells. www.sciencedirect.com
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levels of phosphorylated p38 have been recorded in CD27 CD45RA+ CD4+ T cell phenotype [71]. As p38 activation involves T cell production of pro-inflammatory cytokines (including TNFa) and anti-TNF treatment in rheumatoid arthritis patients depletes CD8+ EMRA T cells [76], it suggests a role for TNFa in the mechanisms generating CD8+ EMRA T cells in vivo. The relationship between cytokine secretion switch and p38 signalingmediated T cell senescence remains to be further defined [71].
A unifying mechanistic explanation of signaling in immunosenescence Lipid rafts (LR), T cells and aging
LR are dynamic cholesterol-rich plasma membrane microdomains that function as organizing centers for
assembly of signaling molecules, protein trafficking and regulation of neurotransmission [77] (Figure 1). In T cells, LR move freely across the plane of the plasma membrane lipid bilayer as discrete entities to assemble the signalosome [78] before or concomitantly with formation of the immune synapse. Whereas some signaling components are constitutive constituents of LR (Lck, Cbp/PAG), others are recruited during activation (TCR, CD28, IL-2R, LAT, PI3K). Our group initially reported alterations in LR in aged humans [39]. Whereas LR were distributed homogeneously in T cells of young subjects, this was not the case in quiescent T cells of older subjects. LR coalescence was altered with aging in TCR CD28 activated T cells. LR poorly coalesced in CD4+ T cells of older subjects but this was less pronounced in CD8+ T cells. Furthermore, recruitment
Figure 2
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Schematic representation of the unified concept of signaling by various receptors on neutrophils (a) and T cells (b) with aging. The signaling molecules shown are only those demonstrated to be altered in aging. Besides the changes in the lipid rafts composition and function which induce alterations in the proximal signaling pathways such as Src kinases and PI3K (feedforward pathways), there are alterations in the feedback regulation pathways mainly mediated by protein tyrosine phosphatases (e.g. SHP-1). These intrinsic changes are influenced and potentiated by the extrinsic milieu of aging such as the reactive oxygen species (ROS) production and inflamm-aging. Current Opinion in Immunology 2014, 29:105–111
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Immune signaling in aging Fulop et al. 109
and activation of Lck, LAT and SHP-1 in LR of aged donors was altered [39,79]. Of significance, CD28 and IL2R were poorly recruited in LR of CD4+ T cells of older subjects. By contrast, these proteins were already located in LR in CD8+ T cells from older subjects before stimulation. These differences may be related to intrinsic regulation of cholesterol metabolism in older subjects [39]. These observations suggested that assembly of the signaling machinery in CD4+ T cells relied largely on recruitment in LR, whereas a preassembly of the signalosome may be present in CD8+ T lymphocytes, as proposed by us and others [8,39,80]. The differential function of LR may be a key factor influencing T cell differentiation. Lipid rafts (LR), neutrophils and aging.
LR are required for neutrophils (PMN) to efficiently fulfill their aggressive protection against intruders (Figure 1). We investigated the role of SHP-1 in aged donors in response to GM-CSF stimulation. Down-regulation of SHP-1 activity was significantly altered with aging. In young donors, SHP-1 was displaced from lipid rafts, but not in the elderly [81]. We observed unbalance of tyrosine/serine phosphorylation of LR-located SHP-1 in PMN of elderly subjects, suggesting that colony-stimulating factor 2 (CSF2) was unable to inhibit serinedependent SHP-1 activity. CSF2 triggered Lyn activation in neutrophils but its activation/translocation into LR was affected with aging. We identified a permanent association of SHP-1 with Lyn in neutrophils of elderly donors that was not reversed by CSF2. Our results suggested alteration of SHP-1 regulation by CSF2 in LR of PMN with aging, explaining the decreased capacity of CSF2 to rescue PMN from apoptosis in the elderly.
Conclusions Increased human longevity represents major social and medical challenges as the incidence of age-related diseases, many of which are influenced by the dysregulation of the immune system, will increase [82]. A large body of data suggests that the immune response is dysregulated in aging. These functional changes may result in serious clinical consequences such as increased infections, cancers and autoimmune diseases. Evidence for alterations in T cell and PMN signaling is widely accepted as an underlying cause of this dysregulation. One unifying cause for these alterations could involve the signaling machinery which combines key signaling and regulatory molecules as well as composition of the plasma membrane (Figures 1 and 2) at least in T cells and PMN as almost no data are available for the other immune cells, however it can be suggested that similar changes may also occur in these cells with aging. Data have provided a basis to pursue studies to understand the molecular mechanisms of T cell signaling deficiencies associated with aging, the involvement of LR, the formation of the immune synapse and the assembly of the signaling machinery. www.sciencedirect.com
Moreover, investigating cholesterol metabolism will help to understand the regulation of cholesterol content in the membrane of T cell during key processes: maturation, activation, differentiation and apoptosis. The role of free radicals in the regulation of membrane integrity, qualitatively and quantitatively for cholesterol content, is of particular importance. We suggest that combined approaches that consist of maintaining adequate early feed-forward activation while down-regulating feedback inhibition would be beneficial in restoring several T cell functions in aging. Current strategies of blocking receptors such as PD-1 are efficiently used in a cancer setting however it is uncertain whether this strategy can be applied in the context of boosting immunity in healthy elderly. Early intervention should reduce the influence of factors that contribute to the increased feedback inhibition and will rescue T cells from exhaustion.
Acknowledgements This work was supported by grants from the Canadian Institutes of Health Research (CIHR) (No. 106634 and No. 106701), the Universite´ de Sherbrooke, and the Research Center on Aging.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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