Abstract
Significance:
Antibacterial defense invokes the innate immune system as a first responder, with neutrophils phagocytozing and forming neutrophil extracellular traps around pathogens in a reactive oxygen species (ROS)-dependent manner. Increased NOX2 activity and mitochondrial ROS production in phagocytic, antigen-presenting cells (APCs) affect local cytokine secretion and proteolysis of antigens for presentation to T cells at the immune synapse. Uncontrolled oxidative post-translational modifications to surface and cytoplasmic proteins in APCs during aging can impair innate immunity.
Recent Advances:
NOX2 plays a role in the maturation of dendritic cells, but paradoxically NOX2 activity has also been shown to promote viral pathogenicity. Accumulating evidence suggests that a reducing environment is essential to inhibit pathogen proliferation, facilitate antigenic processing in the endosomal lumen, and enable an effective immune synapse between APCs and T cells. This suggests that the kinetics and location of ROS production and reducing potential are important for effective innate immunity.
Critical Issues:
During aging, innate immune cells are less well able to phagocytoze, kill bacteria/viruses, and process proteins into antigenic peptides—three key steps that are necessary for developing a specific targeted response to protect against future exposure. Aberrant control of ROS production and impaired Nrf2-dependent reducing potential may contribute to age-associated immune decline.
Future Directions:
Local changes in redox potential may be achieved through adjuvant formulations to improve innate immunity. Further work is needed to understand the timing of delivery for redox modulators to facilitate innate immune cell recruitment, survival, antigen processing and presentation activity without disrupting essential ROS-dependent bacterial killing.
Introduction
The innate immune response is responsible for fast, efficient, and nonspecific clearance of infection and cancers. The clearance function is largely attributed to the activity of phagocytic cells that express pattern recognition receptors (PRRs). These PRRs recognize generic molecular patterns present within or on the surface of microbes, some of which are common with oxidized lipids (12). On recognizing a broad class of pathogens, phagocytes rapidly migrate to the site of infection, ingest and kill pathogens within a phagolysosome (Fig. 1).
NADPH oxidase (NOX) activity is critical for microbial killing by phagocytic cells by generating reactive oxygen species (ROS) that may also react with nitric oxide (NO) to produce cytotoxic reactive nitrogen species within the phagolysosome (66) (Fig. 2). The production of ROS in response to stress is variably reported to be increased in macrophages and dendritic cells (DCs) (from mitochondria) but decreased in neutrophils with age (from NOX). However, the net effect for immune defense is a decline in the ability to effectively remove pathogen (14, 121). This could be interpreted to mean that ROS have a minimal role to play in pathogen removal. Alternatively, these observations may indicate that an overall decrease in immune function with age may relate to the dysregulation of ROS production (wrong time and/or in the wrong place); for example, increased mitochondrial ROS production has been linked to immunosenescence (76) or aberrant regulation of the hydrogen donating antioxidant peptides and enzymes, which results in loss of reducing capacity with age. Table 1 summarizes current knowledge of changes in phagocyte ROS production with age in the respiratory burst and from mitochondria.
Change in Reactive Oxygen Species Production by Phagocytes with Age
BMSC, bone marrow derived stem cell; DC, dendritic cell; fMLP, formyl-methionine-leucine-phenylalanine; IL, interleukin; LPS, lipopolysaccharide; NOX, NADPH oxidase; PBMC, peripheral blood mononuclear cell; ROS, reactive oxygen species.
Opposing the oxidizing side of the redox equation is the capacity to generate reducing equivalents, in part through glycolysis but also via activation of antioxidant gene expression.
Nrf2 is a transcription factor that acts as the master regulator of antioxidant and phase II protective genes. It is responsible for the upregulation of the biosynthetic genes required for synthesis of the major cellular antioxidant glutathione (GSH), including the rate limiting γ-glutamylcysteine ligase. It has been proposed that aging leads to decreased Nrf2 activity, but that it remains sensitive to activation by the electrophilic activator sulforaphane (59), indicating a potential target for restoring age-related immune decline.
This conundrum of variable ROS response and ineffective immunity during aging is the focus of the present review as we explore the relationship between NOX activity, ROS production, thiol donors, and phagocyte function. We highlight how this knowledge may be used to improve the specific, adaptive immune response featuring B and T cells (required for memory, e.g., of vaccination) during aging.
Cells of the Innate Immune System and the Effects of Aging
A range of immune cells play a nonspecific role in engulfing pathogenic or damaged materials in a process known as phagocytosis. There are three major families of professional phagocytes: monocytes/macrophages, granulocytes, and DCs. Neutrophils are the first granulocyte responder cells that arrive at sites of infection within minutes with the goal of killing pathogens through a combination of ROS-mediated oxidative damage and proteolytic degradation (47, 127). They are followed within hours by monocytes that are induced to differentiate into macrophages, for example, by the presence of bacterial fragments such as the peptide formyl-methionine-leucine-phenylalanine (fMLP) and lipopolysaccharide (LPS). However, in the chronic inflammatory disease periodontitis, which increases in frequency with age, neutrophil hyperactivity was observed with low expression of antioxidant genes suggesting a deficiency in an Nrf2-dependent pathway (25).
Each family of phagocytes has some tissue-specific members (some common examples are shown in Table 2). The exception to this is for the bone-marrow-derived cells that are found both within the blood and in tissue. For each of these cell types, emigration from blood into tissue is an important part of their surveillance role to seek out foreign material and tumors.
Major Types and Residency of Specific Phagocytes
cDC, conventional DC; FDCs, follicular DCs; mDCs, myeloid DCs; pDCs, plasmacytoid DCs.
During aging and in the absence of disease, there is no evidence for change in numbers of macrophages or granulocytes although functional deficits in directional migration to infection are observed (105, 123). There are also several subtypes of the phagocytic cells, and the classical definitions, for example, of M1 and M2 macrophages describe M1 as proinflammatory and M2 as playing a role in tissue repair and tumorigenesis. Detailed discussion of these cells is beyond the scope of this review, although it is worth noting that polarization between subtypes is likely to be a continuous process and may, at least indirectly, be ROS dependent (46).
Studies of DC frequency with age are conflicting. Some authors have reported that age is associated with an increase in frequency and maturity of myeloid DC (mDC, recognize a range of pathogens) (24), whereas others have reported no difference in mDC numbers with age (2). The numbers of plasmacytoid DC (pDC) that mediate antiviral responses have been suggested by some authors to be unaffected by age (24). However, others showed an age-associated decrease in pDCs (103). A similar effect is reported for DCs in the spleens of aged mice, which showed reduced frequency of pDC (128). pDCs play an important role in neutralizing virally infected cells by secreting interferons, increasing antigen-presenting capacity, and activating a specific T cell subset, natural killer cells, to kill virally infected cells. Overall, a consistent picture is emerging of a functional decline and decrease in frequency of pDC, but not mDC, in healthy elderly subjects. However, declining health in the elderly associates with loss of mDC. Whether this is cause or consequence has not been explored (54).
Decreases in human Langerhan cell densities have been described in the epidermis of the aging skin (9) again with polarization toward a more mature phenotype (137). Others have shown that follicular DCs (FDCs) in old mice were smaller with lower chemokine CXCL13 expression in response to challenge (117). The FDCs and homing DCs mediate T cell activation, and initiate the first steps of the specific and adaptive immune response. They typically present antigens from invading pathogens to T cells to stimulate the adaptive immune response.
To achieve an antigen-specific response, T cells differentiate into effector T helper 1 (Th1) cells, Th2 cells, and cytotoxic T lymphocytes (CTLs) after contact with antigen-presenting DCs. Effector T cells are important mediators of vaccination responses. Several studies have examined T cell deficits during aging; however, it is possible that defects in DC function with age may underpin the poor vaccination responses typically seen in older adults (116). While increased mitochondrial ROS production during aging may contribution to DC senescence this has not been explored to date. It has been shown that mitochondrial overexpression of catalase inhibited TLR7-mediated activation and crosspresentation, for example, of viral proteins by pDCs to CD8+ T cells without affecting other immune functions (81).
To develop a specific immune response, molecules from foreign pathogens must be presented to the immune system. Macrophages and DCs can serve as antigen-presenting cells (APCs). They phagocytoze the foreign material, process and transport antigenic molecules, for example, derived from ingested pathogens to the lymphoid tissue. They act as a bridge between innate and adaptive immune systems by presenting antigens to naïve B and T cells in lymph nodes in association with costimulatory molecules such as major histocompatibility complex (MHC). T and B cell activation mediate the later adaptive immune response and retain memory of specific antigens from each pathogen that is encountered. Age-associated changes in uptake, processing, cytokine, chemokine, and interferon production, as well as lower expression of costimulatory molecules such as CD86, could contribute to the blunted immune responses, and therefore impaired memory B and T cells to vaccines and infection (10, 24, 72).
Pathogen Recognition by the Innate Immune System: The Effects of Aging
The innate immune response is activated when pathogens and pathogen-associated molecular patterns (PAMPs) are recognized by PRR-bearing cells. They include those with C-type lectin receptor (CLR), scavenger receptor (SR), and Toll-like receptors (TLRs) that are expressed by cells of the innate immune system and on epithelial cells (Table 3). SRs responsible for bacterial clearance also bind oxidized low-density lipoprotein (LDL), for example, CD36, MARCO, and SR1. Due to the similarities between oxidatively damaged host-derived lipids, lipoproteins, and the structural motifs of microbial pathogens, there is often overlap between PAMP and damage-associated molecular pattern (DAMP) recognition (73). In the same way that PAMPs on bacteria are recognized and engulfed, oxidized LDL is also taken up by CD36 and triggers the NLRP3 inflammasome to activate caspase-1, prointerleukin (IL)-1β/pro-IL-18 in macrophages (102). Readers are referred to recent review that explores redox regulation of the NLRP3 inflammasome (1).
Toll Like Receptor Family Distribution Between Phagocytic Cells and Key Ligands That Act as Microbial Pathogen Signals: Effect of Age
PAMPs, pathogen-associated molecular patterns; PRR, pattern recognition receptor; TLR, toll-like receptor.
Oxidized lipids frequently induce an inflammatory response through TLR4 (12, 73, 74). This is at least in part through the binding of oxidized cholesterol to MD-2, an LPS-binding receptor that interacts with TLR4, causing it to dimerize and recruit necessary adaptor proteins (20, 90). This interaction has been shown to induce TLR4-dependent IL-6 and IL-4 production by macrophages (68, 73), and is likely to be a bystander effect of an essential pathway that is normally used for pathogen removal. In contrast, the oxidized phospholipid oxPAPC (oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine) inhibits the ability of innate immune cells to phagocytoze and clear bacteria, increasing disease severity (70).
TLRs are found both as cell surface receptors and as intracellular endosomal membrane receptors, for example, TLR7. Pathogen recognition by surface receptors triggers dimerization, and signals are transduced in the intracellular environment to increase expression of chemokines such as CCL2 and CCL7, thereby facilitating chemotaxis of incoming immune cells to inflammatory sites and phagocytosis.
We have previously reviewed the effect of aging on TLRs in macrophages and did not find any substantive evidence for a change in receptor density with age (30). We proposed that downstream signaling from the macrophage TLRs may be diminished with aging in support of the evidence for age-associated increases in infection (30). Following receptor dimerization after binding to pathogen, intracellular signaling proceeds via Toll/IL-1-like homology receptor (TIR) domain, TIRAP, TRIF, MyD88, and TRAM. Again, evidence is lacking for any age-specific differences in expression of these adaptor molecules during aging (30).
Increased ROS production is widely described during aging, and this associates with an increase in macromolecular oxidation. The lipid oxidation product 4-hydroxynonenal forms adducts with TLR4 and the intracellular MD2 domain cysteine residues, hence preventing the essential dimerization for TLR4 receptor activation. This would be expected in turn to reduce or delay the effector functions of macrophages and impair host defense to infectious agents.
Evidence for the importance of TLR7 cysteine residues for pathogen removal and the negative effect of NOX2-dependent hydrogen peroxide production (presumably after the dismutation of superoxide anion) in macrophages has been reported by To et al. (2017). Endosomal hydrogen peroxide generation was shown to suppress antiviral and humoral signaling networks via modification of Cys98 on TLR7 (111), which inhibits viral products, for example, RNA from binding to TLR7. The authors highlighted an important paradox that emerges from these findings—endosomal ROS are essential for killing but appear to promote viral pathogenicity. Why does such a system exist? The authors suggest that oxidative modification to TLR7 may be necessary to prevent autoimmunity to self-RNA.
Most innate immune cells are short lived; however, engagement of PAMPs with TLR1 in neutrophils prevents apoptosis. In the elderly, reduced expression of TLR1 has been described, implicating a defect in apoptotic regulation with age (93). Cells continue toward apoptosis in the presence of TLR1 agonists but were defective in mitogen-activated protein kinase (MAPK) signaling and chemokine expression. This was attributed to an impairment in glucose uptake and cellular bioenergetics. These findings suggest that neutrophils in the elderly will be less able to kill bacteria based on their shorter life span alone, even if all other functions are intact (13).
Production of reactive oxygen and nitrogen species (RONS) by NOXs, nitric oxide synthase (NOS), and myeloperoxidase (MPO) is central to microbial killing. It is now recognized that PRR activation contributes to ROS production.
Pattern-rich receptors and NOX activation
An adhesion family of G-protein-coupled PRRs has been implicated in pathogen uptake, and results in the activation of NOXs by macrophages through Rac1/2 and p47 phosphorylation (11). In monocytes, protein kinase c theta has been identified as the downstream signaling kinase that activated NOX and phagocytosis of zymosan (fungal) particles after binding to the CLR, dectin 1 (32). After TLR activation, the downstream signaling molecule TRAF6 is pivotal.
TRAF6 lies upstream of MAPK and nuclear factor kappa B (NFκB) activation pathways, both of which are ROS dependent, implicating TRAF6 indirectly in ROS generation. NFκB activation is also required for expression of inducible NOS in phagocytes. A seminal paper by West et al. in 2011 illustrated how TLR signaling via TRAF6 augments ROS by directing mitochondria to phagosomes (124). TRAF6 first translocates to mitochondria, binds to ECSIT (evolutionarily conserved signaling intermediate in Toll pathways), is ubiquitinated, and enhances mitochondrial ROS generation at the phagosome in an Mst1 and MSt2 kinase-specific manner (42, 124). These studies highlight the complexity and potential redundancy of multiple receptors, pathways, and activating signals in different cell types to support effective pathogen removal through ROS production by NOX2 and mitochondria.
RONS Production in the Aging Innate Immune System
The NOX, NOX2, was discovered by Babior in neutrophils in 1979 (39). Since the initial discovery of the multiple component enzyme, NOX2, six further NOX isoforms (NOX1, 3, 4, 5 and DUOX1 and 2) have been described. The organization and activation of the NOX enzyme family have been extensively reviewed, and they share similarities in structure and capacity to produce ROS (8). The expression of different NOX isoforms is cell- and tissue specific. The first ROS to be identified was the superoxide anion radical, which can spontaneously dismutate into hydrogen peroxide under physiological conditions. Hydrogen peroxide potentially regulates the target molecules through reversible or irreversible oxidation of redox-sensitive cysteine residues (97) (Fig. 3). On the contrary, other NOX family members such as NOX4 and DUOX produce hydrogen peroxide directly.
NOX2 was described as essential for the killing of bacterial pathogens by professional defenders in the innate immune system, particularly in neutrophils and macrophages (96). Of the NOX isoforms, NOX2 remains the most significant in terms of quantity and duration of ROS production (84). Despite this, NOX2 has a relatively low Km for oxygen in its resting state. The phosphorylation of different serine residues in p47 by protein kinase c alpha, beta II, delta, and zeta in neutrophils promotes migration to gp91 phox in the membrane, then assembly of the complex (37). Production of the primary radical species from NOX2, superoxide anion radical, is dependent on a supply of NADPH and oxygen (Fig. 1). Superoxide anion radicals are released to the extracellular face of the membrane where they may dismutate to hydrogen peroxide and diffuse across lipid bilayers to mediate intracellular signaling and activate MAPK (129). Conversely, after uptake of pathogen and membrane internalization, superoxide anion radicals are released into the phagosome.
The importance of NOX2 in preventing bacterial infections is highlighted in chronic granulomatous disease (CGD), where patients exhibit persistent bacterial infections (97a). Several mutations have been described in different proteins within the NOX2 complex that give rise to CGD, but ultimately, they all share the same deficit—a failure to produce ROS and to develop an effective phagocytic response.
Bacterial killing would normally take place inside the phagosome. Subsequent fusion with granules rich in enzymes facilitates killing (47). MPO is released from the azurophilic granules into phagosomes where it catalyzes the production of hypohalous acid from hydrogen peroxide and halide ions. Associated with the generation of ROS is an immediate rise followed by a sustained rise of pH in the phagosome, which has been suggested to be an essential part of the killing process (80, 101). An ineffective NOX2 complex, as seen in CGD patients, leads to accumulation of bacteria and the formation of granulomas within tissue.
The literature concerning innate immunity and ROS production by innate immune cells during aging is conflicting as summarized in Table 1 (5, 6, 9, 14, 24, 121). This may relate to study design. There is an inherent difficulty in studying aging in vivo due to the length of time for an aging phenotype to be evident. Most human studies take a cross-sectional snapshot of different aging populations and do not follow the same individual over time. Instead, studies frequently compare groups of older adults with younger adults with the assumption that older people would be physiologically worse off. However, humans who live to older age when surrounded by pathogens may be considered the survivors of biological aging, and this is likely to be a consequence of their genes and environmental mix. Older survivors may express a unique survivor phenotype, and this may not be typical of changes seen during aging in the majority of the population.
Animal model studies of aging enable longitudinal studies to be performed and at the very least, use animals of the same genotype within the same environment. However, despite numerous studies designed to explore NOX2 knockout on the aging process, some describing benefit and others showing increased risk, only two have looked at effects on the immune system. One showed that joint inflammatory and immune responses were enhanced with age in the absence of NOX2, supporting a crucial role of NOX2-dependent ROS generation in the modulation of Th17/Treg cell development during aging (65). In contrast, another study showed the ablation of NOX promoted neutrophil recruitment in the lung but reduced inflammation (139). A third study showed that NOX2-dependent ROS activation was higher in older animals and was enhanced by mitochondrial ROS, suggesting intracellular crosstalk via ROS (63). However, functional outcomes were not reported.
Mitochondrial and NOX-derived ROS in the phagosome during aging
Our understanding of how TLR signaling to ROS production is affected during aging is limited. One study has shown that TRAF6 is less ubiquitinated in lung macrophages from older mice, which are less able to activate NFκB and phagocytoze stimuli (50). This might also be expected to reduce endosomal ROS released because the efficiency of mitochondrial trafficking to the phagosome is impaired; however, mitochondria themselves release more ROS in monocytes with aging (88). The interplay between mitochondrial ROS and NOX activation in phagocytes could contribute to increases in phagosomal ROS during aging (63), which may impede antigen processing. Taken together, these studies suggest a complex interplay between TLR activation, ROS production, and signaling, which may be cell- and receptor specific (57).
ROS and infection in older adults
In human cross-sectional studies, it has been shown that with age, immune defense is compromised. In older adults, there have been conflicting reports about the extent to which impaired ROS production might contribute to an increase in infection rate and failure to kill pathogens effectively. In the neutrophil, few studies have reported an increase in ROS production (14, 15, 86, 100), with no change in MPO products (14), whereas the majority have reported a decrease in ROS production in response to some (e.g., fMLP) but not to other (e.g., zymosan) ligands (13) with aging (Table 1). In a rat model, a similar deficit in neutrophil respiratory burst in response to the bacterial peptide fMLP was reported. This was attributed to an increase in membrane fluidity due to declining cholesterol/phospholipid ratio and increasing PUFA, although it is difficult to appreciate why this might affect the fMLP activation response exclusively (4).
In older adults, infections frequently develop during hospitalization, for example, after a fall. Baehl et al. explored whether the stress associated with hip fracture had any influence on neutrophil function. They showed that the respiratory burst was impaired shortly after the event, and that this effect was sustained despite restoration of phagocytic ability (6).
In monocyte/macrophages, a mixed picture has emerged of a decrease in stimulus-induced ROS production by monocyte NOX (5, 14) with age, although low-level activation may persist with others describing an increase in ROS from mitochondria and from NOX (88, 138).
Circulating monocytes are versatile precursors with the ability to differentiate into a variety of macrophages (43). Different macrophage subsets develop in response to the local cytokine milieu, and play a role in the regulation of innate immune responses to acute and chronic inflammatory stimuli. The M1 macrophage is associated with chronic inflammation, whereas an M2 phenotype promotes healing, tissue remodeling but also cancer. Mitochondrial-derived ROS have been shown as early differentiation signaling to yield M2 tissue-associated macrophages, and it has been proposed that inhibition of mitochondrial ROS, which is elevated during aging, may be a useful cancer target. In summary, dysregulation of macrophage differentiation may lead to defects in wound healing, development of autoimmunity and cancer (35), all common traits in older adults.
Redox Regulation of Phagocyte Function
Innate immune cells have the capacity to sense danger and migrate in a directional manner to pathogens. This is achieved through tethering of leukocytes to the vascular wall local to the inflammatory site. This process is enhanced by circulating oxidized LDL (74). Oxidized phospholipids can modulate vascular endothelial cell adhesion molecule expression, whereas 7-ketocholesterol increases leukocyte MAPK activation and expression of adhesion molecule ligands (45, 110). After adhesion to the endothelium, leukocytes undergo diapedesis into tissue. The first step toward limiting infection or removing damaged tissue is phagocytosis of foreign material. To achieve this, neutrophils exhibit active actin dynamics involving local polymerization and depolymerization, by a ROS-dependent mechanism and oxidation of thiol moieties (99).
The homeostatic control of thiol redox state is supported by transient mixed disulfide formation between cytoskeletal thiols and GSH. To restore thiols and function to actin, deglutathionylation is catalyzed by the enzyme glutaredoxin (Grx, Fig. 3). Grx is a small thiol transferase that removes GSH adducts from proteins (protein -S-SG) using either a monothiol or dithiol mechanism and participates in redox signaling.
Using Grx1-deficient mice, which do not remove GSH from mixed disulfides, it has been shown that NOX2-dependent ROS generation negatively regulates actin polymerization in neutrophils through irreversible glutathionylation (99). Similarly, Grx1-negative neutrophils showed impaired actin polymerization and chemotaxis to sites of inflammation. In addition, adhesion, phagocytosis, and bactericidal activity were all inhibited when Grx1 was absent. In another example of RONS-dependent changes in actin polymerization, Garcia Ortiz et al. (41) have studied the immune synapse, which is a localized clustering of receptors in lipid rafts within different types of immune cells that are required to communicate for an effective immune response. After receptor crosslinking between immune cells, the recruitment of a serine/threonine-specific protein kinase C theta is necessary for the formation of signaling complexes and downstream cytokine transcription.
It has been elegantly demonstrated that NO generated by endothelial NOS controls the clustering of protein kinase C-theta in the immune synapse. The molecular mechanism that underpinned the effect of NO was due to S-nitrosylation of beta-actin on Cys374, which prevented effective polymerization and translocation of protein kinase C (PKC)-theta to the immune synapse. Actin binding to profilin-1 was impaired and PKC theta activation, necessary for NOX activation, was diminished. Collectively, these examples illustrate how a reducing environment may favor phagocytosis and immune activation.
GSH is a cysteine-containing tripeptide that is common to all mammalian cells. Combined with NADH and NADPH, it forms the major source of cellular reducing potential. Under oxidizing conditions, it is rapidly oxidized to form glutathione disulfide (GSSG) or to form mixed disulfides with redox-sensitive thiols in cellular proteins. Through a sequence of reactions with other reducing enzymes/proteins, it is largely responsible for repairing reversibly oxidized thiols (Fig. 3). These are largely under the regulatory control of redox-sensitive transcription factors such as Nrf2. Normally, Nrf2 has a short half-life because it is maintained in a complex with KEAP1 and Cullin 3, which ubiquitinates Nrf2 and transports it to the proteasome for degradation. Under oxidative stress, KEAP1 becomes oxidized, dissociates and Nrf2 accumulates, migrating to the nucleus to promote expression of antioxidant proteins.
As intracellular reducing potential declines in phagocytic cells(122), Nrf2 is activated and more reducing proteins related to the GSH cycle are synthesized; however, this process is impaired during aging (31). GSH- and GSH-related enzyme activities decrease with age (19). Glutathione reductase (GSR) expression is regulated by Nrf2 and catalyzes the reduction of GSSG back to GSH. Interestingly, it has been shown that GSR-null mice exhibit elevated sensitivity to Staphylococcus aureus. Consistent with the findings in Grx1-deficient mice, neutrophil phagocytosis and bacterial killing were impaired in GSR-null mice (130).
In human studies of opsonized bacteria including S. aureus, yeast, and zymosan uptake, a significant reduction in phagocytic ability was observed in neutrophils from older adults (33, 71, 123). One study has shown that phagocytosis of Candida is unaffected by aging (27). None of the studies in humans have explored whether s-nitrosylation or glutathionylation of actin or other cytoskeletal regulatory proteins in neutrophils may contribute to impaired phagocytosis.
Once taken up into a phagosome, microbes are killed by ROS, which serve an additional role in processing of antigenic peptides for surface display by APCs (3, 62). Killing is achieved by fusion of the phagosome with lysosomes that release enzymes such as MPO, lysozyme, and elastase in concert with ROS generated by NOX activity (47, 75). In addition to producing cytotoxic ROS, the acidification of the vacuole to around pH4 is bacteriostatic and may provide an optimal pH for lysosomal cysteine protease activity required for processing of antigens and development of the adaptive immune response (23, 34).
A more recently described phenomenon that operates as the neutrophil dies is the release of neutrophil extracellular traps (NETs). NETs comprise of nuclear material and granule enzymes that entrap bacteria and promote the clearance of microorganisms (104). It has been shown that hypochlorous acid is required and mediates the formation of NETs in humans (85). It is therefore not unexpected that in older adult neutrophils, which may produce less ROS, LPS- and IL-8-induced NET formation exhibited a significant age-related decline (49). Furthermore, GSR-deficient neutrophils display a marked impairment in NET formation. The latter studies suggest that GSR-mediated redox regulation is required for bacterial clearance via NETs (130). The induction of genes that are responsible for GSH homeostasis such as GSR, Grx1, and gamma-glutamyl cysteinyl synthase, for example, by Nrf2 activators may provide an important strategy for restoration of neutrophil-mediated killing efficacy during aging (58, 104).
NOX is required for macrophage polarization
ROS generation by NOX2 has been implicated in the polarization to M2 macrophages, but not M1 macrophages in the mouse (129, 138). As discussed earlier, the activation of MAPK enzymes is important in immune cell activation, and they are involved in macrophage polarization. These include the kinases jun kinase and extracellular receptor kinase, which are redox sensitive. Macrophage differentiation in NOX1/2 knockout animals was impaired with an inability to polarize toward an M2 macrophage phenotype (129). This was associated with a delay in wound healing and the inhibition of tumor growth and metastasis. In summary, these authors suggested that NOX1/2 KO enhances tumor growth. In contrast, a study of p47phox-/- mice showed an increase in production of M2 macrophages that was associated with decreased ROS production (131).
To gain a better physiological understanding of the importance of NOX-dependent ROS signaling for aging and longevity, knockout models of the adapter protein p66Shc have been developed. P66Shc is an adapter protein that links activation of the growth factor receptor and the downstream MAPK cascade, and can be directly activated by H2O2. The p66Shc−/− mouse is unresponsive to growth factor activation and is long lived. It is characterized by a decrease in macrophage ROS production, and is associated with a reduction in inflammation and increased longevity (112). Lower NOX activity in macrophages was suggested to contribute to increased longevity. Further work will be necessary to unravel why different knockdowns that inhibit NOX2 can on the one hand promote a reduction in inflammation and longevity, but on the other hand impair M2 polarization.
Table 4 summarizes our present understanding of the cellular localization of different NOX isoforms within cells that are responsible for immunity. No systematic study has been undertaken to explore whether expression of NOX and DUOX changes in immune cells during aging; however, studies in endothelial cells suggest that NOX4 is upregulated with age and may contribute to paracrine oxidative stress (67, 120).
NADPH Oxidase Localization in Cells Involved in Immune Defense: The Effect of Aging
ND, not determined.
DCs: At the Interface Between the Innate and Adaptive Immune System
DCs are one of a few professional APCs. In common with other phagocytes, they recognize PAMPs and DAMPs from exogenous pathogens and tumor cells in the periphery and phagocytoze them or take them into the cytoplasm by pinocytosis. DC activation by TLR4 ligands induces their homing to lymph nodes where they engage with naïve T cells and promote adaptive immunity (60). Antigen presentation in the lymph nodes is critically controlled to ensure that self-antigens are tolerated and foreign antigens are targeted.
DCs in aging
Three key signals are necessary to induce a T cell response, and these are provided by DCs. These are antigenic peptide loaded onto one of three MHCs (MHC I–III), proinflammatory cytokine secretion, and expression of the costimulatory surface antigens, CD80 and CD86. To produce these signals, DCs must first phagocytoze and process the antigen (Fig. 2).
DCs from elderly individuals have a significantly reduced capacity to phagocytoze pathogens, an impaired chemotactic response to SDF1, increased proinflammatory cytokine secretion after TLR engagement (2), impaired IL-12 and interferon alpha (IFN-α) production by mDCs and pDCs, respectively (24, 103). TLR-mediated expression of CD80 and CD86 is critical for vaccine immunity. Older adult TLR-induced CD80 expression in monocytes is lower than that in young adults, and is associated with an impaired response to vaccination (118). These studies highlight the need to better understand the biology of DC maturation to support strategies that can improve immune response.
After pathogen uptake into a phagolysosome, proteins are degraded by cathepsins, lysozyme, and elastase and ROS, then the digested peptides may become ligands for MHC II receptors within the phagosome membrane. This type of proteolysis creates peptides of 14–20 amino acids in length that are alternatively loaded into MHC II complexes (3). Peptide-loaded MHC-II molecules are delivered to the cell surface either directly or via an intermediate tubular MHC-II storage compartment where they will ultimately activate the classical CD4+ T cell response.
MHC I molecules are usually loaded with peptides derived from cytosolic proteolysis of intracellular antigens for activation of effector CD8+ CTL to promote the killing of virally infected cells. Foreign antigens within the cytoplasm are degraded by the immunoproteasome into peptides of between 8 and 10 amino acids. These are then loaded into MHC I complexes within the ER and presented on the cell surface. However, phagosomes within a subclass of DCs are specialized for crosspresentation, an unusual process by which phagolysosomal protein antigens provide ligands for MHC-I for activation of effector CD8+ CTL.
In older adults, crosspresentation is defective (21, 134). However, in vitro scavenging of ROS partially restores crosspresentation by aged DCs, suggesting that excess phagolysosomal ROS may prevent crosspresentation. In support of an important role for reducing activity in the endosomal lumen, gamma-interferon-inducible lysosomal thiol reductase is a thioredoxin (Trx)-related oxidoreductase found in DCs. It reduces disulfide bonds, promotes unfolding, and assists in MHC class II-restricted antigen processing, for example, through exposing cryptic antigens that are normally present in disulfide crosslinked proteins and also in MHC class I-restricted crosspresentation (125).
Redox regulation of antigen presentation by DCs
The development of a CD8 cytotoxic T cell response through crosspresentation of cytoplasmic peptides is important for tumor immunity and for overcoming viral evasion tactics. In contrast to the inhibitory effects on crosspresentation ascribed to ROS in aging DCs, NOX2 has been proposed to promote crosspresentation that are essential for defense against viral infection. An elegant study of dengue-virus-infected DCs using genome-wide transcription analysis showed three early and discrete responses to infection, STAT1 signaling, NFκB activation, and an Nrf2-dependent oxidative stress response that arose after an increase in NOX2-dependent ROS production. ROS were essential both for inducing apoptosis in infected cells and for activation of bystander cells (83). ROS production by DUOX has also been identified as host protective in mucosal cells against influenza infection, by inhibiting the spliceosome required for RNA processing (107).
In DCs, NOX2 activation changes the endosomal lumen pH to inhibit lysosomal proteases and prevents excessive antigen degradation (62). ROS-mediated oxidation of redox-sensitive thiols on cathepsins will also impede endosomal peptide processing (3), promote endosomal membrane lipid peroxidation, and release antigen into the cytosol for proteasomal degradation (26). Similarly, Kagatani has described how DC maturation is enhanced by sensitizers that oxidize cell surface thiols but inhibited by the reducing agent, N-acetyl cysteine (55). DC maturation under oxidizing conditions was associated with an increase in p38 MAPK signaling.
In contrast, others have shown using p47phox and catalytic subunit NOX2-deficient DC that ROS are required to regulate DC IL-12p70 expression and inhibit inflammation, reducing CD4+ Th1 activation. Similarly, GSH depletion in APCs was correlated with impaired antigen processing and reduced secretion of Th1 cytokines, while an increase in intracellular GSH content increased the expression of inflammatory IL-12 and increased differentiation of naive CD4+ T cells to Th1 cells. Moreover, GSH inhibited the replication of viruses and bacteria (38). O2 •− produced within phagolysosomes rapidly dismutates to H2O2, and regulates PI3K and p38-MAPK signaling pathways, so reducing IL-12p70 expression (53).
Zanoni et al. have demonstrated that oxPAPC and LPS bind to separate domains of caspase 11 in DCs, which leads pro-IL-1β cleavage yielding IL-1β and other synergistic inflammasome-mediated events. LPS alone is able to dimerize TLR-4 and activate pyroptosis; however, the addition of oxPAPC increases DC viability and is thought to prime the inflammasome in such a way as to hyperactivate DCs and increase T cell activation through IL-1β. It is noteworthy to mention that oxPAPC failed to stimulate macrophages; this could in part be explained by DCs existing in more highly primed state than macrophages (136).
Antigen processing is therefore affected by the extent and locality of ROS production or oxidized phospholipid. There is a suggestion that ROS production in DCs may be important at least for crosspresentation in health, but there is ongoing debate about the importance of ROS in these processes (106). In a recent study using gp91−/− mice and the cytochrome inhibitor apocynin, NOX2-dependent ROS formation was not required for maturation of bone-marrow-derived DCs or for T cell activation Similarly, functional analysis of the role of NOX2 in human mDCs showed that NOX2-dependent superoxide anion production did not play a role in DC differentiation, maturation, cytokine production, or induction of T cell proliferation, but was essential for intracellular bacterial killing (44, 106).
Some of these seemingly contradictory studies may be due to a focus on ROS derived from NOX2 only. As described earlier, mitochondrial recruitment and enhancement of ROS production within the endosomal compartment are proving to be of great significance to macrophage function and polarization. As mitochondria leak increasing amounts of ROS with age but without the corresponding pH regulatory potential of NOX2, excessive antigen processing may occur in the endosome, so minimizing crosspresentation by DCs in older adults. The other key component to redox state is the extent of reduced cofactors such as NADPH and thiol-protective GSH present in cell compartments. These are dependent both on metabolism and on Nrf2-dependent gene expression.
In an aging animal model with reduced contact hypersensitivity, Kim et al. showed that poor immune response was likely to be due to poor presentation by DCs through decreased Nrf2-mdeiated antioxidant enzyme expression and GSH synthesis. While transfer of old DCs to young mice impaired the immune response, both the presence of N-acetyl cysteine or sulforaphane to induce GSH synthesis restored the in vivo challenge response (59), highlighting the potential to boost Nrf2 during aging to enhance immune response.
T Cells in Aging
The interface between the innate and adaptive immune responses is at the immune synapse, which is formed between T cells and APCs, such as DCs. An impaired synapse in the elderly could explain increased risk of autoimmunity, infection, and poor vaccination response. Several studies have shown that the ability of macrophages and DCs to activate T cells from older adults and to stimulate T cell proliferation is reduced with aging whether the DCs were from young or older adults suggesting an inherent T cell deficit (72, 133). An effective and sustained T cell response requires a reducing environment (38, 113).
T Cell Recognition of Presented Antigen
Once naive T cells recognize foreign antigenic peptides within an MHC complex on the surface of APCs, they mature into effector T cells. One of the major subsets activated by MHC I carrying peptides is the CD8+ T cell. Activated CD8+ T cells do not prevent infection but target infected cells to reduce, control, and clear intracellular pathogens, either directly by killing infected cells through release of perforin or indirectly by killing infected cells through antimicrobial cytokine release. Similarly, CD4+ Th1 cells do not prevent but help reduce, control, and clear extra- and intracellular pathogens by producing interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α)/-β, IL-2, and IL-3, supporting the activation and differentiation of antibody producing B cells, CD8+ T cells, and macrophages. CD4+ Th2 cells produce IL-4, IL-5, IL-13, IL-6, and IL-10, and support B cell activation and differentiation. Once activated, T cells no longer home to the lymph node, but instead they circulate to the area of infection where they will be triggered by recognition of antigen to release the cytolytic molecule perforin or effector cytokines depending on the T cell type.
The decision to proliferate and differentiate into Th1 or Th2 effector T cells is dependent on energy supply and the cytokines released by DCs. IL-12 and IL-27, for example, are Th1 polarizing signals, whereas the chemokine CCL2 is a Th2 signal, and both yield an antibody response. Lipid metabolism plays a key role in the switch to proinflammatory and autoreactive T cells, in a manner that is intrinsically linked to redox state (16) and aging (113). Knockout of thioredoxin inhibitory protein (TxNIP), that is, the reducing protein Trx (linked to GSH, see Fig. 3) was active, resulting in inhibition of lipid metabolism (82). In addition, fatty acid metabolism at the mitochondrion, which is a net consumer of reducing potential, would be expected to yield increased mitochondrial ROS production, particularly with age. Modulating lipid metabolism, which is intrinsically linked to redox state, is an intense area of investigation for repurposing drugs to modulate aberrant immune responses (51).
Redox regulation of T cell proliferation in response to antigen
The ability of T cells to generate specific response to antigen is dependent on the oxidation state of cell surface and cytoplasmic protein-thiols (91). Intracellular thiols are maintained in their reduced state by a network of redox regulating peptides, proteins, and enzymes such as GSH, Trx, and Trx reductase (Fig. 3).
Yodoi's group were the first to clone cloned human Trx as adult T cell leukemia derived factor produced by HTLV-I transformed cells and then to demonstrate that overexpression of Trx results in resistance to oxidative stress and a possible extension of life span (77, 109). Later studies have shown that overall life span is not affected by overexpression of Trx1, but that the early part life span is improved, particularly in males (92). Further studies that focused on calorie restriction, known to slow aging in animal models, confirmed that the decline in Trx and GSH observed with age is mitigated by calorie restriction, indicating that age-dependent redox change can be mitigated by calorie restriction (19).
We have previously shown that healthy older adults have reduced lymphocyte surface expression of Trx1 and lower circulating plasma Trx1 concentrations. This is associated with reduced proliferation and IL-2 production. Correspondingly, it would be anticipated that more T cell surface cysteines would be oxidized (17). This has been investigated in a systems biology approach to examine the immunopeptidome. It revealed an over-representation of cysteine-containing peptides as T cell epitopes. They comprise between 5% and 10% of the immunopeptidome from a study of 70,000 peptides. Many of the cysteine residues in peptides were oxidized and expressed as S-glutathionylated peptides. This was more prevalent for presentation to virus-specific T cells and may represent a mechanism for evading the virus-specific T cell response (114).
Similarly, it might be predicted that increased peptide glutathionylation would occur due to an increasingly oxidative environment in aging DCs, and that this might also reduce T cell recognition and response to pathogen. Indeed, persistent pathogens like cytomegalovirus that are common in older adults may also evade the effector pathway through peptide glutathionylation. An increase in intracellular DC GSH content stimulated IL-12 and/or IL-27, which in turn induced differentiation of naive CD4+ T cells to Th1 cells (38).
Together, these lines of evidence support the importance of using agents that modulate intracellular redox state in the innate immune DC to improve antigen-specific immunity at the immune synapse. An additional benefit of increased Nrf2 activation for APCs at inflammatory sites is the ability to resist oxidative damage and cell death (122). The timing and extent of reducing versus oxidizing environment are thus predicted to exert a strong influence on and the effectiveness of adaptive immunity.
Therapeutic Targets for Redox Modulation in Vaccination
If dysregulation of redox state plays a role in an impaired immune response during aging, it is anticipated that restoration of the reducing and oxidizing potential at specific sites and times may improve innate immune defense (Fig. 4). Cells of the innate immune system derive from bone marrow where increased ROS production is attributed to resident stromal cells in the aging niche (108). In bone-marrow-derived stem cells (BMSCs), increased expression of NOX during aging is a major source of ROS and associates with senescence. Apocynin, a free radical scavenger and NOX inhibitor, restored mitogenic potential in BMSCs and in vivo (108). Dysregulation of endothelial redox state may also influence BMSCs. This raises the possibility that cells of the immune system are a bystander for aging effects of ROS generated by vascular cells.
However, it is important to remember that ROS are inherently unstable with a half-life of nanoseconds, with limited capacity to diffuse beyond their site of production. Nevertheless, peroxynitrite and hypochlorous acid produced by endothelial cells may diffuse within blood vessels and potentially may oxidize migrating immune cells.
Distant effects of senescent endothelial cells on BMSCs have also been reported, reducing both their stemness and proliferative capacity. Aging endothelial cells express elevated NOX4, produce more ROS, show enhanced activation of NFκB, and secrete proinflammatory cytokines, resembling a senescence-associated secretory phenotype (28, 29). Coculture of senescent human umbilical vein endothelial cells enforced proinflammatory cytokine secretion by bone marrow–mesenchymal stem cells (MSCs) and increased expression of miR-126a-3p, which targets the stemness gene, SOX2 (64).
The antioxidant TEMPOL has been used effectively to maintain endothelial function in aging mice by reducing NOX4 expression and decreasing ROS production; however, whether TEMPOL's protective effects either directly or indirectly via endothelial cells can preserve BM MSC function during aging is unknown (36). Similarly, activation of endothelial SIRT1 in aging mice by SRT1720 was also effective in reducing inflammation, although the mechanism was independent of NOX4 expression but related to increased antioxidant enzyme expression and enhanced cyclooxygenase 2 signaling (40). Targeting the ROS production by NOX4 in the endothelium during aging as a systemic driver of paracrine aging, for example, of vascular or immune cells is an underexplored area that merits further study (67).
After exiting bone marrow and entering the circulation, phagocytes recognize and take up pathogens via PRR. This leads to activation of NOX-dependent intracellular signaling and inflammatory cytokine production (124). There is evidence for molecular mimicry between DAMPs and PAMPs, such that endogenously produced oxidized lipids are also potent PRR activators (74).
Stimulation of macrophages by oxidized cholesteryl esters has been shown to cause lipid buildup through macropinocytosis and the release of inflammatory cytokines (20). This is at least in part through the binding of oxidized cholesteryl to MD-2, an LPS-binding receptor that interacts with TLR-4, causing it to dimerize and recruit necessary adaptor proteins (20, 90), then to increase IL-6 and IL-4 production by macrophages (68, 73). As the purpose of vaccination is to increase the production of cytokines from phagocytes, one potential redox target that could be manipulated to enhance vaccination responses is to present any desired antigen within an oxidized lipid formulation (135). Liposomes synthesized using oxidized lipids could be explored in vaccine adjuvant design, particularly for the development of Th2-biased vaccines for production of neutralizing antibodies.
Another opportunity for redox manipulation in vaccine design is to tackle the role of NOX2 activity in antigen presentation. NOX2 generates superoxide anion radicals in the endosomal lumen of DCs that dismutate to hydrogen peroxide and inhibit proteolytic activity (98), so limiting excessive degradation of peptides. Moreover, in aging increased mitochondrial superoxide production (21) may impair antigen-presenting function due to disulfide crosslink formation (114). In this case, the thiol donor N-acetyl cysteine is protective (21). After activation and homing to lymph nodes for encounter with naïve lymphocytes, increased GSH also positively regulates the later adaptive phase of immune function through increasing IL-12 and IL-27 production, thus promoting a Th1-mediated immune response (38) and viral protection.
Looking Forward
The importance of ROS in adaptive immunity is well documented; however, the redox modulatory potential of adjuvants merits further exploration. From a vaccinology standpoint, oxidized lipids are appealing new areas of adjuvant development and discovery. The oxidation state of the delivery system of vaccine antigens such as liposomes and virosomes could yield more potent adjuvants able to elicit a greater immune response, particularly in older adults.
Using adjuvants, timely manipulation of the redox state with reducing agents, enzymes such as Trx1 (132) or Nrf2 activators may guide the immune response through to T cell interactions with APCs. In combination with other vaccine adjuvant strategies to further extend antigen release times, improve antigen uptake, or prolong depot effects, the addition of redox modulators could offer a new redox-tailored approach to adjuvants.
Footnotes
Funding Information
The authors gratefully acknowledge support from the Biotechnology and Biological Sciences Research Council and Mologic Ltd through CASE funding of a studentship for MCOR (BBSRC BB/M017087/2).