Dendritic Cells and Dysregulated Immunity in the Elderly
Dendritic cells play a central role in generating immunity against foreign antigens and are also crucial in maintenance of tolerance in the periphery and in the mucosa. Chronic inflammation is the underlying cause of most diseases associated with old age. Increased susceptibility to infections particularly of the respiratory mucosa is another characteristic of advancing age. This chapter discusses how the age-associated modifications in dendritic cells functions can account for the above-mentioned aged signatures in particular for humans.
KeywordsWest Nile Virus Aged Mouse Aged Donor Respiratory Viral Infection Common Myeloid Progenitor Cell
Advancing age has a profound effect on the immune system. The capacity to mount effective immune responses against foreign antigens decreases concomitantly as the reactivity towards self increases (Liu et al. 2011). Dysregulation of the immune system at both innate and adaptive levels contributes to the change. Age-associated alterations in the adaptive immune system are more obvious with decline in naïve T cell numbers due to involution of thymus (Brunner et al. 2011). In contrast, the modifications in innate immune system cells are subtle but together cause extensive damage.
Among the innate immune cells, antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages bridge the innate and adaptive immune systems (Steinman 2012); thus age-associated alterations in their functions also impact the functions of downstream adaptive immune cells. The activation of T and B cells, polarization of T helper (Th) cell responses, and generation of effector and memory cells are all governed by APCs particularly DCs as they are initiators of the immune response. Age-associated changes in DC function can thus significantly impact the immune status of the elderly.
6.2 Dendritic Cell Numbers and Phenotype
DCs are rare cells of the immune system which were discovered by Steinman and Cohn (Steinman et al. 1979) in 1979. DCs are derived from hematopoietic stem cells (HSCs) through gradually restricted precursors (Chopin et al. 2012). There are two major subsets of DCs – the myeloid DC (mDC) which are derived from common myeloid progenitor cells (MDP) and the plasmacytoid (PDC) which are lymphoid in origin and are morphologically similar to B plasma cells. Both subsets are widely distributed among all tissues though the myeloid DCs are more abundant than PDCs. An alternative DC developmental circuit occurring after the MDP stage involves monocytes. Under inflammatory conditions, monocytes migrate to the tissues to differentiate into monocyte-derived DCs (Liu and Nussenzweig 2010). This property of monocytes to differentiate into DCs (MDDCs) is used extensively in the laboratory to generate DCs. These MDDCs resemble mDCs in function and phenotype.
Two major populations of DCs have been identified in the blood, the myeloid or conventional DCs (cDCs, to distinguish them from other myeloid DCs present in tissues) and PDCs (Cao and Liu 2007). Extensive characterization of DCs in the circulation has been performed in aged humans. Most reports suggest that numbers and phenotype of cDC populations is not dramatically altered with age; however, various studies did observe a reduction in the number of PDCs in circulation (Jing et al. 2009; Agrawal et al. 2007; Della Bella et al. 2007; Pietschmann et al. 2000; Uyemura et al. 2002; Panda et al. 2010; Perez-Cabezas et al. 2007; Shodell and Siegal 2002; Canaday et al. 2010; Steger et al. 1996). The expression of costimulatory (CD40, CD80, and CD86) and major histocompatibility complex (MHC) markers (HLA-DR) was also comparable between the aged and young cDC and PDC population. However, few studies also reported decreased expression of HLA-DR on cDCs (Shodell and Siegal 2002). In contrast to healthy aged population, the frail elderly populations which reside in nursing homes tended to display more differences in numbers and phenotype of DCs (Jing et al. 2009). Both the numbers and expression of DC activation markers were reduced in this subset of the elderly. In addition to cDC and PDC subsets, MDDCs have also been extensively studied in aging. The generation of MDDCs as well as the expression of DC markers is not reported to be different between aged and young subjects (Agrawal et al. 2007; Uyemura et al. 2002; Steger et al. 1996).
Studies describing DC phenotype and function in tissues from aged subjects are scarce due to the limitation in obtaining the material nevertheless there is sufficient data regarding age-associated changes in DCs in skin and oral cavity. DCs in the skin are myeloid in nature and express Langerin granules and are therefore called Langerhans cells (LCs). A reduction in the number of epidermal LCs in has been reported in elderly subjects (Bhushan et al. 2004). However, in monocytes differentiated into LCs, no significant difference was observed in the numbers or in the expression of activation markers in the aged subjects relative to young (Xu et al. 2012). Another study (Bodineau et al. 2007, 2009) examined intraepithelial LC during chronic periodontitis in elderly patients and reported a decrease in the numbers. They also observed morphological changes in LCs in that LCs from the elderly were more rounded with fewer numbers of dendrites which would affect their T cell stimulation capacity. Thus, it seems DC numbers and phenotype display more age-associated changes in tissues as compared to that in circulation in humans.
Similar to humans, DCs in tissues of mice displayed significant age-associated changes. In general, the cDC numbers in the spleen and lymph nodes were similar to young, whereas the number of cDC in the lungs increased in aged mice (Stout-Delgado et al. 2008; Tan et al. 2012). Several studies in mice have determined the age-associated modifications in DCs in the brain due to the strong correlation between neurodegeneration and immunological changes. Increased levels of CD11+ DCs were observed (Stichel and Luebbert 2007) throughout the brains of older mice while in the young DCs were only visible in the meninges and choroid plexus. These findings were further confirmed by Kaunzner et al. (2012), who showed increased accumulation of DCs in aged brains when compared to younger control animals. Thus, it seems DC numbers can be increased or decreased with age depending on the anatomical location.
6.3 Dendritic Cells and Immunity in the Elderly
6.3.1 TLR and Cytokine Secretion
DCs are key players in generation of immunity to foreign antigens and maintain tolerance to self antigens (Steinman et al. 2000). DCs distributed throughout the body are armed with pathogen recognition receptors (PRRs) which allow them to sense and respond to threats. Engagement of PRRs leads to DC activation characterized by upregulation of several costimulatory and APC surface receptors as well as pro-inflammatory cytokine secretion. PRRs can be divided into different classes such as Toll-like receptors (TLRs), NOD-like receptors (NLRs), and C-type lectin receptors (CLRs) (Kawasaki et al. 2011; Kumar et al. 2011). TLR function, particularly in aged human MDDCs, has been reported to be largely intact at the level of both expression and function (Agrawal et al. 2007; Uyemura et al. 2002). Our own observations (Agrawal et al. 2007) suggest that inflammatory cytokine secretion particularly tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 are increased in response to TLR4 and TLR8. In contrast to MDDCs, intracellular cytokine detection of TLR stimulated cDCs in aged blood revealed significant impairment in the production of TNF-α, IL-6, and IL-12p40 in response to nearly all TLRs (Panda et al. 2010). Other studies with blood myeloid DCs have also reported impairment in IL-12 production though these studies did not observe a reduction in TNF-α and IL-6 (Della Bella et al. 2007). Similar to MDDCs, no significant difference in baseline or inducible cytokine secretion was observed in monocyte-derived LCs from aged or young subjects. A recent study in rhesus macaques (Asquith et al. 2012) observed an increase in the frequency of myeloid DC with age but found reduced secretion of cytokines in response to TLR4, TLR2/6, and TLR7/9. Interestingly, they observed reduced expression of absent in melanoma 2 (AIM2) (Barber 2011) and retinoic acid-inducible gene I (RIG-1) (Rietdijk et al. 2008) in aged animals. Both these receptors are involved in IFN-α production.
6.3.2 Innate Interferon (IFN) Secretion
In keeping with this, the production of IFN-alpha is universally reported to be decreased in aging in response to TLR7, TLR8, or TLR9 (Canaday et al. 2010; Jing et al. 2009; Panda et al. 2010; Sridharan et al. 2011). The PDC subset in the blood is the primary IFN producer against infections. They express the intracellular TLR receptors TLR7 and TLR9 which allow sensing of ssRNA viruses such as influenza and unmethylated DNA motifs from bacteria (Gilliet et al. 2008). The production of IFN from PDCs is very rapid and the amount of IFN produced by these cells can be 100 fold more than other cells (Fitzgerald-Bocarsly 2002). Almost all studies have reported decreased type I IFN secretion by aged PDCs in response to influenza virus, TLR7 ligand Gardiquimod, and TLR9 ligand ODN which may well be responsible for the increased susceptibility of the aged to respiratory viral infections as robust IFN production by PDCs is essential to generate effective immune response against viruses. Reduced IFN secretion by PDCs in aged subjects is due to a number of reasons. Most studies observe a reduction in PDC numbers as well as reduced expression of the TLR7 and TLR9 in PDCs in the blood of aged subjects (Jing et al. 2009; Panda et al. 2009). Our own studies suggest that the defect lies in the signaling pathway downstream of TLRs (Sridharan et al. 2011). We observed impaired phosphorylation of interferon regulatory factor 7 (IRF-7), primary transcription factor required for IFN production (Honda et al. 2005). A recent study (Qian et al. 2011) reports similar deficiency in IFN production from aged PDCs in response to West Nile virus. These studies together suggest that type I IFN secretion from aged PDCs is impaired though the mechanisms may vary with the population studied.
Interestingly, this deficit in IFN production was not restricted to PDCs alone; MDDCs from aged donors also demonstrated significantly impaired IFN secretion in response to influenza virus. Here also, similar to PDCs, secretion of other cytokines was comparable to young (Prakash et al. 2012). Further investigations suggested that the defect was at the epigenetic level. Chromatin immunoprecipitation (ChIP) studies with activator histone, H3K4me3, and repressor histone, H3K9me3, antibodies revealed that the association of IFN promoter with the repressor histone is increased in aged DCs at the basal level which reduces the association of the promoter with activation histone on activation with influenza. Similar reduction in type I IFN secretion in by MDDCs from aged donors was also observed in response to West Nile virus (Qian et al. 2011). Their observations suggested that DCs from older donors had diminished late-phase responses, such as induction of the transcription factors signal transducers and activators of transcription 1 (STAT1) and IRF-7, and lower expression of IRF-1, suggesting defective positive-feedback regulation of type I IFN expression. Altogether, DCs (both PDCs and MDDCs) from aged donors show selective defect in IFN secretion in response to viruses while the secretion of other pro-inflammatory cytokines is largely intact.
In addition to type I IFNs, PDCs also produce type III IFNs – IL28/IL29 or IFN-lambda (Ank et al. 2006, 2008; Yin et al. 2012). These are more recently discovered innate IFNs which have been reported to play a major role in protection against viral infections of the mucosa particularly of the respiratory tract (Mordstein et al. 2010b). In support of this, IL28R knockout mice were reported to be more susceptible to several pneumotropic viruses than IFNAR1-knockout mice (Mordstein et al. 2010a). This is because their induction in DCs utilizes the same pathways as type I IFN however; their mode of action is restricted as the receptor for type III IFN is expressed mainly on epithelial cells of the mucosa (Ank et al. 2006). Thus, IFN-III seems to selectively contribute to innate immunity at mucosal surfaces, which are the most frequent entry sites of viruses. Our group has observed reduced type III IFN secretion from both PDCs and MDDCs from aged in response to influenza virus (Sridharan et al. 2011; Prakash et al. 2012). Reduced IFN-III is also observed in asthmatic individuals (Edwards and Johnston 2011). Thus, impaired secretion of IFN-III by aged DCs could be a major factor in the susceptibility of the elderly to not only respiratory viral infections but also other respiratory disorders such asthma and COPD. Given the selective nature of IFN deficiency observed in aged donors, supplementation of IFN particularly of the type III subtype during viral infections may help reduce the incidence and severity of respiratory viral infections in the elderly provided the response to IFN is not compromised.
Most studies in the elderly are focused on TLRs and there is a scarcity of information on the functions of other PRRs, which are emerging as major players in the immune response. A recent mouse study with influenza infection demonstrates that activation of NOD receptor NLRP3 is impaired in aged mice resulting in reduced IL-1β production (Stout-Delgado et al. 2012). There is reduced expression of ASC, NLRP3, and caspase-1 but increased expression of pro-IL-1β, pro-IL-18, and pro-IL-33 in DCs from aged mice as compared to DCs from young mice. The authors also showed that treatment of mice with nigericin reduced the influenza infection in mice by enhancing the IL-1β production. In another study, the authors have demonstrated that increased generation of danger signals in the thymus activates the caspase-1 via NLRP3 inflammasome resulting in thymic atrophy (Youm et al. 2012). Inhibition of inflammasome activity restored the thymic epithelium and T cell repertoire. One other study has examined the activity of inflammasomes in nonimmune cells. In this study (Mawhinney et al. 2011), the activation of NLRP1 was reported to be enhanced in hippocampus of aged rats resulting in increased secretion of IL-1β and IL-18 which contributed to cognitive decline in the elderly. A recent microarray study (Cribbs et al. 2012) of aged human brain tissue also observed signatures reminiscent of activation of microglia and perivascular macrophages in the aging brain. Almost all innate immune response genes such as TLR signaling, complement components, as well as inflammasome signaling were upregulated in aged brain. These studies suggest that activity of NOD-like receptors may be increased or decreased with age and vary with different receptors and anatomical location.
6.4 Phagocytosis and Migration
Sensing of pathogens by DC leads to antigen uptake by phagocytosis and activation of DCs which upregulates chemokine receptor type 7 (CCR7) receptor on DCs allowing their migration to draining lymph nodes to prime T cells. Our studies indicate that MDDCs from aged are impaired in phosphorylation of AKT, a kinase in the PI3kinase signaling pathway which controls cytoskeletal proteins (Agrawal et al. 2007). This impairment leads to reduced phagocytic uptake of antigens by DCs as well as reduced migration in response to macrophage inflammatory protein-3 (MIP3-β). Similar impairment in migratory capacity of epidermal LCs to TNF-α has also been reported in the elderly (Cumberbatch et al. 2003). However, differentiated monocyte (Ogden et al. 2011) showed migration equivalent to young in response to the chemokine ligand chemokine (C-C motif) ligand 19 (CCL19). A decrease in the capacity of migration of adoptively transferred aged DCs is also observed in aged mice. Reduced CCR7 signaling as well as reduced response to CCL21 was considered to be the primary culprit (Grolleau-Julius et al. 2008). A recent study (Zhao et al. 2011) also observed impaired migration of lung DCs to draining lymph nodes in aged mice in an influenza infection model. They attributed it to increased expression of prostaglandin D2 (PGD2) in mouse lungs. In summary advancing age seems to slow down aged DCs.
6.5 T Cell Priming
Once DCs migrate to lymph nodes they activate CD4+ Th cell, CD8+ T cytotoxic and B cell antibody responses. Almost all studies in humans studying DC-T interaction have been performed using MDDCs. Most studies did not observe a significant difference in the capacity of aged MDDCs to induce T cell proliferation compared to young DCs (Steger et al. 1997; Agrawal et al. 2009, 2012b). This was true for MDDCs and PDCs. However, we did observe (Agrawal et al. 2012b) increased basal level of proliferation and IFN-γ secretion by young CD4+ T cells when cultured with aged MDDCs as compared to young which may be a consequence of increased basal level of DC activation in the elderly. Culture of unstimulated PDCs with CD8+ T cells also resulted in higher basal level of IFN-γ secretion and granzyme and perforin induction (Sridharan et al. 2011). However, influenza-stimulated PDCs from aged subjects were deficient in inducing granzyme and perforin in CD8+ T cells of young subjects due to the reduced secretion of IFN-I and IFN-III by aged PDCs.
DCs also dictate the polarization of Th cells (Manicassamy and Pulendran 2009a). The cytokines secreted by DCs direct the differentiation of Th cells towards Th1/Th2/Th17/Treg/Tfh cells. Evidence suggests that Th1 response is predominant in healthy old subjects which changes to Th2 in the frail elderly probably due to increase in histamine which enhances Th2 polarization (Rafi et al. 2003). Treatment with antihistamine reduced Th2 responses and improved immune function in the frail elderly. Most studies with MDDC-T interaction do not report any change in Th cell cytokine secretion in aged donors. We have observed an increase in basal level of IFN-γ secretion from young Th cells when cultured with aged DC. This increase was more prominent in aged DC-aged T coculture (Agrawal et al. 2012b). We also observed increased differentiation of aged CD4 T cells of aged towards IL-21 secreting T follicular helper (Tfh) cells (Agrawal et al. 2012b). IL-21 affects almost all immune cells of the body and increased secretion of IL-21 is associated with autoimmune diseases (Shekhar and Yang 2012). IL-21 enhances IL-17 production and also increases the differentiation of B cells towards antibody secreting plasma cells thus IL-21 enhances autoantibody production in autoimmune and inflammatory diseases (Sarra et al. 2011). Increased levels of autoantibodies are common in aged individuals (Howard et al. 2006). Furthermore, IL-21 enhances the formation of IL-10 secreting B regulatory cells which suppress immune responses (Yoshizaki et al. 2012). IL-21 also enhances the cytotoxicity of CD8+ T cells and natural killer (NK) cells which may account for increased granzyme and perforin observed in aged CD8+ T cells in the absence of infection. Increased IL-21 was found to enhance the susceptibility of mouse to pneumovirus infection (Spolski et al. 2012). IL-21 also promotes allergic inflammation (Spolski and Leonard 2008). Therefore, increased IL-21 in aging may be detrimental for generating efficient immune responses against infections and enhance the susceptibility of the elderly to respiratory diseases.
6.6 Dendritic Cell and Tolerance
6.6.1 Peripheral Tolerance
DCs are unique among the APCs due to their constitutive low level expression of MHC and costimulatory molecules. This low level expression of MHC allows DCs to sample and present endogenous antigens to T cells and induces the formation of T regulatory (Treg) cells. DCs are therefore crucial for maintenance of tolerance (peripheral and mucosal) in the body (Hu and Wan 2011). Impaired capacity of DCs to maintain tolerance is one of the primary mechanisms of induction of autoimmunity (Agrawal et al. 2012a). Increased response of DCs to self antigens induces the secretion of pro-inflammatory cytokines and decreases the formation of Treg cells. Most studies in aging have not focused on the tolerizing function of DCs. Our own group determined the response of aged MDDCs to self-antigen, human DNA and demonstrated that aged MDDCs secrete significantly higher level of IFN-α and IL-6 compared to young MDDCs (Agrawal et al. 2009). The capacity of aged MDDCs to prime T cells was also enhanced. Increased response to self antigens would enhance chronic inflammation as DCs are constantly sampling self antigens from the surrounding milieu. Investigations into the signaling mechanisms suggest that DCs from aged display increased basal level of activation as evidenced by increased phosphorylation of the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in aged MDDCs. Increased basal level activation of DCs may induce secretion of pro-inflammatory cytokines and other inflammatory mediators from aged DCs. This was confirmed by study of Panda et al. (2010) where they observed increased secretion of TNF and IL-6 from cDCs from aged donors in the absence of stimulation. Our unpublished observations from gene expression analysis using microarrays suggest a pro-inflammatory signature in aged MDDCs.
6.6.2 Mucosal Tolerance
Besides maintaining peripheral tolerance, DCs play a major role in mediating tolerance at mucosal surfaces. The airways and the mucosa are continuously exposed to millions of harmless pathogens, toxins, etc. DCs are present just below the mucosal epithelium and can sense these stimuli via extension of their dendrites as well as via activation of epithelial cells (Allam et al. 2011; Lambrecht and Hammad 2012). The mucosal environment is rich in immunosuppressive cytokines such as transforming growth factor beta (TGF-β) and vitamin A metabolite, retinoic acid (RA), which prevent activation of DCs to these stimuli and results in the generation of Treg cells (Manicassamy and Pulendran 2009b; Feng et al. 2010). Majority of the DCs in the mucosa are myeloid in origin though it has recently been shown DCs in the mucosa can be divided into two subsets: one expressing the CD103+ which are immunosuppressive and induces Tregs and the other CD103 subset which react to pathogens and activate T cells (Haniffa et al. 2012; Ivanov et al. 2012; Nakano et al. 2012; Leepiyasakulchai et al. 2012). Increased activation of DCs from aged donors may disturb the mucosal equilibrium as they may react to harmless antigens to induce chronic inflammation which would result in airway hyperresponsiveness and increase the susceptibility of the elderly to mucosal infections.
In summary, DC function changes significantly with age. Certain responses such as the production of innate IFNs in response to viruses decrease while the basal levels of pro-inflammatory cytokines increase. Reduced IFN secretion impairs the ability of the aged subjects to fight viral infections particularly of the respiratory mucosa, while enhanced basal level of inflammation causes erosion of tolerance both at the peripheral level and in the mucosa. Increased IL-21 secretion further impairs the capacity of the elderly to fight infections. Thus, DCs from aged subjects display defects at multiple levels and therapeutic measures targeting DCs may restore the immune functions in the elderly.
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