The herpes virus affects humans of all ages; however, the elderly (> 65 years old) have an increased susceptibility to these infections and are especially predisposed to complications (13). The increased morbidity and mortality reported in elderly populations are due to several factors that include dysfunctions in the senescent immune system.
We have previously reported that DCs from aged subjects activate the epithelium rendering them more permeable to infections (19). Enhanced basal-level inflammation/activation of cells also allows the dissemination of infections. In summary, we found that AEC functions are significantly impacted with age and, thus, may play a major role in age-associated chronic respiratory diseases and infections. AECs not only form a barrier to prevent the entry of infections but are also well-equipped to deal with, and distinguish between, innocuous and pathogenic inhalants (14). They express TLRs and other pathogen recognition receptors that allow them to sense and respond to pathogens. There is limited information about the changes in AECs' innate immune functions with age. A decrease in ciliary beat capacity and increased secretion of mucin has been reported (15–18). In this study, we demonstrate that TLR3 expression is decreased in AECs from aged mice indicating an impairment in sensing viral nucleic acid exposed during viral replication (Fig. 1). This may be partially responsible for the increased incidence of other viral infections such as influenza and recently COVID-19 in the elderly. In addition, we detected that AECs from aged mice displayed increased basal activation.
DCs serve as the sentinels of the immune response and link innate and adaptive immunity. They are mainly comprised of two major subsets, the DC of myeloid origin and DCs of lymphoid origin. In the murine lung, different DC populations have been recently described, one of the predominant populations includes resident CD11bhigh/CD11chigh cells, also known as conventional DCs (cDCs). The other APC populations analyzed in these animals represent lung macrophages (CD11bhigh/CD11clow) and lymphoid DCs (CD11chigh cells) These cells have specialized antigen-processing capabilities together with co-stimulatory molecules that enable their efficient endocytosis and presentation of endogenous and exogenous antigens to initiate an immune response. DCs detect and respond to pathogens through the expression of pattern recognition receptors (PRRs) (20); (21); (22). PRRs can recognize conserved molecular components or patterns of the pathogen. Examples of PRRs include Toll-like receptors, and Nod-like receptors (20); (21). Exposure of DCs to ligands of all these PRRs results in the production of cytokines that modulate the type of T cell response and functions (20); (22). Deficiencies in human TLR signaling leads to increased severity of several diseases, including sepsis, immunodeficiencies, atherosclerosis, and asthma (5). We have evaluated TLR expression on cDCs, macrophages, and lymphoid DC’s in the context of aging, and observed an age-associated reduced surface expression of TLR3 (Fig. 2). We have previously reported reduced levels of TLRs in whole blood samples and colonic biopsies from older individuals (23), and in macrophages and pDCs from aged mice (24), (25), (26). The expression of TLRs in the context of the lung has not been studied in aging. Our observation that DCs and macrophages from aged mice display reduced TLR-3 expression indicates a possible mechanism rendering the elderly a high-risk population for viral respiratory infections including the recent COVID-19 infection.
APCs are among the first leukocytes to recognize infectious microorganisms. DCs especially are responsible for the surveillance in different tissues and subsequent migration to the lymph nodes where they interact with T-cells to present antigens and trigger the adaptive immune response. Upon encountering the antigen, APCs upregulate several molecules, such as MHC class II to present antigens to CD4+ T cells and provide the required second signal to fully activate these cells (27). In our study MHC class II up-regulation was not altered in lung macrophages after herpes infection, however, it was upregulated in aged DCs (myeloid and lymphoid) at the basal level, indicative of increased baseline activation of DCs that is in keeping with what has been reported for blood DCs in aging (28)(29).
Increased susceptibility to viral infection in the elderly is often ascribed to alteration in T cell functions (30–32). The contribution of the innate immune response that impacts anti-viral defense mechanisms in aging remains largely unexplored. The first line of defense after viral infections consist of robust production of interferons by innate immune cells (4, 33). These interferons have potent anti-viral properties and subsequently regulate innate and adaptive immunity (34). Numerous studies have reported a decreased secretion of IFN-α by aged pDCs, which are the most potent IFN-producing cells (26, 35–37). Besides pDCs, the mDCs subset of DCs is also capable of producing a robust amount of IFNs in response to viral infection (35, 38).
In this study, we showed that IFN-a response from lung supernatant was upregulated after herpes infection in young and adult mice, however aged mice were impaired in their capacity to secrete IFNs in response to viral infection. Our studies (37) as well as reports from others (26, 36, 39–41) have reported decreased production of IFN-a in the aged subjects. In addition to IFN-a, aged DCs were also deficient in the production of IL-1b and IL-22. IL-22 has emerged as a major cytokine that plays a critical role in regulating host defense and epithelial repair responses during viral infection and resolution (42). IL-22 synergizes with IL-17 to induce the secretion of antibacterial proteins and chemokines and also augments cell proliferation and repair following injury. Survival following superinfection with Streptococcus pneumoniae requires IL-22 (43) attributable to the protective effects of IL-22 on the pulmonary epithelium. IL-22 has been shown to protect the airways by increasing transepithelial resistance and promoting bronchial epithelial cell proliferation (44). Although the IL-22R is localized to airway epithelium before infection, it is upregulated at parenchymal sites of lung remodeling induced by influenza (42). Furthermore, treatment of H1N1(PR8/8/34) infected mice with IL-22: Fc. caused a significant reduction in inflammation, neutrophilia, and lung leak. Importantly, this led to improved health outcomes (reduced weight loss, greater activity scores) and decreased mortality (45). IL-22 has also been found to prevent apoptosis through the production of anti-apoptotic proteins, such as Bcl-2 and Bcl-1, in a bleomycin model of lung injury (46). IL-22 is thus integral in protecting the host against the lung damage caused by viral infections and preventing secondary bacterial infections.
CD8 responses are highly dependent on type I IFN-induced signals, as CD8 memory formation in the setting of LCMV infection in the absence of type I IFN signaling is greatly diminished. Consistent with the delayed production of the cytokines, CD4+ and CD8+ T cells showed delayed infiltration into the lungs of aged animals (Figs. 6A and 6B). In a primary infection, herpes-specific cells might play a more prominent role than non-specifically activated cells. Herpes-specific CD8+ T cells were assayed using gB498-505 immunodominant epitopes. Consistent with our hypothesis gB498-505 specific activated CD8+ T cells were consistently higher in young and adult mice compared to aged mice (Figs. 7C and 7D). T-cell responsiveness to type I IFN, commonly produced in viral and bacterial infections, is known to be pivotal for the generation of adaptive immune responses. Our findings from this report identify a reduced IFN-α response in aged mice that may contribute to reduced T-cell responses. Furthermore, the up-regulation of the functional marker CD107 was delayed on CD8+ T cells in aged mice (Fig. 8D). Previous reports had shown no alterations of CD8+ T cells with age in humans and only small changes in mice (47). However, our data coincides with a recent report that demonstrates alterations in the T cell compartment of aged animals infected with the influenza virus (47). T-cell responsiveness to type I IFN, commonly produced in viral and bacterial infections, is known to be pivotal for the generation of adaptive immune responses. Our findings identify a reduced IFN-a response in aging that may contribute to reduced T-cell responses. The reduction in IFN-a is likely due to the reduced expression of TLR-3. Hence, modulating TLR responses may be a beneficial immune intervention strategy for the elderly.
The aged mouse model has proven to be extremely useful in determining the effects of age on the immune responses to HSV-1 infection. In summary, our results demonstrate that HSV infection impairs immunity in old, aged mice and propagates immune senescence. We also demonstrated that herpes-infected mice exhibited perturbations in naive repertoire more profoundly than those seen in aging alone. A highly diverse T-cell population is critical for protection against pathogens. The diversity of the T cell response to infection (i.e., the number of different clonotypes participating) is a better correlate of protection than the magnitude of the response (13, 41–43). As little as a 2- to 3-fold reduction in TCR repertoire diversity dramatically impairs Ag-specific responses (44, 45), and it is the T cell defects in the primary immune response that were identified as a major contributor to immune senescence. Importantly, the observed repertoire alterations were accompanied in herpes virus-positive animals by altered functional responses of the remaining CD8+ T cells and CD4+ T cells and with reduced ability to clear the viral infection. Meanwhile, we found that the absolute number of CD4+ and CD8+ T cells was lower in the older age group compared to the young group and the adult group, which indicated that the CD4+ and CD8+ T cells played important roles in controlling viral infection in aged population.
To address the functional competence of T cells in young adult and aged mice, we analyzed the production of IFN-γ. Remarkably, the increased number of polyfunctional T cells was maintained in young and adult mice but declined in aged mice. In addition, a high frequency of CD107a expressing CD8+ T cells was also identified in the lungs of young, adult, and aged mice post-infection. CD107a is a degranulation marker that has been shown to correlate with cell-mediated cytotoxicity. Accordingly, CD3-mediated stimulation revealed that cytotoxic CD8+ T cells were more frequent in the lungs of young and adult mice, and their high number declined during aging. Since mature (fully primed) DCs efficiently induce cytokine production by CD8+ T cells and the generation of cytotoxic T cells (48), the reduced cytokine production by aged CD8+ T cells might be the result of reduced APC activation as suggested by alteration in MHCI and MHCII up-regulation, potentially leading to delayed clearance of the virus.
In this study, we demonstrated that age affects the immune responses to herpes infection. Alterations in MHCII up-regulation by aged DCs and lung macrophages suggested impairments in their further activation. Remarkably, this correlated with altered levels of cytokines and chemokines, which also correlated with delayed T-cell infiltration. Furthermore, herpes-specific T cells were also reduced in aged animals. These findings correlated with several reports demonstrating that age affects APC, Toll-like receptors expression and function, antigen presentation (defect in the exogenous pathway), and CD8+ stimulating capacity (25, 49–52). Some studies, on the other hand, have not reported defects in DC function in the elderly (53). This might indicate that different populations of APCs in different tissues are affected differently by age. Therefore, the alterations in the APCs are most likely just one step in a large chain of alterations present in the aging immune system. The outcome of delayed virus clearance is probably the addition of various factors and not only involves dysfunction in antigen presentation.
In conclusion, given the significant increase in the elderly population and increased susceptibility to herpes infection, it is becoming imperative to determine the effects of age on the immune system. Identifying the main groups of cells affected by the aging process could aid in further exploring the underlying mechanisms behind the immune alterations and designing better vaccines and adjuvants to boost the immune responses in this important population group.