Influenza virus affects all ages of humans; however, the elderly (>65 years-old) have increased susceptibility to infections and are especially predisposed to complications [6
]. The increased morbidity and mortality reported in elderly populations are due to several factors that include subjacent chronic diseases, such as diabetes and cancer, as well as dysfunctions in the senescent immune system. The mouse model has proven to be an important tool to explore the pathogenesis of influenza, especially due to the similarities to human infections [27
]. The elderly mouse model has also proven to be useful to explore the effects of age on the immune responses to several respiratory infectious agents including Mycobacterium tuberculosis
and influenza virus [18
]. However, most of the studies have focused on the T-cell compartment and indicated that altered T-cell proliferation and memory results in decreased and delayed CTL activity [4
]. Few reports have addressed the role of the aging innate immune system on the protection to influenza virus and only few reports have explored phagocytic functions, NK cell activity, and IFN-α/β production in the aged animals using other pathogens [34
]. In this study, the immune responses to influenza virus in the lungs of aged animals were evaluated. Alterations in APC up-regulation of CD40 correlated with delayed production of cytokines and chemokines in the lungs, which correlated with infiltration of immune cells into these organs. Finally, this correlated with a delayed activation of the adaptive immune responses and subsequent delay in clearance of virus.
The earlier weight loss and enhanced morbidity in aged animals suggests less effective innate immune responses against influenza virus in these mice. Among the cells of the innate immune system, granulocytes, DCs and macrophages are critical players, since these are the first cells to encounter a pathogenic microorganism. Among granulocytes, neutrophils are the predominant population (>90%) and the role of these cells in influenza infection has recently been elucidated [39
]. One mechanism of granulocyte activity is the release of lytic proteins, such as myeloperoxidase, from endocytic granules. Aged animals had delayed lung infiltration kinetics following influenza infection (Fig. ); however, similar levels of MPO were produced by adult and aged mice, except for day 9, where higher levels were detected in the aged group (p < 0.05) (data not shown). This indicates that for the main part, the neutrophil function was not altered in aged animals infected with influenza virus. The higher MPO levels at day 9 might contribute to the enhanced sickness score detected in aged animals at this time point. The data also suggest that the difference in infiltration kinetics may be associated with impaired chemotaxis. Consistent with this hypothesis, the secretion of the chemokine, KC (neutrophil chemoattractant), was delayed in aged mice following influenza virus infection (Fig. ).
Interestingly, the major differences in granulocyte infiltration kinetics were detected between days 11-19 (Fig. ), which corresponds with the resolution phase of the disease. The higher granulocyte infiltration in aged animals also correlated with a prolonged presence of macrophages (Fig. ) and was associated with a higher sickness score and prolonged disease. A prolonged infiltration of the lungs with cells from the inflammatory phase might account for the prolonged disease stage in the animals. To further support this, MPO was higher at day 9 in aged animals and reports that aged populations have a higher tendency to produce inflammatory cytokines, such as TNF-α and IL-α, during infections have been published and correlates with our data (Fig. ) [21
]. In summary, all these immune markers suggest that the immune system can be contributing to the enhanced sickness score detected in the aged group of animals.
APCs are among the first leukocytes to recognize infectious microorganisms. DCs are especially responsible of 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. In the murine lung, different DC populations have been recently described, one of the predominant populations includes resident CD11bhigh
cells, also known as conventional DCs (cDCs) (reviewed in [17
]). The other APC populations analyzed in these animals represent lung macrophages (CD11bhigh
). It is important to note that lung (interstitial) macrophages are different from alveolar macrophages. The later cells suppress the functional characteristics of lung DCs [17
]. Alveolar macrophages in addition to CD11c, express F4/80 [42
]. Both APC populations analyzed in our experiments have low F4/80 expression (data not shown), providing evidence that the analyzed cells were not alveolar macrophages. Upon encountering the antigen, APCs up-regulate several molecules, such as MHC class II and CD40 in order to present antigens to CD4+
T-cells and provide the required second signal to fully activate these cells [43
]. MHC class II up-regulation was not altered neither in lung macrophages nor cDCs from aged mice following influenza infection. However, CD40 up-regulation was delayed in both lung macrophages and cDCs cells, suggesting differences in complete priming of the APCs in aged mice. Fully primed APCs produce cytokines and chemokines that will attract cells to the lungs and subsequently activate them during influenza virus infection [44
]. CD40 interaction with CD40L, is important to fully activate APCs [46
] and the role of CD40 in stimulating the production of IL-12 is well documented (reviewed in [43
]) The concentration of IL-12p70
, the active form of IL-12, was not only significantly higher in the lungs of adult mice, but also spiked earlier compared to aged mice following influenza infection (Fig. ). Considering the importance of CD40 in the induction of IL-12, the delayed CD40 up-regulation in aged animals, most likely contributed to a retarded IL-12p70
production and reduced activation of aged APCs.
In addition to IL-12, activated APCs can produce other pro-inflammatory cytokines, such as IL-1β, IL-1α, and TNF-α (Fig. ) [19
]. Interestingly, not all of these cytokines showed higher concentrations in adult mice compared to aged mice following influenza infection; however, in all cases the peak of cytokine level occurred earlier in adult animals. The higher peak of the pro-inflammatory cytokines detected in elderly animals correlate with reports indicating that elderly populations have enhanced basal levels of these cytokines (e.g. TNF-α) and tendency to produce higher levels upon infection [21
]. Similar to cytokines, the peaks of chemokine production by APCs (MIP-1β, MCP-1, RANTES and KC) to influenza infection were delayed in aged mice. This correlated with the delayed CD40 up-regulation in aged animals, which further suggests a delay in full activation of APCs.
produced by activated APCs stimulates NK cells and CD4+
T-cells to produce IFN-γ [48
]. Consistent with the delayed production of IL-12p70
in the lungs of aged animals to influenza infection, IFN-γ spiked two days later (day 7 vs. day 5) in aged mice compared to adults (Fig. ). Consistent with the delayed production of the chemokines, NK cells, CD4+
T-cells showed delayed infiltration into the lungs of aged animals (Figs. , and ). Furthermore, the up-regulation of the early activation marker CD69 was delayed on NK cells in aged mice (Fig. ). Previous reports had shown no alterations of NK cells with age in humans and only small changes in mice [53
]. However, our data coincides with a recent report that demonstrates alterations in the NK compartment of aged animals infected with influenza virus [40
B-cells showed similar patterns of infiltration between aged and adult mice following influenza infection (Fig. ). The kinetics of this population were delayed compared to CD4+ and CD8+ T-cells (Fig. ), since B-cells started to infiltrate the lungs between days 9 and 11, upon which these cells significantly increased in adult and aged animals (Fig. ). Despite this, surface expression of CD69 was detected by day 3 in adults and day 5 in aged mice (Fig. ), which coincided with an earlier detection of anti-influenza neutralizing antibodies in adult mice compared to aged mice (Fig. ). Interestingly, CD69 up-regulation by CD4+ T-cells was similar between adult and aged mice (Fig. and ), suggesting that the differences in IFN-γ production detected between days 3 to 9 between aged and adult mice is primarily due to secretion of this cytokine by NK cells.
There was a delay in the infiltration of CD4+
T-cells into the lungs of aged mice compared to adult mice (Fig. and ), however, there was little difference in the activation of these cells (Fig. and ). Early CD69 up-regulation by T- lymphocytes in the lungs is dependent on IFNs type I production [58
]. Consistent with this, no differences in IFN-α/β production in the lung supernatants of aged or adult animals were detected. Early CD69 up-regulation by T-cells most likely represented non-specific activation of these cells, which may have inefficiently produced IFN-γ. Furthermore, recent reports suggest that the lung airway environment might also play an important role in the up-regulation and maintenance of CD69 by lymphocytes [60
]. In a primary infection, influenza specific cells, might play a more prominent role than nonspecifically activated cells. To determine this, influenza specific activated (CD69+
T-cells were assayed using HA and NP immunodominant epitopes [61
] conjugated to pentamers of MHC class I molecules. Consistent with our hypothesis that early up-regulation of CD69 by T-cells was not influenza-specific, activated (CD69+
influenza specific T-cells were detected only after day 9 post-infection. Furthermore, the percentage of HA518-526
specific activated CD8+
T-cells were consistently higher in adult mice compared to aged mice (Fig. ). In contrast, the percentage of NP147-155
specific activated CD8+
T-cells were not statistically different between aged and adult animals (Fig. ); nevertheless, adult animal had a higher percentage of CD8+
T-cells between days 9 and 11 post-infection (Fig. ). Considering the delay in virus clearance in aged mice (Fig. ), HA specific CD8+
T-cells most likely play a prime role in virus clearance and the late appearance of these cells in aged mice most likely contributed to the prolonged recovery. Despite that no statistically significant differences were noted by day 15, adult mice showed the tendency to have a higher percentage IL-2, TNF-α and IFN-γ producing CD8+
T-cells regardless of HA or NP stimulation (Fig ). Since mature (fully primed) DCs efficiently induce cytokine production by CD8+
T-cells and the generation of cytotoxic T cells [63
], the reduced cytokine production by aged CD8+
T-cells might be the result of reduced APC activation as suggested by alteration in CD40 up-regulation. This could eventually lead to a delayed virus clearance in the lungs.
Virus titration in the lungs of aged animals showed a delayed virus clearance. However, the data also demonstrated that despite a similar onset of virus between adult and aged animals, the peak was lower in the later set of animals. This might be the result of a reduced homeostasis of epithelial cells of aged animals. Aging reduces the division potential of cells (replicative senescence) both in vivo
and in vitro
. Some of these changes have been only partially explored in the lungs; however, affect most organs in the models used [64
]. A reduced replication capability can be translated in reduced virus production by aged animals during peak days. Even with lower virus titer in the lungs of aged animals, the alterations in the immune responses (innate and adaptive) might account for reduced virus clearance and the enhanced tendency to produce inflammatory cytokines (e.g. TNF-α) [21
] by these animals might be responsible for the enhanced sickness score.
The data in this study shows that age affects the global immune responses to influenza infection. Alterations in CD40 up-regulation by aged cDCs and lung macrophages suggested impairments in their activation. Remarkably, this correlated with altered levels of cytokines (especially IL-12p70
) and chemokines, which also correlated with delayed NK and T-cell infiltration. Furthermore, influenza (HA)-specific T-cells were 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 exogenous pathway) and CD8+ stimulating capacity [50
]. Some studies, on the other hand, have not reported defects in DC function in the elderly [72
]. This might indicate that different populations of APCs at different tissues are affected differently by age. Also, this may be the result of genetic differences between mouse strains. Our data also demonstrated altered humoral immune responses (B-cell activation and HAI titers). 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 final outcome of delayed virus clearance and slow recovery is probably the addition of various factors and not only involve dysfunction in antigen presentation.