Aging is known to augment inflammatory responses, but whether these alterations contribute to death following viral infection is unknown. In the current study, we found that, following either HSV-2 or MCMV infection, aged mice produced higher serum levels of several inflammatory cytokines, in particular IL-17A. Importantly, neutralizing IL-17A in aged mice during HSV-2 or MCMV infection reduced liver injury and increased survival (Fig. , and ). These findings are consistent with prior work showing that IL-17 signaling induces liver injury in non-aging models (Nagata et al., 2008
). IL-17A neutralization also reduced serum levels of other inflammatory cytokines including IL-6 and TNF-α, indicating that elevated IL-17A levels control the production of other cytokines in this experimental system. This is consistent with prior work demonstrating that IL-17A can induce the production of IL-6 by a variety of cells (Fossiez et al., 1996
). Our findings demonstrate that aging induces an aberrant overproduction of IL-17A during systemic viral infection and that this IL-17A overproduction can lead to death secondary to liver damage.
In the aged mice studied here, neutrophil-mediated liver necrosis was responsible for HSV-2-induced death. Prior work in non-aging models has demonstrated that neutrophils are key mediators of liver cell injury (Ramaiah and Jaeschke, 2007
). We found that 24 h after infection, aged virally infected mice, but not their young infected counterparts, exhibited severe hepatocyte necrosis that was associated with neutrophil activation (). Moreover, both liver injury and mortality were blocked by neutrophil depletion (). Both outcomes were driven by IL-17A, as seen by the ability of IL-17A neutralization to reduce levels of MIP-1α (a neutrophil-attracting chemokine) and decrease neutrophil enzyme activity (). These data indicate that neutrophils are a key cellular mediator of IL-17A-dependent death in aged mice with systemic viral infections.
Our study provides evidence that NKT cells are important for the rapid production of IL-17A during HSV-2 infection, because NKT-deficient mice exhibited a much weaker IL-17A response than WT mice (). These results indicate that NKT cells are a major source of IL-17A in our experimental systems but do not exclude the possibility that other cell types with known IL-17-producing capabilities, such as γδ T cells and CD4+
T cells (O’Brien et al., 2009
; Stockinger and Veldhoen, 2007
), contribute to the age-induced IL-17 response in our study. Recent studies have demonstrated that NKT cells produce IL-17A when activated by α-galactosylceramide or LPS instillation (Michel et al., 2007
; Rachitskaya et al., 2008
). However, to our knowledge, the ability of NKT cells to produce IL-17A during a viral infection has not been previously described. Our work suggests that IL-17-producing NKT cells may have pathological consequences during viral infection. Prior work indicates that NKT cells are important for host defenses against infection with HSV and other viruses (Grubor-Bauk et al., 2008
; Grubor-Bauk et al., 2003
). Furthermore, studies with MCMV have indicated that NKT cells are activated via the presence of an accessory cell to maximally induce IFN-γ production during infection (Tyznik et al., 2008
; Wesley et al., 2008
). Our results indicate that NKT cells can respond directly to in vitro
stimulation by HSV-2 to produce IL-17A, although our study does not exclude the possibility that an accessory cell is involved in vivo
. In the future, it will be important to identify the viral components (e.g., viral glycolipids) that stimulate NKT cells to produce IL-17A.
We found that aging increased expression of RORγT, a transcription factor that may control IL-17A production by NKT cells (Michel et al., 2007
; Rachitskaya et al., 2008
). Why aging led to an upregulation of the RORγT gene in NKT cells is unclear. One possible avenue for future investigation is exploration of the role of oxidative stress, as many studies have shown that age-induced phenotypes correlate with increasing levels of oxidative stress [reviewed in (Bokov et al., 2004
Our work provides evidence that two factors (i.e., impaired viral control and aged NKT cells) cooperate in inducing liver injury during viral infection in our experimental models. A recent study demonstrated that aging impairs pDC function and in this way, disrupts the IFN-α response, inhibiting HSV-2 or MCMV clearance in aged mice (Stout-Delgado et al., 2008
). In young infected mice, impeding viral clearance via pDC depletion and adoptively transferring aged, but not young, NKT cells induced greater ALT release than either manipulation alone and imparted a similar phenotype to that seen in aged mice (). Although these young mice became ill, they eventually survived viral infection, suggesting that young hosts contain unidentified protective factors or that aged hosts contain unidentified disease-promoting factors that contribute to mortality during viral infection. A recent study found that IL-17A inhibits NK function, reducing clearance of vaccinia virus (Kawakami et al., 2009
). This study may explain the synergy between impaired viral control and elevated IL-17A levels in our model, as it is possible that the augmented IL-17A response alters other components of the aging immune system, further exacerbating liver injury. Nevertheless, our results indicate that, in aged hosts, viral infection induces liver injury through at least two factors: impaired containment of the virus and aged NKT cells, which exhibit an exaggerated IL-17A response.
In future studies, it will be important to determine whether local or organ-specific infection in aged hosts induces a phenotype similar to that described here in response to systemic viral infections. This may depend on whether NKT cells accumulate with age at local sites of infection. In addition, clinical studies should be conducted to determine whether older people exhibit increased levels of IL-17 during viral infection. A recent study showed that Th17 cells enhance viral persistence in a murine chronic viral infection model (Hou et al., 2009
). If Th17 cells are found to be elevated in aged humans with viral infections, then perhaps age-dependent increases in IL-17 responses may not only contribute to immune pathology during systemic viral infections, but also promote chronic viral infections. In any case, our data clearly show that aging alters the host-virus interaction by inducing liver damage and mortality through a novel pathway involving IL-17A. Thus, anti-IL-17A therapies may hold promise for preventing immune pathology and increasing survival in older individuals with systemic viral infections.