Sensing molecules such as TLRs are a critical component of the innate immune system's surveillance for invading pathogens. The endosomal TLR3 plays an important role in the pathogenesis of WNV infection (36
). Since the development of severe WNV encephalitis is increased in people who are >55 years old, we were interested in determining whether TLR3 plays a role in the pathogenesis of WNV in the aging population. In resting macrophages, the expression of TLR3 in macrophages of the older cohort was reduced in comparison to that of the younger counterparts. The decrease in expression of TLR3 is in agreement with our recent findings of decreased levels and functional defects of TLR1 in humans in the aging population (34
), also reported in aged murine macrophages (28
The reduced expression of TLR3 in resting macrophages from the more-susceptible aged cohort did not initially correlate with the mouse model, where animals lacking TLR3 are more resistant to the development of WNV (36
). However, during WNV infection, we have shown that the expression of TLR3 in the young cohort was reduced, whereas the expression of TLR3 was increased in macrophages from the older counterpart. This reduction of the mRNA level of TLR3 in macrophages from young donors could be detected as early as 1 h postinfection with WNV and may reflect pivotal signaling pathways mediated by WNV during viral entry into phagocytes. One of the characterized surface molecules that has been demonstrated to mediate WNV entry to phagocytes is DC-SIGN (8
). Using RNA interference and a glycosylation-deficient mutant WNV, we have shown here that the interaction between DC-SIGN and the glycosylated WNV-E protein leads to the downregulation of TLR3. Downregulation of TLR3 was not noted in murine macrophages incubated with WNV, as expected, due to species-specific regulation of TLR3 (13
DC-SIGN is a CLR described in the binding and transmission of human immunodeficiency virus type 1 (5
) and mediates cell responses via downstream kinase signaling (4
). Consistent with an earlier report (4
), we found that Lyn, an Src family kinase, was recruited after DC-SIGN ligated with the glycosylated WNV-E protein and that ligation of DC-SIGN increased the phosphorylation of Lyn at the carboxyl terminus, while the phosphorylation at the activation loop of Lyn was unaffected. While the phosphorylation at the carboxyl terminus of Src kinases is mediated by the C-terminal Src kinase, and this change inactivates the activity of Src kinases (26
), the details of how the carboxyl terminus of Lyn is phosphorylated and whether C-terminal kinase is involved in this signaling pathway are currently under investigation.
STAT1 has been implicated as a transcriptional modifier downstream of Src kinases after the engagement of B-cell antigen receptors in a Jak family kinase (JFK)-independent pathway (25
). Since the ligation of DC-SIGN with WNV-E leads to the inactivation of Lyn, we investigated whether this interaction also led to an inactivation of STAT1. Indeed, we found that the WNV-E protein led to a reduction in the phosphorylation of STAT1, consistent with the carboxyl-terminal phosphorylation of Lyn. This alteration is independent of Jak1 and Tyk2 at early time points, strongly suggesting that DC-SIGN utilizes this unconventional Src kinase-STAT pathway to exert its downstream effects.
One of the downstream effects of the Src kinase-STAT pathway is to regulate the expression of TLR3. Our finding that the level of STAT1-bound TLR3 promoter was reduced in macrophages from younger donors after DC-SIGN ligated with WNV-E protein indicates that TLR3 is regulated by STAT1 and that the STAT1-mediated transcription is reduced in the presence of WNV-E protein. In addition, we also found that the Src kinase-STAT pathway is able to modulate the expression of other STAT1-regulated genes, as the mRNA levels of RNaseL, OAS1, CIITA, and IFN-β decreased in macrophages from young individuals after DC-SIGN ligated with the WNV-E protein. These data suggest that the structural E protein of WNV may regulate the antiviral response in addition to the effects of WNV nonstructural proteins reported previously (19
). Our group has recently shown that WNV infection attenuates the activation of human macrophages (14
) and that the WNV-E protein also inhibits murine cellular responses through interaction with RIP1 kinase (2
We were interested in understanding how this pathway may contribute to an age-dependent susceptibility to WNV encephalitis. Interestingly, we found that the association between DC-SIGN and Lyn is impaired when macrophages derived from older individuals were infected with WNV or treated with WNV-E protein. Instead of inactivating Lyn as demonstrated in the young cohort, the ligation of DC-SIGN with glycosylated WNV-E protein led to a decrease of the phosphorylation of Lyn at the carboxyl terminus. The impaired signaling contributes to the unaltered expression of TLR3 and other STAT1-regulated genes within the first hour of infection. However, significant differences between the young and older cohorts were observed at 3 h after infection with WNV. While the inactivation of the Src kinase-STAT pathway downstream of DC-SIGN continues to inhibit the activation of STAT1-regulated genes and the release of cytokines in young individuals, macrophages derived from older individuals induce the release of STAT1-regulated cytokines, including IL-6 and IFN-β. The absence of this effect when macrophages are incubated with WNV cultivated in human cell lines suggests that only the initial interaction with WNV would increase TLR3 levels in macrophages from the elderly.
When the infection with WNV was carried out over 3 days, we found that macrophages from older individuals produced higher levels of cytokines and had higher viral burdens than those of the young cohort. This is exemplified by the elevated levels of both STAT1-regulated and NF-κB-regulated genes, including those for IL-6 and IFN-β and TNF-α, respectively. The increase in TLR3 levels initiated early in infection may trigger an ongoing elevated response that, in combination with other deficits of aged cells, contributes to the increased severity of WNV infection in elderly hosts. Although the studies presented here do not distinguish between hyperresponsiveness to infection and increased permissiveness, the resulting increased level of cytokines may facilitate the pathogenesis of WNV. TNF-α has been shown to facilitate the entry of WNV to the CNS by breaching the blood-brain barrier (36
). Although we have previously shown that IL-6-deficient mice do not show a difference in WNV susceptibility (36
), the elevated level of IL-6 in the elderly could contribute to neuronal injury within the central nervous system in humans (3
It is an apparent paradox that the greater activation of antiviral responses from macrophages is likely to contribute to greater susceptibility to infection in elderly subjects. This may be due in part to the deleterious effects of TNF-α facilitating the entry of WNV to the brain (36
). Impaired innate immune responses noted in aging, such as reduced basal levels of certain immune response genes (27
), may provide an early window with which to establish infection. In addition, macrophages from older adults had a higher viral burden, possibly because they are more permissive to the replication of WNV. The mechanism of permissiveness of the WNV is currently unknown and could be due to the differences in the intracellular trafficking of the WNV-containing vacuoles in macrophages derived from young versus older donors or to other unidentified deficits of cells from elderly donors. Taken together, these results demonstrate one mechanism that may contribute to increased susceptibility in the elderly to infection with WNV or other infectious agents and which may provide clues for enhancing resistance in susceptible populations.