In this report, we show that transgenic mice expressing the viral PCD inhibitor MC159 exhibited enhanced control of VV production in peripheral tissues such as the visceral fat pad and liver, but not in the spleen. The enhanced clearance of VV correlated with increased cell death in the liver and was marked by the increased expression of the chemokine CCL-2/MCP-1. This is accompanied by an early infiltration of γδ T cells, which is consistent with the role of γδ T cells in the innate immune control of VV replication (5
). The differential protection by MC159 in peripheral tissues might reflect the requirement to recruit innate immune effector cells to peripheral sites. In contrast, the abundance of immune effectors might mitigate the requirement for chemokine-driven recruitment of γδ T cells to the infected spleen. Although MC159 is a potent inhibitor of death cytokine-induced apoptosis and programmed necrosis (4
), perforin/granzyme-mediated cell death could bypass inhibition by MC159 to eliminate virus-infected cells in the transgenic mice. This notion is supported by the normal responses of MC159 transgenic mice to lymphocytic choriomeningitis virus (LCMV) (53
), whose clearance requires the coordinated function of perforin/granzyme and Fas (40
). Perforin/granzyme-mediated target cell killing might also explain why MC159 transgenic hepatocytes were protected from LPS-induced liver injury mediated by TNF but remained sensitive to cell-mediated cytotoxicity during VV infections.
MC159 is known predominantly as an inhibitor of death cytokine-induced apoptosis and programmed necrosis (8
). However, MC159 was unable to inhibit apoptosis induced by staurosporine (49
) and by overexpression of caspase-8/FLICE (24
), which bypass the death receptors. Similarly, MC159 transgenic lymphocytes exhibited normal cell death markers, such as annexin V and TUNEL, during VV infection. These results are reminiscent of the lack of inhibition of CD8+
T-cell death by pan-caspase inhibitors during VV infection (35
). Although we cannot rule out the possibility that the transgenic lymphocytes could still undergo programmed necrosis, our results do indicate that the enhanced γδ T-cell infiltration and innate immune control in MC159 transgenic mice during VV infection could not be attributed to inhibition of apoptosis. Since our previous work indicates that lymphocyte activation to various stimuli, including that during LCMV infection, was normal in MC159 transgenic mice (53
), lymphocyte-intrinsic effects are unlikely to account for the enhanced clearance of VV in the MC159 transgenic mice.
How might MC159 expression in the parenchyma facilitate innate immune responses? Our results indicate that transgenic fibroblasts exhibited heightened NF-κB activation in response to TNF and TLR4 stimulation. MC159 appears to specifically affect late-phase NF-κB activation while having little effect on early NF-κB induction. Interestingly, CCL-2 is a transcriptional target of NF-κB (21
), and TNF and TLR4 signaling have been shown to play crucial roles in the innate immune defense against VV (8
). Thus, increased NF-κB signaling to TNF or TLR4 stimulation could explain the increased CCL-2 expression and inflammatory leukocyte infiltration in the transgenic fat pad in response to VV infection. Although enhanced NF-κB activation was also observed for cells of hematopoietic origin (e.g., BMDCs and BMMs), radiation chimeras indicate that enhanced NF-κB activation in hematopoietic cells had a minimal contribution to the enhanced innate immunity against VV infections. Collectively, these results demonstrate an important role for signaling by cells in the parenchyma in innate immune responses to pathogens.
It is noteworthy that another vFLIP, K13 from human herpesvirus 8 (HHV-8), has also been reported to enhance NF-κB activation through an unknown mechanism (12
). Interestingly, we found that MC159 enhanced the binding between RIP1 and TRADD, two essential adaptors for NF-κB activation, by multiple TLRs and TNF receptors (9
). We propose that enhanced interaction between RIP1 and TRADD might underlie the mechanism by which MC159 promotes NF-κB signaling by innate immune receptors. However, the positive effect of MC159 on NF-κB activation was observed only when it is expressed at a low level. When expressed at higher levels, MC159 inhibited NF-κB activation. The inhibitory effect of MC159 at higher expression levels was reminiscent of several previous reports in which MC159 impaired TNF- and PKR-induced NF-κB activation (20
), double-stranded RNA (dsRNA)-mediated interferon regulatory factor 7 (IRF7) signaling (2
), and T-cell activation (54
). The molecular mechanism by which MC159 inhibits NF-κB activation is unclear at the moment. Based on our results, we propose that at low levels of expression, MC159 promotes RIP1 binding to the TRADD. However, as its expression level increases, MC159 might bind to and inhibit other cellular targets that lead to inhibition of NF-κB and other immune functions.
TNF-TNF receptor signaling is particularly important for the innate immune control of poxvirus infections, since TNFR-1−/−
mice were highly susceptible to VV and ectromelia virus infections (8
). In contrast, molluscum contagiosum virus (MCV) is readily controlled in immunocompetent individuals but causes persistent lesions in immunocompromised individuals, such as those infected with HIV (22
). Due to the ability of MC159 to promote or inhibit immune functions, it is tempting to speculate that MC159 might cooperate with other MCV-encoded immunoregulatory molecules to differentially modulate host responses in healthy and immunocompromised individuals.