In this study, we suggest a role for MLK3 in regulating the outcome of influenza infection in vivo. Our study revealed that the genetic ablation of MLK3 was associated with elevated virus production at late time points following infection in vivo. Our data also showed that cultured lung cells from MLK3−/− mice were resistant to influenza virus-mediated cell killing, and were able to sustain release of progeny virions for an extended time period, relative to lung cells from wild-type mice. These in vitro findings suggest that during the course of infection infected cells in the MLK3−/− mice will continue to produce virus progeny for longer periods of time leading to more virions produced per infected cell. Over time, in the absence of MLK3, this will lead to a greater accumulation and spread of virus in the lung.
Histological and flow analysis suggest that MLK3 is not required for proliferation of immune cells or the infiltration of these cells into the lung during infection with influenza. Previous reports on MLK3’s role in cell proliferation and migration have been contradictory (Brancho et al., 2005
; Chadee and Kyriakis, 2004
). MLK3 was suggested to be essential for the proliferation of serum stimulated fibroblasts by Chadee and Kryiakis (Chadee and Kyriakis, 2004
), but studies by Brancho and colleagues on the proliferation and migration of MEFs from MLK3−/− mice suggested that MLK has no role in cell proliferation or migration (Brancho et al., 2005
). Overall, our results are consistent with those of Brancho and colleagues, but not those of Chadee and Kyriakis, suggesting that MLK3 does not impact either of these processes (Brancho et al., 2005
MLK3 is known to be expressed in dendritic cells (DC) (Handley et al., 2007b
) and previous studies have suggested that MLKs may be involved in DC maturation and activation (Handley et al., 2007a
). In our studies, the levels of antigen-specific, cytokine secreting CD8+
T cells were similar in virus-infected MLK3−/− mice versus wild-type mice, suggesting that the function of DC that prime the adaptive immune response is not seriously impaired.
MLK3 is also known to regulate the p38 cascade of the MAPK pathway (Brancho et al., 2005
; Chadee and Kyriakis, 2004
). Previously, it was shown that CD4+
T cells isolated from MKK6 transgenic mice, in which p38 is constitutively active, have increased IFN-γ production (Merritt et al., 2000
; Rincon et al., 1998
). We see a similar trend in CD8+
T cells isolated from MLK3−/− mice during infection with influenza. Conze et al. proposed that increased levels of IFN-γ could augment viral clearance (Conze et al., 2000
). Consistent with this, MLK3−/− mice show clearance in viral titers by day 10 similar to that detected in the wild-type mice. This ability to clear virus could be promoted by the boost in IFN-γ production by antigen specific CD8+
T cells. However, it is important to note that the increased IFN-γ production by T cells is not reflected in the overall IFN-γ levels in the total lung.
CEP1347 treatment of human bronchial epithelial cells has been shown to result in a marked decrease in RANTES release following H3N2 influenza virus infection (Kujime et al., 2000
). In contrast, our findings revealed that RANTES production in lungs of influenza virus-infected MLK3−/− mice was similar to that in wild-type mice. This may reflect either (i) in vivo production of RANTES from non-epithelial cell types, such as T cells and/or (ii) differences related to the virus strain used (H3N2 versus H1N1) and/or (iii) the ability of CEP1347 to inhibit not only MLK3, but also other related family members which may be expressed in bronchial epithelial cells (Maroney et al., 2001
Our in vivo
studies revealed a potential role for MLK3 in viral production during infection with influenza. We observed that virus titers in the lung of MLK3−/− mice were increased in comparison to wild-type mice at late time points following infection. In accordance with our in vitro
data, the increased level of virus production could be a result of increased virus shedding from surviving infected target cells. Enhanced survival of cells lacking MLK3 has been seen previously in other disease models (Hong and Kim, 2007
; Kim et al., 2004
; Sui et al., 2006
; Zhao et al., 2007
Overall, our work clarifies the role of MLK3 during influenza infection and reveals that the loss of MLK3 is associated with increased virus titers in the lung at late time points following experimental infection with influenza virus. Finally, our data suggest that elevated virus titers in MLK3−/− mice may be a result of increased virus replication, as a consequence of the prolonged survival of virus-infected MLK3−/− lung cells.