Abstract
Early molecular responses to Influenza A (FLUA) virus strain A/X-31 H3N2 in macrophages were explored using J774.A1 and RAW 264.7 murine cell lines. NF-kappa B (NFκB) was reported to be central to FLUA host-response in other cell types. Our data showed that FLUA activation of the classical NFκB dependent pathway in these macrophages was minimal. Regulator proteins, IkappaB-alpha and –beta (IκBα, IκBβ), showed limited degradation peaking at 2 h post FLUA exposure and p65 was not observed to translocate from the cytoplasm to the nucleus. Additionally, the non-canonical NFκB pathway was not activated in response to FLUA. The cells did display early increases in TNFα and other inflammatory cytokine and chemokine production. Mitogen activated phosphokinase (MAPK) signaling pathways are also reported to control production of inflammatory cytokines in response to FLUA. The activation of the MAPKs, cJun kinases 1 and 2 (JNK 1/2), extracellular regulated kinases 1 and 2 (ERK 1/2), and p38 were investigated in both cell lines between 0.25 and 3 h post-infection. Each of these kinases showed increased phosphorylation post FLUA exposure. JNK phosphorylation occurred early while p38 phosphorylation appeared later. Phosphorylation of ERK 1/2 occurred earlier in J774.A1 cells compared to RAW 264.7 cells. Inhibition of MAPK activation resulted in decreased production of most FLUA responsive cytokines and chemokines in these cells. The results suggest that in these monocytic cells the MAPK pathways are important in the early response to FLUA.
Highlights
Despite decades of work on vaccines and antivirals, influenza virus infection remains a major health threat
In epithelial cells canonical NF-kappa B (NFkB) activation was observed in response to FLUA infection, as shown by the degradation of NFkB inhibitors IkBa and IkBb visualized by Western blot, by NFkB nuclear translocation visualized by immunofluorescence, or by enhanced transcription factor DNA binding shown by electromobility shift assay or reporter gene assay [34,59,60,61]
There was no substantial decrease in production of viral proteins in the cells that received inhibitor compared to those that did not as judged by densitometry of virus specific bands. In these experiments we examined the degree of activation and kinetics of cell signaling for both of the major NFkB pathways and the Mitogen activated phosphokinase (MAPK) pathways thought to be important for proinflammatory cytokine and chemokine (CK/CHK) production in response to FLUA
Summary
Despite decades of work on vaccines and antivirals, influenza virus infection remains a major health threat. Influenza and pneumonia were listed as the 8th leading cause of death in the USA for the last several years [1,2,3]. Together, they represent a huge cost to the U.S economy, estimated in 2005 to be $40 billion [4]. Vaccines are less effective in influenza susceptible populations such as the very young and the very old. Understanding the specific biochemical pathways controlling influenza induced immune functions such as cytokine and chemokine (CK/CHK) production may lead to the development of improved therapies
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