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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.

RAW 264.7 and J774.1 murine macrophage cells are widely used to model responses to infectious diseases; therefore they were selected to explore early molecular responses to FLUA. NF‐kappa B is reported to be central to FLUA host‐response in other cell types. Previous data show that FLUA (X31/H3N2) does not activate the classical NF‐kappa B dependent pathway in these macrophages. Regulator proteins, I kappa B ‐alpha and ‐beta, are not degraded in response to FLUA infection, and p65 does not translocate from the cytoplasm to the nucleus. However, the cells display the expected increase in TNF‐alpha and other inflammatory cytokine and chemokine secretions. Mitogen Activated Phosphokinase (MAPK) signaling pathways are also reported to control production of inflammatory cytokines in response to FLUA. The activation of the MAPK pathway intermediates p38, Janus Kinases 1 and 2 (JNK 1/2), and Extracellular Regulated Kinases 1 and 2 (ERK 1/2) were investigated in both cell lines between 0.25 and 3 hours post‐infection. Each of these kinases showed increased phosphorylation post FLUA infection. JNK phosphorylation occurred early while p38 phosphorylation appeared late. Phosphorylation of ERK 1/2 occurred earlier in J774.1 cells as compared to RAW 264.7 cells. The results suggest that in these monocytic cells the MAPK pathways are important in the early pathogen activated molecular pattern (PAMP) response to FLUA.

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