Activation of an Immunoregulatory and Antiviral Gene Expression Program in Poly(I:C)-Transfected Human Neutrophils

Neutrophils, historically known for their involvement in acute inflammation, also serve critical protective functions as they directly interact with products of bacterial infection through several surface receptor proteins, including Toll-like receptors (TLRs). Neutrophils are also targets for infection by many different DNA and RNA viruses, but how they do recognize and respond to viral components is poorly understood. In this regard, polyinosinic:polycytidylic acids [poly(I:C)] is a synthetic mimetic of viral double-stranded RNA (dsRNA) that is known to interact either with endosomal TLR3 – not expressed by human neutrophils – or with cytoplasmic RNA helicases such as melanoma differentiation-associated gene 5 (MDA5) and retinoic acid–inducible gene I (RIG-I). In this study, we report that intracellularly administered poly(I:C) stimulates human neutrophils to specifically express elevated mRNA levels encoding type I interferons, immunoregulatory cytokines and chemokines, such as TNFa, IL-12p40, CXCL10, CXCL8, CCL4 and CCL20, as well as classical interferon-responsive genes (IRG), including IFIT1, ISG15, PKR and IRF7. Investigations into the mechanisms whereby transfected poly(I:C) promotes gene expression in neutrophils uncovered a crucial involvement of the NF-kB-, MAPK-, PKR- and TANK-binding kinase (TBK1)/IRF3-signaling transduction pathways, as illustrated by the use of specific pharmacological inhibitors. Consistent with the requirement of the cytoplasmic dsRNA pathway for antiviral signaling, human neutrophils were found to constitutively express significant levels of both MDA5 and RIG-I, but not TLR3. Accordingly, neutrophils isolated from MDA5 deficient mice had a partial impairment in the production of IFNb and TNFa upon infection with encephalomyocarditis virus (S. McCartney and M. Colonna). Taken together, our data demonstrate, for the first time, that neutrophils are potentially able to activate anti-viral responses via helicase recognition, establishing neutrophils as important innate cells at the frontline of virus immunity.