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

Abstract

Neutrophils, historically known for their involvement in acute inflammation, are also targets for infection by many different DNA and RNA viruses. However, the mechanisms by which they recognize and respond to viral components are poorly understood. Polyinosinic:polycytidylic acid (poly(I:C)) is a synthetic mimetic of viral 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 IFNs, immunoregulatory cytokines, and chemokines, such as TNF-α, IL-12p40, CXCL10, CXCL8, CCL4, and CCL20, as well as classical IFN-responsive genes (IRG), including IFIT1 (IFN-induced protein with tetratricopeptide repeats 1)/IFN-stimulated gene (ISG)56, G1P2/ISG15, PKR (dsRNA-dependent protein kinase), and IFN-regulatory factor (IRF)7. Investigations into the mechanisms whereby transfected poly(I:C) promotes gene expression in neutrophils uncovered a crucial involvement of the MAPK-, PKR-, NF-κB-, and TANK (TNF receptor-associated NF-κB kinase)-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 IFN-β and TNF-α upon infection with encephalomyocarditis virus. Taken together, our data demonstrate that neutrophils are able to activate antiviral responses via helicase recognition, thus acting at the frontline of immunity against viruses.

Induction and regulatory function of miR-9 in human monocytes and neutrophils exposed to proinflammatory signals

“…S2), as previously reported in other cell types (11), demonstrating that the lack of miR-9 induction was not due to a general failure of monocytes to activate the TRIF-dependent pathway downstream TLR3. In agreement with the lack of TLR3 expression in human PMN (13), poly(I:C) had no effect on miR-9 expression in this cell type (Fig. 3A).…”

IFN-β Expression Is Directly Activated in Human Neutrophils Transfected with Plasmid DNA and Is Further Increased via TLR-4–Mediated Signaling

“…In fact, RNA polymerase III recognizes AT-rich DNA and consequently synthesizes uncapped 59 triphosphatebearing RNA, which, in turn, serves as an agonist for retinoic acidinducible gene (RIG-I) (39,40), which is constitutively expressed by human neutrophils (Fig. 6A) (5). Once again, a specific RNA polymerase III inhibitor-based approach, using ML-60218 (39,40), did not suppress the induction of IFN-b mRNA exerted by plasmid DNA or poly(dA:dT) electroporation (Supplemental Fig.…”

“…The latter include, among others, the TLRs (2), which are constitutively expressed and functional in human neutrophils, with the exception of TLR3 (3)(4)(5). For instance, TLR4, which is the specific receptor for endotoxin (6), potently triggers TNF-a, CXCL8, CCL3, CCL4, CCL19, CCL20, IL-1ra, and IL-12p40 mRNA expression and production in neutrophils stimulated with LPS (7).…”

Neutrophils and interferon-α-producing cells: who produces interferon in lupus?

“…Low-density PMNs present features of both immature and activated PMNs and have a decreased phagocytic potential, but it is still unknown whether they represent a distinct cell population. It should be noted that another study has shown that human PMNs isolated from healthy donors also produce IFN-β mRNA upon stimulation with transfected poly I:C [15], although IFN-α/β secretion was not detected. Likewise, mouse PMNs infected with encephalomyocarditis virus express IFN-β mRNA, a mechanism partially dependent on the cytoplasmic RNA helicase MDA5.…”