IFN\u3b1 enhances the production of IL-6 by human neutrophils activated via TLR8.

Abstract

PURPOSE Recently, we reported that human neutrophils produce biologically active IL-6 when incubated with agonists activating TLR8, a receptor recognizing viral single strand RNA. Herein, we investigated the effect of IFN\u3b1, a cytokine known to modulate the early innate immune responses toward viral and bacterial infections, on the production of IL-6 by TLR8-activated neutrophils. METHODS Human neutrophils isolated from healthy donors or systemic lupus erythematosus (SLE) patients by negative selection using immunomagnetic beads (99.7 \ub1 0.2 % purity), were incubated for up to 20 h with or without 5 \u3bcM R848 (a TLR8 agonist), in the presence or the absence of 1000 U/ml IFN\u3b1. mRNA expression and cytokine production were then measured by, respectively, RT-qPCR and ELISA, while C/EBP\u3b2 transcription factor recruitment at the IL-6 genomic locus was investigated by chromatin immunoprecipitation (ChIP) assays. RESULTS In this study, we demonstrate that IFN\u3b1 potently enhances the production of IL-6 in neutrophils incubated with R848. Such an effect is not caused by an IFN\u3b1-dependent induction of TLR7, another receptor for R848, but, rather, by an increased release of TNF\u3b1, which in turn amplifies IL-6 expression. Endogenous TNF\u3b1, in fact, was shown to promote an augmented synthesis of the IkB\u3b6 coactivator and an enhanced recruitment of C/EBP\u3b2 to the IL-6 promoter. Moreover, our data uncover that neutrophils from active SLE patients, displaying an IFN-induced gene expression signature, produce increased amounts of both IL-6 and TNF\u3b1 in response to R848 as compared to healthy donors. DISCUSSION Altogether, data clarify the molecular bases of the IFN\u3b1-dependent enhancement of IL-6 production in TLR8-activated neutrophils. More in general, we show that TLR8 ligands, IFN\u3b1 and TNF\u3b1, three players often coexisting in many diseases of viral or autoimmune origin, promote a strong production of IL-6 in human neutrophils, placing the same neutrophils among potential targets for immunotherapeutic interventions