Genome wide characterization of a STAT1-independent antiviral and immunoregulatory transcriptional program induced by IFNβ and TNFα reveals non-canonical STAT2 and IRF9 pathways

Interferon (IFN) β and Tumor Necrosis Factor (TNF) α are key players in immunity against pathogens as well as in the development of autoinflammatory and autoimmune diseases. Accordingly, their molecular pathways have attracted much interest as therapeutic targets. Compelling evidence has shown that the antiviral and inflammatory transcriptional response induced by IFNβ is reprogrammed by crosstalk with TNFα. IFNβ typically induces interferon-stimulated genes by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway leading to activation of the canonical ISGF3 transcriptional complex, composed of STAT1, STAT2 and IRF9. The signaling pathways engaged downstream of the combination of IFNβ and TNFα remain elusive, but previous observations suggested the existence of a response independent of STAT1. Here, using genome-wide transcriptional analysis by RNASeq, we observed a broad antiviral and immunoregulatory response initiated in the absence of STAT1 upon IFNβ and TNFα costimulation. Additional stratification of this transcriptional response with respect to the role of STAT2 and IRF9 revealed that they mediate the expression of a wide spectrum of genes. While a subset of genes was regulated by the concerted action of STAT2 and IRF9, other gene sets were independently regulated by STAT2 or IRF9. Collectively, our data supports a model in which STAT2 and IRF9 act through non-canonical parallel pathways to regulate distinct pool of genes in response to IFNβ and TNFα. This study provides novel insights into the molecular pathways leading to antiviral and immunoregulatory gene expression in conditions where elevated levels of both IFNβ and TNFα are present. To investigate the transcriptional program triggered by IFNβ and TNFα independently of STAT1, the human STAT1-deficient U3A cell line (derived from the 2ftGH fibrosarcoma cell line, McKendry R, et al.Proc Natl Acad Sci U S A. 1991;88(24):11455-9) were efficiently transfected with Control (Ctrl), STAT2 or IRF9 targeting RNAi and further left untreated or stimulated with a combination of IFNβ (1000U/ml) +TNFα (10ng/ml) for 24h. Total RNA was isolated and analyzed by RNA sequencing (n=3 for each group) on an Illumina HiSeq2500 platform. Bioinformatics analysis was performed to determine genes differentially regulated upon IFNβ and TNFα costimulation. Moreover, the role of STAT2 and IRF9 in the regulation of these genes was analyzed. Results of RNASeq were validated by qRT-PCR.