IRF8 and IRF3 cooperatively regulate rapid interferon-β induction in human blood monocytes

Abstract

Robust and rapid induction of interferon-␤ (IFN-␤) in monocytes after pathogenic stimulation is a hallmark of innate immune responses. Here, we reveal the molecular mechanism underlying this key property that is exclusive to human blood monocytes. We found that IFN-␤ was produced rapidly in primary human monocytes as a result of cooperation between the myeloid-specific transcription factor IRF8 and the ubiquitous transcription factor IRF3. Knockdown of IRF8 in monocytes abrogated IFN-␤ transcription, whereas reintroduction of IRF8 into the IRF8 ؊/؊ 32Dcl3 murine myeloid cell line reinstated IFN-␤ transcription. Moreover, we provide evidence that IRF8 constitutively binds to the ETS/IRF composite element of the IFN-␤ promoter region together with PU.1 in vivo. Furthermore we uncovered a requirement for IRF3, a master regulator of IFN-␤ production, as a previously un-indentified interaction partner of IRF8. We mapped the protein- IntroductionProduction of interferon-␤ (IFN-␤) by the host is an essential first line of defense against both viral and nonviral pathogens. [1][2][3] As well as its involvement in innate immunity, IFN-␤ is an important initiator and modulator of the adaptive immune response. [4][5][6] For example, IFN-␤ influences the differentiation, maturation, and migration of dendritic cells. 7 In addition, it regulates antibody production and antigen-mediated apoptosis of B cells, 8 alongside promoting the generation of CD4 ϩ Th1 responses, 9 and the expansion of antigen-specific CD8 ϩ T cells. 10 Interestingly, Stockinger et al have recently demonstrated that IFN-␤ production contributes to Listeria monocytogenes-induced lethality in mice. 11 As such, IFN-␤ seems able to induce immunopathology as well as influence the resolution of infections, thus making it important to understand the mechanisms linking pathogen stimulation of immune cells with the production of IFN-␤.IFN-␤ transcription is initiated after cellular recognition of pathogen-associated molecular patterns by pattern recognition receptors. 12 Many cell types express IFN-␤ after microbial exposure, [13][14][15] although the underlying mechanisms vary. In murine embryonic fibroblasts, IFN-␤ induction is mediated by the activation of both IRF3 and IRF7. 16,17 In contrast, induction of IFN-␤ in murine plasmacytoid dendritic cells occurs after CpG recognition by Toll-like receptor 9 and is entirely dependent on IRF7, 18 whereas in murine macrophages transcription of IFN-␤ is dependent on IRF3 alone. 19 IRF3 is a ubiquitous cytosolic transcription factor that translocates to the nucleus on activation, 20 where it is able to induce IFN-␤ expression. However, this process takes more than 6 hours to produce detectable levels of IFN-␤ mRNA in HeLa cells, with levels peaking even later at 9 to 19 hours. 21 Monocytes are myeloid cells of the immune system that play a key role in the rapid innate response to pathogens, including the production of IFN-␤. 11,22 However, the molecular mechanisms underlying the rapid induction of IFN-␤ in monocytes hav...

A dual cis-regulatory code links IRF8 to constitutive and inducible gene expression in macrophages

“…5D) confirmed that the Ifnb1 gene promoter was constitutively bound by IRF8 and that PU.1 association with it was largely IRF8 dependent. Binding of IRF8 and PU.1 to the IFNb promoter in human and mouse monocytes was previously mapped to promoter sites resembling PWM1 (a composite ETS/IRF site that differs from PWM1 because of a 4-nt spacer between the 59-GGAA-39 ETS consensus and the 59-GAAA-39 IRF consensus) and PWM4 (Li et al 2011) and is fully consistent with our ChIP-seq data. Moreover, constitutive IRF8/PU.1 binding was shown to be required for IRF3 recruitment, thus explaining reduced IFNb induction in cells depleted of IRF8 (Li et al 2011).…”

Zebrafish IRF1 Regulates IFN Antiviral Response through Binding to IFNϕ1 and IFNϕ3 Promoters Downstream of MyD88 Signaling

“…First, the spleen and head kidney of zebrafish were collected for total RNA extraction at 4,8,12,20,32, or 48 h postinfection with SVCV. mRNAs were made using the polyATtract mRNA isolation system (Promega) and used for construction of a 1 3 10 6 CFU BacterioMatch II two-hybrid system library, according to the manufacturer's instructions (Agilent).…”

“…In addition to IRF3/7, accumulating evidence suggests that IRF1, IRF5, and IRF8 trigger IFN responses in cell-specific fashions (5)(6)(7)(8). IRF1 is the founder member of the IRF family and initially was identified as a positive regulator directly binding to type I IFN gene promoters (9).…”

Zebrafish IRF1, IRF3, and IRF7 Differentially Regulate IFNΦ1 and IFNΦ3 Expression through Assembly of Homo- or Heteroprotein Complexes

“…IRF8 directs the IFN response, together with IRF3/7, in DCs (18) and with IRF3 in human blood monocytes (19). IRF3, IRF5, and IRF7 coordinately act in myeloid DCs downstream of mitochondrial antiviral signaling protein signaling (20).…”