Interferon (IFN) β plays a critical role in the first line of defense against viruses, through its ability to induce a broad antiviral transcriptional response in virtually all cell types [1
]. IFNβ also possesses key immunoregulatory functions that determine the outcome of the adaptive immune response against pathogens [1
]. Over the years, in vitro and in vivo studies aimed at characterizing the mechanisms and the functional outcomes of IFNβ signaling were mostly performed in relation to single cytokine stimulation. This unlikely reflects physiological settings, as a plethora of cytokines are secreted in a specific situation. As a consequence, a cell rather simultaneously responds to a cocktail of cytokines to foster the appropriate transcriptional program. Response to IFNβ is no exception and is very context-dependent, particularly regarding the potential crosstalk with other cytokines. Elevated levels of IFNβ and Tumor Necrosis Factor (TNF) are found during the host response to viruses. Aberrant increased levels of both cytokines is also associated with a number of autoinflammatory and autoimmune diseases such as Systemic Lupus Erythematosus (SLE), psoriasis, and Sjögren’s syndrome [3
]. While the cross-regulation of IFNβ and TNF is well documented [4
], the functional crosstalk between these two cytokines remains poorly known.
IFNβ typically acts through binding to the IFNAR receptor (IFNAR1 and IFNAR2) leading to the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway involving JAK1- and Tyk2-mediated phosphorylation of STAT1 and STAT2, and to a lesser extent other STAT members in a cell-specific manner [7
]. Phosphorylated STAT1 and STAT2, together with IFN Regulatory Factor (IRF) 9, form the IFN-stimulated gene factor 3 (ISGF3) complex that binds to the consensus IFN-stimulated response element (ISRE) sequences in the promoter of hundreds of IFN stimulated genes (ISGs) [9
]. Formation of the ISGF3 complex is considered a hallmark of the engagement of the type I IFN response and, consequently, the requirement of STAT1 in a specific setting has become a marker of the engagement of type I IFN signaling [7
]. However, in recent years, this paradigm has started to be challenged with accumulating evidence demonstrating the existence of non-canonical JAK-STAT signaling that mediates type I IFN responses [8
Synergism between IFNβ and TNF was shown to enhance the antiviral response to Vesicular Stomatitis Virus (VSV), Myxoma virus, and paramyxovirus infections [12
]. Gene expression analyses showed that IFNβ and TNF synergistically regulate hundreds of genes induced by individual cytokines alone, but also drive a specific delayed transcriptional program composed of genes that are either not responsive to IFNβ or TNF separately or are only responsive to either one of the cytokine [4
]. The signaling mechanisms engaged downstream of the costimulation with IFNβ and TNF remained elusive, but it is implicitly assumed that the fate of the gene expression response requires that both IFNβ- and TNF-induced signaling pathways exhibit significant crosstalk. Analysis of the enrichment of specific transcription factors binding sites in the promoters of a panel of genes synergistically induced by IFNβ and TNF failed to give a clue about the specificity of the transcriptional regulation of these genes [13
]. We previously showed that the DUOX2
gene belongs to the category of delayed genes that are remarkably induced to high levels in response to the combination of IFNβ and TNF in lung epithelial cells [8
]. We found that DUOX2
expression required STAT2 and IRF9 but not STAT1, suggesting that STAT2 and IRF9 activities might segregate in an alternative STAT1-independent pathway that could be involved in gene regulation downstream of IFNβ and TNF [14
In the present study, we aimed to fully characterize the transcriptional profile of the delayed response to IFNβ and TNF that occurs independently of STAT1 and evaluate the role of STAT2 and IRF9 in the regulation of this response. We found that the costimulation by IFNβ and TNF induces a broad set of antiviral and immunoregulatory genes in the absence of STAT1. We also report the differential regulation of distinct subsets of IFNβ− and TNF-induced genes by STAT2 and IRF9. While IFNβ and TNF act in part through the concerted action of STAT2 and IRF9, specific sets of genes were only regulated by either STAT2 or IRF9. Altogether, our findings uncovered non-canonical STAT2 and/or IRF9-dependent pathways that coexist to regulate distinct pools of antiviral and immunoregulatory genes in a context of IFNβ and TNF crosstalk.
Previous studies have described that IFNβ and TNF synergize to elicit a specific delayed transcriptional program that differs from the one induced by either cytokine alone [13
]. The mechanisms underlying the transcriptional induction of genes specifically regulated by IFNβ and TNF remain poorly defined. The present study was specifically designed to document the functional relevance of a previously observed delayed gene expression induced by IFNβ in the presence of TNF in the absence of STAT1 [14
] and to document the role of STAT2 and IRF9 in this response.
The observation that STAT2 and IRF9 activation in response to IFNβ + TNF is reduced in STAT1-deficient U3A cells compared to the wild-type 2ftGH parental cells and that IFNβ + TNF-mediated induction of the STAT2- and IRF9-dependent CXCL10
promoter exhibits partial dependence on STAT1 support a model in which a canonical ISGF3 pathway is engaged downstream of the costimulation. Importantly, it also implied the existence of a STAT2- and/or IRF9-dependent transcriptional response occurring in the absence of STAT1. The human STAT1-deficient U3A cell model offered a unique opportunity to specifically pinpoint this STAT1-independent response. In this model, genome wide RNA sequencing highlighted that the transcriptional program induced by IFNβ + TNF in the absence of STAT1 encompasses a wide range of immunoregulatory and antiviral functions. The functional relevance of this response was confirmed by the observation that the treatment with IFNβ + TNF induced an antiviral state capable of restricting VSV replication in the absence of STAT1. This points to a significant role of the STAT1-independent pathway in the establishment of the antiviral state induced by the synergistic action of IFNβ and TNF that enhances the restriction of VSV (Figure 4
D and [12
]), Myxoma virus [13
], and paramyxoviruses [14
]. Although previous reports have shown that type I IFNs, mostly IFNα, alone can trigger STAT1-independent responses [8
], we neither observed establishment of an IFNβ-induced antiviral state against VSV, nor activation of the CXCL10
promoter in the absence of STAT1 in our model (Figure 4
D and Figure 6
We previously reported that IFNβ + TNF induces the DUOX2
gene via a STAT2- and IRF9-dependent pathway in the absence of STAT1 [14
]. To what extent this pathway contributes to the STAT1-independent transcriptional response elicited by IFNβ + TNF remained to be addressed. Here, we demonstrate that IFNβ + TNFα-induced DEGs segregate into seven categories that reflect distinct contributions of STAT2 and/or IRF9, thereby highlighting an unexpected heterogeneity of the STAT1-independent pathways engaged downstream of IFNβ + TNF. Importantly, only one anecdotic gene was found in categories implying inverse regulation by STAT2 and IRF9 (categories H and I) pointing to convergent functions of STAT2 and IRF9 when both are engaged in gene regulation. We can rule out that these distinct regulation mechanisms reflect specific induction profiles by IFNβ + TNF as CXCL10
all exhibit synergistic induction by IFNβ + TNF, but are differentially regulated by STAT2 and/or IRF9; while CXCL10
is dependent on STAT2 and IRF9, IL33
is independent on STAT2 and IRF9, and CCL20
are STAT2-independent but IRF9-dependent (Figure 5
; Supplemental Table S1
). Consistent with our previous observation [14
], we found several STAT1-independent genes positively regulated by STAT2 and IRF9 (Category A). DEGs in this category encompass most of the functions induced in response to IFNβ + TNF, with the notable exception of cell cycle and growth arrest and inflammasome and receptor signaling functions. Genes negatively regulated by STAT2 and IRF9 were also identified (Category B). Formation of an alternative STAT2/IRF9-containing complex mediating gene expression in the absence of STAT1 [37
] has been reported, but with limited DNA-binding affinity for the typical ISRE sequence [37
]. The existence of a STAT2/IRF9 complex is also supported by our recent observation of a high affinity of IRF9 for STAT2 with an equilibrium dissociation constant (Kd) of 10 nM [42
]. A recent report of experiments, performed in murine bone marrow-derived macrophages proposes a model in which murine STAT2/IRF9 complex drives basal expression of ISGs, while IFNβ-inducible expression of ISGs depends on a switch to the ISGF3 complex [43
]. This differs from our results as silencing of either STAT2 or IRF9 did not alter basal gene expression (Supplemental Figure S1
). Further analysis of the CXCL10
promoter demonstrates a restricted usage of ISRE sites by the STAT2/IRF9 pathway compared to the ISGF3 pathway. Further large-scale studies will be needed to identify the parameters allowing binding of ISGF3, but not STAT2/IRF9, to specific ISRE sequences upon IFNβ + TNF.
The observation that some IFNβ + TNF-induced genes were solely dependent on STAT2 (either positively or negatively) but not on IRF9, (Categories D and E) is a rare genome wide demonstration of gene regulation by STAT2 independently of STAT1 and IRF9. Previous reports have identified ISGF3-independent, STAT2-dependent genes but the association with IRF9 was not formally excluded [44
]. STAT2 was shown to associate with STAT3 and STAT6, but it is not clear whether IRF9 is also part of these alternative complexes [44
]. Transcriptional module analyses demonstrated that the functional distribution of genes negatively regulated by STAT2 is very limited compared to other categories; only a virus-sensing module was enriched in this category. In contrary, IRF9-independent genes positively regulated by STAT2 mediate broader antiviral and immunoregulatory functions.
ISGF3-independent functions of IRF9 have been proposed based on the study of IRF9 deficiencies [11
]. However, IRF9 target genes in these contexts have been barely documented. Intriguingly, Li et al. [49
] studied IFNα-induced genes and their dependency on the ISGF3 subunits. While they confirmed previous studies showing that IFNα can trigger a delayed and sustained ISG response via an ISGF3-independent pathway, it is very striking that they did not find STAT1- and STAT2-independent but IRF9-dependent genes. All identified IRF9-dependent genes were either STAT2- or STAT1-dependent. This result greatly differs with our study. Here, we found several IFNβ + TNF-induced DEGs independent of STAT1 and STAT2, but positively or negatively regulated by IRF9 (Categories F and G). Typically, IRF9 is considered a positive regulator of gene transcription. However, our findings are consistent with recent reports documenting the role of IRF9 in the negative regulation of the TRIF/NF-κB transcriptional response [50
] or the expression of SIRT1 in acute myeloid leukemia cells [51
]. The molecular mechanisms underlying gene regulation by IRF9 without association with either STAT1 or STAT2 remain to be elucidated. To the best of our knowledge, no alternative IRF9-containing complex has yet been described.
Our analysis showed that a large number of genes were induced by IFNβ + TNF independently of STAT2 and IRF9 (Category C). All transcriptional modules were enriched in this category pointing to a major role of this pathway in the establishment of a host defense and immunoregulatory response. The STAT2 and IRF9 independent genes does not solely reflects induction by TNF alone. For instance, APOBEC3G
that is amongst the STAT2- and IRF9- independent genes is not induced by TNF alone (Figure 2
A). While NF-κB, a downstream effector of the TNF receptor, is an obvious candidate for the regulation of these DEGs, this might fall short in explaining the synergistic action of IFNβ + TNF as we did not observe enhanced NF-κB activation compared to TNF alone [14
]. Alternatively, the potential role of AP-1 is supported by the finding that the AP-1 transcription network module is enriched amongst IFNβ + TNF-induced DEGs. However, this module is not restricted to genes regulated independently of STAT2 and IRF9. It is also worth noting that two modules of IRF2-target genes were enriched, although again not specifically in the STAT2- and IRF9-independent category. A similar crosstalk was reported between IFNα and TNF in macrophages resulting in increased colocalized recruitment of IRF1 and p65 to the promoter of a subset of genes [52
]. However, while IRF1 was found synergistically induced by IFNβ + TNF at early stages (Figure 1
), we did not observe significant induction of IRF1 in the absence of STAT1 by RNASeq (Supplemental Table S1
) or qRT-PCR (data not shown), suggesting that IRF1 is unlikely to be involved in our system. Further studies will be required to uncover these STAT2- and IRF9-independent pathways.
This study provides novel insight into the molecular pathways leading to delayed antiviral and immunoregulatory gene expression in conditions where elevated levels of both IFNβ and TNF are present. Altogether our results demonstrate that in addition to the engagement of an ISGF3-dependent canonical response, a broad transcriptional program is elicited independently of STAT1, and support a model in which STAT2 and IRF9 contribute to the regulation of this response through non-canonical parallel pathways involving their concerted or independent action (Figure 7
Consistent with accumulating evidence [8
], these distinct STAT2 and IRF9 actions most likely result from the formation of specific complexes that coexist with ISGF3 upon IFNβ and TNF stimulation. Studies are underway to biochemically solve the complexity of the dynamic and specific mechanisms of activation of the alternative STAT2 and/or IRF9-containing complexes in a wild-type cell context to further characterize the transcriptional response induced by IFNβ and TNF.