Céline Molle

IL-27 synthesis induced by TLR ligation critically depends on IFN regulatory factor 3.

IL-27 is a heterodimeric cytokine composed of EBV-induced gene 3 and p28. Produced by dendritic cells (DCs) in response to TLR ligands, IL-27 recently emerged as a key regulator of inflammatory responses. In this study, we first demonstrate that Toll/IL-1R-containing adaptor inducing IFN-β and its associated IFN regulatory factor (IRF) 3 transcription factor are critically involved in IL-27p28 expression in mouse DCs stimulated by TLR ligands. We then show that IL-27 serum levels are dramatically reduced in IRF3−/− upon LPS injection, indicating a critical role for IRF3 in TLR4-mediated IL-27 production in vivo. We identified an IRF3-binding site within the IL-27p28 promoter region which is required for IL-27p28 gene activation in reporter gene assays. In human DCs, IL-27p28 mRNA was preferentially induced by Toll/IL-1R-containing adaptor inducing IFN-β-coupled TLR ligands and following CMV infection. Furthermore, chromatin immunoprecipitation studies demonstrate that IRF3 is recruited to the endogenous p28 promoter in TLR4-stimulated human DCs. We conclude that IRF3 activation is a master switch for IL-27 synthesis.

Critical Role of the IFN-Stimulated Gene Factor 3 Complex in TLR-Mediated IL-27p28 Gene Expression Revealing a Two-Step Activation Process

In myeloid dendritic cells, activation of the IL-27p28 gene is selectively induced by ligands of TLR4 or TLR3, both coupled to the Toll/IL-1R–related domain-containing adaptor-inducing IFN/IFN regulatory factor (IRF)3 pathway. In response to both ligands, autocrine type 1 IFN production was required for optimal IL-27p28 expression. Type I IFN signaling was necessary for sustained IRF1 activation and formation of the IRF9-containing IFN-stimulated gene factor 3 complex. Indeed, we demonstrated that IRF1 and IRF9 are sequentially activated and recruited to the IL-27p28 IFN-stimulated regulatory element site. Involvement of IRF1 and IRF9 in the induction of IL-27p28 was confirmed in vitro and upon in vivo exposure to TLR ligands. Thus, in response to TLR4 or TLR3 ligation, the initial induction of the IL-27p28 gene depends on the recruitment of IRF1 and IRF3, whereas transcriptional amplification requires recruitment of the IFN-stimulated gene factor 3 complex. These results highlight the complex molecular interplay between TLRs and type I IFNs for the control of IL-27 synthesis.

Tristetraprolin regulation of interleukin 23 mRNA stability prevents a spontaneous inflammatory disease

Tristetraprolin deficiency results in enhanced IL-23 via dysregulated mRNA decay that leads to an inflammatory syndrome characterized by cachexia, myeloid hyperplasia, dermatitis, and erosive arthritis.

Interferon regulatory factor 3 controls interleukin-17 expression in CD8 T lymphocytes

Significance Interferon regulatory factor (IRF) 3 is one of the key transcription factors implicated in innate antiviral responses. In recent years its role in shaping adaptive immune responses through activation of a specific transcriptional program in antigen-presenting cells has been appreciated. In this work we show that within CD8 T cells, IRF3 interacts with the transcriptional network that controls their polarization, thereby limiting the capacity of these cells to produce IL-17. These findings shed light on the functions of IRF3 in adaptive immune responses. IFN regulatory factor (IRF) 3 plays a key role in innate responses against viruses. Herein we assessed its contribution to T-cell activation. We observed that poly(I:C)-induced IRF3 activation in CD8 T cells represses IL-17 expression in a type I IFN-independent fashion. Even in the absence of poly(I:C), polyclonally activated naïve IRF3−/− CD8 T cells expressed high levels of IL-17 and IL-23R in comparison with wild-type cells. Furthermore, IRF3−/− OT1 cells adoptively transferred into wild-type hosts also produced higher IL-17 levels upon immunization than their wild-type counterparts. This phenotype could be reversed by ectopic expression of IRF3, confirming that this effect is intrinsic to T cells. We show that IRF3 directly interacts with RORγt in the cytoplasm through its IRF interaction domain and limits its ability to bind and transactivate the IL-17 promoter. These observations uncover an unexpected role of IRF3 in the control of CD8 T-cell polarization.

A conventional protein kinase C inhibitor targeting IRF-3-dependent genes differentially regulates IL-12 family members.

Protein kinase C (PKC) isoforms play a critical role in the regulation of innate immune responses. We have previously demonstrated that conventional PKC (cPKC) α is involved in interferon regulatory factor 3 (IRF-3) activation and IFN-β synthesis. Herein, we investigated the role of cPKCs in the regulation of IL-12 family members expression mediated by the Toll-like receptor 3 (TLR3) and TLR4. First, inhibition of cPKCs activity in human DCs by a cPKC-specific inhibitor, Gö6976 downregulated the expression of IL-12p70 and IL-27p28 but not IL-12/IL-23p40, IL-23, IL-27EBI3 induced by LPS or poly(I:C). Furthermore, reporter gene assays in RAW 264.7 macrophages showed that cPKCs regulate IL-12p35 and IL-27p28 promoter activities since Gö6976 repressed LPS and poly(I:C)-mediated transcriptional activities of IL-12p35 and IL-27p28. In contrast, no effect was observed with IL-12/IL-23p40 and IL-23p19 reporter constructs. These results prompted us to study the role of IRF-3 on IL-23 expression. Bone marrow-derived DC (BMDCs) from IRF-3(-/-) mice produced comparable levels of IL-23 induced by both LPS and poly(I:C) as compared to wild type BMDCs, indicating that IRF-3 is not involved in IL-23 production. Finally, BMDCs from PKCα(-/-) mice displayed a reduced synthesis of IL-27 induced by poly(I:C). Collectively, these data identify cPKCs as critical components that control IRF-3-dependent IL-12p35 and IL-27p28 gene expression downstream of TLR3 and TLR4.

Activation of the endoplasmic reticulum stress sensor IRE1α by the vaccine adjuvant AS03 contributes to its immunostimulatory properties

The oil-in-water emulsion Adjuvant System 03 (AS03) is one of the few adjuvants used in licensed vaccines. Previous work indicates that AS03 induces a local and transient inflammatory response that contributes to its adjuvant effect. However, the molecular mechanisms involved in its immunostimulatory properties are ill-defined. Upon intramuscular injection in mice, AS03 elicited a rapid and transient downregulation of lipid metabolism-related genes in the draining lymph node. In vitro, these modifications were associated with profound changes in lipid composition, alteration of endoplasmic reticulum (ER) morphology and activation of the unfolded protein response pathway. In vivo, treatment with a chemical chaperone or deletion of the ER stress sensor kinase IRE1α in myeloid cells decreased AS03-induced cytokine production and its capacity to elicit high affinity antigen-specific antibodies. In summary, our results indicate that IRE1α is a sensor for the metabolic changes induced by AS03 in monocytic cells and may constitute a canonical pathway that could be exploited for the design of novel vaccine adjuvants.Adjuvants: ER stress is critical for adjuvant efficacyAdjuvants are the ‘secret sauce’ of vaccines—by stimulating the innate arm of the immune system they potentiate activation of adaptive immunity; however, their mode of action is incompletely understood. A team led by Stanislas Goriely at the Université Libre de Bruxelles studied the functional basis of a major class of clinically-applied adjuvant—AS03—an oil-in-water emulsion adjuvant used in human influenza vaccines. Using an in vivo mouse model and cell lines they observe that AS03 triggered downregulation of genes controlling lipid metabolism. At the same time there was an increase in an ER-stress signature likely as a result of accumulation of intracellular lipid content in myeloid cells such as macrophages. ER stress was critical for the production of inflammatory cytokines by macrophages and the subsequent generation of robust and specific antibody responses following vaccination. Selective enhancement of ER stress might therefore lead to superior vaccines while minimizing side effects.