Faculty Bibliography

Immune health requires innate and adaptive immune cells to engage precisely balanced pro- and anti-inflammatory forces. We employ the concept of chemical immunophenotypes to classify small molecules functionally or mechanistically according to their patterns of effects on primary innate and adaptive immune cells. The high-specificity, low-toxicity cyclin dependent kinase 8 (CDK8) inhibitor DCA exerts a distinct tolerogenic profile in both innate and adaptive immune cells. DCA promotes Treg and Th2 differentiation, while inhibiting Th1 and Th17 differentiation, in both murine and human cells. This unique chemical immunophenotype led to mechanistic studies showing that DCA promotes Treg differentiation in part by regulating a previously undescribed CDK8-GATA3-FOXP3 pathway that regulates early pathways of Foxp3 expression. These results highlight previously unappreciated links between Treg and Th2 differentiation and extend our understanding of the transcription factors that regulate Treg differentiation and their temporal sequencing. These findings have significant implications for future mechanistic and translational studies of CDK8 and CDK8 inhibitors

In contrast to the common role of histone deacetylases (HDACs) for gene repression, HDAC activity provides a required positive function for IFN-stimulated gene (ISG) expression. Here, we show that HDAC1/2 as components of the Sin3A complex are required for ISG transcriptional elongation but not for recruitment of RNA polymerase or transcriptional initiation. Transcriptional arrest by HDAC inhibition coincides with failure to recruit the epigenetic reader Brd4 and elongation factor P-TEFb due to sequestration of Brd4 on hyperacetylated chromatin. Brd4 availability is regulated by an equilibrium cycle between opposed acetyltransferase and deacetylase activities that maintains a steady-state pool of free Brd4 available for recruitment to inducible promoters. An ISG expression signature is a hallmark of interferonopathies and other autoimmune diseases. Combined inhibition of HDAC1/2 and Brd4 resolved the aberrant ISG expression detected in cells derived from patients with two inherited interferonopathies, ISG15 and USP18 deficiencies, defining a novel therapeutic approach to ISG-associated autoimmune diseases.

Objective To study the outcome of patients with antiphospholipid syndrome (APS) after oral anticoagulant treatment cessation. Methods We performed a retrospective study of patients with APS experiencing cessation of oral anticoagulant and enrolled in a French multicentre observational cohort between January 2014 and January 2016. The main outcome was the occurrence of recurrent thrombotic event after oral anticoagulation cessation. Results Forty four APS patients interrupted oral anticoagulation. The median age was 43 (27-56) years. The median duration of anticoagulation was 21 (9-118) months. Main causes of oral anticoagulant treatment cessation were switch from vitamin K antagonists to aspirin in 15 patients, prolonged disappearance of antiphospholipid antibodies in ten, bleeding complications in nine and a poor therapeutic adherence in six. Eleven (25%) patients developed a recurrent thrombotic event after oral anticoagulation cessation, including three catastrophic APS and one death due to lower limb ischemia. Antihypertensive treatment required at time of oral anticoagulants cessation seems to be an important factor associated with recurrent thrombosis after oral anticoagulant cessation (15.2% in patients with no relapse versus 45.5% in patients with recurrent thrombosis, p = 0.038). Oral anticoagulant treatment was re-started in 18 (40.9%) patients. Conclusion The risk of a new thrombotic event in APS patients who stopped their anticoagulation is high, even in those who showed a long lasting disappearance of antiphospholipid antibodies. Except for the presence of treated hypertension, this study did not find a particular clinical or biological phenotype for APS patients who relapsed after anticoagulation cessation. Any stopping of anticoagulant in such patients should be done with caution.

Dendritic cells (DCs) are crucial for mediating immune responses but, when deregulated, also contribute to immunological disorders, such as autoimmunity. The molecular mechanism underlying the function of DCs is incompletely understood. In this study, we have identified TANK-binding kinase 1 (TBK1), a master innate immune kinase, as an important regulator of DC function. DC-specific deletion of Tbk1 causes T cell activation and autoimmune symptoms and also enhances antitumor immunity in animal models of cancer immunotherapy. The TBK1-deficient DCs have up-regulated expression of co-stimulatory molecules and increased T cell-priming activity. We further demonstrate that TBK1 negatively regulates the induction of a subset of genes by type I interferon receptor (IFNAR). Deletion of IFNAR1 could largely prevent aberrant T cell activation and autoimmunity in DC-conditional Tbk1 knockout mice. These findings identify a DC-specific function of TBK1 in the maintenance of immune homeostasis and tolerance.

Sensing invading pathogens early in infection is critical for establishing host defense. Two cytosolic RIG-like RNA helicases, RIG-I and MDA5, are key to type I interferon (IFN) induction in response to viral infection. Mounting evidence suggests that another viral RNA sensor, protein kinase R (PKR), may also be critical for IFN induction during infection, although its exact contribution and mechanism of action are not completely understood. Using PKR-deficient cells, we found that PKR was required for type I IFN induction in response to infection by vaccinia virus lacking the PKR antagonist E3L (VVDeltaE3L), but not by Sendai virus or influenza A virus lacking the IFN-antagonist NS1 (FluDeltaNS1). IFN induction required the catalytic activity of PKR, but not the phosphorylation of its principal substrate, eIF2alpha, or the resulting inhibition of host translation. In the absence of PKR, IRF3 nuclear translocation was impaired in response to MDA5 activators, VVDeltaE3L and encephalomyocarditis virus, but not during infection with a RIG-I-activating virus. Interestingly, PKR interacted with both RIG-I and MDA5; however, PKR was only required for MDA5-mediated, but not RIG-I-mediated, IFN production. Using an artificially activated form of PKR, we showed that PKR activity alone was sufficient for IFN induction. This effect required MAVS and correlated with IRF3 activation, but no longer required MDA5. Nonetheless, PKR activation during viral infection was enhanced by MDA5, as virus-stimulated catalytic activity was impaired in MDA5-null cells. Taken together, our data describe a critical and non-redundant role for PKR following MDA5, but not RIG-I, activation to mediate MAVS-dependent induction of type I IFN through a kinase-dependent mechanism.

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19ARF as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14ARF expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.

Juvenile myelomonocytic leukemia, acute myeloid leukemia (AML), and other myeloproliferative neoplasms (MPNs) are genetically heterogeneous but frequently display activating mutations in Ras GTPases and activation of signal transducer and activator of transcription 3 (STAT3). Altered STAT3 activity is observed in up to 50% of AML correlating with poor prognosis. Activated STAT proteins, classically associated with tyrosine phosphorylation, support tumor development as transcription factors, but alternative STAT functions independent of tyrosine phosphorylation have been documented, including roles for serine-phosphorylated STAT3 in mitochondria supporting transformation by oncogenic Ras. We examined requirements for STAT3 in experimental murine K-Ras-dependent hematopoietic neoplasia. We show that STAT3 is phosphorylated on S727 but not Y705 in diseased animals. Moreover, a mouse with a point mutation abrogating STAT3 S727 phosphorylation displayed delayed onset and decreased disease severity with significantly extended survival. Activated K-Ras required STAT3 for cytokine-independent growth of myeloid progenitors in vitro, and mitochondrially restricted STAT3 and STAT3-Y705F, both transcriptionally inert mutants, supported factor-independent growth. STAT3 was dispensable for growth of normal or K-Ras-mutant myeloid progenitors in response to cytokines. However, abrogation of STAT3-S727 phosphorylation impaired factor-independent malignant growth. These data document that serine-phosphorylated mitochondrial STAT3 supports neoplastic hematopoietic cell growth induced by K-Ras.

STAT proteins bind DNA as dimers to regulate gene expression. Cooperative recruitment of pairs of dimers (tetramers) to adjacent DNA sites has also been documented. In this issue, Lin et al. (2012) examined tetramer function in vivo and showed that STAT5 tetramers function primarily as transcriptional activators.

Signal transducers and activators of transcription (STATs) and interferon regulatory factors (IRFs) share common target genes. Here we show that the Irf7 gene is regulated by transcription factors STAT1 and IRF9 in response to the type II interferon (IFN) IFN-gamma. IRF7 cooperated with STAT1 and IRF1 to stimulate the expression of a subset of IFN-gamma-induced STAT1 target genes. IRF7-mediated control of the Gbp2 gene required the presence and basal activity of the S/T kinase TANK-binding kinase 1 (TBK1), whereas the binding of IRF7 to the Gbp2 promoter did not. Analysis of RNA polymerase II (Pol II) recruitment to the Gbp2 promoter revealed a role for IRF7 at later stages of the IFN-gamma response. In support of the role of IRF7 in establishing an effective antibacterial response, IFN-gamma-pretreated Irf7(-/-) macrophages showed an increased bacterial burden after infection with Listeria monocytogenes. Our data thus describe a biologically relevant basal activity of TBK1 and identify IRF7 as a novel player in the IFN-gamma response.