Transcription Factor IRF3 Research Articles

Generation and characterization of the Eµ-Irf8 mouse model

In mature B-cell malignancies, chromosomal translocations often juxtapose an oncogenic locus to the regulatory regions of the immunoglobulin genes. These genomic rearrangements can associate with specific clinical/pathological sub-entities and inform diagnosis and treatment decisions. Recently, we characterized the t(14;16)(q32;q24) in diffuse large B-cell lymphoma (DLBCL), and showed that it targets the transcription factor IRF8, which is also somatically mutated in ~10% of DLBCLs. IRF8 regulates innate and adaptive immune responses mediated by myeloid/monocytic and lymphoid cells. While the role of IRF8 in human myeloid/dendritic-cell disorders is well established, less is known of its contribution to the pathogenesis of mature B-cell malignancies. To address this knowledge gap, we generated the Eµ-Irf8 mouse model, which mimics the IRF8 deregulation associated with t(14;16) of DLBCL. Eµ-Irf8 mice develop normally and display peripheral blood cell parameters within normal range. However, Eµ-Irf8 mice accumulate pre-pro-B-cells and transitional B-cells in the bone marrow and spleen, respectively, suggesting that the physiological role of Irf8 in B-cell development is amplified. Notably, in Eµ-Irf8 mice, the lymphomagenic Irf8 targets Aicda and Bcl6 are overexpressed in mature B-cells. Yet, the incidence of B-cell lymphomas is not increased in the Eµ-Irf8 model, even though their estimated survival probability is significantly lower than that of WT controls. Together, these observations suggest that the penetrance on the Irf8-driven phenotype may be incomplete and that introduction of second genetic hit, a common strategy in mouse models of lymphoma, may be necessary to uncover the pro-lymphoma phenotype of the Eµ-Irf8 mice.

IRF5 guides monocytes toward an inflammatory CD11c+ macrophage phenotype and promotes intestinal inflammation.

Mononuclear phagocytes (MNPs) are vital for maintaining intestinal homeostasis but, in response to acute microbial stimulation, can also trigger immunopathology, accelerating recruitment of Ly6Chi monocytes to the gut. The regulators that control monocyte tissue adaptation in the gut remain poorly understood. Interferon regulatory factor 5 (IRF5) is a transcription factor previously shown to play a key role in maintaining the inflammatory phenotype of macrophages. Here, we investigate the impact of IRF5 on the MNP system and physiology of the gut at homeostasis and during inflammation. We demonstrate that IRF5 deficiency has a limited impact on colon physiology at steady state but ameliorates immunopathology during Helicobacter hepaticus-induced colitis. Inhibition of IRF5 activity in MNPs phenocopies global IRF5 deficiency. Using a combination of bone marrow chimera and single-cell RNA-sequencing approaches, we examined the intrinsic role of IRF5 in controlling colonic MNP development. We demonstrate that IRF5 promotes differentiation of Ly6Chi monocytes into CD11c+ macrophages and controls the production of antimicrobial and inflammatory mediators by these cells. Thus, we identify IRF5 as a key transcriptional regulator of the colonic MNP system during intestinal inflammation.

IRF4 instructs effector Treg differentiation and immune suppression in human cancer

The molecular mechanisms responsible for the high immunosuppressive capacity of CD4+ Tregs in tumors are not well known. High-dimensional single-cell profiling of T cells from chemotherapy-naive individuals with non-small-cell lung cancer identified the transcription factor IRF4 as specifically expressed by a subset of intratumoral CD4+ effector Tregs with superior suppressive activity. In contrast to the IRF4- counterparts, IRF4+ Tregs expressed a vast array of suppressive molecules, and their presence correlated with multiple exhausted subpopulations of T cells. Integration of transcriptomic and epigenomic data revealed that IRF4, either alone or in combination with its partner BATF, directly controlled a molecular program responsible for immunosuppression in tumors. Accordingly, deletion of Irf4 exclusively in Tregs resulted in delayed tumor growth in mice while the abundance of IRF4+ Tregs correlated with poor prognosis in patients with multiple human cancers. Thus, a common mechanism underlies immunosuppression in the tumor microenvironment irrespective of the tumor type.

Molecular cloning and functional characterization of duck DEAD (Asp-Glu-Ala-Asp) box RNA helicase 3 (DDX3X)

DEAD (Asp-Glu-Ala-Asp) box RNA helicase 3 (DDX3X) is demonstrated to have crucial functions in the antiviral immune response. To our knowledge, little information focuses on the function of duck DDX3X. In this study, duck DDX3X (duDDX3X) was cloned and its role in the type I interferon (IFN) signaling pathway was investigated using duck embryo fibroblast (DEF) cells. Full-length duDDX3X cDNA encodes 652 amino acid residues and contains a DEADc domain and a HELICc domain. According to tissue distribution analysis, duDDX3X mRNA was widely expressed in different tissues, especially the spleen and the liver. Overexpression of duDDX3X in DEF cells induced IFN-β by activating transcription factors IRF1 and NF-κB. Knockdown of duDDX3X in DEF cells with siRNA significantly reduced IFN-β expression induced by poly(I:C), a double-stranded RNA (dsRNA) analog. Our results demonstrated that duck DDX3X was involved in the dsRNA-mediated type I IFN signaling pathway in DEF cells.

Identifying mediators of STING-dependent TB vaccine immunity

Abstract Tuberculosis (TB) is the top infectious killer in the world. The only current licensed vaccine for TB, Mycobacterium bovis Bacille Calmette-Guérin (BCG), has not proven reliably effective against adult pulmonary TB in all studies. It furthermore cannot be administered safely to immunocompromised individuals as it is a live attenuated vaccine. Protein subunit vaccines offer increased safety over live attenuated vaccines. Many recently developed protein subunit TB vaccines use toll-like receptor-stimulating adjuvants that induce potent inflammatory responses, but it is uncertain whether they can induce long-lasting memory protection against an intracellular bacterium such as TB. Cyclic-di-nucleotides are an alternative adjuvant that target the cytosolic immunosurveillance pathway mediated by STING. Recently, a STING-activating cyclic-di-nucleotide (CDN)-adjuvanted TB protein subunit vaccine was shown to elicit an effective memory T cell response against TB challenge in mice. Intriguingly, intranasal delivery of the vaccine provided superior protection compared to subcutaneous administration of the CDN-adjuvanted vaccine or BCG. Protection induced by mucosal administration correlates with a potent Th17 response rather than the Th1-dominated CD4+ T cell response induced by subcutaneous vaccination. Protection is partially dependent on IL-17 and type I interferon, while the transcription factor STAT6 is dispensable. The Th17 response and protection both precedes Th1 influx into the lungs and also persists after the Th1 response wanes. We are currently determining whether the transcription factor IRF3 and cytokine IFNγ are also mediators of Th17 memory induction and protection against TB infection.

Conventional type 2 dendritic cells and natural killer cells mediate control of early metastatic seeding

Abstract The multistep progression of transformed primary tumors to metastatic dissemination and seeding of distant site is a lethal determinant of disease. Although the role of immune cells and immune sensors in promoting or restricting metastatic growth is well described, whether immune cells are capable of detecting and controlling metastatic cells during peripheral tissue seeding is unknown. Here we examine if local immune cells can confer early anti-metastatic control in the lung using intracardiac injection to mimic metastatic spread. Using a syngeneic mouse model of YUMMER1.7 melanoma expressing GFP and luciferase (YMR1.7-G/L), we demonstrate that lung resident conventional type 2 dendritic cells (cDC2) and extravascular NK cells confer host anti-tumor immunity during the first three days following intracardiac injection. Rapid production of IFN-γ by NK cells serves an essential role to limit metastatic burden. Using mice deficient in innate sensors and signaling pathways, we demonstrate that the transcription factors IRF3 and IRF7 are required for early metastatic control in a type I IFN-independent manner. In contrast, ablation of adaptive immune cells had no effect to initial metastatic burden. Collectively our results highlight a novel cDC2 - NK cell axis that orchestrates a non-redundant innate immune response program to limit initial metastatic burden in the lung.

Th17 immunity in the colon is controlled by two novel subsets of colon-specific mononuclear phagocytes

Abstract Immune responses in the intestine are coordinated by specialized populations of mononuclear phagocytes. Although the functions of intestine-resident mononuclear phagocytes and the different cell subsets that constitute these populations are thought uniform across the entire intestine, it is clear that the anatomy, microbes, and immunological demands are distinct for small and large intestine. Whether these distinctions also include organ-specific mononuclear phagocyte populations, or that these have organ-specific roles in immunity are unknown. Here we identify two novel subsets of colon-specific mononuclear phagocytes, a dendritic cell (DC) subset and a macrophage subset, both of which are exclusive to the colon and are not found in small intestine. These colon-specific DCs and macrophages co-expressed CD14 and CD24, markers which are generally singularly expressed by intestinal DCs and macrophages and commonly used to distinguish between these two cell types. Colon-specific CD14+CD24+ DCs and macrophages failed to express macrophage markers CX3CR1, F4/80, CD64 and CD88, and surprisingly, both the DC and macrophage subset were dependent on the transcription factor IRF4. We further find that colon and small intestine have distinct antigen presenting cell (APC) requirements for Th17 immunity and that while colon-specific CD14+CD24+ APCs were essential for Th17 immunity in the colon, this role in small intestine was mediated by different APC subsets. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.

Suppression of Inflammasome Activation by IRF8 and IRF4 in cDCs is Critical for T cell Priming

Abstract Activation of inflammasome leads to pyroptotic cell death thereby eliminating the replicative niche of virulent pathogens, a process integral to innate immunity. While inflammasome-associated cytokines such as IL-1β and IL-18 have an established role in T cell function, whether inflammasome activation in dendritic cells (DCs) is critical for T cell priming is not clear. Here, we discover that inflammasome activation in conventional DCs (cDCs) is detrimental to the induction of adaptive immunity. We found that lymphoid organ resident cDCs actively suppress inflammasome activation to avoid pyroptotic cell death. This protection from inflammasome-induced cell death is critical for both CD4 and CD8 T cell priming. Transcription factors IRF8 and IRF4, in cDC1s and cDC2s respectively, mediate this suppression of inflammasome activation by limiting the expression of inflammasome-associated genes. We show that reduced expression of IRF8 leads to aberrant inflammasome activation in cDC1s which hampers subsequent CD8 T cell priming. Conversely, overexpression of either of IRF4 or IRF8 is sufficient to inhibit inflammasome activation in macrophages. These results uncover the molecular mechanism of inflammasome suppression in cDCs, and ascribe a novel post-developmental role for IRF4 and IRF8 in cDC function.

Suppression of Inflammasome Activation by IRF8 and IRF4 in cDCs Is Critical for T Cell Priming

SUMMARYInflammasome activation leads to pyroptotic cell death, thereby eliminating the replicative niche of virulent pathogens. Although inflammasome-associated cytokines IL-1β and IL-18 have an established role in T cell function, whether inflammasome activation in dendritic cells (DCs) is critical for T cell priming is not clear. Here, we find that conventional DCs (cDCs) suppress inflammasome activation to prevent pyroptotic cell death, thus preserving their ability to prime both CD4 and CD8 T cells. Transcription factors IRF8 and IRF4, in cDC1s and cDC2s, respectively, mediate suppression of inflammasome activation by limiting the expression of inflammasome-associated genes. Overexpression of IRF4 or IRF8 inhibits inflammasome activation in macrophages, while reduced expression of IRF8 leads to aberrant inflammasome activation in cDC1s and hampers their ability to prime CD8 T cells. Thus, activation of inflammasome in DCs is detrimental to adaptive immunity, and our results reveal that cDCs use IRF4 and IRF8 to suppress this response.

TLR2-Tpl2-dependent ERK Signaling Drives Opposite IL-12 Regulation in Dendritic Cells and Macrophages

Abstract This study investigated responses to TLR2-driven ERK signaling in dendritic cells versus macrophages. TLR2 signaling was induced with Pam3Cys, and the role of ERK signaling was interrogated pharmacologically with a MEK1/2 inhibitor (U0126) or genetically using bone-marrow-derived macrophages or dendritic cells from Tpl2−/− mice. We assessed cytokine production via ELISA and mRNA levels by qRT-PCR. In macrophages, blockade of ERK signaling by pharmacologic or genetic approaches inhibited IL-10 production and increased IL-12p40 production significantly. In dendritic cells, blockade of ERK signaling similarly inhibited IL-10 production but decreased IL-12p40 production, opposite to the effect of ERK signaling blockade in macrophages. This difference in IL-12p40 regulation correlated with differential expression of transcription factors cFos and IRF1, which are known to regulate IL-12. Thus, the impact of ERK signaling in response to TLR2 stimulation differs between macrophages and dendritic cells, potentially regulating their distinctive functions in the immune system. ERK-mediated suppression of IL-12p40 in macrophages may prevent excess inflammation and associated tissue damage following TLR2-stimuation, while ERK-mediated induction of IL-12p40 in dendritic cells may promote priming of Th1 responses. Greater understanding of the role that ERK signaling plays in different immune cell types may inform the development of host-directed therapy for a number of infectious pathogens.

Internal N6-methyladenosine modification controls the innate immune response to viral RNA

Abstract Internal N6-methyladenosine (m6A) modification is one of the most prevalent and abundant modifications of RNA. The m6A methylation is catalyzed by host methyltransferases (METTL3 and METTL14) and can be reversibly removed by host demethylases (FTO and ALKBH5). Recent studies have revealed that RNA of many DNA and RNA viruses contain m6A methylation and that viral m6A can play a pro-viral or anti-viral role. However, the biological functions of viral RNA m6A methylation remain poorly understood. In this study, we found that genome and antigenome of human respiratory syncytial virus (RSV), a non-segmented negative-sense RNA virus, are m6A methylated and that viral m6A methylation positively regulates RSV replication and gene expression. Interestingly, virion RNAs purified from RSV grown in METTL3-knockout cells induced significantly higher type I interferon responses than virion RNAs purified from RSV grown in wild type cells, demonstrating that viral RNA m6A methylation inhibits innate immunity. Similarly, inactivation of m6A methylation in RSV genome and/or antigenome by site-directed mutagenesis induced significantly higher innate immune responses than wild type RSV RNA. Mechanistically, these m6A-unmodified or m6A-deficient RSV RNAs trigger a higher expression of pattern recognition receptors (such as RIG-I and MDA5), enhance their binding affinity to the innate immune RNA sensors, and enhance the phosphorylation of downstream transcription factor IRF3, leading to an enhanced activation of type I interferon signaling pathway. Taken together, our study identified a novel biological function of viral m6A methylation, which is to serves as a molecular marker for host innate immunity to discriminate self from nonself RNAs.

RNA-binding protein HuR promotes Th17 cell differentiation and targeting HuR reduces autoimmune neuroinflammation

Abstract Dysregulated Th17 cell differentiation is associated with autoimmune diseases such as multiple sclerosis, which has no curative treatment. Understanding the molecular mechanisms of regulating Th17 cell differentiation will help in identifying a novel therapeutic target for treating Th17 cell-mediated diseases. Here, we investigated the cell-intrinsic processes by which RNA-binding protein HuR orchestrates Th17 cell fate decisions by post-transcriptionally regulating transcription factor Irf4 and Runx1 and receptor Il12rb1 expression, in turn promoting Th17 cell and Th1-like Th17 cell differentiation. Knockout of HuR impaired the transcriptome of Th17 cells characterized by reduced levels of RORγt, IRF4, RUNX1, and T-bet, thereby reducing the number of pathogenic IL-17+IFN-γ+CD4+ T cells during experimental autoimmune encephalomyelitis (EAE). In keeping with the fact that HuR increased the expression of adhesion molecule VLA-4 on Th17 cells, knockout of HuR impaired Th17 cell migration to the central nervous system and abolished the disease. Accordingly, targeting HuR by its inhibitor, DHTS, suppressed Th17 cell differentiation and reduced EAE severity. In sum, we uncovered the molecular mechanism underlying HuR’s regulation of Th17 cell differentiation and functions, underscoring the therapeutic value of HuR as a novel treatment for autoimmune neuroinflammation. (Supported by NIH R01 AI119135)

Ablation of IRF4 in donor T cells eliminates GVHD while partially preserving GVT in experimental bone marrow transplantation

Abstract Objective We discovered that the transcription factor IRF4 governs T cell function in heart transplantation (Immunity 47:1114). Here, using a bone marrow (BM) transplantation model, we studied the role of IRF4 in regulating T cell function in graft-vs-host disease (GVHD) and graft-vs-tumor (GVT). Methods 9 Gy irradiated Balb/c recipients were i.v. injected: 1) with 5 × 106 T cell-depleted CD45.1+ B6 BM cells; 2) with or without 0.1 × 106 Balb/c A20-luciferase lymphoma cells; and 3) with or without 1 × 106 CD45.2+ wild type (WT), Irf4−/−, or Irf4−/−Foxp3sf/Y (Treg-deficient Irf4−/−) B6 donor T cells. GVHD, GVT, survival, and donor T cell states were determined. Results In the absence of A20 infusion, all recipients without T cell transfer (n=10), or transferred with Irf4−/− (n=10) or Irf4−/−Foxp3sf/Y (n=5) donor T cells survived long-term (>60 days post BM transplantation). Recipients transferred with WT donor T cells had developed lethal GVHD [mean survival time (MST) = 9.5±2 days; n =8]. Among A20 infused groups, recipients without T cell transfer died due to lymphoma progression (MST=22±2.80 days; n =15). Transfer of Irf4−/− donor T cells led to a moderate GVT effect and significantly prolonged survival (MST=33.4 ± 11.22 days; n =15; p<0.001 vs no T cell). Transferred WT donor T cells exerted potent GVT activity but caused lethal GVHD (12.56 ± 6.02 days; n = 18). Mechanistically, Irf4−/− donor T cells developed into effector T cells, but barely infiltrated into GVHD tissues and were prone to apoptosis. Conclusions IRF4 ablation in donor T cells eliminates GVHD in a Treg independent manner while partially preserving GVT. This reveals the possibility to separate GVHD and GVT effects by modulating transcription factors in T cells.

Abstract B50: ERK MAPK inhibition enhances the immunogenicity of KRAS-mutant colorectal cancer

Abstract The three RAS genes are the most commonly mutated oncogenes in cancer and are refractory to conventional therapies. Oncogenic mutation of KRAS in 44% of colorectal cancers (CRC) leads to aberrant activation of downstream effector pathways, including the ERK MAPK signaling cascade, which can lead to an immunosuppressive tumor microenvironment. Single-agent therapies targeting the ERK MAPK cascade or immune checkpoint (PD-1/PD-L1) have been mostly unsuccessful in KRAS-mutant CRC, and efforts are under way to identify effective combination strategies. We employed an unbiased RNA-seq approach to identify gene signatures significantly upregulated upon ERK MAPK pathway inhibition in a panel of KRAS-mutant CRC cell lines. We found that treatment with either the MEK inhibitor trametinib or ERK inhibitor Vx-11e induced upregulation of interferon gene signatures and JAK/STAT pathway signaling components. Inhibition of MEK or ERK also transcriptionally increased levels of MHC Class I and antigen presentation machinery. Upregulation of MHC Class I was reversed with either pharmacologic inhibition of JAK/STAT signaling or genetic knockdown of the transcription factor IRF1. We propose that combining MAPK inhibition with checkpoint immunotherapy may provide an effective treatment in KRAS-mutant CRC tumors in vivo and that potential synergy may be due to enhancing the immunogenicity of tumors through priming of interferon response pathways and antigen presentation machinery. Citation Format: Meagan B. Ryan, Ferran Fece de la Cruz, Leanne G. Ahronian, Sarah Phat, David T. Myers, Heather A. Shahzade, Catriona Hong, Ryan B. Corcoran. ERK MAPK inhibition enhances the immunogenicity of KRAS-mutant colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B50.

Interferon regulatory factor 5 expression in myeloid cells and T cells negatively impacts the outcome of visceral leishmaniasis

Abstract Visceral leishmaniasis (VL) is a potentially fatal disease caused by Leishmania donovani and/or L. infantum. VL is characterized by hepatosplenomegaly, immune suppression, and chronic inflammation. We have previously reported that the transcription factor IRF-5 largely contributes to the induction of inflammation and the development of splenomegaly in mice infected with L. donovani. Moreover, Irf5−/− mice only develop limited IFNγ+ CD4 T responses and are more susceptible to infection. However, the cellular source of IRF-5 responsible for these effects is yet unknown. IRF-5 is constitutively expressed in several cells, such as macrophages, B cells, and dendritic cells. We have recently reported that CD4 T cells also express IRF-5 during chronic VL. Here, we investigate IRF-5 function in myeloid and CD4 T cells during VL, using cell-specific knockout mice. First, we show that IRF-5 expression in CD11c+ cells is required for the generation of splenomegaly and leads to increased disease susceptibility. We also demonstrate that IRF-5 expression in CD11c+ cells is not crucial for the development of Th1 responses. Moreover, we demonstrate that IRF-5 activation in Th1 cells results in cell death during chronic L. donovani infection. Triggering of TLR7 by apoptotic cell material promotes IRF-5 activation in Th1 cells. This results in the upregulation of death receptor 5 and caspase 8, making IFNγ+ CD4 T cells more prone to cell death. Because chronic inflammation and tissue disruption are common characteristics of persistent infections, the TLR7- IRF-5 pathway in CD4 T cells may represent a novel mechanism aimed at protecting tissues against sustained inflammation.

Lipopolysaccharide restricts murine norovirus infection in macrophages mainly through NF-kB and JAK-STAT signaling pathway

The inflammasome machinery has recently been recognized as an emerging pillar of innate immunity. However, little is known regarding the interaction between the classical interferon (IFN) response and inflammasome activation in response to norovirus infection. We found that murine norovirus (MNV-1) infection induces the transcription of IL-1β, a hallmark of inflammasome activation, which is further increased by inhibition of IFN response, but fails to trigger the release of mature IL-1β. Interestingly, pharmacological inflammasome inhibitors do not affect viral replication, but slightly reverse the inflammasome activator lipopolysaccharide (LPS)-mediated inhibition of MNV replication. LPS efficiently stimulates the transcription of IFN-β through NF-ĸB, which requires the transcription factors IRF3 and IRF7. This activates downstream antiviral IFN-stimulated genes (ISGs) via the JAK-STAT pathway. Moreover, inhibition of NF-ĸB and JAK-STAT signaling partially reverse LPS-mediated anti-MNV activity, suggesting additional antiviral mechanisms activated by NF-ĸB. This study reveals additional insight in host defense against MNV infection.

RNA-Binding Protein HuR Promotes Th17 Cell Differentiation and Can Be Targeted to Reduce Autoimmune Neuroinflammation.

Dysregulated Th17 cell differentiation is associated with autoimmune diseases such as multiple sclerosis, which has no curative treatment. Understanding the molecular mechanisms of regulating Th17 cell differentiation will help find a novel therapeutic target for treating Th17 cell-mediated diseases. In this study, we investigated the cell-intrinsic processes by which RNA-binding protein HuR orchestrates Th17 cell fate decisions by posttranscriptionally regulating transcription factors Irf4 and Runx1 and receptor Il12rb1 expression, in turn promoting Th17 cell and Th1-like Th17 cell differentiation in C57BL/6J mice. Knockout of HuR altered the transcriptome of Th17 cells characterized by reducing the levels of RORγt, IRF4, RUNX1, and T-bet, thereby reducing the number of pathogenic IL-17+IFN-γ+CD4+ T cells in the spleen during experimental autoimmune encephalomyelitis. In keeping with the fact that HuR increased the abundance of adhesion molecule VLA-4 on Th17 cells, knockout of HuR impaired splenic Th17 cell migration to the CNS and abolished the disease. Accordingly, targeting HuR by its inhibitor DHTS inhibited splenic Th17 cell differentiation and reduced experimental autoimmune encephalomyelitis severity. In sum, we uncovered the molecular mechanism of HuR regulating Th17 cell functions, underscoring the therapeutic value of HuR for treatment of autoimmune neuroinflammation.

STING Activation and its Role in IFN Production

The immune system’s ability to respond to foreign DNA is a highly conserved mechanism. One crucial part of this mechanism is Stimulator of Interferon Genes (STING), a central innate immune protein located in the endoplasmic reticulum. It is a pattern recognition receptor that is activated in response to intracellular infection and, ultimately, mediates type 1 interferon (IFN) production. Encoded by the gene TMEM173, STING is expressed in T‐cells, monocytes, natural killer cells, and dermal fibroblasts.. It is composed of four transmembrane helices and a cytoplasmic ligand binding and signaling domain, which form a domain‐swapped dimer assembly. The STING pathway is initiated when double‐stranded DNA (dsDNA) is sensed, causing cyclic‐GMP‐AMP‐synthase (cGAS) to cyclize GTP and ATP and produce 2′3′‐cyclic‐GMP‐AMP (cGAMP), the main ligand that activates STING. Upon STING activation, the ligand‐binding domain closes, inducing a 180 degree rotation of the ligand‐binding domain relative to the transmembrane domain. The C‐terminal tail of STING has been found to autoinhibit the protein by blocking the polymerization interface of inactive STING, preventing auto activation. The conformational change made in response to cGAMP releases the CTT, exposing the polymer interface and allowing disulfide link formation. Alternatively, STING can be activated by bacterial cyclic‐di‐GMP in a cooperative manner, with cyclic‐di‐GMP serving as a partial antagonist of the cGAMP signaling pathway. Structural and biochemical studies have provided elegant insight into the mechanism of differential of STING by cGAMP and cDi‐GMP. STING activates tank binding kinase 1 (TBK1) which phosphorylates transcription factor IRF‐3 and causes it to translocate to the nucleus. Gain‐of‐function mutations of TMEM173 causing elevated transcription of IFNs have been proven to cause autoimmune syndromes such as systemic lupus erythematosus and, most recently, three mutations of TMEM173 have been found to directly cause STING‐associated vasculopathy with onset in infants (SAVI). New research proposes a therapeutic strategy that uses molecules that induces a different STING conformation than cGAMP‐bound STING as a STING inhibitor. This method would prevent constitutive STING activation in autoimmune syndromes while being less immunosuppressive than previously proposed therapies involving STING inhibition. The Walton High School MSOE Center for Biomolecular Modeling SMART Team has designed a 3‐D model of STING using a recently published cryo‐electron microscopy structure that reveals the transmembrane domain structure to further investigate and examine the structure‐function relationship of STING.Support or Funding InformationMSOE Center for Biomolecular Modeling

Unique Roles for Streptococcus pneumoniae Phosphodiesterase 2 in Cyclic di-AMP Catabolism and Macrophage Responses.

Cyclic di-AMP (c-di-AMP) is an important signaling molecule for pneumococci, and as a uniquely prokaryotic product it can be recognized by mammalian cells as a danger signal that triggers innate immunity. Roles of c-di-AMP in directing host responses during pneumococcal infection are only beginning to be defined. We hypothesized that pneumococci with defective c-di-AMP catabolism due to phosphodiesterase deletions could illuminate roles of c-di-AMP in mediating host responses to pneumococcal infection. Pneumococci deficient in phosphodiesterase 2 (Pde2) stimulated a rapid induction of interferon β (IFNβ) expression that was exaggerated in comparison to that induced by wild type (WT) bacteria or bacteria deficient in phosphodiesterase 1. This IFNβ burst was elicited in mouse and human macrophage-like cell lines as well as in primary alveolar macrophages collected from mice with pneumococcal pneumonia. Macrophage hyperactivation by Pde2-deficient pneumococci led to rapid cell death. STING and cGAS were essential for the excessive IFNβ induction, which also required phagocytosis of bacteria and triggered the phosphorylation of IRF3 and IRF7 transcription factors. The select effects of Pde2 deletion were products of a unique role of this enzyme in c-di-AMP catabolism when pneumococci were grown on solid substrate conditions designed to enhance virulence. Because pneumococci with elevated c-di-AMP drive aberrant innate immune responses from macrophages involving hyperactivation of STING, excessive IFNβ expression, and rapid cytotoxicity, we surmise that c-di-AMP is pivotal for directing innate immunity and host-pathogen interactions during pneumococcal pneumonia.

Role of IRF8 in immune cells functions, protection against infections, and susceptibility to inflammatory diseases.

The transcription factor IRF8 (ICSBP) is required for the development and maturation of myeloid cells (dendritic cells, monocytes, macrophages), and for expression of intrinsic anti-microbial function such as antigen capture, processing and presentation to lymphoid cells, and for activation of these cells in response to cytokines and pro-inflammatory stimuli (IFN-γ, IFN-β, LPS). IRF8 deficiency in humans causes a severe primary immunodeficiency presenting as susceptibility to infections, complete or severe depletion of blood dendritic cells (DC) subsets, depletion of CD14+ and CD16+ monocytes and reduced numbers and impaired activity of NK cells. In genome-wide association studies (GWAS), sequence variants near IRF8 are significant risk factors for multiple chronic inflammatory diseases in humans including inflammatory bowel disease, lupus, rheumatoid arthritis, multiple sclerosis, and several others. Recent studies have cataloged all the genes bound by and transcriptionally activated by IRF8 in myeloid cells, either alone or in combination with other transcription factors (PU.1, IRF1, STAT1) at steady state and in response to pro-inflammatory stimuli. This IRF1/IRF8 regulome comprises immune pathways such as antigen processing and presentation pathways, expression of costimulatory molecules, cytokines and chemokines, response to stimuli such as cytokine receptors, pathogen-associated molecular pattern receptors, TLRs and nucleotide-binding oligomerization domain-like receptor signaling pathways, and small antiviral GTPases. Members of the IRF8/IRF1 regulome are over-represented amongst genes in which mutations cause primary immunodeficiencies, and are specifically enriched at GWAS loci associated with chronic inflammatory diseases in humans. These recent studies highlight a critical role of IRF8 in the activity of several immune cell types for protection against infections, but also in pathological inflammation associated with common human inflammatory conditions.