Transcription Factor IRF3 Research Articles

Overexpression of miR-101 May Target DUSP1 to Promote the Cartilage Degradation in Rheumatoid Arthritis.

This study aimed to explore crucial genes that contribute to the development of rheumatoid arthritis (RA). Three GSE77298, GSE55457, and GSE55235 data sets were used to analyze the differentially expressed genes (DEGs) between RA synovial membrane tissue samples and normal synovial membrane tissue samples. Then, the functional enrichment analysis and protein-protein interactions (PPIs) construction were performed for DEGs. Subsequently, submodule analysis and regulatory network that contained transcription factors (TFs), microRNAs, and their targets were conducted. Finally, small-molecule drugs related to the DEGs were predicted. A total of 173 upregulated and 54 downregulated DEGs identified in at least 2 of 3 data sets. TYROBP, CTSS, MMP9, CXCR4, and CXCL10 were both highlighted in the PPI and submodule networks. In addition, miR-101, IRF1 TF, DUSP1, and CXCR4 had high degree in the regulatory network, and regulation pairs of miR-101-DUSP1 and IRF1 TF-CXCR4 were obtained. Drugs such as alemtuzumab and marimastat were negatively related to expression of the DEGs and might be useful drugs for RA treatment. In addition, most DEGs were involved in innate immune response (e.g., TYROBP, CCL5, CXCL10, FCGR1A, and FCGR3B) and phagosome pathway (e.g., CTSS). We suggested that miR-101 that regulated DUSP1, IRF1 TF that regulated CXCR4, as well as DEGs as TYROBP and CTSS might contribute to the RA pathogenesis. In addition, anti-inflammatory agent alemtuzumab and matrix metalloproteinase inhibitor marimastat might be useful drugs for RA treatment through functioning on their target genes.

1741-P: Overexpression of the Type 1 Diabetes-Associated IFIH1 A946T Single Nucleotide Polymorphism in Coxsackievirus B Infection

Type 1 diabetes (T1D) is a multifactorial autoimmune disease with a strong genetic basis. However, evidence suggests a role for environmental factors such as viral infections in triggering disease onset. One viral infection that is highly correlated with T1D is the Coxsackievirus B (CVB) serotype. Sensing of CVB is mediated by the melanoma differentiation-associated protein 5 (MDA5), a cytosolic sensor of dsRNA, which is encoded by the IFIH1 gene. Stimulation of the MDA5 signaling pathway activates the transcription factors IRF3 and NFκB p65, which induce type I IFNs synthesis. Type I IFNs can signal in an autocrine manner through the JAK/STAT pathway to initiate an antiviral transcriptional program. Single nucleotide polymorphisms (SNP) in the IFIH1 gene such as rs1990760, which results in a non-synonymous mutation that changes alanine at position 946 to a threonine, is highly associated with increased risk for T1D. Studies in human PBMCs and mice have demonstrated that the A946T SNP results in an increased sensitivity to viral ligands and subsequently a stronger downstream IFN response. We hypothesize that cells overexpressing the type I diabetes-associated variant 946T will have enhanced downstream signaling and subsequent type I IFN secretion in response to viral ligands and CVB infection. To test this, we stably overexpressed MDA5 with the 946A or 946T variants in HeLa cells. We anticipate that signaling events downstream of CVB infection and synthetic ligand poly (I:C) treatment, such as levels of phosphorylated IRF3 and NFκB p65 will be increased in cells expressing the T1D-associated allele 946T in contrast to cells overexpressing the non-risk allele 946A. Corroborating the increase in IRF3 and NFκB p65 pathway activation, we expect to observe enhanced production and secretion of type I IFNs were measured using HEK Blue IFN reporter cells that may partly explain how CVB infections can trigger autoimmune diabetes. Disclosure J.P. Taylor: None. H.M. Tse: None. Funding National Institutes of Health

Differential Roles of the mTOR-STAT3 Signaling in Dermal γδ T Cell Effector Function in Skin Inflammation

SUMMARYDermal γδT cells play critical roles in skin homeostasis and inflammation. However, the underlying molecular mechanisms by which these cells are activated have not been fully understood. Here, we show that the mechanistic or mammalian target of rapamycin (mTOR) and STAT3 pathways are activated in dermal γδT cells in response to innate stimuli such as interleukin-1β (IL-1β) and IL-23. Although both mTOR complex 1 (mTORC1) and mTORC2 are essential for dermal γδT cell proliferation, mTORC2 deficiency leads to decreased dermal γδT17 cells. It appears that mitochondria-mediated oxidative phosphorylation is critical in this process. Notably, although the STAT3 pathway is critical for dermal Vγ4T17 effector function, it is not required for γδ6T17 cells. Transcription factor IRF-4 activation promotes dermal γδT cell IL-17 production by linking IL-1β and IL-23 signaling. The absence of mTORC2 in dermal γδT cells, but not STAT3, ameliorates skin inflammation. Taken together, our results demonstrate that the mTOR-STAT3 signaling differentially regulates dermal γδT cell effector function in skin inflammation.

Physiological expression of miR-130a during differentiation of CD34+ human hematopoietic stem cells results in the inhibition of monocyte differentiation

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner, thereby determining their degradation or inhibiting translation. They are involved in processes such as proliferation, differentiation and apoptosis by fine-tuning the expression of genes underlying such events. The expression of specific miRNAs is involved in hematopoietic differentiation and their deregulation contributes to the development of hematopoietic malignancies such as acute myeloid leukemia (AML). miR-130a is over-expressed in AML. Here we show that miR-130a is physiologically expressed in myeloblasts and down-regulated during monocyte differentiation. Gain- and loss-of-function experiments performed on CD34+ human hematopoietic stem cells confirmed that expression of miR-130a inhibits monocyte differentiation by interfering with the expression of key transcription factors HOXA10, IRF8, KLF4, MAFB and PU-1. The data obtained in this study highlight that the correct modulation of miR-130a is necessary for normal differentiation to occur and confirming that deregulation of this miRNA might underlie the differentiation block occurring in AML.

Porcine deltacoronavirus nucleocapsid protein antagonizes IFN-β production by impairing dsRNA and PACT binding to RIG-I

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in newborn piglets. Previous studies have suggested that PDCoV infection antagonizes RIG-I-like receptor (RLR)-mediated IFN-β production to evade host innate immune defense, and PDCoV-encoded nonstructural protein nsp5 and accessory protein NS6 are associated with this process. However, whether the structural protein(s) of PDCoV also antagonize IFN-β production remains unclear. In this study, we found that PDCoV nucleocapsid (N) protein, the most abundant viral structural protein, suppressed Sendai virus (SEV)-induced IFN-β production and transcription factor IRF3 activation, but did not block IFN-β production induced by overexpressing RIG-I/MDA5. Furthermore, study revealed that PDCoV N protein interacted with RIG-I and MDA5 in an in vitro overexpression system and evident interactions between N protein and RIG-I could be detected in the context of PDCoV infection, which interfered with the binding of dsRNA and protein activator of protein kinase R (PACT) to RIG-I. Together, our results demonstrate that PDCoV N protein is an IFN antagonist and utilizes diverse strategies to attenuate RIG-I recognition and activation.

Bisphenol A promotes macrophage proinflammatory subtype polarization via upregulation of IRF5 expression in vitro

Exposure to environmental endocrine-disrupting chemical Bisphenol-A (BPA) is closely associated with an imbalance of immune homeostasis, but the underlying mechanisms are not fully understood. In the present study, the effects of BPA on the polarization of mouse peritoneal macrophages were investigated in vitro. Environmentally relevant low concentrations of BPA treatment under M1 type polarization conditions increased the number of M1 subtype macrophages, the gene expression of M1 phenotypic marker CD11c and the activity and gene expression of M1 functional marker iNOS, as well as the production of pro-inflammatory cytokines. However, The same dose BPA treatment under M2 type polarization conditions reduced the number of M2 subtype macrophages, the gene expression of M2 phenotypic marker CD206 and the activity and gene expression of M2 functional marker Arg-1, along with the production of anti-inflammatory cytokines. We also identified that the expression of transcription factor IRF5 was upregulated by BPA exposure in M1 macrophages under M1 type polarization conditions. Our results demonstrate that BPA promotes macrophage polarization toward proinflammatory M1 subtype and M1 activity, associated with upregulated expression of IRF5, while BPA inhibits macrophage toward anti-inflammatory M2 subtype polarization. These findings provide new insight into the link between exposure to BPA and impairment of immune functions.

Interferon regulatory factor 3 plays a role in macrophage responses to interferon-γ

IFN-γ produced during viral infections activates the IFN-γ receptor (IFNGR) complex for STAT1 transcriptional activity leading to expression of Interferon Regulatory Factors (IRF). Simultaneous activation of TBK/IKKε via TLR3 during viral infections activates the transcription factor IRF3. Together these transcription factors contributes to expression of intracellular proteins (e.g. ISG49, ISG54) and secreted proteins (e.g. IFN-β, IP-10, IL-15) that are essential to innate antiviral immunity. Here we examined the role of IRF3 in expression of innate anti-viral proteins produced in response to IFN-γ plus TLR3 agonist. Wild-type (WT) and IRF3KO RAW264.7 cells, each with ISG54-promoter-luciferase reporter vectors, were stimulated with IFN-γ, poly I:C, or both together. ISG54 promoter activity was significantly reduced in IRF3KO RAW264.7 cells responding to IFN-γ, poly I:C, or IFN-γ plus poly I:C, compared with WT RAW264.7 cells. These data were confirmed with western blot and qRT-PCR. Primary macrophages and dendritic cells (DCs) from IRF3KO mice also showed decreased ISG54 in response to IFN-γ, poly I:C, or IFN-γ plus poly I:C compared with those from WT mice. Moreover, pharmacological inhibition of TBK/IKKε significantly reduced ISG54 promoter activity in response to IFN-γ, poly I:C, or IFN-γ plus poly I:C. Similarly, expression of ISG49 and IL-15, but not IP-10, was impaired in IRF3KO RAW264.7 cells responding to IFN-γ or poly I:C, which also had impaired STAT1 phosphorylation and IRF1 expression. These data show that IRF3 contributes to IFN-γ/IFNGR signaling for expression of innate anti-viral proteins in macrophages.

Bach2 attenuates T cell receptor-dependent transcription to fine-tune regulatory T cell differentiation

Abstract The differentiation of regulatory T (Treg) cells is strictly controlled by T cell receptor (TCR) signals. However, the downstream regulators of this process are incompletely understood. Here we have found that Bach2 blocks the genomic binding of the TCR-induced transcription factor IRF4, attenuating TCR-dependent transcriptional programs to fine-tune Treg cell development and differentiation. In the absence of Bach2 we observed an increase in TCR-driven Treg cell outcomes, including the enhanced differentiation of effector Treg cells and thymic Treg precursor cells, and a reduction in peripheral Treg cell differentiation. Additional loss of the TCR-responsive transcription factor IRF4 was sufficient to normalise frequencies of each of these Treg cell populations in the absence of Bach2. Transcriptomic analysis identified significant deregulation of gene expression in Bach2-deficient cells which was dependent upon IRF4 expression. Assessing genome-wide occupancy of these transcription factors, we found Bach2 to restrict access of IRF4 to most of its genomic binding sites. Together, these data indicate that Bach2 inhibits IRF4-dependent transcription by blocking its access to their shared binding sites. Our work reveals Bach2 and IRF4 to drive opposing programs in Treg cell development and differentiation. Bach2 maintains the functional quiescence of Treg cells, in large part by blocking the genomic binding of IRF4 to attenuate the transcriptional program of TCR signalling.

Regulation of GLI3 expression by TLR4 signaling

Abstract While acute inflammation is an important host response to infections, chronic inflammation has emerged as a mechanism driving many diseases including autoimmune and malignant diseases. Monocytes are a subset of immune cells that secrete inflammatory cytokines in response to infections through toll-like receptor (TLR) signaling. The cross-talk between TLR signaling and other pathways has been shown to be important for downstream effector functions. We identify a novel role for the transcription factor GLI3, a negative regulator of Hedgehog signaling, in mediating TLR4-induced inflammation. Stimulation of monocytes with LPS increases GLI3 mRNA and protein expression. This occurred in a dose- and time-dependent manner. Investigation of the signaling mechanism suggests that LPS induced GLI3 occurs through the adaptor protein TRIF (but not MyD88). Targeting of transcription factors NFkb, AP-1 and IRF3 by pharmaceutical inhibitors suggests that TLR4-mediated GLI3 is regulated by IRF3. Transfection of our monocyte cell lines with shRNA targeting GLI3 suggests a regulatory role of transcription factor GLI3 in mRNA expression of essential cytokines involved in inflammation including IL-6 and CCL2. We generated conditional GLI3 knockout in mice myeloid cells using LysM-cre mediated recombination. IFA-elicited macrophages from mice with conditional GLI3 knockout driven by LysM-cre stimulated with LPS show reduced IL-6 and CCL2 secretion compared to macrophages from wild-type littermates. Taken together, these data suggest that GLI3 plays a role in innate immunity by regulating TLR4-induced inflammation.

RAS P21 Protein Activator 3 (RASA3) specifically promotes pathogenic T helper 17 cell generation by repressing T helper 2-biased programs

Abstract Th17 cells that produce the signature cytokine interleukin 17 (IL-17) can be both pathogenic and non-pathogenic. Pathogenic-Th17 (pTh17) cells drive inflammation and immune-pathology, but whether pTh17 cells are a Th17-subset whose generation is under specific molecular control remains unaddressed. We found that Ras p21 protein activator 3 (RASA3), a GTPase activating protein, was highly expressed by IL-1β/IL-6/IL-23 induced-pTh17 cells relative to TGFβ1/IL-6 induced-non-pTh17 cells and was required specifically for pTh17 generation in vitro and in vivo. Mice conditionally deficient for Rasa3 in T cells showed less pathology during experimental autoimmune encephalomyelitis. By RNA-seq analysis, Rasa3-deficient T cells were found to acquire a Th2-biased program that dominantly trans-suppressed pTh17 cell generation via interleukin 4 production. The Th2-bias of Rasa3-deficient T cells was due to aberrantly elevated transcription factor IRF4 expression. Furthermore, RASA3 promoted proteasome-mediated IRF4 protein degradation by facilitating interaction of IRF4 with E3-ubiquitin ligase Cbl-b. Therefore, a RASA3-IRF4-Cbl-b pathway specifically directs pTh17 cell generation by balancing reciprocal Th17-Th2 programs. These findings indicate that a distinct molecular program directs pTh17 generation and reveals targets for treating pTh17-related pathology and diseases.

Role of NLRC5 and IRF1 in the induction of MHC class I

Abstract MHC class I and MHC class II plays a major role in adaptive immune responses through activation of CD8+ T cells or CD4+ T cells respectively, by presenting intracellular or extracellular antigens. The MHC class I transactivator (CITA), NLRC5 was recently identified as a key transcriptional regulator of MHC class I and related genes. Although promoters of both MHC class I and class II genes share similar cis-regulatory elements, the specificity of NLRC5 to MHC class I gene transactivation remains unclear. To delineate the specificity of NLRC5 in NLRC5-dependent MHC classs I transactivation, we performed co-immunoprecipitation with different deletion mutants of IRF1. The results were further confirmed in mice lacking Irf1 and Nlrc5. We found that the transcription factor IRF1 associates with NLRC5, enabling efficient transactivation of MHC class I and related genes. Double deficiency of Irf1 and Nlrc5 resulted in the severe reduction of MHC class I and related gene expression in vitro and in vivo. Therefore, IRF1 is a key component that determine the specificity of NLRC5-dependent MHC class I gene transactivation, and their involvement in the CITA enhanceosome is critical for MHC class I-dependent immune responses. Further studies on the interaction of IRF1 and NLRC5 would shed light on the potential therapeutic intervention on the MHC class I related diseases such as auto-immune disorders, cancer immunity and in organ transplantation

Epigenetic control of early dendritic cell lineage specification by the transcription factor IRF8 in mice

AbstractDendritic cells (DCs), which are vital for immune responses, are derived from bone marrow hematopoietic stem cells via common DC progenitors (CDPs). DC lineage fate decisions occurring at stages much earlier than CDPs have recently been recognized, yet the mechanism remains elusive. By single-cell RNA-sequencing, in vivo cell transfer experiments, and an assay for transposase-accessible chromatin sequencing using wild-type, IRF8-GFP chimera knock-in or IRF8-knockout mice, we demonstrate that IRF8 regulates chromatin at the lymphoid-primed multipotent progenitor (LMPP) stage to induce early commitment toward DCs. A low but significant expression of IRF8, a transcription factor essential for DC and monocyte development, was initiated in a subpopulation within LMPPs. These IRF8+ LMPPs were derived from IRF8– LMPPs and predominantly produced DCs, especially classical DC1s, potentially via known progenitors, such as monocyte-DC progenitors, CDPs, and preclassical DCs. IRF8+ LMPPs did not generate significant numbers of monocytes, neutrophils, or lymphocytes. Although IRF8– and IRF8+ LMPPs displayed very similar global gene expression patterns, the chromatin of enhancers near DC lineage genes was more accessible in IRF8+ LMPPs than in IRF8– LMPPs, an epigenetic change dependent on IRF8. The majority of the genes epigenetically primed by IRF8 were still transcriptionally inactive at the LMPP stage, but were highly expressed in the downstream DC lineage populations such as CDPs. Therefore, early expression of the key transcription factor IRF8 changes chromatin states in otherwise multipotent progenitors, biasing their fate decision toward DCs.

834 Rational design of recombinant modified vaccinia virus Ankara for cancer immunotherapy

Poxviruses are large cytoplasmic DNA viruses and modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia strain. Intratumoral (IT) delivery of inactivated MVA (iMVA) eradicates injected tumors and generates systemic antitumor immunity, which synergizes with immune chekpoint blockade antibodies (Dai et al. Science Immunology, 2017). The antitumor effects induced by iMVA requires CD8+ T cells and CD103+ dendritic cells (DCs), and is dependent on cGAS/STING. We hypothesize that MVA encodes viral inhibitor(s) of the cGAS/STING pathway and therefore deleting such inhibitor(s) from MVA would improve the antitumor effects of the virus. We conducted a systematic screening of vaccinia early genes for potential inhibitors of the cGAS/STING pathway and identified several candidates. Among them, the vaccinia E5 protein encoded by the E5R gene has a dominant inhibitory function. Infection of bone marrow-derived DCs (BMDCs) with MVAΔE5R induces 80-fold higher IFN-β secretion compared with MVA and 10-fold higher IFN-β secertion compared with iMVA. IT MVAΔE5R results in strong antitumor effects that is dependent on the cGAS/STING/IFNAR pathway. MVA also expresses another virulence factor C7, which is encoded by the C7L gene. C7 binds to the transcription factors IRF3 and Stat2 and thereby inhibits type I IFN production and IFNAR signaling. IT recombinant MVA with deletion of both C7L and E5R, and with expression of two transgenes human Flt3L, a growth factor for DCs, and murine OX40L, a costimulatory factor for T cells, results in enhanced antitumor immunity compared with iMVA in murine B16-F10 melanoma model and other tumor models. Our results demonstrate the feasibility of dramatically improving MVA-based cancer immunotherapeutics through rational design.

Transcription factors IRF8 and PU.1 are required for follicular B cell development and BCL6-driven germinal center responses

The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells.

Engineering a haematopoietic stem cell niche by revitalizing mesenchymal stromal cells.

Haematopoietic stem cells (HSCs) are maintained by bone marrow (BM) niches in vivo1,2, but the ability of niche cells to maintain HSCs ex vivo is markedly diminished. Expression of niche factors by Nestin-GFP+ mesenchymal-derived stromal cells (MSCs) is downregulated upon culture, suggesting that transcriptional rewiring may contribute to this reduced HSC maintenance potential. Using an RNA sequencing screen, we identified 5 transcription factors (Klf7, Ostf1, Xbp1, Irf3, Irf7) that restored HSC niche function in cultured BM-derived MSCs. These revitalized MSCs (rMSCs) exhibited enhanced synthesis of HSC niche factors while retaining their mesenchymal differentiation capacity. In contrast to HSCs co-cultured with control MSCs, HSCs expanded with rMSCs showed higher repopulation capacity and protected lethally irradiated recipient mice. Competitive reconstitution assays revealed ~7-fold expansion of functional HSCs by rMSCs. rMSCs prevented the accumulation of DNA damage in cultured HSCs, a hallmark of ageing and replication stress. Analysis of the reprogramming mechanisms uncovered a role for myocyte enhancer factor 2c (Mef2c) in the revitalization of MSCs. These results provide insight in the transcriptional regulation of the niche with implications for stem cell-based therapies.

Structural mechanism of vaccinia virus protein C6 mediated inactivation of transcription factors IRF3 and IRF7 via inhibition of TBK1 mediated scaffold protein‐protein interactions

Interferons (IFNs) are signaling proteins with potent antiviral and immune stimulatory effects. IFN signaling is initiated after host cell pattern recognition receptors (PRRs) recognize pathogen‐associated molecular patterns (PAMPs) present in many viral proteins. This event in turn generates a complex signaling cascade involving numerous proteins, activation of various transcription factors (TF), such as IFN‐regulatory factors (IRF) 3 and 7. These TFs subsequently translocate to the nucleus to elevate the transcription of type I IFNs, cytokines, and chemokines. Viruses such as Vaccinia virus (VACV) have evolved many strategies to interfere with the IFNs or their mechanism of action. Specifically, VACV encodes a small 16kDa protein, C6, that inhibits the activation of IRF3/7 via binding with an upstream kinase, TBK1, and its adaptor proteins TANK, NAP1, and SINTBAD in order to interfere with the production of IFNβ. Binding with these adaptor proteins, C6 prevents TBK1/IKKɛ‐dependent activation of IRF3 and IRF7 allowing VACV to evade immune recognition. To elucidate the structural mechanism of how C6 mediated inhibition of these adaptor protein/TBK1 interactions can block the activation of IRF3 and IRF7, we have chosen C6 and TANK as canonical binding partners. We have identified the binding regions between C6 and TANK by utilizing co‐expression/co‐purification studies of C6 and TANK proteins, and co‐crystallization trials are ongoing. The immune evasive strategy exploited by VACV can be used to develop novel therapies, such as C6 mimetics to provide TBK1 inhibition. The development of therapeutics depends immensely on a detailed structural analysis of the interactions between C6 and adaptor proteins. Our structural studies of C6 and TBK1 adaptor proteins will provide useful insights in developing superior therapies against malignancies, infectious diseases, and autoimmune disorders.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Histone modifications underlie monocyte dysregulation in patients with systemic sclerosis, underlining the treatment potential of epigenetic targeting

Background and objectiveSystemic sclerosis (SSc) is a severe autoimmune disease, in which the pathogenesis is dependent on both genetic and epigenetic factors. Altered gene expression in SSc monocytes, particularly of...

Arc/Arg3.1 defines dendritic cells and Langerhans cells with superior migratory ability independent of phenotype and ontogeny in mice.

The key function of migratory dendritic cells (migDCs) is to take up antigens in peripheral tissues and migrate to draining lymph nodes (dLN) to initiate immune responses. Recently, we discovered that in the mouse immune system activity-regulated cytoskeleton associated protein/activity-regulated gene 3.1 (Arc/Arg3.1) is exclusively expressed by migDCs and is a central driver of fast inflammatory migration. However, the frequency of Arc/Arg3.1-expressing cells in different migDC subsets and Langerhans cells (LCs), their phylogenetic origin, transcription factor dependency, and functional role remain unclear. Here, we found that Arc/Arg3.1+ migDCs derived from common DC precursors and radio-resistant LCs. We detected Arc/Arg3.1+ migDCs in varying frequencies within each migDC subset and LCs. Consistently, they showed superiority in inflammatory migration. Arc/Arg3.1 expression was independent of the transcription factors Irf4 or Batf3 in vivo. In intradermal Staphylococcus aureus infection that relies on inflammatory antigen transport, Arc/Arg3.1 deletion reduced T-cell responses. By contrast, Arc/Arg3.1 deficiency did not hamper the immune response to systemic Listeria monocytogenes infection, which does not require antigen transport. Thus, Arc/Arg3.1 expression is independent of ontogeny and phenotype and although it is restricted to a small fraction within each migDC subset and LCs, Arc/Arg3.1+ migDCs are important to facilitate infectious migration.

Structural basis of STING binding with and phosphorylation by TBK1.

The invasion of mammalian cytoplasm by microbial DNA from infectious pathogens or by self DNA from the nucleus or mitochondria represents a danger signal that alerts the host immune system1. Cyclic GMP-AMP synthase(cGAS) is a sensor of cytoplasmic DNA that activates the type-I interferon pathway2. On binding to DNA, cGAS is activated to catalyse the synthesis of cyclic GMP-AMP (cGAMP) from GTP and ATP3. cGAMP functions as a second messenger that binds to and activates stimulator of interferon genes (STING)3-9. STING then recruits and activates tank-binding kinase 1 (TBK1), which phosphorylates STING and the transcription factor IRF3 to induce type-I interferons and other cytokines10,11. However, how cGAMP-bound STING activates TBK1 and IRF3 is not understood. Here we present the cryo-electron microscopy structure of human TBK1 in complex with cGAMP-bound, full-length chicken STING. The structure reveals that the C-terminal tail of STING adopts a β-strand-like conformation and inserts into a groove between the kinase domain of one TBK1 subunit and the scaffold and dimerization domain of the second subunit in the TBK1 dimer. In this binding mode, the phosphorylation site Ser366 in the STING tail cannot reach the kinase-domain active site of bound TBK1, which suggests that STING phosphorylation by TBK1 requires the oligomerization of both proteins. Mutational analyses validate the interaction mode between TBK1 and STING and support a model in which high-order oligomerization of STING and TBK1, induced by cGAMP, leads to STING phosphorylation by TBK1.

Transformation of mature mouse B cells into malignant plasma cells in vitro via introduction of defined genetic elements

An experimental system where defined alterations in gene function or gene expression levels in primary B cells would result in the development of transformed plasma cells in vitro would be useful in order to facilitate studies of the underlying molecular mechanisms of plasma cell malignancies. Here, such a system is described in which primary murine B cells rapidly become transformed into surface CD138+ , IgM-/low , CD19- IgM-secreting plasma cells as a result of expression of the transcription factors IRF4 and MYC together with simultaneous expression of BMI1, mutated p53 or silencing of p19Arf , and suppression of intrinsic apoptosis through expression of BCLXL. Analysis of gene expression patterns revealed that this combination of transforming genes resulted in expression of a number of genes previously associated with terminally differentiated B cells (plasma cells) and myeloma cells, whereas many genes associated with mature B cells and B-cell lymphomas were not expressed. Upon transplantation, the transformed cells preferentially localized to the bone marrow, presenting features of a plasma cell malignancy of the IgM isotype. The present findings may also be applicable in the development of novel methods for production of monoclonal antibodies.