Transcription Factor IRF3 Research Articles

The transcription factor E2A can bind to and cleave single-stranded immunoglobulin heavy chain locus DNA

Class switch recombination (CSR) changes the constant region of the immunoglobulin heavy chain (IgH), and somatic hypermutation (SH) introduces point mutations in the variable regions of the antibody genes. Both these processes that optimize antibody responses of B lymphocytes are initiated by the enzyme Activation Induced cytidine Deaminase (AID). Here we have searched for CSR or SH coupled activities of the transcription factor E2A, since E2A is in a complex with AID and the transcription factors PAX5, ETS1 and IRF4 on key sequences of the Igh locus in B lymphocytes activated to CSR and SH. We report that E2A in contrast to other described transcription factors binds sequence specifically also to single-stranded DNA. The binding of E2A to single-stranded DNA has a strong sequence preference for one strand of a site in the intronic enhancer of the Igh locus. Furthermore, E2A was also found to cleave single-stranded DNA. The sequence profile of substrates cleaved by E2A is coupled to the sequences of substrates and products of AID, suggesting that E2A has a role not only in targeting of AID to switch regions and SH parts of antibody genes but also in cleavage of DNA at these sites.

Manipulation of innate immune signaling pathways by SARS-CoV-2 non-structural proteins.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic, induces an unbalanced immune response in the host. For instance, the production of type I interferon (IFN) and the response to it, which act as a front-line defense against virus invasion, are inhibited during SARS-CoV-2 infection. In addition, tumor necrosis factor alpha (TNF-α), a proinflammatory cytokine, is upregulated in COVID-19 patients with severe symptoms. Studies on the closely related betacoronavirus, SARS-CoV, showed that viral proteins such as Nsp1, Orf6 and nucleocapsid protein inhibit IFN-β production and responses at multiple steps. Given the conservation of these proteins between SARS-CoV and SARS-CoV-2, it is not surprising that SARS-CoV-2 deploys similar immune evasion strategies. Here, we carried out a screen to examine the role of individual SARS-CoV-2 proteins in regulating innate immune signaling, such as the activation of transcription factors IRF3 and NF-κB and the response to type I and type II IFN. In addition to established roles of SARS-CoV-2 proteins, we report that SARS-CoV-2 proteins Nsp6 and Orf8 inhibit the type I IFN response but at different stages. Orf6 blocks the translocation of STAT1 and STAT2 into the nucleus, whereas ORF8 inhibits the pathway in the nucleus after STAT1/2 translocation. SARS-CoV-2 Orf6 also suppresses IRF3 activation and TNF-α-induced NF-κB activation.

Single-Cell Multi-Omics Analysis Reveals the Role of CX3CR1+ GNLY+ CD8+ T Cells in Improving Survival of Patients with Relapse/Refractory Hematologic Malignancies after Haplo+Cord HSCT

Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for hematological malignancies. Previous works have shown superior survival outcomes of sequential transplantation of haploidentical stem cell and unrelated cord blood (haplo+cord HSCT) among recipients with relapsed/refractory leukemia, compared with single cord HSCT. However, the underlying mechanisms of immune reconstitution remain largely unexplored. Methods we first performed scRNA-seq and scATAC-seq on bone marrow mononuclear cells (BMMCs) from nine patients with relapsed/refractory leukemia underwent haplo+cord or single cord HSCT to profile the immune landscapes at single cell resolution. Second, we compared the differences of gene expression and chromatin accessibility between the subsets of immune cells. Finally, by integrating multi-omics data, we explored the gene expression regulation mechanism behind the differences between two transplant group. Results We observed distinct composition and function of global immune landscapes (T, B, natural killer cells and monocytes) between two transplant strategies, where immune cells from haplo+cord HSCT involved in interferon signaling pathway and response to virus. Analysis on T cells revealed two cytotoxic CD8+ T cells populations, enriched in haplo+cord HSCT (expressing GNLY, GZMB and PRF1) and enriched in single cord HSCT (expressing CXCR4 and GZMK) respectively. Interestingly, the augmented expression of GNLY and CX3CR1 suggested a dual property of effector and memory of haplo+cord HSCT enriched CD8+ T cells that potentially offers long-term protection for patients post-transplantation. The transcription factor IRF7, IRF9 and TBX21 played the key role on regulating immune signal transduction of CD8+ T cell subsets after haplo+cord HSCT, which might be the mechanism of transcriptional regulation for cytotoxic CD8+T cell function. Finally, single cell multi-omics analysis delineate regulatory network controlling lineage-specific gene expression after HSCT, illustrated by the example of GNLY expression in CD8+ T cells regulated by an enhancer cluster within CTCF-mediated chromatin loop. Conclusions These data collectively constitute the transcriptomic and epigenetic landscape of bone marrow post-transplantation in hematopoietic malignancies at single-cell resolution, demonstrating that a CX3CR1+ GNLY+ CD8+ T cells characterized by effector and memory property served the improved survival of patients receiving haplo+cord HSCT. keywords Single cell multi-omics, CX3CR1+ GNLY+ CD8+T cells, haplo+cord HSCT, single cord HSCT, relapsed/refractory leukemia

SHP-2 Regulates Myeloid Cell Differentiation, Anti-Tumor Responses and Innate Immune Memory

PD-1 is a T cell checkpoint inhibitor and the most extensively exploited therapeutic target of cancer immunotherapy. PD-1-mediated T cell inactivation is attributed to the function of SHP-2 phosphatase, which is activated by recruitment to PD-1 cytoplasmic tail. However, T cell-specific SHP-2 deletion did not improve anti-tumor immunity or altered responses to anti-PD-1 immunotherapy. Previously, we determined that PD-1 is also expressed in common myeloid progenitors and granulocyte/macrophage progenitors, which accumulate during cancer-driven emergency myelopoiesis and give rise to immunosuppressive myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAMs). In tumor-bearing mice with myeloid-specific PD-1 ablation, accumulation of MDSCs was prevented, while output of differentiated effector myeloid cells with monocytic lineage dominance was increased. However, the molecular mechanism remains elusive. It is also unknown whether a canonical PD-1:SHP-2 axis is operative in myeloid cells. Temporal activation of SHP-2 is critical for myeloid cell fate. Gain of function SHP-2 mutations, resulting in constitutive phosphatase activation, prevent myeloid differentiation and lead to the accumulation of immature myelocytes and development of leukemia. HOXA proteins, members of the HOX family of transcription factors involved in normal hematopoiesis, are targets of SHP-2, which dephosphorylates HOXA and prevents derepressing of HOXA target genes and myeloid cell differentiation. In addition, SHP-2 regulates the function of IFN regulatory factor (IRF)-8, a transcription factor that has a decisive role in myeloid progenitor differentiation toward monocytes and DCs. IRF8 phosphorylation is mandatory for its nuclear translocation and initiation of gene transcription. The cytoplasmic-nuclear shuttling of IRF8 is regulated by SHP-2, which dephosphorylates IRF8 and prevents its nuclear localization and transcriptional activation. We generated mice with conditional targeting of the Ptpn11 gene (encoding for Shp-2) in T cells (Shp2f/fLckCre) or myeloid cells (Shp2f/fLysMCre), and found that Shp2f/fLysMCre but not Shp2f/fLckCre mice had diminished tumor growth. As determined by RNA-sequencing, this was paralleled by the presence of inflammatory neutrophils and tumor-associated macrophages (TAMs) with molecular signatures of enhanced differentiation, phagocytosis and antigen-processing and presentation. SHP-2 deficient TAMs also had increased monocyte and dendritic cells (DCs) specification transcription factors, chemokine and cytokine production, and expression of immunostimulatory molecules that promote T cell recruitment and activation. Moreover, monocytes from tumor-bearing Shp2f/fLysMCre mice suppressed tumor growth after transfer to naïve recipients indicating development of innate immune memory, named trained immunity. In bone marrow myelocytes, SHP-2 deletion enhanced GM-CSF-mediated phosphorylation of the transcription factors HOXA10 and IRF8 that promote myeloid differentiation and monocytic/moDC lineage commitment, respectively. In contrast, in WT bone marrow myelocytes GM-CSF induced PD-1 expression, phosphorylation and interaction with SHP-2, the Src family kinase Lyn, and GM-CSF receptor beta chain, indicating that the PD-1:SHP-2 axis targets a key pathway of myelocyte differentiation. Our results reveal a previously unidentified mechanistic role of SHP-2 and the PD-1:SHP-2 axis in regulating myeloid cell differentiation in the context of cancer and provide evidence that SHP-2 poses a signaling restrain to myeloid differentiation and monocyte lineage commitment resulting in a myeloid landscape that suppresses anti-tumor immunity. This process is hijacked by the PD-1/PD-L1 pathway.

IMiDs Synergize with EP300 Inhibition to Disrupt the Ikzf/MYC/IRF4 Axis in Multiple Myeloma

Multiple myeloma (MM) is a heterogeneous plasma cell malignancy for which current therapies eventually fail. In myeloma, endogenous and translocated superenhancers drive the expression of the lineage-defining transcription factor IRF4 and oncogene MYC, respectively - both considered "undruggable". Successful standard-of-care immunomodulatory imide drugs (IMiDs) degrade superenhancer-binding pioneer factors IKAROS and AIOLOS; however, depletion of IKAROS and AIOLOS does not always correlate with a successful IMiD response, and patients treated with IMiDs ultimately relapse. We show responses to the IMiD Pomalidomide (POM) are directly correlated with downregulation of IRF4 and MYC. In IMiD-resistant cells and tumors, IRF4 and MYC downregulation could be achieved with novel coactivator-targeting drugs including the BET inhibitor JQ1 and the CBP/EP300 inhibitor GNE-781, but these were toxic and lacked a therapeutic window in vivo. We show how POM combined with GNE781 synergize and downregulate MYC and IRF4 levels, killing myeloma cells in vitro and in vivo. However, resistance to this combination was induced by expression of the AP-1 factor BATF, which directly maintained IRF4 expression and myeloma cell survival. These results identify a potent therapeutic combination and a hereto unrealized mechanism of IMiD resistance in multiple myeloma.

Non-canonical NF-κB contributes to endothelial pyroptosis and atherogenesis dependent on IRF-1

Cell inflammation and death are closely linked processes contributing to endothelial dysfunction, which plays a critical role in atherogenesis. Activation of the NLRP3 inflammasome causes pyroptosis, the Gasdermin D (GSDMD)-mediated inflammatory cell death. The non-canonical NF-κB pathway has been implicated in inflammation; however, its role in NLRP3 inflammasome-mediated endothelial dysfunction has not been investigated. This study investigated a role for the non-canonical NF-κB pathway in regulating endothelial pyroptosis as it relates to atherogenesis. Immunohistochemistry indicated inflammasome activation in the endothelial cells (EC) of human atherosclerotic arteries. Flow cytometry and Western blot analysis revealed that oxidized low-density lipoprotein (oxLDL) activated the NLRP3 inflammasome, concomitant with the activation of non-canonical NF-κB in primary human aortic EC. Interference of NF-κB inducing kinase (NIK), the key regulator of the non-canonical pathway, significantly attenuated oxLDL- or LPS/ATP-induced NLRP3 inflammasome activation, pyroptosis, IL-1β, and IL-18 secretion. In contrast, overexpression of NIK exacerbated these responses. Chromatin immunoprecipitation revealed that activation of the non-canonical NF-κB pathway upregulated the transcription factor IRF-1 through RelB/p52 binding to its promoter region at -782/-770. In addition to the known target CASP1, RNA sequencing further identified GSDMD as a target gene of IRF-1. IRF-1 but not RelB/p52 interacted with the GSDMD promoter at -526/-515 and the CASP1 promoter at -11/10 to promote the expression and CASP1-mediated activation of GSDMD. Consistent with the observations in cultured endothelium, endothelial-specific deficiency of NIK or IRF-1 attenuated atherosclerosis in high-fat diet-fed Apoe-null mice. These data demonstrate that the non-canonical NF-κB pathway contributes to NLRP3 inflammasome-mediated endothelial pyroptosis and the development of atherosclerosis through GSDMD activation in a manner dependent on IRF-1. Further investigation may facilitate the identification of specific therapeutic targets for atherosclerotic heart diseases.

EIF4A3 Promotes RNA Viruses' Replication by Inhibiting Innate Immune Responses.

Viral infection activates the type I interferons (IFNs) and cellular antiviral responses. Eukaryotic initiation factor 4A-III (eIF4A3) has been shown to promote influenza A virus (IAV) replication by promoting viral mRNA splicing and spliced mRNA nuclear export. Here, we identified eIF4A3 as a negative regulator of virus-triggered type I IFN induction. Our study found that eIF4A3 promoted multiple RNA viruses' replication by binding to IFN regulatory factor 3 (IRF3) and impaired the interaction between tank-binding kinase 1 (TBK1) and IRF3, leading to attenuation of the phosphorylation of IRF3 by TBK1, the formation of IRF3 dimer, and the nuclear translocation of IRF3. This impaired its biological functions in the nucleus, which blocked IRF3 binding to interferon-stimulated response element (ISRE) and the interaction of IRF3 and CBP/p300, resulting in inhibiting the transcription of IFN-β and downstream IFN-stimulated genes (ISGs), thereby impairing innate antiviral immune responses against RNA viruses. These findings reveal a previously unknown function of eIF4A3 in host innate immunity and establish a mechanistic link between eIF4A3 and IRF3 activation that expands potential therapeutic strategies for viral infectious diseases. IMPORTANCE Production of type I IFN is pivotal for the cellular antiviral immunity. Virus infection leads to the activation of transcription factor IRF3 and subsequent production of type I IFN to eliminate viral infection. Thus, the regulation of IRF3 activity is an important way to affect type I IFN production. IRF3 activation requires phosphorylation, dimerization, and nuclear translocation. Here, we first reported that eIF4A3, a member of DEAD box family, served as a negative regulator of antiviral innate immune responses by inhibiting IRF3 activation. Mechanistically, eIF4A3 binds to IRF3 to impair the recruitment of IRF3 by TBK1, which is independent of eIF4A3 ATP binding, ATPase, and RNA helicase activities. Our study delineates a common mechanism of eIF4A3 promoting replication of different RNA viruses and provides important insights into the negative regulation of host antiviral innate immune responses against virus infections.

Gene Co-Expression Network Modular Analysis Reveals Altered Immune Mechanisms in HIV-HAND.

Although the introduction of HAART has completely changed the natural course of HIV infection, the number of chronic forms of HIV-associated neurocognitive disorder (HAND) has risen. It is estimated that up to half of subjects undergoing HAART therapy exhibit mild cognitive impairments. In the current study, we apply the gene co-expression network modular analysis, a well-established system biology approach, to the gene expression profiles of cases from the National NeuroAIDS Tissue Consortium (NNTC). We observed a negative enrichment for genes associated with the control of immune responses and putatively regulated by the transcription factors IRF8 and SPI1 and by both type I and II interferons. Our study provides evidence of altered immune responses, which are likely associated with the occurrence of HAND in the absence of HIV encephalitis (HIVE).

When Smart Beta Meets Machine Learning and Portfolio Optimization

<h3>Abstract</h3> Exposure of human cells to interferon-γ (IFNγ) results in a mitotically heritable yet reversible state called long-term transcriptional memory. We previously identified the clustered GBP genes as strongly primed by IFNγ. Here we discovered that in primed cells, both interferon-responsive transcription factors STAT1 and IRF1 target chromatin with accelerated kinetics upon re-exposure to IFNγ, specifically at promotors of primed genes. Priming does not alter the degree of IFNγ-induced STAT1 activation or nuclear import, indicating that memory does not alter upstream JAK-STAT signalling. We found STAT1 to be critical to establish transcriptional memory but in a manner that is independent of mere transcription activation. Interestingly, while Serine 727 phosphorylation of STAT1 was maintained during the primed state, STAT1 is not required for the heritability of GBP gene memory. Our results suggest that memory of interferon exposure constitutes a STAT1-mediated, heritable state that is established during priming. This renders GBP genes poised for subsequent STAT1 and IRF1 binding and accelerated gene activation upon a secondary interferon exposure.

EMBO young Investigator LectureStimulation of plasmacytoid Dendritic Cells by the Yellow fever vaccine

Although representing a rare cell type of the immune system, plasmacytoid dendritic cells (pDCs) are the most important source of type I interferon (IFN-I) upon viral infection. These high secretion levels of IFN-I in response to viruses not only have direct inhibitory effects on viral replication, but also activate many other immune cells, such as natural killer cells, B cells, T cells and myeloid DCs, therefore strengthening adaptive immune mechanisms. We investigated the mechanism by which the yellow fever vaccine 17D, a widely-used live attenuated vaccine, stimulates human pDCs to produce IFN-I. We showed that productive infection of human pDCs by cell-free 17D triggered a RIG-I-like receptor (RLR)-dependent IFN-I response. The RIG-I downstream kinase TBK1 and the transcription factor IRF3 were phosphorylated, suggesting that the whole RLR signaling cascade was activated in pDCs. By contrast, pDCs sensing of 17D-infected cells induced a TLR7-dependent, replication-independent IFN-I response. We characterized in detail the mechanisms by which 17D-infected cells stimulate pDCs. Cells producing immature 17D particles were more potent at stimulating pDCs than cells releasing mature virions. Additionally, cells replicating a release-deficient 17D mutant or a 17D subgenomic RNA lacking structural protein-coding sequences participated in pDC stimulation. These viral RNAs free of viral core components were transported to pDCs in an exosome-independent manner. Our study reveals that human pDCs are armed to sense replicative viral RNA, and, depending on its delivery route, a virus is able to stimulate the TLR or the RLR signaling pathway in the same target cell. Our work also provides new insights into the mechanisms by which infected cells stimulate pDCs to induce an antiviral response.

Chromatin structure undergoes global and local reorganization during murine dendritic cell development and activation

Classical dendritic cells (cDCs) are essential for immune responses and differentiate from hematopoietic stem cells via intermediate progenitors, such as monocyte-DC progenitors (MDPs) and common DC progenitors (CDPs). Upon infection, cDCs are activated and rapidly express host defense-related genes, such as those encoding cytokines and chemokines. Chromatin structures, including nuclear compartments and topologically associating domains (TADs), have been implicated in gene regulation. However, the extent and dynamics of their reorganization during cDC development and activation remain unknown. In this study, we comprehensively determined higher-order chromatin structures by Hi-C in DC progenitors and cDC subpopulations. During cDC differentiation, chromatin activation was initially induced at the MDP stage. Subsequently, a shift from inactive to active nuclear compartments occurred at the cDC gene loci in CDPs, which was followed by increased intra-TAD interactions and loop formation. Mechanistically, the transcription factor IRF8, indispensable for cDC differentiation, mediated chromatin activation and changes into the active compartments in DC progenitors, thereby possibly leading to cDC-specific gene induction. Using an infection model, we found that the chromatin structures of host defense-related gene loci were preestablished in unstimulated cDCs, indicating that the formation of higher-order chromatin structures prior to infection may contribute to the rapid responses to pathogens. Overall, these results suggest that chromatin structure reorganization is closely related to the establishment of cDC-specific gene expression and immune functions. This study advances the fundamental understanding of chromatin reorganization in cDC differentiation and activation.

Abstract 2091: Macrophage PI3Kg control of chromatin accessibility and transcription factor activity promotes immune suppression and tumor progression

Abstract Tumor development and progression are associated with immune suppression and evasion as well as angiogenesis, fibrosis, and metastasis. Macrophages play critical roles in tumor progression by controlling these events and are the focus of efforts to develop novel cancer therapeutics. We previously showed that PI(3)Kinase γ, a class Ib phosphatidyl inositol 3, 4, 5 kinase that is expressed primarily in myeloid lineage cells, controls tumor progression by serving as a critical switch between immune stimulation and suppression. PI3Kγ also promotes angiogenesis, fibrosis and metastasis. PI3Kγ signaling in macrophages promotes transcription of tumor-promoting cytokines and factors such as Pdgf, Tgfb, Ccl2, Plau, Mmps and Arg-1 and inhibits expression of Il12, Tnfa, Nos2 and other pro-inflammatory factors to stimulate tumor progression and immune evasion. Genetic or pharmaceutical inhibition of PI3Kγ reverses these events and suppress tumor growth. In order to develop novel strategies to control macrophage contributions to cancer progression, we investigated in detail how this kinase controls gene expression. We found that PI3Kγ deletion or inhibition with a selective inhibitor IPI-549, which is currently in late stage clinical development for cancer therapy, stimulates the expression and secretion of IL12, IL6, IL1β, NOS, IFNα, and TNFα, while reducing the secretion and expression of IL10, CCL2, PDGFβ, ARG-1, TGFβ and other factors. Gene expression analysis indicates that PI3Kγ inhibition in macrophages rapidly stimulates transcription of NFκB and IFNγ driven pro-inflammatory pathways as well as nuclear translocation of transcription factors STAT1, IRF1, IRF3, TBK1 and NFκB and inhibition of CEBPβ nuclear activity. To further understand the impact of PI3Kγ on transcription in macrophages, we performed ATAC seq on WT and PI3Kγ null macrophages stimulated with LPS/IFNγ in order to determine the impact of PI3Kγ activity and inhibition on chromatin accessibility. We found that PI3Kγ deletion promoted more open chromatin sites for IRF, FRA I, Pu.1, and NFκB binding, as well as fewer open chromatin sites for AP1 (FOS/JUN), IRF8, and CEBP sites. Importantly, we identified overall chromatin accessibility in the promoters of some NFκB regulated genes, such as Tnfα and Il6, as well as novel open chromatin sites in the promoters for Il12b and Il1b in PI3Kγ null macrophages. We also identified novel closed chromatin sites in the promoters of Il10, Plau and Ccl2, genes that are downregulated upon PI3Kγ inhibition. Further studies to identify the mechanisms by which PI3Kγ regulates transcription are investigating PI3Kγ controls of epigenetic modifiers and transcription factors are ongoing. These studies should lead to the identification of new approaches to regulate tumor associated macrophages to inhibit tumor progression. Citation Format: Hui Chen, Jingwen Liao, Christina Flores, Diana Hargreaves, Judith Varner. Macrophage PI3Kg control of chromatin accessibility and transcription factor activity promotes immune suppression and tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2091.

Abstract 124: PRDX4 links ER-specific redox vulnerability to innate immunity in pancreatic cancer

Abstract The prognosis of pancreatic ductal carcinoma (PDAC) remains dismal, with an overall 5-year survival rate of 7%. Immunotherapy (IT) has provided new hope for other hard-to-treat cancers, but PDAC has shown striking resistance to IT by orchestrating mechanisms of immune escape. This spurs an interest to identify novel strategies to improve PDAC sensitivity to IT. We recently unveiled that perturbating redox homeostasis by targeting the antioxidant protein peroxiredoxin 4 (PRDX4) render pancreatic cancer cells highly vulnerable. PRDX4 supports redox homeostasis by metabolizing H2O2 in the endoplasmic reticulum (ER), and its loss leads to oxidative stress and toxicity. Interestingly, cell death induced by PRDX4 depletion is accompanied by DNA damage and accumulation of cytosolic DNA, a potent primer for immune therapy. In line with that, PRDX4 knockdown results in the transcriptional upregulation of inflammatory genes, including type I interferons (IFNs), primary cytokines and interferon-stimulated genes (ISGs). Importantly, our data indicate that this induction is dependent on the critical regulator of the innate immune responses, STING, as treatment with a STING inhibitor, or a STING knockdown, substantially blunts their upregulation. It is known that following DNA damage detection, the ER-localized STING translocates to the Golgi, where it associates with the kinase TBK1 and mediates the induction of inflammatory target genes, depending on IRF3 and NF-κB transcription factors. We observe an activation of downstream STING effectors upon PRDX4 loss, and co-depletion with NFκB significantly rescues the upregulation of those genes. As such, we propose that PRDX4 links ER-specific redox vulnerability to innate immunity signaling in PDAC. Since PRDX4 loss causes increased DNA damage and inflammatory signaling, our preliminary data suggest that PRDX4 could be a novel therapeutic target that may act synergistically with immune checkpoint inhibitors. Citation Format: Lucie Malbeteau, Pallavi Jain, Shane Harding, Bradley Wouters, Marianne Koritzinsky. PRDX4 links ER-specific redox vulnerability to innate immunity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 124.

Gene Set Enrichment Analysis Reveals That Fucoidan Induces Type I IFN Pathways in BMDC.

Fucoidan, a sulfated polysaccharide extracted from brown seaweed, has been proposed to effectively treat and prevent various viral infections. However, the mechanisms behind its antiviral activity are not completely understood. We investigate here the global transcriptional changes in bone marrow-derived dendritic cells (BMDCs) using RNA-Seq technology. Through both analysis of differentially expressed genes (DEG) and gene set enrichment analysis (GSEA), we found that fucoidan-treated BMDCs were enriched in virus-specific response pathways, including that of SARS-CoV-2, as well as pathways associated with nucleic acid-sensing receptors (RLR, TLR, NLR, STING), and type I interferon (IFN) production. We show that these transcriptome changes are driven by well-known regulators of the inflammatory response against viruses, including IRF, NF-κB, and STAT family transcription factors. Furthermore, 435 of the 950 upregulated DEGs are classified as type I IFN-stimulated genes (ISGs). Flow cytometric analysis additionally showed that fucoidan increased MHCII, CD80, and CD40 surface markers in BMDCs, indicative of greater antigen presentation and co-stimulation functionality. Our current study suggests that fucoidan transcriptionally activates PRR signaling, type I IFN production and signaling, ISGs production, and DC maturation, highlighting a potential mechanism of fucoidan-induced antiviral activity.

Correlation of the transcription factors IRF4 and BACH2 with the abnormal NFATC1 expression in T cells from chronic myeloid leukemia patients

ABSTRACT Objective T cell dysfunction is a common characteristic of patients with myeloid leukemia and is closely related to clinical efficacy and prognosis. In order to clarify the mechanisms leading to the T cell dysfunction, we characterized the gene expression profile of T cells from chronic myelogenous leukemia (CML) patients by microarray analysis and investigated the related regulating pathway. Methods We employed gene expression profiling, bioinformatics and real-time quantitative reverse transcription PCR (RT-qPCR) to detect genes differentially expressed in CML patients versus healthy donors. Results There were 1704 genes differentially expressed between CD3+ T cells from CML patients and healthy donors, including 868 up-regulated genes and 836 down-regulated genes, which mostly related to T cell functional pathways. In particular, lower expression of NFATC1, a member of the TCR signaling pathway, was detected in CD3+ T cells from CML patients. We further found that the expression of IRF4 and BACH2, transcription factors that potentially regulate NFATC1, in CD3+ T cells from CML patients was significantly lower than that in healthy donors. Conclusion We for the first time observed the altered gene expression profiles of CD3+ T cells from CML patients, and the results suggested that IRF4, BACH2 and NFATC1 may be involved in regulating T cell dysfunction in CML patients in the form of a transcriptional regulatory network. These findings may provide potential targets for tyrosine kinase inhibitors in combination with other targeted immunotherapies .

Elucidation of the inhibitory effect of (+)-hopeaphenol on polyinosinic–polycytidylic acid-induced innate immunity activation in human cerebral microvascular endothelial cells

Drug development for regulating the innate immune system is important for the prevention and treatment of autoinflammatory and autoimmune diseases. In this context, we investigated the effect of resveratrol derivatives on the inflammatory reactions in the brain. Resveratrol, which can be found in Vitis plants in the form of oligomers, exhibits neuroprotective effects; however, its regulatory effects on innate immunity are still unclear. We examined the effects of (+)-hopeaphenol, a resveratrol tetramer, and its derivatives on the polyinosinic–polycytidylic acid (poly IC)-induced production of interferon (IFN)-β and C-X-C motif chemokine 10 (CXCL10) in the cultured human cerebral microvascular endothelial cell line hCMEC/D3. (+)-Hopeaphenol (1–10 μM) inhibited the poly IC-induced production of not only CXCL10 but also retinoic acid-inducible gene-I in a dose-dependent manner and significantly reduced the poly IC-induced IFN-β gene expression and protein release from hCMEC/D3 cells by inhibiting the phosphorylation of p65 but not that of the interferon regulatory transcription factor IRF3. A docking study indicated a high affinity of (+)-hopeaphenol for p65. These results suggest that (+)-hopeaphenol can regulate the innate immune system by inhibiting the poly IC/IFN-β/CXCL10 signaling axis via suppression of the phosphorylation of the transcription factor NF-ĸB.

Blimp1 controls GC B cell expansion and exit through regulating cell cycle progression and key transcription factors BCL6 and IRF4

Abstract Blimp1 is required for plasma cell differentiation and antibody production. Blimp1 is also expressed in some dark zone germinal center (GC) B cells (GCBC), however, the exact role of Blimp1 in regulating GC B cell function is not clear. To investigate this, we generated chimeric mice using equal mixtures of bone marrow (BM) from Blimp1+/+ CD19Cre and Blimp1fl/fl CD19Cre mice. In these BM chimeras, Blimp1 sufficient and deficient B cells compete in the same environment, allowing us to examine the cell-intrinsic function of Blimp1 in GCBC, independent of environmental factors such as antibody or antigen levels. We discovered striking B cell-intrinsic roles for Blimp1. Blimp1 deficient B cells quickly dominate GCs and persist for a much longer time compared to the wild type cells. Mechanistically, Blimp1 deficiency increases the percentage of GCBC in the S phase. This difference seems independent of positive selection signals, because Blimp1 deficient GCBC express normal levels of c-Myc, a marker of positive selection. Importantly, Blimp1 deficient GCBC fail to downregulate BCL6 and upregulate IRF4, suggesting that Blimp1 induction in GCBC controls the expression of these key transcription factors, which are required for exit from the GC program. Consistent with this, memory B cells derived from the Blimp1 deficient B cells were only slightly increased, despite the fact that Blimp1 deficient GCBC are highly expanded and persistent. In addition, Blimp1 deficiency also reduced class-switching to IgG1 in response to NP-CGG immunization. These findings revealed unique antibody- and antigen-independent functions of Blimp1 in regulating GCBC responses that impact long-lived immune compartments.

STING is an intrinsic checkpoint inhibitor that restrains the TH17 cell pathogenic program.

SUMMARYExternal and intrinsic factors regulate the transcriptional profile of T helper 17 (TH17) cells, thereby affecting their pathogenic potential and revealing their context-dependent plasticity. The stimulator of interferon genes (STING), a component of the intracellular DNA-sensing pathway, triggers immune responses but remains largely unexplored in T cells. Here, we describe an intrinsic role of STING in limiting the TH17 cell pathogenic program. We demonstrate that non-pathogenic TH17 cells express higher levels of STING than those activated under pathogenic conditions. Activation of STING induces interleukin-10 (IL-10) production in TH17 cells, decreasing IL-17A and IL-23R expression in a type I interferon (IFN)-independent manner. Mechanistically, STING-induced IL-10 production partially requires aryl hydrocarbon receptor (AhR) signaling, while the decrease of IL-17A expression occurs due to a reduction of Rorγt transcriptional activity. Our findings reveal a regulatory function of STING in the TH17 cell activation program, proposing it as a valuable target to limit TH17-cell-mediated inflammation.

Tandem Mass Tag-based proteomics analysis reveals the vital role of inflammation in traumatic brain injury in a mouse model

Proteomics is a powerful tool that can be used to elucidate the underlying mechanisms of diseases and identify new biomarkers. Therefore, it may also be helpful for understanding the detailed pathological mechanism of traumatic brain injury (TBI). In this study, we performed Tandem Mass Tag-based quantitative analysis of cortical proteome profiles in a mouse model of TBI. Our results showed that there were 302 differentially expressed proteins in TBI mice compared with normal mice 7 days after injury. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that these differentially expressed proteins were predominantly involved in inflammatory responses, including complement and coagulation cascades, as well as chemokine signaling pathways. Subsequent transcription factor analysis revealed that the inflammation-related transcription factors NF-κB1, RelA, IRF1, STAT1, and Spi1 play pivotal roles in the secondary injury that occurs after TBI, which further corroborates the functional enrichment for inflammatory factors. Our results suggest that inflammation-related proteins and inflammatory responses are promising targets for the treatment of TBI.

Flt3L, LIF, and IL-10 combination promotes the selective in vitro development of ESAMlow cDC2B from murine bone marrow.

The development of two conventional dendritic cells (DC) subsets (cDC1 and cDC2) and the plasmacytoid DC (pDC) in vivo and in cultures of bone marrow (BM) cells is mediated by the growth factor Flt3L. However, little is known about the factors that direct the development of the individual DC subsets. Here, we describe the selective in vitro generation of murine ESAMlow CD103- XCR1- CD172a+ CD11b+ cDC2 from BM by treatment with a combination of Flt3L, LIF, and IL-10 (collectively named as FL10). FL10 promotes common dendritic cell progenitors (CDP) proliferation in the cultures, similar to Flt3L and CDP sorted and cultured in FL10 generate exclusively cDC2. These cDC2 express the transcription factors Irf4, Klf4, and Notch2, and their growth is reduced using BM from Irf4-/- mice, but the expression of Batf3 and Tcf4 is low. Functionally they respond to TLR3, TLR4, and TLR9 signals by upregulation of the surface maturation markers MHC II, CD80, CD86, and CD40, while they poorly secrete proinflammatory cytokines. Peptide presentation to TCR transgenic OT-II cells induced proliferation and IFN-γ production that was similar to GM-CSF-generated BM-DC and higher than Flt3L-generated DC. Together, our data support that FL10 culture of BM cells selectively promotes CDP-derived ESAMlow cDC2 (cDC2B) development and survival in vitro.