Interferon Regulatory Factor 5 Research Articles

Early macrophage response to obesity encompasses Interferon Regulatory Factor 5 regulated mitochondrial architecture remodelling

Adipose tissue macrophages (ATM) adapt to changes in their energetic microenvironment. Caloric excess, in a range from transient to diet-induced obesity, could result in the transition of ATMs from highly oxidative and protective to highly inflammatory and metabolically deleterious. Here, we demonstrate that Interferon Regulatory Factor 5 (IRF5) is a key regulator of macrophage oxidative capacity in response to caloric excess. ATMs from mice with genetic-deficiency of Irf5 are characterised by increased oxidative respiration and mitochondrial membrane potential. Transient inhibition of IRF5 activity leads to a similar respiratory phenotype as genomic deletion, and is reversible by reconstitution of IRF5 expression. We find that the highly oxidative nature of Irf5-deficient macrophages results from transcriptional de-repression of the mitochondrial matrix component Growth Hormone Inducible Transmembrane Protein (GHITM) gene. The Irf5-deficiency-associated high oxygen consumption could be alleviated by experimental suppression of Ghitm expression. ATMs and monocytes from patients with obesity or with type-2 diabetes retain the reciprocal regulatory relationship between Irf5 and Ghitm. Thus, our study provides insights into the mechanism of how the inflammatory transcription factor IRF5 controls physiological adaptation to diet-induced obesity via regulating mitochondrial architecture in macrophages.

IRF8: Mechanism of Action and Health Implications.

Interferon regulatory factor 8 (IRF8) is a transcription factor of the IRF protein family. IRF8 was originally identified as an essentialfactor for myeloid cell lineage commitment and differentiation. Deletion of Irf8 leads to massive accumulation of CD11b+Gr1+ immature myeloid cells (IMCs), particularly the CD11b+Ly6Chi/+Ly6G− polymorphonuclear myeloid-derived suppressor cell-like cells (PMN-MDSCs). Under pathological conditions such as cancer, Irf8 is silenced by its promoter DNA hypermethylation, resulting in accumulation of PMN-MDSCs and CD11b+ Ly6G+Ly6Clo monocytic MDSCs (M-MDSCs) in mice. IRF8 is often silenced in MDSCs in human cancer patients. MDSCs are heterogeneous populations of immune suppressive cells that suppress T and NK cell activity to promote tumor immune evasion and produce growth factors to exert direct tumor-promoting activity. Emerging experimental data reveals that IRF8 is also expressed in non-hematopoietic cells. Epithelial cell-expressed IRF8 regulates apoptosis and represses Osteopontin (OPN). Human tumor cells may use the IRF8 promoter DNA methylation as a mechanism to repress IRF8 expression to advance cancer through acquiring apoptosis resistance and OPN up-regulation. Elevated OPN engages CD44 to suppress T cell activation and promote tumor cell stemness to advance cancer. IRF8 thus is a transcription factor that regulates both the immune and non-immune components in human health and diseases.

Transcription-independent regulation of STING activation and innate immune responses by IRF8 in monocytes

Sensing of cytosolic DNA of microbial or cellular/mitochondrial origin by cGAS initiates innate immune responses via the adaptor protein STING. It remains unresolved how the activity of STING is balanced between a productive innate immune response and induction of autoimmunity. Here we show that interferon regulatory factor 8 (IRF8) is essential for efficient activation of STING-mediated innate immune responses in monocytes. This function of IRF8 is independent of its transcriptional role in monocyte differentiation. In uninfected cells, IRF8 remains inactive via sequestration of its IRF-associated domain by its N- and C-terminal tails, which reduces its association with STING. Upon triggering the DNA sensing pathway, IRF8 is phosphorylated at Serine 151 to allow its association with STING via the IRF-associated domain. This is essential for STING polymerization and TBK1-mediated STING and IRF3 phosphorylation. Consistently, IRF8-deficiency impairs host defense against the DNA virus HSV-1, and blocks DNA damage-induced cellular senescence. Bone marrow-derived mononuclear cells which have an autoimmune phenotype due to deficiency of Trex1, respond to IRF-8 deletion with reduced pro-inflammatory cytokine production. Peripheral blood mononuclear cells from systemic lupus erythematosus patients are characterized by elevated phosphorylation of IRF8 at the same Serine residue we find to be important in STING activation, and in these cells STING is hyper-active. Taken together, the transcription-independent function of IRF8 we describe here appears to mediate STING activation and represents an important regulatory step in the cGAS/STING innate immune pathway in monocytes.

Regulation of microglial activation in stroke in aged mice: a translational study.

Numerous neurochemical changes occur with aging and stroke mainly affects the elderly. Our previous study has found interferon regulatory factor 5 (IRF5) and 4 (IRF4) regulate neuroinflammation in young stroke mice. However, whether the IRF5-IRF4 regulatory axis has the same effect in aged brains is not known. In this study, aged (18-20-month-old), microglial IRF5 or IRF4 conditional knockout (CKO) mice were subjected to a 60-min middle cerebral artery occlusion (MCAO). Stroke outcomes were quantified at 3d after MCAO. Flow cytometry and ELISA were performed to evaluate microglial activation and immune responses. We found aged microglia express higher levels of IRF5 and lower levels of IRF4 than young microglia after stroke. IRF5 CKO aged mice had improved stroke outcomes; whereas worse outcomes were seen in IRF4 CKO vs. their flox controls. IRF5 CKO aged microglia had significantly lower levels of IL-1β and CD68 than controls; whereas significantly higher levels of IL-1β and TNF-α were seen in IRF4 CKO vs. control microglia. Plasma levels of TNF-α and MIP-1α were decreased in IRF5 CKO vs. flox aged mice, and IL-1β/IL-6 levels were increased in IRF4 CKO vs. controls. The anti-inflammatory cytokines (IL-4/IL-10) levels were higher in IRF5 CKO, and lower in IRF4 CKO aged mice vs. their flox controls. IRF5 and IRF4 signaling drives microglial pro- and anti-inflammatory response respectively; microglial IRF5 is detrimental and IRF4 beneficial for aged mice in stroke. IRF5-IRF4 axis is a promising target for developing new, effective therapeutic strategies for the cerebral ischemia.

Who on IRF are you? IRF8 deficiency redirects cDC1 lineage commitment

Interferon regulatory factor 8 (IRF8) has long been associated with conventional dendritic cell type I (cDC1) development. In a recent study, Lança et al. demonstrate that IRF8 is also crucial in cells already committed to the cDC1 lineage. Here, deletion of IRF8 from the XCR1-expressing pre-cDC1 stage onward leads to a loss of commitment and reprogramming of the cells toward a cDC2-like phenotype.

Expression of NAC1 Restrains the Memory Formation of CD8+ T Cells during Viral Infection.

Nucleus accumbens-associated protein 1 (NAC1) is a transcription co-factor that has been shown to possess multiple roles in stem cell and cancer biology. However, little is known about its roles in regulation of the immune system. In the current study, we observed that expression of NAC1 impacted the survival of CD8+ T cells in vitro. NAC1−/− CD8+ T cells displayed lower metabolism, including reduced glycolysis and oxidative phosphorylation. In vivo, compared with wild-type (WT) mice, NAC1−/− mice produced a lower response to vaccinia virus (VACV) infection, and viral antigen (Ag)-specific CD8+ T cells decreased more slowly. Additionally, we observed that the NAC1−/− mice demonstrated a stronger memory formation of viral Ag-specific CD8+ T cells post-viral infection. Mechanically, we identified that compared with WT CD8+ T cells, the Interferon Regulatory Factor 4 (IRF4), a key transcription factor in T cell development, was highly expressed in NAC1−/− CD8+ T cells, insinuating that IRF4 could be a critical regulatory target of NAC1 in the memory formation of CD8+ T cells. Our results indicate that NAC1 restrains the memory formation of CD8+ T cells by modulating IRF4, and targeting NAC1 may be exploited as a new approach to boosting CD8+ T cell memory.

Postbiotics engage IRF4 in adipocytes to promote sex-dependent changes in blood glucose during obesity.

Postbiotics are microbial‐derived components or metabolites that can influence host immunity and metabolism. Some postbiotics can improve blood glucose control and lower inflammation during bacterial or nutritional stress. Bacterial cell wall‐derived muramyl dipeptide (MDP) is a potent insulin‐sensitizing postbiotic that engages NOD2, RIPK2, and requires interferon regulatory factor 4 (IRF4) to lower inflammation and improve blood glucose. However, the sex‐dependent effects of this postbiotic and the cell type required for IRF4 to cause inflammatory versus glycemic responses to MDP were unknown. Here, we measured how MDP injection altered glucose tolerance and adipose tissue inflammation during low‐level endotoxemia and high fat diet (HFD)‐induced obesity in male and female adipocyte‐specific IRF4 knockout mice (AdipoIRF4fl/fl) compared to WTfl/fl mice. Adipocyte IRF4 was required for the blood glucose‐lowering effects of MDP during endotoxemia and HFD‐induced obesity in male mice. However, MDP did not alter blood glucose in female WTfl/fl and AdipoIRF4fl/f mice during endotoxemia. Unexpectedly, female HFD‐fed AdipoIRF4fl/f mice had lower blood glucose after MDP treatment compared to WTfl/fl mice. MDP lowered inflammatory gene expression in adipose tissue of HFD‐fed WTfl/fl and AdipoIRF4fl/fl mice of both sexes. Therefore, MDP‐mediated lowering of adipose inflammation does not require adipocyte IRF4 and was independent of sex. Together, these data show that injection of MDP, an insulin‐sensitizing postbiotic, lowers adipose tissue inflammation in male and female mice, but lower adipose inflammation is not always associated with improved blood glucose. The blood glucose‐lowering effect of the postbiotic MDP and dependence on adipocyte IRF4 is sex‐dependent.

TARC and Septin 7 can be better monitoring biomarkers than CX3CL1, sICAM5, and IRF5 in children with seizure-free epilepsy with monotherapy and drug-resistant epilepsy

Aim: To evaluate i) the relationship between epilepsy and inflammation by analyzing the levels of thymus activation-regulated chemokine (TARC), and interferon regulatory factor 5 (IRF5) in healthy controls, patients with epilepsy on monotherapy and polytherapy, ii) the levels of sICAM5, chemokine (c-x3-c motif) ligand 1 (CX3CL1), and septin 7 (SEPT7) which are important in both inflammation and synaptic formation. Methods: Patients who were seizure-free with monotherapy (epilepsy group-1), patients with drug-resistant epilepsy (epilepsy group-2), and healthy controls were included. Demographical data, disease durations, and medications were noted. Measurements were made by commercial ELISA kits. Results: The numbers of epilepsy group-1, epilepsy group-2, and healthy controls were 23, 20, and 21, respectively. TARC levels were significantly lower in healthy controls than in both epilepsy groups. Higher TARC levels than 0.58 pg/ml indicated epilepsy with a sensitivity of 81.8% and specificity of 84.0%. SEPT7 levels were significantly higher in epilepsy group-1 than in those epilepsy group-2. A negative correlation was found between SEPT7 levels and disease duration as is the case for the correlation between SEPT7 and average seizure duration. A positive correlation was found between IRF5 and CX3CL1 levels, SEPT7 and IRF5 levels, and IRF5 and sICAM5 levels. Conclusions: We suggest that TARC is a promising biomarker, even in a heterogeneous epilepsy group not only for drug-resistance epilepsy but also for seizure-free epilepsy with monotherapy. Additionally, drug resistance, longer disease, and longer seizure durations are related to lower levels of SEPT7, which has an essential role in immunological functions and dendritic morphology.

IRF8 deficiency induces the transcriptional, functional, and epigenetic reprogramming of cDC1 into the cDC2 lineage

Conventional dendritic cells (cDCs) consist of two major functionally and phenotypically distinct subsets, cDC1 and cDC2, whose development is dependent on distinct sets of transcription factors. Interferon regulatory factor 8 (IRF8) is required at multiple stages of cDC1 development, but its role in committed cDC1 remains unclear. Here, we used Xcr1-cre to delete Irf8 in committed cDC1 and demonstrate that Irf8 is required for maintaining the identity of cDC1. In the absence of Irf8, committed cDC1 acquired the transcriptional, functional, and chromatin accessibility properties of cDC2. This conversion was independent of Irf4 and was associated with the decreased accessibility of putative IRF8, Batf3, and composite AP-1-IRF (AICE)-binding elements, together with increased accessibility of cDC2-associated transcription-factor-binding elements. Thus, IRF8 expression by committed cDC1 is required for preventing their conversion into cDC2-like cells.

Kinetic Changes in B7 Costimulatory Molecules and IRF4 Expression in Human Dendritic Cells during LPS Exposure.

A key aspect of the inflammatory phenomenon is the involvement of costimulatory molecules expressed by antigen-presenting cells (APCs) and their ability to secrete cytokines to set instructions for an adaptive immune response and to generate tolerance or inflammation. In a novel integrative approach, we aimed to evaluate the kinetic expression of the membrane and soluble B7 costimulatory molecules CD86, ICOS-L, PDL1, PDL2, the transcription factor Interferon Regulatory Factor 4 (IRF4), and the cytokines produced by monocyte-derived dendritic cells (Mo-DCs) after challenging them with different concentrations of stimulation with E. coli lipopolysaccharide (LPS) for different lengths of time. Our results showed that the stimuli concentration and time of exposure to an antigen are key factors in modulating the dynamic expression pattern of membrane and soluble B7 molecules and cytokines.

Elevated TNF-α Leads to Neural Circuit Instability in the Absence of Interferon Regulatory Factor 8.

Interferon regulatory factor 8 (IRF8) is a transcription factor necessary for the maturation of microglia, as well as other peripheral immune cells. It also regulates the transition of microglia and other immune cells to a pro-inflammatory phenotype. Irf8 is also a known risk gene for multiple sclerosis and lupus, and it has recently been shown to be downregulated in schizophrenia. While most studies have focused on IRF8-dependent regulation of immune cell function, little is known about how it impacts neural circuits. Here, we show by RNAseq from Irf8 -/- male and female mouse brains that several genes involved in regulation of neural activity are dysregulated. We then show that these molecular changes are reflected in heightened neural excitability and a profound increase in susceptibility to lethal seizures in male and female Irf8 -/- mice. Finally, we identify that TNF-α is elevated specifically in microglia in the CNS, and genetic or acute pharmacological blockade of TNF-α in the Irf8 -/- CNS rescued the seizure phenotype. These results provide important insights into the consequences of IRF8 signaling and TNF-α on neural circuits. Our data further suggest that neuronal function is impacted by loss of IRF8, a factor involved in neuropsychiatric and neurodegenerative diseases.SIGNIFICANCE STATEMENT Here, we identify a previously unknown and key role for interferon regulator factor 8 (IRF8) in regulating neural excitability and seizures. We further determine that these effects on neural circuits are through elevated TNF-α in the CNS. As IRF8 has most widely been studied in the context of regulating the development and inflammatory signaling in microglia and other immune cells, we have uncovered a novel function. Further, IRF8 is a risk gene for multiple sclerosis and lupus, IRF8 is dysregulated in schizophrenia, and elevated TNF-α has been identified in a multitude of neurologic conditions. Thus, elucidating these IRF8 and TNF-α-dependent effects on brain circuit function has profound implications for understanding underlying, therapeutically relevant mechanisms of disease.

Interferon Regulatory Factor 4 (IRF4) Promotes Lipopolysaccharide-Induced Colonic Mucosal Epithelial Cell Proliferation by Regulating Macrophage Polarization

Introduction: Ulcerative colitis (UC) is a chronic disease characterized by diffuse inflammation of the mucosa of colon and rectum. Interferon regulatory factor 4 (IRF4) mediates macrophage anti-inflammatory phenotype (alternatively activated macrophages [M2]). This study aimed to investigate the mechanism of IRF4 in lipopolysaccharide (LPS)-induced colonic mucosal epithelial cell proliferation via the regulation of macrophage polarization. Methods: Human bone marrow-derived macrophages were subjected to interleukin 4 (IL-4) induction. M2 macrophages were identified using flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). IRF4 expression in M2 macrophages was detected using Western blot and qRT-PCR. IRF4 expression was silenced in M2 macrophages. IL-10 mRNA expression and protein level were detected using qRT-PCR and Western blot. The binding relation between IRF4 and IL-10 was verified using dual-luciferase and chromatin immunoprecipitation assays. Macrophages under different treatments were cocultured with LPS-induced human colonic mucosal epithelial cells. The levels of inflammatory factors (TNF-α, IL-6, and IL-1β) were detected using enzyme-linked immunosorbent assay. The proliferation of inflammatory cells was measured using Cell Counting Kit-8 assay, and the healing of inflammatory cells was detected using wound healing assay. Results: M2 macrophages alleviated LPS-induced inflammatory responses. IRF4 bound to IL-10 and promoted IL-10 expression. Inhibition of IRF4 reduced IL-10 expression and attenuated the alleviating effect of M2 macrophages on inflammatory responses. Inhibition of IRF4 combined with IL-10 overexpression enhanced the promoting effect of M2 macrophages on inflammatory healing. Conclusion: IRF4 promoted colonic mucosal epithelial cell proliferation by increasing IL-10 expression and regulating macrophage polarization to M2 phenotype, which might be related to UC mucosal healing.

Preventive Intrathecal Injection of Bupivacaine Alleviated Microglia Activation and Neuropathic Pain in a Rat Model of Chronic Constriction Injury

Spinal microglia are crucial to neuronal hyper-excitability and pain hypersensitivity. The local anesthetic bupivacaine is commonly used for both peripheral and spinal anesthesia. The pain-relief effects resulting from the peripheral and systemic administration of bupivacaine have been previously reported. In this study, the preventive effects of intrathecal bupivacaine administration against neuropathic pain were revealed in a rat model of sciatic nerve chronic constriction injury (CCI). Using a CCI rat model, pain hypersensitivity, characterized by mechanical allodynia and thermal hyperalgesia, correlated well with microglia M1 polarization, activation and pro-inflammatory cytokine expression in both spinal cord dorsal horns and sciatic nerves. Bupivacaine attenuated pain behaviors and inflammatory alternations. We further identified that the Interferon Regulatory Factor 5 (IRF5)/P2X Purinoceptor 4 (P2X4R) and High Mobility Group Box 1 (HMGB1)/Toll-Like Receptor 4 (TLR4)/NF-κB inflammatory axes may each play pivotal roles in the acquisition of microglia M1 polarization and pro-inflammatory cytokine expression under CCI insult. The relief of pain paralleled with the suppression of microglia M1 polarization, elevation of microglia M2 polarization, and inhibition of IRF5/P2X4R and HMGB1/TLR4/NF-κB in both the spinal cord dorsal horns and sciatic nerve. Our findings provide molecular and biochemical evidence for the anti-neuropathic effect of preventive bupivacaine.

TIRAP/Mal Positively Regulates TLR8-Mediated Signaling via IRF5 in Human Cells

Toll-like receptor 8 (TLR8) recognizes single-stranded RNA of viral and bacterial origin as well as mediates the secretion of pro-inflammatory cytokines and type I interferons by human monocytes and macrophages. TLR8, as other endosomal TLRs, utilizes the MyD88 adaptor protein for initiation of signaling from endosomes. Here, we addressed the potential role of the Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) in the regulation of TLR8 signaling in human primary monocyte-derived macrophages (MDMs). To accomplish this, we performed TIRAP gene silencing, followed by the stimulation of cells with synthetic ligands or live bacteria. Cytokine-gene expression and secretion were analyzed by quantitative PCR or Bioplex assays, respectively, while nuclear translocation of transcription factors was addressed by immunofluorescence and imaging, as well as by cell fractionation and immunoblotting. Immunoprecipitation and Akt inhibitors were also used to dissect the signaling mechanisms. Overall, we show that TIRAP is recruited to the TLR8 Myddosome signaling complex, where TIRAP contributes to Akt-kinase activation and the nuclear translocation of interferon regulatory factor 5 (IRF5). Recruitment of TIRAP to the TLR8 signaling complex promotes the expression and secretion of the IRF5-dependent cytokines IFNβ and IL-12p70 as well as, to a lesser degree, TNF. These findings reveal a new and unconventional role of TIRAP in innate immune defense.

Autoimmune Cytopenias in Common Variable Immunodeficiency Are a Diagnostic and Therapeutic Conundrum: An Update.

Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency (PID). CVID is a heterogenous condition and clinical manifestations may vary from increased susceptibility to infections to autoimmune manifestations, granulomatous disease, polyclonal lymphoproliferation, and increased risk of malignancy. Autoimmune manifestations may, at times, be the first and only clinical presentation of CVID, resulting in diagnostic dilemma for the treating physician.Autoimmune cytopenias (autoimmune haemolytic anaemia and/or thrombocytopenia) are the most common autoimmune complications seen in patients with CVID. Laboratory investigations such as antinuclear antibodies, direct Coomb’s test and anti-platelet antibodies may not be useful in patients with CVID because of lack of specific antibody response. Moreover, presence of autoimmune cytopenias may pose a significant therapeutic challenge as use of immunosuppressive agents can be contentious in these circumstances. It has been suggested that serum immunoglobulins must be checked in all patients presenting with autoimmune cytopenia such as immune thrombocytopenia or autoimmune haemolytic anaemia.It has been observed that patients with CVID and autoimmune cytopenias have a different clinical and immunological profile as compared to patients with CVID who do not have an autoimmune footprint. Monogenic defects have been identified in 10-50% of all patients with CVID depending upon the population studied. Monogenic defects are more likely to be identified in patients with CVID with autoimmune complications. Common genetic defects that may lead to CVID with an autoimmune phenotype include nuclear factor kappa B subunit 1 (NF-kB1), Lipopolysaccharide (LPS)-responsive beige-like anchor protein (LRBA), cytotoxic T lymphocyte antigen 4 (CTLA4), Phosphoinositide 3-kinase (PI3K), inducible T-cell costimulatory (ICOS), IKAROS and interferon regulatory factor-2 binding protein 2 (IRF2BP2). In this review, we update on recent advances in pathophysiology and management of CVID with autoimmune cytopenias.

Obese Skeletal Muscle-Expressed Interferon Regulatory Factor 4 Transcriptionally Regulates Mitochondrial Branched-Chain Aminotransferase Reprogramming Metabolome.

In addition to the significant role in physical activity, skeletal muscle also contributes to health through the storage and use of macronutrients associated with energy homeostasis. However, the mechanisms of regulating integrated metabolism in skeletal muscle are not well-defined. Here, we compared the skeletal muscle transcriptome from obese and lean control subjects in different species (human and mouse) and found that interferon regulatory factor 4 (IRF4), an inflammation-immune transcription factor, conservatively increased in obese subjects. Thus, we investigated whether IRF4 gain of function in the skeletal muscle predisposed to obesity and insulin resistance. Conversely, mice with specific IRF4 loss in skeletal muscle showed protection against the metabolic effects of high-fat diet, increased branched-chain amino acids (BCAA) level of serum and muscle, and reprogrammed metabolome in serum. Mechanistically, IRF4 could transcriptionally upregulate mitochondrial branched-chain aminotransferase (BCATm) expression; subsequently, the enhanced BCATm could counteract the effects caused by IRF4 deletion. Furthermore, we demonstrated that IRF4 ablation in skeletal muscle enhanced mitochondrial activity, BCAA, and fatty acid oxidation in a BCATm-dependent manner. Taken together, these studies, for the first time, established IRF4 as a novel metabolic driver of macronutrients via BCATm in skeletal muscle in terms of diet-induced obesity.

Abstract 3164: Detection and CAR T-cell directed targeting of inflammation-responsive Wnt pathway receptors on multiple myeloma cancer stem-like cells

Abstract Multiple myeloma (MM) is the second most common hematological malignancy characterized by a malignant expansion of abnormal antibody-producing cells. Despite available treatments, relapsed disease is a common occurrence with patients receiving survival predictions of less than one year. Relapsed MM is often refractory to multiple lines of therapy and occurs due to the uncontrolled regeneration of a population of malignant cells displaying progenitor-cell-like phenotypes. Selectively targeting this cancer stem cell (CSC) population provides an effective way to limit incidence of relapsed disease, improving the prognosis for high-risk patients. In MM, the defining characteristics of CSCs remain poorly understood, though signaling pathways promoting ‘stemness’ that are often deregulated in CSCs, such as interleukin-6 (IL-6) signaling, as well as canonical and non-canonical Wnt signaling are also aberrantly activated in MM. In this context, the central objective of this project was to explore the functionality of two proteins which have been shown to be downstream targets of IL-6/JAK2/STAT3 signaling and potentiate Wnt signaling, the embryonic G-protein coupled receptor LGR4 and the fetal-oncogene tyrosine kinase like orphan receptor-1 (ROR1), as CSC biomarker candidates, and explore the feasibility of CSC targeting therapeutics for MM. Receptor expression was assessed by quantitative PCR and flow cytometry in primary plasma cell dyscrasia patient samples across the spectrum of disease, as well as in human myeloma cell lines (HMCLs). Whole transcriptome sequencing analyses were carried out in HMCLs induced to overexpress LGR4, which enriched genes associated with ion transport and embryonic development. Cell adhesion and extracellular matrix genes were downregulated, along with genes associated with metabolic and mitochondrial pathways. Results of ROR1 gene expression and flow cytometry studies suggest that a subset of MM patient samples harbor a low-frequency ROR1-positive cell population, and expression is enriched in an in vitro model of acquired drug resistance. Overexpression of interferon regulatory factor-4 (IRF4) - a myeloma cell-enriched transcription factor that we recently showed promotes myeloma regeneration - also contributed to increased human ROR1 protein levels. HMCLs with high endogenous ROR1 expression were sensitive to in vitro killing by anti-ROR1 CAR T-cell treatment, which employs an scFv generated from the fully humanized ROR1-specific monoclonal antibody cirmtuzumab, at effector:target cell ratios (ET) as low as 0.33:1. Together, these results suggest a central role for Wnt signaling in MM stem cell phenotypes and activation of embryonic transcriptional pathways, while also paving the way towards development of clinical therapies that target a CSC population in MM. Citation Format: Lara C. Avsharian, Christopher S. Oh, Ashni A. Vora, Jayde Fernandez, Caitlin Shin, Navyaa Sinha, Charles Prussak, Leslie Crews. Detection and CAR T-cell directed targeting of inflammation-responsive Wnt pathway receptors on multiple myeloma cancer stem-like cells [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 3164.

A RAC-alpha serine/threonine-protein kinase (CgAKT1) involved in the synthesis of CgIFNLP in oyster Crassostrea gigas

The RAC-alpha serine/threonine-protein kinase (AKT) is one of the most important protein kinases involved in many biological processes in eukaryotes. In the present study, a novel AKT homologue named CgAKT1 was identified from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgAKT1 cDNA was of 1482 bp encoding a peptide with 493 amino acid residues. There were classical domains in the predicted CgAKT1 protein, including an N-terminal pleckstrin homology domain, a central catalytic domain and a C-terminal hydrophobic domain. The mRNA transcripts of CgAKT1 were detected in all the examined tissues of C. gigas with higher level in gills (8.24-fold of that in mantle, p < 0.05) and haemocytes (3.62-fold of that in mantle, p < 0.05). After poly (I:C) stimulation, the mRNA expression of CgAKT1 decreased significantly in haemocytes from 3 h (0.44-fold of that in the control group, p < 0.001) to 24 h (0.20-fold of that in the control group, p < 0.001), and then increased significantly at 48 h (3.65-fold of that in the control group, p < 0.05). The expression level of CgAKT1 mRNA increased significantly at 6 h after rCgIFNLP stimulation, which was 3.60-fold of that in the control group (p < 0.001). The Alexa Fluor 488 positive signals of CgAKT1 protein were found to be distributed in the cytoplasm and cell membrane of haemocytes, while those in the cytoplasm became weaker after poly (I:C) stimulation. In CgAKT1-RNAi oysters, the mRNA expression of cyclic GMP-AMP synthase (CgcGAS) and TANK-binding kinase 1 (CgTBK1) did not change significantly, but the mRNA expression level of stimulator of interferon gene (CgSTING), interferon regulatory factor-1 (CgIRF-1), interferon regulatory factor-8 (CgIRF-8) and IFN-like protein (CgIFNLP) increased significantly, which was 1.40-fold, 1.53-fold, 1.72-fold and 1.99-fold of that in EGFP-RNAi oysters (p < 0.05), respectively. In CgIFNLP-RNAi oysters, the transcripts of CgAKT1 decreased significantly compared to those in EGFP-RNAi oysters (0.16-fold, p < 0.01). Moreover, the expression of p-CgTBK1, CgSTING and CgIFNLP at the protein level in the oysters treated with p-AKT1 activator (SC-79) was significantly suppressed after poly (I:C) stimulation. After the transfection of CgAKT1, the expression of p-cGAS protein in HEK293T cells increased significantly, while the cyclic GMP-AMP in the cells and the interferon (IFN-β) in the cell culture fluid decreased significantly compared with that in the control group. These results indicated that CgAKT1 might play a negative role in antiviral immunity of oyster by regulating the synthesis of CgIFNLP.

Dihydromyricetin ameliorates diet-induced obesity and promotes browning of white adipose tissue by upregulating IRF4/PGC-1α

BackgroundPromoting the browning of white adipose tissue (WAT) is a promising approach for the treatment of obesity and related comorbidities because it increases energy expenditure. In this study, we investigated whether Dihydromyricetin (DHM), a flavonoid component, could ameliorate diet-induced obesity through promoting the browning of WAT.MethodsMale C57BL/6 J mice were received a high-fat diet (HFD) to induce obesity and subsequently were treated with DHM (100 mg/kg/day) or vehicle for 4 weeks. The effects of DHM on weight reduction and metabolic phenotype improvement were observed in the mice. The expression of genes and protein involved in browning of WAT were assessed in inguinal WAT (iWAT) of the mice. Then, the effect of DHM on the inducing browning program was verified in adipocytes differentiated from stromal vascular fraction (SVF) cells of mouse iWAT. Finally, the mechanism by which DHM improves the browning of WAT was explored using RNA-seq and luciferase reporter assay.ResultsWe find that DHM reduces body weight, decreases WAT mass, improves glucose and lipid metabolic disorders, and ameliorates hepatic steatosis in diet-induced obese (DIO) mice. Further studies show that DHM induces WAT browning, which is manifested by increased expression of uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and enhanced mitochondrial activity in iWAT and primary adipocytes. In addition, we also find that DHM enhances interferon regulatory factor 4 (IRF4) expression, which is a key transcriptional regulator of PGC-1α.ConclusionOur findings identify that DHM prevents obesity by inducing the browning of WAT through the upregulation of IRF4/PGC-1α, which may have potential therapeutic implications for the treatment of obesity.

STOPTRAFFIC-1: A phase I/II trial of SX-682 in combination with nivolumab for refractory RAS-mutated microsatellite stable (MSS) metastatic colorectal cancer (mCRC).

TPS3638 Background: Refractory RAS mutated MSS mCRC represents a critical unmet need with minimal response to immune checkpoint blockade (ICB). Preclinical CRC models reveal that KRAS mutations activate the CXCR2 axis promoting an immunosuppressive tumor microenvironment (TME). This occurs via KRAS repression of interferon regulatory factor 2 (IRF2) resulting in upregulation of CXCL3 chemokines, which bind CXCR2 and recruit myeloid-derived suppressor cells (MDSC; Liao et al, Cancer Cell 2019; Grover et al, Cancer Discovery, 2022). SX-682 is a novel oral small-molecule inhibitor of the CXCR1/2 chemokine receptors involved in MDSC-recruitment to the TME. This proof-of-concept study investigates whether de novo immunotherapy resistance in MSS RAS mutated mCRC can be overcome by treatment with a small molecule CXCR1/2 antagonist in combination with anti-PD-1 therapy. Methods: STOPTRAFFIC-1 (NCT04599140, enrolling) is a phase I/II, open-label, dose-escalation/dose expansion study of SX-682 in combination with nivolumab to evaluate safety and clinical activity for patients (pts) with refractory RAS ( KRAS and NRAS) mutated MSS mCRC. Key eligibility criteria: patients (pts) with MSS mCRC with measurable disease, progression or intolerance to at least 2 prior lines of standard therapy, and ECOG 0 or 1. Pts will receive SX-682 (5 dose levels: 25 mg, 50 mg, 100mg, 200mg, or 400 mg by mouth twice daily) administered in an 8-week cycle with intravenous nivolumab (480 mg) on days 1 and 29 of each cycle. Adverse events are assessed according to CTCAE v5.0. Response assessments (per RECIST) occur every 8 weeks. In dose escalation, pts enter a 3-week monotherapy safety run-in of SX-682 followed by 3-week combination with nivolumab for a six-week dose-limiting toxicity (DLT) period. Cohorts 1-4 have been completed without DLT. Enrollment to cohort 5 began in January 2022. Following determination of maximum tolerated dose (MTD), dose expansion design is a Simon's optimal two-stage design. Efficacy will be assessed in the first 15 pts with a requirement of at least 2 responses in order to enroll 14 additional pts (N=29) in the second stage. Pre- and on-treatment tissue biopsies will be collected in the expansion phase. The primary objectives are to determine the safety profile of SX-682 alone and in combination with nivolumab, including the MTD, recommended phase 2 dose, and the DLT. The secondary objectives include overall response rate, progression-free survival, overall survival, and pharmacokinetic profiles of SX-682. Translational analyses include correlations of clinical outcomes with genomic and immune biomarkers from paired tissue and plasma samples. Clinical trial information: NCT04599140.