Molecular mechanisms involved in alcohol craving, IRF3, and endoplasmic reticulum stress: a multi-omics study.

Alcohol use disorder (AUD) is the most prevalent substance use disorder worldwide. Acamprosate and naltrexone are anti-craving drugs used in AUD pharmacotherapy. However, molecular mechanisms underlying their anti-craving effect remain unclear. This study utilized a patient-derived induced pluripotent stem cell (iPSC)-based model system and anti-craving drugs that are used to treat AUD as "molecular probes" to identify possible mechanisms associated with alcohol craving. We examined the pathophysiology of craving and anti-craving drugs by performing functional genomics studies using iPSC-derived astrocytes and next-generation sequencing. Specifically, RNA sequencing performed using peripheral blood mononuclear cells from AUD patients with extreme values for alcohol craving intensity prior to treatment showed that inflammation-related pathways were highly associated with alcohol cravings. We then performed a genome-wide assessment of chromatin accessibility and gene expression profiles of induced iPSC-derived astrocytes in response to ethanol or anti-craving drugs. Those experiments identified drug-dependent epigenomic signatures, with IRF3 as the most significantly enriched motif in chromatin accessible regions. Furthermore, the activation of IRF3 was associated with ethanol-induced endoplasmic reticulum (ER) stress which could be attenuated by anti-craving drugs, suggesting that ER stress attenuation might be a target for anti-craving agents. In conclusion, we found that craving intensity was associated with alcohol consumption and treatment outcomes. Our functional genomic studies suggest possible relationships among craving, ER stress, IRF3 and the actions of anti-craving drugs.

The STING-IRF3 Signaling Pathway, Mediated by Endoplasmic Reticulum Stress, Contributes to Impaired Myocardial Autophagic Flux After Ischemia/Reperfusion.

ABSTRACT: This study aimed to determine whether endoplasmic reticulum (ER) stress is involved in impaired autophagy after myocardial ischemia/reperfusion (M-I/R) and elucidate the underlying mechanisms. The expression levels of stimulator of interferon gene (STING) and interferon regulatory transcription factor 3 (IRF3) phosphorylation increased in M-I/R heart tissues and hypoxia-treated/reoxygenation-treated H9c2 cells. The ER stress inhibitor 4-phenylbutyric acid (4-PBA) significantly suppressed the stimulation of STING-IRF3 transcription and alleviated cardiac dysfunction caused by M-I/R injury. In addition, 4-PBA reversed ischemia-induced/reperfusion-induced autophagic flux dysfunction, as demonstrated by a decrease in p 62 and LC3 levels. Similarly, the protective effect of STING deficiency on myocardial cell damage was achieved by the recovery of autophagic flux. Conversely, the protective effect of 4-PBA against hypoxia/reoxygenation injury in cardiomyocytes was offset by STING overexpression, wherein the activated STING-IRF3 pathway promoted the expression of Rubicon (a negatively-regulated autophagic molecule) by binding to the Rubicon promoter. Rubicon ablation effectively counteracts the adverse effects of STING overexpression in cardiomyocytes. The data showed that STING-IRF3 signaling of ER stress receptors is particularly important in the progression of physiological M-I/R caused by the inhibition of autophagic flow in vivo and in vitro.

Berberine inhibits low shear stress-induced vascular endothelial inflammation via decreasing phosphorylation of Akt and IRF3.

BACKGROUND: Low shear stress (LSS) is closely related to vascular endothelial inflammation and the development of atherosclerosis. Berberine (BBR), a natural compound isolated from Coptis chinensis, has been reported to exert anti-inflammatory and antiatherosclerotic effects. However, the role of berberine in low shear stress-induced endothelial inflammation remains unclear. METHODS: The role of berberine in low shear stress-induced vascular endothelial inflammation was investigated in human umbilical vein endothelial cells (HUVECs) using a plate flow chamber in vitro and in mice with an established LSS model by partial ligation of the carotid artery in vivo. RESULTS: First, in vitro experiments demonstrated that BBR significantly decreased the expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) and the phosphorylation of Akt in HUVECs induced by low shear stress. Moreover, BBR significantly inhibited the low shear stress-mediated phosphorylation of IRF3 and its translocation to the nucleus. Notably, Akt overexpression markedly reversed the inhibitory effects of BBR on LSS-induced IRF3 activation and ICAM-1 expression. Moreover, in vivo experiments showed that BBR markedly decreased intimal ICAM-1 and IRF3 in the LSS areas of partially ligated carotid arteries in mice; however, EC-specific Akt overexpression mediated by adeno-associated viruses abolished the anti-inflammatory effect of BBR. CONCLUSION: Taken together, our findings suggest that BBR treatment attenuates LSS-induced vascular endothelial inflammation by decreasing the activation of the Akt/IRF3 signalling pathway.

Microglial AIM2 alleviates antiviral-related neuro-inflammation in mouse models of Parkinson's disease.

Inflammasome involvement in Parkinson's disease (PD) has been intensively investigated. Absent in melanoma 2 (AIM2) is an essential inflammasome protein known to contribute to the development of several neurological diseases. However, a specific role for AIM2 in PD has not been reported. In this study, we investigated the effect of AIM2 in the N-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced PD model by use of various knockout and bone marrow chimeric mice. The mechanism of action for AIM2 in PD was assessed by RNA-sequencing and in vitro primary microglial transfection. Results were validated in the A30P transgenic mouse model of PD. In the MPTP mouse model, AIM2 activation was found to negatively regulate neuro-inflammation independent of the inflammasome. Microglial AIM2 deficiency exacerbated behavioral and pathological features of both MPTP-induced and transgenic PD mouse models. Mechanistically, AIM2 reduced cyclic GMP-AMP synthase (cGAS)-mediated antiviral-related inflammation by inhibition of AKT-interferon regulatory factor 3 (IRF3) phosphorylation. These results demonstrate microglial AIM2 to inhibit the antiviral-related neuro-inflammation associated with PD and provide for a foundation upon which to identify new therapeutic targets for treatment of the disease.

Therapeutic targeting of STING-TBK1-IRF3 signalling ameliorates chronic stress induced depression-like behaviours by modulating neuroinflammation and microglia phagocytosis.

Stress is well known to contribute to the development of both neurological and psychiatric diseases. In the central nervous system, a role for STING (stimulator of interferon genes) in modulating immunological responses has been widely suggested, and this protein possesses both neurotoxic and neuroprotective properties. However, the potential role of the STING signalling pathway and the underlying regulatory mechanism in chronic stress have not been well established. In this study, C57BL/6 mice were subjected to intermittent restraint stress for 14 days (6 h/day), and sucrose preference, elevated plus maze, and tail suspension tests were performed by mice subjected to chronic restraint stress (RST). Here, we showed that RST mice displayed depression-like behaviours, accompanied by increased levels of proinflammatory cytokines in the brain. We also observed remarkably decreased levels of the pathway components STING, p-TBK1 (phospho-TANK-binding kinase-1), and p-IRF3 (phospho-interferon regulatory factor-3) in the hippocampus and the prefrontal cortex of RST mice. Significant reductions in STING fluorescence intensity were also observed in the hippocampus and the prefrontal cortex of RST mice. Next, fluorescently labelled latex beads, flow cytometry, and CD68-positive cell counts were utilized to evaluate the phagocytic abilities of microglia in vivo and in vitro. Importantly, our results first indicated that activation of the STING pathway by administration of the STING agonist 2'3-cGAMP enhanced microglial phagocytosis and suppressed the release of the proinflammatory cytokines TNF-α, IL-6, and IL-1ß in the brains of RST mice, which further led to antidepressant effects. Based on the results of our study, the amelioration of stress-driven depression-like behaviours by activation of the STING pathway is associated with the suppression of neuroinflammation and enhanced phagocytosis.

Plasmacytoid Dendritic Cells Mediate Myocardial Ischemia/Reperfusion Injury by Secreting Type I Interferons.

Background We previously demonstrated that ischemically injured cardiomyocytes release cell-free DNA and HMGB1 (high mobility group box 1 protein) into circulation during reperfusion, activating proinflammatory responses and ultimately exacerbating reperfusion injury. We hypothesize that cell-free DNA and HMGB1 mediate myocardial ischemia-reperfusion injury by stimulating plasmacytoid dendritic cells (pDCs) to secrete type I interferon (IFN-I). Methods and Results C57BL/6 and interferon alpha receptor-1 knockout mice underwent 40 minutes of left coronary artery occlusion followed by 60 minutes of reperfusion (40'/60' IR) before infarct size was evaluated by 2,3,5-Triphenyltetrazolium chloride-Blue staining. Cardiac perfusate was acquired in ischemic hearts without reperfusion by antegrade perfusion of the isolated heart. Flow cytometry in pDC-depleted mice treated with multiple doses of plasmacytoid dendritic cell antigen-1 antibody via intraperitoneal injection demonstrated plasmacytoid dendritic cell antigen-1 antibody treatment had no effect on conventional splenic dendritic cells but significantly reduced splenic pDCs by 60%. pDC-depleted mice had significantly smaller infarct size and decreased plasma interferon-α and interferon-ß compared with control. Blockade of the type I interferon signaling pathway with cyclic GMP-AMP synthase inhibitor, stimulator of interferon genes antibody, or interferon regulatory factor 3 antibody upon reperfusion similarly significantly attenuated infarct size by 45%. Plasma levels of interferon-α and interferon-ß were significantly reduced in cyclic GMP-AMP synthase inhibitor-treated mice. Infarct size was significantly reduced by >30% in type I interferon receptor monoclonal antibody-treated mice and interferon alpha receptor-1 knockout mice. In splenocyte culture, 40'/0' cardiac perfusate treatment stimulated interferon-α and interferon-ß production; however, this effect disappeared in the presence of cyclic GMP-AMP synthase inhibitor. Conclusions Type I interferon production is stimulated following myocardial ischemia by cardiogenic cell-free DNA/HMGB1 in a pDC-dependent manner, and subsequently activates type I interferon receptors to exacerbate reperfusion injury. These results identify new potential therapeutic targets to attenuate myocardial ischemia-reperfusion injury.

IRF3 Inhibits Neutrophil Recruitment in Mice Infected with Pseudomonas aeruginosa.

Pseudomonas aeruginosa is the major cause of morbidity and mortality in patients with ventilator-associated pneumonia. Interferon regulatory factor 3 (IRF3) is a transcription factor that plays an important role in the immune response to viral infection via the IRF3/IFN-ß signaling pathway. Controversial data exist regarding the role of IRF3 in immune cell recruitment during bacterial infections. IRF3 has been shown to promote neutrophil recruitment and bacterial clearance in mice infected with P. aeruginosa by inducing the production of specific chemokines and cytokines. In contrast, our study showed that IRF3 knockout (KO) mice infected with P. aeruginosa exhibited greater survival rates, demonstrated enhanced bacterial clearance, and showed significantly increased neutrophil recruitment to the lungs, when compared with the wild-type (WT) mice. The peritoneal lavage fluid collected from IRF3 KO mice 4 h after intraperitoneal injection with P. aeruginosa or 3% thioglycolate contained a significantly increased number of neutrophils. Furthermore, neutrophils from the bone marrow (BM) of IRF3 KO mice showed greater adhesiveness to the extracellular matrix when compared with those of WT mice, post-P. aeruginosa infection. In addition, IRF3 induced the expression of target genes in WT neutrophils infected with P. aeruginosa. These findings indicate that IRF3 exacerbates P. aeruginosa-induced mortality in mice by inhibiting neutrophil adhesion and recruitment to the lungs. Together, these data indicate that the inhibition of IRF3 might provide a possible mechanism for controlling P. aeruginosa infections.

Anti-viral and anti-inflammatory mechanisms of the innate immune transcription factor interferon regulatory factor 3: relevance to human CNS diseases.

Interferon regulatory factor 3 (IRF3) is a transcription factor critical in the induction of antiviral immunity. IRF3 is activated following stimulation of cell membrane or cytosolic nucleic acid sensors and is essential in the induction of the IFNß gene. Most cells constitutively express IRF3 in vitro, but little is known about the regulation of expression of IRF3 in vivo. Immunohistochemical analysis of selected human and mouse tissues demonstrated that IRF3 expression is highly organ- and cell-type specific, showing high expression in certain epithelial cells. In the CNS, while ependymal cells are strongly positive, brain parenchyma has little detectable IRF3 immunoreactivity. The importance of IRF3 in antiviral immunity has been demonstrated by the requirement for IRF3 in suppressing viral replication, but also by the demonstration that virus degrades IRF3 protein in infected cells. Furthermore, HIV-infected microglia in human CNS show abnormal IRF3+ aggregates, indicative of aberrant protein processing in vivo. In addition to antiviral immunity, IRF3 also plays a critical role in the modulation of neuroinflammation. A combination of dominant-negative and over-expression strategies in vitro as well as transgenic expression of IRF3 in vivo demonstrated that IRF3 plays a major role in modulating glial cytokine expression, i.e., suppression of proinflammatory cytokines and promotion of anti-inflammatory or immunoregulatory cytokines. These observations together suggest that IRF3 is a crucial regulator of immune responses against pathogen- and damage-associated molecules. We review recent literature on the molecular pathways of IRF3 activation and function of IRF3 and discuss their implications for CNS diseases.

Differential regulation of human papillomavirus type 8 by interferon regulatory factors 3 and 7.

The genus ß human papillomavirus (HPV) type 8 is associated with nonmelanoma skin cancer in patients with epidermodysplasia verruciformis, and evidence for its protumorigenic potential in the general population increases. To date, strategies to suppress genus ß HPV infections are limited. Interferon regulatory factors IRF-3 and IRF-7 play key roles in the activation of the innate immune response to viral infections. In this study, we show for the first time that both IRF-3 and IRF-7 regulate transcription of a papillomavirus, but with opposing effects. IRF-7, expressed in the suprabasal layers of human epidermis, increased HPV8 late promoter activity via direct binding to viral DNA. UV-B light-induced activation of the HPV8 promoter involved IRF-7 as a downstream effector. In contrast, IRF-3, expressed in all layers of human epidermis, induced strong HPV8 suppression in primary keratinocytes. IRF-3-mediated suppression prevailed over IRF-7-induced HPV8 transcription. Unlike the E6 oncoprotein of the mucosal high-risk HPV16, the HPV8 E6 protein did not bind to IRF-3 and only weakly antagonized its activity. Strong antiviral activity was also observed, when keratinocytes were treated with potent IRF-3 activators, poly(I:C) or RNA bearing 5' phosphates. In conclusion, we show that IRF-3 activation induces a state of cell-autonomous immunity against HPV in primary human keratinocytes. Our study suggests that local application of IRF-3-activating compounds might constitute an attractive novel therapeutic strategy against HPV8-associated diseases, particularly in epidermodysplasia verruciformis patients.