Single-cell sequencing of PBMC characterizes the altered transcriptomic landscape of classical monocytes in BNT162b2-induced myocarditis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been the most dangerous threat to public health worldwide for the last few years, which led to the development of the novel mRNA vaccine (BNT162b2). However, BNT162b2 vaccination is known to be associated with myocarditis. Here, as an attempt to determine the pathogenesis of the disease and to develop biomarkers to determine whether subjects likely proceed to myocarditis after vaccination, we conducted a time series analysis of peripheral blood mononuclear cells of a patient with BNT162b2-induced myocarditis. Single-cell RNA sequence analysis identified monocytes as the cell clusters with the most dynamic changes. To identify distinct gene expression signatures, we compared monocytes of BNT162b2-induced myocarditis with monocytes under various conditions, including SARS-CoV-2 infection, BNT162b2 vaccination, and Kawasaki disease, a disease similar to myocarditis. Representative changes in the transcriptomic profile of classical monocytes include the upregulation of genes related to fatty acid metabolism and downregulation of transcription factor AP-1 activity. This study provides, for the first time, the importance of classical monocytes in the pathogenesis of myocarditis following BNT162b2 vaccination and presents the possibility that vaccination affects monocytes, further inducing their differentiation and infiltration into the heart.

Human immune and gut microbial parameters associated with inter-individual variations in COVID-19 mRNA vaccine-induced immunity.

COVID-19 mRNA vaccines induce protective adaptive immunity against SARS-CoV-2 in most individuals, but there is wide variation in levels of vaccine-induced antibody and T-cell responses. However, the mechanisms underlying this inter-individual variation remain unclear. Here, using a systems biology approach based on multi-omics analyses of human blood and stool samples, we identified several factors that are associated with COVID-19 vaccine-induced adaptive immune responses. BNT162b2-induced T cell response is positively associated with late monocyte responses and inversely associated with baseline mRNA expression of activation protein 1 (AP-1) transcription factors. Interestingly, the gut microbial fucose/rhamnose degradation pathway is positively correlated with mRNA expression of AP-1, as well as a gene encoding an enzyme producing prostaglandin E2 (PGE2), which promotes AP-1 expression, and inversely correlated with BNT162b2-induced T-cell responses. These results suggest that baseline AP-1 expression, which is affected by commensal microbial activity, is a negative correlate of BNT162b2-induced T-cell responses.

Expression of SARS-CoV-2 receptor angiotensin-converting enzyme 2 by activating protein-1 in human mast cells.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection activates mast cells and induces a cytokine storm, leading to severe Coronavirus disease in 2019 (COVID-19). SARS-CoV-2 employs angiotensin-converting enzyme 2 (ACE2) for cell entry. In the present study, the expression of ACE2 and its mechanism in activated mast cells were studied utilizing the human mast cell line, HMC-1 cells and it was elucidated whether dexamethasone used as a treatment for COVID-19 could regulate ACE2 expression. Here we documented for the first time that levels of ACE2 were increased by stimulation of phorbol 12-myristate 13-acetate and A23187 (PMACI) in HMC-1 cells. Increased levels of ACE2 were significantly diminished by treatment with Wortmannin, SP600125, SB203580, PD98059, or SR11302. The expression of ACE2 was most significantly reduced by the activating protein (AP)-1 inhibitor SR11302. PMACI stimulation enhanced the expression of the transcription factor AP-1 for ACE2. In addition, levels of transmembrane protease/serine subfamily member 2 (TMPRSS2) and tryptase were increased in PMACI-stimulated HMC-1 cells. However, dexamethasone significantly lowered levels of ACE2, TMPRSS2, and tryptase generated by PMACI. Treatment with dexamethasone also reduced activation of signaling molecules linked to ACE2 expression. According to these findings, levels of ACE2 were up-regulated through activation of AP-1 in mast cells, suggesting that suppressing ACE2 levels in mast cells would be a therapeutic approach to lessen the harm caused by COVID-19.

Porcine deltacoronavirus E protein induces interleukin-8 production via NF-κB and AP-1 activation.

Infection induces the production of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) and interleukin-6 (IL-6). Although they facilitate local antiviral immunity, their excessive release leads to life-threatening cytokine release syndrome, exemplified by the severe cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In the present study, we found that interleukin-8 (IL-8) was upregulated by PDCoV infection. We then demonstrated that PDCoV E protein induced IL-8 production and that the TM domain and the C-terminal domain of the E protein were important for IL-8 production. Subsequently, we showed here that deleting the AP-1 and NF-κB binding motif in porcine IL-8 promoter abrogated its activation, suggesting that IL-8 expression was dependent on AP-1 and NF-κB. Furthermore, PDCoV E induced IL-8 production, which was also dependent on the NF-κB pathway through activating nuclear factor p65 phosphorylation and NF-κB inhibitor alpha (IκBα) protein phosphorylation, as well as inducing the nuclear translocation of p65, eventually resulting in the promotion of IL-8 production. PDCoV E also activated c-fos and c-jun, both of which are members of the AP-1 family. These findings provide new insights into the molecular mechanisms of PDCoV-induced IL-8 production and help us further understand the pathogenesis of PDCoV infection.

Hesperetin from Root Extract of Clerodendrum petasites S. Moore Inhibits SARS-CoV-2 Spike Protein S1 Subunit-Induced NLRP3 Inflammasome in A549 Lung Cells via Modulation of the Akt/MAPK/AP-1 Pathway.

Inhibition of inflammatory responses from the spike glycoprotein of SARS-CoV-2 (Spike) by targeting NLRP3 inflammasome has recently been developed as an alternative form of supportive therapy besides the traditional anti-viral approaches. Clerodendrum petasites S. Moore (C. petasites) is a Thai traditional medicinal plant possessing antipyretic and anti-inflammatory activities. In this study, C. petasites ethanolic root extract (CpEE) underwent solvent-partitioned extraction to obtain the ethyl acetate fraction of C. petasites (CpEA). Subsequently, C. petasites extracts were determined for the flavonoid contents and anti-inflammatory properties against spike induction in the A549 lung cells. According to the HPLC results, CpEA significantly contained higher amounts of hesperidin and hesperetin flavonoids than CpEE (p < 0.05). A549 cells were then pre-treated with either C. petasites extracts or its active flavonoids and were primed with 100 ng/mL of spike S1 subunit (Spike S1) and determined for the anti-inflammatory properties. The results indicate that CpEA (compared with CpEE) and hesperetin (compared with hesperidin) exhibited greater anti-inflammatory properties upon Spike S1 induction through a significant reduction in IL-6, IL-1ß, and IL-18 cytokine releases in A549 cells culture supernatant (p < 0.05). Additionally, CpEA and hesperetin significantly inhibited the Spike S1-induced inflammatory gene expressions (NLRP3, IL-1ß, and IL-18, p < 0.05). Mechanistically, CpEA and hesperetin attenuated inflammasome machinery protein expressions (NLRP3, ASC, and Caspase-1), as well as inactivated the Akt/MAPK/AP-1 pathway. Overall, our findings could provide scientific-based evidence to support the use of C. petasites and hesperetin in the development of supportive therapies for the prevention of COVID-19-related chronic inflammation.

Modulation of viral replication, apoptosis and antiviral response by induction and mutual regulation of EGR and AP-1 family genes during coronavirus infection.

Coronaviruses have evolved a variety of strategies to exploit normal cellular processes and signalling pathways for their efficient reproduction in a generally hostile cellular environment. One immediate-early response gene (IEG) family, the AP-1 gene family, was previously shown to be activated by coronavirus infection. In this study, we report that another IEG family, the EGR family, is also activated in cells infected with four different coronaviruses in three genera, i.e. gammacoronavirus infectious bronchitis virus (IBV), alphacoronaviruses porcine epidemic diarrhoea virus (PEDV) and human coronavirus-229E (HCoV-229E), and betacoronavirus HCoV-OC43. Knockdown of EGR1 reduced the expression of cJUN and cFOS, and knockdown of cJUN and/or cFOS reduced the expression of EGR1, demonstrating that these two IEG families may be cross-activated and mutual regulated. Furthermore, ERK1/2 was identified as an upstream kinase, and JNK and p38 as inhibitors of EGR1 activation in coronavirus-infected cells. However, upregulation of EGR family genes, in particular EGR1, appears to play a differential role in regulating viral replication, apoptosis and antiviral response. EGR1 was shown to play a limited role in regulation of coronavirus replication, and an anti-apoptotic role in cells infected with IBV or PEDV, but not in cells infected with HCoV-229E. Upregulation of EGR1 may also play a differential role in the regulation of antiviral response against different coronaviruses. This study reveals a novel regulatory network shared by different coronaviruses in the immediate-early response of host cells to infection.

ORAI1 Limits SARS-CoV-2 Infection by Regulating Tonic Type I IFN Signaling.

ORAI1 and stromal interaction molecule 1 (STIM1) are the critical mediators of store-operated Ca2+ entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca2+ signaling, STIM1 is also involved in regulation of the type I IFN (IFN-I) response. To examine their potential role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated ORAI1 and STIM1 knockout human HEK293-angiotensin-converting enzyme 2 cells and checked their responses. STIM1 knockout cells showed strong resistance to SARS-CoV-2 infection as a result of enhanced IFN-I response. On the contrary, ORAI1 deletion induced high susceptibility to SARS-CoV-2 infection. Mechanistically, ORAI1 knockout cells showed reduced homeostatic cytoplasmic Ca2+ concentration and severe impairment in tonic IFN-I signaling. Transcriptome analysis showed downregulation of multiple antiviral signaling pathways in ORAI1 knockout cells, likely because of reduced expression of the Ca2+-dependent transcription factors of the AP-1 family and MEF2C Accordingly, modulation of homeostatic Ca2+ concentration by pretreatment with ORAI1 blocker or agonist could influence baseline IFNB expression and resistance to SARS-CoV-2 infection in a human lung epithelial cell line. Our results identify a novel role of ORAI1-mediated Ca2+ signaling in regulating the tonic IFN-I levels, which determine host resistance to SARS-CoV-2 infection.

The single-cell epigenomic and transcriptional landscape of immunity to influenza vaccination.

Emerging evidence indicates a fundamental role for the epigenome in immunity. Here, we mapped the epigenomic and transcriptional landscape of immunity to influenza vaccination in humans at the single-cell level. Vaccination against seasonal influenza induced persistently diminished H3K27ac in monocytes and myeloid dendritic cells (mDCs), which was associated with impaired cytokine responses to Toll-like receptor stimulation. Single-cell ATAC-seq analysis revealed an epigenomically distinct subcluster of monocytes with reduced chromatin accessibility at AP-1-targeted loci after vaccination. Similar effects were observed in response to vaccination with the AS03-adjuvanted H5N1 pandemic influenza vaccine. However, this vaccine also stimulated persistently increased chromatin accessibility at interferon response factor (IRF) loci in monocytes and mDCs. This was associated with elevated expression of antiviral genes and heightened resistance to the unrelated Zika and Dengue viruses. These results demonstrate that vaccination stimulates persistent epigenomic remodeling of the innate immune system and reveal AS03's potential as an epigenetic adjuvant.

TAK1/AP-1-Targeted Anti-Inflammatory Effects of Barringtonia augusta Methanol Extract.

Barringtonia augusta methanol extract (Ba-ME) is a folk medicine found in the wetlands of Thailand that acts through an anti-inflammatory mechanism that is not understood fully. Here, we examine how the methanol extract of Barringtonia augusta (B. augusta) can suppress the activator protein 1 (AP-1) signaling pathway and study the activities of Ba-ME in the lipopolysaccharide (LPS)-treated RAW264.7 macrophage cell line and an LPS-induced peritonitis mouse model. Non-toxic concentrations of Ba-ME downregulated the mRNA expression of cytokines, such as cyclooxygenase and chemokine ligand 12, in LPS-stimulated RAW264.7 cells. Transfection experiments with the AP-1-Luc construct, HEK293T cells, and luciferase assays were used to assess whether Ba-ME suppressed the AP-1 functional activation. A Western blot assay confirmed that C-Jun N-terminal kinase is a direct pharmacological target of Ba-ME action. The anti-inflammatory effect of Ba-ME, which functions by ß-activated kinase 1 (TAK1) inhibition, was confirmed by using an overexpression strategy and a cellular thermal shift assay. In vivo experiments in a mouse model of LPS-induced peritonitis showed the anti-inflammatory effect of Ba-ME on LPS-stimulated macrophages and acute inflammatory mouse models. We conclude that Ba-ME is a promising anti-inflammatory drug targeting TAK1 in the AP-1 pathway.

Induction of the Proinflammatory Chemokine Interleukin-8 Is Regulated by Integrated Stress Response and AP-1 Family Proteins Activated during Coronavirus Infection.

Infection induces the production of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) and IL-6. Although they facilitate local antiviral immunity, their excessive release leads to life-threatening cytokine release syndrome, exemplified by the severe cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this study, we investigated the roles of the integrated stress response (ISR) and activator protein-1 (AP-1) family proteins in regulating coronavirus-induced IL-8 and IL-6 upregulation. The mRNA expression of IL-8 and IL-6 was significantly induced in cells infected with infectious bronchitis virus (IBV), a gammacoronavirus, and porcine epidemic diarrhea virus, an alphacoronavirus. Overexpression of a constitutively active phosphomimetic mutant of eukaryotic translation initiation factor 2α (eIF2α), chemical inhibition of its dephosphorylation, or overexpression of its upstream double-stranded RNA-dependent protein kinase (PKR) significantly enhanced IL-8 mRNA expression in IBV-infected cells. Overexpression of the AP-1 protein cJUN or its upstream kinase also increased the IBV-induced IL-8 mRNA expression, which was synergistically enhanced by overexpression of cFOS. Taken together, this study demonstrated the important regulatory roles of ISR and AP-1 proteins in IL-8 production during coronavirus infection, highlighting the complex interactions between cellular stress pathways and the innate immune response.

Transcriptional Regulation of Thrombin-Induced Endothelial VEGF Induction and Proangiogenic Response.

Thrombin, the ligand of the protease-activated receptor 1 (PAR1), is a well-known stimulator of proangiogenic responses in vascular endothelial cells (ECs), which are mediated through the induction of vascular endothelial growth factor (VEGF). However, the transcriptional events underlying this thrombin-induced VEGF induction and angiogenic response are less well understood at present. As reported here, we conducted detailed promotor activation and signal transduction pathway studies in human microvascular ECs, to decipher the transcription factors and the intracellular signaling events underlying the thrombin and PAR-1-induced endothelial VEGF induction. We found that c-FOS is a key transcription factor controlling thrombin-induced EC VEGF synthesis and angiogenesis. Upon the binding and internalization of its G-protein-coupled PAR-1 receptor, thrombin triggers ERK1/2 signaling and activation of the nuclear AP-1/c-FOS transcription factor complex, which then leads to VEGF transcription, extracellular secretion, and concomitant proangiogenic responses of ECs. In conclusion, exposure of human microvascular ECs to thrombin triggers signaling through the PAR-1-ERK1/2-AP-1/c-FOS axis to control VEGF gene transcription and VEGF-induced angiogenesis. These observations offer a greater understanding of endothelial responses to thromboinflammation, which may help to interpret the results of clinical trials tackling the conditions associated with endothelial injury and thrombosis.

Mining of high throughput screening database reveals AP-1 and autophagy pathways as potential targets for COVID-19 therapeutics.

The recent global pandemic of the Coronavirus disease 2019 (COVID-19) caused by the new coronavirus SARS-CoV-2 presents an urgent need for the development of new therapeutic candidates. Many efforts have been devoted to screening existing drug libraries with the hope to repurpose approved drugs as potential treatments for COVID-19. However, the antiviral mechanisms of action of the drugs found active in these phenotypic screens remain largely unknown. In an effort to deconvolute the viral targets in pursuit of more effective anti-COVID-19 drug development, we mined our in-house database of approved drug screens against 994 assays and compared their activity profiles with the drug activity profile in a cytopathic effect (CPE) assay of SARS-CoV-2. We found that the autophagy and AP-1 signaling pathway activity profiles are significantly correlated with the anti-SARS-CoV-2 activity profile. In addition, a class of neurology/psychiatry drugs was found to be significantly enriched with anti-SARS-CoV-2 activity. Taken together, these results provide new insights into SARS-CoV-2 infection and potential targets for COVID-19 therapeutics, which can be further validated by in vivo animal studies and human clinical trials.

Longitudinal transcriptome analyses show robust T cell immunity during recovery from COVID-19.

Understanding the processes of immune regulation in patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for improving treatment. Here, we performed longitudinal whole-transcriptome RNA sequencing on peripheral blood mononuclear cell (PBMC) samples from 18 patients with coronavirus disease 2019 (COVID-19) during their treatment, convalescence, and rehabilitation. After analyzing the regulatory networks of differentially expressed messenger RNAs (mRNAs), microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) between the different clinical stages, we found that humoral immunity and type I interferon response were significantly downregulated, while robust T-cell activation and differentiation at the whole transcriptome level constituted the main events that occurred during recovery from COVID-19. The formation of this T cell immune response might be driven by the activation of activating protein-1 (AP-1) related signaling pathway and was weakly affected by other clinical features. These findings uncovered the dynamic pattern of immune responses and indicated the key role of T cell immunity in the creation of immune protection against this disease.

Absence of Mal/TIRAP Results in Abrogated Imidazoquinolinones-Dependent Activation of IRF7 and Suppressed IFNß and IFN-I Activated Gene Production.

Activation of TLR7 by small imidazoquinoline molecules such as R848 or R837 initiates signaling cascades leading to the activation of transcription factors, such as AP-1, NF-κB, and interferon regulatory factors (IRFs) and afterward to the induction of cytokines and anti-viral Type I IFNs. In general, TLRs mediate these effects by utilizing different intracellular signaling molecules, one of them is Mal. Mal is a protein closely related to the antibacterial response, and its role in the TLR7 pathways remains poorly understood. In this study, we show that Mal determines the expression and secretion of IFNß following activation of TLR7, a receptor that recognizes ssRNA and imidazoquinolines. Moreover, we observed that R848 induces Mal-dependent IFNß production via ERK1/2 activation as well as the transcription factor IRF7 activation. Although activation of TLR7 leads to NF-κB-dependent expression of IRF7, this process is independent of Mal. We also demonstrate that secretion of IFNß regulated by TLR7 and Mal in macrophages and dendritic cells leads to the IP-10 chemokine expression. In conclusion, our data demonstrate that Mal is a critical regulator of the imidazoquinolinones-dependent IFNß production via ERK1/2/IRF7 signaling cascade which brings us closer to understanding the molecular mechanism's regulation of innate immune response.

Isodeoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber Linn., inhibits pro-inflammatory mediators' production through both NF-κB and AP-1 pathways in LPS-activated macrophages.

Isodeoxyelephantopin (IDET) has been identified as an anti-tumor natural constituent whose anti-tumor activity and mechanism have been widely investigated. Since the occurrence and development of cancer usually accompany with inflammation, and tumor signaling shares many components with inflammation signaling, the agents with anti-tumor activity are likely to possess anti-inflammation potential. Thus, the current study aims to demonstrate the anti-inflammatory activity along with the underlying mechanism of IDET in lipopolysaccharide (LPS)-primed macrophages. By using Griess method and ELISA, we found that in both bone marrow derived macrophages and alveolar macrophage cell line, IDET, at relatively low concentrations (0.75, 1.5 and 3 µM), could inhibit LPS-induced expression of various pro-inflammatory mediators including nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS), interleukin (IL)-6, monocyte chemotactic protein-1 (MCP-1) and IL-1ß. Meanwhile, in activated MH-S cells, the inhibitory action of IDET on mRNA expression levels of these cytokines was also detected using qPCR. Mechanistically, the effects of IDET on two key inflammatory signalings, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) pathways, were determined in LPS-activated MH-S cells by reporter gene along with western blot assays. On the one hand, IDET suppressed NF-κB signaling via down-regulating phosphorylation and degradation of inhibitor of NF-κB (IκB)-α and the subsequent p65 translocation. On the other hand, IDET dampened AP-1 signaling through attenuating phosphorylation of both c-jun N-terminal kinase 1/2 (JNK1/2) and extracellular signal regulated kinase 1/2 (ERK1/2). Our study indicates that IDET might be a promising constituent from the anti-inflammatory herb Elephantopus scaber Linn. in mitigating inflammatory conditions, especially respiratory inflammation.