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1.
Front Immunol ; 12: 791753, 2021.
Article in English | MEDLINE | ID: covidwho-1639168

ABSTRACT

Background: Infection of SARS-CoV-2 may cause acute respiratory syndrome. It has been reported that SARS-CoV-2 nucleocapsid protein (N-protein) presents early in body fluids during infection. The direct involvement of N-protein in lung injury is poorly understood. Methods: Recombinant N-protein was pretreated with polymyxin B, a lipopolysaccharide (LPS)-neutralizing agent. C57BL/6, C3H/HeJ (resistant to LPS), and C3H/HeN (control for C3H/HeJ) mice were exposed to N-protein via intratracheal administration to examine acute lung injury. In vitro, bone marrow-derived macrophages (BMDMs) were cultured with N-protein to study phosphorylation of nuclear factor kappa B (NF-ĸB) p65, macrophage polarization, and expression of proinflammatory cytokines. Results: N-protein produced acute lung injury in C57BL/6 mice, with elevated protein permeability, total cell count, neutrophil infiltration, and proinflammatory cytokines in the bronchioalveolar lavage. N-protein also induced lung injury in both C3H/HeJ and C3H/HeN mice, indicating that the effect could not be attributed to the LPS contamination. N-protein triggered phosphorylation of NF-ĸB p65 in vitro, which was abolished by both N-protein denaturation and treatment with an antibody for N-protein, demonstrating that the effect is N-protein specific. In addition, N-protein promoted M1 macrophage polarization and the expression of proinflammatory cytokines, which was also blocked by N-protein denaturation and antibody for N-protein. Furthermore, N-protein induced NF-ĸB p65 phosphorylation in the lung, while pyrrolidine dithiocarbamate, an NF-ĸB inhibitor, alleviated the effect of N-protein on acute lung injury. Conclusions: SARS-CoV-2 N-protein itself is toxic and induces acute lung injury in mice. Both N-protein and NF-ĸB pathway may be therapeutic targets for treating multi-organ injuries in Coronavirus disease 2019 (COVID-19).


Subject(s)
Acute Lung Injury/virology , COVID-19 , Coronavirus Nucleocapsid Proteins/toxicity , NF-kappa B/metabolism , Acute Lung Injury/metabolism , Animals , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Phosphoproteins/toxicity , SARS-CoV-2
2.
Sci Rep ; 12(1): 696, 2022 01 13.
Article in English | MEDLINE | ID: covidwho-1621270

ABSTRACT

Despite encouraging preclinical data, therapies to reduce ARDS mortality remains a globally unmet need, including during the COVID-19 pandemic. We previously identified extracellular nicotinamide phosphoribosyltransferase (eNAMPT) as a novel damage-associated molecular pattern protein (DAMP) via TLR4 ligation which regulates inflammatory cascade activation. eNAMPT is tightly linked to human ARDS by biomarker and genotyping studies in ARDS subjects. We now hypothesize that an eNAMPT-neutralizing mAb will significantly reduce the severity of ARDS lung inflammatory lung injury in diverse preclinical rat and porcine models. Sprague Dawley rats received eNAMPT mAb intravenously following exposure to intratracheal lipopolysaccharide (LPS) or to a traumatic blast (125 kPa) but prior to initiation of ventilator-induced lung injury (VILI) (4 h). Yucatan minipigs received intravenous eNAMPT mAb 2 h after initiation of septic shock and VILI (12 h). Each rat/porcine ARDS/VILI model was strongly associated with evidence of severe inflammatory lung injury with NFkB pathway activation and marked dysregulation of the Akt/mTORC2 signaling pathway. eNAMPT neutralization dramatically reduced inflammatory indices and the severity of lung injury in each rat/porcine ARDS/VILI model (~ 50% reduction) including reduction in serum lactate, and plasma levels of eNAMPT, IL-6, TNFα and Ang-2. The eNAMPT mAb further rectified NFkB pathway activation and preserved the Akt/mTORC2 signaling pathway. These results strongly support targeting the eNAMPT/TLR4 inflammatory pathway as a potential ARDS strategy to reduce inflammatory lung injury and ARDS mortality.


Subject(s)
Acute Chest Syndrome/metabolism , Mechanistic Target of Rapamycin Complex 2/metabolism , NF-kappa B/metabolism , Nicotinamide Phosphoribosyltransferase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/physiology , Animals , Antibodies, Neutralizing/metabolism , Biomarkers/metabolism , COVID-19/metabolism , Disease Models, Animal , Inflammation/metabolism , Lipopolysaccharides/metabolism , Lung/metabolism , Male , Rats , Rats, Sprague-Dawley , SARS-CoV-2/pathogenicity , Swine
3.
Signal Transduct Target Ther ; 6(1): 167, 2021 04 24.
Article in English | MEDLINE | ID: covidwho-1585891

ABSTRACT

The ongoing 2019 novel coronavirus disease (COVID-19) caused by SARS-CoV-2 has posed a worldwide pandemic and a major global public health threat. The severity and mortality of COVID-19 are associated with virus-induced dysfunctional inflammatory responses and cytokine storms. However, the interplay between host inflammatory responses and SARS-CoV-2 infection remains largely unknown. Here, we demonstrate that SARS-CoV-2 nucleocapsid (N) protein, the major structural protein of the virion, promotes the virus-triggered activation of NF-κB signaling. After binding to viral RNA, N protein robustly undergoes liquid-liquid phase separation (LLPS), which recruits TAK1 and IKK complex, the key kinases of NF-κB signaling, to enhance NF-κB activation. Moreover, 1,6-hexanediol, the inhibitor of LLPS, can attenuate the phase separation of N protein and restrict its regulatory functions in NF-κB activation. These results suggest that LLPS of N protein provides a platform to induce NF-κB hyper-activation, which could be a potential therapeutic target against COVID-19 severe pneumonia.


Subject(s)
COVID-19/metabolism , Coronavirus Nucleocapsid Proteins/metabolism , NF-kappa B/metabolism , RNA, Viral/metabolism , SARS-CoV-2/metabolism , Signal Transduction , A549 Cells , Acrylates/pharmacology , Animals , COVID-19/drug therapy , COVID-19/pathology , Chlorocebus aethiops , HEK293 Cells , HeLa Cells , Humans , Inflammation/drug therapy , Inflammation/metabolism , Inflammation/pathology , Phosphoproteins/metabolism , Vero Cells
4.
Sci Rep ; 11(1): 13464, 2021 06 29.
Article in English | MEDLINE | ID: covidwho-1500743

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus disease 2019 (COVID-19) that emerged in human populations recently. Severely ill COVID-19 patients exhibit the elevation of proinflammatory cytokines, and such an unbalanced production of proinflammatory cytokines is linked to acute respiratory distress syndrome with high mortality in COVID-19 patients. Our study provides evidence that the ORF3a, M, ORF7a, and N proteins of SARS-CoV-2 were NF-κB activators. The viral sequence from infected zoo lions belonged to clade V, and a single mutation of G251V is found for ORF3a gene compared to all other clades. No significant functional difference was found for clade V ORF3a, indicating the NF-κB activation is conserved among COVID-19 variants. Of the four viral proteins, the ORF7a protein induced the NF-κB dictated proinflammatory cytokines including IL-1α, IL-1ß, IL-6, IL-8, IL-10, TNF-α, and IFNß. The ORF7a protein also induced IL-3, IL-4, IL-7, IL-23. Of 15 different chemokines examined in the study, CCL11, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL26, CCL27, and CXCL9 were significantly upregulated by ORF7. These cytokines and chemokines were frequently elevated in severely ill COVID-19 patients. Our data provide an insight into how SARS-CoV-2 modulates NF-κB signaling and inflammatory cytokine expressions. The ORF7a protein may be a desirable target for strategic developments to minimize uncontrolled inflammation in COVID-19 patients.


Subject(s)
Cytokines/metabolism , NF-kappa B/metabolism , SARS-CoV-2/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , COVID-19/pathology , COVID-19/virology , Chemokines/genetics , Chemokines/metabolism , Cytokines/genetics , HeLa Cells , Humans , Point Mutation , SARS-CoV-2/isolation & purification , Sequence Alignment , Severity of Illness Index , Up-Regulation , Viral Matrix Proteins/genetics , Viral Proteins/genetics , Viroporin Proteins/chemistry , Viroporin Proteins/genetics , Viroporin Proteins/metabolism
5.
Front Immunol ; 12: 727861, 2021.
Article in English | MEDLINE | ID: covidwho-1477822

ABSTRACT

Inflammatory response is a host-protective mechanism against tissue injury or infections, but also has the potential to cause extensive immunopathology and tissue damage, as seen in many diseases, such as cardiovascular diseases, neurodegenerative diseases, metabolic syndrome and many other infectious diseases with public health concerns, such as Coronavirus Disease 2019 (COVID-19), if failure to resolve in a timely manner. Recent studies have uncovered a superfamily of endogenous chemical molecules that tend to resolve inflammatory responses and re-establish homeostasis without causing excessive damage to healthy cells and tissues. Among these, the monocyte chemoattractant protein-induced protein (MCPIP) family consisting of four members (MCPIP-1, -2, -3, and -4) has emerged as a group of evolutionarily conserved molecules participating in the resolution of inflammation. The focus of this review highlights the biological functions of MCPIP-1 (also known as Regnase-1), the best-studied member of this family, in the resolution of inflammatory response. As outlined in this review, MCPIP-1 acts on specific signaling pathways, in particular NFκB, to blunt production of inflammatory mediators, while also acts as an endonuclease controlling the stability of mRNA and microRNA (miRNA), leading to the resolution of inflammation, clearance of virus and dead cells, and promotion of tissue regeneration via its pleiotropic effects. Evidence from transgenic and knock-out mouse models revealed an involvement of MCPIP-1 expression in immune functions and in the physiology of the cardiovascular system, indicating that MCPIP-1 is a key endogenous molecule that governs normal resolution of acute inflammation and infection. In this review, we also discuss the current evidence underlying the roles of other members of the MCPIP family in the regulation of inflammatory processes. Further understanding of the proteins from this family will provide new insights into the identification of novel targets for both host effectors and microbial factors and will lead to new therapeutic treatments for infections and other inflammatory diseases.


Subject(s)
Gene Expression Regulation/genetics , Inflammation Mediators/metabolism , Inflammation/immunology , Ribonucleases/immunology , SARS-CoV-2/immunology , Transcription Factors/immunology , Animals , Apoptosis/genetics , COVID-19/immunology , Humans , Inflammation/pathology , Mice , NF-kappa B/metabolism , RNA Processing, Post-Transcriptional/genetics , Transcriptional Activation/immunology , Ubiquitination
6.
Viruses ; 13(10)2021 10 15.
Article in English | MEDLINE | ID: covidwho-1470998

ABSTRACT

Nuclear transport and vesicle trafficking are key cellular functions involved in the pathogenesis of RNA viruses. Among other pleiotropic effects on virus-infected host cells, ivermectin (IVM) inhibits nuclear transport mechanisms mediated by importins and atorvastatin (ATV) affects actin cytoskeleton-dependent trafficking controlled by Rho GTPases signaling. In this work, we first analyzed the response to infection in nasopharyngeal swabs from SARS-CoV-2-positive and -negative patients by assessing the gene expression of the respective host cell drug targets importins and Rho GTPases. COVID-19 patients showed alterations in KPNA3, KPNA5, KPNA7, KPNB1, RHOA, and CDC42 expression compared with non-COVID-19 patients. An in vitro model of infection with Poly(I:C), a synthetic analog of viral double-stranded RNA, triggered NF-κB activation, an effect that was halted by IVM and ATV treatment. Importin and Rho GTPases gene expression was also impaired by these drugs. Furthermore, through confocal microscopy, we analyzed the effects of IVM and ATV on nuclear to cytoplasmic importin α distribution, alone or in combination. Results showed a significant inhibition of importin α nuclear accumulation under IVM and ATV treatments. These findings confirm transcriptional alterations in importins and Rho GTPases upon SARS-CoV-2 infection and point to IVM and ATV as valid drugs to impair nuclear localization of importin α when used at clinically-relevant concentrations.


Subject(s)
Active Transport, Cell Nucleus/drug effects , Atorvastatin/pharmacology , COVID-19/drug therapy , Ivermectin/pharmacology , SARS-CoV-2/drug effects , alpha Karyopherins/metabolism , A549 Cells , Actin Cytoskeleton/drug effects , Animals , Antiviral Agents/pharmacology , Cell Line, Tumor , Chlorocebus aethiops , Drug Repositioning , HeLa Cells , Humans , NF-kappa B/metabolism , Vero Cells , rho GTP-Binding Proteins/metabolism
7.
J Immunol ; 207(10): 2521-2533, 2021 11 15.
Article in English | MEDLINE | ID: covidwho-1468558

ABSTRACT

Many patients with coronavirus disease 2019 in intensive care units suffer from cytokine storm. Although anti-inflammatory therapies are available to treat the problem, very often, these treatments cause immunosuppression. Because angiotensin-converting enzyme 2 (ACE2) on host cells serves as the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), to delineate a SARS-CoV-2-specific anti-inflammatory molecule, we designed a hexapeptide corresponding to the spike S1-interacting domain of ACE2 receptor (SPIDAR) that inhibited the expression of proinflammatory molecules in human A549 lung cells induced by pseudotyped SARS-CoV-2, but not vesicular stomatitis virus. Accordingly, wild-type (wt), but not mutated (m), SPIDAR inhibited SARS-CoV-2 spike S1-induced activation of NF-κB and expression of IL-6 and IL-1ß in human lung cells. However, wtSPIDAR remained unable to reduce activation of NF-κB and expression of proinflammatory molecules in lungs cells induced by TNF-α, HIV-1 Tat, and viral dsRNA mimic polyinosinic-polycytidylic acid, indicating the specificity of the effect. The wtSPIDAR, but not mutated SPIDAR, also hindered the association between ACE2 and spike S1 of SARS-CoV-2 and inhibited the entry of pseudotyped SARS-CoV-2, but not vesicular stomatitis virus, into human ACE2-expressing human embryonic kidney 293 cells. Moreover, intranasal treatment with wtSPIDAR, but not mutated SPIDAR, inhibited lung activation of NF-κB, protected lungs, reduced fever, improved heart function, and enhanced locomotor activities in SARS-CoV-2 spike S1-intoxicated mice. Therefore, selective targeting of SARS-CoV-2 spike S1-to-ACE2 interaction by wtSPIDAR may be beneficial for coronavirus disease 2019.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Anti-Inflammatory Agents/therapeutic use , COVID-19/therapy , Lung/immunology , Peptides/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , A549 Cells , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/immunology , Cytokines/metabolism , Female , HEK293 Cells , Humans , Inflammation Mediators/metabolism , Locomotion , Male , Mice , Molecular Targeted Therapy , NF-kappa B/metabolism , Peptides/genetics , Peptides/therapeutic use , Signal Transduction , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
8.
Oxid Med Cell Longev ; 2021: 5513868, 2021.
Article in English | MEDLINE | ID: covidwho-1467753

ABSTRACT

COVID-19 is a widespread global pandemic with nearly 185 million confirmed cases and about four million deaths. It is caused by an infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which primarily affects the alveolar type II pneumocytes. The infection induces pathological responses including increased inflammation, oxidative stress, and apoptosis. This situation results in impaired gas exchange, hypoxia, and other sequelae that lead to multisystem organ failure and death. As summarized in this article, many interventions and therapeutics have been proposed and investigated to combat the viral infection-induced inflammation and oxidative stress that contributes to the etiology and pathogenesis of COVID-19. However, these methods have not significantly improved treatment outcomes. This may partly be attributable to their inability at restoring redox and inflammatory homeostasis, for which molecular hydrogen (H2), an emerging novel medical gas, may complement. Herein, we systematically review the antioxidative, anti-inflammatory, and antiapoptotic mechanisms of H2. Its small molecular size and nonpolarity allow H2 to rapidly diffuse through cell membranes and penetrate cellular organelles. H2 has been demonstrated to suppress NF-κB inflammatory signaling and induce the Nrf2/Keap1 antioxidant pathway, as well as to improve mitochondrial function and enhance cellular bioenergetics. Many preclinical and clinical studies have demonstrated the beneficial effects of H2 in varying diseases, including COVID-19. However, the exact mechanisms, primary modes of action, and its true clinical effects remain to be delineated and verified. Accordingly, additional mechanistic and clinical research into this novel medical gas to combat COVID-19 complications is warranted.


Subject(s)
COVID-19 , Hydrogen/therapeutic use , Oxidative Stress/drug effects , SARS-CoV-2/metabolism , Signal Transduction/drug effects , COVID-19/drug therapy , COVID-19/metabolism , Humans , Inflammation/drug therapy , Inflammation/metabolism , Kelch-Like ECH-Associated Protein 1/metabolism , NF-E2-Related Factor 2/metabolism , NF-kappa B/metabolism
9.
J Virol ; 95(23): e0139621, 2021 11 09.
Article in English | MEDLINE | ID: covidwho-1434896

ABSTRACT

Emerging evidence suggests that endothelial activation plays a central role in the pathogenesis of acute respiratory distress syndrome (ARDS) and multiorgan failure in patients with coronavirus disease 2019 (COVID-19). However, the molecular mechanisms underlying endothelial activation in COVID-19 patients remain unclear. In this study, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins that potently activate human endothelial cells were screened to elucidate the molecular mechanisms involved in endothelial activation. It was found that nucleocapsid protein (NP) of SARS-CoV-2 significantly activated human endothelial cells through Toll-like receptor 2 (TLR2)/NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Remarkably, though the protein sequences of N proteins from coronaviruses are highly conserved, only NP from SARS-CoV-2 induced endothelial activation. The NPs from other coronaviruses such as SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HUB1-CoV, and influenza virus H1N1 did not activate endothelial cells. These findings are consistent with the results from clinical investigations showing broad endotheliitis and organ injury in severe COVID-19 patients. In conclusion, the study provides insights on SARS-CoV-2-induced vasculopathy and coagulopathy and suggests that simvastatin, an FDA-approved lipid-lowering drug, may help prevent the pathogenesis and improve the outcome of COVID-19 patients. IMPORTANCE Coronavirus disease 2019 (COVID-19), caused by the betacoronavirus SARS-CoV-2, is a worldwide challenge for health care systems. The leading cause of mortality in patients with COVID-19 is hypoxic respiratory failure from acute respiratory distress syndrome (ARDS). To date, pulmonary endothelial cells (ECs) have been largely overlooked as a therapeutic target in COVID-19, yet emerging evidence suggests that these cells contribute to the initiation and propagation of ARDS by altering vessel barrier integrity, promoting a procoagulative state, inducing vascular inflammation and mediating inflammatory cell infiltration. Therefore, a better mechanistic understanding of the vasculature is of utmost importance. In this study, we screened the SARS-CoV-2 viral proteins that potently activate human endothelial cells and found that nucleocapsid protein (NP) significantly activated human endothelial cells through TLR2/NF-κB and MAPK signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Our results provide insights on SARS-CoV-2-induced vasculopathy and coagulopathy, and suggests that simvastatin, an FDA-approved lipid-lowering drug, may benefit to prevent the pathogenesis and improve the outcome of COVID-19 patients.


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/virology , SARS-CoV-2 , Signal Transduction , Simvastatin/pharmacology , COVID-19/virology , Cell Line , Human Umbilical Vein Endothelial Cells , Humans , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Toll-Like Receptor 2/metabolism
10.
Viruses ; 13(9)2021 09 21.
Article in English | MEDLINE | ID: covidwho-1430984

ABSTRACT

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has expanded into a global pandemic, with more than 220 million affected persons and almost 4.6 million deaths by 8 September 2021. In particular, Europe and the Americas have been heavily affected by high infection and death rates. In contrast, much lower infection rates and mortality have been reported generally in Africa, particularly in the sub-Saharan region (with the exception of the Southern Africa region). There are different hypotheses for this African paradox, including less testing, the young age of the population, genetic disposition, and behavioral and epidemiological factors. In the present review, we address different immunological factors and their correlation with genetic factors, pre-existing immune status, and differences in cytokine induction patterns. We also focus on epidemiological factors, such as specific medication coverage, helminth distribution, and malaria endemics in the sub-Saharan region. An analysis combining different factors is presented that highlights the central role of the NF-κB signaling pathway in the African paradox. Importantly, insights into the interplay of different factors with the underlying immune pathological mechanisms for COVID-19 can provide a better understanding of the disease and the development of new targets for more efficient treatment strategies.


Subject(s)
COVID-19/epidemiology , COVID-19/virology , Host-Pathogen Interactions , SARS-CoV-2/physiology , Africa/epidemiology , Angiotensin-Converting Enzyme 2/metabolism , Biomarkers , COVID-19/immunology , COVID-19/metabolism , Comorbidity , Cytokines/metabolism , Disease Susceptibility , Geography, Medical , Global Health , Humans , Mortality , NF-kappa B/metabolism , Population Surveillance , Signal Transduction
11.
Proc Natl Acad Sci U S A ; 118(38)2021 09 21.
Article in English | MEDLINE | ID: covidwho-1392993

ABSTRACT

COVID-19 induces a robust, extended inflammatory "cytokine storm" that contributes to an increased morbidity and mortality, particularly in patients with type 2 diabetes (T2D). Macrophages are a key innate immune cell population responsible for the cytokine storm that has been shown, in T2D, to promote excess inflammation in response to infection. Using peripheral monocytes and sera from human patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and a murine hepatitis coronavirus (MHV-A59) (an established murine model of SARS), we identified that coronavirus induces an increased Mφ-mediated inflammatory response due to a coronavirus-induced decrease in the histone methyltransferase, SETDB2. This decrease in SETDB2 upon coronavirus infection results in a decrease of the repressive trimethylation of histone 3 lysine 9 (H3K9me3) at NFkB binding sites on inflammatory gene promoters, effectively increasing inflammation. Mφs isolated from mice with a myeloid-specific deletion of SETDB2 displayed increased pathologic inflammation following coronavirus infection. Further, IFNß directly regulates SETDB2 in Mφs via JaK1/STAT3 signaling, as blockade of this pathway altered SETDB2 and the inflammatory response to coronavirus infection. Importantly, we also found that loss of SETDB2 mediates an increased inflammatory response in diabetic Mϕs in response to coronavirus infection. Treatment of coronavirus-infected diabetic Mφs with IFNß reversed the inflammatory cytokine production via up-regulation of SETDB2/H3K9me3 on inflammatory gene promoters. Together, these results describe a potential mechanism for the increased Mφ-mediated cytokine storm in patients with T2D in response to COVID-19 and suggest that therapeutic targeting of the IFNß/SETDB2 axis in T2D patients may decrease pathologic inflammation associated with COVID-19.


Subject(s)
Coronavirus/metabolism , Diabetes Mellitus, Type 2/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Inflammation Mediators/metabolism , Inflammation/virology , Macrophages/metabolism , Animals , COVID-19/immunology , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Cytokine Release Syndrome , Cytokines/metabolism , Diabetes Mellitus, Type 2/genetics , Female , Histone-Lysine N-Methyltransferase/genetics , Humans , Inflammation/metabolism , Inflammation/physiopathology , Male , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , SARS-CoV-2/metabolism , Signal Transduction
12.
Signal Transduct Target Ther ; 5(1): 218, 2020 10 03.
Article in English | MEDLINE | ID: covidwho-1387198

Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Cardiac Glycosides/pharmacology , Gene Expression Regulation/drug effects , Host-Pathogen Interactions/drug effects , Animals , Antiviral Agents/chemistry , Betacoronavirus/pathogenicity , Biological Products/chemistry , Biological Products/pharmacology , Bufanolides/chemistry , Bufanolides/pharmacology , COVID-19 , Cardiac Glycosides/chemistry , Cell Survival/drug effects , Chlorocebus aethiops , Chloroquine/chemistry , Chloroquine/pharmacology , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Digoxin/chemistry , Digoxin/pharmacology , High-Throughput Screening Assays , Host-Pathogen Interactions/genetics , Humans , Janus Kinases/antagonists & inhibitors , Janus Kinases/genetics , Janus Kinases/metabolism , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , NF-E2-Related Factor 2/antagonists & inhibitors , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , NF-kappa B/metabolism , Pandemics , Phenanthrenes/chemistry , Phenanthrenes/pharmacology , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , SARS-CoV-2 , Signal Transduction , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Sodium-Potassium-Exchanging ATPase/genetics , Sodium-Potassium-Exchanging ATPase/metabolism , Vero Cells , Virus Replication/drug effects
13.
Phytomedicine ; 92: 153729, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1373222

ABSTRACT

BACKGROUND: Inflammation-mediated lung injury is a major cause of health problems in many countries and has been the leading cause of morbidity/mortality in intensive care units. In the current COVID-19 pandemic, the majority of the patients experienced serious pneumonia resulting from inflammation (Acute respiratory distress syndrome/ARDS). Pathogenic infections cause cytokine release syndrome (CRS) by hyperactivation of immune cells, which in turn release excessive cytokines causing ARDS. Currently, there are no standard therapies for viral, bacterial or pathogen-mediated CRS. PURPOSE: This study aimed to investigate and validate the protective effects of Dehydrozingerone (DHZ) against LPS induced lung cell injury by in-vitro and in-vivo models and to gain insights into the molecular mechanisms that mediate these therapeutic effects. METHODS: The therapeutic activity of DHZ was determined in in-vitro models by pre-treating the cells with DHZ and exposed to LPS to stimulate the inflammatory cascade of events. We analysed the effect of DHZ on LPS induced inflammatory cytokines, chemokines and cell damage markers expression/levels using various cell lines. We performed gene expression, ELISA, and western blot analysis to elucidate the effect of DHZ on inflammation and its modulation of MAPK and NF-κB pathways. Further, the prophylactic and therapeutic effect of DHZ was evaluated against the LPS induced ARDS model in rats. RESULTS: DHZ significantly (p < 0.01) attenuated the LPS induced ROS, inflammatory cytokine, chemokine gene expression and protein release in macrophages. Similarly, DHZ treatment protected the lung epithelial and endothelial cells by mitigating the LPS induced inflammatory events in a dose-dependent manner. In vivo analysis showed that DHZ treatment significantly (p < 0.001) mitigated the LPS induced ARDS pathophysiology of increase in the inflammatory cells in BALF, inflammatory cytokine and chemokines in lung tissues. LPS stimulated neutrophil-mediated events, apoptosis, alveolar wall thickening and alveolar inflammation were profoundly reduced by DHZ treatment in a rat model. CONCLUSION: This study demonstrates for the first time that DHZ has the potential to ameliorate LPS induced ARDS by inhibiting cytokine storm and oxidative through modulating the MAPK and NF-κB pathways. This data provides pre-clinical support to develop DHZ as a potential therapeutic agent against ARDS.


Subject(s)
COVID-19 , Respiratory Distress Syndrome , Animals , Cytokine Release Syndrome , Endothelial Cells/metabolism , Humans , Lipopolysaccharides , Lung/metabolism , NF-kappa B/metabolism , Oxidative Stress , Pandemics , Rats , Respiratory Distress Syndrome/drug therapy , SARS-CoV-2 , Styrenes
14.
J Mol Graph Model ; 108: 107968, 2021 11.
Article in English | MEDLINE | ID: covidwho-1373131

ABSTRACT

NF-κB is a central regulator of immunity and inflammation. It is suggested that the inflammatory response mediated by SARS-CoV-2 is predominated by NF-κB activation. Thus, NF-κB inhibition is considered a potential therapeutic strategy for COVID-19. The aim of this study was to identify potential anti-inflammation lead molecules that target NF-κB using a quantitative structure-activity relationships (QSAR) model of currently used and investigated anti-inflammatory drugs as the basis for screening. We applied an integrated approach by starting with the inflammation-based QSAR model to screen three libraries containing more than 220,000 drug-like molecules for the purpose of finding potential drugs that target the NF-κB/IκBα p50/p65 (RelA) complex. We also used QSAR models to rule out molecules that were predicted to be toxic. Among screening libraries, 382 molecules were selected as potentially nontoxic and were analyzed further by short and long molecular dynamics (MD) simulations and free energy calculations. We have discovered five hit ligands with highly predicted anti-inflammation activity and nearly no predicted toxicities which had strongly favorable protein-ligand interactions and conformational stability at the binding pocket compared to a known NF-κB inhibitor (procyanidin B2). We propose these hit molecules as potential NF-κB inhibitors which can be further investigated in pre-clinical studies against SARS-CoV-2 and may be used as a scaffold for chemical optimization and drug development efforts.


Subject(s)
COVID-19 , Quantitative Structure-Activity Relationship , Drug Discovery , Humans , Inflammation/drug therapy , NF-kappa B/metabolism , SARS-CoV-2
16.
Front Immunol ; 12: 683879, 2021.
Article in English | MEDLINE | ID: covidwho-1369666

ABSTRACT

Diseases caused by pathogenic bacteria in animals (e.g., bacterial pneumonia, meningitis and sepsis) and plants (e.g., bacterial wilt, angular spot and canker) lead to high prevalence and mortality, and decomposition of plant leaves, respectively. Melatonin, an endogenous molecule, is highly pleiotropic, and accumulating evidence supports the notion that melatonin's actions in bacterial infection deserve particular attention. Here, we summarize the antibacterial effects of melatonin in vitro, in animals as well as plants, and discuss the potential mechanisms. Melatonin exerts antibacterial activities not only on classic gram-negative and -positive bacteria, but also on members of other bacterial groups, such as Mycobacterium tuberculosis. Protective actions against bacterial infections can occur at different levels. Direct actions of melatonin may occur only at very high concentrations, which is at the borderline of practical applicability. However, various indirect functions comprise activation of hosts' defense mechanisms or, in sepsis, attenuation of bacterially induced inflammation. In plants, its antibacterial functions involve the mitogen-activated protein kinase (MAPK) pathway; in animals, protection by melatonin against bacterially induced damage is associated with inhibition or activation of various signaling pathways, including key regulators such as NF-κB, STAT-1, Nrf2, NLRP3 inflammasome, MAPK and TLR-2/4. Moreover, melatonin can reduce formation of reactive oxygen and nitrogen species (ROS, RNS), promote detoxification and protect mitochondrial damage. Altogether, we propose that melatonin could be an effective approach against various pathogenic bacterial infections.


Subject(s)
Anti-Bacterial Agents/pharmacology , Inflammasomes/metabolism , Melatonin/pharmacology , Sepsis/metabolism , Signal Transduction/drug effects , Animals , Humans , Inflammasomes/drug effects , Mitogen-Activated Protein Kinases/drug effects , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/drug effects , NF-kappa B/metabolism , Plant Leaves , Reactive Oxygen Species , Sepsis/genetics , Sepsis/immunology
17.
Viruses ; 13(8)2021 08 04.
Article in English | MEDLINE | ID: covidwho-1359300

ABSTRACT

Transcriptomics, proteomics and pathogen-host interactomics data are being explored for the in silico-informed selection of drugs, prior to their functional evaluation. The effectiveness of this kind of strategy has been put to the test in the current COVID-19 pandemic, and it has been paying off, leading to a few drugs being rapidly repurposed as treatment against SARS-CoV-2 infection. Several neglected tropical diseases, for which treatment remains unavailable, would benefit from informed in silico investigations of drugs, as performed in this work for Dengue fever disease. We analyzed transcriptomic data in the key tissues of liver, spleen and blood profiles and verified that despite transcriptomic differences due to tissue specialization, the common mechanisms of action, "Adrenergic receptor antagonist", "ATPase inhibitor", "NF-kB pathway inhibitor" and "Serotonin receptor antagonist", were identified as druggable (e.g., oxprenolol, digoxin, auranofin and palonosetron, respectively) to oppose the effects of severe Dengue infection in these tissues. These are good candidates for future functional evaluation and clinical trials.


Subject(s)
Antiviral Agents/therapeutic use , Dengue/drug therapy , Transcriptome , Adenosine Triphosphatases/antagonists & inhibitors , Adrenergic Antagonists/pharmacology , Adrenergic Antagonists/therapeutic use , Antiviral Agents/pharmacology , Brain/metabolism , Computer Simulation , Dengue/blood , Dengue/genetics , Dengue/metabolism , Drug Discovery , Drug Evaluation, Preclinical , Drug Repositioning , Humans , Liver/metabolism , Metabolic Networks and Pathways/drug effects , NF-kappa B/metabolism , Serotonin Antagonists/pharmacology , Serotonin Antagonists/therapeutic use , Severe Dengue/blood , Severe Dengue/drug therapy , Severe Dengue/genetics , Severe Dengue/metabolism , Spleen/metabolism
18.
Front Immunol ; 12: 716075, 2021.
Article in English | MEDLINE | ID: covidwho-1359192

ABSTRACT

The existence of asymptomatic and re-detectable positive coronavirus disease 2019 (COVID-19) patients presents the disease control challenges of COVID-19. Most studies on immune responses in COVID-19 have focused on moderately or severely symptomatic patients; however, little is known about the immune response in asymptomatic and re-detectable positive (RP) patients. Here we performed a comprehensive analysis of the transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) from 48 COVID-19 patients which included 8 asymptomatic, 13 symptomatic, 15 recovered and 12 RP patients. The weighted gene co-expression network analysis (WGCNA) identified six co-expression modules, of which the turquoise module was positively correlated with the asymptomatic, symptomatic, and recovered COVID-19 patients. The red module positively correlated with symptomatic patients only and the blue and brown modules positively correlated with the RP patients. The analysis by single sample gene set enrichment analysis (ssGSEA) revealed a lower level of IFN response and complement activation in the asymptomatic patients compared with the symptomatic, indicating a weaker immune response of the PBMCs in the asymptomatic patients. In addition, gene set enrichment analysis (GSEA) analysis showed the enrichment of TNFα/NF-κB and influenza infection in the RP patients compared with the recovered patients, indicating a hyper-inflammatory immune response in the PBMC of RP patients. Thus our findings could extend our understanding of host immune response during the progression of COVID-19 disease and assist clinical management and the immunotherapy development for COVID-19.


Subject(s)
Asymptomatic Diseases , COVID-19/immunology , Carrier State/immunology , Leukocytes, Mononuclear/immunology , SARS-CoV-2/immunology , Transcriptome/genetics , Adult , Carrier State/virology , Complement Activation/immunology , Female , Gene Expression Profiling , Humans , Inflammation/immunology , Influenza, Human/complications , Interferons/blood , Interferons/immunology , Male , Middle Aged , NF-kappa B/metabolism , Transcriptome/immunology , Tumor Necrosis Factor-alpha/metabolism , Young Adult
19.
J Virol ; 95(17): e0074721, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1356909

ABSTRACT

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is bringing an unprecedented health crisis to the world. To date, our understanding of the interaction between SARS-CoV-2 and host innate immunity is still limited. Previous studies reported that SARS-CoV-2 nonstructural protein 12 (NSP12) was able to suppress interferon-ß (IFN-ß) activation in IFN-ß promoter luciferase reporter assays, which provided insights into the pathogenesis of COVID-19. In this study, we demonstrated that IFN-ß promoter-mediated luciferase activity was reduced during coexpression of NSP12. However, we could show NSP12 did not affect IRF3 or NF-κB activation. Moreover, IFN-ß production induced by Sendai virus (SeV) infection or other stimulus was not affected by NSP12 at mRNA or protein level. Additionally, the type I IFN signaling pathway was not affected by NSP12, as demonstrated by the expression of interferon-stimulated genes (ISGs). Further experiments revealed that different experiment systems, including protein tags and plasmid backbones, could affect the readouts of IFN-ß promoter luciferase assays. In conclusion, unlike as previously reported, our study showed SARS-CoV-2 NSP12 protein is not an IFN-ß antagonist. It also rings the alarm on the general usage of luciferase reporter assays in studying SARS-CoV-2. IMPORTANCE Previous studies investigated the interaction between SARS-CoV-2 viral proteins and interferon signaling and proposed that several SARS-CoV-2 viral proteins, including NSP12, could suppress IFN-ß activation. However, most of these results were generated from IFN-ß promoter luciferase reporter assay and have not been validated functionally. In our study, we found that, although NSP12 could suppress IFN-ß promoter luciferase activity, it showed no inhibitory effect on IFN-ß production or its downstream signaling. Further study revealed that contradictory results could be generated from different experiment systems. On one hand, we demonstrated that SARS-CoV-2 NSP12 could not suppress IFN-ß signaling. On the other hand, our study suggests that caution needs to be taken with the interpretation of SARS-CoV-2-related luciferase assays.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase , Interferon-beta , Promoter Regions, Genetic , SARS-CoV-2 , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , HEK293 Cells , Humans , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon-beta/antagonists & inhibitors , Interferon-beta/biosynthesis , Interferon-beta/genetics , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , SARS-CoV-2/genetics , SARS-CoV-2/metabolism
20.
J Virol ; 95(17): e0074721, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1350002

ABSTRACT

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is bringing an unprecedented health crisis to the world. To date, our understanding of the interaction between SARS-CoV-2 and host innate immunity is still limited. Previous studies reported that SARS-CoV-2 nonstructural protein 12 (NSP12) was able to suppress interferon-ß (IFN-ß) activation in IFN-ß promoter luciferase reporter assays, which provided insights into the pathogenesis of COVID-19. In this study, we demonstrated that IFN-ß promoter-mediated luciferase activity was reduced during coexpression of NSP12. However, we could show NSP12 did not affect IRF3 or NF-κB activation. Moreover, IFN-ß production induced by Sendai virus (SeV) infection or other stimulus was not affected by NSP12 at mRNA or protein level. Additionally, the type I IFN signaling pathway was not affected by NSP12, as demonstrated by the expression of interferon-stimulated genes (ISGs). Further experiments revealed that different experiment systems, including protein tags and plasmid backbones, could affect the readouts of IFN-ß promoter luciferase assays. In conclusion, unlike as previously reported, our study showed SARS-CoV-2 NSP12 protein is not an IFN-ß antagonist. It also rings the alarm on the general usage of luciferase reporter assays in studying SARS-CoV-2. IMPORTANCE Previous studies investigated the interaction between SARS-CoV-2 viral proteins and interferon signaling and proposed that several SARS-CoV-2 viral proteins, including NSP12, could suppress IFN-ß activation. However, most of these results were generated from IFN-ß promoter luciferase reporter assay and have not been validated functionally. In our study, we found that, although NSP12 could suppress IFN-ß promoter luciferase activity, it showed no inhibitory effect on IFN-ß production or its downstream signaling. Further study revealed that contradictory results could be generated from different experiment systems. On one hand, we demonstrated that SARS-CoV-2 NSP12 could not suppress IFN-ß signaling. On the other hand, our study suggests that caution needs to be taken with the interpretation of SARS-CoV-2-related luciferase assays.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase , Interferon-beta , Promoter Regions, Genetic , SARS-CoV-2 , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , HEK293 Cells , Humans , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon-beta/antagonists & inhibitors , Interferon-beta/biosynthesis , Interferon-beta/genetics , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , SARS-CoV-2/genetics , SARS-CoV-2/metabolism
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