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1.
Sci Immunol ; 7(68): eabn8014, 2022 02 18.
Article in English | MEDLINE | ID: covidwho-1651046

ABSTRACT

Current coronavirus disease 2019 (COVID-19) vaccines effectively reduce overall morbidity and mortality and are vitally important to controlling the pandemic. Individuals who previously recovered from COVID-19 have enhanced immune responses after vaccination (hybrid immunity) compared with their naïve-vaccinated peers; however, the effects of post-vaccination breakthrough infections on humoral immune response remain to be determined. Here, we measure neutralizing antibody responses from 104 vaccinated individuals, including those with breakthrough infections, hybrid immunity, and no infection history. We find that human immune sera after breakthrough infection and vaccination after natural infection broadly neutralize SARS-CoV-2 (severe acute respiratory coronavirus 2) variants to a similar degree. Although age negatively correlates with antibody response after vaccination alone, no correlation with age was found in breakthrough or hybrid immune groups. Together, our data suggest that the additional antigen exposure from natural infection substantially boosts the quantity, quality, and breadth of humoral immune response regardless of whether it occurs before or after vaccination.


Subject(s)
Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , COVID-19 Vaccines/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Vaccination , Adult , Aged , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/epidemiology , COVID-19/immunology , Chlorocebus aethiops , Enzyme-Linked Immunosorbent Assay , Humans , Immunogenicity, Vaccine , Middle Aged , Phagocytosis , SARS-CoV-2/growth & development , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/immunology , THP-1 Cells , Time Factors , Vero Cells , Viral Load
2.
Cell Rep Med ; 3(2): 100510, 2022 02 15.
Article in English | MEDLINE | ID: covidwho-1636907

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOCs) exhibit escape from neutralizing antibodies, causing concern about vaccine effectiveness. However, while non-neutralizing cytotoxic functions of antibodies are associated with improved disease outcome and vaccine protection, Fc effector function escape from VOCs is poorly defined. Furthermore, whether VOCs trigger Fc functions with altered specificity, as has been reported for neutralization, is unknown. Here, we demonstrate that the Beta VOC partially evades Fc effector activity in individuals infected with the original (D614G) variant. However, not all functions are equivalently affected, suggesting differential targeting by antibodies mediating distinct Fc functions. Furthermore, Beta and Delta infection trigger responses with significantly improved Fc cross-reactivity against global VOCs compared with D614G-infected or Ad26.COV2.S-vaccinated individuals. This suggests that, as for neutralization, the infecting spike sequence affects Fc effector function. These data have important implications for vaccine strategies that incorporate VOCs, suggesting these may induce broader Fc effector responses.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Immunoglobulin Fc Fragments/immunology , SARS-CoV-2/immunology , /immunology , Adult , Aged , COVID-19/blood , COVID-19/prevention & control , COVID-19/virology , Cohort Studies , Cross Reactions , Female , HEK293 Cells , Humans , Jurkat Cells , Male , Middle Aged , Neutralization Tests , Protein Binding , Spike Glycoprotein, Coronavirus/immunology , THP-1 Cells , Treatment Outcome , Vaccination/methods
3.
EBioMedicine ; 75: 103803, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1587923

ABSTRACT

BACKGROUND: The Coronavirus Disease 2019 (COVID-19) pandemic has been a great threat to global public health since 2020. Although the advance on vaccine development has been largely achieved, a strategy to alleviate immune overactivation in severe COVID-19 patients is still needed. The NLRP3 inflammasome is activated upon SARS-CoV-2 infection and associated with COVID-19 severity. However, the processes by which the NLRP3 inflammasome is involved in COVID-19 disease remain unclear. METHODS: We infected THP-1 derived macrophages, NLRP3 knockout mice, and human ACE2 transgenic mice with live SARS-CoV-2 in Biosafety Level 3 (BSL-3) laboratory. We performed quantitative real-time PCR for targeted viral or host genes from SARS-CoV-2 infected mouse tissues, conducted histological or immunofluorescence analysis in SARS-CoV-2 infected mouse tissues. We also injected intranasally AAV-hACE2 or intraperitoneally NLRP3 inflammasome inhibitor MCC950 before SARS-CoV-2 infection in mice as indicated. FINDINGS: We have provided multiple lines of evidence that the NLRP3 inflammasome plays an important role in the host immune response to SARS-CoV-2 invasion of the lungs. Inhibition of the NLRP3 inflammasome attenuated the release of COVID-19 related pro-inflammatory cytokines in cell cultures and mice. The severe pathology induced by SARS-CoV-2 in lung tissues was reduced in Nlrp3-/- mice compared to wild-type C57BL/6 mice. Finally, specific inhibition of the NLRP3 inflammasome by MCC950 alleviated excessive lung inflammation and thus COVID-19 like pathology in human ACE2 transgenic mice. INTERPRETATION: Inflammatory activation induced by SARS-CoV-2 is an important stimulator of COVID-19 related immunopathology. Targeting the NLRP3 inflammasome is a promising immune intervention against severe COVID-19 disease. FUNDING: This work was supported by grants from the Bureau of Frontier Sciences and Education, CAS (grant no. QYZDJ-SSW-SMC005 to Y.G.Y.), the key project of the CAS "Light of West China" Program (to D.Y.) and Yunnan Province (202001AS070023 to D.Y.).


Subject(s)
COVID-19 , Lung , Macrophages , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Animals , COVID-19/genetics , COVID-19/immunology , COVID-19/pathology , Disease Models, Animal , Humans , Lung/immunology , Lung/pathology , Lung/virology , Macrophages/immunology , Macrophages/pathology , Macrophages/virology , Male , Mice , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , SARS-CoV-2/genetics , THP-1 Cells
4.
Front Immunol ; 12: 750969, 2021.
Article in English | MEDLINE | ID: covidwho-1551506

ABSTRACT

The COVID-19 is an infectious disease caused by SARS-CoV-2 infection. A large number of clinical studies found high-level expression of pro-inflammatory cytokines in patients infected with SARS-CoV-2, which fuels the rapid development of the disease. However, the specific molecular mechanism is still unclear. In this study, we found that SARS-CoV-2 Nsp5 can induce the expression of cytokines IL-1ß, IL-6, TNF-α, and IL-2 in Calu-3 and THP1 cells. Further research found that Nsp5 enhances cytokine expression through activating the NF-κB signaling pathway. Subsequently, we investigated the upstream effectors of the NF-κB signal pathway on Nsp5 overexpression and discovered that Nsp5 increases the protein level of MAVS. Moreover, Nsp5 can promote the SUMOylation of MAVS to increase its stability and lead to increasing levels of MAVS protein, finally triggering activation of NF-κB signaling. The knockdown of MAVS and the inhibitor of SUMOylation treatment can attenuate Nsp5-mediated NF-κB activation and cytokine induction. We identified a novel role of SARS-CoV-2 Nsp5 to enhance cytokine production by activating the NF-κB signaling pathway.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Coronavirus 3C Proteases/immunology , Cytokines/biosynthesis , NF-kappa B/metabolism , SARS-CoV-2/immunology , Sumoylation/physiology , Adaptor Proteins, Signal Transducing/genetics , Animals , COVID-19/immunology , Cell Line , Chlorocebus aethiops , Enzyme Activation/drug effects , HEK293 Cells , Humans , Immunity, Innate/immunology , Interleukin-1beta/biosynthesis , Interleukin-2/biosynthesis , Interleukin-6/biosynthesis , Signal Transduction/physiology , Sumoylation/drug effects , THP-1 Cells , Tumor Necrosis Factor-alpha/biosynthesis , Vero Cells
5.
Front Immunol ; 12: 767981, 2021.
Article in English | MEDLINE | ID: covidwho-1528824

ABSTRACT

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to constitute a serious public health threat worldwide. Protective antibody-mediated viral neutralization in response to SARS-CoV-2 infection has been firmly characterized. Where the effects of the antibody response are generally considered to be beneficial, an important biological question regarding potential negative outcomes of a SARS-CoV-2 antibody response has yet to be answered. We determined the distribution of IgG subclasses and complement activation levels in plasma from convalescent individuals using in-house developed ELISAs. The IgG response towards SARS-CoV-2 receptor-binding domain (RBD) after natural infection appeared to be mainly driven by IgG1 and IgG3 subclasses, which are the main ligands for C1q mediated classical complement pathway activation. The deposition of the complement components C4b, C3bc, and TCC as a consequence of SARS-CoV-2 specific antibodies were depending primarily on the SARS-CoV-2 RBD and significantly correlated with both IgG levels and disease severity, indicating that individuals with high levels of IgG and/or severe disease, might have a more prominent complement activation during viral infection. Finally, freshly isolated monocytes and a monocyte cell line (THP-1) were used to address the cellular mediated inflammatory response as a consequence of Fc-gamma receptor engagement by SARS-CoV-2 specific antibodies. Monocytic Fc gamma receptor charging resulted in a significant rise in the secretion of the pro-inflammatory cytokine TNF-α. Our results indicate that SARS-CoV-2 antibodies might drive significant inflammatory responses through the classical complement pathway and via cellular immune-complex activation that could have negative consequences during COVID-19 disease. We found that increased classical complement activation was highly associated to COVID-19 disease severity. The combination of antibody-mediated complement activation and subsequent cellular priming could constitute a significant risk of exacerbating COVID-19 severity.


Subject(s)
Antibodies, Viral/blood , COVID-19/immunology , Complement System Proteins/immunology , Immunoglobulin G/blood , SARS-CoV-2/immunology , COVID-19/blood , Complement Activation , Cytokines/immunology , Humans , Inflammation/immunology , Receptors, IgG/immunology , THP-1 Cells
6.
PLoS One ; 16(11): e0259732, 2021.
Article in English | MEDLINE | ID: covidwho-1518359

ABSTRACT

Mesenchymal stem cell derived extracellular vesicles (MSC-EVs) are bioactive particles that evoke beneficial responses in recipient cells. We identified a role for MSC-EV in immune modulation and cellular salvage in a model of SARS-CoV-2 induced acute lung injury (ALI) using pulmonary epithelial cells and exposure to cytokines or the SARS-CoV-2 receptor binding domain (RBD). Whereas RBD or cytokine exposure caused a pro-inflammatory cellular environment and injurious signaling, impairing alveolar-capillary barrier function, and inducing cell death, MSC-EVs reduced inflammation and reestablished target cell health. Importantly, MSC-EV treatment increased active ACE2 surface protein compared to RBD injury, identifying a previously unknown role for MSC-EV treatment in COVID-19 signaling and pathogenesis. The beneficial effect of MSC-EV treatment was confirmed in an LPS-induced rat model of ALI wherein MSC-EVs reduced pro-inflammatory cytokine secretion and respiratory dysfunction associated with disease. MSC-EV administration was dose-responsive, demonstrating a large effective dose range for clinical translation. These data provide direct evidence of an MSC-EV-mediated improvement in ALI and contribute new insights into the therapeutic potential of MSC-EVs in COVID-19 or similar pathologies of respiratory distress.


Subject(s)
Acute Lung Injury/complications , Acute Lung Injury/virology , COVID-19/pathology , Extracellular Vesicles/metabolism , Mesenchymal Stem Cells/metabolism , Pneumonia/complications , Pneumonia/virology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Disease Models, Animal , Extracellular Vesicles/ultrastructure , Humans , Immunomodulation , Male , Models, Biological , Pneumonia/pathology , Rats, Sprague-Dawley , SARS-CoV-2/physiology , Signal Transduction , THP-1 Cells
7.
Front Immunol ; 12: 732298, 2021.
Article in English | MEDLINE | ID: covidwho-1506693

ABSTRACT

Immune modulating therapies and vaccines are in high demand, not least to the recent global spread of SARS-CoV2. To achieve efficient activation of the immune system, professional antigen presenting cells have proven to be key coordinators of such responses. Especially targeted approaches, actively directing antigens to specialized dendritic cells, promise to be more effective and accompanied by reduced payload due to less off-target effects. Although antibody and glycan-based targeting of receptors on dendritic cells have been employed, these are often expensive and time-consuming to manufacture or lack sufficient specificity. Thus, we applied a small-molecule ligand that specifically binds Langerin, a hallmark receptor on Langerhans cells, conjugated to a model protein antigen. Via microneedle injection, this construct was intradermally administered into intact human skin explants, selectively loading Langerhans cells in the epidermis. The ligand-mediated cellular uptake outpaces protein degradation resulting in intact antigen delivery. Due to the pivotal role of Langerhans cells in induction of immune responses, this approach of antigen-targeting of tissue-resident immune cells offers a novel way to deliver highly effective vaccines with minimally invasive administration.


Subject(s)
Antigens, CD/metabolism , Antigens/administration & dosage , Green Fluorescent Proteins/administration & dosage , Langerhans Cells/metabolism , Lectins, C-Type/metabolism , Mannose-Binding Lectins/metabolism , Animals , Antigens/immunology , Antigens/metabolism , COS Cells , Chlorocebus aethiops , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Injections, Intradermal , Langerhans Cells/immunology , Ligands , Miniaturization , Nanomedicine , Needles , Protein Binding , Protein Transport , Proteolysis , THP-1 Cells , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunology , Vaccines, Subunit/metabolism
8.
Viruses ; 13(10)2021 10 15.
Article in English | MEDLINE | ID: covidwho-1470996

ABSTRACT

Infections with viral pathogens are widespread and can cause a variety of different diseases. In-depth knowledge about viral triggers initiating an immune response is necessary to decipher viral pathogenesis. Inflammasomes, as part of the innate immune system, can be activated by viral pathogens. However, viral structural components responsible for inflammasome activation remain largely unknown. Here we analyzed glycoproteins derived from SARS-CoV-1/2, HCMV and HCV, required for viral entry and fusion, as potential triggers of NLRP3 inflammasome activation and pyroptosis in THP-1 macrophages. All tested glycoproteins were able to potently induce NLRP3 inflammasome activation, indicated by ASC-SPECK formation and secretion of cleaved IL-1ß. Lytic cell death via gasdermin D (GSDMD), pore formation, and pyroptosis are required for IL-1ß release. As a hallmark of pyroptosis, we were able to detect cleavage of GSDMD and, correspondingly, cell death in THP-1 macrophages. CRISPR-Cas9 knockout of NLRP3 and GSDMD in THP-1 macrophages confirmed and strongly support the evidence that viral glycoproteins can act as innate immunity triggers. With our study, we decipher key mechanisms of viral pathogenesis by showing that viral glycoproteins potently induce innate immune responses. These insights could be beneficial in vaccine development and provide new impulses for the investigation of vaccine-induced innate immunity.


Subject(s)
Immunity, Innate/immunology , Inflammasomes/immunology , Macrophages/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , Viral Envelope Proteins/immunology , Viral Fusion Proteins/immunology , Cell Line, Tumor , Cytomegalovirus/immunology , Hepacivirus/immunology , Humans , Interleukin-1beta/biosynthesis , Interleukin-1beta/immunology , Pyroptosis/immunology , SARS Virus/immunology , SARS-CoV-2/immunology , THP-1 Cells
9.
Int Immunopharmacol ; 100: 108125, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1401542

ABSTRACT

Mucosal barrier alterations may play a role in the pathogenesis of several diseases, including COVID-19. In this study we evaluate the association between bacterial translocation markers and systemic inflammation at the earliest time-point after hospitalization and at the last 72 h of hospitalization in survivors and non-survivors COVID-19 patients. Sixty-six SARS-CoV-2 RT-PCR positive patients and nine non-COVID-19 pneumonia controls were admitted in this study. Blood samples were collected at hospital admission (T1) (Controls and COVID-19 patients) and 0-72 h before hospital discharge (T2, alive or dead) to analyze systemic cytokines and chemokines, lipopolysaccharide (LPS) concentrations and soluble CD14 (sCD14) levels. THP-1 human monocytic cell line was incubated with plasma from survivors and non-survivors COVID-19 patients and their phenotype, activation status, TLR4, and chemokine receptors were analyzed by flow cytometry. COVID-19 patients presented higher IL-6, IFN-γ, TNF-α, TGF-ß1, CCL2/MCP-1, CCL4/MIP-1ß, and CCL5/RANTES levels than controls. Moreover, LPS and sCD14 were higher at hospital admission in SARS-CoV-2-infected patients. Non-survivors COVID-19 patients had increased LPS levels concomitant with higher IL-6, TNF-α, CCL2/MCP-1, and CCL5/RANTES levels at T2. Increased expression of CD16 and CCR5 were identified in THP-1 cells incubated with the plasma of survivor patients obtained at T2. The incubation of THP-1 with T2 plasma of non-survivors COVID-19 leads to higher TLR4, CCR2, CCR5, CCR7, and CD69 expression. In conclusion, the coexistence of increased microbial translocation and hyperinflammation in patients with severe COVID-19 may lead to higher monocyte activation, which may be associated with worsening outcomes, such as death.


Subject(s)
COVID-19/immunology , Inflammation/etiology , Lipopolysaccharides/blood , Monocytes/physiology , SARS-CoV-2 , Aged , Aged, 80 and over , Bacterial Translocation , COVID-19/mortality , Female , Hospitalization , Humans , Inflammation Mediators/blood , Male , Middle Aged , Severity of Illness Index , THP-1 Cells
11.
Cell ; 184(3): 628-642.e10, 2021 02 04.
Article in English | MEDLINE | ID: covidwho-1385216

ABSTRACT

SARS-CoV-2 infection causes more severe disease in pregnant women compared to age-matched non-pregnant women. Whether maternal infection causes changes in the transfer of immunity to infants remains unclear. Maternal infections have previously been associated with compromised placental antibody transfer, but the mechanism underlying this compromised transfer is not established. Here, we used systems serology to characterize the Fc profile of influenza-, pertussis-, and SARS-CoV-2-specific antibodies transferred across the placenta. Influenza- and pertussis-specific antibodies were actively transferred. However, SARS-CoV-2-specific antibody transfer was significantly reduced compared to influenza- and pertussis-specific antibodies, and cord titers and functional activity were lower than in maternal plasma. This effect was only observed in third-trimester infection. SARS-CoV-2-specific transfer was linked to altered SARS-CoV-2-antibody glycosylation profiles and was partially rescued by infection-induced increases in IgG and increased FCGR3A placental expression. These results point to unexpected compensatory mechanisms to boost immunity in neonates, providing insights for maternal vaccine design.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , Immunoglobulin G/immunology , Maternal-Fetal Exchange/immunology , Placenta/immunology , Pregnancy Complications, Infectious/immunology , SARS-CoV-2/immunology , Adult , Female , Humans , Infant, Newborn , Pregnancy , Pregnancy Trimester, Third/immunology , Receptors, IgG/immunology , THP-1 Cells
12.
Signal Transduct Target Ther ; 6(1): 308, 2021 08 18.
Article in English | MEDLINE | ID: covidwho-1364579

ABSTRACT

Cytokine storm induced by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a major pathological feature of Coronavirus Disease 2019 (COVID-19) and a crucial determinant in COVID-19 prognosis. Understanding the mechanism underlying the SARS-CoV-2-induced cytokine storm is critical for COVID-19 control. Here, we identify that SARS-CoV-2 ORF3a and host hypoxia-inducible factor-1α (HIF-1α) play key roles in the virus infection and pro-inflammatory responses. RNA sequencing shows that HIF-1α signaling, immune response, and metabolism pathways are dysregulated in COVID-19 patients. Clinical analyses indicate that HIF-1α production, inflammatory responses, and high mortalities occurr in elderly patients. HIF-1α and pro-inflammatory cytokines are elicited in patients and infected cells. Interestingly, SARS-CoV-2 ORF3a induces mitochondrial damage and Mito-ROS production to promote HIF-1α expression, which subsequently facilitates SARS-CoV-2 infection and cytokines production. Notably, HIF-1α also broadly promotes the infection of other viruses. Collectively, during SARS-CoV-2 infection, ORF3a induces HIF-1α, which in turn aggravates viral infection and inflammatory responses. Therefore, HIF-1α plays an important role in promoting SARS-CoV-2 infection and inducing pro-inflammatory responses to COVID-19.


Subject(s)
COVID-19/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mitochondria/metabolism , SARS-CoV-2/metabolism , Signal Transduction , Viroporin Proteins/metabolism , A549 Cells , Animals , Chlorocebus aethiops , HEK293 Cells , HeLa Cells , Humans , Mitochondria/pathology , RNA-Seq , THP-1 Cells , Vero Cells
13.
Front Immunol ; 12: 669103, 2021.
Article in English | MEDLINE | ID: covidwho-1348487

ABSTRACT

Targeted therapeutics for the treatment of coronavirus disease 2019 (COVID-19), especially severe cases, are currently lacking. As macrophages have unique effector functions as a first-line defense against invading pathogens, we genetically armed human macrophages with chimeric antigen receptors (CARs) to reprogram their phagocytic activity against SARS-CoV-2. After investigation of CAR constructs with different intracellular receptor domains, we found that although cytosolic domains from MERTK (CARMERTK) did not trigger antigen-specific cellular phagocytosis or killing effects, unlike those from MEGF10, FcRγ and CD3ζ did, these CARs all mediated similar SARS-CoV-2 clearance in vitro. Notably, we showed that CARMERTK macrophages reduced the virion load without upregulation of proinflammatory cytokine expression. These results suggest that CARMERTK drives an 'immunologically silent' scavenger effect in macrophages and pave the way for further investigation of CARs for the treatment of individuals with COVID-19, particularly those with severe cases at a high risk of hyperinflammation.


Subject(s)
COVID-19/drug therapy , COVID-19/immunology , Immunotherapy, Adoptive , Macrophages/immunology , SARS-CoV-2/immunology , Virion/immunology , Animals , COVID-19/genetics , Chlorocebus aethiops , Humans , Phagocytosis , SARS-CoV-2/genetics , THP-1 Cells , Vero Cells , Virion/genetics
14.
Nat Commun ; 12(1): 4664, 2021 08 02.
Article in English | MEDLINE | ID: covidwho-1338538

ABSTRACT

Excessive inflammatory responses induced upon SARS-CoV-2 infection are associated with severe symptoms of COVID-19. Inflammasomes activated in response to SARS-CoV-2 infection are also associated with COVID-19 severity. Here, we show a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation. N protein facilitates maturation of proinflammatory cytokines and induces proinflammatory responses in cultured cells and mice. Mechanistically, N protein interacts directly with NLRP3 protein, promotes the binding of NLRP3 with ASC, and facilitates NLRP3 inflammasome assembly. More importantly, N protein aggravates lung injury, accelerates death in sepsis and acute inflammation mouse models, and promotes IL-1ß and IL-6 activation in mice. Notably, N-induced lung injury and cytokine production are blocked by MCC950 (a specific inhibitor of NLRP3) and Ac-YVAD-cmk (an inhibitor of caspase-1). Therefore, this study reveals a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation and induces excessive inflammatory responses.


Subject(s)
COVID-19/metabolism , Coronavirus Nucleocapsid Proteins/metabolism , Inflammasomes/metabolism , Inflammation/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , SARS-CoV-2/metabolism , Animals , COVID-19/virology , Cells, Cultured , Cytokines/metabolism , HEK293 Cells , Humans , Inflammasomes/genetics , Lung Injury/genetics , Lung Injury/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Phosphoproteins/metabolism , Protein Binding , SARS-CoV-2/physiology , THP-1 Cells
15.
EMBO J ; 40(18): e108249, 2021 09 15.
Article in English | MEDLINE | ID: covidwho-1323479

ABSTRACT

SARS-CoV-2 is an emerging coronavirus that causes dysfunctions in multiple human cells and tissues. Studies have looked at the entry of SARS-CoV-2 into host cells mediated by the viral spike protein and human receptor ACE2. However, less is known about the cellular immune responses triggered by SARS-CoV-2 viral proteins. Here, we show that the nucleocapsid of SARS-CoV-2 inhibits host pyroptosis by blocking Gasdermin D (GSDMD) cleavage. SARS-CoV-2-infected monocytes show enhanced cellular interleukin-1ß (IL-1ß) expression, but reduced IL-1ß secretion. While SARS-CoV-2 infection promotes activation of the NLRP3 inflammasome and caspase-1, GSDMD cleavage and pyroptosis are inhibited in infected human monocytes. SARS-CoV-2 nucleocapsid protein associates with GSDMD in cells and inhibits GSDMD cleavage in vitro and in vivo. The nucleocapsid binds the GSDMD linker region and hinders GSDMD processing by caspase-1. These insights into how SARS-CoV-2 antagonizes cellular inflammatory responses may open new avenues for treating COVID-19 in the future.


Subject(s)
COVID-19/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Nucleocapsid/metabolism , Phosphate-Binding Proteins/metabolism , Pyroptosis/physiology , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/immunology , COVID-19/pathology , COVID-19/virology , Caspase 1/immunology , Caspase 1/metabolism , HEK293 Cells , Host-Pathogen Interactions , Humans , Inflammasomes/immunology , Inflammasomes/metabolism , Interleukin-1beta/immunology , Interleukin-1beta/metabolism , Intracellular Signaling Peptides and Proteins/immunology , Mice , Monocytes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Phosphate-Binding Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , THP-1 Cells
16.
Cell Death Dis ; 12(8): 732, 2021 07 23.
Article in English | MEDLINE | ID: covidwho-1322464

ABSTRACT

Severe coronavirus disease 2019 (COVID-19) is characterized by symptoms of lymphopenia and multiorgan damage, but the underlying mechanisms remain unclear. To explore the function of N6-methyladenosine (m6A) modifications in COVID-19, we performed microarray analyses to comprehensively characterize the m6A epitranscriptome. The results revealed distinct global m6A profiles in severe and mild COVID-19 patients. Programmed cell death and inflammatory response were the major biological processes modulated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Further, RBM15, a major m6A methyltransferase, was significantly elevated and positively correlated with disease severity. Silencing RBM15 drastically reduced lymphocyte death in vitro. Knockdown of RBM15 remarkably suppressed the expression levels of multitarget genes related to programmed cell death and inflammatory response. This study shows that SARS-CoV-2 infection alters the m6A epitranscriptome of lymphocytes, particularly in the case of severe patients. RBM15 regulated host immune response to SARS-CoV-2 by elevating m6A modifications of multitarget genes. These findings indicate that RBM15 can serve as a target for the treatment of COVID-19.


Subject(s)
Adenosine/analogs & derivatives , COVID-19/genetics , RNA Processing, Post-Transcriptional , RNA-Binding Proteins/metabolism , Transcriptome , Adenosine/metabolism , COVID-19/pathology , Cell Line, Tumor , Epigenesis, Genetic , Female , Humans , Lymphocytes/metabolism , Male , Middle Aged , RNA-Binding Proteins/genetics , THP-1 Cells
17.
Front Immunol ; 12: 683800, 2021.
Article in English | MEDLINE | ID: covidwho-1305645

ABSTRACT

The major cause of death in SARS-CoV-2 infected patients is due to de-regulation of the innate immune system and development of cytokine storm. SARS-CoV-2 infects multiple cell types in the lung, including macrophages, by engagement of its spike (S) protein on angiotensin converting enzyme 2 (ACE2) receptor. ACE2 receptor initiates signals in macrophages that modulate their activation, including production of cytokines and chemokines. IL-1R-associated kinase (IRAK)-M is a central regulator of inflammatory responses regulating the magnitude of TLR responsiveness. Aim of the work was to investigate whether SARS-CoV-2 S protein-initiated signals modulate pro-inflammatory cytokine production in macrophages. For this purpose, we treated PMA-differentiated THP-1 human macrophages with SARS-CoV-2 S protein and measured the induction of inflammatory mediators including IL6, TNFα, IL8, CXCL5, and MIP1a. The results showed that SARS-CoV-2 S protein induced IL6, MIP1a and TNFα mRNA expression, while it had no effect on IL8 and CXCL5 mRNA levels. We further examined whether SARS-CoV-2 S protein altered the responsiveness of macrophages to TLR signals. Treatment of LPS-activated macrophages with SARS-CoV-2 S protein augmented IL6 and MIP1a mRNA, an effect that was evident at the protein level only for IL6. Similarly, treatment of PAM3csk4 stimulated macrophages with SARS-CoV-2 S protein resulted in increased mRNA of IL6, while TNFα and MIP1a were unaffected. The results were confirmed in primary human peripheral monocytic cells (PBMCs) and isolated CD14+ monocytes. Macrophage responsiveness to TLR ligands is regulated by IRAK-M, an inactive IRAK kinase isoform. Indeed, we found that SARS-CoV-2 S protein suppressed IRAK-M mRNA and protein expression both in THP1 macrophages and primary human PBMCs and CD14+ monocytes. Engagement of SARS-CoV-2 S protein with ACE2 results in internalization of ACE2 and suppression of its activity. Activation of ACE2 has been previously shown to induce anti-inflammatory responses in macrophages. Treatment of macrophages with the ACE2 activator DIZE suppressed the pro-inflammatory action of SARS-CoV-2. Our results demonstrated that SARS-CoV-2/ACE2 interaction rendered macrophages hyper-responsive to TLR signals, suppressed IRAK-M and promoted pro-inflammatory cytokine expression. Thus, activation of ACE2 may be a potential anti-inflammatory therapeutic strategy to eliminate the development of cytokine storm observed in COVID-19 patients.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/immunology , Cytokine Release Syndrome/immunology , Interleukin-1 Receptor-Associated Kinases/metabolism , Macrophages/immunology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Gene Expression Regulation , Humans , Immunity, Innate , Inflammation Mediators/metabolism , Interleukin-1 Receptor-Associated Kinases/genetics , Interleukin-6/genetics , Interleukin-6/metabolism , Lipopolysaccharides/immunology , Macrophages/virology , Protein Binding , THP-1 Cells , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
18.
Theranostics ; 11(15): 7379-7390, 2021.
Article in English | MEDLINE | ID: covidwho-1266907

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel strain of highly contagious coronaviruses that infects humans. Prolonged fever, particularly that above 39.5 °C, is associated with SARS-CoV-2 infection. However, little is known about the pathological effects of fever caused by SARS-CoV-2. Methods: Primary bovine alveolar macrophages (PBAMs), RAW264.7 mouse macrophages, and THP-1 human cells were transfected with plasmids carrying the genes encoding the SARS-CoV-2 spike (S) protein or receptor-binding domain (RBD). Proteins in the macrophages interacting with S-RBD at 39.5 °C or 37 °C were identified by immunoprecipitation-mass spectrometry. Glutathione S-transferase pulldown, surface plasmon resonance, and immunofluorescence were performed to evaluate the transient receptor potential vanilloid 2 (TRPV2) interaction with SARS-CoV-2-S-RBD at 39.5 °C. Using an RNA sequencing-based approach, cytokine gene expression induced by SARS-CoV-2 S transfection at 39.5 °C and 37.5 °C in primary alveolar macrophages was measured. Fluo-4 staining and enzyme-linked immunosorbent assays were used to assess the regulatory function of TRPV2 in intracellular Ca 2+ and cytokines under SARS-CoV-2-S-RBD at 39.5 °C. Additionally, cytokine release was examined after TRPV2 knockdown with shRNA oligonucleotides or inhibition using the SKF-96365 antagonist. Results: We identified an interaction between the primary alveolar macrophage receptor TRPV2 and S-RBD under febrile conditions. Febrile temperature promotes Ca2+ influx through SARS-CoV-2 infection in PBAMs, further activates the NF-κB p65 signaling pathway, and enhances the secretion of cytokines. Furthermore, knockdown or antagonist (with SKF-96365) of TRPV2 significantly decreased the release of cytokines that drive the inflammatory response. Conclusion: Collectively, our findings identified TRPV2 as a receptor of SARS-CoV-2 in conditions of febrile temperature, providing insight into critical interactions of SARS-CoV-2 with macrophages, as well as a useful resource and potential drug target for coronavirus disease 2019.


Subject(s)
COVID-19/virology , Fever/virology , Macrophages/metabolism , Macrophages/virology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , TRPV Cation Channels/metabolism , Virus Internalization , Animals , Calcium/metabolism , Cattle , Cells, Cultured , Cytokines/metabolism , Humans , Imidazoles/pharmacology , Kinetics , Macrophages/drug effects , Mice , NF-kappa B/metabolism , Protein Binding/drug effects , RAW 264.7 Cells , SARS-CoV-2/drug effects , Signal Transduction/drug effects , THP-1 Cells , Temperature , Virus Internalization/drug effects
19.
ChemMedChem ; 16(15): 2345-2353, 2021 08 05.
Article in English | MEDLINE | ID: covidwho-1248684

ABSTRACT

The C-type lectin receptor DC-SIGN mediates interactions with envelope glycoproteins of many viruses such as SARS-CoV-2, ebola, and HIV and contributes to virus internalization and dissemination. In the context of the recent SARS-CoV-2 pandemic, involvement of DC-SIGN has been linked to severe cases of COVID-19. Inhibition of the interaction between DC-SIGN and viral glycoproteins has the potential to generate broad spectrum antiviral agents. Here, we demonstrate that mannose-functionalized poly-l-lysine glycoconjugates efficiently inhibit the attachment of viral glycoproteins to DC-SIGN-presenting cells with picomolar affinity. Treatment of these cells leads to prolonged receptor internalization and inhibition of virus binding for up to 6 h. Furthermore, the polymers are fully bio-compatible and readily cleared by target cells. The thermodynamic analysis of the multivalent interactions reveals enhanced enthalpy-driven affinities and promising perspectives for the future development of multivalent therapeutics.


Subject(s)
Antiviral Agents/pharmacology , Cell Adhesion Molecules/antagonists & inhibitors , Glycoconjugates/pharmacology , Lectins, C-Type/antagonists & inhibitors , Receptors, Cell Surface/antagonists & inhibitors , Virus Attachment/drug effects , Antiviral Agents/chemical synthesis , Antiviral Agents/metabolism , Cell Adhesion Molecules/metabolism , Glycoconjugates/chemical synthesis , Glycoconjugates/metabolism , Humans , Lectins, C-Type/metabolism , Mannose/analogs & derivatives , Mannose/metabolism , Mannose/pharmacology , Microbial Sensitivity Tests , Polylysine/analogs & derivatives , Polylysine/metabolism , Polylysine/pharmacology , Protein Binding/drug effects , Receptors, Cell Surface/metabolism , SARS-CoV-2/drug effects , THP-1 Cells , Thermodynamics , Viral Envelope Proteins/antagonists & inhibitors , Viral Envelope Proteins/metabolism
20.
J Innate Immun ; 13(6): 345-358, 2021.
Article in English | MEDLINE | ID: covidwho-1245277

ABSTRACT

Regulation of proinflammatory cytokine expression is critical in the face of single-stranded RNA (ssRNA) virus infections. Many viruses, including coronavirus and influenza virus, wreak havoc on the control of cytokine expression, leading to the formation of detrimental cytokine storms. Understanding the regulation and interplay between inflammatory cytokines is critical to the identification of targets involved in controlling the induction of cytokine expression. In this study, we focused on how the antiviral cytokine interleukin-27 (IL-27) regulates signal transduction downstream of Toll-like receptor 7 (TLR7) and TLR8 ligation, which recognize endosomal single-stranded RNA. Given that IL-27 alters bacterial-sensing TLR expression on myeloid cells and can inhibit replication of single-stranded RNA viruses, we investigated whether IL-27 affects expression and function of TLR7 and TLR8. Analysis of IL-27-treated THP-1 monocytic cells and THP-1-derived macrophages revealed changes in mRNA and protein expression of TLR7 and TLR8. Although treatment with IL-27 enhanced TLR7 expression, only TLR8-mediated cytokine secretion was amplified. Furthermore, we demonstrated that imiquimod, a TLR7 agonist, inhibited cytokine and chemokine production induced by a TLR8 agonist, TL8-506. Delineating the immunomodulatory role of IL-27 on TLR7 and TLR8 responses provides insight into how myeloid cell TLR-mediated responses are regulated during virus infection.


Subject(s)
Interleukin-27/immunology , Macrophages/immunology , Monocytes/immunology , Toll-Like Receptor 7/immunology , Toll-Like Receptor 8/immunology , Cytokines/immunology , Humans , Immunomodulation , Inflammation , RNA, Messenger/metabolism , Signal Transduction , THP-1 Cells , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/genetics , Toll-Like Receptor 8/metabolism
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