A Dual-Fluorescence Labeling Pseudovirus for Real-Time Imaging of Single SARS-CoV-2 Entry in Respiratory Epithelial Cells

Abstract The pseudovirus strategy makes studies of highly pathogenic viruses feasible without the restriction of high-level biosafety facility, thus greatly contributing to virology and being used in research of SARS-CoV-2. Here, we generated a dual-color pseudo-SARS-CoV-2 virus using an HIV-1 pseudovirus production system and the SARS-CoV-2 spike (S) glycoprotein, of which the membrane was labeled with lipophilic dye (DiO) and the genomic RNA-related viral protein R (Vpr) of the viral core were fused with mCherry. With this dual-color labeling strategy, not only the movement of the whole virus but also the fate of the labeled components can be traced. The pseudovirions were applied to track viral entry at a single particle level in four types of the human respiratory cells: nasal epithelial cells (HNEpC), pulmonary alveolar epithelial cells (HPAEpiC), bronchial epithelial cells (BEP-2D), and oral epithelial cells (HOEC). Pseudo-SARS-CoV-2 entered into the host cell and released viral core into the cytoplasm，which clearly indicates that the host entry mainly occurred through endocytosis. The infection efficiency was found to be correlated with the expression of the known receptor of SARS-CoV-2, angiotensin-converting 2 (ACE2) on the host cell surface. We believe that the dual-color fluorescence labeled pseudovirus system created in this study can be a useful tool in SARS-CoV-2/COVID-19 for many purposes.

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GRP78 and Integrins Play Different Roles in Host Cell Invasion During Mucormycosis

10.1101/2020.04.29.069666 ◽

2020 ◽

Author(s):

Abdullah Alqarihi ◽

Teclegiorgis Gebremariam ◽

Yiyou Gu ◽

Marc Swidergall ◽

Sondus Alkhazraji ◽

...

Keyword(s):

Epithelial Cells ◽

Host Cell ◽

Hematologic Malignancies ◽

Alveolar Epithelial Cells ◽

Host Factors ◽

Pulmonary Infections ◽

Integrin Β1 ◽

Nasal Epithelial Cell ◽

Alveolar Epithelial ◽

Pulmonary Mucormycosis

AbstractMucormycosis, caused by Rhizopus species, is a life-threatening fungal infection that occurs in patients immunocompromised by diabetic ketoacidosis (DKA), cytotoxic chemotherapy, immunosuppressive therapy, hematologic malignancies or severe trauma. Inhaled Rhizopus spores cause pulmonary infections in patients with hematologic malignancies, while patients with DKA are much more prone to rhinoorbital/cerebral mucormycosis. Here we show that R. delemar interacts with glucose-regulated protein 78 (GRP78) on nasal epithelial cells via its spore coat protein CotH3 to invade and damage the nasal epithelial cell. Expression of the two proteins is significantly enhanced by high glucose, iron and ketone body levels (hallmark features of DKA), potentially leading to frequently lethal rhinoorbital/cerebral mucormycosis. In contrast, R. delemar CotH7 recognizes integrin β1 as a receptor on alveolar epithelial cells causing the activation of epidermal growth factor receptor (EGFR) leading to host cell invasion. Anti-integrin β1 antibodies inhibit R. delemar invasion of alveolar epithelial cells and protect mice from pulmonary mucormycosis. Our results show that R. delemar interacts with different mammalian receptors depending on the host cell type. Susceptibility of patients with DKA primarily to rhinoorbital/cerebral disease can be explained by host factors typically present in DKA and known to upregulate CotH3 and nasal GRP78 thereby trapping the fungal cells within the rhino-orbital milieu, leading to subsequent invasion and damage. Our studies highlight that mucormycosis pathogenesis can potentially be overcome by the development of novel customized therapies targeting niche-specific host receptors or their respective fungal ligands.ImportanceMucormycosis caused by Rhizopus species is a fungal infection with often fatal prognosis. Inhalation of spores is the major route of entry, with nasal and alveolar epithelial cells among the first cells that encounter the fungi. In patients with hematologic malignancies or those undergoing cytotoxic chemotherapy, Rhizopus causes pulmonary infections. On the other hand, DKA patients predominantly suffer from rhinoorbital/cerebral mucormycosis. The reason for such disparity in disease types by the same fungus is not known. Here we show that, the unique susceptibility of DKA subjects to rhinoorbital/cerebral mucormycosis is likely due to specific interaction between nasal epithelial cell GRP78 and fungal CotH3, the expression of which increase in the presence of host factors present in DKA. In contrast, pulmonary mucormycosis is initiated via interaction of inhaled spores expressing CotH7 with integrin β1 receptor which activates EGFR to induce fungal invasion of host cells. These results introduce plausible explanation to disparate disease manifestations in DKA versus hematologic malignancy patients and provide a foundation for development of therapeutic interventions against these lethal forms of mucormycosis.

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Role of diminished epithelial GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2000.279.3.l487 ◽

2000 ◽

Vol 279 (3) ◽

pp. L487-L495 ◽

Cited By ~ 37

Author(s):

Paul J. Christensen ◽

Marc B. Bailie ◽

Richard E. Goodman ◽

Aidan D. O'Brien ◽

Galen B. Toews ◽

...

Keyword(s):

Epithelial Cells ◽

Pulmonary Fibrosis ◽

Animal Studies ◽

Protective Role ◽

Alveolar Epithelial Cells ◽

Type Ii ◽

Alveolar Epithelial ◽

Gm Csf ◽

High Level

Evidence derived from human and animal studies strongly supports the notion that dysfunctional alveolar epithelial cells (AECs) play a central role in determining the progression of inflammatory injury to pulmonary fibrosis. We formed the hypothesis that impaired production of the regulatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) by injured AECs plays a role in the development of pulmonary fibrosis. To test this hypothesis, we used the well-characterized model of bleomycin-induced pulmonary fibrosis in rats. GM-CSF mRNA is expressed at a constant high level in the lungs of untreated or saline-challenged animals. In contrast, there is a consistent reduction in expression of GM-CSF mRNA in the lung during the first week after bleomycin injury. Bleomycin-treated rats given neutralizing rabbit anti-rat GM-CSF IgG develop increased fibrosis. Type II AECs isolated from rats after bleomycin injury demonstrate diminished expression of GM-CSF mRNA immediately after isolation and in response to stimulation in vitro with endotoxin compared with that in normal type II cells. These data demonstrate a defect in the ability of type II epithelial cells from bleomycin-treated rats to express GM-CSF mRNA and a protective role for GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis.

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GRP78 and Integrins Play Different Roles in Host Cell Invasion during Mucormycosis

mBio ◽

10.1128/mbio.01087-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 4

Author(s):

Abdullah Alqarihi ◽

Teclegiorgis Gebremariam ◽

Yiyou Gu ◽

Marc Swidergall ◽

Sondus Alkhazraji ◽

...

Keyword(s):

Epithelial Cells ◽

Host Cell ◽

Hematologic Malignancies ◽

Alveolar Epithelial Cells ◽

Host Factors ◽

Host Cell Invasion ◽

Pulmonary Infections ◽

Integrin Β1 ◽

Alveolar Epithelial ◽

Pulmonary Mucormycosis

ABSTRACT Mucormycosis, caused by Rhizopus species, is a life-threatening fungal infection that occurs in patients immunocompromised by diabetic ketoacidosis (DKA), cytotoxic chemotherapy, immunosuppressive therapy, hematologic malignancies, or severe trauma. Inhaled Rhizopus spores cause pulmonary infections in patients with hematologic malignancies, while patients with DKA are much more prone to rhinoorbital/cerebral mucormycosis. Here, we show that Rhizopus delemar interacts with glucose-regulated protein 78 (GRP78) on nasal epithelial cells via its spore coat protein CotH3 to invade and damage the nasal epithelial cells. Expression of the two proteins is significantly enhanced by high glucose, iron, and ketone body levels (hallmark features of DKA), potentially leading to frequently lethal rhinoorbital/cerebral mucormycosis. In contrast, R. delemar CotH7 recognizes integrin β1 as a receptor on alveolar epithelial cells, causing the activation of epidermal growth factor receptor (EGFR) and leading to host cell invasion. Anti-integrin β1 antibodies inhibit R. delemar invasion of alveolar epithelial cells and protect mice from pulmonary mucormycosis. Our results show that R. delemar interacts with different mammalian receptors depending on the host cell type. Susceptibility of patients with DKA primarily to rhinoorbital/cerebral disease can be explained by host factors typically present in DKA and known to upregulate CotH3 and nasal GRP78, thereby trapping the fungal cells within the rhinoorbital milieu, leading to subsequent invasion and damage. Our studies highlight that mucormycosis pathogenesis can potentially be overcome by the development of novel customized therapies targeting niche-specific host receptors or their respective fungal ligands. IMPORTANCE Mucormycosis caused by Rhizopus species is a fungal infection with often fatal prognosis. Inhalation of spores is the major route of entry, with nasal and alveolar epithelial cells among the first cells that encounter the fungi. In patients with hematologic malignancies or those undergoing cytotoxic chemotherapy, Rhizopus causes pulmonary infections. On the other hand, DKA patients predominantly suffer from rhinoorbital/cerebral mucormycosis. The reason for such disparity in disease types by the same fungus is not known. Here, we show that the unique susceptibility of DKA subjects to rhinoorbital/cerebral mucormycosis is likely due to specific interaction between nasal epithelial cell GRP78 and fungal CotH3, the expression of which increases in the presence of host factors present in DKA. In contrast, pulmonary mucormycosis is initiated via interaction of inhaled spores expressing CotH7 with integrin β1 receptor, which activates EGFR to induce fungal invasion of host cells. These results introduce a plausible explanation for disparate disease manifestations in DKA versus those in hematologic malignancy patients and provide a foundation for development of therapeutic interventions against these lethal forms of mucormycosis.

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Coronil, a Tri-Herbal Formulation, Attenuates Spike-Protein-Mediated SARS-CoV-2 Viral Entry into Human Alveolar Epithelial Cells and Pro-Inflammatory Cytokines Production by Inhibiting Spike Protein-ACE-2 Interaction

Journal of Inflammation Research ◽

10.2147/jir.s298242 ◽

2021 ◽

Vol Volume 14 ◽

pp. 869-884

Author(s):

Acharya Balkrishna ◽

Swati Haldar ◽

Hoshiyar Singh ◽

Partha Roy ◽

Anurag Varshney

Keyword(s):

Epithelial Cells ◽

Inflammatory Cytokines ◽

Viral Entry ◽

Alveolar Epithelial Cells ◽

Spike Protein ◽

Alveolar Epithelial ◽

Herbal Formulation ◽

Human Alveolar Epithelial Cells ◽

Pro Inflammatory Cytokines

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Temporal Pore Formation-Mediated Egress from Macrophages and Alveolar Epithelial Cells by Legionella pneumophila

Infection and Immunity ◽

10.1128/iai.68.11.6431-6440.2000 ◽

2000 ◽

Vol 68 (11) ◽

pp. 6431-6440 ◽

Cited By ~ 92

Author(s):

O. A. Terry Alli ◽

Lian-Yong Gao ◽

Lisa L. Pedersen ◽

Steven Zink ◽

Marina Radulic ◽

...

Keyword(s):

Epithelial Cells ◽

Host Cell ◽

Legionella Pneumophila ◽

Pore Formation ◽

Alveolar Epithelial Cells ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Intracellular Replication ◽

Alveolar Epithelial ◽

Bacterial Replication

ABSTRACT Legionella pneumophila does not induce apoptosis in the protozoan host, but induces pore formation-mediated cytolysis after termination of intracellular replication (L.-Y. Gao and Y. Abu Kwaik, Environ. Microbiol. 2:79–90, 2000). In contrast to this single mode of killing of protozoa, we have recently proposed a biphasic model by which L. pneumophila kills macrophages, in which the first phase is manifested through the induction of apoptosis during early stages of the infection, followed by an independent and temporal induction of necrosis during late stages of intracellular replication. Here we show that, similar to the protozoan host, the induction of necrosis and cytolysis of macrophages by L. pneumophila is mediated by the pore-forming toxin or activity. This activity is temporally and maximally expressed only upon termination of bacterial replication and correlates with cytolysis of macrophages and alveolar epithelial cells in vitro. We have identified five L. pneumophila mutants defective in the pore-forming activity. The phagosomes harboring the mutants do not colocalize with the late endosomal or lysosomal marker Lamp-1, and the mutants replicate intracellularly similar to the parental strain. Interestingly, despite their prolific intracellular replication, the mutants are defective in cytotoxicity and are “trapped” within and fail to lyse and egress from macrophages and alveolar epithelial cells upon termination of intracellular replication. However, the mutants are subsequently released from the host cell, most likely due to apoptotic death of the host cell. Data derived from cytotoxicity assays, confocal laser scanning microscopy, and electron microscopy confirm the defect in the mutants to induce necrosis of macrophages and the failure to egress from the host cell. Importantly, the mutants are completely defective in acute lethality (24 to 48 h) to intratracheally inoculated A/J mice. We conclude that the pore-forming activity of L. pneumophila is not required for phagosomal trafficking or for intracellular replication. This activity is expressed upon termination of bacterial replication and is essential to induce cytolysis of infected macrophages to allow egress of intracellular bacteria. In addition, this activity plays a major role in pulmonary immunopathology in vivo.

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SARS-CoV-2 Exploits Sexually Dimorphic and Adaptive IFN and TNFa Signaling to Gain Entry into Alveolar Epithelium

10.1101/2021.07.23.453505 ◽

2021 ◽

Author(s):

Filippo Giancotti ◽

Yan Wang ◽

Sreeharsha Gurrapu ◽

Hong Chen ◽

Sara Laudato ◽

...

Keyword(s):

Epithelial Cells ◽

Viral Entry ◽

Alveolar Epithelium ◽

Alveolar Epithelial Cells ◽

Sexually Dimorphic ◽

Jak Inhibitors ◽

Alveolar Cells ◽

Alveolar Epithelial ◽

Tnf Α ◽

Simultaneous Inhibition

Infection of the alveolar epithelium constitutes a bottleneck in the progression of COVID-19 to SARS presumably due to the paucity of viral entry receptors in alveolar epithelial type 1 and 2 cells. We have found that the male alveolar epithelial cells express twice as many ACE2 and TMPRSS2 entry receptors as the female ones. Intriguingly, IFN and TNF-α signaling are preferentially active in male alveolar cells and induce binding of the cognate transcription factors to the promoters and lung-active enhancers of ACE2 and TMPRSS2. Cotreatment with IFN-I and III dramatically increases expression of the receptors and viral entry in alveolar epithelial cells. TNFα and IFN-II, typically overproduced during the cytokine storm, similarly collaborate to induce these events. Whereas JAK inhibitors suppress viral entry induced by IFN-I/III, simultaneous inhibition of IKK/NF-κB is necessary to block viral entry induced by TNFα and IFN II. In addition to explaining the increased incidence of SARS in males, these findings indicate that SARS-Cov-2 hijacks epithelial immune signaling to promote infection of the alveolar epithelium and suggest that JAK inhibitors, singly and in combination with NF-KB inhibitors, may exhibit efficacy in preventing or treating COVID-19 SARS.

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Caspase-4 Mediates Restriction of Burkholderia pseudomallei in Human Alveolar Epithelial Cells

Infection and Immunity ◽

10.1128/iai.00868-19 ◽

2019 ◽

Vol 88 (3) ◽

Author(s):

Chanya Srisaowakarn ◽

Matsayapan Pudla ◽

Marisa Ponpuak ◽

Pongsak Utaisincharoen

Keyword(s):

Epithelial Cells ◽

Burkholderia Pseudomallei ◽

A549 Cells ◽

Alveolar Epithelial Cells ◽

Lung Epithelial Cells ◽

Host Cells ◽

Bacterial Killing ◽

Content Type ◽

Alveolar Epithelial ◽

High Level

ABSTRACT Melioidosis is an infectious disease with a high mortality rate responsible for community-acquired sepsis in Southeast Asia and Northern Australia. The causative agent of this disease is Burkholderia pseudomallei, a Gram-negative bacterium that resides in soil and contaminated natural water. After entering into host cells, the bacteria escape into the cytoplasm, which has numerous cytosolic sensors, including the noncanonical inflammatory caspases. Although the noncanonical inflammasome (caspase-11) has been investigated in a murine model of B. pseudomallei infection, its role in humans, particularly in lung epithelial cells, remains unknown. We, therefore, investigated the function of caspase-4 (ortholog of murine caspase-11) in intracellular killing of B. pseudomallei. The results showed that B. pseudomallei induced caspase-4 activation at 12 h postinfection in human alveolar epithelial A549 cells. The number of intracellular B. pseudomallei bacteria was increased in the absence of caspase-4, suggesting its function in intracellular bacterial restriction. In contrast, a high level of caspase-4 processing was observed when cells were infected with lipopolysaccharide (LPS) mutant B. pseudomallei. The enhanced bacterial clearance in LPS-mutant-infected cells is also correlated with a higher degree of caspase-4 activation. These results highlight the susceptibility of the LPS mutant to caspase-4-mediated intracellular bacterial killing.

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Apoptosis in Macrophages and Alveolar Epithelial Cells during Early Stages of Infection by Legionella pneumophila and Its Role in Cytopathogenicity

Infection and Immunity ◽

10.1128/iai.67.2.862-870.1999 ◽

1999 ◽

Vol 67 (2) ◽

pp. 862-870 ◽

Cited By ~ 97

Author(s):

Lian-Yong Gao ◽

Yousef Abu Kwaik

Keyword(s):

Epithelial Cells ◽

Host Cell ◽

Legionella Pneumophila ◽

Alveolar Epithelial Cells ◽

Alveolar Epithelial ◽

Early Stages ◽

Induction Of Apoptosis ◽

Bacterial Replication ◽

Induced Apoptosis ◽

Nuclear Apoptosis

ABSTRACT The hallmark of Legionnaires’ disease is intracellular replication of Legionella pneumophila within cells in the alveolar spaces. Cytopathogenicity of this bacterium to the host cell has been well demonstrated, but the mechanisms of host cell death due to infection by L. pneumophila are not well understood. In this study, induction of apoptosis in macrophages and alveolar epithelial cells by L. pneumophila during early stages of infection was confirmed by using multiple criteria, including DNA fragmentation by agarose gel electrophoresis, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, surface exposure of phosphatidylserine, and cellular morphology by transmission electron microscopy. Induction of nuclear apoptosis in L. pneumophila-infected macrophages is mediated by activation of the caspase cascade death machinery. We provide genetic and biochemical evidence thatL. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells does not require intracellular bacterial replication or new protein synthesis. In addition, extracellularL. pneumophila is capable of inducing apoptosis. Furthermore, induction of apoptosis by L. pneumophilacorrelates with cytopathogenicity. We conclude that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells plays an important role in cytopathogenicity to the host cell during early stages of infection.

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