Autophagy inhibition suppresses Newcastle disease virus-induced cell death by inhibiting viral replication in human breast cancer cells.

Newcastle disease virus (NDV) is an oncolytic virus which selectively replicates in cancer cells without harming normal cells. Autophagy is a cellular mechanism that breaks down unused cytoplasmic constituents into nutrients. In previous studies, autophagy enhanced NDV-induced oncolysis in lung cancer and glioma cells. However, the effect of autophagy inhibition on NDV-induced oncolysis in breast cancer cells remains unknown. This study aimed to examine the effect of autophagy inhibition on NDV-induced oncolysis in human breast cancer cells, MCF7. To inhibit autophagy, we knocked down the expression of the autophagy protein beclin-1 (BECN1) by short interfering RNA (siRNA). The cells were infected with the recombinant NDV strain AF2240 expressing green fluorescent protein. We found that NDV induced autophagy and knockdown of BECN1 significantly reduced the NDV-induced autophagy in MCF7 cells. Importantly, BECN1 knockdown significantly suppressed cell death by inhibiting viral replication, as observed at 24 h post infection. Overall, our data suggest that autophagy inhibition may not be a suitable strategy to enhance NDV oncolytic efficacy against breast cancer.

Knockdown of the Autophagy Protein Beclin-1 Does Not Affect Innate Cytokine Production in Human Lung Epithelial Cells during Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in young children, globally. Autophagy is a cellular degradation process that mediates cell survival. Studies using mouse models have demonstrated that inhibiting autophagy affects the production of cytokines triggered by RSV. However, the effect of autophagy on RSV-induced cytokine production in human cells remains inadequately studied. Our previous research showed that inhibiting autophagy using pharmacological inhibitors did not affect the innate cytokine production in human lung epithelial cells (BEAS-2B) following RSV infection. In this study, we sought to validate these findings using a more specific approach, employing short-interfering RNA (siRNA) to target the important autophagy protein Beclin-1 (Bec-1). Prior to measuring cytokine production, we confirmed that silencing Bec-1 with siRNA effectively suppressed autophagy without affecting cell viability. Our results revealed that inhibiting autophagy through Bec-1 knockdown did not affect the production of innate cytokines CXCL8 and CCL5 in BEAS-2B cells during RSV infection, consistent with our previous findings using pharmacological inhibitors. Overall, our data suggest that targeting autophagy may not be an effective strategy for alleviating RSV-induced airway inflammation.

Role of interleukin-1 and MyD88-dependent signaling in rhinovirus infection.

Rhinoviral infection is an important trigger of acute inflammatory exacerbations in patients with underlying airway disease. We have previously established that interleukin-1ß (IL-1ß) is central in the communication between epithelial cells and monocytes during the initiation of inflammation. In this study we explored the roles of IL-1ß and its signaling pathways in the responses of airway cells to rhinovirus-1B (RV-1B) and further determined how responses to RV-1B were modified in a model of bacterial coinfection. Our results revealed that IL-1ß dramatically potentiated RV-1B-induced proinflammatory responses, and while monocytes did not directly amplify responses to RV-1B alone, they played an important role in the responses observed with our coinfection model. MyD88 is the essential signaling adapter for IL-1ß and most Toll-like receptors. To examine the role of MyD88 in more detail, we created stable MyD88 knockdown epithelial cells using short hairpin RNA (shRNA) targeted to MyD88. We determined that IL-1ß/MyD88 plays a role in regulating RV-1B replication and the inflammatory response to viral infection of airway cells. These results identify central roles for IL-1ß and its signaling pathways in the production of CXCL8, a potent neutrophil chemoattractant, in viral infection. Thus, IL-1ß is a viable target for controlling the neutrophilia that is often found in inflammatory airway disease and is exacerbated by viral infection of the airways.

Inhibition of Autophagy Does Not Affect Innate Cytokine Production in Human Lung Epithelial Cells During Respiratory Syncytial Virus Infection.

Human respiratory syncytial virus (RSV) is one of the major causes of childhood acute lower respiratory tract infection worldwide. Autophagy is an intracellular pathway involved in nutrient recycling. Recently, autophagy has been reported to play a role in regulating host cytokine response to several viruses, including vesicular stomatitis virus and human immunodeficiency virus. Previous in vivo studies using mouse model has shown that inhibition of autophagy reduces RSV-induced cytokine production. However, the role of autophagy in modulating RSV-induced cytokine response in human cells has not been reported. We investigated the role of autophagy in regulating the production of the cytokines C-X-C motif ligand 8 (CXCL8) and C-C motif ligand 5 (CCL5), in RSV-infected human bronchial epithelium BEAS-2B cells. Fluorescent microscopic analysis showed that RSV infection induced autophagosome formation in BEAS-2B cells. This autophagy inducing ability of RSV was further confirmed by flow cytometry. The effects of pharmacological inhibition of autophagy by SAR405 or chloroquine on cell death and cytokine release were quantified using lactate dehydrogenase assay and enzyme-linked immunosorbent assay (ELISA), respectively. We found that SAR405 or chloroquine did not cause cell death. Importantly, ELISA analysis showed that pharmacological inhibition of autophagy by SAR405 or chloroquine did not affect the productions of both CXCL5 and CXCL8. In contrast to the previous studies using mouse model, our data suggest that pharmacological inhibition of autophagy may not be a suitable strategy in controlling RSV-induced airway inflammation.

Antimicrobial activity and mode of action of 1,8-cineol against carbapenemase-producing Klebsiella pneumoniae.

Antimicrobial resistance remains one of the most challenging issues that threatens the health of people around the world. Plant-derived natural compounds have received considerable attention for their potential role to mitigate antibiotic resistance. This study was carried out to assess the antimicrobial activity and mode of action of a monoterpene, 1,8-cineol (CN) against carbapenemase-producing Klebsiella pneumoniae (KPC-KP). Results showed that resazurin microplate assay and time-kill analysis revealed bactericidal effects of CN at 28.83 mg/mL. Zeta potential showed that CN increased the surface charge of bacteria and an increase of outer membrane permeability was also detected. CN was able to cause leakage of proteins and nucleic acids in KPC-KP cells upon exposure to CN and ethidium bromide influx/efflux experiment showed the uptake of ethidium bromide into the cell; this was attributed to membrane damage. CN was also found to induce oxidative stress in CN-treated KPC-KP cells through generation of reactive oxygen species which initiated lipid peroxidation and thus damaging the bacterial cell membrane. Scanning and transmission electron microscopies further confirmed the disruption of bacterial cell membrane and loss of intracellular materials. In this study, we demonstrated that CN induced oxidative stress and membrane damage resulting in KPC-KP cell death.

Phosphoinositide-3 kinase inhibition modulates responses to rhinovirus by mechanisms that are predominantly independent of autophagy.

Human rhinoviruses (HRV) are a major cause of exacerbations of airways disease. Aspects of cell signalling responses to HRV infection remain unclear, particularly with regard to signalling via PI3K, and the PI3K-dependent pathway, autophagy. We investigated the roles of PI3K and autophagy in the responses of epithelial cells to major and minor group HRV infection. The PI3K inhibitor 3-MA, commonly used to inhibit autophagy, markedly reduced HRV-induced cytokine induction. Further investigation of potential targets of 3-MA and comparison of results using this inhibitor to a panel of general and class I-selective PI3K inhibitors showed that several PI3Ks cooperatively regulate responses to HRV. Targeting by siRNA of the autophagy proteins Beclin-1, Atg7, LC3, alone or in combination, or targeting of the autophagy-specific class III PI3K had at most only modest effects on HRV-induced cell signalling as judged by induction of proinflammatory cytokine production. Our data indicate that PI3K and mTOR are involved in induction of proinflammatory cytokines after HRV infection, and that autophagy has little role in the cytokine response to HRV or control of HRV replication.