Investigating the role of autophagy in regulating innate cytokine response of human lung epithelial cells to respiratory syncytial virus
Human respiratory syncytial virus (RSV) is one of the leading causes of childhood acute lower respiratory tract infection in Malaysia. It is responsible for significant morbidity and mortality among children, the elderly and individuals with chronic respiratory illnesses worldwide. Despite years...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2021
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/104589/1/FBSB%202021%2029%20IR.pdf |
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| Summary: | Human respiratory syncytial virus (RSV) is one of the leading causes of
childhood acute lower respiratory tract infection in Malaysia. It is responsible for
significant morbidity and mortality among children, the elderly and individuals
with chronic respiratory illnesses worldwide. Despite years of effort, currently
there are neither licensed vaccines nor specific antiviral drugs against RSV.
The severity of RSV-acquired diseases is predominantly caused by an
overexuberant inflammatory response to the virus. Thus, a complete
understanding of all the mechanisms that regulate cytokine production during
RSV infection is crucial to further refine the therapeutic strategies to alleviate
the excessive RSV-induced inflammatory response. Autophagy has recently
been linked to the regulation of host cytokine responses to several viruses,
including the vesicular stomatitis virus and the human immunodeficiency virus.
In vivo studies using mouse model have shown that inhibiting autophagy
attenuates the production of RSV-induced cytokines. However, the involvement
of autophagy in the innate cytokine response of RSV-infected human cells has
not been reported. Lung epithelial cells are known to be the main site of RSV
infection and replication. Therefore, the main aim of this study was to
determine the potential role of autophagy in regulating the production of RSVinduced
innate cytokine C-X-C motif ligand 8 (CXCL8) and C-C motif ligand 5
(CCL5) production in lung epithelial BEAS-2B cells using both pharmacological
inhibitors and short-interfering RNA knockdown approaches. It was found that
RSV infection induced autophagy in BEAS-2B cells, as measured by CytoID®
Autophagy Kit-based fluorescence microscopy and flow cytometry analyses.
Inhibition of autophagy was performed using both pharmacological inhibitors
and short-interfering RNA knockdown approaches. To confirm that autophagy
inhibition does not affect cell viability, lactate dehydrogenase (LDH) assay was
conducted. It was observed that inhibition of autophagy by the pharmacological
inhibitors SAR405 and chloroquine (CQ); and siRNA-mediated knockdown of
the autophagy protein Beclin-1 (Bec-1) did not kill the BEAS-2B cells. Human respiratory syncytial virus (RSV) is one of the leading causes of
childhood acute lower respiratory tract infection in Malaysia. It is responsible for
significant morbidity and mortality among children, the elderly and individuals
with chronic respiratory illnesses worldwide. Despite years of effort, currently
there are neither licensed vaccines nor specific antiviral drugs against RSV.
The severity of RSV-acquired diseases is predominantly caused by an
overexuberant inflammatory response to the virus. Thus, a complete
understanding of all the mechanisms that regulate cytokine production during
RSV infection is crucial to further refine the therapeutic strategies to alleviate
the excessive RSV-induced inflammatory response. Autophagy has recently
been linked to the regulation of host cytokine responses to several viruses,
including the vesicular stomatitis virus and the human immunodeficiency virus.
In vivo studies using mouse model have shown that inhibiting autophagy
attenuates the production of RSV-induced cytokines. However, the involvement
of autophagy in the innate cytokine response of RSV-infected human cells has
not been reported. Lung epithelial cells are known to be the main site of RSV
infection and replication. Therefore, the main aim of this study was to
determine the potential role of autophagy in regulating the production of RSVinduced
innate cytokine C-X-C motif ligand 8 (CXCL8) and C-C motif ligand 5
(CCL5) production in lung epithelial BEAS-2B cells using both pharmacological
inhibitors and short-interfering RNA knockdown approaches. It was found that
RSV infection induced autophagy in BEAS-2B cells, as measured by CytoID®
Autophagy Kit-based fluorescence microscopy and flow cytometry analyses.
Inhibition of autophagy was performed using both pharmacological inhibitors
and short-interfering RNA knockdown approaches. To confirm that autophagy
inhibition does not affect cell viability, lactate dehydrogenase (LDH) assay was
conducted. It was observed that inhibition of autophagy by the pharmacological
inhibitors SAR405 and chloroquine (CQ); and siRNA-mediated knockdown of
the autophagy protein Beclin-1 (Bec-1) did not kill the BEAS-2B cells. |
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