Periodic Reporting for period 1 - BatSECoV (Canonical and non-canonical secretory mechanisms of cytokines in bat and human cell cultures in response to coronavirus infection: a comparative study)
Berichtszeitraum: 2021-09-01 bis 2023-08-31
The majority of coronaviruses (CoV) originally emerged from bats including highly pathogenic SARS- and MERS-CoV. Bats evolved unique innate immune mechanisms that are likely responsible for the lack of immune pathology. One key process of the immune response is the secretion of cytokines, like interleukins (IL) and type I interferons (IFNs), however molecular details of cytokine secretions and their influence on the outcome of virus infections have not been studied in context of reservoir host species like bats.
The aim of this research proposal is to determine the molecular mechanisms involved in the secretion of IL-1β and IFN in human and bat cells that could explain why bats have developed a tolerance against coronavirus infections. The training-through-research of the candidate will consist in analyzing the secretion of IFNs and IL-1β in cell culture supernatants of bat and human CoV-infected cells subjected to knockdowns of different molecules implicated in canonical or non-canonical secretory pathways, as well as activation and interactions between receptors of both secretory pathways. The multidisciplinary nature of the project is strong, involving a combination of well-developed molecular biology, cell biology, and virology.
The results will show in-depth mechanistic insights into bat cytokine secretion, which still has some gaps in its knowledge, and will serve as a promising platform for the development of new antiviral strategies to fight emerging zoonotic viruses. This topic is of critical importance considering the context of the recent COVID-19 pandemic, which caused a tremendous impact in the European society at the sociological and economical levels. This issue is in line with the EU strategy listed Societal Changes of H2020 for the health, demographic change and wellbeing.
The aim of this research proposal is to determine the molecular mechanisms involved in the secretion of IL-1β and IFN in human and bat cells that could explain why bats have developed a tolerance against coronavirus infections. The training-through-research of the candidate will consist in analyzing the secretion of IFNs and IL-1β in cell culture supernatants of bat and human CoV-infected cells subjected to knockdowns of different molecules implicated in canonical or non-canonical secretory pathways, as well as activation and interactions between receptors of both secretory pathways. The multidisciplinary nature of the project is strong, involving a combination of well-developed molecular biology, cell biology, and virology.
The results will show in-depth mechanistic insights into bat cytokine secretion, which still has some gaps in its knowledge, and will serve as a promising platform for the development of new antiviral strategies to fight emerging zoonotic viruses. This topic is of critical importance considering the context of the recent COVID-19 pandemic, which caused a tremendous impact in the European society at the sociological and economical levels. This issue is in line with the EU strategy listed Societal Changes of H2020 for the health, demographic change and wellbeing.
The work done by the candidate from the beginning to the end of the project compromises the following research activities:
- Generation of a THP1 expression human ACE2 receptor as a model to evaluate cytokine secretion in human monocytes
- Generation and analysis of a SARS-CoV-2 construct expressing GFP as reporter gene
- Study of expression kinetics of IFNs in human and bat lung cell lines during SARS-CoV-2 infection
- Generation of human and bat knock-out cell lines for molecules involved in autophagy (LC3B, SEC22B)
- Study of the influence of autophagy in secretion of cytokines in bat and human lung cells
- Optimization of a microscopy platform for live-cell imaging
- Analysis of vesicle trafficking from endoplasmic reticulum and autophagosomes in human and bat cells infected with SARS-CoV-2
- Co-localization of receptors involved in secretory autophagy by co-immunoprecipitations and confocal microscopy
- Evaluation of stress granule formation in bats during SARS-CoV-2 infection.
Up to date, the results obtained have shown:
- Several human cell lines, such as THP1 expressing human ACE2 receptor and A549-hACE2 with LC3B and SEC22B knocked-out have been successfully established within the host institute.
- Bats and humans have similar type I IFN expression kinetics during RVFV infection
- Opposite to this, bats have higher and earlier expression of type I IFNs (IFNβ, IFNε) in response to SARS-CoV-2 expression.
- Autophagosomes (marked by LC3B expression) co-localized with SEC22B in SARS-CoV-2 infected bat cells, which suggests a strong secretory autophagy response.
- Bats overexpress galectin-8, an intermediary SNARE receptor involved in secretory autophagy
- Bats lack stress granule formation during SARS-CoV-2 infection
The dissemination of the results will be done through publications in open-access peer-reviewed international journals, participation in the European Researcher’s Night, contribution in scientific congresses, university seminars in the country of origin of the candidate, open articles in the host institute website and social networks (Twitter).
- Generation of a THP1 expression human ACE2 receptor as a model to evaluate cytokine secretion in human monocytes
- Generation and analysis of a SARS-CoV-2 construct expressing GFP as reporter gene
- Study of expression kinetics of IFNs in human and bat lung cell lines during SARS-CoV-2 infection
- Generation of human and bat knock-out cell lines for molecules involved in autophagy (LC3B, SEC22B)
- Study of the influence of autophagy in secretion of cytokines in bat and human lung cells
- Optimization of a microscopy platform for live-cell imaging
- Analysis of vesicle trafficking from endoplasmic reticulum and autophagosomes in human and bat cells infected with SARS-CoV-2
- Co-localization of receptors involved in secretory autophagy by co-immunoprecipitations and confocal microscopy
- Evaluation of stress granule formation in bats during SARS-CoV-2 infection.
Up to date, the results obtained have shown:
- Several human cell lines, such as THP1 expressing human ACE2 receptor and A549-hACE2 with LC3B and SEC22B knocked-out have been successfully established within the host institute.
- Bats and humans have similar type I IFN expression kinetics during RVFV infection
- Opposite to this, bats have higher and earlier expression of type I IFNs (IFNβ, IFNε) in response to SARS-CoV-2 expression.
- Autophagosomes (marked by LC3B expression) co-localized with SEC22B in SARS-CoV-2 infected bat cells, which suggests a strong secretory autophagy response.
- Bats overexpress galectin-8, an intermediary SNARE receptor involved in secretory autophagy
- Bats lack stress granule formation during SARS-CoV-2 infection
The dissemination of the results will be done through publications in open-access peer-reviewed international journals, participation in the European Researcher’s Night, contribution in scientific congresses, university seminars in the country of origin of the candidate, open articles in the host institute website and social networks (Twitter).
This project supposes a pioneer study in the field of bat immunology since this is the first study analyzing mechanisms of cytokine secretion in bats. Therefore, the project has a strong novelty component that will significantly increase the knowledge of bat immune response.
The results will show which secretory pathways are being used in human and bat cells to secrete pro-inflammatory cytokines in response to SARS-CoV-2 infection. We expect a differential outcome about the mechanisms used by bats to secrete cytokines. This outcome is based in our working hypothesis, which uses two recent findings to support it. These facts are:
- SARS-CoV-2 infection in humans produces a higher number of free autophagosomes that are not associated with lysosomes. This implies that more autophagosomes are available to be targeted towards the cell membrane for secretion.
- Bats have enhanced autophagy responses when compared with humans, which suggest that bats might use a higher number of autophagosomes for such effect.
Therefore, considering these two facts, we believe that the secretion of cytokines in bats would happen mostly through the non-canonical secretory autophagy pathway. This ability might contribute towards reaching a better antiviral state that why bats tolerate viral infections better than humans.
These results will have major implications at the wellbeing of the European society since we will increase the knowledge of the immune response against SARS-CoV-2 at the cytokine secretion level. Considering that this project involves the use of human cells we will perform a screening analysis of pro-inflammatory cytokines secreted during SARS-CoV-2 infection. Cytokine secretion in humans infected with SARS-CoV-2 is a process of key importance because of the ‘cytokine storm’ that some COVID-19 patients develop. This event typically produces an overproduction of pro-inflammatory cytokines that exacerbates the immune response. Causing higher mortality. A better understanding about how these cytokines are secreted in humans may possibly lead to the development of treatments as part of future projects to prevent cytokine secretion using selective autophagy inhibitors, depending on which secretory pathways are involved in human cells.
The results will show which secretory pathways are being used in human and bat cells to secrete pro-inflammatory cytokines in response to SARS-CoV-2 infection. We expect a differential outcome about the mechanisms used by bats to secrete cytokines. This outcome is based in our working hypothesis, which uses two recent findings to support it. These facts are:
- SARS-CoV-2 infection in humans produces a higher number of free autophagosomes that are not associated with lysosomes. This implies that more autophagosomes are available to be targeted towards the cell membrane for secretion.
- Bats have enhanced autophagy responses when compared with humans, which suggest that bats might use a higher number of autophagosomes for such effect.
Therefore, considering these two facts, we believe that the secretion of cytokines in bats would happen mostly through the non-canonical secretory autophagy pathway. This ability might contribute towards reaching a better antiviral state that why bats tolerate viral infections better than humans.
These results will have major implications at the wellbeing of the European society since we will increase the knowledge of the immune response against SARS-CoV-2 at the cytokine secretion level. Considering that this project involves the use of human cells we will perform a screening analysis of pro-inflammatory cytokines secreted during SARS-CoV-2 infection. Cytokine secretion in humans infected with SARS-CoV-2 is a process of key importance because of the ‘cytokine storm’ that some COVID-19 patients develop. This event typically produces an overproduction of pro-inflammatory cytokines that exacerbates the immune response. Causing higher mortality. A better understanding about how these cytokines are secreted in humans may possibly lead to the development of treatments as part of future projects to prevent cytokine secretion using selective autophagy inhibitors, depending on which secretory pathways are involved in human cells.