Roles of mitochondria in modulating chicken innate immunity following newcastle disease virus infection
Mitochondria have been established as having a vital role in innate immunity. Mitochondrial antiviral signalling (MAVS) protein is a mitochondrial protein proven to modulate the production of interferons and pro-inflammatory cytokines as well as initiating apoptosis in viral infection. Newcastle...
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| Format: | Thesis |
| Language: | English |
| Published: |
2019
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/98627/1/IB%202021%204%20-%20IR.pdf |
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| Summary: | Mitochondria have been established as having a vital role in innate immunity.
Mitochondrial antiviral signalling (MAVS) protein is a mitochondrial protein proven to
modulate the production of interferons and pro-inflammatory cytokines as well as
initiating apoptosis in viral infection. Newcastle disease (ND) is a common threat to the
poultry industry globally with genotype VII Newcastle disease virus (NDV) strains
becoming one of the prominent virulent NDV strains. Antiviral innate immune response
involve various pattern recognition receptors (PRR) such as Toll-like receptors (TLR),
retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5
(MDA5) to identify invading microorganism via their pathogen-associated molecular
patterns (PAMP) like double-stranded RNA (dsRNA), bacterial lipopolysaccharide
(LPS) and viral proteins. This leads to further downstream processes in the signalling
cascade of the innate immune system. In this study, CARD adaptor inducing IFN-β
(CARDIF), a prominent mitochondrial adaptor molecule linking RIG-I and MDA5 is
amplified and exogenously expressed in two chicken cell lines prior to IBS002/11 strain
(genotype VII NDV) infection. The gene expression experiment was conducted to assess
whether the presence of CARDIF affected the production of type I interferons and proinflammatory
cytokines in both cell lines following NDV strain IBS002/11 infection,
besides its role in the induction of apoptosis. The contribution of mitochondrial DNA
(mtDNA) in chicken innate immunity was also assessed following NDV infection. The
assessments of cytokines expressions and mtDNA level were conducted via qPCR assay,
while the determination of cell proliferation and apoptosis were conducted via MTS and
JC-1 assays. Morphological evaluation was conducted by transmission electron
micrography (TEM). All experiments were carried out on two cells lines; DF-1 and HD-
11. The DF-1 cell line is a spontaneously transformed chicken embryo fibroblast while
HD-11 is a chicken macrophage-like cell line. The CARDIF gene was successfully
amplified via PCR and cloned into the pcDNA6V5-His B plasmid, a mammalian
expression vector to produce the plasmid pcDNA6/CARDIF. Meanwhile, two truncated
genes of the original CARDIF gene were also amplified and cloned into the expression vector yielding pcDNA6/ΔCARD (putative CARDIF lacking the CARD domain) and
pcDNA6/ΔTM (putative CARDIF lacking the TM domain). The effects of exogenous
expression of CARDIF on the production of type I and II IFNs and pro-inflammatory
cytokines in both cell lines following NDV infection were conducted via quantitative
polymerase chain reaction (qPCR). Despite upregulated levels of type I IFN (IFN-α and
IFN-β) following NDV infection observed in both cell lines as the effect of exogenous
expression of CARDIF, the expression of IFN-α occurred at a much later time point (72
h) in both cell lines, showing that the production of IFN-α was less affected by the
presence of the CARDIF gene in comparison to IFN-β. HD-11 cells exhibited a greater
magnitude of IFN-β upregulation compared to DF-1 cells. The transfected CARDIF gene
also upregulated the expression of IFN-γ (type II IFN) as well as IL-18 in HD-11 cells
while the same molecules were downregulated in DF-1 cells. CXCLi2, a chemokine was
upregulated in both cell lines while IL-1β was found to be upregulated in DF-1 cells. The
presence of CARDIF resulted in the decreased number of viral copies over the treatment
period in contrast to both truncated CARDIF genes. Both truncated genes caused an
increase in the viral copy number over the treatment period. Subsequently, the mtDNA
levels were detected to be higher (P<0.05) in infected cells compared to the control. Two
mtDNA genes were used in the qPCR assay i.e. Cyt b and COIII whereby the higher
level of mtDNA expression in infected cells means leakage of the mtDNA into the
cytosol. In this study, the leakage of mtDNA to the surroundings assisted in triggering
the production of pro-inflammatory cytokines as well as type I IFN. The proliferation
percentage of the cells was assessed via the MTS assay in two conducts, the conventional
and co-treatment method. A continuous decline of HD-11 cells in contrast to the DF-1
cells in the existence of the CARDIF gene was observed with the co-treatment method
considered as a better approach in determining the percentage of cell proliferation
following virulent NDV infection. The occurrence of CARDIF gene also initiated the
onset of apoptosis as a measure to curb viral infection. The assessment was conducted
via the mitochondrial membrane potential (Δψ) assay (JC-1 assay). The results of JC-1
assay supported those of the MTS assay whereby the decline of cell proliferation
percentage in the MTS assay is in agreement with the increased percentage of apoptotic
cells in the JC-1 assay. The results of the cell proliferation and mitochondrial membrane
potential assays were further confirmed by visual assessment of the cell morphology in
TEM. Noticeable hallmarks of apoptosis such as convolution of nuclear membrane,
condensed chromatin and the presence of lipid droplets were identified in both cell lines.
Therefore, this study concluded that the presence of CARDIF, a mitochondrial adaptor
molecule managed to prompt the production of type I and type II IFNs, as well other proinflammatory
cytokines during infection with virulent NDV strain. The presence of the
exogenous gene also showed better impact on the cell component of the immune system
(HD-11 cells) compared to the stromal cell line (DF-1 cells). The exogenous gene
managed to induce apoptosis in both cell lines, with HD-11 cells being more susceptible
following NDV infection. |
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