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Comparative characterization of two interleukin-6 family members in redlip mullet (Liza haematocheila) and insights to their functions in regulating inflammation and apoptosis through STAT3 signaling during pathogenic invasion

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Abstract
The immune system is pivotal in protecting the organism from the exogenous pathogens and other damage associated reactions and molecules. Interleukins are a subgroup of cytokines that can modulate the immune system and involves in critical biological processes. The interleukin 6 family or IL-6 family, in short, has a number of key players in inflammation and other immune responses. IL-6, IL-11, IL-27, IL-31, Leptin, Leukemia Inhibitory Factor (LIF), Cardiotrophin-1 and Ciliary neurotrophic factor (CNTF) are among them. IL-6 acts as antiinflammatory cytokine through soluble IL-6R receptors. Moreover, IL-6 is serving as a proinflammatory cytokine with trans-signaling, through gp130. IL-11 continues as antiinflammatory and anti-apoptotic cytokine during the inflammation.
In the view of signal transduction accomplishes by both IL-6 and IL-11, they can bind to the specific transmembrane receptor, IL-6 or IL-11 receptor alpha denoted as IL-6Rα or IL-11Rα. Thereby they can activate the JAK-STAT3 pathway. IL6/ IL-6Rα or IL-11/IL-11Rα heterodimers trigger the activation of gp130 in the canonical signaling cascade. The complex of IL protein/ its corresponding receptor and gp130 protein further activates JACK family tyrosine kinases. This catalyzes phosphorylation and translocation of STAT3. The translocated STAT3 can activate transcription of target genes.
The current study was conducted to characterize teleostean IL-6 and IL-11 orthologues from redlip mullet and determine their functional role in mullet kidney cells. Online bioinformatics tools and databases were used together with software to identify characteristic features of the sequences of redlip mullet IL-6 and IL-11. Expression levels of both IL-6 and IL-11 in different tissues of redlip mullet were examined. Groups of healthy redlip mullets were challenged with lipopolysaccharide (LPS), Lactococcus garviae and polyinosinicpolycytidylic acid (poly I:C). mRNA was extracted, synthesized cDNA was subjected to quantitative real-time PCR in order to determine expression levels of both interleukins underimmune stimulants. Recombinant IL-6 or IL-11 proteins was overexpressed in Escherichia coli BL21 strain and purified using pMAL™ protein fusion and purification system. The purified IL-6 or IL-11 proteins were treated to mullet kidney cells in separate experiments with MBP control. LPS was used to induce inflammation in the cells. Cells were harvested in 0, 1, 3, 6 and 12-hour time points and subjected to mRNA extraction. First strand cDNA was synthesized and qPCR was employed to determine the TNF-α, IL-1β, BCL2 and BAX gene transcription levels in mullet kidney cells during the experiment. Furthermore, the recombinant two interleukins were treated to murine macrophage RAW 264.7 cells to investigate the effect on nitric oxide production.
The redlip mullet IL-6 and IL-11 are composed with 218 and 200 of amino acids respectively. Their predicted molecular weights were 24.6 kDa and 23.168 kDa respectively.
The Multiple sequence alignment indicates that both sequences are barely conserved during the evolution. Both of IL-6 and IL-11 of redlip mullet composed with four bundle architecture of α-helixes, as other corresponding orthologues. Identity–similarity matrix demonstrated that a higher identity with fish counterparts but less with other phyla.
Phylogenetic analysis demonstrated that IL-6 and IL-11 have clustered with their fish counterparts respectively. This is the first attempt to clone and characterize both recombinant IL-6 and IL-11 from redlip mullet Liza haematocheila. The treatment of recombinant protein into mullet kidney cells has demonstrated that recombinant IL-6 involves in upregulating proinflammatory TNF-α, IL-1β significantly. Recombinant IL-11 protein can downregulate the pro-inflammatory TNF-α, IL-1β, and apoptotic BCL2, BAX gene expression in mullet kidney cells. Furthermore, the effect of overexpression of IL-11on STAT-3 signaling in mullet kidney cells was investigated. The experiment revealed that a high dose of IL-11 negatively effects on the transcription of STAT-3 signaling components such as IL-11R, STAT3. However, the transcription of the SOCS3 gene also downregulated by the IL-11 treatment. Additionally, theresults of the current study revealed recombinant IL-6 can induce the inflammatory NO production in Murine RAW 264.7 cells in-vitro, while recombinant IL-11 can reduce the NO production. The overexpression of IL-11 may significantly change the conditions of mullet kidney cells and RAW264.7 cells. Therefore the cells may change the transcription of downstream genes to make a balance in the next stage.
Author(s)
Kasthuri Arachchige Thilina Duminda Weerasingh
Issued Date
2019
Awarded Date
2019. 8
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/common/orgView/000000009017
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
Lee, Je Hee
Table Of Contents
Acknowledgments. iv
Summary v
Table of content . viii
List of tables . x
List of figures xi
1. Introduction 1
1.1 Cytokines, Interleukins, and IL-6 family 1
1.2 Interleukin 6 . 1
1.3 Interleukin 11 . 2
1.4 Signal transduction of IL-6 and IL-11 . 3
1.5 Significance of STAT3 transcription factor in the mediation of inflammation . 5
1.6 Redlip mullet aquaculture in South Korea . 6
2. Material and methods . 7
2.1 Rearing of fish and tissue collection 7
2.2 Transcriptomic database construction 7
2.3 Bioinformatics analysis 7
2.4 Immune challenge experiment . 8
2.5 Total RNA isolation and cDNA synthesis . 8
2.6 Analysis of spatial and temporal IL-6 and IL-11expression variation by real-time PCR (qRT-PCR) . 8
2.7 Plasmid construction for recombinant LhIL-6 and LhIL-11a production 9
2.8 Overexpression, and purification of rLhIL-6 and rLhIL-11a 10
2.8 rLhIL-11a treatment in mullet kidney cells . 11
2.8.1 Primary culture of mullet kidney cells 11
2.8.2 rLhIL-6 or rLhIL-11a treatment and qRT-PCR for pro-inflammatory and apoptotic gene expression. . 12
2.9 NO production assay and MTT assay 13
3. Results and Discussion 14
3.1 LhIL-11a sequence analysis . 14
3.2 Homology analysis of LhIL-6 and LhIL-11a . 15
(B) Identity similarity of LhIL-11a 16
3.3 Sequence alignment of LhIL-6 and LhIL-11a with other known homologs . 16
Multiple 17
3.4 phylogenetic relationship reconstruction of LhIL-6 and LhIL-11a 18
3.5 Three dimensional (3D) modeling of LhIL-6 and LhIL-11a tertiary structure. 19
3.6 Expression of LhIL-6 and LhIL-11a in different redlip mullet tissues 20
3.7 LhIL-6 and LhIL-11a expression under simulated pathogenic stress in mullet. 22
3.8 Bacterial overexpression and recombinant protein purification 26
3.9 Biological activity of rLhIL-6 and rLhIL-11a on red lip mullet kidney cells . 27
3.9.1 Regulation of pro-inflammatory cytokine gene expression 27
3.9.2 Regulation of anti-apoptotic BCL2 and pro-apoptotic BAX gene expression . 30
3.9.3 Feedback transcription response of STAT3 signaling cascade components to IL-11a ligand 34
3.10 NO production assay in murine macrophage (RAW264.7) cells . 36
4. Conclusion . 39
References 40
Degree
Master
Publisher
제주대학교 대학원
Citation
Kasthuri Arachchige Thilina Duminda Weerasingh. (2019). Comparative characterization of two interleukin-6 family members in redlip mullet (Liza haematocheila) and insights to their functions in regulating inflammation and apoptosis through STAT3 signaling during pathogenic invasion
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General Graduate School > Marine Life Sciences
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