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Original Article

Production of IL-1β and Inflammasome with Up-Regulated Expressions of NOD-Like Receptor Related Genes in Toxoplasma gondii-Infected THP-1 Macrophages

Jia-Qi Chu1, Ge Shi2, Yi-Ming Fan2, In-Wook Choi3, Guang-Ho Cha3, Yu Zhou4,*, Young-Ha Lee3,*, Juan-Hua Quan4,*
The Korean Journal of Parasitology 2016;54(6):711-717.
Published online: December 31, 2016

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*Corresponding author (ahdg2005@126.com; yhalee@cnu.ac.kr; qjh0880@163.com)
• Received: October 6, 2016   • Revised: December 5, 2016   • Accepted: December 5, 2016

Copyright © 2016 by The Korean Society for Parasitology and Tropical Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Production of IL-1β and Inflammasome with Up-Regulated Expressions of NOD-Like Receptor Related Genes in Toxoplasma gondii-Infected THP-1 Macrophages
Korean J Parasitol. 2016;54(6):711-717.   Published online December 31, 2016
DOI: https://doi.org/10.3347/kjp.2016.54.6.711
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Production of IL-1β and Inflammasome with Up-Regulated Expressions of NOD-Like Receptor Related Genes in Toxoplasma gondii-Infected THP-1 Macrophages
Korean J Parasitol. 2016;54(6):711-717.   Published online December 31, 2016
DOI: https://doi.org/10.3347/kjp.2016.54.6.711
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Production of IL-1β and Inflammasome with Up-Regulated Expressions of NOD-Like Receptor Related Genes in Toxoplasma gondii-Infected THP-1 Macrophages
Image Image Image Image
Fig. 1 T. gondii triggers the expression and secretion of mature IL-1β. (A, B) THP-1 macrophages were mock-infected or infected with RH strain of T. gondii at an MOI of 10 for 4 hr and 8 hr. IL-1β expression levels were quantified by western blotting (A) and qRT-PCR (B). (C) Culture supernatants were collected, and IL-1β levels were measured in the supernatants by ELISA. Results represent the means ± SD from a representative of 3 independent experiments (*P<0.05 compared with the control).
Fig. 2 T. gondii infection induces caspase-1 activation and elevates NLRP1, NLRP3, NLRC4, and AIM2. (A) THP-1 macrophages were mock-infected or infected with RH strain of T. gondii using different MOIs for 8 hr. Cells were collected, and caspase-1 expression levels were quantified by western blotting. The α-tubulin was used as a loading control. (B) THP-1 macrophages were mock-infected or infected with RH strain of T. gondii at an MOI of 10. Cells were harvested at 4 hr or 8 hr post infection, RNA was extracted, and NLRP1, NLRP3, NLRC4, and AIM2 expressions were determined by qRT-PCR. HPRT1 was used as an internal control. (C) THP-1 macrophages were mock-infected or infected with RH strain of T. gondii at an MOI of 10 for 4 hr or 8 hr. NLRP3 and NLRC4 protein levels in the cells were quantified by western blotting. The α-tubulin was used as a loading control. A representative of 3 independent replicates with similar results is shown (*P<0.05, compared with the control).
Fig. 3 T. gondii upregulates ASC mRNA and protein levels and caspase-1 mRNA levels. (A) THP-1 macrophages were mock-infected or infected with RH strain of T. gondii at an MOI of 10 for 4 hr or 8 hr. ASC protein levels in the cells were quantified by western blotting. The α-tubulin was used as a loading control. (B) THP-1 macrophages were mock-infected or infected with RH strain of T. gondii at an MOI of 10. Cells were harvested at 4 hr or 8 hr post infection, RNA was extracted, and ASC and caspase-1 gene expression was determined by qRT-PCR. HPRT1 was used as an internal control. A representative of 3 independent replicates with similar results is shown (*P<0.05 compared with the control).
Fig. 4 T. gondii infection regulates a subset of NLRP genes and NAIP. THP-1 macrophages were mock-infected or infected with RH strain of T. gondii at an MOI of 10. Cells were harvested at 4 hr or 8 hr post infection, RNA was extracted, and the expression of various genes was determined by qRT-PCR. HPRT1 was used as an internal control. A representative of 3 independent replicates with similar results is shown (*P<0.05, compared with the control).

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Production of IL-1β and Inflammasome with Up-Regulated Expressions of NOD-Like Receptor Related Genes in Toxoplasma gondii-Infected THP-1 Macrophages

Primer sequences used for real-time quantitative reverse transcriptase PCR (qRT-PCR) in this study

Gene name GeneBank accession no. Primer sequence (5′-3′) Product size (bp)
IL-1β NM_000576.2 F-CCACAGACCTTCCAGGAGAA
R-GTGATCGTACAGGTGCATCG		 121
NLRP1 NM_014922.4 F-ATACGAAGCCTTTGGGGACT
R-ACAAAGCAGAGACCCGTGTT		 148
NLRP3 XM_011544055.2 F-AAAGGAAGTGGACTGCGAGA
R-TTCAAACGACTCCCTGGAAC		 129
NLRC4 XM_017004619.1 F-GGAAAGTGCAAGGCTCTGAC
R-TGTCTGCTTCCTGATTGTGC		 129
AIM2 XM_005245616.4 F-AGCCTGAACAGAAACAGATGG
R-CTTCTTGGGTCTCAAACGTGA		 120
ASC NM_145182.2 F-CTGACGGATGAGCAGTACCA
R-CAAGTCCTTGCAGGTCCAGT		 108
CASP1 XM_017018396.1 F-GGGGTACAGCGTAGATGTGAA
R-CTTCCCGAATACCATGAGACA		 137
NLRP6 XM_017017253.1 F-CTGTTCTGAGCTACTGCGTGAG
R-AGGCTCTTCTTCTTCTTCTCCTG		 100
NLRP7 XM_011526601.2 F-TAACCCGTAGCACCTGTCATC
R-GGTCTTCTTCCCAATGAAAGC		 101
NLRP8 NM_001317000.1 F-CGCTGGTGTGCTTTCTACTTC
R-GGTCGGGTTTGGACATAATCT		 130
NLRP9 XM_011526894.2 F-CTAGCCTCTCCCAGTCTGACAT
R-GCGATGTCTTCACAAACTTCAC		 121
NLRP10 XM_011520043.2 F-GTCACGGTGGAGGCTCTATTT
R-CGAGAGTTGTCTTTCCAGTGC		 100
NLRP11 NM_001297743.1 F-TAGATTGCTGCACGACCTTG
R-GGCACAGACCCACGAGTATT		 135
NLRP12 XM_017027467.1 F-CGACCTTTACCTGACCAACAA
R-AGGTCCATCCCAAATAACCAG		 114
NLRP13 NM_001321057.1 F-ATGGTGTGTTGGACCGTATGT
R-GCCAAATCTACCTCTGCTGT		 140
NLRP14 XM_011520044.1 F-CCGCTTGTACTTGTCTGAAGC
R-GCCTCCATCTACTGGTGTGAA		 122
NAIP XM_011543413.2 F-AGTACTTTTTCGACCACCCAGA
R-TAGTTGGCACCTGTGATTTGTC		 135
HPRT1 NM_000194.2 F-GACCAGTCAACAGGGGACAT
R-CTGCATTGTTTTGCCAGTGT		 111
Table 1 Primer sequences used for real-time quantitative reverse transcriptase PCR (qRT-PCR) in this study

Table 1

PHD : Parasites, Hosts and Diseases

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