Please wait a minute...
Journal of Integrative Agriculture  2023, Vol. 22 Issue (10): 3148-3158    DOI: 10.1016/j.jia.2023.06.016
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |
CRISPR/Cas9-mediated knockout of SLC15A4 gene involved in the immune response in bovine rumen epithelial cells
JIANG Mao-cheng1, HU Zi-xuan2, WANG Ke-xin1, YANG Tian-yu1, LIN Miao1, 3, 4, ZHAN Kang1, 3, 4#, ZHAO Guo-qi1, 3, 4#
1 Institute of Animal Culture Collection and Application, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R.China
2 Faculty of Health Sciences, University of Macau, Macau 999078, P.R.China
3 Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou 225009, P.R.China
4 Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education, Yangzhou University, Yangzhou 225009, P.R.China
Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      
摘要  

本研究的目的是确定SLC15A4胞壁酰二肽MDP)介导的奶牛瘤胃上皮细胞(BRECs)炎症反应中的作用。首先,检测了10 μg mL-1 MDP处理后BRECs中促炎因子mRNA表达量的变化。RT-qPCR结果显示,在MDP刺激下,促炎因子(IL-1βIL-6TNF-α)的mRNA表达量显著增加(P < 0.001)。此外,通过慢病毒包装、转染、筛选和细胞单克隆培养获得了SLC15A4-KO细胞系。为了进一步了解SLC15A4的潜在功能,我们利用转录组数据揭示了野生型BRECsSLC15A4-KO之间的基因变化。鉴定出5个下调的促炎基因和13个下调的趋化因子基因与炎症反应有关。同时,下调的基因主要富集在NF-κBMAPK信号通路中。RT-qPCR的结果也证实了这些变化。为了进一步确定WTSLC15A4-KO BREC如何参与炎症反应的机制,我们研究了添加MDPBRECs的炎症反应。与对照相比,添加10 μg mL-1 MDP处理WT BRECSLC15A4-KO后,我们的研究结果表明,与野生型BRECs相比,SLC15A4-KO BRECs降低了炎症反应中(IL-6TNF-αCXCL2CXCL3CXCL9CCL2)和蛋白质(p-p65p-p44/42)的mRNA表达量(P < 0.05)。在本实验中,CRISPR-Cas9系统用于敲除奶牛瘤胃上皮细胞中的SLC15A4基因,其作用通过MDP诱导的BRECs炎症反应得到证实。这项工作将为研究MDP的促炎机制及其在奶牛亚急性瘤胃酸中毒防治工作中的应用提供理论依据。



Abstract  

The objective of this study was to determine the role of SLC15A4 in the muramyl dipeptide (MDP)-mediated inflammatory response of bovine rumen epithelial cells (BRECs).  First, changes in the mRNA expression of pro-inflammatory factor genes in BRECs following 10 μg mL–1 MDP treatments were examined.  RT-qPCR results showed that the expression of pro-inflammatory factor (IL-1β, IL-6, and TNF-α) mRNAs were significantly increased under MDP stimulation (P<0.001).  Moreover, SLC15A4-Knockout (SLC15A4-KO) cells were obtained through lentivirus packaging, transfection, screening, and cell monoclonal culture.  In order to gain further insight into the potential function of SLC15A4, we utilized transcriptome data, which revealed a change in the genes between WT-BRECs and SLC15A4-KO.  Five down-regulated pro-inflammatory genes and 13 down-regulated chemokine genes related to the inflammatory response were identified.  Meanwhile, the down-regulated genes were mostly enriched in the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways.  The results of RT-qPCR also verified these detected changes.  To further determine the mechanism of how WT and SLC15A4-KO BRECs are involved in inflammatory responses, we investigated the inflammatory responses of cells exposed to MDP.  WT-BRECs and SLC15A4-KO were treated with a culture medium containing 10 μg mL–1 MDP, in comparison to a control without MDP.  Our results show that SLC15A4-KO BRECs had reduced the expression of genes (IL-6, TNF-α, CXCL2, CXCL3, CXCL9, and CCL2) and proteins (p-p65 and p-p44/42) from the MDP-mediated inflammatory response compared to WT-BRECs (P<0.05).  In this experiment, CRISPR-Cas9 was used to KO the di/tripeptide transporter SLC15A4, and its role was confirmed via the MDP-induced inflammatory response in BRECs.  This work will provide a theoretical basis for studying the pro-inflammatory mechanism of MDP and its application in the prevention and treatment of subacute rumen acidosis in dairy cows.

Keywords:  SLC15A4        CRISPR/Cas9        immune response        proton-coupled oligopeptide transporter (POT) families        MDP  
Received: 14 February 2023   Accepted: 21 April 2023
Fund: This study was supported by the National Natural Science Foundation of China (31972589), the earmarked fund for China Agriculture Research System (CARS-36) and the Postgraduate Research & Practice Innovation Program of Jiangsu Province, China (KYCX21-3283).
About author:  JIANG Mao-cheng, E-mail: jmcheng1993@163.com; #Correspondence ZHAN Kang, E-mail: kzhan@yzu.edu.cn; ZHAO Guo-qi, Tel: +86-514-87997195, E-mail: gqzhao@yzu.edu.cn

Cite this article: 

JIANG Mao-cheng, HU Zi-xuan, WANG Ke-xin, YANG Tian-yu, LIN Miao, ZHAN Kang, ZHAO Guo-qi. 2023. CRISPR/Cas9-mediated knockout of SLC15A4 gene involved in the immune response in bovine rumen epithelial cells. Journal of Integrative Agriculture, 22(10): 3148-3158.

Andrade A I, Chavez E G, Bautista C R, Ovalle C O, Cabrera M A R, Lagunes A G. 2021. Influence of prebiotic activity of Agave salmiana fructans on mucus production and morphology changes in colonic epithelium cell of healthy wistar rats. Frontiers in Plant Science12, 717460.

Bissa B, Beedle A M, Govindarajan R. 2016. Lysosomal solute carrier transporters gain momentum in research. Clinical Pharmacology & Therapeutics100, 431–436.

Blasius A L, Arnold C N, Georgel P, Rutschmann S, Xia Y, Lin P, Ross C, Li X H, Smart N G, Beutler B. 2010. SLC15A4AP-3, and Hermansky-Pudlak syndrome proteins are required for Toll-like receptor signaling in plasmacytoid dendritic cells. Proceedings of the National Academy of Sciences of the United States of America107, 19973–19978.

Buyse M, Tsocas A, Walker F, Merlin D, Bado A. 2002. PepT1-mediated fMLP transport induces intestinal inflammation in vivoAmerican Journal of Physiology283, C1795–C1800.

Chen G, Shaw M H, Kim Y G, Nunez G. 2009. NOD-like receptors: Role in innate immunity and inflammatory disease. Annual Review of Pathology (Mechanisms of Disease), 4, 365–398.

Cobb R E, Wang Y, Zhao H. 2015. High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synthetic Biology4, 723–728.

Coulombe F, Divangahi M, Veyrier F, de Leseleuc L, Gleason J L, Yang Y B, Kelliher M A, Pandey A K, Sassetti C M, Reed M B, Behr M A. 2009. Increased NOD2-mediated recognition of N-glycolyl muramyl dipeptide. Journal of Experimental Medicine206, 1709–1716.

Dai H, Ma N, Chang G, Aabdin Z U, Shen X. 2022. Long-term high-concentrate diet feeding induces apoptosis of rumen epithelial cells and inflammation of rumen epithelium in dairy cows. Animal Biotechnology33, 289–296.

Dalmasso G, Nguyen H T, Charrier-Hisamuddin L, Yan Y, Laroui H, Demoulin B, Sitaraman S V, Merlin D. 2010. PepT1 mediates transport of the proinflammatory bacterial tripeptide L-Ala-gamma-D-Glu-meso-DAP in intestinal epithelial cells. American Journal of Physiology299, G687–G696.

Edick A M, Audette J, Burgos S A. 2021. CRISPR-Cas9-mediated knockout of GCN2 reveals a critical role in sensing amino acid deprivation in bovine mammary epithelial cells. Journal of Dairy Science104, 1123–1135.

Girardin S E, Boneca I G, Viala J, Chamaillard M, Labigne A, Thomas G, Philpott D J, Sansonetti P J. 2003. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. Journal of Biological Chemistry278, 8869–8872.

van der Goot A T, Pearce M M P, Leto D E, Shaler T A, Kopito R R. 2018. Redundant and antagonistic roles of XTP3B and OS9 in decoding glycan and non-glycan degrons in ER-associated degradation. Molecular Cell70, 516–530.

Han J W, Zheng H F, Cui Y, Sun L D, Ye D Q, Hu Z, Xu J H, Cai Z M, Huang W, Zhao G P, Xie H F, Fang H, Lu Q J, Xu J H, Li X P, Pan Y F, Deng D Q, Zeng F Q, Ye Z Z, Zhang X Y, et al. 2009. Genome-wide association study in a Chinese Han population identifies nine new susceptibility loci for systemic lupus erythematosus. Nature Genetics41, U1234–U1298.

Han L, Zhang F, Liu Y, Yu J, Zhang Q, Ye X, Song H, Zheng C, Han B. 2022. Uterus globulin associated protein 1 (UGRP1) binds podoplanin (PDPN) to promote a novel inflammation pathway during Streptococcus pneumoniae infection. Clinical and Translational Medicine12, e850.

Han L, Zhang M, Xing Z, Coleman D N, Liang Y, Loor J J, Yang G. 2020. Knockout of butyrophilin subfamily 1 member A1 (BTN1A1) alters lipid droplet formation and phospholipid composition in bovine mammary epithelial cells. Journal of Animal Science and Biotechnology11, 72.

He C F, Liu Y S, Cheng Y L, Gao J P, Pan T M, Han J W, Quan C, Sun L D, Zheng H F, Zuo X B, Xu S X, Sheng Y J, Yao S, Hu W L, Li Y, Yu Z Y, Yin X Y, Zhang X J, Cui Y, Yang S. 2010. TNIP1SLC15A4ETS1RasGRP3 and IKZF1 are associated with clinical features of systemic lupus erythematosus in a Chinese Han population. Lupus19, 1181–1186.

Hitotsumatsu O, Ahmad R C, Tavares R, Wang M, Philpott D, Turer E E, Lee B L, Shiffin N, Advincula R, Malynn B A, Werts C, Ma A. 2008. The ubiquitin-editing enzyme A20 restricts nucleotide-binding oligomerization domain containing 2-triggered signals. Immunity28, 381–390.

Hu Y J, Song F F, Jiang H D, Nunez G, Smith D E. 2018. SLC15A2 and SLC15A4 mediate the transport of bacterially derived di/tripeptides to enhance the nucleotide-binding oligomerization domain-dependent immune response in mouse bone marrow-derived macrophages. Journal of Immunology201, 652–662.

Jin J, Hu H B, Li H Y S, Yu J Y, Xiao Y C, Brittain G C, Zou Q, Cheng X H, Mallette F A, Watowich S S, Sun S C. 2014. Noncanonical NF-kappa B pathway controls the production of type I interferons in antiviral innate immunity. Immunity40, 342–354.

Kent-Dennis C, Aschenbach J R, Griebel P J, Penner G B. 2020. Effects of lipopolysaccharide exposure in primary bovine ruminal epithelial cells. Journal of Dairy Science103, 9587–9603.

Kieser K J, Kagan J C. 2017. Multi-receptor detection of individual bacterial products by the innate immune system. Nature Reviews Immunology17, 376–390.

Lee J, Tattoli I, Wojtal K A, Vavricka S R, Philpott D J, Girardin S E. 2009. pH-dependent internalization of muramyl peptides from early endosomes enables Nod1 and Nod2 signaling. Journal of Biological Chemistry284, 23818–23829.

Lee P Y, Kumagai Y, Li Y, Takeuchi O, Yoshida H, Weinstein J, Kellner E S, Nacionales D, Barker T, Kelly-Scumpia K, van Rooijen N, Kumar H, Kawai T, Satoh M, Akira S, Reeves W H. 2008. TLR7-dependent and Fc gamma R-independent production of type I interferon in experimental mouse lupus. Journal of Experimental Medicine205, 2995–3006.

Li Q, Lee C H, Peters L A, Mastropaolo L A, Thoeni C, Elkadri A, Schwerd T, Zhu J, Zhang B, Zhao Y Z, Hao K, Dinarzo A, Hoffman G, Kidd B A, Murchie R, Al Adham Z, Guo C H, Kotlarz D, Cutz E, Walters T D, et al. 2016. Variants in TRIM22 that affect NOD2 signaling are associated with very-early-onset inflammatory bowel disease. Gastroenterology150, 1196–1207.

Marina-Garcia N, Franchi L, Kim Y G, Hu Y, Smith D E, Boons G J, Nunez G. 2009. Clathrin- and dynamin-dependent endocytic pathway regulates muramyl dipeptide internalization and NOD2 activation. Journal of Immunology182, 4321–4327.

Nakamura N, Lill J R, Phung Q, Jiang Z, Bakalarski C, de Maziere A, Klumperman J, Schlatter M, Delamarre L, Mellman I. 2014. Endosomes are specialized platforms for bacterial sensing and NOD2 signalling. Nature509, 240–244.

Niu Q, Zheng H, Zhang L, Qin F, Facemire L, Zhang G, Cao F, Zhang K Q, Huang X, Yang J, He L, Liu C. 2015. Knockout of the adp gene related with colonization in Bacillus nematocida B16 using customized transcription activator-like effectors nucleases. Microbial Biotechnology8, 681–692.

Sanjana N E, Shalem O, Zhang F. 2014. Improved vectors and genome-wide libraries for CRISPR screening. Nature Methods11, 783–784.

Sasawatari S, Okamura T, Kasumi E, Tanaka-Furuyama K, Yanobu-Takanashi R, Shirasawa S, Kato N, Toyama-Sorimachi N. 2011. The SLC15A4 regulates TLR9 and NOD1 functions in the innate immune system and promotes colitis in mice. Gastroenterology140, 1513–1525.

Smith D E, Clemencon B, Hediger M A. 2013. Proton-coupled oligopeptide transporter family SLC15: Physiological, pharmacological and pathological implications. Molecular Aspects of Medicine34, 323–336.

Solcan N, Kwok J, Fowler P W, Cameron A D, Drew D, Iwata S, Newstead S. 2012. Alternating access mechanism in the POT family of oligopeptide transporters. EMBO Journal31, 3411–3421.

Steele M A, Penner G B, Chaucheyras-Durand F, Guan L L. 2016. Development and physiology of the rumen the lower gut: Targets for improving gut health. Journal of Dairy Science99, 4955–4966.

Strober W, Murray P J, Kitani A, Watanabe T. 2006. Signalling pathways and molecular interactions of NOD1 and NOD2Nature Reviews Immunology6, 9–20.

Tian H, Luo J, Zhang Z, Wu J, Zhang T, Busato S, Huang L, Song N, Bionaz M. 2018. CRISPR/Cas9-mediated Stearoyl-CoA Desaturase 1 (SCD1) deficiency affects fatty acid metabolism in goat mammary epithelial cells. Journal of Agricultural and Food Chemistry66, 10041–10052.

Trosvik P, de Muinck E J, Rueness E K, Fashing P J, Beierschmitt E C, Callingham K R, Kraus J B, Trew T H, Moges A, Mekonnen A, Venkataraman V V, Nguyen N. 2018. Multilevel social structure and diet shape the gut microbiota of the gelada monkey, the only grazing primate. Microbiome6, 84.

Tyshkovskiy A, Bozaykut P, Borodinova A A, Gerashchenko M V, Ables G P, Garratt M, Khaitovich P, Clish C B, Miller R A, Gladyshev V N. 2019. Identification and application of gene expression signatures associated with lifespan extension. Cell Metabolism30, 573–593.

Vavricka S R, Musch M W, Chang J E, Nakagawa Y, Phanvijhitsiri K, Waypa T S, Merlin D, Schneewind O, Chang E B. 2004. HPepT1 transports muramyl dipeptide, activating NF-kappaB stimulating IL-8 secretion in human colonic Caco2/bbe cells. Gastroenterology127, 1401–1409.

Wang C, Ahlford A, Jarvinen T M, Nordmark G, Eloranta M L, Gunnarsson I, Svenungsson E, Padyukov L, Sturfelt G, Jonsen A, Bengtsson A A, Truedsson L, Eriksson C, Rantapaa-Dahlqvist S, Sjowall C, Julkunen H, Criswell L A, Graham R R, Behrens T W, Kere J, et al. 2013. Genes identified in Asian SLE GWASs are also associated with SLE in Caucasian populations. European Journal of Human Genetics21, 994–999.

Wu S P Smith D E. 2013. Impact of intestinal PepT1 on the kinetics dynamics of N-formyl-methionyl-leucyl-phenylalanine, a bacterially-produced chemotactic peptide. Molecular Pharmaceutics10, 677–684.

Zhan K, Gong X, Chen Y, Jiang M, Yang T, Zhao G. 2019. Short-chain fatty acids regulate the immune responses via G protein-coupled receptor 41 in bovine rumen epithelial cells. Frontiers in Immunology10, 2042.

[1] OUYANG Chun-zheng, YE Fan, WU Qing-jun, WANG Shao-li, Neil CRICKMORE, ZHOU Xu-guo, GUO Zhao-jiang, ZHANG You-jun. CRISPR/Cas9-based functional characterization of PxABCB1 reveals its roles in the resistance of Plutella xylostella (L.) to Cry1Ac, abamectin and emamectin benzoate[J]. >Journal of Integrative Agriculture, 2023, 22(10): 3090-3102.
No Suggested Reading articles found!