Skip to main content
NCBI home page
Cellular & Molecular Biology Letters logoLink to Cellular & Molecular Biology Letters
. 2014 Oct 29;19(4):623–637. doi: 10.2478/s11658-014-0217-1

The effect of buffalo CD14 shRNA on the gene expression of TLR4 signal pathway in buffalo monocyte/macrophages

Xiangping Li 1,✉, Meiqing Li 1, Shihai Huang 2, Shuye Qiao 1, Zhaoxian Qin 3, Chao Kang 2, Deshun Shi 1,✉
PMCID: PMC6275898  PMID: 25355240

Abstract

CD14 plays a crucial role in the inflammatory response to lipopolysaccharide (LPS), which interacts with TLR4 and MD-2 to enable cell activation, resulting in inflammation. Upstream inhibition of the inflammation pathway mediated by bacterial LPS, toll-like receptor 4 (TLR4) and cluster of differentiation antigen 14 (CD14) was proven to be an effective therapeutic approach for attenuating harmful immune activation. To explore the effect of CD14 downregulation on the expression of TLR4 signaling pathway-related genes after LPS stimulation in buffalo (Bubalus bubalis) monocyte/macrophages, effective CD14 shRNA sequences were screened using qRT-PCR and FACS analysis with buffalo CD14 shRNA lentiviral recombinant plasmids (pSicoRGFP-shRNA) and buffalo CD14 fusion expression plasmids (pDsRed-N1-buffalo CD14) co-transfected into HEK293T cells via liposomes. Of the tested shRNAs, shRNA-1041 revealed the highest knockdown efficiency (p < 0.01). When buffalo peripheral blood monocyte/macrophages were infected with shRNA-1041 lentivirus and stimulated with LPS, the expression of endogenous CD14 was significantly decreased by CD14 shRNA (p < 0.01), and the mRNA expression levels of TLR4, IL-6 and TNF-α were also significantly downregulated compared to the control groups (p < 0.01). These results demonstrated that the knockdown of endogenous CD14 had clear regulatory effects on the signal transduction of TLR4 after stimulation with LPS. These results may provide a better understanding of the molecular mechanisms of CD14 regulation in the development of several buffalo diseases.

Keywords: Buffalo CD14 gene, shRNA, TLR4 signal pathway, Gene expression, Monocyte/macrophages

Full Text

The Full Text of this article is available as a PDF (3.6 MB).

Abbreviations used

CD14

cluster of differentiation antigen 14

IL-6

interleukin-6

LPS

lipopolysaccharide

MOI

multiplicity of infection

NF-KB

nuclear factor kappaB

shRNA

short hair RNA

TLR

toll-like receptor

TNF-α

tumor necrosis factor-α

Contributor Information

Xiangping Li, Email: xiangpingli@163.com.

Deshun Shi, Email: ardsshi@gxu.edu.cn.

References

  • 1.Simmons DL, Tan S, Tenen DG, Nicholson-Weller A, Seed B. Monocyte antigen CD14 is a phospholipid anchored membrane protein. Blood. 1989;73:284–289. [PubMed] [Google Scholar]
  • 2.Ziegler-Heitbrock H, Ulevitch R. CD14: cell surface receptor and differentiation marker. Immunol. Today. 1993;14:121–125. doi: 10.1016/0167-5699(93)90212-4. [DOI] [PubMed] [Google Scholar]
  • 3.Le Roy D, Di Padova F, Adachi Y, Glauser MP, Calandra T, Heumann D. Critical role of lipopolysaccharide-binding protein and CD14 in immune responses against Gram-negative bacteria. J. Immunol. 2001;167:2759–2765. doi: 10.4049/jimmunol.167.5.2759. [DOI] [PubMed] [Google Scholar]
  • 4.Verbon A, Dekkers PE, ten Hove T, Hack CE, Pribble JP, Turner T, Souza S, Axtelle T, Hoek FJ, van Deventer SJ. IC14, an anti-CD14 antibody, inhibits endotoxin-mediated symptoms and inflammatory responses in humans. J. Immunol. 2001;166:3599–3605. doi: 10.4049/jimmunol.166.5.3599. [DOI] [PubMed] [Google Scholar]
  • 5.Krombach F, Münzing S, Allmeling AM, Gerlach JT, Behr J, Dörger M. Cell size of alveolar macrophages: an interspecies comparison. Environ. Health Perspect. 1997;105:1261. doi: 10.1289/ehp.97105s51261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Swangchan-Uthai T, Lavender C R, Cheng Z, Fouladi-Nashta AA, Wathes DC. Time course of defense mechanisms in bovine endometrium in response to lipopolysaccharide 1. Biol. Reprod. 2012;87:135. doi: 10.1095/biolreprod.112.102376. [DOI] [PubMed] [Google Scholar]
  • 7.Miyake K. Innate recognition of lipopolysaccharide by toll-like receptor 4-MD-2. Trends Microbiol. 2004;12:186–192. doi: 10.1016/j.tim.2004.02.009. [DOI] [PubMed] [Google Scholar]
  • 8.Miyake K. Innate immune sensing of pathogens and danger signals by cell surface toll-like receptors. Semin. Immunol. 2007;19:3–10. doi: 10.1016/j.smim.2006.12.002. [DOI] [PubMed] [Google Scholar]
  • 9.Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell. 2010;140:805–820. doi: 10.1016/j.cell.2010.01.022. [DOI] [PubMed] [Google Scholar]
  • 10.Islam MA, Cinar MU, Uddin MJ, Tholen E, Tesfaye D, Looft C, Schellander K. Expression of toll-like receptors and downstream genes in lipopolysaccharide-induced porcine alveolar macrophages. Vet. Immunol. Immunopathol. 2012;146:62–73. doi: 10.1016/j.vetimm.2012.02.001. [DOI] [PubMed] [Google Scholar]
  • 11.Nagaoka I, Hirota S, Niyonsaba F, Hirata M, Adachi Y, Tamura H, Heumann D. Cathelicidin family of antibacterial peptides CAP18 and CAP11 inhibit the expression of TNF-alpha by blocking the binding of LPS to CD14 (+) cells. J. Immunol. 2001;167:3329–3338. doi: 10.4049/jimmunol.167.6.3329. [DOI] [PubMed] [Google Scholar]
  • 12.Loyi T, Kumar H, Nandi S, Mathapati BS, Patra M, Pattnaik B. Differential expression of pro-inflammatory cytokines in endometrial tissue of buffaloes with clinical and sub-clinical endometritis. Res. Vet. Sci. 2013;94:336–340. doi: 10.1016/j.rvsc.2012.09.008. [DOI] [PubMed] [Google Scholar]
  • 13.He Y, Reichow S, Ramamoorthy S, Ding X, Lathigra R, Craig JC, Sobral BW, Schurig GG, Sriranganathan N, Boyle SM. Brucella melitensis triggers time-dependent modulation of apoptosis and downregulation of mitochondrion-associated gene expression in mouse macrophages. Infect. Immun. 2006;74:5035–5046. doi: 10.1128/IAI.01998-05. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Kiku Y, Ozawa T, Kushibiki S, Sudo M, Kitazaki K, Abe N, Takahashi H, Hayashi T. Decrease in bovine CD14 positive cells in colostrum is associated with the incidence of mastitis after calving. Vet. Res. Commun. 2010;34:197–203. doi: 10.1007/s11259-009-9339-8. [DOI] [PubMed] [Google Scholar]
  • 15.Lichtman SN, Wang J, Lemasters JJ. LPS receptor CD14 participates in release of TNF-α in RAW 264.7 and peritoneal cells but not in Kupffer cells. Am. J. Physiol-gastr. L. 1998;275:G39–G46. doi: 10.1152/ajpgi.1998.275.1.G39. [DOI] [PubMed] [Google Scholar]
  • 16.Buczynski MW, Stephens DL, Bowers-Gentry RC, Grkovich A, Deems RA, Dennis EA. TLR-4 and sustained calcium agonists synergistically produce eicosanoids independent of protein synthesis in RAW264. 7 cells. J. Biol. Chem. 2007;282:22834–22847. doi: 10.1074/jbc.M701831200. [DOI] [PubMed] [Google Scholar]
  • 17.Avni D, Ernst O, Philosoph A, Zor T. Role of CREB in modulation of TNF and IL-10 expression in LPS-stimulated RAW264. 7 macrophages. Mol. Immunol. 2010;47:1396–1403. doi: 10.1016/j.molimm.2010.02.015. [DOI] [PubMed] [Google Scholar]
  • 18.Lei M, Jiao H, Liu T, Du L, Cheng Y, Zhang D, Hao Y, Man C, Wang F. siRNA targeting mCD14 inhibits TNF-α, MIP-2, and IL-6 secretion and NO production from LPS-induced RAW264. 7 cells. Appl. Microbiol. Biotechnol. 2011;92:115–124. doi: 10.1007/s00253-011-3371-7. [DOI] [PubMed] [Google Scholar]
  • 19.Cronin JG, Turner ML, Goetze L, Bryant CE, Sheldon IM. Tolllike receptor 4 and MYD88-dependent signaling mechanisms of the innate immune system are essential for the response to lipopolysaccharide by epithelial and stromal cells of the bovine endometrium. Biol. Reprod. 2011;86:51. doi: 10.1095/biolreprod.111.092718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Hannon GJ. RNA interference. Nature. 2002;418:244–251. doi: 10.1038/418244a. [DOI] [PubMed] [Google Scholar]
  • 21.Moffat J, Sabatini DM. Building mammalian signaling pathways with RNAi screens. Nat. Rev. Mol. Cell. Biol. 2006;7:177–187. doi: 10.1038/nrm1860. [DOI] [PubMed] [Google Scholar]
  • 22.Meade B, Dowdy S. The road to therapeutic RNA interference (RNAi): Tackling the 800 pound siRNA delivery gorilla. Discov. Med. 2009;8:253. [PubMed] [Google Scholar]
  • 23.Kafri T, Blömer U, Peterson DA, Gage FH, Verma IM. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat. Genet. 1997;17:314–317. doi: 10.1038/ng1197-314. [DOI] [PubMed] [Google Scholar]
  • 24.Olivier M, Berthon P, Chastang J, Cordier G, Lantier F. Establishment and characterisation of ovine blood monocyte-derived cell lines. Vet. Immunol. Immunopathol. 2001;82:139–151. doi: 10.1016/s0165-2427(01)00330-0. [DOI] [PubMed] [Google Scholar]
  • 25.Belosevic M, Hanington PC, Barreda DR. Development of goldfish macrophages in vitro. Fish Shellfish Immunol. 2006;20:152–171. doi: 10.1016/j.fsi.2004.10.010. [DOI] [PubMed] [Google Scholar]
  • 26.Wang XH, Wang Y, Diao F, Lu J. RhoB is involved in lipopolysaccharide-induced inflammation in mouse in vivo and in vitro. J. Physiol. Biochem. 2013;69:189–197. doi: 10.1007/s13105-012-0201-z. [DOI] [PubMed] [Google Scholar]
  • 27.Song Y, Dou H, Gong W, Liu X, Yu Z, Li E, Tan R, Hou Y. Bis-N-norgliovictin, a small-molecule compound from marine fungus, inhibits LPS-induced inflammation in macrophages and improves survival in sepsis. Eur. J. Pharmacol. 2013;705:49–60. doi: 10.1016/j.ejphar.2013.02.008. [DOI] [PubMed] [Google Scholar]
  • 28.Haziot A, Ferrero E, Köntgen F, Hijiya N, Yamamoto S, Silver J, Stewart CL, Goyert SM. Resistance to endotoxin shock and reduced dissemination of Gram-negative bacteria in CD14-deficient mice. Immunity. 1996;4:407–414. doi: 10.1016/s1074-7613(00)80254-x. [DOI] [PubMed] [Google Scholar]
  • 29.Liu J, Bátkai S, Pacher P, Harvey-White J, Wagner JA, Cravatt BF, Gao B, Kunos G. Lipopolysaccharide induces anandamide synthesis in macrophages via CD14/MAPK/phosphoinositide 3-kinase/NF-κB independently of platelet-activating factor. J. Biol. Chem. 2003;278:45034–45039. doi: 10.1074/jbc.M306062200. [DOI] [PubMed] [Google Scholar]
  • 30.Sanz G, Pérez E, Jiménez-Marín A, Mompart F, Morera L, Barbancho M, Llanes D, Garrido JJ. Molecular cloning, chromosomal location, and expression analysis of porcine CD14. Dev. Comp. Immunol. 2007;31:738–747. doi: 10.1016/j.dci.2006.10.006. [DOI] [PubMed] [Google Scholar]
  • 31.Gomes N, Brunialti M, Mendes M, Freudenberg M, Galanos C, Salomao R. Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood. Braz. J. Med. Biol. Res. 2010;43:853–858. doi: 10.1590/s0100-879x2010007500078. [DOI] [PubMed] [Google Scholar]
  • 32.Mansouri-Attia N, Oliveira LJ, Forde N, Fahey AG, Browne JA, Roche JF, Sandra O, Reinaud P, Lonergan P, Fair T. Pivotal role for monocytes/macrophages and dendritic cells in maternal immune response to the developing embryo in cattle. Biol. Reprod. 2012;87:123. doi: 10.1095/biolreprod.112.101121. [DOI] [PubMed] [Google Scholar]

Articles from Cellular & Molecular Biology Letters are provided here courtesy of BMC

RESOURCES