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

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.

Abbreviations

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

References

  1. Simmons, D.L., Tan. S., Tenen, D.G., Nicholson-Weller, A. and Seed, B. Monocyte antigen CD14 is a phospholipid anchored membrane protein. Blood 73 (1989) 284–289.

    CAS  PubMed  Google Scholar 

  2. Ziegler-Heitbrock, H. and Ulevitch, R. CD14: cell surface receptor and differentiation marker. Immunol. Today 14 (1993) 121–125.

    CAS  PubMed  Article  Google Scholar 

  3. Le Roy, D., Di Padova, F., Adachi, Y., Glauser, M.P., Calandra, T. and Heumann, D. Critical role of lipopolysaccharide-binding protein and CD14 in immune responses against Gram-negative bacteria. J. Immunol. 167 (2001) 2759–2765.

    PubMed  Article  Google Scholar 

  4. Verbon, A., Dekkers, P.E., ten Hove, T., Hack, C.E., Pribble, J.P., Turner, T., Souza, S., Axtelle, T., Hoek, F.J. and van Deventer, S.J. IC14, an anti-CD14 antibody, inhibits endotoxin-mediated symptoms and inflammatory responses in humans. J. Immunol. 166 (2001) 3599–3605.

    CAS  PubMed  Article  Google Scholar 

  5. Krombach, F., Münzing, S., Allmeling, A.M., Gerlach, J.T., Behr, J. and Dörger, M. Cell size of alveolar macrophages: an interspecies comparison. Environ. Health Perspect. 105 (1997) 1261.

    PubMed Central  PubMed  Article  Google Scholar 

  6. Swangchan-Uthai, T., Lavender, C. R., Cheng, Z., Fouladi-Nashta, A.A. and Wathes, D.C. Time course of defense mechanisms in bovine endometrium in response to lipopolysaccharide 1. Biol. Reprod. 87 (2012) 135.

    PubMed  Article  Google Scholar 

  7. Miyake, K. Innate recognition of lipopolysaccharide by toll-like receptor 4-MD-2. Trends Microbiol. 12 (2004) 186–192.

    CAS  PubMed  Article  Google Scholar 

  8. Miyake, K. Innate immune sensing of pathogens and danger signals by cell surface toll-like receptors. Semin. Immunol. 19 (2007) 3–10.

    CAS  PubMed  Article  Google Scholar 

  9. Takeuchi, O. and Akira, S. Pattern recognition receptors and inflammation. Cell 140 (2010) 805–820.

    CAS  PubMed  Article  Google Scholar 

  10. Islam, M.A., Cinar, M.U., Uddin, M.J., Tholen, E., Tesfaye, D., Looft, C. and Schellander, K. Expression of toll-like receptors and downstream genes in lipopolysaccharide-induced porcine alveolar macrophages. Vet. Immunol. Immunopathol. 146 (2012) 62–73.

    CAS  PubMed  Article  Google Scholar 

  11. Nagaoka, I., Hirota, S., Niyonsaba, F., Hirata, M., Adachi, Y., Tamura, H. and 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. 167 (2001) 3329–3338.

    CAS  PubMed  Article  Google Scholar 

  12. Loyi, T., Kumar, H., Nandi, S., Mathapati, B.S., Patra, M. and Pattnaik, B. Differential expression of pro-inflammatory cytokines in endometrial tissue of buffaloes with clinical and sub-clinical endometritis. Res. Vet. Sci. 94 (2013) 336–340.

    CAS  PubMed  Article  Google Scholar 

  13. He, Y., Reichow, S., Ramamoorthy, S., Ding, X., Lathigra, R., Craig, J.C., Sobral, B.W., Schurig, G.G., Sriranganathan, N. and Boyle, S.M. Brucella melitensis triggers time-dependent modulation of apoptosis and downregulation of mitochondrion-associated gene expression in mouse macrophages. Infect. Immun. 74 (2006) 5035–5046.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  14. Kiku, Y., Ozawa, T., Kushibiki, S., Sudo, M., Kitazaki, K., Abe, N., Takahashi, H. and Hayashi, T. Decrease in bovine CD14 positive cells in colostrum is associated with the incidence of mastitis after calving. Vet. Res. Commun. 34 (2010) 197–203.

    PubMed  Article  Google Scholar 

  15. Lichtman, S.N., Wang, J. and Lemasters, J.J. 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. 275 (1998) G39–G46.

    CAS  Google Scholar 

  16. Buczynski, M.W., Stephens, D.L., Bowers-Gentry, R.C., Grkovich, A., Deems, R.A. and Dennis, E.A. TLR-4 and sustained calcium agonists synergistically produce eicosanoids independent of protein synthesis in RAW264. 7 cells. J. Biol. Chem. 282 (2007) 22834–22847.

    CAS  PubMed  Article  Google Scholar 

  17. Avni, D., Ernst, O., Philosoph, A. and Zor, T. Role of CREB in modulation of TNF and IL-10 expression in LPS-stimulated RAW264. 7 macrophages. Mol. Immunol. 47 (2010) 1396–1403.

    CAS  PubMed  Article  Google Scholar 

  18. Lei, M., Jiao, H., Liu, T., Du, L., Cheng, Y., Zhang, D., Hao, Y., Man, C. and 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. 92 (2011) 115–124.

    CAS  PubMed  Article  Google Scholar 

  19. Cronin, J.G., Turner, M.L., Goetze, L., Bryant, C.E. and Sheldon, I.M. 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. 86 (2011) 51.

    Article  Google Scholar 

  20. Hannon, G.J. RNA interference. Nature 418 (2002) 244–251.

    CAS  PubMed  Article  Google Scholar 

  21. Moffat, J. and Sabatini, D.M. Building mammalian signaling pathways with RNAi screens. Nat. Rev. Mol. Cell. Biol. 7 (2006) 177–187.

    CAS  PubMed  Article  Google Scholar 

  22. Meade, B. and Dowdy, S. The road to therapeutic RNA interference (RNAi): Tackling the 800 pound siRNA delivery gorilla. Discov. Med. 8 (2009) 253.

    PubMed  Google Scholar 

  23. Kafri, T., Blömer, U., Peterson, D.A., Gage, F.H. and Verma, I.M. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat. Genet. 17 (1997) 314–317.

    CAS  PubMed  Article  Google Scholar 

  24. Olivier, M., Berthon, P., Chastang, J., Cordier, G. and Lantier, F. Establishment and characterisation of ovine blood monocyte-derived cell lines. Vet. Immunol. Immunopathol. 82 (2001) 139–151.

    CAS  PubMed  Article  Google Scholar 

  25. Belosevic, M., Hanington, P.C. and Barreda, D.R. Development of goldfish macrophages in vitro. Fish Shellfish Immunol. 20 (2006) 152–171.

    CAS  PubMed  Article  Google Scholar 

  26. Wang, X.H., Wang, Y., Diao, F. and Lu, J. RhoB is involved in lipopolysaccharide-induced inflammation in mouse in vivo and in vitro. J. Physiol. Biochem. 69 (2013) 189–197.

    CAS  PubMed  Article  Google Scholar 

  27. Song, Y., Dou, H., Gong, W., Liu, X., Yu, Z., Li, E., Tan, R. and 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. 705 (2013) 49–60.

    CAS  PubMed  Article  Google Scholar 

  28. Haziot, A., Ferrero, E., Köntgen, F., Hijiya, N., Yamamoto, S., Silver, J., Stewart, C.L. and Goyert, S.M. Resistance to endotoxin shock and reduced dissemination of Gram-negative bacteria in CD14-deficient mice. Immunity 4 (1996) 407–414.

    CAS  PubMed  Article  Google Scholar 

  29. Liu, J., Bátkai, S., Pacher, P., Harvey-White, J., Wagner, J.A., Cravatt, B.F., Gao, B. and Kunos, G. Lipopolysaccharide induces anandamide synthesis in macrophages via CD14/MAPK/phosphoinositide 3-kinase/NF-κB independently of platelet-activating factor. J. Biol. Chem. 278 (2003) 45034–45039.

    CAS  PubMed  Article  Google Scholar 

  30. Sanz, G., Pérez, E., Jiménez-Marín, A., Mompart, F., Morera, L., Barbancho, M., Llanes, D. and Garrido, J.J. Molecular cloning, chromosomal location, and expression analysis of porcine CD14. Dev. Comp. Immunol. 31 (2007) 738–747.

    CAS  PubMed  Article  Google Scholar 

  31. Gomes, N., Brunialti, M., Mendes, M., Freudenberg, M., Galanos, C. and 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. 43 (2010) 853–858.

    CAS  PubMed  Article  Google Scholar 

  32. Mansouri-Attia, N., Oliveira, L.J., Forde, N., Fahey, A.G., Browne, J.A., Roche, J.F., Sandra, O., Reinaud, P., Lonergan, P. and Fair, T. Pivotal role for monocytes/macrophages and dendritic cells in maternal immune response to the developing embryo in cattle. Biol. Reprod. 87 (2012) 123.

    PubMed  Article  Google Scholar 

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Correspondence to Xiangping Li or Deshun Shi.

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Li, X., Li, M., Huang, S. et al. The effect of buffalo CD14 shRNA on the gene expression of TLR4 signal pathway in buffalo monocyte/macrophages. Cell Mol Biol Lett 19, 623–637 (2014). https://doi.org/10.2478/s11658-014-0217-1

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  • DOI: https://doi.org/10.2478/s11658-014-0217-1

Keywords

  • Buffalo CD14 gene
  • shRNA
  • TLR4 signal pathway
  • Gene expression
  • Monocyte/macrophages