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.
[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. Search in Google Scholar
[2] Ziegler-Heitbrock, H. and Ulevitch, R. CD14: cell surface receptor and differentiation marker. Immunol. Today 14 (1993) 121–125. http://dx.doi.org/10.1016/0167-5699(93)90212-410.1016/0167-5699(93)90212-4Search in 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. http://dx.doi.org/10.4049/jimmunol.167.5.275910.4049/jimmunol.167.5.2759Search in Google Scholar PubMed
[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. http://dx.doi.org/10.4049/jimmunol.166.5.359910.4049/jimmunol.166.5.3599Search in Google Scholar PubMed
[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. http://dx.doi.org/10.1289/ehp.97105s5126110.1289/ehp.97105s51261Search in Google Scholar PubMed PubMed Central
[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. http://dx.doi.org/10.1095/biolreprod.112.10237610.1095/biolreprod.112.102376Search in Google Scholar PubMed
[7] Miyake, K. Innate recognition of lipopolysaccharide by toll-like receptor 4-MD-2. Trends Microbiol. 12 (2004) 186–192. http://dx.doi.org/10.1016/j.tim.2004.02.00910.1016/j.tim.2004.02.009Search in Google Scholar PubMed
[8] Miyake, K. Innate immune sensing of pathogens and danger signals by cell surface toll-like receptors. Semin. Immunol. 19 (2007) 3–10. http://dx.doi.org/10.1016/j.smim.2006.12.00210.1016/j.smim.2006.12.002Search in Google Scholar PubMed
[9] Takeuchi, O. and Akira, S. Pattern recognition receptors and inflammation. Cell 140 (2010) 805–820. http://dx.doi.org/10.1016/j.cell.2010.01.02210.1016/j.cell.2010.01.022Search in Google Scholar PubMed
[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. http://dx.doi.org/10.1016/j.vetimm.2012.02.00110.1016/j.vetimm.2012.02.001Search in Google Scholar PubMed
[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. http://dx.doi.org/10.4049/jimmunol.167.6.332910.4049/jimmunol.167.6.3329Search in Google Scholar PubMed
[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. http://dx.doi.org/10.1016/j.rvsc.2012.09.00810.1016/j.rvsc.2012.09.008Search in Google Scholar PubMed
[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. http://dx.doi.org/10.1128/IAI.01998-0510.1128/IAI.01998-05Search in Google Scholar PubMed PubMed Central
[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. http://dx.doi.org/10.1007/s11259-009-9339-810.1007/s11259-009-9339-8Search in Google Scholar PubMed
[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. 10.1152/ajpgi.1998.275.1.G39Search in Google Scholar PubMed
[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. http://dx.doi.org/10.1074/jbc.M70183120010.1074/jbc.M701831200Search in Google Scholar PubMed
[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. http://dx.doi.org/10.1016/j.molimm.2010.02.01510.1016/j.molimm.2010.02.015Search in Google Scholar PubMed
[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. http://dx.doi.org/10.1007/s00253-011-3371-710.1007/s00253-011-3371-7Search in Google Scholar PubMed
[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. http://dx.doi.org/10.1095/biolreprod.111.09271810.1095/biolreprod.111.092718Search in Google Scholar PubMed PubMed Central
[20] Hannon, G.J. RNA interference. Nature 418 (2002) 244–251. http://dx.doi.org/10.1038/418244a10.1038/418244aSearch in Google Scholar PubMed
[21] Moffat, J. and Sabatini, D.M. Building mammalian signaling pathways with RNAi screens. Nat. Rev. Mol. Cell. Biol. 7 (2006) 177–187. http://dx.doi.org/10.1038/nrm186010.1038/nrm1860Search in Google Scholar PubMed
[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. Search in 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. http://dx.doi.org/10.1038/ng1197-31410.1038/ng1197-314Search in 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. http://dx.doi.org/10.1016/S0165-2427(01)00330-010.1016/S0165-2427(01)00330-0Search in 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. http://dx.doi.org/10.1016/j.fsi.2004.10.01010.1016/j.fsi.2004.10.010Search in 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. http://dx.doi.org/10.1007/s13105-012-0201-z10.1007/s13105-012-0201-zSearch in 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. http://dx.doi.org/10.1016/j.ejphar.2013.02.00810.1016/j.ejphar.2013.02.008Search in 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. http://dx.doi.org/10.1016/S1074-7613(00)80254-X10.1016/S1074-7613(00)80254-XSearch in 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. http://dx.doi.org/10.1074/jbc.M30606220010.1074/jbc.M306062200Search in Google Scholar PubMed
[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. http://dx.doi.org/10.1016/j.dci.2006.10.00610.1016/j.dci.2006.10.006Search in Google Scholar PubMed
[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. http://dx.doi.org/10.1590/S0100-879X201000750007810.1590/S0100-879X2010007500078Search in 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. http://dx.doi.org/10.1095/biolreprod.112.10112110.1095/biolreprod.112.101121Search in Google Scholar PubMed
© 2014 University of Wrocław, Poland
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
