Phenotypic characterization of swine peripheral blood monocyte-derived macrophages and ex vivo infection with Salmonella enterica serovar Typhimurium
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Macrophages are critical mediators of the inflammatory process, playing a relevant role in the pathogenesis of Salmonella Typhimurium. The protocols for isolation, culture, and differentiation of monocytes into macrophages and their interaction with Salmonella are well established in humans and murine models, but little information is available in swine. The aims of this study were to establish an efficient protocol for macrophage culture and to evaluate the interaction of the invA mutant strain and the wild type (WT) Salmonella Typhimurium with porcine macrophages. Peripheral blood monocyte-derived macrophages from pigs were obtained, separated by density-gradient centrifugation, and cultured in Teflon vials for 10 days. After the differentiation period, cultures consisted of 92.4% CD14+ cells. In addition, these cells showed phagocytic ability, demonstrated by the presence of the same amount of WT and invA mutant Salmonella Typhimurium 1 h after interaction with macrophages. The early cytotoxic effect was Salmonella pathogenicity island (SPI)-dependent, in which log-phase WT strains were more efficient (p < 0.01) than the invA mutant strain at inducing the death of macrophages.
KeywordsSalmonella Typhimurium Pigs Macrophage Cytotoxicity
This work was supported by the FAPEMIG (grant numbers: APQ-02189-13) and PRPq-UFMG. RMCG, RLS, and OAMF are recipients of fellowships from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil). The authors thank the Program for Technological Development in Tools for Health-RPT-FIOCRUZ for the use of its facilities.
Compliance with ethical standards
This experimental protocol has been reviewed and approved by the Ethics in Animal Use Committee at the Universidade Federal de Minas Gerais (CEUA/UFMG protocol number 249/2015).
- 1.Donné E, Pasmans F, Boyen F, van Immerseel F, Adriaensen C, Hernalsteens JP, Ducatelle R, Haesebrouck F (2005) Survival of Salmonella serovar Typhimurium inside porcine monocytes is associated with complement binding and suppression of the production of reactive oxygen species. Vet Microbiol 107:205–214CrossRefGoogle Scholar
- 3.Eze JI, Onunkwo JI, Shoyinka SVO et al (2010) Haematological profiles of pigs raised under intensive management system in south-eastern Nigeria. Nig Vet J 31(2):115–123Google Scholar
- 4.Adenkola AY, Ayo JO, Asala OO (2011) Variations in haematological parameters and erythrocyte osmotic fragility of pigs during hot-dry and harmattan season in northern Guinea savanna zone of Nigeria. Niger J Physiol Sci 26:113–118Google Scholar
- 6.Poppe C, Smart N, Khakhria R et al (1998) Salmonella typhimurium DT104: a virulent and drug resistant pathogen. Can Vet J 39:559–565Google Scholar
- 8.Buchmeier NA, Heffron F (1989) Intracellular survival of wild type Salmonella Typhimurium and macrophage-sensitive mutants in diverse populations of macrophages. Infect Immun 57:1–7Google Scholar
- 16.Franchi L, Amer A, Body-Malapel M, Kanneganti TD, Özören N, Jagirdar R, Inohara N, Vandenabeele P, Bertin J, Coyle A, Grant EP, Núñez G (2006) Cytosolic flagellin requires Ipaf for activation of caspase-1 and interleukin 1β in Salmonella-infected macrophages. Nat Immunol 7(6):576–582CrossRefGoogle Scholar
- 20.Winter SE, Thiennimitr P, Nuccio SP, Haneda T, Winter MG, Wilson RP, Russell JM, Henry T, Tran QT, Lawhon SD, Gomez G, Bevins CL, Russmann H, Monack DM, Adams LG, Baumler AJ (2009) Contribution of flagellin pattern recognition to intestinal inflammation during Salmonella enterica serotype Typhimurium infection. Infect Immun 77(5):1904–1916CrossRefGoogle Scholar
- 32.Lundberg U, Vinatzer U, Berdnik D et al (1999) Growth phase-regulated induction of Salmonella-induced macrophage apoptosis correlates with transient expression of SPI-1 genes. J Bacteriol 181:3433–3437Google Scholar