Advertisement

Phenotypic characterization of swine peripheral blood monocyte-derived macrophages and ex vivo infection with Salmonella enterica serovar Typhimurium

  • Carlos Eduardo Real Pereira
  • Márcio Sobreira Silva Araújo
  • Juliana Pinto da Silva Mol
  • José Paulo Hiroji Sato
  • Amanda Gabrielle de Souza Daniel
  • Olindo Assis Martins-Filho
  • Renato Lima Santos
  • Roberto Mauricio Carvalho GuedesEmail author
Veterinary Microbiology - Research Paper
  • 7 Downloads

Abstract

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)-[1]dependent, in which log-phase WT strains were more efficient (p < 0.01) than the invA mutant strain at inducing the death of macrophages.

Keywords

Salmonella Typhimurium Pigs Macrophage Cytotoxicity 

Notes

Funding sources

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).

References

  1. 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
  2. 2.
    Maria-Pilar JB, Dudal S, Dornand J, Gross A (2005) Cellular bioterrorism: how Brucella corrupts macrophage physiology to promote invasion and proliferation. Clin Immunol 114:227–238CrossRefGoogle Scholar
  3. 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. 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
  5. 5.
    Berends BR, Urlings HA, Snijders JMA, Van Knapen F (1996) Identification and quantification of risk factors in animal management and transport regarding Salmonella spp. in pigs. Int J Food Microbiol 30:37–53CrossRefGoogle Scholar
  6. 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
  7. 7.
    Santos RL, Baumler AJ (2004) Cell tropism of Salmonella enterica. Int J Med Microbiol 294:225–233CrossRefGoogle Scholar
  8. 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
  9. 9.
    Santos RL, Tsolis RM, Bäumler AJ et al (2001) Salmonella enterica serovar Typhimurium induces cell death in bovine monocyte-derived macrophages by early sipB-dependent and delayed sipB-independent mechanisms. Infect Immun 69(4):2293–2301CrossRefGoogle Scholar
  10. 10.
    Misselwitz B, Kreibich SK, Rout S, Stecher B, Periaswamy B, Hardt WD (2011) Salmonella enterica serovar Typhimurium binds to HeLa cells via Fim-mediated reversible adhesion and irreversible type three secretion system 1–mediated docking. Infect Immun 79:330–341CrossRefGoogle Scholar
  11. 11.
    Patel JC, Galan JE (2006) Differential activation and function of Rho GTPases during Salmonella-host cell interactions. J Cell Biol 175:453–463CrossRefGoogle Scholar
  12. 12.
    Cain RJ, Hayward RD, Koronakis V (2008) Deciphering interplay between Salmonella invasion effectors. PLoS Pathog 4:e1000037CrossRefGoogle Scholar
  13. 13.
    Galan JE, Ginocchio C, Costeas P (1992) Molecular and functional characterization of the Salmonella invasion gene invA: homology of invA to members of a new protein family. J Bacteriol 174(13):4338–4349CrossRefGoogle Scholar
  14. 14.
    Lilic M, Quezada CM, Stebbins CE (2010) A conserved domain in type III secretion links the cytoplasmic domain of InvA to elements of the basal body. Acta Crystallogr D Biol Crystallogr 66(6):709–713CrossRefGoogle Scholar
  15. 15.
    van der Velden AW, Dougherty JT, Starnbach MN (2008) Down-modulation of TCR expression by Salmonella enterica serovar Typhimurium. J Immunol 180(8):5569–5574CrossRefGoogle Scholar
  16. 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
  17. 17.
    Miao EA, Alpuche-Aranda CM, Dors M, Clark AE, Bader MW, Miller SI, Aderem A (2006) Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1β via Ipaf. Nat Immunol 7(6):569–575CrossRefGoogle Scholar
  18. 18.
    Boyen F, Pasmans F, Van Immerseel F et al (2006a) Salmonella Typhimurium SPI-1 genes promote intestinal but not tonsillar colonization in pigs. Microbes Infec 8(14–15):2899–2907CrossRefGoogle Scholar
  19. 19.
    Stojiljkovic I, Bäumler AJ, Heffron F (1995) Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster. J Bacteriol 177(5):1357–1366CrossRefGoogle Scholar
  20. 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
  21. 21.
    Saldarriaga OA, Velásquez JI, Ossa JE, Rugeles MT (2003) Standardization of bovine macrophage monolayers and isolation and culture of trypanosomes. Mem Inst Oswaldo Cruz 98:269–271CrossRefGoogle Scholar
  22. 22.
    Bueno R, Mello MN, Menezes CAS, Dutra WO, Santos RL (2005) Phenotypic, functional, and quantitative characterization of canine peripheral blood monocyte-derived macrophages. Mem Inst Oswaldo Cruz 100(5):521–524CrossRefGoogle Scholar
  23. 23.
    Oluwole OO, Omitogun GO (2016) Haematological traits of nigerian indigenous pig and its hybrid (50% large white× 50 NIP) at post weaning ages. Am J Mol Biol 6(1):45–52CrossRefGoogle Scholar
  24. 24.
    Aladi NO, Okeudo NJ, Okoli IC, Akanno EC (2008) Reproductive and hematological characteristics of the Nigerian indigenous and large white pigs in a humid tropical environment. Asian J Anim Vet Advcs 3(1):17–23CrossRefGoogle Scholar
  25. 25.
    Riber U, Lind P (1999) Interaction between Salmonella Typhimurium and phagocytic cells in pigs: phagocytosis, oxidative burst and killing in polymorphonuclear leukocytes and monocytes. Vet Immunol Immunopathol 67(3):259–270CrossRefGoogle Scholar
  26. 26.
    Boyen F, Pasmans F, Donné E, van Immerseel F, Adriaensen C, Hernalsteens JP, Ducatelle R, Haesebrouck F (2006b) Role of SPI-1 in the interactions of Salmonella Typhimurium with porcine macrophages. Vet Microbiol 113(1):35–44CrossRefGoogle Scholar
  27. 27.
    Brumell JH, Grinstein S (2004) Salmonella redirects phagosomal maturation. Curr Opin Microbiol 7:78–84CrossRefGoogle Scholar
  28. 28.
    Figueira R, Holden DW (2012) Functions of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system effectors. Microbiology 158:1147–1161CrossRefGoogle Scholar
  29. 29.
    McGourty K, Thurston TL, Matthews SA, Pinaud L, Mota LJ, Holden DW (2012) Salmonella inhibits retrograde trafficking of mannose-6-phosphatereceptors and lysosome function. Science 338:963–967CrossRefGoogle Scholar
  30. 30.
    D’Costa VM, Braun V, Landekic M, Shi R, Proteau A, McDonald L, Cygler M, Grinstein S, Brumell JH (2015) Salmonella disrupts host endocytic trafficking by SopD2-mediated inhibition of Rab7. Cell Rep 12(9):1508–1518CrossRefGoogle Scholar
  31. 31.
    Mariathasan S, Newton K, Monack DM, Vucic D, French DM, Lee WP, Roose-Girma M, Erickson S, Dixit VM (2004) Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430:213–218CrossRefGoogle Scholar
  32. 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
  33. 33.
    Lee SH, Galan JE (2004) Salmonella type III secretion-associated chaperones confer secretion-pathway specificity. Mol Microbiol 51:483–495CrossRefGoogle Scholar

Copyright information

© Sociedade Brasileira de Microbiologia 2019

Authors and Affiliations

  • Carlos Eduardo Real Pereira
    • 1
  • Márcio Sobreira Silva Araújo
    • 2
  • Juliana Pinto da Silva Mol
    • 1
  • José Paulo Hiroji Sato
    • 1
  • Amanda Gabrielle de Souza Daniel
    • 1
  • Olindo Assis Martins-Filho
    • 2
  • Renato Lima Santos
    • 1
  • Roberto Mauricio Carvalho Guedes
    • 1
    Email author
  1. 1.Departmento de Clínica e Cirúrgia Veterinária, Escola de VeterináriaUniversidade Federal de Minas GeraisBelo HorizonteBrazil
  2. 2.Laboratório de Biomarcadores de Diagnóstico e MonitoraçãoCentro de Pesquisas René Rachou-FiocruzBelo HorizonteBrazil

Personalised recommendations