Parasitology Research

, Volume 118, Issue 6, pp 1841–1848 | Cite as

Increased phagocytosis and growth inhibition of Encephalitozoon cuniculi by LPS-activated J774A.1 murine macrophages

  • J. R. González-Machorro
  • L. E. Rodríguez-Tovar
  • R. Gómez-Flores
  • A. Soto-Dominguez
  • H. Rodríguez-Rocha
  • A. Garcia-García
  • P. Tamez-Guerra
  • U. Castillo-VelázquezEmail author
Immunology and Host-Parasite Interactions - Original Paper


Encephalitozoon cuniculi is an obligate macrophage parasite of vertebrates that commonly infects rodents, monkeys, dogs, birds, and humans. In the present study, we aimed to assess the phagocytosis and intracellular survival of E. cuniculi spores using untreated and lipopolysaccharide (LPS)-activated J774A.1 murine macrophages and assess the macrophage viability. The experimental groups comprised untreated spores, spores killed by heat treatment at 90 °C, and spores killed by treatment with 10% formalin. LPS-activated macrophages significantly increased the phagocytosis of spores and reduced their intracellular growth after 24 and 48 h (P < 0.01); however, after 72 h, we observed an increase in spore replication but no detectable microbicidal activity. These results indicate that LPS activation enhanced E. cuniculi phagocytosis between 24 and 48 h of treatment, but the effect was lost after 72 h, enabling parasitic growth. This study contributes to the understanding of the phagocytosis and survival of E. cuniculi in murine macrophages.


Encephalitozoon cuniculi Phagocytosis Macrophages Intracellular survival LPS activation Nitric oxide 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Abu-Akkada SS, El Kerdany ED, Mady RF, Diab RG, Khedr GA, Ashmawy KI, Lotfy WM (2015) Encephalitozoon cuniculi infection among immunocompromised and immunocompetent humans in Egypt. Iran J Parasitol 10(4):561–570 Google Scholar
  2. Bohne W, Böttcher K, Groß U (2011) The parasitophorous vacuole of Encephalitozoon cuniculi: biogenesis and characteristics of the host cell–pathogen interface. Int J Med Microbiol 301(5):395–399. Google Scholar
  3. Castillo-Velázquez U, Aranday-Cortés E, Gutiérrez-Pabello JA (2011) Alternative activation modifies macrophage resistance to Mycobacterium bovis. Vet Microbiol 151(1):51–59. Google Scholar
  4. Chan J, Xing Y, Magliozzo RS, Bloom BR (1992) Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages. J Exp Med 175(4):1111–1122. Google Scholar
  5. Chiu FL, Lin JK (2008) Tomatidine inhibits iNOS and COX-2 through suppression of NF-κB and JNK pathways in LPS-stimulated mouse macrophages. FEBS Lett 582(16):2407–2412. Google Scholar
  6. Couzinet S, Cejas E, Schittny J, Deplazes P, Weber R, Zimmerli S (2000) Phagocytic uptake of Encephalitozoon cuniculi by nonprofessional phagocytes. Infect Immun 68(12):6939–6945 Google Scholar
  7. Del Aguila C, Croppo GP, Moura H, Da Silva AJ, Leitch GJ, Moss DM, Wallace S, Slemenda SB, Pieniazek NJ, Visvesvara GS (1998) Ultrastructure, immunofluorescence, Western blot, and PCR analysis of eight isolates of Encephalitozoon (Septata) intestinalis established in culture from sputum and urine samples and duodenal aspirates of five patients with AIDS. J Clin Microbiol 36(5):1201–1208 Google Scholar
  8. Del Aguila C, Izquierdo F, Granjab AG, Hurtado C, Fenoya S, Fresno M, Revilla Y (2006) Encephalitozoon microsporidia modulates p53-mediated apoptosis in infected cells. Int J Parasitol 36(8):869–876. Google Scholar
  9. Didier ES, Bowers LC, Martin AD, Kuroda MJ, Khan IA, Didier PJ (2010) Reactive nitrogen and oxygen species, and iron sequestration contribute to macrophage-mediated control of Encephalitozoon cuniculi (Phylum Microsporidia) infection in vitro and in vivo. Microbes Infect 12(14):1244–1251. Google Scholar
  10. Didier ES, Orenstein JM, Aldras A, Bertucci D, Rogers LB, Janney FA (1995) Comparison of three staining methods for detecting microsporidia in fluids. J Clin Microbiol 33(12):3138–3145 Google Scholar
  11. Didier ES, Varner PW, Didier PJ, Aldras AM, Millichamp NJ, Murphy-Corb M, Bohm R, Shadduck JA (1994) Experimental microsporidiosis in immunocompetent and immunodeficient mice and monkeys. Folia Parasitol 41(1):1–11 Google Scholar
  12. Didier ES, Weiss LM (2012) Microsporidiosis: not just in AIDS patients. Curr Opinion Infect Dis 24(5):490–495. Google Scholar
  13. Fischer J, Suire C, Hale-Donze H (2008) Toll-like receptor 2 recognition of the microsporidia Encephalitozoon spp. induces nuclear translocation of NF-κB and subsequent inflammatory responses. Infect Immun 76(10):4737–4744. Google Scholar
  14. Franzen C, Müller A, Hartmann P, Salzberger B (2005) Cell invasion and intracellular fate of Encephalitozoon cuniculi (Microsporidia). Parasitology 130(03):285–292. Google Scholar
  15. Gazzinelli RT, Oswald IP, Hieny S, James SL, Sher A (1992) The microbicidal activity of interferon-γ-treated macrophages against Trypanosoma cruzi involves an L-arginine-dependent, nitrogen oxide-mediated mechanism inhibitable by interleukin-10 and transforming growth factor-β. Eur J Immunol 22(19):2501–2506. Google Scholar
  16. Gomez-Flores R, Tamez-Guerra R, Tucker SD, Mehta RT (1997) Bidirectional effects of IFN-γ on growth of Mycobacterium avium complex in murine peritoneal macrophages. J Interf Cytokine Res 17(6):331–336. Google Scholar
  17. Harcourt-Brown FM (2004) Encephalitozoon cuniculi infection in rabbits. Semin Avian Exot Pet Med 13(2):86–93. Google Scholar
  18. Hermeling S, Crommelin DJ, Schellekens H, Jiskoot W (2004) Structure-immunogenicity relationships of therapeutic proteins. Pharm Res 21(6):897–903. Google Scholar
  19. Hibbs JB Jr, Taintor RR, Vavrin Z, Rachlin EM (1988) Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem Biophys Res Commun 157(1):87–94. Google Scholar
  20. Joseph J, Sharma S (2009) In vitro culture of various species of microsporidia causing keratitis: evaluation of three immortalized cell lines. Ind J Med Microbiol 27(1):35–39 Google Scholar
  21. Kawai T, Akira S (2007) Signaling to NF-κB by Toll-like receptors. Trends Mol Med 13(11):460–469. Google Scholar
  22. Keeling PJ (2003) Congruent evidence from α-tubulin and β-tubulin gene phylogenies for a zygomycete origin of microsporidia. Fungal Genet Biol 38(3):298–309. Google Scholar
  23. Koudela B, Kucerova S, Hudcovic T (1999) Effect of low and high temperatures on infectivity of Encephalitozoon cuniculi spores suspended in water. Folia Parasitol 46(3):171–174 Google Scholar
  24. Künzel F, Gruber A, Tichy A, Edelhofer R, Nell B, Hassan J, Leschnika M, Joachim A (2008) Clinical symptoms and diagnosis of encephalitozoonosis in pet rabbits. Vet Parasitol 151(2):115–124. Google Scholar
  25. Ladapo TA, Nourse P, Pillay K, Frean J, Birkhead M, Poonsamy B, Gajjar P (2014) Microsporidiosis in pediatric renal transplant patients in Cape Town, South Africa: two case reports. Pediatr Transplant 18(7):E220–E226. Google Scholar
  26. Leech MSJGJ, Shaw AP, Moura H, Visvesvara GS (1999) Susceptibility to apoptosis is reduced in the microsporidia-infected host cell. J Eukaryot Microbiol 46(5):34S–35S Google Scholar
  27. Leiro J, Ortega M, Sanmartı́n ML, Ubeira FM (2000) Non-specific responses of turbot (Scophthalmus maximus L.) adherent cells to microsporidian spores. Vet Immunol Immunopathol 75(1–2):81–95. Google Scholar
  28. Levaditi C, Nicolau S, Schoen R (1923) L’agent étiologique de l’encéphalite épizootique du lapin (Encephalitozoon cuniculi). CR Soc Biol Paris 89:984–986Google Scholar
  29. Lorsbach RB, Murphy WJ, Lowenstein CJ, Snyder SH, Russell SW (1993) Expression of the nitric oxide synthase gene in mouse macrophages activated for tumor cell killing. Molecular basis for the synergy between interferon-gamma and lipopolysaccharide. J Biol Chem 268(3):1908–1913 Google Scholar
  30. Mathews A, Hotard A, Hale-Donze H (2009) Innate immune responses to Encephalitozoon species infections. Microb Infect 11(12):905–911. Google Scholar
  31. Mathis A (2000) Microsporidia: emerging advances in understanding the basic biology of these unique organisms. Int J Parasitol 30(7):795–804. Google Scholar
  32. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1–2):55–63. Google Scholar
  33. Murray HW, Carl FN (1999) Macrophage microbicidal mechanisms in vivo: reactive nitrogen versus oxygen intermediates in the killing of intracellular visceral Leishmania donovani. J Exp Med 189(4):741–746. Google Scholar
  34. Peuvel-Fanget I, Polonais V, Brosson D, Texier C, Kuhn L, Peyret P, Vivarés C, Delbac F (2006) EnP1 and EnP2, two proteins associated with the Encephalitozoon cuniculi endospore, the chitin-rich inner layer of the microsporidian spore wall. Int J Parasitol 36(3):309–318. Google Scholar
  35. Rönnebäumer K, Gross U, Bohne W (2008) The nascent parasitophorous vacuole membrane of Encephalitozoon cuniculi is formed by host cell lipids and contains pores which allow nutrient uptake. Eukaryot Cell 7(6):1001–1008. Google Scholar
  36. Salát J, Braunfuchsová P, Kopecký J, Ditrich O (2002) Role of CD4+ and CD8+ T lymphocytes in the protection of mice against Encephalitozoon intestinalis infection. Parasitol Res 88(7):603–608. Google Scholar
  37. Spalter SH, Kaveri SV, Bonnin E, Mani JC, Cartron JP, Kazatchkine MD (1999) Normal human serum contains natural antibodies reactive with autologous ABO blood group antigens. Blood 93(12):4418–4424 Google Scholar
  38. Valencakova A, Halanova M (2012) Immune response to Encephalitozoon infection review. Comp Immunol Microbiol Infect Dis 35(1):1–7. Google Scholar
  39. Visvesvara GS (2002) In vitro cultivation of microsporidia of clinical importance. Clin Microbiol Rev 15(3):401–413. Google Scholar
  40. Waller T (1979) Sensitivity of Encephalitozoon cuniculi to various temperatures, disinfectants and drugs. Lab Anim 13(3):227–230. Google Scholar
  41. Weidner E (1975) Interactions between Encephalitozoon cuniculi and macrophages. Parasitol Res 47(1):1–9. Google Scholar
  42. Williams BAP (2009) Unique physiology of host–parasite interactions in microsporidia infections. Cell Micobiol 11(11):1551–1560. Google Scholar
  43. Wright JH, Craighead EM (1922) Infectious motor paralysis in young rabbits. J Exp Med 36(1):135–140 Google Scholar
  44. Yaoqian PAN, Shuai W, Xingyou LIU, Ruizhen LI, Yuqian SUN (2015) Seroprevalence of Encephalitozoon cuniculi in humans and rabbits in China. Iran J Parasitol 10(2):290–295 Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • J. R. González-Machorro
    • 1
  • L. E. Rodríguez-Tovar
    • 1
  • R. Gómez-Flores
    • 2
  • A. Soto-Dominguez
    • 3
  • H. Rodríguez-Rocha
    • 3
  • A. Garcia-García
    • 1
  • P. Tamez-Guerra
    • 2
  • U. Castillo-Velázquez
    • 1
    Email author
  1. 1.Facultad de Medicina Veterinaria y Zootecnia, Departamento de Inmunología Veterinaria, Campus de Agricultura y Ciencias BiológicasUniversidad Autónoma de Nuevo LeónEscobedoMexico
  2. 2.Facultad de Ciencias Biológicas, Departamento de Microbiología e InmunologíaUniversidad Autónoma de Nuevo LeónSan Nicolás de los GarzaMexico
  3. 3.Facultad de Medicina, Departamento de HistologíaUniversidad Autónoma de Nuevo LeónMonterreyMexico

Personalised recommendations