Impact of primary mouse macrophage cell types on Leishmania infection and in vitro drug susceptibility
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Primary mouse macrophages are frequently used to provide an in vitro intracellular model to evaluate antileishmanial drug efficacy. The present study compared the phenotypic characteristics of Swiss, BALB/c, and C57BL/6 mouse bone marrow-derived macrophages and peritoneal exudate cells using different stimulation and adherence protocols upon infection with a Leishmania infantum laboratory strain and two clinical isolates. Evaluation parameters were susceptibility to infection, permissiveness to amastigote multiplication, and impact on drug efficacy. Observed variations in infection of peritoneal exudate cells can mostly be linked to changes in the inflammatory cytokine profiles (IL-6, TNF-α, KC/GRO) rather than to differences in initial production of nitric oxide and reactive oxygen species. Optimization of the cell stimulation and adherence conditions resulted in comparable infection indices among peritoneal exudate cells and the various types of bone marrow-derived macrophages. BALB/c-derived bone marrow-derived macrophages were slightly more permissive to intracellular amastigote replication. Evaluation of antileishmanial drug potency in the various cell systems revealed minimal variation for antimonials and paromomycin, and no differences for miltefosine and amphotericin B. The study results allow to conclude that drug evaluation can be performed in all tested primary macrophages as only marginal differences are observed in terms of susceptibility to infection and impact of drug exposure. Combined with some practical considerations, the use of 24-h starch-stimulated, 48-h adhered, Swiss-derived peritoneal exudate cells can be advocated as an efficient, reliable, relatively quick, and cost-effective tool for routine drug susceptibility testing in vitro whenever the use of primary cells is feasible.
KeywordsLeishmania Host cell Primary macrophage Drug susceptibility
The authors thank Pim-Bart Feijens, Margot Desmet, Mandy Vermont, and An Matheeussen for the excellent technical assistance and Dr. Laurence Lachaud (CNRL, Montpellier, France) for providing us the LEM3049 clinical isolate. We also thank Dr. Hannelie Korf (KU Leuven) for her help with the multiplex ELISA.
This work was funded by the Research Fund Flanders (G051812N, 12I0317N, and 1136417N) and a research fund of the University of Antwerp (TT-ZAPBOF 33049). LMPH is a partner of the Antwerp Drug Discovery Network (ADDN, www.addn.be) and the Excellence Centre “Infla-Med” (www.uantwerpen.be/infla-med).
Compliance with ethical standards
The use of laboratory rodents was carried out in strict accordance to all mandatory guidelines (EU directives, including the Revised Directive 2010/63/EU on the Protection of Animals used for Scientific Purposes that came into force on 01/01/2013, and the declaration of Helsinki in its latest version) and was approved by the ethical committee of the University of Antwerp, Belgium (UA-ECD 2016–54 (02/09/2016)).
Conflict of interest
The authors declare that they have no conflict of interest.
- da Costa Val A (2004) Tratamento da leishmaniose visceral canina com antimonial pentavalente encapsulado em lipossomas Tese de Doutorado em Ciência Animal, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, 125pGoogle Scholar
- Denys A, Udalova IA, Smith C, Williams LM, Ciesielski CJ, Campbell J, Andrews C, Kwaitkowski D, Foxwell BMJ (2002) Evidence for a dual mechanism for IL-10 suppression of TNF-alpha production that does not involve inhibition of p38 mitogen-activated protein kinase or NF-kappa B in primary human macrophages. J Immunol 168:4837–4845CrossRefGoogle Scholar
- Depke M, Breitbach K, Dinh Hoang Dang K, Brinkmann L, Salazar MG, Dhople VM, Bast A, Steil L, Schmidt F, Steinmetz I, Völker U (2014) Bone marrow-derived macrophages from BALB/c and C57BL/6 mice fundamentally differ in their respiratory chain complex proteins, lysosomal enzymes and components of antioxidant stress systems. J Proteome 103:72–86CrossRefGoogle Scholar
- Hendrickx S, Guerin PJ, Caljon G, Croft SL, Maes L (2016) Evaluating drug resistance in visceral leishmaniasis: the challenges. Parasitology 1–11Google Scholar
- Howes A, Taubert C, Blankley S, Spink N, Wu X, Graham CM, Zhao J, Saraiva M, Ricciardi-Castagnoli P, Bancroft GJ, O’Garra A (2016) Differential production of type I IFN determines the reciprocal levels of IL-10 and proinflammatory cytokines produced by C57BL/6 and BALB/c macrophages. J Immunol 197:2838–2853CrossRefGoogle Scholar
- Kaushik RS, Uzonna JE, Zhang Y, Gordon JR, Tabel H (2000) Innate resistance to experimental African trypanosomiasis: differences in cytokine (TNF-alpha, IL-6, IL-10 and IL-12) production by bone marrow-derived macrophages from resistant and susceptible mice. Cytokine 12:1024–1034CrossRefGoogle Scholar
- Koniordou M, Patterson S, Wyllie S, Seifert K (2017) Snapshot profiling of the antileishmanial potency of lead compounds and drug candidates against intracellular Leishmania donovani amastigotes, with a focus on human-derived host cells. Antimicrob Agents Chemother 61Google Scholar
- Matte C (2016) Exploitation of the host cell membrane fusion machinery by Leishmania is part of the infection process. 12Google Scholar
- Mookerjee Basu J et al (2006) Sodium antimony gluconate induces generation of reactive oxygen species and nitric oxide via phosphoinositide 3-kinase and mitogen-activated protein kinase activation in Leishmania donovani-infected macrophages. Antimicrobial Agents Chemother 50:1788–1797CrossRefGoogle Scholar
- Santos JL, Andrade AA, Dias AA, Bonjardim CA, Reis LF, Teixeira SM, Horta MF (2006) Differential sensitivity of C57BL/6 (M-1) and BALB/c (M-2) macrophages to the stimuli of IFN-gamma/LPS for the production of NO: correlation with iNOS mRNA and protein expression. J Interf Cytokine Res 26:682–688CrossRefGoogle Scholar
- Vermeersch M, da Luz RI, Tote K, Timmermans JP, Cos P, Maes L (2009) In vitro susceptibilities of Leishmania donovani promastigote and amastigote stages to antileishmanial reference drugs: practical relevance of stage-specific differences. Antimicrob Agents Chemother 53:3855–3859CrossRefGoogle Scholar
- Weischenfeldt J, Porse B (2008) Bone marrow-derived macrophages (BMM): isolation and applications. CSH protocols 2008:pdb.prot5080Google Scholar