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Effect of Dexamethasone, LPS, or INFγ on the production of TNFα, IL-12, IL-6, IL-10, CXCL8, CXCL10, and CLC3, by bovine macrophages infected in vitro with Mycobacterium avium paratuberculosis.

René Ramírez-García1, Mauricio Rojas2, Beatriz Peña-Arboleda3 and Juan G. Maldonado-Estrada4*
  • 1 Universidad CES, Veterinary Medicine, Colombia
  • 2 Universidad de Antioquia, Grupo de Inmunología Celular e Inmunogenética, Instituto de Investigaciones Médicas, Facultad de Medicina, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia, Colombia
  • 3 Universidad de Antioquia, Grupo Reproducción, Colombia
  • 4 University of Antioquia, School of Veterinary Medicine, Colombia

In this study primary monocyte-derived macrophages from bovine were used for intracellular proliferation of Mycobacterium avium subespeceis paratuberculosis (MAP) in vitro. Macrophages derived from peripheral blood monocyte (MDM) from a donor negative for the IS900 Map specific sequence, were infected with a reference MAP strain at a Dose of infection (DOI) of 5:1 during 2 h. The MDM were then incubated with IFNγ (3x106 U/500 ul), LPS (10 ng/ml), Dexamethasone (1 ug/ml) or medium alone / 24 h. After infection the MDM were evaluated at 0, 6, 72 and 120 h after culture for detecting proliferation of MAP by amplification of the IS900 fragment by real-time PCR. The production of pro-inflammatory cytokines (IL6, IL12, and TNFα), anti-inflammatory cytokines (IL10), and chemokines (CXCL 8, CXCL10, and CCL3) in culture supernatants during each period of incubation was performed by Luminex. Data were analyzed by Tukey test. The amplification of the IS900 segment was detected in MDM after infection at each time of incubation regardless of the stimulus (P>0.05). MDM that were treated with Dexamethasone did show reduced production of TNF-α (at 72 and 120h), IL-6 (at 120h) and CCL3 (at 72 and 120h), but showed increased production of IL12 and IP10 (at 120h) (P<0.01). MDM that were treated with IFNγ did produced significantly more TNF-α (at 6 y 72h), IL-12 (at 120h), and IP-10 (at 72 and 120h) (P <0.01). MDM that were treated with LPS did produce significantly more TNF-α (at 6 and 72h), IL-12 and IP10 (at 120h) (P <0.01). Discussion In this work we provide evidence on the multiplication of MAP in primary MDM obtained from bovine negative to infection by MAP. In addition, the effect of three factors affecting the microenvironment of the macrophages in vivo was tested in vitro. These factors are commonly related to stress (Dexamethasone), infection (LPS) or a Th1 immune response (IFN-γ). Moreover, the profile of MAP proliferation in MDM infected in vitro was similar in control MDM compared to MDM stimulated with Dexamethasone. Glucocorticoids (GC) can exert its effects on macrophages cultured in vitro depending on the dose: at low doses (nanomolar range) GC can induce increased adhesion, chimiotaxis, phagocytosis and cytokine production, whereas at high doses (micromolar range) it can induce immunosupression (Zhou et al, 2010). In our study we used GC in a range of doses causing immussupresion, a fact that could explain the capability of MDM treated with Dexamethasone to allow a greater MAP proliferation when it was compared to MDM treated with IFN-γ or LPS. In this work proliferation of MAP in primary bovine MDM was evidenced, as it was the ability of infection by MAP to modify the functional profile of cytokine production by these macrophages. The infected MDM were viable at least until the final time of evaluation at 120 h post-infection. Anti-inflammatory cytokines (IL-10) and proinflammatory (TNF-α, IL-6, and IL-12), as well as chemiokines (CXCL8, CCL3, and CXCL10), were quantified in MDM supernatants. In summary, the results of our study show that primary bovine MDM can become infected by MAP in vitro and can survive after the infection at leas up to 120 h. These MDM were viable as evidenced by their production of cytokines and cemokines. They were also able to maintain an active multiplication of MAP as it was evidenced by the results on the amplification of the IS900 fragment by real-time PCR results. Further studies are requiered for studying the relationship between the cytokine and chemokines tested in this work and the way they control or allow MAP survival in vitro.

Acknowledgements

Authors thank´s Vicerrectoría de Investigación at University of Antioquia for financial support (Mediana cuantía 2007, Sostenibilidad 2014-2015). Special thanks to Dr. Carlos Muskus and PECET Research Group at University of Antioquia, to University CES, Colombian Institute for Tropical Medicine (ICMT) and Giovanny Torres for technical support in real-time PCR. We also thank Ronald Peláez for technical assistance in data analysis on gene expression. Special thanks to GICIC group.

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Keywords: bovine macrophages, Bovine Paratuberculosis, Chemokines, Intracellular cytokines, intracellular pathogens

Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.

Presentation Type: Oral Presentation

Topic: Veterinary and Comparative Immunology

Citation: Ramírez-García R, Rojas M, Peña-Arboleda B and Maldonado-Estrada JG (2015). Effect of Dexamethasone, LPS, or INFγ on the production of TNFα, IL-12, IL-6, IL-10, CXCL8, CXCL10, and CLC3, by bovine macrophages infected in vitro with Mycobacterium avium paratuberculosis.. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00329

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Received: 01 Jun 2015; Published Online: 15 Sep 2015.

* Correspondence: Dr. Juan G Maldonado-Estrada, University of Antioquia, School of Veterinary Medicine, Medellin, Antioquia, 050031, Colombia, juan.maldonado@udea.edu.co