A more pro-inflammatory environment is generated in nervous cells cultures in the simultaneous presence of HIV-1 and Toxoplasma gondii, even with a lower parasite replication
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1
Instituto Venezolano de Investigaciones Científicas, Centro de Medicina Experimental, Venezuela
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2
Universidad Central de Venezuela, Escuela de Medicina "J.M. Vargas", Venezuela
Background
HIV-1 is able to invade central nervous system (CNS) from early stages of infection and cause alterations in its functionality, with signs and symptoms in the infected patient that range from subtle/mild (minor cognitive motor disorder-MCMD) to severe (HIV-associated dementia-HAD) (1). On the other hand, T. gondii, in immunocompetent hosts, causes infections that use to be chronic/asymptomatic, showing special tropism for CNS (2). But, in immunodeficiency states, as in HIV-1-induced AIDS, parasite infection could reactivate and produce harmful and even deadly disease, as toxoplasmic encephalitis (3). We are interested in studying CNS responses in the presence of both HIV-1 and T. gondii, and understanding how these can mediate neuroprotective or neurotoxic effects. As models of rodents nervous cells has proven useful to study neurotoxic effects of both pathogens (4-8), we used primary cultures of rat nervous cells to evaluate production of cytokines and nitric oxide.
Methods
Primary Cultures of Nervous Cells: We used primary cultures of Sprague-Dawley rat-fetal brain cells, obtained at 17-19 days of gestation, and cultured in 24 wells plaques (250.000 cells/well), in RPMI-10%FCS (Group A-Control), or in the presence of 1.9 x 10exp9 copies/ml of HIV-1-IIIB (Group B-HIV), of 1.000 viable tachyzoites/well of T. gondii-RH strain (Group C-T. gondii) or in the simultaneous presence of HIV and T. gondii (Group D-HIV/T. gondii), for 3, 5 and 7 days.
Determination of Cytokine Levels: culture supernatants were harvested at the end of incubation periods, and stored at -80° C, until the moment of cytokine determination. IL-1α, IL-6, IL-10 and TNFα were determined by flow cytometry, with a FACScalibur cytometer, using and following instructions of a commercial Kit (Cytometric Bead Array, Rat Flex Sets, Becton Dickinson).
Nitric Oxide Determination: nitric oxide levels were determined in culture supernatants, using Griess method, as described (9).
Nervous cells and Free Tachyzoites counts: nervous cells were counted after detaching by trypsin treatment; free tachyzoites were counted in the precipitate (pellet) of centrifuged culture supernatants. Counts were made by microscopy examination, using Trypan Blue and Neubauer chamber.
Statistic: to compare Gropus, ANOVA or Kruskal-Wallis tests were used, as appropriate, with Bonferroni’s or Dunn’s multiple comparison tests, respectively, considering as significantly p< 0.05.
Results
Significant higher levels of IL-1α, IL-6, IL-10, TNFα and nitric oxide were associated with HIV-1-stimulation (see Fig. 1 and 2, Group B vs. A). In cultures in the presence of HIV-1, levels of IL-1α and nitric oxide were significant higher as days advanced, while TNFα levels diminished significantly. T. gondii-infection alone (Group C) didn’t induce significant production of any cytokine or nitric oxide (see Fig. 1 and 2, Group C vs. A). But when HIV and T. gondii were co-culture, production of IL-1α was significantly enhanced; whilst production of IL-10 and TNFα were significantly lowered (see Fig. 1, Group D vs. B). When nervous cells were cultured in the exclusive presence of T. gondii (Group C), counts of free tachyzoites increased significantly, as days of culture augmented (see Fig. 3B), evidencing active parasite replication and progression of T. gondii-infection. But, when HIV-1 was simultaneous present, although by day 3 parasite counts were not significant different of those on the condition with the parasite alone (see Fig. 3A, Group D vs. C, day 3), on days 5 and 7 there were no significant modification in the counts of free tachyzoites in Group D (see Fig. 3B, Group D, counts on days 5 and 7 were not significant different to counts on day 3) and these counts were significant lower than those in Group C (see Fig. 3A, Group D vs. C, days 5 and 7), showing an inhibition of parasite replication in the simultaneous presence of HIV-1. Viable nervous cell counts in Group A (Control) showed a significant increment in its values, from day 3 to days 5 and 7, evidencing an active replication of cells (See Fig. 4B and Table 1). In the presence of T. gondii, by day 5, a significant increment in nervous cell counts was observed, showing also replication of nervous cells (See Fig. 4B and Table 1, Group C, day 5 vs. day 3), but counts were significant lower than those in Control (See Fig. 4A, day 5, Group C vs. A), showing that replication was partially counteracted by parasite infection. Moreover, by day 7, counts significantly diminished (See Fig. 4B and Table 1, Group C, day 7 vs. day 5) and were also significant lower than those in Control (See Fig. 4A, day 7, Group C vs. A), evidencing that cell destruction, in this stage of parasite infection, overpassed cell replication. The presence of only HIV-1 was associated with significant lower viable nervous cell counts from day 5 of culture (See Fig. 4A, days 5 and 7, Group B vs. A), but when HIV-1 and T. gondii were co-culture, nervous cells counts were the lowest (See Fig. 4A, days 3, 5 and 7, Group D vs. A, B and D, p<0.05), evidencing more cell destruction, this is, potentiation of neurotoxicity, in the simultaneous presence of HIV-1 and T. gondii.
Conclusions
In our experiments, augmented production of IL-1α, IL-6, IL-10, TNFα and nitric oxide in the exclusive presence of HIV-1 in nervous cells cultures, in conditions were no productive viral infection is expected (rat nervous cells are not susceptible to HIV-1 infection), stresses in the fact that the only interaction of the virus/viral molecules with nervous cells or its receptors (as chemokine receptors expressed in nervous cells, CCR5 and CXCR4) is capable of induce cellular responses that change the microenvironment, with serious consequences for cellular function and survival. The produced factors include important known proinflammatory mediators. It is outstanding, that in the simultaneous presence of HIV and T. gondii, while enhanced production of IL-6 and nitric oxide were maintained, production of IL-1α was significant higher and that of the immunomodulatory cytokine, IL-10, was significant lower, suggesting potentiation of the proinflammatory environment. TNFα production was also significantly diminished in this condition, showing a dynamic and complex modification of cultures environment in the presence of both pathogens. On the other hand, T. gondii infection alone showed to be destructive to nervous cells. T. gondii tachyzoites actively replicate within infected cells, until the parasite number is so high that induce cell lysis. The observed dynamics of nervous cells counts in T. gondii condition, evidences the complexity of the phenomenon, with cell proliferation and cell destruction in the same system, and how, only at the ending of cultures, in the context of parasite infection, cell destruction overcomes cell proliferation. In the simultaneous presence of HIV-1, parasite infection was significatively inhibited. This, in part, may be explained for the proinflammatory environment (cytokines, nitric oxide) induced in the presence of the virus, that has been demonstrated to be deleterious for the parasite. The exclusive presence of HIV-1, was also associated with significant lower nervous cells counts. High levels of nitric oxide are neurotoxic for brain cells, and this could explain in part the cellular destruction. The induction of a pro-inflammatory cytokine envinronment, could also account for cell destruction. But, in the simultaneous presence of HIV-1 and T. gondii, although parasite infection was significantly inhibited, more nervous cell destruction was observed, suggesting enhancement of neurotoxicity in the presence of both pathogens. We must remember than in this condition, a more pro-inflammatory set of mediators (high nitric oxide and IL-6 production, higher IL-1α, lower IL-10) is induced, and this could account for augmented nervous cell destruction. Our results stress on the importance that simultaneous presence of both pathogens could represent for patients in the setting of co-infection, and how central nervous system could be more affected in this situation, even with a lower parasite replication.
Acknowledgements
This work was suported by grants from FONACIT (G-2005000823, Misión Ciencia Scholarship, Programa de Incentivo Institucional) and IVIC (Scholarships, CEA, Dirección)
References
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Figures Legends
Figure 1. Production of cytokines in primary cultures of rat nervous cells in the presence of HIV-1 and/or T. gondii: levels of (A) IL-1α, (B) IL-6, (C) IL-10 and (D) TNFα (mean ± standard deviation), in primary cultures of Sprague-Dawley rat-fetal brain cells, cultured in RPMI-10%FCS (Control), or in the presence of 1.9 x 10exp9 copies/ml of HIV-1-IIIB (HIV), of 1.000 viable tachyzoites/well of T. gondii-RH strain (T. gondii) or in the simultaneous presence of HIV and T. gondii (HIV/T. gondii), for 3, 5 and 7 days. Cytokine levels are expressed in pg/ml.
Figure 2. Production of nitric oxide in primary cultures of rat nervous cells in the presence of HIV-1 and/or T. gondii: nitric oxide, measured as levels of NO2- (mean ± standard deviation), in primary cultures of Sprague-Dawley rat-fetal brain cells, cultured in RPMI-10%FCS (Control), or in the presence of 1.9 x 10exp9 copies/ml of HIV-1-IIIB (HIV), of 1.000 viable tachyzoites/well of T. gondii-RH strain (T. gondii) or in the simultaneous presence of HIV and T. gondii (HIV/T. gondii), for 3, 5 and 7 days. (A) Differences between groups, each day, are analyzed; (B) changes into each group, as days of culture advance, are analyzed. Levels of NO2- are expressed as µM.
Figure 3. Free tachyzoites counts in primary cultures of rat nervous cells in the presence of T. gondii or “T. gondii and HIV-1”: Counts of free tachyzoites (mean ± standard deviation), in primary cultures of Sprague-Dawley rat-fetal brain cells, cultured in RPMI-10%FCS, in the presence of 1.000 viable tachyzoites/well of T. gondii-RH strain (T. gondii) or in the simultaneous presence of T. gondii and HIV-1IIIB (1.9 x 10exp9 copies/ml) (HIV/T. gondii), for 3, 5 and 7 days. (A) Differences between groups, each day, are analyzed; ; (B) changes into each group, as days of culture advance, are analyzed.
Figure 4. Nervous cell counts in primary cultures of rat nervous cells in the presence of HIV-1 and/or T. gondii: Counts of viable nervous cells (mean ± standard deviation), in primary cultures of Sprague-Dawley rat-fetal brain cells, cultured in RPMI-10%FCS (Control), or in the presence of 1.9 x 109 copies/ml of HIV-1IIIB (HIV), of 1.000 viable tachyzoites/well of T. gondii-RH strain (T. gondii) or in the simultaneous presence of HIV and T. gondii (HIV/T. gondii), for 3, 5 and 7 days. (A) Differences between groups, each day, are analyzed; (B) changes into each group, as days of culture advance, are analyzed.
Keywords:
HIV-1,
Toxoplasma gondii,
proinflammatory cytokines,
Central Nervous System,
Nitric Oxide,
Coinfection
Conference:
15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.
Presentation Type:
Abstract
Topic:
Host-pathogen interactions
Citation:
Escobar
EE and
Alfonzo
MA
(2013). A more pro-inflammatory environment is generated in nervous cells cultures in the simultaneous presence of HIV-1 and Toxoplasma gondii, even with a lower parasite replication.
Front. Immunol.
Conference Abstract:
15th International Congress of Immunology (ICI).
doi: 10.3389/conf.fimmu.2013.02.01122
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Received:
31 Jul 2013;
Published Online:
22 Aug 2013.
*
Correspondence:
Mr. Edwin E Escobar, Instituto Venezolano de Investigaciones Científicas, Centro de Medicina Experimental, Caracas, Distrito Capital, 1010, Venezuela, edscobar@gmail.com