Abstract Microorganisms killing by dendritic cells (DCs) is an important effector mechanism during innate immune response, as it can avoid dissemination of infection during migration of these cells toward draining lymph nodes. However, this function depends on pattern recognition receptors (PRRs) to which the microorganism will bind in these cells. Regarding this, TLR9 activation, by stimulating the oxidative metabolism, induces increase in microbicidal activity of these cells. Accordingly, we showed that DCs treatment with a TLR9 agonist results in an increase in fungicidal activity of these cells against the fungus Paracoccidioides brasiliensis (Pb), which however, was not associated to higher H2O2 levels. Paracoccidioides brasiliensis, dendritic cells, TLR9, fungicidal activity, H2O2. Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil, whose etiologic agent is the thermodimorphic fungus of the genus Paracoccidioides, comprising the cryptic species S1, PS2, PS3 of Paracoccidioides brasiliensis (Pb) and the specie Paracoccidioides lutzii.1–3 Among the mechanisms of immune response against the fungus, those played by dendritic cells (DCs) deserve to be highlighted. Similarly, to other phagocytic cells, DCs release potent cytotoxic molecules that enable them to efficiently destroy microorganisms. This function represents an important effector mechanism during the innate immune response. The greater or lesser capacity of DCs to destroy the various microorganisms may result in differences in the dissemination of the infection during the migration of these cells from the periphery to the secondary lymphoid organs.4–6 In a previous study (unpublished data) we have detected that DCs do not release adequate levels of H2O2 in response to Pb and consequently do not display efficient fungicidal activity toward this fungus. The type of PRR to which the fungus binds can determine its fate within DCs. In this context, TLR9 activation results in an increase in peroxide formation and consequent microorganisms killing by DCs.7 Here, we found that TLR9 activation in human DCs results in an increase in fungicidal activity of these cells toward high and low virulent strains of Pb. However, this process was not associated with increase in H2O2 levels. Peripheral blood was collected from healthy volunteer individuals after signature of informed consent form. Human DCs were generated from purified monocytes (CD14+ cells isolated by magnetic-activated cell sorting (MACS) (Human CD14 Microbeads, Kit: Miltenyi Biotec Inc, Auburn, CA, USA). Monocytes pellet were suspended in RPMI 1640 medium (Sigma-Aldrich, ST. Louis, MO, USA) supplemented with 2 mM (millimolar) of L-glutamine (Sigma-Aldrich), 40 mg/ml gentamicin and 10% inactivated fetal bovine serum (complete culture medium), seeded in six-well tissue culture plates (5 × 106 cells /ml) and allowed to adhere for 2 h at 37°C in an atmosphere of 5% CO2. After this period, supernatants were removed and cultures were incubated with complete culture medium containing 80 ng/mL (nanogram/milliliter) of rH IL-4 and 80 ng/ml of rH GM-CSF (R&D Systems, Inc, Minneapolis, MN, USA) during 7 days for DCs generation. After this period, loosely adherent cells were collected, checked for viability (trypan blue staining), seeded in to 24-well tissue culture plates (106cells/mL), treated with 5 μg /ml of a TLR9 agonist (CPG-ODN-1826) (InvivoGen, San Diego, CA, USA) or a TLR9 antagonist (10 μg / ml- microgram/milliliter) (CPG-ODN-2088) (InvivoGen) for 24 h, challenged for 4 h and 24 h with high (Pb18) and low (Pb265) virulent strains of Pb (cultured as previously8) at concentrations of 2 × 105 yeast/ml of complete culture medium (DCs: yeast ratio of 5:1) and evaluated for fungicidal activity as follow: cocultures were washed with RPMI medium at 37°C to eliminate non-phagocytosed yeasts and after, washed with sterile ice water to allow cells lysis and release of fungi that were phagocytosed. The suspensions resultant of these lysates were considered as experimental cultures. We also performed cultures constituted only by the suspension of fungi and Erro de tradução that was submmitted to the same procedures of experimental cultures and was called control cultures. These cultures were necessary to account for the amounts of fungi that are killed spontaneously during the incubation period, i.e., without the effect of DCs. After the washing process (final volume: 2 ml) control and experimental cultures were plated in triplicates in Petri dishes (100 μl/dish- microliter) containing BHI-agar culture medium (OXOID, LTD, England) at the concentration of 47 g/l (grams/liter) plus 4% horse serum, 50 μg/ml gentamycin and 5% growth factor or aqueous extract.9 After 8 days of BOD incubation at 36°C, the colony forming units (cfu) contained in the control and experimental plaques were counted and the percentage of fungicidal activity calculated using the following formula: 1- (mean of the colony-forming units counted in the experimental plaques/mean of the colony forming units counted in the control plates) × 100. The same cocultures used for evaluating fungicidal activity were also tested for H2O2 production measured by the horseradish peroxidase-phenol red oxidation method.10 However, in this assay, DCs were seeded in to 96-well tissue culture plates (106 cells/ml), and the challenge was made by a fungi suspension (2 × 105 yeast/ml) diluted in a solution of phenol red solution containing 140 mM NaCl; 10 mM phosphate buffer pH7; 5.5 mM dextrose; 0.56 mM phenol red and 0.01 mg/ml of horseradish peroxidase (Sigma-Aldrich). This solution allows that, after H2O2 release, a colorimetric reaction occurs, whose analysis reveals the levels of this metabolite. Thus, after challenge, the colorimetric reaction was stopped by the addition of 0.01 ml of 1 N NaOH and absorbance was determined on an automatic ELISA reader (620 nm filter). The detected absorbance was transformed into nanomoles by using a standard curve constructed with H2O2 serially diluted from 0.25 to 4 nanomoles. The results were expressed as nanomoles /1 × 105 DCs. Differences among results were calculated by analysis of variance (ANOVA) Tukey Kramer test by using GraphPad Prism 5.0 software. The level of significance was set at P < .05. As expected, we found that unstimulated cells had low fungicidal activity towards Pb18 and mainly Pb265. However, after DCs stimulation with a TLR9 agonist a significant increase in the activity was detected in response to both strains and in both periods. It is worthnoting, however, that untreated DCs display higher fungicidal activity toward Pb18 compared to the Pb265, while TLR9 agonist treated DCs show higher fungicidal activity towards the Pb265, mainly in the 4 h period. The specificity of the TLR9 activating role on the activity of DCs was supported by assays in which the cells were incubated with a receptor antagonist. In these experiments fungicidal activity was similar to that detected in unstimulated DCs (Fig. 1). Figure 1. View largeDownload slide Fungicidal activity of DCs unstimulated (Pb18 and Pb265 4 and 24 h) or stimulated with a TLR9 receptor agonist (5 μg/ml) or TLR9 antagonist receptor (10 μg/ml) for 24 h and challenged with Pb18 and Pb265 for 4 and 24 h. The results are expressed by mean ± standard deviation and were obtained from cultures of six subjects. *P < .05× respective unstimulated DCs + P < .05× respective Pb18 & P < 0.05× respective Pb18. Figure 1. View largeDownload slide Fungicidal activity of DCs unstimulated (Pb18 and Pb265 4 and 24 h) or stimulated with a TLR9 receptor agonist (5 μg/ml) or TLR9 antagonist receptor (10 μg/ml) for 24 h and challenged with Pb18 and Pb265 for 4 and 24 h. The results are expressed by mean ± standard deviation and were obtained from cultures of six subjects. *P < .05× respective unstimulated DCs + P < .05× respective Pb18 & P < 0.05× respective Pb18. Results regarding H2O2 production can be analyzed in Figure 2 (A–D). We found that all unchallenged cultures produced low levels of the metabolite, even after agonist or antagonist treatment. However, after challenge with Pb 18 (A) and Pb265 (C, D) a significant increase of the metabolite production was detected (exception for the challenge with Pb 18 for 24 h (B), in which metabolite increase was not significant). However, unexpectedly, this production remained unchanged when cells were pretreated with the agonist before challenge. Figure 2. View largeDownload slide H2O2 production by DCs unstimulated or stimulated with a TLR9 receptor agonist (5 μg/ml) or with a TLR9 antagonist receptor (10 μg/ml) for 24 h and challenged with Pb18 (A, B) and Pb265 (C, D) for 4 and 24 h. The results are expressed by mean ± standard deviation and were obtained from cultures of six subjects. *P < .05× respective unchallenged DCs. Figure 2. View largeDownload slide H2O2 production by DCs unstimulated or stimulated with a TLR9 receptor agonist (5 μg/ml) or with a TLR9 antagonist receptor (10 μg/ml) for 24 h and challenged with Pb18 (A, B) and Pb265 (C, D) for 4 and 24 h. The results are expressed by mean ± standard deviation and were obtained from cultures of six subjects. *P < .05× respective unchallenged DCs. In conclusion, our data showed that DCs stimulation with a TLR9 agonist results in an increase in the fungicidal activity of these cells towards both strains tested. However, it is interesting to note that untreated DCs display higher fungicidal activity toward the Pb18 compared to the Pb265, while TLR9 agonist treated DCs show higher fungicidal activity towards the Pb265. Higher fungicidal activity against Pb18 in comparison to Pb265 could occur due to differences already in the phagocytosis process. If DCs phagocytosed more yeasts from Pb18 compared to Pb265, subsequently more yeasts are submitted to fungicidal activity. Unfortunately, during the present study, we were not able to establish the phagocytic index of infected DCs. However, we performed a parallel assay, in which the supernatants from the two cocultures (containing fungi that were not phagocytosed) were plated. We found that the number of colony forming units were similar for the two cocultures supernatants. Thus, even if by an indirect way, we can consider that DCs equally phagocytose Pb18 and Pb265. That way, differences in fungicidal activity can not be atributed to differences in phagocytosis indexes. One possible explanation for these results would be the differences in the concentrations of β-glucans present on the surface of the two strains. In previous studies, using monocytes, we found that fungicidal activity was higher for Pb265. The explanation was that this strain by having a higher amount of β-glucans on its surface, compared to Pb18, stimulates increased production of TNF-α, which activates cells to release higher H2O2 levels and consequently to display greater fungicidal activity.11 However, in the case of DCs (untreated), an opposite mechanism could be suggested. Higher concentrations of β-glucans in Pb265 surface may induce its binding to PRRs, such as dectin-1, whose activation signals result in lower fungicidal activity. However, in treated DCs, stimulation of TLR9 can induce increased expression of other PRRs, in which Pb265 binds through other PAMPS, different from glucans but also present in higher concentrations compared to Pb18, whose activation signals result in increased fungicidal activity. According to this idea, other examples of positive cross talk between CpG and other fungal PAMPs, such as mannoproteins, have been demonstrated in BMDCs.12 Our results agree with others showing that TLR9 stimulation positively modulates DCs activity.13 Regarding specifically to microbicidal activity it was shown that growth of Salmonella typhimurium in human DCs was inhibited by stimulation of these cells with CpG-DNA, a TLR9 receptor agonist.7 This effect was mediated by an increase in reactive oxygen species production. In addition, they showed that DCs increase their capacity to mature and present antigens to CD4 T cells. Thus, our study should continue in order to evaluate whether TLR9 activation also results in maturation and increase in antigen presentation by DCs, since studies in our lab have already shown that DCs/Pb interaction does not result in maturation of these cells.8 Our results also corroborate studies with other fungi, such as Cryptococcus neoformans,14Candida albicans and Saccharomyces cerevisiae,15Aspergillus fumigatus16 and mainly with Paracoccidioides brasiliensis, that have shown the protector effect of TLR9 by using experimental models of the infection.17–18 Therein, our results contribute to the literature showing that the TLR9 protector effect during fungal infection is, among other factors, associated with its ability to modulate dendritic cell activity. The effect of TLR9 has been attributed to its ability to activate the NADPH-oxidase system with a consequent increase in reactive oxygen species production. Our results, however, did not confirm this association, as increase in DCs capacity to kill the fungus, after TLR9 activation, was not accompanied by higher H2O2 levels. This result is not expected, as in monocytes, we established a direct association between the levels of this metabolite and fungicidal activity, which led us to conclude that it is involved in effective fungus killing displayed by these cells, when activated by cytokines.19 However, in the case of DCs, we can suggest that other toxic molecules can be responsible for the increase in fungicidal activity after TLR9 stimulation. Further experiments will be necessary to prove this hypothesis. Acknowledgment We thank the healthy volunteers for their willingness to participate in this study. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. The authors contributed equally to this study. References 1. 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Trends Microbiol . 2002 ; 10 : 425 – 461 . Google Scholar CrossRef Search ADS PubMed 6. Serbina NV , Salazar-Mather TP , Biron CA , Kuziel WA , Pamer EG . TNF/iNOS-producing dendritic cells mediate innate immune defense against bacterial infection . Immunity . 2003 ; 19 : 59 – 70 . Google Scholar CrossRef Search ADS PubMed 7. Lahiri A , Das P , Vani J , Shaila MS , Chakravortty D . TLR 9 activation in dendritic cells enhances salmonella killing and antigen presentation via involvement of the reactive oxygen species . Plos One . 2010 ; 5 : e13772 . Google Scholar CrossRef Search ADS PubMed 8. Fernandes RK , Bachiega TF , Rodrigues DR et al. Correction: Paracoccidioides brasiliensis interferes on dendritic cells maturation by inhibiting PGE2 production . Plos One . 2015 ; 10 : e0131380 . Google Scholar CrossRef Search ADS PubMed 9. Kurita N , Sano A , Coelho KI , Takeo K , Nishimura K , Miyaji M . An improved culture medium for detecting live yeast phase cells of Paracoccidioides brasiliensis . J Med Vet Mycol . 1993 ; 31 : 201 – 205 . Google Scholar CrossRef Search ADS PubMed 10. Pick E , Keisari Y . A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture . J Immunol Methods . 1980 ; 38 : 161 – 170 . Google Scholar CrossRef Search ADS PubMed 11. Calvi SA , Peraçoli MT , Mendes RP et al. Effect of cytokines on the in vitro fungicidal activity of monocytes from paracoccidioidomycosis patients . Microbes Infect . 2003 ; 5 : 107 – 113 . Google Scholar CrossRef Search ADS PubMed 12. Dan JM , Wang JP , Lee CK , Levitz SM . Cooperative stimulation of dendritic cells by Cryptococcus neoformans mannoproteins and CpG oligodeoxynucleotides . PLoS One . 2008 ; 3 : e2046 . Google Scholar CrossRef Search ADS PubMed 13. Gowda NM , Wu X , Gowda DC . TLR9 and MyD88 are crucial for the development of protective immunity to malaria . J Immunol . 2012 ; 188 : 5073 – 5085 . Google Scholar CrossRef Search ADS PubMed 14. Zhang Y , Wang F , Bhan U et al. TLR9 signaling is required for generation of the adaptive immune protection in Cryptococcus neoformans-infected lungs . Am J Pathol . 2010 ; 177 : 754 – 765 . Google Scholar CrossRef Search ADS PubMed 15. Kasperkovitz PV , Khan NS , Tam JM , Mansour MK , Davids PJ , Vyas JM . Toll-like receptor 9 modulates macrophage antifungal effector function during innate recognition of Candida albicans and Saccharomyces cerevisiae . Infect Immun . 2011 ; 79 : 4858 – 4867 . Google Scholar CrossRef Search ADS PubMed 16. Kasperkovitz PV , Cardenas ML , Vyas JM . TLR9 is actively recruited to Aspergillus fumigatus phagosomes and requires the N-terminal proteolytic cleavage domain for proper intracellular trafficking . J Immunol . 2010 ; 185 : 7614 – 7622 . Google Scholar CrossRef Search ADS PubMed 17. Morais EA , Chame DF , Melo EM et al. TLR 9 involvement in early protection induced by immunization with rPb27 against paracoccidioidomycosis . Microbes Infect . 2016 ; 18 : 137 – 147 . Google Scholar CrossRef Search ADS PubMed 18. Menino JF , Saraiva M , Gomes-Alves AG et al. TLR9 activation dampens the early inflammatory response to Paracoccidioides brasiliensis, impacting host survival . Plos Negl Trop Dis . 2013 ; 25 : e2317 . Google Scholar CrossRef Search ADS 19. Carmo JP , Dias-Melicio LA , Calvi SA , Peraçoli MT , Soares AM . TNF-alpha activates human monocytes for Paracoccidioides brasiliensis killing by an H2O2-dependent mechanism . Med Mycol . 2006 ; 44 : 363 – 368 . Google Scholar CrossRef Search ADS PubMed © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: email@example.com
Medical Mycology – Oxford University Press
Published: Dec 8, 2017
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