TNF-alpha and CD8(+) T Cells Mediate the Beneficial Effects of Nitric Oxide Synthase-2 Deficiency in Pulmonary Paracoccidioidomycosis

Background: Nitric oxide (NO), a key antimicrobial molecule, was previously shown to exert a dual role in paracoccidioidomycosis, an endemic fungal infection in Latin America. in the intravenous and peritoneal models of infection, NO production was associated with efficient fungal clearance but also with non-organized granulomatous lesions. Because paracoccidioidomycosis is a pulmonary infection, we aimed to characterize the role of NO in a pulmonary model of infection.Methodology/Principal Findings: C57Bl/6 wild type (WT) and iNOS(-/-) mice were i.t. infected with 1x10(6) Paracoccidioides brasiliensis yeasts and studied at several post-infection periods. Unexpectedly, at week 2 of infection, iNOS(-/-) mice showed decreased pulmonary fungal burdens associated with an M2-like macrophage profile, which expressed high levels of TGF-beta impaired ability of ingesting fungal cells. This early decreased fungal loads were concomitant with increased DTH reactions, enhanced TNF-alpha synthesis and intense migration of activated macrophages, CD4(+) and CD8(+) T cells into the lungs. By week 10, iNOS(-/-) mice showed increased fungal burdens circumscribed, however, by compact granulomas containing elevated numbers of activated CD4(+) T cells. Importantly, the enhanced immunological reactivity of iNOS(-/-) mice resulted in decreased mortality rates. in both mouse strains, depletion of TNF-alpha led to non-organized lesions and excessive influx of inflammatory cells into the lungs, but only the iNOS(-/-) mice showed increased mortality rates. in addition, depletion of CD8(+) cells abolished the increased migration of inflammatory cells and decreased the number of TNF-alpha and IFN-gamma CD4(+) and CD8(+) T cells into the lungs of iNOS(-/-) mice.Conclusions/Significance: Our study demonstrated that NO plays a deleterious role in pulmonary paracoccidioidomycosis due to its suppressive action on TNF-alpha production, T cell immunity and organization of lesions resulting in precocious mortality of mice. It was also revealed that uncontrolled fungal growth can be overcome by an efficient immune response.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ São Paulo, Inst Ciencias Biomed, Dept Imunol, BR-05508 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilHosp Sirio Libanes São Paulo, Dept Patol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFAPESP: 04/14518-2FAPESP: 2011/51258-2Web of Scienc

iNOS activity controls the influx of activated mononuclear phagocytes and T cells to the lungs.

<p>Flow cytometry characterization of lung infiltrating leucocytes (LIL) from iNOS^−/− and WT mice after i.t. infection with 1×10⁶<i>P. brasiliensis</i> yeast cells. Lungs of iNOS^−/− and WT mice (n = 6–8) were excised, washed in PBS, minced, and digested enzymatically. At weeks 2 (A, C) and 10 (B, D) after infection, lung cell suspensions were obtained and stained as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002325#s2" target="_blank">Materials and Methods</a>. (A, B) Phenotypic characterization of CD11b⁺ mononuclear phagocytes expressing CD80, CD86 and CD40 and dendritic cells expressing high levels of CD11c and CD86 (CD11c^highCD86^high). An increased presence of mononuclear phagocytes was observed in deficient mice, while the number of dendritic cells was equivalent in the lungs of both mouse strains. (C, D) Characterization of T cell subsets by flow cytometry in LIL obtained at weeks 2 (C) and 10 (D) after infection. To characterize the expansion of regulatory T cells in LIL, surface staining of CD25⁺ and intracellular FoxP3 expression were back-gated on the CD4⁺ T cell population. The acquisition and analysis gates were restricted to macrophages (A, B) or lymphocytes (C, D). The data represent the mean ± SEM of the results from 6–8 mice per group and are representative of two independent experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01) and *** (<i>P</i><0.001), compared with WT mice.</p

Neutralization of TNF-α is more detrimental to iNOS^−/− than WT mice.

<p>TNF-α neutralization leads to increased influx of activated cells to the lungs, increased mortality and non-organized lesions in iNOS-deficient mice. WT and iNOS^−/− mice were treated with anti-TNF-α mAb (MP6 XT 22) or normal rat IgG (control) and i.t. infected with 1×10⁶ fungal cells. Number and activation of lymphocytes (A) and mononuclear phagocytes (B) present in pulmonary lesions at the 2^nd week postinfection of anti-TNF-α treated and untreated mice were determined by flow cytometry. (C) Survival times of anti-TNF-α treated and untreated mice. TNF-α depleted iNOS^−/− mice showed a decreased survival time, which was significantly different (* <i>P</i><0.05) from all other studied groups. The results are representative of three independent experiments (n = 6–7). (D) Photomicrographs of pulmonary lesions developed by IgG-treated (D, superior panels) and TNF-α-depleted (B, lower panels) mice at the week 8 of infection. Anti-TNF-α treatment of iNOS-deficient mice resulted in less organized, fungi rich, confluent lesions, occupying the largest part of lung tissue. H&E stained lesions (100×).</p

In pulmonary paracoccidioidomycosis, iNOS-deficiency alters fungal loads and synthesis of cytokines.

<p>(A, B) Recovery of CFUs from the lungs and liver of iNOS^−/− and WT control mice infected i.t. with 1×10⁶ yeasts. Early in infection (week 2), the lack of NO production results in lower fungal loads but at a late period (week 10), an increased pulmonary fungal burden was observed. The bars represent means ± SEM of log₁₀ CFU obtained from groups of six to eight mice. The results are representative of three experiments with equivalent results. (C, D) Levels of type 1 and type 2 cytokines in lungs homogenates of iNOS^−/− and WT mice (n = 6–8). At the 2^nd (C) and 10^th (D) weeks after i.t. infection, lungs from iNOS^−/− and WT mice were collected and disrupted in 5.0 mL of PBS and supernatants were analyzed for cytokines content by capture ELISA. The bars depict means ± SEM of cytokine levels (6–8 animals per group). * (<i>P</i><0.05) and *** (<i>P</i><0.001) compared with WT controls.</p

CD8⁺ T cells control the early influx of inflammatory cells to the lungs of iNOS-deficient mice.

<p>Groups (n = 6–7) of WT and iNOS^−/− mice were given 200 µg of anti-CD8α mAbs (H-35) or normal rat IgG (controls) by the i.p. route, 48 and 24 h before infection, and 150 µg of the anti-CD8α mAbs or rat IgG at days 6 and 12 of infection with 1×10⁶<i>P. brasiliensis</i>. The severity of infection was assessed by organ CFU counts (A) at the 2^nd week of infection. The phenotypes of lung infiltrating lymphocytes (B), mononuclear phagocytes (C), and CD11c⁺ dendritic cells isolated by magnetic beads (D) were characterized by flow cytometry. The results are representative of two experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001).</p

INOS^−/− macrophages are refractory to IFN-γ and TNF-α activation and have an impaired phagocytic activity.

<p>(A) For fungicidal assays, peritoneal macrophages from WT and iNOS ^−/− C57BL/6 mice were cultivated in the presence or absence of recombinant IFN-γ (40 ng/ml), TNF-α (20 ng/ml) or 1-methyl-DL-tryptophan (1 MT, 1 mM), a specific inhibitor of 2,3 indoleamine dioxygenase. Cultures were infected with <i>P. brasiliensis</i> yeasts in a macrophage∶yeast ratio of 12.5∶1. After 48 h, macrophages were lysed, and viable yeasts determined using a CFU assay. All analyzes were done with five wells per condition in three independent experiments. (B) For phagocytic assays, macrophages were infected with heat-inactivated, FITC labeled, <i>P. brasiliensis</i> yeasts at a macrophage∶yeast ratio of 1∶1 for 2 h at 37°C in 5% CO₂ to allow fungi adhesion and ingestion. Some macrophage cultures were treated with IFN-γ (40 ng/ml), or TNF-α (20 ng/ml) overnight, before infection. Macrophages were washed, cells detached from plastic, and labeled with anti-F4/80 (APC) antibodies. The cell suspensions were immediately read on a FACScalibur cytometer. All analyzes were done with five wells per condition in three independent experiments. (C) A quenching assay employing a trypan blue solution (TB, 250 µg/mL) was used to distinguish internalized from surface-bound yeasts (FITC- labeled <i>P.brasiliensis</i> particles). Phagocytic assays were performed as above described, and adherent/ingested cells measured using the FL1 and FL4 channels of a FACscalibur cytometer. Cell suspensions were then treated with a TB solution for quenching the green surface-bound fluorescence on macrophages and samples were again analyzed. APC-labeled macrophages were gated, and FL1 and FL3 channels used to discriminate ingested (green fluorescent, FL1) from adherent (red fluorescent, FL3) yeasts. All analyzes were done with five wells per condition in three independent experiments. The bars depict means ± SEM ** (<i>P</i><0.01) and *** (<i>P</i><0.001) compared with WT controls.</p

iNOS−/− mice show a better disease outcome than WT mice.

<p>Positive DTH reactions and well-organized lesions appear to compensate the increased fungal burdens, and are associated with increased survival times of iNOS^−/− mice. (A) iNOS ^−/− and WT mice were i.t. infected with 1×10⁶<i>P.brasiliensis</i> yeasts and DTH reactions measured 2 and 10 weeks later. Uninfected mice were used as control. DTH reactions were evaluated in control and infected mice by the footpad swelling 48 h after the s.c. injection of 25 µl of a soluble <i>P. brasiliensis</i> antigen. The bars depict means ± SE of footpad swelling. (n = 6–8). (B) Photomicrographs of pulmonary lesions of WT (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002325#pntd-0002325-g004" target="_blank">Fig. 4B</a>, upper panel), and iNOS^−/− (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002325#pntd-0002325-g004" target="_blank">Fig. 4B</a>, lower panel) at weeks 10 of infection with 1×10⁶<i>P. brasiliensis</i> yeasts. Both mouse strains developed extensive lesions, but organized lesions were only observed in NO-deficient mice (HE, 100×). (C) Survival times of iNOS^−/− and WT control mice (n = 6–8) after i.t. infection with 1×10⁶<i>P. brasiliensis</i> yeast cells was determined in a period of 300 days. The data are representative of three independent experiments with equivalent results. * (<i>P</i><0.05), and *** (<i>P</i><0.001) compared with WT controls.</p

Depletion of CD8⁺ T cells alters the numbers of TNF-α⁺, IFN-γ⁺ and IL-4⁺ T cells in the lungs of iNOS^−/− and WT mice.

<p>Groups (n = 6–7) of WT and iNOS^−/− mice were treated with 200 µg of anti-CD8α mAbs (H-35) or normal rat IgG (controls) and i.t. infected with 1×10⁶<i>P. brasiliensis</i>. The presence of TNF-α⁺, IFN-γ⁺ and IL-4⁺ CD4⁺ and CD8⁺ T cells in the lung infiltrating leukocytes was assessed by intracellular cytokine staining by flow cytometry at week 2 after infection. Lung cells were re-stimulated in vitro with PMA/ionomycin for 6 h and subjected to intracellular staining for TNF-α, IFN-γ and IL-4. The lymphocyte population was gated by forward/side scatters. Results are from one experiment and are representative of two independent experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001).</p

INOS^−/− macrophages express high levels of TGF-β arginase1 and TNF-α.

<p>Uninfected and <i>P. brasiliensis</i> infected macrophages were used to characterize the expression of iNOS, ARG1, TGF-β, IL-12, TNF-α and IDO mRNA by quantitative Real-Time PCR. Total RNA was extracted using Trizol reagent, reverse transcribed, and cDNA amplified. Real-time PCR was performed using TaqMan universal master mix. Amplified products were normalized to the amount of GAPDH products from macrophages. All analyzes were done with five wells per condition in three independent experiments. The bars depict means ± SEM ** (<i>P</i><0.01) and *** (<i>P</i><0.001) compared with WT controls.</p