Fabrine S. M. Tristão

CCR5-Dependent Regulatory T Cell Migration Mediates Fungal Survival and Severe Immunosuppression

Paracoccidioidomycosis, a debilitating pulmonary mycosis, is caused by the dimorphic fungus Paracoccidioides brasiliensis. The infection results in the formation of granulomas containing viable yeast cells that are the fungal sources for disease reactivation. Because CD4+CD25+ regulatory T cells (Tregs) are in the lesions of patients with paracoccidioidomycosis, the migration of Treg cells is dependent on the axis chemokine-chemokine receptors, and CCR5 ligands are produced in P. brasiliensis-induced lesions, we investigated the role of CCR5 in the control of the infection. The results showed that CCR5−/− mice are more efficient in controlling fungal growth and dissemination and exhibited smaller granulomas than wild-type (WT) mice. In the absence of CCR5, the percentage of CD4+CD25+ T cells expressing Foxp3, glucocorticoid-induced TNFR (GITR), CD103, CD45low, and CTLA-4 in the granulomas was significantly decreased. Interestingly, P. brasiliensis infection resulted in an absence of T cell proliferation in response to Con A in WT but not CCR5−/− mice that was abrogated by anti-CTLA-4 mAb and anti-GITR mAb. Moreover, the adoptive transfer of CD4+CD25+ but not CD4+CD25− T cells from infected WT to infected CCR5−/− mice resulted in a significant increase in fungal load. Overall, CCR5 is a key receptor for the migration of Treg cells to the site of P. brasiliensis infection, leading to down-modulation of effector immune response and the long-term presence of the fungus in the granulomas. Thus, a tight control of Treg cell migration to the granulomatous lesions could be an important mechanism for avoiding exacerbation and reactivation of the disease.

5-Lipoxygenase Activity Increases Susceptibility to Experimental Paracoccidioides brasiliensis Infection

ABSTRACT Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the thermodimorphic fungus Paracoccidioides brasiliensis. Leukotrienes and lipoxins are lipid mediators produced after 5-lipoxygenase (5-LO) activation that exhibit pro- and anti-inflammatory roles, respectively. Here, we have investigated the contribution of 5-LO enzymatic activity in PCM using an experimental model of P. brasiliensis infection. B6.129 wild-type (B6.129) and 5-LO-deficient (5-LO−/−) mice were intravenously inoculated with a virulent strain of P. brasiliensis (Pb18), and the survival rate of the infected mice was investigated on different days after yeast infection. 5-LO−/− mice exhibited an increased survival rate associated with a decreased number of CFU. The resistance of 5-LO−/− during PCM was associated with augmented nitric oxide (NO) production and the formation of compact granulomas. In addition, the absence of 5-LO was associated with a diminished number of CD4+ CD25+ regulatory T cells, higher levels of gamma interferon and interleukin-12, and increased T-bet (a T-box transcription factor that directs Th1 lineage commitment) mRNA levels in the lungs. Taken together, our results show for the first time that 5-LO enzymatic activity increases susceptibility to P. brasiliensis, suggesting that this pathway may be a potential target for therapeutic intervention during PCM.

Cell-Free Antigens from Paracoccidioides brasiliensis Drive IL-4 Production and Increase the Severity of Paracoccidioidomycosis

The thermally dimorphic fungus Paracoccidioides brasiliensis (Pb) is the causative agent of paracoccidioidomycosis (PCM), one of the most frequent systemic mycosis that affects the rural population in Latin America. PCM is characterized by a chronic inflammatory granulomatous reaction, which is consequence of a Th1-mediated adaptive immune response. In the present study we investigated the mechanisms involved in the immunoregulation triggered after a prior contact with cell-free antigens (CFA) during a murine model of PCM. The results showed that the inoculation of CFA prior to the infection resulted in disorganized granulomatous lesions and increased fungal replication in the lungs, liver and spleen, that paralleled with the higher levels of IL-4 when compared with the control group. The role of IL-4 in facilitating the fungal growth was demonstrated in IL-4-deficient- and neutralizing anti-IL-4 mAb-treated mice. The injection of CFA did not affect the fungal growth in these mice, which, in fact, exhibited a significant diminished amount of fungus in the tissues and smaller granulomas. Considering that in vivo anti-IL-4-application started one week after the CFA-inoculum, it implicates that IL-4-CFA-induced is responsible by the mediation of the observed unresponsiveness. Further, the characterization of CFA indicated that a proteic fraction is required for triggering the immunosuppressive mechanisms, while glycosylation or glycosphingolipids moieties are not. Taken together, our data suggest that the prior contact with soluble Pb antigens leads to severe PCM in an IL-4 dependent manner.

Effects of Antimicrobial Photodynamic Therapy on Transforming Growth Factor-β1 Levels in the Gingival Crevicular Fluid

BACKGROUND AND OBJECTIVE Antimicrobial photodynamic therapy (aPDT) has been proposed as an adjunctive therapy to scaling and root planing (SRP). The transforming growth factor-β1 (TGF-β) has been considered as an anti-inflammatory cytokine, and its levels in the gingival crevicular fluid (GCF) could monitor the periodontal repair. This study evaluated the adjunct effect of aPDT compared with SRP, analyzing the TGF-β levels in GCF after nonsurgical and surgical therapy in chronic periodontitis patients. METHODS Fifteen patients, presenting bilaterally lower molars with class III furcation lesions, were selected. Each pair of teeth was randomly assigned to a control group (CG) or test group (TG). Initially, SRP was performed in the CG, and SRP + aPDT in the TG. Forty-five days later, flap surgery plus SRP, and flap surgery plus SRP + aPDT were performed in CG and TG, respectively. GCF was collected and an enzyme-linked immunosorbent assay (ELISA) test was conducted to determine the amount and concentration of TGF-β in the GCF at baseline, 45 days post-initial therapy, and 21 days after surgery. RESULTS Statistically significant differences between groups were found in relation to GCF volume 21 days after the surgical procedures (p=0.03) and TGF-β concentration in GCF 45 days post-initial therapy (p=0.04), favoring the TG. CONCLUSIONS There was an additional effect of the aPDT protocol compared with SRP for the TGF-β concentration in GCF 45 days after nonsurgical therapy, and for the GCF volume 21 days after surgical therapy.

B Cell-Deficient Mice Display Enhanced Susceptibility to Paracoccidioides brasiliensis Infection

Paracoccidioidomycosis (PCM) is a chronic granulomatous disease caused by the thermally dimorphic fungus Paracoccidioidesbrasiliensis. T helper 1 (Th1)-mediated immunity is primarily responsible for acquired resistance during P. brasiliensis infection. On the contrary, the susceptibility is associated with occurrence of type-2 immunity (Th2), which is characterized by IL-4 release, B cell activation, and production of antibodies. Although antibodies are frequently associated with severe PCM, it is not clear whether they contribute to susceptibility or merely constitute a marker of infection stage. Here, we assessed the function of B cells during experimental P. brasiliensis infection in mice, and our results showed that B cell-knockout (BKO) mice are more susceptible than their wild-type littermate controls (C57BL/6, WT). The BKO mice showed higher mortality rate, increased number of colony-forming units in the lungs, and larger granulomas than WT mice. In the absence of B cells, we observed high levels of IL-10, whereas IFN-γ, TNF-α, and IL-4 levels were similar between both groups. Finally, we showed that transference of WT immune serum to BKO mice resulted in diminished infiltration of inflammatory cells and better organization of the pulmonary granulomas. Taken together, these data suggest that B cells are effectively involved in the control of P. brasiliensis growth and organization of the granulomatous lesions observed during the experimental PCM.

Role of Interleukin (IL)-18 in experimental paracoccidioidomycosis

Interleukin (IL)-18 has been regarded as a Th1 type cytokine involved in many fungal and parasitic infections. Since there have been no studies, as of yet, evaluating the role of this cytokine in paracoccidioidomycosis (PCM), we assessed the function of IL-18 by using an experimental PCM model. Our results showed that IL-18 knockout (IL-18 -/-) BALB/c were more resistant to Paracoccidioides brasiliensis than their littermate controls (WT). In fact, mortality rate was higher in WT mice and in the first month of infection, the number of colony forming units of the etiologic agent recovered from the lungs was greater in WT mice. In histopathological analyses, well-formed granulomas were seen in both WT and IL-18(-/-) mice. However, substantial differences were observed at the second month of infection when epithelioid cells predominated in the lesions of IL-18(-/-) mice, which could infer that IL-18 postpones pulmonary healing. The levels of IL-10 were significantly higher in IL-18 sufficient mice at early stages of infection and therefore account for the delayed fungal clearance observed in WT mice. TNF-alpha augmented later in the infection of WT mice, seemingly to compensate high levels of IL-10. Our results demonstrated that IL-18 has a critical role in protecting BALB/c mice against disseminated PCM.

Th17-inducing cytokines IL-6 and IL-23 are crucial for granuloma Formation during experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), a chronic granulomatous disease caused by the thermally dimorphic fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii, has the highest mortality rate among systemic mycosis. The T helper 1-mediated immunity is primarily responsible for acquired resistance during P. brasiliensis infection, while susceptibility is associated with Th2 occurrence. Th17 is a population of T CD4+ cells that, among several chemokines and cytokines, produces IL-17A and requires the presence of IL-1, IL-6, and TGF-β for differentiation in mice and IL-23 for its maintenance. Th17 has been described as an arm of the immune system that enhances host protection against several bacterial and fungal infections, as Pneumocystis carinii and Candida albicans. In this study, we aimed to evaluate the Th17 immune response and the role of Th17-associated cytokines (IL-6, IL-23, and IL-17A) during experimental PCM. First, we observed that P. brasiliensis infection [virulent yeast strain 18 of P. brasiliensis (Pb18)] increased the IL-17A production in vitro and all the evaluated Th17-associated cytokines in the lung tissue from C57BL/6 wild-type mice. In addition, the deficiency of IL-6, IL-23, or IL-17 receptor A (IL-17RA) impaired the compact granuloma formation and conferred susceptibility during infection, associated with reduced tumor necrosis factor-α, IFN-γ, and inducible nitric oxide synthase enzyme expression. Our data suggest that IL-6 production by bone marrow-derived macrophages (BMDMs) is important to promote the Th17 differentiation during Pb18 infection. In accordance, the adoptive transfer of BMDMs from C57BL/6 to infected IL-6−/− or IL-17RA−/− mice reduced the fungal burden in the lungs compared to nontransferred mice and reestablished the pulmonary granuloma formation. Taken together, these results suggest that Th17-associated cytokines are involved in the modulation of immune response and granuloma formation during experimental PCM.

Human Leucocyte Antigen-G (HLA-G) and Its Murine Functional Homolog Qa2 in the Trypanosoma cruzi Infection

Genetic susceptibility factors, parasite strain, and an adequate modulation of the immune system seem to be crucial for disease progression after Trypanosoma cruzi infection. HLA-G and its murine functional homolog Qa2 have well-recognized immunomodulatory properties. We evaluated the HLA-G 3′ untranslated region (3′UTR) polymorphic sites (associated with mRNA stability and target for microRNA binding) and HLA-G tissue expression (heart, colon, and esophagus) in patients presenting Chagas disease, stratified according to the major clinical variants. Further, we investigated the transcriptional levels of Qa2 and other pro- and anti-inflammatory genes in affected mouse tissues during T. cruzi experimental acute and early chronic infection induced by the CL strain. Chagas disease patients exhibited differential HLA-G 3′UTR susceptibility allele/genotype/haplotype patterns, according to the major clinical variant (digestive/cardiac/mixed/indeterminate). HLA-G constitutive expression on cardiac muscle and colonic cells was decreased in Chagasic tissues; however, no difference was observed for Chagasic and non-Chagasic esophagus tissues. The transcriptional levels of Qa2 and other anti and proinflammatory (CTLA-4, PDCD1, IL-10, INF-γ, and NOS-2) genes were induced only during the acute T. cruzi infection in BALB/c and C57BL/6 mice. We present several lines of evidence indicating the role of immunomodulatory genes and molecules in human and experimental T. cruzi infection.

The left lung is preferentially targeted during experimental paracoccidioidomycosis in C57BL/6 mice

Paracoccidioidomycosis (PCM) is a chronic systemic mycosis caused by the inhalation of the thermally dimorphic fungus Paracoccidioides brasiliensis as well as the recently described P. lutzii. Because the primary infection occurs in the lungs, we investigated the differential involvement of the right and left lungs in experimental P. brasiliensis infection. Lungs were collected from C57BL/6 mice at 70 days after intravenous infection with 1×106 yeast cells of a virulent strain of P. brasiliensis (Pb18). The left lung, which in mice is smaller and has fewer lobes than the right lung, yielded increased fungal recovery associated with a predominant interleukin-4 response and diminished synthesis of interferon-γ and nitric oxide compared with the right lung. Our data indicate differential involvement of the right and left lungs during experimental PCM. This knowledge emphasizes the need for an accurate, standardized protocol for tissue collection during studies of experimental P. brasiliensis infection, since experiments using the same lungs favor the collection of comparable data among different mice.

Differential behavioral and glial responses induced by dopaminergic mechanisms in the iNOS knockout mice

HIGHLIGHTSiNOS deficient mice shows altered basal exploratory behavior.Either haloperidol or reserpine induces different effects in iNOS deficient mice.There is decreased activation of astrocytes and microglia in iNOS deficient mice.iNOS deficient mice showed decreased astroglial reaction induced by reserpine. ABSTRACT The interaction between distinctive nitric oxide synthase (NOS) isoforms and the dopamine system provides new avenues to the development of pharmacological tools for the pathophysiological conditions of the dopaminergic system. Our aim was to investigate the influences of dopamine‐induced effects in inducible NOS knockout (iNOS KO) mice. In order to characterize iNOS KO mice phenotype, the animals were submitted to the basal analyses of motor, sensorimotor and sensorial abilities. Pharmacological challenging of the dopaminergic system included the investigation of amphetamine‐induced prepulse inhibition (PPI) disruption, haloperidol‐induced catalepsy, reserpine‐induced oral involuntary movements and hyperlocomotion induced by amphetamine in reserpine treated mice. The iNOS KO mice showed significant reduction of spontaneous motor activity, but there was no significant difference in sensorimotor or sensorial responses of iNOS KO mice compared to wild type (WT). Regarding the dopaminergic system, iNOS KO mice showed a significant increase of haloperidol‐induced catalepsy. This effect was confirmed through an iNOS pharmacological inhibitor (1400W) in WT mice. In addition, iNOS KO reserpine treated mice showed reduced oral involuntary movements and amphetamine‐induced hyperlocomotion. Knowing that iNOS is mainly expressed in glial cells we analyzed the immunoreactivity (ir) for GFAP (astrocyte marker) and IBA‐1 (microglial marker) in the striatum, an area enrolled in motor planning among other functions. iNOS KO presented reduced GFAP‐ir and IBA‐1‐ir compared with WT. Reserpine treatment increased GFAP‐ir in both WT and iNOS KO. However, these effects were slighter in iNOS KO. Activated state of microglia was increased by reserpine only in WT mice. Our results further demonstrated that the absence of iNOS interfered with dopamine‐mediated behavioral and molecular responses. These results increase the understanding of the dopamine and NO system interaction, which is useful for the management of the dopamine‐related pathologies.