Pulmonary paracoccidioidomycosis in AhR deficient hosts is severe and associated with defective Treg and Th22 responses

© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.AhR is a ligand-activated transcription factor that plays an important role in the innate and adaptive immune responses. In infection models, it has been associated with host responses that promote or inhibit disease progression. In pulmonary paracoccidioidomycosis, a primary fungal infection endemic in Latin America, immune protection is mediated by Th1/Th17 cells and disease severity with predominant Th2/Th9/Treg responses. Because of its important role at epithelial barriers, we evaluate the role of AhR in the outcome of a pulmonary model of paracoccidioidomycosis. AhR-/- mice show increased fungal burdens, enhanced tissue pathology and mortality. During the infection, AhR-/- mice have more pulmonary myeloid cells with activated phenotype and reduced numbers expressing indoleamine 2,3 dioxygenase 1. AhR-deficient lungs have altered production of cytokines and reduced numbers of innate lymphoid cells (NK, ILC3 and NCR IL-22). The lungs of AhR-/- mice showed increased presence Th17 cells concomitant with reduced numbers of Th1, Th22 and Foxp3+ Treg cells. Furthermore, treatment of infected WT mice with an AhR-specific antagonist (CH223191) reproduced the main findings obtained in AhR-/- mice. Collectively our data demonstrate that in pulmonary paracoccidioidomycosis AhR controls fungal burden and excessive tissue inflammation and is a possible target for antifungal therapy.This work was supported by a grant from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP-grant to VLGC 2011/51258-2 and 2016/23189-0; fellowship to EFA 2014/18668-2; grant to FVL 2018/14762-3; fellowship to NWP 2019-09278-8), European Union H2020 ERA project (No 667824 – EXCELLtoINNOV) to MV and Conselho Nacional de Pesquisas (CNPq).info:eu-repo/semantics/publishedVersio

Transcriptional profiling of a fungal granuloma reveals a low metabolic activity of Paracoccidioides brasiliensis yeasts and an actively regulated host immune response

Granulomas are important immunological structures in the host defense against the fungus Paracoccidioides brasiliensis, the main etiologic agent of Paracoccidioidomycosis (PCM), a granulomatous systemic mycosis endemic in Latin America. We have performed transcriptional and proteomic studies of yeasts present in the pulmonary granulomas of PCM aiming to identify relevant genes and proteins that act under stressing conditions. C57BL/6 mice were infected with 1x106 yeasts and after 8- and 12-weeks of infection, granulomatous lesions were obtained for extraction of fungal and murine RNAs and fungal proteins. Dual transcriptional profiling was done comparing lung cells and P. brasiliensis yeasts from granulomas with uninfected lung cells and the original yeast suspension used in the infection, respectively. Mouse transcripts indicated a lung malfunction, with low expression of genes related to muscle contraction and organization. In addition, an increased expression of transcripts related to the activity of neutrophils, eosinophils, macrophages, lymphocytes as well as an elevated expression of IL-1β, TNF-α, IFN-γ, IL-17 transcripts were observed. The increased expression of transcripts for CTLA-4, PD-1 and arginase-1, provided evidence of immune regulatory mechanisms within the granulomatous lesions. Also, our results indicate iron as a key element for the granuloma to function, where a high number of transcripts related to fungal siderophores for iron uptake was observed, a mechanism of fungal virulence not previously described in granulomas. Furthermore, transcriptomics and proteomics analyzes indicated a low fungal activity within the granuloma, as demonstrated by the decreased expression of genes and proteins related to energy metabolism and cell cycle

Cross-sectional analysis of students and school workers reveals a high number of asymptomatic SARS-CoV-2 infections during school reopening in Brazilian cities

Brazil experienced one of the most prolonged periods of school closures, and reopening could have exposed students to high rates of SARS-CoV-2 infection. However, the infection status of students and school workers at the time of the reopening of schools located in Brazilian cities is unknown. Here we evaluated viral carriage by RT-PCR and seroprevalence of anti-SARS-CoV-2 antibodies (IgM and IgG) by immunochromatography in 2259 individuals (1139 students and 1120 school workers) from 28 schools in 28 Brazilian cities. We collected the samples within 30 days after public schools reopened and before the start of vaccination campaigns. Most students (n = 421) and school workers (n = 446) had active (qRT-PCR + IgM− IgG− or qRT-PCR + IgM + IgG−/+) SARS-CoV-2 infection. Regression analysis indicated a strong association between the infection status of students and school workers. Furthermore, while 45% (n = 515) of the students and 37% (n = 415) of the school workers were neither antigen nor antibody positive in laboratory tests, 16% of the participants (169 students and 193 school workers) were oligosymptomatic, including those reinfected. These individuals presented mild symptoms such as headache, sore throat, and cough. Notably, most of the individuals were asymptomatic (83.9%). These results indicate that many SARS-CoV-2 infections in Brazilian cities during school reopening were asymptomatic. Thus, our study highlights the need to promote a coordinated public health effort to guarantee a safe educational environment while avoiding exacerbating pre-existent social inequalities in Brazil, reducing social, mental, and economic losses for students, school workers, and their families

Correction: A Human Trypanosome Suppresses CD8+ T Cell Priming by Dendritic Cells through the Induction of Immune Regulatory CD4+ Foxp3+ T Cells.

[This corrects the article DOI: 10.1371/journal.ppat.1005698.]

The IDO–AhR Axis Controls Th17/Treg Immunity in a Pulmonary Model of Fungal Infection

In infectious diseases, the enzyme indoleamine 2,3 dioxygenase-1 (IDO1) that catalyzes the tryptophan (Trp) degradation along the kynurenines (Kyn) pathway has two main functions, the control of pathogen growth by reducing available Trp and immune regulation mediated by the Kyn-mediated expansion of regulatory T (Treg) cells via aryl hydrocarbon receptor (AhR). In pulmonary paracoccidioidomycosis (PCM) caused by the dimorphic fungus Paracoccidioides brasiliensis, IDO1 was shown to control the disease severity of both resistant and susceptible mice to the infection; however, only in resistant mice, IDO1 is induced by TGF-β signaling that confers a stable tolerogenic phenotype to dendritic cells (DCs). In addition, in pulmonary PCM, the tolerogenic function of plasmacytoid dendritic cells was linked to the IDO1 activity. To further evaluate the function of IDO1 in pulmonary PCM, IDO1-deficient (IDO1−/−) C57BL/6 mice were intratracheally infected with P. brasiliensis yeasts and the infection analyzed at three postinfection periods regarding several parameters of disease severity and immune response. The fungal loads and tissue pathology of IDO1−/− mice were higher than their wild-type controls resulting in increased mortality rates. The evaluation of innate lymphoid cells showed an upregulated differentiation of the innate lymphoid cell 3 phenotype accompanied by a decreased expansion of ILC1 and NK cells in the lungs of infected IDO1−/− mice. DCs from these mice expressed elevated levels of costimulatory molecules and cytokine IL-6 associated with reduced production of IL-12, TNF-α, IL-1β, TGF-β, and IL-10. This response was concomitant with a marked reduction in AhR production. The absence of IDO1 expression caused an increased influx of activated Th17 cells to the lungs with a simultaneous reduction in Th1 and Treg cells. Accordingly, the suppressive cytokines IL-10, TGF-β, IL-27, and IL-35 appeared in reduced levels in the lungs of IDO1−/− mice. In conclusion, the immunological balance mediated by the axis IDO/AhR is fundamental to determine the balance between Th17/Treg cells and control the severity of pulmonary PCM

Tolerogenic Plasmacytoid Dendritic Cells Control <i>Paracoccidioides brasiliensis</i> Infection by Inducting Regulatory T Cells in an IDO-Dependent Manner

<div><p>Plasmacytoid dendritic cells (pDCs), considered critical for immunity against viruses, were recently associated with defense mechanisms against fungal infections. However, the immunomodulatory function of pDCs in pulmonary paracoccidiodomycosis (PCM), an endemic fungal infection of Latin America, has been poorly defined. Here, we investigated the role of pDCs in the pathogenesis of PCM caused by the infection of 129Sv mice with 1 x 10⁶ <i>P</i>. <i>brasiliensis-</i>yeasts. <i>In vitro</i> experiments showed that <i>P</i>. <i>brasiliensis</i> infection induces the maturation of pDCs and elevated synthesis of TNF-α and IFN-β. The <i>in vivo</i> infection caused a significant influx of pDCs to the lungs and increased levels of pulmonary type I IFN. Depletion of pDCs by a specific monoclonal antibody resulted in a less severe infection, reduced tissue pathology and increased survival time of infected mice. An increased influx of macrophages and neutrophils and elevated presence of CD4⁺ and CD8⁺ T lymphocytes expressing IFN-γ and IL-17 in the lungs of pDC-depleted mice were also observed. These findings were concomitant with decreased frequency of Treg cells and reduced levels of immunoregulatory cytokines such as IL-10, TGF-β, IL-27 and IL-35. Importantly, <i>P</i>. <i>brasilienis</i> infection increased the numbers of pulmonary pDCs expressing indoleamine 2,3-dioxygenase-1 (IDO), an enzyme with immunoregulatory properties, that were reduced following pDC depletion. In agreement, an increased immunogenic activity of infected pDCs was observed when IDO-deficient or IDO-inhibited pDCs were employed in co-cultures with lymphocytes Altogether, our results suggest that in pulmonary PCM pDCs exert a tolerogenic function by an IDO-mediated mechanism that increases Treg activity.</p></div

pDC depletion determines increased presence of pulmonary macrophages, neutrophils and activated T lymphocytes.

<p>pDC-depleted and control groups were inoculated i.t. with 1 × 10⁶ <i>P</i>. <i>brasiliensis</i> yeasts and cell phenotypes determined at weeks 2 and 8 after infection. Lungs of both mouse groups (n = 4–5) were excised and digested enzymatically. Cell suspensions were obtained and stained as described in Materials and Methods. The stained cells were analyzed immediately on a FACSCanto II equipment with gating of lymphocytes or granulocytes, as judged from FSC and SSC scatters. Gated CD4⁺ and CD8⁺ cells were also analyzed for the expression of activation and deactivation markers. (A) Representative FACS plots demonstrating the gating strategy for lymphocytes, macrophages and neutrophils. (B) Number of total leukocytes. (C) Frequency and number of neutrophils CD11b⁺F4/80^-Gr1⁺, and (D) macrophages F4/80⁺CD11b⁺. (E) Total and activated CD4⁺ T and (F) CD8⁺ T cells. Markers of the suppressive activity of T cells (CTLA4, GITR, ICOS, PD-L1) were also measured by flow cytometry at weeks 2 (G) and 8 (H) after infection. One hundred thousand cells were counted and the data expressed as frequency and number of positive cells. Data are expressed as means ± SE of the mean and are representative of three independent experiments. *<i>p</i> < 0.05.</p