Silver Nanoparticle-Embedded Carbon Nitride: Antifungal Activity on Candida albicans and Toxicity toward Animal Cells.

The development of engineered nanomaterials has been considered a promising strategy to control oral infections. In this study, silver-embedded carbon nitrides (Ag@g-CN) were synthesized and tested against Candida albicans, investigating their antifungal action and biocompatibility in animal cells. Ag@g-CN was synthesized by a simple one-pot thermal polymerization technique and characterized by various analytical techniques. X-ray diffraction (XRD) analysis revealed slight alterations in the crystal structure of g-CN upon the incorporation of Ag. Fourier transform infrared (FT-IR) spectroscopy confirmed the presence of Ag-N bonds, indicating successful silver incorporation and potential interactions with g-CN's amino groups. UV-vis spectroscopy demonstrated a red shift in the absorption edge of Ag@g-CN compared with g-CN, attributed to the surface plasmon resonance effect of silver nanoparticles. Field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) confirmed the 2D layered sheet like morphology of both materials. The Ag 3d peaks found in X-ray photoelectron spectroscopy (XPS) confirmed the presence of metallic Ag0 nanoparticles in Ag@g-CN. The Ag@g-CN materials exhibited high antifungal activity against reference and oral clinical strains of C. albicans, with minimal inhibitory concentration (MIC) ranges between 16-256 µg/mL. The mechanism of Ag@g-CN on C. albicans was attributed to the disruption of the membrane integrity and disturbance of the biofilm. In addition, the Ag@g-CN material showed good biocompatibility in the fibroblastic cell line and in Galleria mellonella, with no apparent cytotoxicity observed at a concentration up to 1000 µg/mL. These findings demonstrate the potential of the Ag@g-CN material as an effective and safe antifungal agent for the treatment of oral fungal infections.

Blocking the CTLA-4 and PD-1 pathways during pulmonary paracoccidioidomycosis improves immunity, reduces disease severity, and increases the survival of infected mice.

Immune checkpoint pathways, i.e., coinhibitory pathways expressed as feedback following immune activation, are crucial for controlling an excessive immune response. Cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death protein-1 (PD-1) are the central classical checkpoint inhibitory (CPI) molecules used for the control of neoplasms and some infectious diseases, including some fungal infections. As the immunosuppression of severe paracoccidioidomycosis (PCM), a chronic granulomatous fungal disease, was shown to be associated with the expression of coinhibitory molecules, we hypothesized that the inhibition of CTLA-4 and PD-1 could have a beneficial effect on pulmonary PCM. To this end, C57BL/6 mice were infected with Paracoccidioides brasiliensis yeasts and treated with monoclonal antibodies (mAbs) α-CTLA-4, α-PD-1, control IgG, or PBS. We verified that blockade of CTLA-4 and PD-1 reduced the fungal load in the lungs and fungal dissemination to the liver and spleen and decreased the size of pulmonary lesions, resulting in increased survival of mice. Compared with PBS-treated infected mice, significantly increased levels of many pro- and anti-inflammatory cytokines were observed in the lungs of α-CTLA-4-treated mice, but a drastic reduction in the liver was observed following PD-1 blockade. In the lungs of α-CPI and IgG-treated mice, there were no changes in the frequency of inflammatory leukocytes, but a significant reduction in the total number of these cells was observed. Compared with PBS-treated controls, α-CPI- and IgG-treated mice exhibited reduced pulmonary infiltration of several myeloid cell subpopulations and decreased expression of costimulatory molecules. In addition, a decreased number of CD4+ and CD8+ T cells but sustained numbers of Th1, Th2, and Th17 T cells were detected. An expressive reduction in several Treg subpopulations and their maturation and suppressive molecules, in addition to reduced numbers of Treg, TCD4+, and TCD8+ cells expressing costimulatory and coinhibitory molecules of immunity, were also detected. The novel cellular and humoral profiles established in the lungs of α-CTLA-4 and α-PD-1-treated mice but not in control IgG-treated mice were more efficient at controlling fungal growth and dissemination without causing increased tissue pathology due to excessive inflammation. This is the first study demonstrating the efficacy of CPI blockade in the treatment of pulmonary PCM, and further studies combining the use of immunotherapy with antifungal drugs are encouraged.

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-1ß, 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.

The immunosuppressive activity of myeloid-derived suppressor cells in murine Paracoccidioidomycosis relies on Indoleamine 2,3-dioxygenase activity and Dectin-1 and TLRs signaling.

Paracoccidioidomycosis (PCM) is a systemic mycosis with a high incidence in Latin America. Prior studies have demonstrated the significance of the enzyme Indoleamine 2,3-dioxygenase (IDO-1) in the immune regulation of PCM as well as the vital role of myeloid-derived suppressor cells (MDSCs) in moderating PCM severity. Additionally, Dectin-1 and Toll-Like Receptors (TLRs) signaling in cancer, infection, and autoimmune diseases have been shown to impact MDSC-IDO-1+ activity. To expand our understanding of MDSCs and the role of IDO-1 and pattern recognition receptors (PRRs) signaling in PCM, we generated MDSCs in vitro and administered an IDO-1 inhibitor before challenging the cells with Paracoccidioides brasiliensis yeasts. By co-culturing MDSCs with lymphocytes, we assessed T-cell proliferation to examine the influence of IDO-1 on MDSC activity. Moreover, we utilized specific antibodies and MDSCs from Dectin-1, TLR4, and TLR2 knockout mice to evaluate the effect of these PRRs on IDO-1 production by MDSCs. We confirmed the importance of these in vitro findings by assessing MDSC-IDO-1+ in the lungs of mice following the fungal infection. Taken together, our data show that IDO-1 expression by MDSCs is crucial for the control of T-cell proliferation, and the production of this enzyme is partially dependent on Dectin-1, TLR2, and TLR4 signaling during murine PCM.

Myeloid-derived suppressor cells are associated with impaired Th1 and Th17 responses and severe pulmonary paracoccidioidomycosis which is reversed by anti-Gr1 therapy.

Previous studies on paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America, revealed that host immunity is tightly regulated by several suppressive mechanisms mediated by tolerogenic plasmacytoid dendritic cells, the enzyme 2,3 indoleamine dioxygenase (IDO-1), and regulatory T-cells (Tregs). IDO-1 orchestrates local and systemic immunosuppressive effects through the recruitment and activation of myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid cells possessing a potent ability to suppress T-cell responses. However, the involvement of MDSCs in PCM remains uninvestigated. The presence, phenotype, and immunosuppressive activity of MDSCs were evaluated at 96 h, 2 weeks, and 8 weeks of pulmonary infection in C57BL/6 mice. Disease severity and immune responses were assessed in MDSC-depleted and nondepleted mice using an anti-Gr1 antibody. Both monocytic-like MDSCs (M-MDSCs) and polymorphonuclear-like MDSCs (PMN-MDSCs) massively infiltrated the lungs during Paracoccidioides brasiliensis infection. Partial reduction of MDSC frequency led to a robust Th1/Th17 lymphocyte response, resulting in regressive disease with a reduced fungal burden on target organs, diminishing lung pathology, and reducing mortality ratio compared with control IgG2b-treated mice. The suppressive activity of MDSCs on CD4 and CD8 T-lymphocytes and Th1/Th17 cells was also demonstrated in vitro using coculture experiments. Conversely, adoptive transfer of MDSCs to recipient P. brasiliensis-infected mice resulted in a more severe disease. Taken together, our data showed that the increased influx of MDSCs into the lungs was linked to more severe disease and impaired Th1 and Th17 protective responses. However, protective immunity was rescued by anti-Gr1 treatment, resulting in a less severe disease and controlled tissue pathology. In conclusion, MDSCs have emerged as potential target cells for the adjuvant therapy of PCM.