Dataset Information

Experimental Therapy of Paracoccidioidomycosis Using P10-Primed Monocyte-Derived Dendritic Cells Isolated From Infected Mice.

ABSTRACT: Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin American caused by the thermodimorphic fungi of the genus Paracoccidioides spp. Notably, a Th1 immune response is required to control PCM. In this context, dendritic cells (DCs) seem to be essential players in capture, processing and presentation of Paracoccidioides antigens to naïve T cells and their further activation. We have previously demonstrated that differentiated DCs from bone marrow cells, pulsed with the immunoprotective peptide 10 (P10), effectively control experimental PCM immunocompetent and immunosuppressed mice. However, this procedure may not be infeasible or it is limited for the therapy of human patients. Therefore, we have sought a less invasive but equally effective approach that would better mimics the autologous transplant of DC in a human patient. Here, we isolated and generated monocyte differentiated dendritic cells (MoDCs) from infected mice, pulsed them with P-10, and used them in the therapy of PCM in syngeneic mice. Similar to the results using BMDCs, the P10-pulsed MoDCs stimulated the proliferation of CD4⁺ T lymphocytes, induced a mixed production of Th₁/Th₂ cytokines and decreased the fungal burden in murine lungs in the setting of PCM. The process of differentiating MoDCs derived from an infected host, and subsequently used for therapy of PCM is much simpler than that for obtaining BMDCs, and represents a more reasonable approach to treat patients infected with Paracoccidioides. The results presented suggest that P10-primed MoDC may be a promising strategy to combat complicated PCM as well as to significantly shorten the lengthy requirements for treatment of patients with this fungal disease.

SUBMITTER: Silva LBR

PROVIDER: S-EPMC6685297 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Experimental Therapy of Paracoccidioidomycosis Using P10-Primed Monocyte-Derived Dendritic Cells Isolated From Infected Mice.

Silva Leandro B R LBR Taira Cleison L CL Dias Lucas S LS Souza Ana C O ACO Nosanchuk Joshua D JD Travassos Luiz R LR Taborda Carlos P CP

Frontiers in microbiology 20190731

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin American caused by the thermodimorphic fungi of the genus <i>Paracoccidioides</i> spp. Notably, a Th1 immune response is required to control PCM. In this context, dendritic cells (DCs) seem to be essential players in capture, processing and presentation of <i>Paracoccidioides</i> antigens to naïve T cells and their further activation. We have previously demonstrated that differentiated DCs from bone marrow cells, pulsed with the immunop ...[more]

PMID: 31417520

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Project description:Advances in fundamental and applied immunology research often originate from pilot studies utilizing animal models. While cattle represent an ideal model for disease pathogenesis and vaccinology research for a number of human disease, optimized bovine culture models have yet to be fully established. Monocyte-derived dendritic cells (MoDC) are critical in activating adaptive immunity and are an attractive subset for experimental and clinical applications. The use of serum-supplemented culture medium in this ex vivo approach is undesirable as serum contains unknown quantities of immune-modulating components and may induce unwanted immune responses if not autologous. Here, we describe a standardized protocol for generating bovine MoDC in serum-free medium (AIM-V) and detail the MoDC phenotype, cytokine profile, and metabolic signature achieved using this culture methodology. MoDC generated from adult, barren cattle were used for a series of experiments that evaluated the following culture conditions: medium type, method of monocyte enrichment, culture duration, and concentration of differentiation additives. Viability and yield were assessed using flow cytometric propidium iodide staining and manual hemocytometer counting, respectively. MoDC phenotype and T cell activation and proliferation were assessed by flow cytometric analysis of surface markers (MHC class II, CD86, CD14, and CD205), and CD25 and CFSE respectively. Cytokine secretion was quantified using a multiplex bovine cytokine panel (IL-1α, IL-1β, IL-8, IL-10, IL-17A, IFN-γ, MIP-1α, TNF-α, and IL-4). Changes in cell metabolism following stimulation were analyzed using an Extracellular Flux (XFe96) Seahorse Analyzer. Data were analyzed using paired t-tests and repeated measures ANOVA. Immature MoDC generated in serum-free medium using magnetic-activated cell sorting with plate adhesion to enrich monocytes and cultured for 4 days have the following phenotypic profile: MHC class II+++, CD86+, CD205++, and CD14-. These MoDC can be matured with PMA and ionomycin as noted by increased CD86 and CD40 expression, increased cytokine secretion (IL-1α, IL-10, MIP-1α, and IL-17A), a metabolic switch to aerobic glycolysis, and induction of T cell activation and proliferation following maturation. Cultivation of bovine MoDC utilizing our well-defined culture protocol offers a serum-free approach to mechanistically investigate mechanisms of diseases and the safety and efficacy of novel therapeutics for both humans and cattle alike.

Dataset Information

Experimental Therapy of Paracoccidioidomycosis Using P10-Primed Monocyte-Derived Dendritic Cells Isolated From Infected Mice.

Publications

Experimental Therapy of Paracoccidioidomycosis Using P10-Primed Monocyte-Derived Dendritic Cells Isolated From Infected Mice.

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