Itraconazole in combination with neutrophil depletion reduces the expression of genes related to pulmonary fibrosis in an experimental model of paracoccidioidomycosis.

Itraconazole (ITC) is the drug of choice for treating paracoccidioidomycosis (PCM); nonetheless, patients with the chronic form of this mycosis develop fibrosis, a residual pulmonary abnormality, even after treatment. Recently, we observed that the depletion of neutrophils with a specific monoclonal antibody (mAb-anti-Ly6G) during the chronic stages of PCM was associated with a decrease in the fungal burden, the inflammatory response and a reduction of fibrosis. Herein, we aimed to evaluate the effect of ITC in combination with the mAb-anti-Ly6G in an experimental model of pulmonary PCM. BALB/c male mice were challenged with Paracoccidioides brasiliensis yeasts and treated with the mAb-anti-Ly6G and/or ITC at 4th week post-infection (p.i.) and then sacrificed at 12th week p.i. to assess neutrophil subpopulations, fungal load, collagen, expression of fibrosis- and pro-inflammatory-related genes and histopathology. We observed that combination of ITC/mAb-anti-Ly6G favored the control of infection and diminished the inflammatory response. Of note, such therapeutic strategy reduced the expression of IL-1ß, IL-6, IL-17, IL-10, TNF-α, TGF-ß1, TGF-ß3, GATA-3, RORc, Ahr, MMP-1α, MMP-8 MMP-15, TIMP-1, and TIMP-2 genes in an additive manner compared to those mice treated with the mAb or ITC alone. Interestingly, ITC induced an increase of type-II neutrophils even in those mice treated with the mAb-anti-Ly6G. These results indicate that combination ITC/mAb-anti-Ly6G reduced the infection and pulmonary fibrosis through down-regulation of inflammatory and pro-fibrotic genes. Additionally, we confirmed the immunomodulatory properties of this antifungal in vivo. This work emphasizes the importance of exploring new potential combination treatments to treat fungal infections.

Bone marrow-derived mesenchymal stem cells transplantation alters the course of experimental paracoccidioidomycosis by exacerbating the chronic pulmonary inflammatory response.

Several studies have shown the potential use of bone marrow mesenchymal stem cells (BM-MSCs) as a therapeutic approach to infectious diseases. Since BM-MSCs can exert antimicrobial properties and influence the immune response against pathogens, our aim was to study the antimicrobial therapeutic potential of BM-MSCs in an experimental model of paracoccidioidomycosis (PCM). BM-MSCs were isolated from BALB/c donor mice. Paracoccidioides brasiliensis-infected male BALB/c mice were injected with purified BM-MSCs at 8th week post-infection. Mice were sacrificed at 12th week post-infection. Homing of BM-MSCs was confirmed by cellular labeling with fluorescent lipophilic dye and detected by flow cytometry. We found that, in comparison with nontransplanted infected animals, BM-MSCs-treated and P. brasiliensis-infected mice showed a significant increase in (i) fungal burdens, (ii) neutrophils, eosinophils and M2 macrophages counts, and (iii) interleukin (IL)-6, IL-9, GM-CSF, CXCL1, CXCL9, and CCL5 levels, while presenting a decrease in M1 macrophages and Treg cells in lungs. In addition, the histopathological analysis of the lungs showed an increased inflammatory process. This is the first study to our knowledge that evaluates the effects of BM-MSCs treatment in PCM. Our results indicate that the immunoregulatory function of BM-MSCs may be triggered by the interaction with P. brasiliensis, which exacerbates chronic pulmonary inflammatory response.

Impaired anti-fibrotic effect of bone marrow-derived mesenchymal stem cell in a mouse model of pulmonary paracoccidioidomycosis.

Bone marrow-derived mesenchymal stem cells (BMMSCs) have been consider as a promising therapy in fibrotic diseases. Experimental models suggest that BMMSCs may be used as an alternative therapy to treat chemical- or physical-induced pulmonary fibrosis. We investigated the anti-fibrotic potential of BMMSCs in an experimental model of lung fibrosis by infection with Paracoccidioides brasiliensis. BMMSCs were isolated and purified from BALB/c mice using standardized methods. BALB/c male mice were inoculated by intranasal infection of 1.5x106 P. brasiliensis yeasts. Then, 1x106 BMMSCs were administered intra venous at 8th week post-infection (p.i.). An additional group of mice was treated with itraconazole (ITC) two weeks before BMMSCs administration. Animals were sacrificed at 12th week p.i. Histopathological examination, fibrocytes counts, soluble collagen and fibrosis-related genes expression in lungs were evaluated. Additionally, human fibroblasts were treated with homogenized lung supernatants (HLS) to determine induction of collagen expression. Histological analysis showed an increase of granulomatous inflammatory areas in BMMSCs-treated mice. A significant increase of fibrocytes count, soluble collagen and collagen-3α1, TGF-ß3, MMP-8 and MMP-15 genes expression were also observed in those mice. Interestingly, when combined therapy BMMSCs/ITC was used there is a decrease of TIMP-1 and MMP-13 gene expression in infected mice. Finally, human fibroblasts stimulated with HLS from infected and BMMSCs-transplanted mice showed a higher expression of collagen I. In conclusion, our findings indicate that late infusion of BMMSCs into mice infected with P. brasiliensis does not have any anti-fibrotic effect; possibly because their interaction with the fungus promotes collagen expression and tissue remodeling.

Depletion of Neutrophils Promotes the Resolution of Pulmonary Inflammation and Fibrosis in Mice Infected with Paracoccidioides brasiliensis.

Chronic stages of paracoccidioidomycosis (PCM) are characterized by granulomatous lesions which promote the development of pulmonary fibrosis leading to the loss of respiratory function in 50% of patients; in addition, it has been observed that neutrophils predominate during these chronic stages of P. brasiliensis infection. The goal of this study was to evaluate the role of the neutrophil during the chronic stages of experimental pulmonary PCM and during the fibrosis development and tissue repair using a monoclonal specific to this phagocytic cell. Male BALB/c mice were inoculated intranasally with 1.5x106 P. brasiliensis yeast cells. A monoclonal antibody specific to neutrophils was administered at 4 weeks post-inoculation followed by doses every 48h during two weeks. Mice were sacrificed at 8 and 12 weeks post-inoculation to assess cellularity, fungal load, cytokine/chemokine levels, histopathological analysis, collagen and expression of genes related to fibrosis development. Depletion of neutrophils was associated with a significant decrease in the number of eosinophils, dendritic cells, B cells, CD4-T cells, MDSCs and Treg cells, fungal load and levels of most of the pro-inflammatory cytokines/chemokines evaluated, including IL-17, TNF-α and TGF-ß1. Recovery of lung architecture was also associated with reduced levels of collagen, high expression of TGF-ß3, matrix metalloproteinase (MMP)-12 and -14, and decreased expression of tissue inhibitor metalloproteinase (TIMP)-2, and MMP-8. Depletion of neutrophils might attenuate lung fibrosis and inflammation through down-regulating TGF-ß1, TNF-α, IL-17, MMP-8 and TIMP-2. These results suggest that neutrophil could be considered as a therapeutic target in pulmonary fibrosis induced by P. brasiliensis.

Depletion of Neutrophils Exacerbates the Early Inflammatory Immune Response in Lungs of Mice Infected with Paracoccidioides brasiliensis.

Neutrophils predominate during the acute phase of the Paracoccidioides brasiliensis infection. Herein, we determined the role of the neutrophil during the early stages of experimental pulmonary paracoccidioidomycosis using a monoclonal antibody (mAb) specific for neutrophils. Male BALB/c mice were inoculated intranasally with 1.5 × 10(6) or 2 × 10(6) P. brasiliensis yeast cells. The mAb was administered 24 h before infection, followed by doses every 48 h until mice were sacrificed. Survival time was evaluated and mice were sacrificed at 48 h and 96 h after inoculation to assess cellularity, fungal load, cytokine/chemokine levels, and histopathological analysis. Neutrophils from mAb-treated mice were efficiently depleted (99.04%). Eighty percent of the mice treated with the mAb and infected with 1.5 × 10(6) yeast cells died during the first two weeks after infection. When mice were treated and infected with 2 × 10(6) yeast cells, 100% of them succumbed by the first week after infection. During the acute inflammatory response significant increases in numbers of eosinophils, fungal load and levels of proinflammatory cytokines/chemokines were observed in the mAb-treated mice. We also confirmed that neutrophils are an important source of IFN-γ and IL-17. These results indicate that neutrophils are essential for protection as well as being important for regulating the early inflammatory immune response in experimental pulmonary paracoccidioidomycosis.