Estudo da resposta inflamatória em pulmões de camundongos imunizados com Pb27r submetidos à infecção desafio por Paracoccidioides brasiliensis

Abstract

Paracoccidioides brasiliensis is a thermal dimorphic fungus and known to be the etiologic agent of a systemic mycosis called paracoccidioidomycosis (PCM). Described in 1908 by Adolf Lutz, this mycosis is rural and sub urban endemic disease, prevalent in tropical and subtropical areas of Latin America, with 80% of cases reported in Brazil. The infection may be acquired by the respiratory route through inhalation of infecting particles or spores, produced by the fungus mycelial form and transported by air. Primary infection usually occurs in the lungs and may also lead to chronic pulmonary insufficiency, resulting from the fibrosis development, which occurs simultaneously with the inflammation and leukocyte infiltration. Among antigens of P. brasiliensis, is especially remarkable Pb27 protein, an antigen which is extensively studied and has shown significant potential to induce a protective immune response and as marker for immunodiagnosis of PCM. In this work, this antigen was used to determine the pulmonary inflammatory response induced by immunization with the protein Pb27r. To this end, animals were immunized with the protein Pb27r and subsequently infected with 3 x 105 yeast cells of the virulent strain of P brasiliensis (PB18) and received chemotherapeutic treatment 30 days after challenge infection. The UFCs were determined were determined 30 and 90 days after infection and in both the immunization had a protective effect. In addition, antibody levels produced by the animals before and during infection were determined by ELISA assays. Histological staining (He and Masson's trichrome), were used in the classification of inflammatory infiltrates and the detection of pulmonary fibrosis. The techniques of Immunohistochemistry and Western blot were used in a qualitative and quantificative form (respectively) for the marking of TLR2 and 4 receptors, chemokine receptor CCR7, active Caspase 3, VEGF and its receptor VEGFR-2 and for the detection of collagen fibers Type I. Levels of pro and anti-inflammatory cytokines and eNOS and iNOS enzymes were also detected using the technique of RT-PCR. The joint analysis of the results showed that the recombinant protein Pb27 acts positively modulating the mechanisms of inflammatory response and consequently induces protection of immunized individuals. The immunization has the ability to induce greater protection and restore pulmonary homeostasis after challenge infection and also actively restore the pulmonary vasculature, nitric oxide production by endothelial cells and prevent the exacerbation of inflammation and lung fibrosis.