Enhanced apoptosis, inflammatory cellularity, collagen deposition, and interaction between fibroblasts and Leishmania amastigotes in undamaged ear skin of dogs with leishmaniosis

Fibroblasts are located close to the area of skin inoculation of Leishmania promastigotes. They are a potential cellular target for early parasite infection, harboring amastigotes of Leishmania spp. This study aimed to determine the apoptosis in fibroblasts, and to correlate these results with inflammation, parasite load, AgNOR (Argyrophilic Nucleolar Organizer Region) index, and clinical features in Leishmania-affected dogs. Fragments from the undamaged ear skin of 16 Leishmania-infected and seven uninfected dogs were evaluated by histomorphometry and immunohistochemical analysis, which correlated fibroblast apoptosis to clinical manifestation and parasite load. Ultra-thin sections were examined under transmission electronic microscopy (TEM). When applying immunohistochemical analysis, Leishmania amastigotes were only found in clinically affected dogs. The cellularity of the inflammatory infiltrate and the AgNOR index (fibroblasts and inflammatory infiltrate) were higher in clinically affected dogs. The collagen deposition score was statistically significantly higher in Leishmania-infected dogs. The apoptotic index of inflammatory cells and fibroblasts proved to be higher in clinically affected dogs. From an ultrastructural point of view, apoptotic cells shrank, while the nuclear chromatin and cytoplasm condensed. Amastigotes were observed within inflammatory cells (neutrophils and macrophages) and in the inner portions of fibroblasts. Fibroblast apoptosis was related to both the increase in the parasite load and the intensity of the inflammatory response. Histomorphometric assessments (inflammation, parasite load, AgNOR index, and apoptosis) and clinical manifestations were also associated. Collagen deposition was positively correlated with AgNOR expression and the apoptotic index (inflammatory cell and fibroblast). Therefore, fibroblast apoptosis contributes to the infection process, pathogenesis, and chronicity of canine leishmaniosis. Moreover, fibroblasts may well provide an escape mechanism for immune defenses against Leishmania.