Resistance to P. brasiliensis Experimental Infection of Inbred Mice Is Associated with an Efficient Neutrophil Mobilization and Activation by Mediators of Inflammation

Abstract

Paracoccidioidomycosis (PCM) is a systemic fungal infection, endemic in Brazil, that leads to severe morbidity and even mortality if not correctly treated. Patients may respond differently to PCM depending on the pattern of the acquired immune response developed. The onset of protective immune response is notably mediated by neutrophils (PMN) that play an important role through directly killing the fungi and also by interacting with other cell types to modulate the acquired protective immune response that may follow. In that way, this study aimed to present and compare different experimental models of PCM (intraperitoneal and subcutaneous) regarding PMN production and maturation inside femoral bone marrow and also PMN infiltration in peritoneal and subcutaneous exudates of resistant and susceptible mice. We also assessed the fungal colony forming units and the levels of soluble inflammatory mediators (LTB4, KC, IFN-γ, GM-CSF, and IL-10) inside subcutaneous air-pouches to compare the efficiency of the PMN present at this site in relation to the two main neutrophil functions: initial lysis of the invading pathogen and modulation of the acquired immune response. P. brasiliensis inoculated intraperitoneally was able to disseminate to the bone marrow of susceptible mice, causing a more marked alteration of PMN production and maturation than that observed after resistant mice infection by the same route. Subcutaneous air-pouch inoculation of P. brasiliensis elicited a controlled and limited infection that produced a PMN-rich exudate, thus favoring the study of the interaction between the fungus and the neutrophils. Susceptible mice produced higher numbers of PMN; however, these cells were less effective in killing the fungi. Inflammatory cytokines were more pronounced in resistant mice, which supports their PCM raised resistance.

Figure 1

Kinetics of PMN population in bone marrow following intraperitoneal inoculation of Pb: (a) immature PMN population after intraperitoneal inoculation; (b) mature PMN population after intraperitoneal inoculation; (c) total PMN population after intraperitoneal route; A/J and B10.A: resistant and susceptible mice, respectively.

Figure 2

Relative neutrophilic population inside bone marrow between intraperitoneal and subcutaneous routes of Pb infection after 15 days: (a) mature cells in A/J resistant mice; (b) mature cells in B10.A susceptible mice; (c) total number of cells in resistant mice; (d) total number of cells in susceptible mice.

Figure 3

Neutrophilic population in the peripheral blood of resistant A/J or susceptible B.10A mice: (a) kinetics of PMN in mice that had Pb inoculated intraperitoneally; (b) relative population of PMN in mice that suffered intraperitoneal and subcutaneous injection of Pb.

Figure 4

Neutrophilic infiltrates in peritoneum and subcutaneous exudate of A/J resistant and B10.A susceptible mice: (a) kinetics of PMN infiltration in peritoneum; (b) kinetics of PMN infiltration inside subcutaneous air-pouches.

Figure 5

Histological appearance of marrow: (a) conspicuous presence of Pb yeasts in susceptible mice 90 days after intraperitoneal infection; (b) evident neutropoiesis along mature megakaryocytes in susceptible mice after 90 days of intraperitoneal infection.

Figure 6

Number of viable Pb and concentration of soluble mediators after 13 days of subcutaneous inoculation in A/J resistant and B.10A susceptible mice: (a) colony forming units of Pb; levels of (b) GM-CSF, (c) IFN-γ, (d) LTB4, (e) IL-10, and (f) KC.