Abstract

Introduction and Aims: Traditionally NK cells have been associated with resistance to viral infection and tumors. However, recent studies have shown the involvement of these cells in fungal infections. The aim of this study was to evaluate the capability of NK cells to recognize and kill Paracocciodioides brasiliensis yeast cells. Methods and Results: NK cells from healthy donors and paracoccidioidomycosis (PCM) patients were purified from peripheral blood mononuclear cells (PBMC) by immunomagnetic positive selection. The isolated NK cells were incubated for 48 hours with P. brasiliensis yeast cells (strains: Pb18 - virulent and Pb265 - avirulent) in a ratio of 1:250 (yeast:NK), with or without addition of recombinant IL-15. After incubation, the supernatants were plated on BHI media, and the numbers of CFUs were counted. To determine whether the cytotoxic activity was dependent on granules, in some experiments the cells were treated with SrCl (for 18 hours), concanamicin A (CMA - 4 hours) or EGTA (in the co-cultures). The results showed that cells were able to kill both strains of P.brasiliensis and the addition of IL-15 increased this capacity. The direct cytotoxicity was granule-dependent, but perforin-independent. Moreover, we observed that NK cells express large amounts of granulysin and release this protein when in contact with yeast cells. One possible mechanism for the recognition of P. brasiliensis by NK cells is through the TLR2 and CR3. Therefore, we evaluate the expression of these receptors on NK cells stimulated with both strains of P. brasiliensis for 24 hours. It was found that NK cells express TLR2 and CR3 on their surface, and that the stimulus with IL-15 increases the expression of TLR2. Furthermore, the analysis showed an augmented expression of activation markers (CD25 and CD69) by stimulated cells, mainly in healthy controls. Conclusions: NK cells are capable of direct recognition and killing of P. brasiliensis yeast cells. The direct cytotoxic activity was dependent on granules but independent on perforin. Granulysin is the possible mediator of the cytotoxic effect. Furthermore, our data showed that NK cells express TLR2 and CR3, which may be responsible for the recognition of yeast cells through the b-glucan present in the yeasts cell-walls.਀ Financial support: CNPq and FAPESP਀㰀⼀昀漀渀琀㸀㰀⼀瀀㸀㰀戀爀㸀㰀戀㸀䬀攀礀眀漀爀搀㨀 㰀⼀戀㸀☀渀戀猀瀀㬀一愀琀甀爀愀氀 䬀椀氀氀攀爀 挀攀氀氀猀Ⰰ 倀愀爀愀挀漀挀挀椀搀椀漀椀洀礀挀漀猀椀猀Ⰰ 挀礀琀漀琀漀砀椀挀椀琀礀㰀⼀琀搀㸀㰀⼀琀爀㸀㰀⼀琀愀戀氀攀㸀㰀⼀琀爀㸀㰀⼀琀搀㸀㰀⼀琀愀戀氀攀㸀㰀⼀戀漀搀礀㸀㰀⼀栀琀洀氀㸀