Development of In Vitro Macrophage System to Evaluate Phagocytosis and Intracellular Fate of Penicillium marneffei Conidia

Abstract

Penicillium marneffei is a pathogenic fungus that can cause a life-threatening systemic mycosis in the immunocompromised hosts. We established the model for the phagocytosis of P. marneffei conidia by RAW264.7 murine macrophages and designated the fate of P. marneffei in RAW264.7 cells with respect to persistence, phagosome-lysosome-fusion. And we impaired the immune status of mouse and compared the fate and phagosome-lysosome-fusion of P. marneffei in immunocompetent and immunosuppressed mouse peritoneal macrophages cells. We found that conidia could germinate and survive in macrophages. Within 30 min and up to 2 h of heat-killed conidia internalization, the majority of all phagosome types were labeled for the EEA1 (endosomal markers) and LAMP-1 (lysosomal markers), respectively. But both the percentages of LAMP-1 and EEA1 that associated with live conidia were significantly lower than that with heat-killed conidia. Administration of cyclophosphamide resulted in a significant suppression of macrophages function (phagocytic and fungicidal) against P. marneffei that were not apparently seen. Our data provide the evidence that (i) intracellular conversion of P. marneffei conidia into yeast cells still could be observed in macrophages. (ii) Phagosomes containing live Penicillium marneffei conidia might inhibit the phagosome-lysosome-fusion and result to no acidification surrounding the organisms. (iii) Immunity impaired by cyclophosphamide could not influence the function, including phagocytosis, fungicidal activity and phagosome-lysosome-fusion, of macrophages against P. marneffei.

<p>Murine Macrophage Requires CD11b to Recognize <em>Talaromyces marneffei</em></p>

“…A strain of T. marneffei SUMS0152 (IFM52703) was obtained from the Fungi Research Center of Sun Yat-sen Memorial Hospital and the experimental protocol was approved by the Medical Ethics Committee of Sun Yatsen University. 14 The animal studies were reviewed and approved by the Animal Research and Care Committee of Sun Yat-sen University (SCXK Guangdong 2009-0011), and followed the guidelines for the welfare of the laboratory animals (Laboratory animal-Guideline for ethical review of animal welfare, GB/T3589-2009).…”

“…Peritoneal macrophages (pMacs) were collected from the control and immunosuppressed BALB/c mice after the third injection by peritoneal lavage as described. 14 Mouse RAW264.7 macrophages were preserved in the Fungi Research Center of Sun Yat-sen Memorial Hospital.…”

“…The cells in each well were added with non-opsonized FITC-labeled T. marneffei at a ratio of 5:1 (conidia spore to macrophage) and the phagocytosis rate was determined using flow cytometry as conventional method. 14 In addition, the CD11b specific or control siRNAtransfected RAW264.7 cells were tested for their phagocytosis.…”

The in vitro fungicidal activity of human macrophages against Penicillium marneffei is suppressed by dexamethasone

“…Besides, our previous study has found the exact contribution of the acidification of the phagolysosome and the activation of lysosomal enzymes in anti-fungi [19].…”

“…P. marneffei conidia were labeled with fluorescein isothiocyanate (FITC) as previously described [19]. MDM were seeded in 6-well plates at a concentration of 1 Â 10 6 cells/ml with and without dexamethasone pretreatment, and infected with P. marneffei conidia at a multiplicity of infection (MOI) of 1:5.…”