INTRODUCTION
Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones & Samson, comb. nov 2011, previously called Paecilomyces lilacinus (Thom) Samson 1974 is a filamentous, asexual hyaline fungus. The new genera Purpureocilliumwas established after a recent molecular and morphological study suggested that P. lilacinus was not related to the Paecilomyces genus1.
Purpureocillium lilacinus is widely considered as a cosmopolitan, saprophytic fungus frequently detected in the environmental soil samples; it can cause deterioration of grains, food, and paper. The fungus can also be recovered from contaminated skin creams and lotions used clinically, and from clinical materials such as catheters and plastic implants. Currently, it is considered an important opportunistic pathogen in both immunocompromised and immunocompetent hosts2,3. It has been found parasitizing insects and nematodes, and hence, some researchers have described a potential use of this fungus as a biocontrol agent4. It can also cause infection in other animals such as cats5.
Purpureocillium lilacinum is one of the causal agents of hyalohyphomycosis, a mycotic infection caused by a group of fungi including Acremonium spp, Beauveria spp, Fusarium spp, Scopulariopsis spp, andPaecilomyces spp. In this condition, the fungi are observed in the affected tissues as septate hyphae with pigmentless cell walls6,7. Most clinical manifestations of P. lilacinumhyalohyphomycosis are associated with ocular, cutaneous, or subcutaneous infections and the major risk factors are organ transplantations, corticosteroid therapy, primary immunodeficiency, diabetes mellitus, acquired immunodeficiency syndrome, intraocular lens implantation, and ophthalmic surgery2,3,8,9. No effective treatment has been established for this infection, and antifungal agents, including amphotericin B, flucytosine, and fluconazole, have often provided unsatisfactory results3. However, some of the so-called new azoles, such as posaconazole, ravuconazole, and voriconazole, have recently demonstrated good activity against P. lilacinum in vitro10. In many cases, a combination of antifungal agents or application of these agents together with surgical treatment was necessary to induce remission2,10.
An in vivo study using an animal model has demonstrated that P. lilacinum virulence is generally low, as evidenced by the high inoculum (106-107 conidia per animal) and immunosuppression required to establish a successful infection3. On the other hand, we have previously shown that both immunocompetent and immunosuppressed mice that intravenously received about 104 P. lilacinum conidia developed the infection: fungal structures were observed and fungal cells were recovered from different organs2,11. These data indicate the need of additional studies to better comprehend the real invasive capability of P. lilacinum.
The antifungal immune response, although exhibiting certain species-specific variations, is generally initiated by phagocytic cells. Neutrophils, macrophages, and monocytes are important antifungal effector cells. Additional effector cells, including neutrophils and monocytes, are recruited to the sites of infection by inflammatory signals such as cytokines, chemokines, and complement components12.
Professional antigen-presenting cells (APCs) belong to the host innate immune system and are represented mainly by macrophages and dendritic cells (DCs). These cells capture and process antigens, express lymphocyte costimulatory molecules, migrate to lymphoid organs, and secrete cytokines to initiate immune response13. DCs are important components of the immune system; they provide the first line of defense and are therefore essential for the onset of a strong immune response to several incoming pathogens14,15. DCs play an instrumental role in linking innate and adaptive responses against a variety of pathogenic fungi including Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans12.
This study aimed to analyze the in vitro interaction between P. lilacinum conidia and two types of human APCs, macrophages and DCs, to help elucidate the pathogenesis of infection caused by this fungus.
METHODS
A clinical P. lilacinum isolate from the nasal sinus, which was kindly provided by Dr. Annette W. Fothergill (Fungus Testing Laboratory, University of Texas Health Science Center, San Antonio, USA), was grown on potato-dextrose agar (Difco, Detroit, MI, USA) at room temperature for 14 days. Spores were collected by scraping the colonies, suspended in 50mM phosphate-buffered saline (PBS), pH 7.2, chilled to 4°C, and heated to 37°C. The suspension was then centrifuged at 200 × g for 30min, and the number of conidia in the resultant supernatant (rich in conidia but free of hyphae) was estimated by microscopy using a Neubauer hemocytometer16. All the conidial suspensions were freshly prepared for each experiment.
Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats of eight peripheral blood samples from healthy donors screened for human immunodeficiency virus (HIV) and hepatitis B virus (kindly provided by Serviço de Hemoterapia, Hospital Universitário Clementino Fraga Filho, RJ, Brazil). The cells were isolated using a Ficoll-Hypaque 1077 gradient (Sigma, St. Louis, MO, USA)17. Briefly, cells were washed, resuspended in Roswell Park Memorial Institute (RPMI) medium containing L-glutamine and penicillin-streptomycin (Sigma), and quantified using the Neubauer hemocytometer. Monocytes were separated from lymphocytes by cold aggregation during 30min17. The cells were resuspended in fresh RPMI medium containing 10% fetal bovine serum (Hyclone®; Thermo Scientific, South Logan, UT, USA) and seeded at 2 × 105 cells/well into eight-well chamber slides (Lab-Tek™ Nunc International, Rochester, NY, USA) and at 1 × 106/tube into Falcon® polystyrene tubes (Becton Dickinson Company, Franklin Lakes, NJ, USA) for cell phenotype evaluation by flow cytometry. For differentiation into macrophages, monocytes were incubated at 37°C in a humidified incubator with a 5% CO2/95% air mixture (model MC0-19AIC-UV; Etten Leur, The Netherlands) for 7 to 10 days. For differentiation into DCs, monocytes were incubated in the presence of 100U/mL recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF; Peprotech, Rocky Hill, NJ, USA) and 1,000U/mL recombinant human interleukin-4 (rhIL-4; Peprotech) as described above17,18. For phenotypic evaluation, cells were washed with 200µl PBS containing 0.1% bovine serum albumin and 0.01% sodium azide and stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies against cluster of differentiation 14 (CD14) and peridinin-chlorophyll protein-cyanine dye (PerCP-Cy5.5)-conjugated CD209, also known as dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)19–21, on ice for 60min. Cells were washed and analyzed using an Accuri C6 flow cytometer (Accuri Cytometers Inc., Ann Arbor, MI, USA) and the FlowJo™ software (Tree Star, Ashland, OR, USA).
The interaction experiments were conducted with three different ratios of conidia to human cells (5:1, 2:1, and 1:1). Briefly, the conidial suspension was added to each well of the chamber slides at the desired concentration, and incubated with APCs (macrophages or DCs) differentiated from monocytes of each donor for 1h, 6h, and 24h at 37°C in a 5% CO2 atmosphere. Subsequently, the cells were washed gently with sterile PBS at room temperature to remove extracellular conidia, fixed with methanol for 3min, stained with Giemsa solution (Sigma) for 15min, and then examined under a light microscope (model Axiophot, Carl Zeiss Microscopy GmbH, Göttingen, Germany). Control APCs without conidia were treated and evaluated in the same manner. Quantification was performed by counting 100 fields on duplicate coverslips and the results were expressed as follows: % of infected cells = [(APC with fungus – control) ÷ control] × 100%22. The data were analyzed by the Student’s t-test and the difference at p < 0.05 was considered statistically significant.
RESULTS
Immunophenotyping of the cells was performed to monitor the differentiation of monocytes to macrophages and DCs. The differentiated DCs were characterized by the high expression of CD209 (DC-SIGN) and low expression of CD14 (Figure 1). In contrast, the differentiated macrophages displayed high expression of CD14 and low levels of CD209 (Figure 1).
FIGURE 1- Percentages of CD14- and CD209-expressing cells among macrophages and dendritic cells differentiated from monocytes isolated from buffy coat samples of eight healthy donors. CD: cluster of differentiation.
For all three tested ratios of conidia to human cells (5:1, 2:1, and 1:1), phagosome-like structures containing conidia could be observed inside the cells, indicating that conidia were phagocytized by APCs (Figure 2). The control APCs presented typical morphology (Figures 2A and 2B). The results obtained with the 5:1 ratio showed that conidia were phagocytized by APCs, similar to the findings for the 2:1 sample (Figures 2C and 2D). However, because of the excessive number of conidia inside and outside APCs, it was not possible to quantify the internalized conidia.
FIGURE 2- Macrophages (A, C, E, G, I) and dendritic cells (B, D, F, H, J) derived from monocytes isolated from human mononuclear cells were infected in vitro with conidia of a Purpureocillium lilacinum human isolate, stained using Giemsa staining, and analyzed by light microscopy at ×1,000 magnification. Macrophages (A) and dendritic cells (DCs; B) incubated without the fungi were used as negative controls and did not show any changes throughout the experiment. The conidia were phagocytized by macrophages and DCs, as evidenced by the presence of phagosome-like structures containing conidia (→) after 1h of interaction at the 2:1 (C and D) and 1:1 (E and F) ratios of conidia to human cells. At 6h of contact using the 1:1 ratio, the internalization gradually increased and some conidia became inflated (→) inside macrophages (G) and DCs (H) and at 24h of interaction, the conidia with germ tubes (?) and hyphae (→) were observed in macrophages (I) and DCs (J). Scale bar, 10µm.
For the 1:1 ratio of conidia to human cells, the infection could be followed for 1h, 6h, and 24h (Figures 2Eto 2J). Within 1h of interaction, P. lilacinum conidia were internalized by macrophages (Figure 2E) and DCs (Figure 2F); at 6h, the internalization gradually increased and some conidia became inflated (Figures 2G and 2H). After 24h interaction, macrophages and DCs presented inflated conidia that formed germ tubes and hyphae (Figures 2I and 2J); in many cells, they developed into septate hyphae and finally destroyed both macrophages and DCs (data not shown). This pattern of infection was observed for APCs from all the donors.
We also analyzed the percentage of infected APCs (macrophages and DCs) after 6-h interaction, because at this time point, the infection was well established and conidia were clearly observed inside the cells (Figures 2G and 2H). No significant differences between the infected macrophages and DCs containing P. lilacinum conidia were detected at both ratios analyzed (1:1 and 2:1, conidia:human cells) (Figure 3).
FIGURE 3- Phagocytosis of Purpureocillium lilacinum conidia by human macrophages and dendritic cells (DCs). The results are presented as the mean ± standard deviation of the percentage of infected cells after 6-h interaction between conidia and human cells at the ratios of 2:1 and 1:1. No significant differences were detected between the percentages of infected macrophages and DCs.
DISCUSSION
In the present study, we performed in vitro analysis of the interaction between P. lilacinum conidia and human professional APCs derived from human monocytes, and demonstrated that fungal conidia were capable of infecting and destroying both macrophages and DCs.
After a 1h interaction period, we found conidia inside human APCs as described previously with Penicillium marneffei, Fusarium solani, F. oxysporum, and Verticillium nigrescens23,24. A recent study showed that phagocytosis and cell death of Aspergillus fumigatus, A. terreus, and A. flavus occurred within macrophages and DCs after 30min of interaction25.
Here, we observed that, once internalized, the conidia swelled over time and started producing germ tubes in an attempt to generate mycelia. The ability to produce mycelia and sporulate in the infected tissue is a peculiar feature of P. lilacinum26. According to Latgé, conidial swelling is a prerequisite step for the development of hyphae27. This researcher showed that inhaled conidia of A. fumigatus could reach the alveoli and that, at this stage of infection, Aspergillus germinated and showed the growth of small germ tubes, followed by the generation of hyphal fragments28. Furthermore, conidia can germinate in monocytes, suggesting an essential role of phagocytic cells such as neutrophils, in containing conidia that resist intracellular killing29–31.
Previous studies have also demonstrated the ability of DCs to ingest latent A. fumigatus that develop swollen conidia and hyphae32,33. Macrophages and DCs have been described as efficient phagocytic cells with regard to Histoplasma capsulatum, Cryptococcus neoformans, Candida albicans, and A. fumigatus34and, until this study, also to P. lilacinum.
After 24h of interaction with human cells, the conidia of P. lilacinum destroyed macrophages and DCs, demonstrating that the fungus was able to remain viable and active inside both types of APCs, evading the innate cell defense responses by an as-yet unknown mechanism. A similar phenomenon has been observed for Fusarium solani, one of the species phenotypically related to P. lilacinum1, after phagocytosis by human macrophages (unpublished observations).
Purpureocillium lilacinum is generally described as a fungus of low virulence, but our group has demonstrated its capacity to infect murine macrophages and to produce mycelium within 24h of in vitrointeraction16. The present observations with human cells indicate the rapid germination of P. lilacinumconidia and complete destruction of all macrophages and DCs, in sharp contrast to the data obtained for Aspergillus spp., including A. fumigatus, which were efficiently killed by human monocyte-derived macrophages within 120min29. This difference is interesting considering that A. fumigatus is likely more invasive than P. lilacinum, causing infection that often leads to fatal invasive aspergillosis in humans27.
Previous studies on hyalohyphomycosis caused by P. lilacinum have focused on clinical manifestations, treatment, prognosis, and drug-susceptibility testing2, while little is known about the fungus interaction with the host. To the best of our knowledge, this is the first study to address the fate of P. lilacinum after phagocytosis by human APCs. We are currently investigating the immune evasion mechanisms of P. lilacinum, in particular the factors that support its survival within the host cells and are responsible for the pathogenicity of infection. The elucidation of these mechanisms will certainly help in the development of new antifungal treatment strategies.
In conclusion, P. lilacinum conidia were capable of infecting and destroying both macrophages and DCs, clearly demonstrating the ability of this fungus to invade human phagocytic cells.
FINANCIAL SUPPORT
This study was partly financed by the Programa de Fomento à Pesquisa (FOPESQ) Universidade Federal Fluminense (UFF) and Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq). The authors MLPP and DCMS are MSc students at Instituto de Pesquisa Clínica Evandro Chagas Evandro Chagas Clinical Research Institute, Fundação Oswaldo Cruz (FIOCRUZ). The authors ICCS and ECLN are MSc students at UFF.
