Abstract
These studies demonstrate that pathogenic fungi (dermatophytic, subcutaneous, and systemic) have the ability to produce eicosanoids both from simple metabolites and from arachidonic acid. Host-derived eicosanoids have been previously demonstrated to enhance fungal colonization and atopic disease development. Thus, fungus-derived eicosanoids represent a potential class of novel virulence factors.
Eicosanoids are potent regulators of host immune responses (14). Eicosanoids are oxygenated metabolites of dihomo-γ-linolenic, arachidonic, or eicosanopentaenoic acid and include the prostaglandins and leukotrienes. Prostaglandins (e.g., prostaglandin D2 [PGD2], PGE2, and PGF2α) are produced via the initial action of prostaglandin G/H synthase (cyclooxygenase) followed by specific prostaglandin synthases. Leukotrienes are produced via a lipoxygenase (LOX) followed by conversion into leukotriene B4 (LTB4) and the cysteinyl leukotrienes (LTC4, LTD4, and LTE4) by different synthases and hydrolases. Prostaglandins can inhibit Th1 type immune responses, chemokine production, phagocytosis, and lymphocyte proliferation (3, 9, 11, 14, 17, 19, 20). Leukotrienes are potent leukocyte chemotactic factors (7). Prostaglandins and leukotrienes can also promote Th2 type responses and tissue eosinophilia (4, 7, 11, 14, 18). In the context of antifungal immunity, chronic or disseminating fungal infections will result if the early Th1/Th2 balance of cellular immunity is shifted away from Th1 toward Th2 type responses (16). Thus, enhanced prostaglandin production during fungal infection could be an important factor in promoting fungal colonization and chronic infection.
Host cells are one source of eicosanoids during fungal infection; however, another potential source of eicosanoids is the fungal pathogen itself. We recently reported that the pathogenic yeasts Cryptococcus neoformans and Candida albicans produce prostaglandins de novo and when fed exogenous arachidonic acid (AA) (13). In addition, yeast-derived PGE could inhibit lymphocyte proliferation, decrease tumor necrosis factor alpha production, and augment interleukin-10 production (13). Our objective was to determine if eicosanoid production extended to other pathogenic fungi, including dermatophytes (Epidermophyton floccosum, Fusarium dimerum, Microsporum audouinii, Microsporum canis, and Trichophyton rubrum), subcutaneous pathogens (Sporothrix schenckii), and systemic pathogens (Absidia corymbifera, Aspergillus fumigatus, Histoplasma capsulatum, Blastomyces dermatitidis, Penicillium spp., Rhizopus spp., and Rhizomucor pusillus).
To examine eicosanoid production in various species of pathogenic fungi, PGE2, PGD2, PGF2α, LTB4, and cysteinyl leukotriene (CysLT) levels from culture supernatants were measured. H. capsulatum and B. dermatitidis were grown for 7 days at 37°C in RPMI medium while shaking. All others were grown for 7 days at 25°C in RPMI medium while shaking. The cultures were incubated for an additional 2 h with 1 mM AA (Cayman Chemicals, Ann Arbor, Mich.). Culture supernatants from both AA-fed and non-AA-fed fungi were analyzed for eicosanoid production using enzyme-linked immunosorbent assay kits (Cayman Chemicals) for PGE2, PGD2, PGF2α, LTB4, and CysLT (detects LTC4, LTD4, and LTE4) according to the manufacturer’s instructions. The cultures without AA measure the (endogenous) production of eicosanoids by fungi in the absence of exogenous fatty acid substrates (RPMI medium is a defined medium devoid of fatty acids). All fungal strains grown in RPMI medium alone produced PGE2, PGD2, PGF2α, LTB4, and CysLT (Table 1). In the presence of exogenous AA, approximately 10-fold more of each eicosanoid was detected in the cultures (Table 2). C. neoformans and C. albicans also produced both prostaglandins and leukotrienes from exogenous AA. These data demonstrate that all of these pathogenic fungi have the ability to convert exogenous AA into both LOX- and prostaglandin G/H synthase-derived eicosanoids. Thus, pathogenic fungi have the ability to produce eicosanoids both from simple metabolites and from exogenous AA.
TABLE 1.
Eicosanoid levels in culture supernatants of pathogenic fungia
| Fungal species | Strainb | Mean (SE) eicosanoid level (pg/ml)
|
||||
|---|---|---|---|---|---|---|
| Prostaglandins
|
Leukotrienes
|
|||||
| PGE2 | PGD2 | PGF2α | CysLT | LTB4 | ||
| Absidia corymbifera | ATCC 66271 | 67 (1) | 109 (28) | 12 (1) | 256 (17) | 21 (2) |
| Aspergillus fumigatus | ATCC 13073 | 75 (19) | 248 (140) | 4 (1) | 230 (89) | 12 (3) |
| Blastomyces dermatitidis | UM | 113 (8) | 144 (31) | 8 (2) | 273 (11) | 21 (3) |
| Epidermophyton floccosum | ATCC 52061 | 76 (22) | 115 (25) | 3 (1) | 204 (2) | 15 (1) |
| Epidermophyton floccosum | UM | 66 (5) | 79 (20) | 2 (1) | 207 (13) | 8 (0) |
| Fusarium dimerum | ATCC 62876 | 24 (5) | 35 (14) | 6 (1) | 29 (9) | 3 (1) |
| Histoplasma capsulatum | UM | 86 (0) | 100 (4) | 13 (0) | 241 (78) | 25 (4) |
| Microsporum audouinii | UM | 39 (9) | 90 (0) | 6 (2) | 229 (68) | 30 (1) |
| Microsporum canis | ATCC 42559 | 22 (16) | 111 (24) | 4 (1) | 168 (33) | 13 (1) |
| Penicillium citrinum | ATCC 76113 | 121 (24) | 119 (14) | 7 (0) | 32 (17) | 17 (2) |
| Penicillium notatum | ATCC 24655 | 36 (2) | 123 (33) | 9 (1) | 30 (10) | 24 (3) |
| Penicillium piscarium | ATCC 12109 | 74 (38) | 128 (12) | 7 (2) | 18 (5) | 7 (1) |
| Penicillium spp. | UM | 72 (25) | 127 (18) | 9 (0) | 29 (5) | 5 (1) |
| Rhizomucor pusillus | ATCC 36606 | 40 (14) | 78 (34) | 19 (1) | 44 (1) | 19 (2) |
| Rhizopus spp. | UM | 30 (11) | 132 (13) | 11 (2) | 12 (0) | 7 (2) |
| Sporothrix schenckii | ATCC 24646 | 137 (2) | 164 (1) | 9 (1) | 33 (5) | 9 (2) |
| Sporothrix schenckii | UM | 111 (0) | 100 (1) | 10 (0) | 37 (9) | 14 (1) |
| Trichophyton rubrum | ATCC 18760 | 81 (22) | 146 (1) | 5 (1) | 7 (6) | 12 (3) |
| Trichophyton rubrum | UM | 95 (6) | 220 (43) | 2 (0) | 6 (0) | 14 (2) |
Fungi were cultured for 7 days in RPMI medium (which does not contain any fatty acids or eicosanoids).
American Type Culture Collection catalog number or reference standard from The University of Michigan Medical Microbiology Laboratory (UM).
TABLE 2.
Eicosanoid levels in culture supernatants from arachidonic acid-fed pathogenic fungia
| Fungal species | Strainb | Mean (SE) eicosanoid level (pg/ml)
|
||||
|---|---|---|---|---|---|---|
| Prostaglandin
|
Leukotriene
|
|||||
| PGE2 | PGD2 | PGF2α | CysLT | LTB4 | ||
| Absidia corymbifera | ATCC 66271 | 2,044 (48) | 2,068 (425) | 996 (129) | 4,832 (683) | 1,679 (266) |
| Aspergillus fumigatus | ATCC 13073 | 2,413 (58) | 1,192 (202) | 1,611 (300) | 1,398 (290) | 379 (19) |
| Blastomyces dermatitidis | UM | 1,720 (141) | 1,121 (243) | 839 (142) | 987 (142) | 467 (9) |
| Epidermophyton floccosum | ATCC 52061 | 1,302 (170) | 1,733 (360) | 1,736 (176) | 3,233 (442) | 942 (0) |
| Epidermophyton floccosum | UM | 617 (98) | 1,002 (224) | 782 (85) | 1,427 (79) | 356 (38) |
| Fusarium dimerum | ATCC 62876 | 1,262 (45) | 514 (83) | 534 (129) | 382 (77) | 142 (23) |
| Histoplasma capsulatum | UM | 1,825 (171) | 1,211 (306) | 973 (239) | 1,951 (213) | 869 (89) |
| Microsporum audouinii | UM | 1,886 (111) | 1,511 (327) | 1,516 (205) | 2,130 (376) | 777 (53) |
| Microsporum canis | ATCC 42559 | 345 (67) | 1,120 (159) | 762 (159) | 1,198 (240) | 330 (47) |
| Penicillium citrinum | ATCC 76113 | 1,073 (144) | 662 (4) | 954 (257) | 346 (48) | 387 (31) |
| Penicillium notatum | ATCC 24655 | 454 (98) | 1,179 (128) | 891 (157) | 491 (72) | 564 (111) |
| Penicillium piscarium | ATCC 12109 | 1,148 (70) | 1,434 (19) | 1,480 (432) | 1,337 (169) | 910 (81) |
| Penicillium spp. | UM | 1,413 (281) | 822 (31) | 1,096 (116) | 566 (159) | 496 (21) |
| Rhizomucor pusillus | ATCC 36606 | 378 (35) | 261 (79) | 3,250 (250) | 467 (83) | 186 (54) |
| Rhizopus spp. | UM | 868 (133) | 1,139 (88) | 750 (298) | 592 (127) | 406 (86) |
| Sporothrix schenckii | ATCC 24646 | 1,755 (21) | 1,833 (87) | 1,015 (34) | 1,677 (278) | 1,280 (50) |
| Sporothrix schenckii | UM | 690 (171) | 961 (28) | 776 (42) | 629 (131) | 498 (40) |
| Trichophyton rubrum | ATCC 18760 | 970 (109) | 1,409 (76) | 1,189 (371) | 534 (24) | 612 (81) |
| Trichophyton rubrum | UM | 2,287 (244) | 1,716 (567) | 1,180 (132) | 2,304 (583) | 1,082 (171) |
| Cryptococcus neoformansc | 52D | 211 (42) | 814 (165) | 373 (92) | 508 (141) | 139 (26) |
| Candida albicansc | CHN1 | 23 (10) | 562 (41) | 325 (45) | 726 (132) | 28 (7) |
Fungi were cultured for 7 days in RPMI medium, and then 1 mM AA was added for 2 h.
American Type Culture Collection catalog number or reference standard from The University of Michigan Medical Microbiology Laboratory (UM).
C. neoformans and C. albicans were cultured for 3 days in Sabouraud dextrose broth, spun out, resuspended in RPMI medium containing 1 mM AA, and incubated for 2 h.
Fungal infections are most notable for their chronicity (e.g., dermatophyte infections) and nonprotective or injurious inflammatory responses (e.g., dermatophytoses, subcutaneous mycoses, and vaginal candidiasis). In mammals, prostaglandins and leukotrienes can play a dual role in the pathogenesis of inflammatory diseases, both promoting and counteracting inflammatory processes (7, 12). All fungal pathogens examined produced prostaglandins and leukotrienes in the absence and presence of extracellular AA (Tables 1 and 2), thereby representing the potential for both de novo and “trans-species” metabolic production of eicosanoids during infection (during infection, exogenous AA could be generated via the action of fungal phospholipases on host phospholipids) (5). Fungal leukotrienes could enhance the acute phase of an inflammatory response, while fungal prostaglandins could locally down-regulate the innate effector phase or protective Th1 response to the infection. The result of fungal eicosanoid production would be an immunologic “tolerance” of the infection with or without acute inflammation in an otherwise immunocompetent host, leading to chronic fungal colonization.
Why do fungi produce eicosanoids? Since the fungi reported in this work represent a diverse group of organisms, one possibility is that there is a link between eicosanoid production and fungal growth. We previously reported that indomethacin and etodolac (prostaglandin G/H synthase inhibitors) could inhibit the growth of C. neoformans and C. albicans (13). We have also observed that nordihydroguaiaretic acid (a LOX inhibitor) can also inhibit the growth of these fungi (data not shown).
The precise role of fungal eicosanoids in the pathogenesis of fungal diseases remains to be determined, but the potential link between fungal eicosanoids and the development of atopic diseases in the host is intriguing. The role of eicosanoids in the pathogenesis of allergy and asthma is well documented (2–4, 7, 8, 11, 13, 14, 16, 21). Fungi elicit a variety of allergic diseases, including asthma, sinusitis, allergic bronchopulmonary mycoses, and hypersensitivity pneumonitis (6, 10, 15). As demonstrated in this study, fungi produce immunologically active mediators that can promote the development and manifestation of atopic responses to the infection itself or fungal antigens. By analogy, schistosome-derived PGD2 was recently reported to play a role in the immune deviation that occurs following skin infection by this parasite (1). Thus, the discovery that pathogenic fungi produce eicosanoids opens up a new realm of investigation for virulence mechanisms in fungal pathogenesis and also for fungal eicosanoids as potential cofactors of atopic diseases.
Acknowledgments
We thank Susan Salo and Carl Pierson of the University of Michigan Medical Microbiology Laboratory for providing the fungal reference standards and Deirdra Williams and Cara Chrisman for technical assistance.
This work was supported by a New Investigator Award in Molecular Pathogenic Mycology from the Burroughs-Wellcome Fund (G.B.H.). M.C.N. is also supported by NIH-NIAID training grant T32AI07528.
Editor: T. R. Kozel
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