Pathogenic yeasts Cryptococcus neoformans and Candida albicans produce immunomodulatory prostaglandins

Enhanced prostaglandin production during fungal infection could be an important factor in promoting fungal colonization and chronic infection. Host cells are one source of prostaglandins; however, another potential source of prostaglandins is the fungal pathogen itself. Our objective was to determine if the pathogenic yeasts Cryptococcus neoformans and Candida albicans produce prostaglandins and, if so, to begin to define the role of these bioactive lipids in yeast biology and disease pathogenesis. C. neoformans and C. albicans both secreted prostaglandins de novo or via conversion of exogenous arachidonic acid. Treatment with cyclooxygenase inhibitors dramatically reduced the viability of the yeast and the production of prostaglandins, suggesting that an essential cyclooxygenase like enzyme may be responsible for fungal prostaglandin production. A PGE series lipid was purified from both C. albicans and C. neoformans and was biologically active on both fungal and mammalian cells. Fungal PGE(x) and synthetic PGE(2) enhanced the yeast-to-hypha transition in C. albicans. Furthermore, in mammalian cells, fungal PGE(x) down-modulated chemokine production, tumor necrosis factor alpha production, and splenocyte proliferation while up-regulating interleukin 10 production. These are all activities previously documented for mammalian PGE(2). Thus, eicosanoids are produced by pathogenic fungi, are critical for growth of the fungi, and can modulate host immune functions. The discovery that pathogenic fungi produce and respond to immunomodulatory eicosanoids reveals a virulence mechanism that has potentially great implications for understanding the mechanisms of chronic fungal infection, immune deviation, and fungi as disease cofactors.     

Mechanisms of the proinflammatory response of endothelial cells to Candida albicans infection

Endothelial cells can influence significantly the host inflammatory response against blood-borne microbial pathogens. Previously, we found that endothelial cells respond to in vitro infection with Candida albicans by secreting interleukin 8 (IL-8) and expressing E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). We have now examined the mechanisms mediating this endothelial cell response. We determined that C. albicans stimulated endothelial cells to synthesize tumor necrosis factor alpha (TNF-alpha), which in turn induced these infected cells to secrete IL-8 and express E-selectin by an autocrine mechanism. Expression of VCAM-1 was mediated not only by TNF-alpha but also by IL-1alpha and IL-1beta, all of which were synthesized by endothelial cells in response to C. albicans. These three cytokines remained primarily cell associated rather than being secreted. Candidal induction of ICAM-1 expression was independent of TNF-alpha, IL-1alpha, and IL-1beta. These observations demonstrate that different proinflammatory endothelial cell responses to C. albicans are induced by distinct mechanisms. A clear understanding of these mechanisms is important for therapeutically modulating the endothelial cell response to C. albicans and perhaps other opportunistic pathogens that disseminate hematogenously.     

Candida albicans stimulates cytokine production and leukocyte adhesion molecule expression by endothelial cells.

Endothelial cells have the potential to influence significantly the host immune response to blood-borne microbial pathogens, such as Candida albicans. We investigated the ability (of this organism to stimulate endothelial cell responses relevant to host defense in vitro. Infection with C. albicans induced endothelial cells to express mRNAs encoding E-selectin, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, interleukin 6, interleukin 8, monocyte chemoattractant protein 1, and inducible cyclooxygenase (cox2). All three leukocyte adhesion molecule proteins were expressed on the surfaces of the endothelial cells after 8 h of exposure to C. albicans. An increase in secretion of all three cytokines was found after 12 h of infection. Cytochalasin D inhibited accumulation of the endothelial cell cytokine and leukocyte adhesion molecule mRNAs in response to C. albicans, suggesting that endothelial cell phagocytosis of the organism is required to induce this response. Live Candida tropicalis, Candida glabrata, a nongerminating strain of C. albicans, and killed C. albicans did not stimulate the expression of any of the cytokine or leukocyte adhesion molecule mRNAs. These findings indicate that a factor associated with live, germinating C. albicans is required for induction of endothelial cell mRNA expression. Furthermore, since endothelial cells phagocytize killed C. albicans, phagocytosis is likely necessary but not sufficient for this organism to stimulate mRNA accumulation. In conclusion, the secretion of proinflammatory cytokines and expression of leukocyte adhesion molecules by endothelial cells in response to C. albicans could enhance the host defense against this organism by contributing to the recruitment of activated leukocytes to sites of intravascular infection.     

Characterization of prostaglandin E2 production by Candida albicans

Candida albicans produces lipid metabolites that are functionally similar to host prostaglandins. These studies, using mass spectrometry, demonstrate that C. albicans produces authentic prostaglandin E(2) (PGE(2)) from arachidonic acid. Maximal PGE(2) production was achieved at 37 degrees C in stationary-phase culture supernatants and in cell-free lysates generated from stationary-phase cells. Interestingly, PGE(2) production is inhibited by both nonspecific cyclooxygenase and lipoxygenase inhibitors but not by inhibitors specific for the cyclooxygenase 2 isoenzyme. The C. albicans genome does not possess a cyclooxygenase homolog; however, several genes that may play a role in prostaglandin production from C. albicans were investigated. It was found that a C. albicans fatty acid desaturase homolog (Ole2) and a multicopper oxidase homolog (Fet3) play roles in prostaglandin production, with ole2/ole2 and fet3/fet3 mutant strains exhibiting reduced PGE(2) levels compared with parent strains. This work demonstrates that the synthesis of PGE(2) in C. albicans proceeds via novel pathways.     

Invasive phenotype of Candida albicans affects the host proinflammatory response to infection

Candida albicans is a major opportunistic pathogen in immunocompromised patients. Production of proinflammatory cytokines by host cells in response to C. albicans plays a critical role in the activation of immune cells and final clearance of the organism. Invasion of host cells and tissues is considered one of the virulence attributes of this organism. The purpose of this study was to investigate whether the ability of C. albicans to invade host cells and tissues affects the proinflammatory cytokine responses by epithelial and endothelial cells. In this study we used the invasion-deficient RIM101 gene knockout strain DAY25, the highly invasive strain SC5314, and highly invasive RIM101-complemented strain DAY44 to compare the proinflammatory cytokine responses by oral epithelial or endothelial cells. Using a high-throughput approach, we found both qualitative and quantitative differences in the overall inflammatory responses to C. albicans strains with different invasive potentials. Overall, the highly invasive strains triggered higher levels of proinflammatory cytokines in host cells than the invasion-deficient mutant triggered. Significant differences compared to the attenuated mutant were noted in interleukin-1alpha (IL-1alpha), IL-6, IL-8, and tumor necrosis factor alpha in epithelial cells and in IL-6, growth-related oncogene, IL-8, monocyte chemoattractant protein 1 (MCP-1), MCP-2, and granulocyte colony-stimulating factor in endothelial cells. Our results indicate that invasion of host cells and tissues by C. albicans enhances the host proinflammatory response to infection.     

Candida albicans triggers interleukin-8 secretion by oral epithelial cells

Oropharyngeal candidiasis is a frequent opportunistic infection associated with immunocompromised hosts. Candida albicans is the principal species responsible for this infection. Production of interleukin-8 (IL-8), by oral epithelial cells can be expected to play a major role in the recruitment and activation of professional phagocytes at the infected site. The purpose of this study was to determine whether C. albicans triggers secretion of IL-8 by oral epithelial cells in vitro and investigate mechanisms of host cell-fungal interactions that trigger such responses. Oral epithelial cell lines (SCC4, SCC15, and OKF6/TERT-2) as well as primary gingival epithelial cells were used. Epithelial cells were cocultured with C. albicans, strains SC5314, ATCC28366 or ATCC32077, for 24-48 hr, and supernatants were analyzed for IL-8 content by ELISA. A germination-deficient mutant (efg1/efg1 cph1/cph1), otherwise isogenic to strain SC5314, was used to assess the requirement for germination in triggering IL-8 responses. In order to ascertain whether direct contact of yeast with host cells is required to trigger cytokine production, epithelial cells were separated from yeast using cell culture inserts. To test whether IL-8 secretion is dependent on IL-1alpha activity, epithelial cells were challenged with viable C. albicans in the presence or absence of neutralizing anti-IL-1alpha antibody or IL-1ra, and IL-8 secretion was measured in the supernatants. All cell lines and primary cultures responded to C. albicans with an increase in IL-8 secretion. IL-8 responses were contact-dependent, strain-specific, required yeast viability and germination into hyphae, and were in part autoregulated by IL-1alpha.     

Gamma interferon protects endothelial cells from damage by Candida albicans by inhibiting endothelial cell phagocytosis.

Once Candida albicans comes in contact with endothelial cells, it induces cellular injury. This endothelial cell injury may be a mechanism by which blood-borne organisms escape from the intravascular compartment and invade the tissue parenchyma during hematogenous infection. We have been investigating the ability of cytokines to modulate endothelial cell injury caused by C. albicans. Previously we reported that pretreatment of endothelial cells with gamma interferon (IFN-gamma) protects these cells from candidal injury in vitro. In the current study, we examined potential mechanisms of the cytoprotective effects of IFN-gamma. Time course experiments demonstrated that maximal reduction in candidal injury of endothelial cells occurred after the endothelial cells had been exposed to IFN-gamma for at least 72 h. In other studies, we determined that IFN-gamma reduced endothelial cell phagocytosis of C. albicans by 41.3% compared with that of untreated endothelial cells (P < 0.01). Since endothelial cell phagocytosis of C. albicans is required for damage to occur, inhibition of phagocytosis is likely a mechanism by which IFN-gamma protects endothelial cells from candidal injury. We also found that the cytoprotective effect of IFN-gamma is not mediated by reducing access of the organisms to intracellular endothelial cell iron or by upregulating the synthesis of reactive oxygen intermediates (which could potentially reduce the ability of C. albicans to injure endothelial cells). Thus, inhibiting endothelial cell phagocytosis of C. albicans may be a mechanism by which IFN-gamma augments the host defense against hematogenously disseminated candidal infections.     

Bradykinin-stimulated prostaglandin E2 production by endothelial cells and its modulation by antiinflammatory compounds

Prostaglandin production was studied in cultures of pig aorta endothelial cells using radioimmunoassay, radiochromatography, and smooth muscle bioassay. PGE₂ was produced in higher concentrations than other prostaglandins. Bradykinin produced a rapid dose-related stimulation of PGE₂ production. These results provided the basis for establishment of a simplified test system for investigating new compounds which alter prostaglandin synthesis and might therefore affect inflammatory responses. It was also observed that these endothelial cells do not metabolize prostaglandins via 15-hydroxyprostaglandin dehydrogenase.     

Inhibition of hydrophobic protein-mediated Candida albicans attachment to endothelial cells during physiologic shear flow

Adhesion interactions during hematogenous dissemination of Candida albicans likely involve a complex array of host and fungal factors. Possible C. albicans factors include changes in cell surface hydrophobicity and exposed antigens that have been shown in static adhesion assays to influence attachment events. We used a novel in vitro shear analysis system to investigate host-pathogen interactions and the role of fungal cell surface hydrophobicity in adhesion events with human endothelial cells under simulated physiologic shear. Endothelial monolayers were grown in capillary tubes and tested with and without interleukin-1 beta activation in buffered medium containing human serum. Hydrophobic and hydrophilic stationary-phase C. albicans yeast cells were infused into the system under shear flow and found to adhere with widely varying efficiencies. The average number of adherent foci was determined from multiple fields, sampled via video microscopy, between 8 and 12 min after infusion. Hydrophobic C. albicans cells demonstrated significantly more heterotypic binding events (Candida-endothelial cell) and greater homotypic binding events (Candida-Candida) than hydrophilic yeast cells. Cytokine activation of the endothelium significantly increased binding by hydrophobic C. albicans compared to unactivated host cells. Preincubation of hydrophobic yeast cells with a monoclonal antibody against hydrophobic cell wall proteins significantly blocked adhesion interactions with the endothelial monolayers. Because the antibody also blocks C. albicans binding to laminin and fibronectin, results suggest that vascular adhesion events with endothelial cells and exposed extracellular matrix may be blocked during C. albicans dissemination. Future studies will address the protective efficacy of blocking or redirecting blood-borne fungal cells to favor host defense mechanisms.     

Requirement of cyclooxygenase-2 expression and prostaglandins for human prostate cancer cell invasion

The PC-3 Low Invasive cells and the PC-3 High Invasive cells were used to investigate the correlation of the COX-2 expression and its arachidonic acid metabolites, prostaglandins, with their invasiveness through Matrigel using a Boyden chamber assay. The COX-2 expression in PC-3 High Invasive cells was approximately 3-fold higher than in PC-3 Low Invasive cells while the COX-1 expression was similar in both cell sublines. When incubated with arachidonic acid, PGE2 was the major prostaglandin produced by these cells. PC-3 High Invasive cells produced PGE2 approximately 2.5-fold higher than PC-3 Low Invasive cells. PGD2 was the second most abundant prostaglandin produced by these cells. Both indomethacin (a nonspecific COX inhibitor) and NS-398 (a specific COX-2 inhibitor) inhibited the production of prostaglandins and the cell invasion. PGE2 alone did not induce the cell invasion of PC-3 Low Invasive cells. However, PGE2 reversed the inhibition of cell invasion by NS-398 and enhanced the cell invasion of the PC-3 High Invasive cells. In contrast, PGD2 slightly inhibited the cell invasion. These results suggest that in the PC-3 Low Invasive cells, COX-2-derived PGE2 may not be sufficient to induce cell invasion while in the PC-3 High Invasive cells, PGE2 may be sufficient to act as an enhancer for the cell invasion. Further, PGD2 may represent a weak inhibitor and counteracts the effect of PGE2 in the cell invasion.     

Live Candida albicans suppresses production of reactive oxygen species in phagocytes

Production of reactive oxygen species (ROS) is an important aspect of phagocyte-mediated host responses. Since phagocytes play a crucial role in the host response to Candida albicans, we examined the ability of Candida to modulate phagocyte ROS production. ROS production was measured in the murine macrophage cell line J774 and in primary phagocytes using luminol-enhanced chemiluminescence. J774 cells, murine polymorphonuclear leukocytes (PMN), human monocytes, and human PMN treated with live C. albicans produced significantly less ROS than phagocytes treated with heat-killed C. albicans. Live C. albicans also suppressed ROS production in murine bone marrow-derived macrophages from C57BL/6 mice, but not from BALB/c mice. Live C. albicans also suppressed ROS in response to external stimuli. C. albicans and Candida glabrata suppressed ROS production by phagocytes, whereas Saccharomyces cerevisiae stimulated ROS production. The cell wall is the initial point of contact between Candida and phagocytes, but isolated cell walls from both heat-killed and live C. albicans stimulated ROS production. Heat-killed C. albicans has increased surface exposure of 1,3-beta-glucan, a cell wall component that can stimulate phagocytes. To determine whether surface 1,3-beta-glucan exposure accounted for the difference in ROS production, live C. albicans cells were treated with a sublethal dose of caspofungin to increase surface 1,3-beta-glucan exposure. Caspofungin-treated C. albicans was fully able to suppress ROS production, indicating that suppression of ROS overrides stimulatory signals from 1,3-beta-glucan. These studies indicate that live C. albicans actively suppresses ROS production in phagocytes in vitro, which may represent an important immune evasion mechanism.     

The classical CD14⁺⁺ CD16⁻ monocytes, but not the patrolling CD14⁺ CD16⁺ monocytes, promote Th17 responses to Candida albicans

In the present study, we investigated the functional differences between cluster of differentiation (CD)14(++) CD16(-) and CD14(+) CD16(+) monocytes during anti-Candida host defense. CD14(++) CD16(-) are the "classical" monocytes and represent the majority of circulating monocytes in humans, while CD14(+) CD16(+) monocytes patrol the vasculature for maintenance of tissue integrity and repair. Both monocyte subsets inhibited the germination of live Candida albicans, and there was no difference in their capacity to phagocytose and kill Candida. Although production of IL-6 and IL-10 induced by C. albicans was found to be similar between monocyte subsets, IL-1β and prostaglandin E2 (PGE2) production was higher in CD14(++) CD16(-) compared with CD14(+) CD16(+) monocytes. In line with the increased production of IL-1β and PGE2, central mediators for inducing Th17 responses, CD14(++) CD16(-) monocytes induced greater Th17 responses upon stimulation with heat-killed C. albicans yeast. The percentage of cells that expressed mannose receptor (MR) was higher in the CD14(++) CD16(-) monocyte subset, and MR-specific stimulation induced higher Th17 responses only in co-cultures of CD14(++) CD16(-) monocytes and CD4 lymphocytes. In conclusion, both monocyte subsets have potent innate antifungal properties, but only CD14(++) CD16(-) monocytes are capable of inducing a potent Th17 response to C. albicans, an important component of antifungal host defense.     

Interaction of the mucosal barrier with accessory immune cells during fungal infection

The mucosal epithelium is of central importance in host defence and immune surveillance, as it is the primary cell layer that initially encounters environmental microorganisms. Induction of antifungal innate immune responses depends on recognition of fungal components by host pattern recognition receptors. Members of the Toll-like receptor family have emerged as key sensors that recognize fungal pathogens and trigger defence responses. During oral infection with the fungal pathogen Candida albicans, a large number of cytokines is secreted by oral epithelial cells, which in turn activate myeloid cells in the submucosal layers to clear the invading pathogen. Recent data provide novel insights into the complex molecular mechanisms of innate immune responses initiated by cooperation between epithelial cells and neutrophils. In this review, we discuss the role of epithelial TLRs and how the immunological crosstalk between C. albicans-infected oral epithelium and neutrophils protects the mucosal surface from fungal invasion and cell injury.     

Role of endothelial dysfunction in the development of severe sepsis--the pathomechanism, evaluation by laboratory tests and new therapeutic strategies

Tumor necrosis factor (TNF) is critically involved in biological responses against various insults. TNF excessively produced by monocytes or macrophages activates endothelial cells and neutrophils, thereby inducing endothelial cell injury. Endothelial cells are capable of inhibiting TNF production by producing prostaglandins that inhibit TNF production. Sensory neurons play an important role in promotion of the endothelial production of prostaglandins by releasing calcitonin gene-related peptide. Neutrophils activated by TNF release a huge amount of neutrophil elastase that is capable of decreasing endothelial prostaglandin production. Consequently, TNF production is enhanced, leading to the development of multi-organ failure in sepsis. E-selectin, an endothelial leukocyte adhesion molecule, is released from the endothelial cell membrane by the action of TNF and exists as soluble E-selectin in plasma. The detection of increases in plasma levels of soluble E-selectin in patients with systemic inflammatory response syndrome predicts the imminent onset of acute respiratory syndrome. Early detection of increases in plasma levels of soluble E selectin by a rapid assay system, developed by the authors, enables early effective treatment of patients with sepsis.     

Expression of prostaglandin H synthase (cyclooxygenase) in Hodgkin''s mononuclear and Reed-Sternberg cells. Functional resemblance between H-RS cells and histiocytes or interdigitating reticulum cells.

The synthesis of prostaglandin, prostacyclin, and thromboxane, which requires the enzyme prostaglandin H (PGH) synthase (cyclooxygenase), is a general property of histiocytes, monocytes, and Langerhans cells. Previously the authors reported the production of prostaglandin E2 in a Hodgkin's cell line, KM-H2, and suggested that these cells therefore have a functional similarity to histiocyte-related cells. The present study confirms that the Hodgkin's neoplastic (Reed-Sternberg, H-RS) cells in tissue are also capable of producing prostaglandins by demonstrating the presence of PGH synthase in these cells in B5-fixed, paraffin-embedded tissue sections. It was found that the H-RS cells from seven of ten patients with Hodgkin's disease were stained variously with anti-PGH synthase antibodies. In normal and reactive lymphoid tissues, anti-PGH synthase staining was restricted to histiocytes, endothelial cells, and interdigitating reticulum cells. Thus, this study provides further evidence for a possible relationship between H-RS cells and histiocytes or interdigitating reticulum cells; this relationship has also been supported by information obtained in extensive immunologic, biochemical, and cell-differentiation studies. The secretion of PGH-synthase products, especially prostaglandin E2, in H-RS cells may play a major role in the regulation of cellular immunity in patients with Hodgkin's disease.     

Bioactive interleukin-1alpha is cytolytically released from Candida albicans-infected oral epithelial cells.

Oral epithelial cells are primary targets of Candida albicans in the oropharynx and may regulate the inflammatory host response to this pathogen. This investigation studied the mechanisms underlying interleukin-1alpha (IL-1alpha) release by oral epithelial cells and the role of IL-1alpha in regulating the mucosal inflammatory response to C. albicans. Infected oral epithelial cells released processed IL-1alpha protein in culture supernatants. The IL-1alpha generated was stored intracellularly and was released upon cell lysis. This was further supported by the fact that different C. albicans strains induced variable IL-1alpha release, depending on their cytolytic activity. IL-1alpha from C. albicans-infected oral epithelial cells upregulated proinflammatory cytokine secretion (IL-8 and GM-CSF) in uninfected oral epithelial or stromal cells. Our studies suggest that production of IL-1alpha, IL-8 and GM-CSF may take place in the oral mucosa in response to lytic infection of epithelial cells with C. albicans. This process can act as an early innate immune surveillance system and may contribute to the clinicopathologic signs of infection in the oral mucosa.     

Taenia crassiceps cysticercosis: A role for prostaglandin E2 in susceptibility

Several biological factors are involved in susceptibility and resistance to murine cysticercosis. A substantial body of evidence implies prostaglandins as potent regulators of immune responses during parasitic diseases. Here we evaluated the role played by prostaglandin E2 in cysticercosis. Mice were treated in vivo with prostaglandin E2 or with indomethacin (a prostaglandin E2 synthesis inhibitor) before infection. Parasite growth was enhanced by prostaglandin treatment, which provoked poor Con-A responses, low Th1-type cytokines secretion, and high levels of IL-6 and IL-10. In contrast, mice receiving indomethacin showed a reduction in parasite load parallel to a strong Con-A response and high levels of IL-2 and IFN-γ, concomitantly with a decrease in IL-4, IL-6 and IL-10 production. Indirect in vitro studies suggest that an important source of prostaglandin E2 production could be related to host's adherent cells. However, prostaglandin E2 from parasite origin cannot be discarded.     

Adherence of Candida albicans to endothelial cells is inhibited by prostaglandin I2.

The adherence of Candida species yeast cells to bovine and human endothelium was measured. The adherence phenomenon is a saturable event which can be altered by the release of prostaglandin I2 (PGI2 or prostacyclin) by endothelial cells. Release of PGI2 by endothelium has a protective effect, reducing Candida albicans yeast cell adherence to endothelial cells in a dose-dependent manner. Conversely, C. albicans appears to inhibit the release of PGI2 by endothelial cells and thus perhaps is capable of increasing the number of yeast cells which adhere to endothelium. Furthermore, a long-acting analog of PGI2, carbacyclin, reduced the number of yeast cells adhering to human platelet aggregates. Thus, it is possible that the release of PGI2 by endothelium has a protective effect against the hematogenous dissemination of C. albicans and its ability to adhere to vascular endothelium and fibrin-platelet matrices.     

Immunogenicity of adult mesenchymal stem cells: lessons from the fetal allograft

Herein we review recent data that support host tolerance of allogeneic adult mesenchymal stem cells (MSC). Evidence is emerging that donor MSC deploy a very powerful array of mechanisms that allow escape from host allogeneic responses. These mechanisms include limited expression of alloantigen by the stem cell and cell contact-dependent and -independent mechanisms. MSC modulate host dendritic cell and T cell function, promoting induction of suppressor or regulatory T cells. These effects are complemented by the induction of divisional arrest anergy in T cells and by stem cell production of soluble immunomodulatory factors, including interleukin-10, transforming growth factor-beta, prostaglandin E2, and hepatocyte growth factor. In addition, MSC express the enzyme indoleamine 2,3-dioxygenase, which creates a tryptophan-depleted milieu that promotes immunosuppression. We propose that these observations show striking similarity to emerging data on the maternal acceptance of the fetal allograft. This comparison suggests new approaches to determine the contribution of different mechanisms to the successful use of MSC in regenerative medicine.