Host Defences Against Fusarium and Scedosporium spp In vitro Studies

Most studies of host defences against filamentous fungi have focused to A. fumigatus and to Rhizopus oryzae (Diamond & Clark, 1982; Schaffner et al., 1982; Washburn et al., 1987). Knowledge of the host response against Fusarium and Scedosporium spp. is rather limited and less well understood.

Since Fusarium and Scedosporium spp. share some common features with A. fumigatus, such as utilizing the same portals of entry into human body as well as causing life-threatening and therapy-refractory infections especially in immuno-compromised patients, immune mechanisms similar to A. fumigatus probably apply to the two fungal genera. The main line of host defence against A. fumigatus consists of phagocytes, including circulating polymorphonuclear leukocytes (PMNs) and MNCs as well as monocyte-derived macrophages (MDMs). Although MNCs and macrophages can damage hyphae, the bulk of this role appears to fall upon the PMNs (Waldorf, 1989). Phagocytes are capable of damaging hyphal elements through oxygen-dependent and independent mechanisms. The oxygen-dependent mechanisms consist of a series of reactions starting with the production of superoxide anion (O2-), which is dismutated into hydrogen peroxide (H2O2). Myeloperoxidase (MPO) then catalyzes the conversion of H2O2 and halides to generate hypohalides and chloramines, which exert potent antifungal activities (Babior, 2000; Hampton et al., 1998). Cationic peptides (defensins and cathelici-dins) are part of the oxygen-independent pathway of phagocytic cells during the innate host response to fungi (De Lucca & Walsh, 1999; Ramanathan et al., 2002; Yang et al., 2002). A large body of evidence reveals that a variety of cytokines, chemokines and growth factors play an important role in the host response connecting the innate with the adaptive immunity and to the pathogenesis of filamentous fungal infections (Romani, 2004).

Recognition of fungal elements by macrophages occurs through Toll-like receptors and other surface receptors. These molecules transduce the signal of activation into the nucleus and induce expression of pro-inflammatory and other cytokines. However, no knowledge exists on the recognition activation of cells, intracellular transduction and gene expression in response to Fusarium and Scedosporium spp. Cytokines such as interleukin-6 (IL-6) and interleukin-8 (IL-8) but not tumor necrosis factor-a (TNF-a) are released by the phagocytes in response to F solani and S. prolificans.

The immune response to Fusarium spp. may be species-dependent. Specifically in vitro studies have shown that human PMNs elicit higher oxidative burst in response to serum-opsonized hyphae of F. solani than of F. oxysporum. In addittion, PMNs induce much less damage to F. solani hyphae than to those of F. oxysporum. This finding indicates that F solani hyphae are more resistant to the innate immune response than F. oxysporum hyphae. The increased incidence of invasive disease due to F. solani might be alternatively explained as arising from a higher level of environmental exposure of patients to this species than to F. oxysporum. However, it would seem extremely unlikely that there is no link between the apparently superior resistance of F. solani hyphae to MNC damage and the finding that this is the most frequently encountered of the two species in invasive infections of neutropenic patients (Winn et al., 2003).

Th1-type cytokines have exhibited certain enhancing activities on antifungal phagocytic responses. In this regard, effects of IL-15 on antifungal responses of human PMNs against Fusarium spp. have been studied. IL-15 did not affect PMN oxidative respiratory burst evaluated as O2- production in response to F. solani and F. oxysporum. However, IL-15 increased interleukin-8 (IL-8) release from PMNs challenged by F. solani hyphae, but not by F. oxysporum hyphae. Additionaly, release of TNF-a was not affected by the use of IL-15 (Winn et al., 2005).

Pulmonary alveolar macrophages (PAMs), the primary phagocytic cells of pulmonary host defence during neutropenia, exhibited fungicidal activity against conidia of F. solani and achieved a time-dependent increase in killing. In addition, when PAMs were incubated with DAMB or ABLC, it was found that ABLC and, to a lesser degree, DAMB additively augments the fungicidal activity of PAMs against conidia of F. solani (Roilides et al., 2006).

Different immunomodulatory effects of amphotericin B formulations (deoxycholate, liposomal, lipid complex and colloidal dispersion amphotericin B) on oxidative antifungal activities of human MNCs and PMNs against F. solani hyphae have been determined. Specifically, MNCs and PMNs pretreated with all amphotericin B formulations induce increased F. solani hyphal damage; while, ABLC appears most effective. Further, the effects of amphotericin B formulations on PMN-induced hyphal damage are significantly higher than those on MNC-induced damage. In contrast, O2- production by MNCs or PMNs upon hyphal challenge is not stimulated by amphotericin B formulations (Dotis, J., Simitsopoulou, M., Dalakiouridou, M., Konstantinou, T., Walsh, T.J., Roilides, E. Comparative study of the effects of amphotericin B formulations on antifungal activity of human phagocytes against Aspergillus fumigatus and F. solani, submitted).

In vitro studies have shown that S. apiospermum and S. prolificans conidia and hyphae are susceptible to phagocytes in a manner comparable to A. fumigatus with minor differences (Gil-Lamaignere et al., 2001, 2003). Specifically, MDMs are able to phagocytose Scedosporium conidia similarly to the phagocytosis of Aspergillus, despite the much bigger size of S. prolificans conidia. Additionally, MDMs inhibit germination of S. prolificans conidia less efficiently, as compared to A. fumigatus (Gil-Lamaignere et al., 2001). In vitro studies have demonstrated that phagocytes are capable of exhibiting sufficient oxidative burst to control S. prolificans strains in the presence of serum. In the absence of serum, however, the production of O2- appears to be lessened (Gil-Lamaignere et al., 2001). The way the opsonization status affects the oxidative burst in response to S. prolificans remains unclear and merits further investigation. Isolates of S. prolificans tested in vitro have been damaged in an effector cell-target ratio-dependent manner when challenged with both kinds of phagocytes. Moreover, phagocytes have tended to induce more damage to S. prolificans than to A. fumigatus (Gil-Lamaignere et al., 2001).

Further insight into the immunopathogenesis of Scedosporium spp. infection has been gained through in vitro studies of the phagocytic cell responses to amphotericin B-resistant and susceptible S. apiospermum isolates. Accordingly, it has been found that macrophages are able to phagocytose S. apiospermum conidia, damage hyphae in an effector cell-target ratio-dependent manner and release O2- in response to serum-opsonized hyphae. It has also been observed that hyphae of the two strains with different amphotericin B-susceptibility patterns have different levels of susceptibility to MPO products. This phenomenon, although not fully elucidated, may be related to the various levels of pathogenicity and antifungal drug resistance of S. apiospermum (Gil-Lamaignere et al., 2003).

ABLC has been reported that it displays a significant additive effect with PMNs against S. prolificans and S. apiospermum in vitro (Gil-Lamaignere et al., 2002a). Similarly, in another in vitro study, triazoles used in combination with PMNs have caused significant additive increase in the damage of the hyphae of S. prolificans and S. apiospermum. Furthermore, under certain conditions synergism has been noted between triazoles and PMNs against S. prolificans hyphae. Interestingly, the synergis-tic activity has been observed at the low concentrations of the antifungals used. This finding may be of particular importance especially in immunocompromised patients when a triazole reaches its trough level in plasma where such synergy may prevent fungal regrowth (Gil-Lamaignere et al., 2002b). Regardless of the mechanisms behind these collaborative effects, the findings from these studies would support the concomitant administration of antifungals and PMN transfusions to persistently neutropenic patients with invasive scedosporiosis. However, no cases of scedosporio-sis have been reported that have been treated with PMN transfusions up to date.

Immunosuppression constitutes a significant risk factor for the surge of invasive fungal infections. In this regard, a number of studies have aimed to assess the immu-nomodulatory utility of cytokines in confronting emerging fungal pathogens (Steinbach & Perfect, 2003). S. prolificans has been shown to induce significantly more TNF and IL-6 release by human MNCs, as compared to Aspergillus spp. This could be attributed to the specific composition of the S. prolificans cell wall, although its exact composition is not known, which may yield more potent stimulatory molecules. Speculatively, this could be associated with the virulence of the specific fungus (Warris et al., 2005).

Besides it has been shown that the presence of IL-15 significantly enhances PMN-induced hyphal damage and oxidative respiratory burst of S. prolificans but not S. apiospermum. Additionally, IL-15 increases IL-8 release from PMNs challenged by S. prolificans whereas release of TNF is not affected. The inability of IL-15 to exhibit enhanced damage of S. apiospermum hyphae is in concordance with its greatest intrinsic virulence in humans. These findings suggest that IL-15 has species-specific enhancing effects on antifungal activities of PMNs against Scedosporium spp. Further, some of the cytokine-induced effects have been shown that are the result of direct actions on effector activities of PMNs while others, related to the increased release of IL-8, acting in an autocrine way on PMNs, result in enhanced indirect antifungal actions (Winn et al., 2005).

Among the cytokines studied that enhance PMN antifungal activity against Scedosporium spp. are interferon-y (IFN-y) and GM-CSF (Gil-Lamaignere et al., 2005). IFN-y is produced endogenously primarily by T cells and is a potent activator of MNC-macrophages and PMNs (Gaviria et al., 1999b). This cytokine induces the Th1 response, which favours resistance to fungal disease, regulates the gene expression of NADPH oxidase subunits at the transcriptional level and potentiates the synthesis of antimicrobial peptides in macrophages (Chaves et al., 1996; Amezaga et al., 1992; Cassatella et al., 1990). GM-CSF acts on early as well as on late stages of haematopoiesis and increases the number of cells of the macrophage-monocyte system. This cytokine has been found to enhance phagocytosis, oxidative burst, increase the number and membrane binding of several classes of surface receptors on PMNs and inhibit PMN apoptosis (Rodriguez-Adrian et al., 1998; Giles, 1998;

Armitage, 1998). Treatment of PMNs with the combination of IFN and GM-CSF had broader effects on Scedosporium spp. enhancing PMN functions while cytokines alone had no effect. Despite the poor effect of either cytokine alone on the PMN oxi-dative burst after 22 h, the combined treatment showed enhancement of oxidative burst in response to opsonized S. apiospermum hyphae. Similarly, after incubation with cytokines for 2 h only the combination significantly enhanced the oxidative burst against serum-opsonized and non-opsonized hyphae of Scedosporium spp. Thus, in this study it has been demonstrated that IFN and GM-CSF exhibit a significant time-and species-dependent capability to enhance PMN activity against Scedosporium spp. (Gil-Lamaignere et al., 2005).

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