Relevance of platelets and complement for the pathogenesis of invasive fungal infections

Invasive fungal infections (IFI) are associated with a high lethality of affected patients, making advanced knowledge about the antifungal immune reaction an urgent need. Aspergillus sp. and mucormycetes (with Lichtheimia sp. as a predominant representative) are the moulds that cause the majority of severe human cases. Therapeutic regimens are of limited success, and a deeper understanding of the relevant pathomechanisms is needed to develop new options for intervention. Thrombocytes were recently recognized to fulfil immune functions in the antimicrobial host defence. A linkage between thrombocytes and invading fungi seems obvious, since thrombocytopenia is a risk factor for IFI and, conversely, the fungal infection affects the platelet function inducing thrombosis as well as thrombocyte loss. Recent own experiments demonstrated that Aspergillus secretes soluble factors, which induce thrombocyte activation and subsequently the deposition of complement proteins on the platelet surface. The consequences of this Aspergillus-induced opsonization of thrombocytes for the pathogenesis of IFI are as yet unknown, but may include a contribution to both thrombocytopenia and thrombosis, two important hallmarks of IFI. However, complement also represents a main player to network different innate immune weapons (e.g. granulocytes, monocytes, dendritic cells, cytokines/chemokines). As a consequence, fungus-induced complement deposition on platelets could also have positive effects by supporting inflammation, immune defence, and clearance. Thus, complement binding on platelets might have profound effects on the pathogenesis of invasive fungal infections and should be investigated thoroughly. We plan to clarify the effect of Aspergillus-induced complement deposition on platelets for the pathogenesis and innate immune activity in fungal infection. The results will be compared with that of the mucormycete Lichtheimia sp. To achieve this aim we will: a) investigate in detail the extent and the mechanisms how Aspergillus-derived secretory products trigger complement deposition on platelets, and compare them with Lichtheimia sp; b) analyse the putative consequences of mould-induced opsonization of platelets with complement: inflammation: can opsonised platelets attract immune cells to the site of infection and stimulate their activity and functions? thrombosis: can opsonization of platelets contribute to formation of thrombi in the blood vessels? loss and clearance of activated thrombocytes: can complement directly mediate lysis of activated thrombocytes or support the phagocytotic clearance of these opsonised platelets?

Aspergilli (e.g. A. fumigatus) and mucormycetes (e.g. Lichtheimia corymbifera) are a common inducer of opportunistic infections, affecting mainly immunocompromised patients. The high morbidity and mortality rates underline the need for new antifungal approaches, e.g. supportive therapies to strengthen the immune response. We studied therefore the role of platelets and complement in fungal infections, which are both important innate immunity elements. Previous experiments revealed platelet activation by direct contact with the hyphal-associated polysaccharide galactosaminogalactan (GAG). By screening culture supernatants (SN) from various fungal species, we could show that also secreted GAG may be relevant to trigger platelet-associated immune reactions or thrombosis. This is of particular relevance since this mechanism enables systemic inflammation, immune activation and thrombosis, also distant from the site of infection. This effect is strictly species-dependent with A. fumigatus and flavus isolates being potent platelet stimulators, while SN of other Aspergillus species and all tested mucormycete species did not significantly induce platelet activation. The capacity of culture SN to activate platelets was dependent on the deposition of secreted GAG on the platelet surface as shown by flow cytometry and confocal microscopy. This deposition of fungus-derived GAG on the platelet surface results in the recognition of the platelets to be foreign and, as a consequence, in platelet opsonisation by the complement cascade. We could prove that fragments of the complement factor C3 cover the platelet surface; in addition activation of the terminal complement pathway could be revealed by the presence of the lytic C5b-9 complex on the platelets. The lectin pathway could be identified to be the relevant complement activation pathway, a result that is interesting in regard of putative complement-modulating therapeutics. We demonstrated that opsonized platelets show reduced viability and better interaction with complement receptor-bearing granulocytes. Furthermore, complement activation results in shedding of pro-inflammatory microparticles from the platelets and thus fuels the systemic inflammation. By the FWF project important interactions between fungi, platelets and complement could be ascertained that have tremendous consequences for pathogenesis and antifungal immune reactions in invasive mycoses.