Complementary Roles of the Classical and Lectin Complement Pathways in the Defense against Aspergillus fumigatus

Abstract

Aspergillus fumigatus infections are associated with a high mortality rate for immunocompromised patients. The complement system is considered to be important in protection against this fungus, yet the course of activation is unclear. The aim of this study was to unravel the role of the classical, lectin, and alternative pathways under both immunocompetent and immunocompromised conditions to provide a relevant dual-perspective on the response against A. fumigatus. Conidia (spores) from a clinical isolate of A. fumigatus were combined with various human serum types (including serum deficient of various complement components and serum from umbilical cord blood). We also combined this with inhibitors against C1q, mannose-binding lectin (MBL), and ficolin-2 before complement activation products and phagocytosis were detected by flow cytometry. Our results showed that alternative pathway amplified complement on A. fumigatus, but required classical and/or lectin pathway for initiation. In normal human serum, this initiation came primarily from the classical pathway. However, with a dysfunctional classical pathway (C1q-deficient serum), lectin pathway activated complement and mediated opsonophagocytosis through MBL. To model the antibody-decline in a compromised immune system, we used serum from normal umbilical cords and found MBL to be the key complement initiator. In another set of experiments, serum from patients with different kinds of immunoglobulin insufficiencies showed that the MBL lectin pathway contribution was highest in the samples with the lowest IgG/IgM binding. In conclusion, lectin pathway appears to be the primary route of complement activation in the absence of anti-A. fumigatus antibodies, whereas in a balanced immune state classical pathway is the main activator. This suggests a crucial role for the lectin pathway in innate immune protection against A. fumigatus in immunocompromised patients.

The complement system in Aspergillus fumigatus infections and its crosstalk with pentraxins

“…Furthermore, human neutrophils failed to engulf A. fumigatus conidia in vitro in the presence of heat-inactivated serum, indicating that this process did not involve IgG-mediated phagocytosis [50]. These observations were expanded and revisited in a study by Rosbjerg et al [52] who reported AP amplification on the dormant conidia of A. fumigatus that was, however, conditioned to initiation of the CP/LP. The relative contribution of the CP and LP to deposition of C4b, C3b, and MAC as well as phagocytosis by human neutrophils in vitro was primarily set by the serum content of IgM, in that C1q was the main initiator of complement (via the CP) in the presence of normal human serum (NHS, that contains physiological levels of IgM), and MBL proved to be the key trigger for complement activation (via the LP) in the presence of serum from umbilical cord (UC) and a patient with X-linked agammaglobulinemia in IgG replacement therapy, both IgG-competent but poor in IgM.…”

“…The relative contribution of the CP and LP to deposition of C4b, C3b, and MAC as well as phagocytosis by human neutrophils in vitro was primarily set by the serum content of IgM, in that C1q was the main initiator of complement (via the CP) in the presence of normal human serum (NHS, that contains physiological levels of IgM), and MBL proved to be the key trigger for complement activation (via the LP) in the presence of serum from umbilical cord (UC) and a patient with X-linked agammaglobulinemia in IgG replacement therapy, both IgG-competent but poor in IgM. Furthermore, UC sera with low titers of MBL had reduced complement activation on A. fumigatus, as opposed to normal levels of activation in MBLcompetent NHS [52]. While questioning the previously proposed C2 bypass mechanism [47] and, possibly, underestimating the contribution of the AP (i.e., heatinactivated conidia were used throughout the study that likely exposed PAMPs as a consequence of thermal damage to the outer cell wall), this report highlighted the role of MBL and C1q as key initiators of the complement response against A. fumigatus.…”

“…In this regard, dormant conidia are acknowledged to activate mainly the AP (here schematically depicted as hydrolysis of C3 to C3b, scaffolding unit of the AP C3 convertase, and C3a, potent anaphylatoxin) [46,47], while there is growing activation of the LP/CP as conidia swell and germinate [50]. CP activation is mainly driven by immune complexes (with IgM prevailing over IgG), and the LP is sustained by MBL and, to a lesser extent, ficolins (especially, ficolin-2) [52]. The complement-dependent disposal of the fungal pathogen is mostly mediated by opsonic and phagocytic mechanisms, with direct membrane lysis (i.e., by MAC formation) playing a minor role, due to thickness and resistance of the cell wall [23].…”

“…A. fumigatus has evolved several strategies to evade the complement response, including secretion of proteases (i.e., Alp1 [94] and Mep1p [96]) that targets key components of the system, such as C3 and C1q (as well as C4 and C5, not shown in the figure), and recruitment of inhibitors of the AP (i.e., factor H, fH) [89] and CP/LP (i.e., C4BP) [90]. contributed to complement activation in conditions of C1q deficiency and MBL competence [52]. In this regard, a synergism was reported between ficolin-2 and PTX3 in the recognition of and complement deposition on dormant conidia (further discussed in the 'PTX3 in the immune response to A. fumigatus and its crosstalk with complement' section), which was mostly apparent in the absence of both C1q and MBL, indicating that ficolin-2, likely via a crosstalk with PTX3, participates in the LP response to A. fumigatus [55].…”

Platelet Thrombus Formation in eHUS is Prevented by Anti-MBL2

“…The lectin pathway, one of three known molecular pathways of complement activation (classical, alternative and mannose-binding lectin (MBL)), also plays an important role in innate immune protection against A. fumigatus in immunocompromised patients [14], the pathophysiology of atherosclerosis in humans [15], myocardial infarction, coagulation, brain ischemic injury, and the innate immune response to pneumococcal infection in mice [16][17][18]. We previously demonstrated that injection of (Stx-2 leads to fibrin deposition in mouse glomeruli that was largely blocked by the co-injection of anti-MBL-antibody 3F8 [19,20].…”