Person:

Stahl, Gregory L.

Mannose-binding lectin (MBL), a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid (\beta) peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to A(\beta) are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.

The complement system plays a key role in host defense against pneumococcal infection. Three different pathways, the classical, alternative and lectin pathways, mediate complement activation. While there is limited information available on the roles of the classical and the alternative activation pathways of complement in fighting streptococcal infection, little is known about the role of the lectin pathway, mainly due to the lack of appropriate experimental models of lectin pathway deficiency. We have recently established a mouse strain deficient of the lectin pathway effector enzyme mannan-binding lectin associated serine protease-2 (MASP-2) and shown that this mouse strain is unable to form the lectin pathway specific C3 and C5 convertases. Here we report that MASP-2 deficient mice (which can still activate complement via the classical pathway and the alternative pathway) are highly susceptible to pneumococcal infection and fail to opsonize Streptococcus pneumoniae in the none-immune host. This defect in complement opsonisation severely compromises pathogen clearance in the lectin pathway deficient host. Using sera from mice and humans with defined complement deficiencies, we demonstrate that mouse ficolin A, human L-ficolin, and collectin 11 in both species, but not mannan-binding lectin (MBL), are the pattern recognition molecules that drive lectin pathway activation on the surface of S. pneumoniae. We further show that pneumococcal opsonisation via the lectin pathway can proceed in the absence of C4. This study corroborates the essential function of MASP-2 in the lectin pathway and highlights the importance of MBL-independent lectin pathway activation in the host defense against pneumococci.

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes.

Objective: Ischemic heart disease is the leading cause of death worldwide. The complement system plays a major role in inflammation and tissue injury following myocardial ischemia and reperfusion (MI/R) injury. Systemic C5 inhibition in clinical studies has resulted in mixed results and the role of earlier complement components (e.g., C3a), upstream from C5 cleavage, has not been elucidated for MI/R injury. Therefore, we evaluated the role of C5 or C3a in a mouse model of MI/R injury. Methods: We performed experimental MI/R with 30 min of ischemia and 4 hr of reperfusion in 8–12 wk old C57BL/6 (WT) mice. Systemic C5 or C3a inhibition was performed with an anti-C5 monoclonal antibody (BB5.1) 30 min prior to reperfusion or with a C3a receptor antagonist (C3aRA). Since the C3aRA induces neutropenia that resolves within 120 min, we administered C3aRA at two different time points in two separate groups: 30 min prior to reperfusion within the neutropenic time frame and 120 min prior to reperfusion, when the neutropenia had resolved, but C3aRA remained active. Following MI/R, cardiac function was assessed via echocardiography, serum troponin I concentrations were measured as an index of myocardial cell death and myocardial inflammation was determined via myocardial polymorphonuclear leukocyte (PMN) infiltration. Results: In wild type mice, MI/R significantly decrease myocardial ejection fraction and increased serum troponin I levels and myocardial PMN infiltration compared to sham-operated animals. Systemic C5 inhibition, 30 min prior to reperfusion, significantly protected mice from MI/R injury, confirming an important role for C5 in murine MI/R injury. Treatment with the C3aRA, 30 min prior to reperfusion (i.e., within the neutropenic time frame), protected mice significantly from MI/R related injury. In contrast, administration of the C3aRA 120 min prior to reperfusion, when the neutropenia had resolved, but C3aRA remained active, did not prevent MI/R injury. Conclusions: These results confirm an important role for C5 cleavage in murine MI/R injury. At the same time, they suggest a minimal role for C3a, since neutropenia rather than C3a receptor antagonism appears to be responsible for C3aRA related amelioration in MI/R injury. While C5 inhibition in the clinical setting of MI/R does not appear to be therapeutic, our results raise the possibility that inhibition of either C5a or C5b-9 may be more advantageous than inhibition of C3a or complete inhibition of C5 in humans.

There is considerable evidence that influenza A virus (IAV) promotes adherence, colonization, and superinfection by S. pneumoniae (Spn) and contributes to the pathogenesis of otitis media (OM). The complement system is a critical innate immune defense against both pathogens. To assess the role of the complement system in the host defense and the pathogenesis of acute pneumococcal OM following IAV infection, we employed a well-established transtympanically-induced mouse model of acute pneumococcal OM. We found that antecedent IAV infection enhanced the severity of acute pneumococcal OM. Mice deficient in complement C1qa (C1qa−/−) or factor B (Bf −/−) exhibited delayed viral and bacterial clearance from the middle ear and developed significant mucosal damage in the eustachian tube and middle ear. This indicates that both the classical and alternative complement pathways are critical for the oto-immune defense against acute pneumococcal OM following influenza infection. We also found that Spn increased complement activation following IAV infection. This was characterized by sustained increased levels of anaphylatoxins C3a and C5a in serum and middle ear lavage samples. In contrast, mice deficient in the complement C5a receptor (C5aR) demonstrated enhanced bacterial clearance and reduced severity of OM. Our data support the concept that C5a-C5aR interactions play a significant role in the pathogenesis of acute pneumococcal OM following IAV infection. It is possible that targeting the C5a-C5aR axis might prove useful in attenuating acute pneumococcal OM in patients with influenza infection.

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.

Background: The obligate intracellular bacterial pathogen Coxiella burnetii causes the zoonosis Q fever. The intracellular niche of C. burnetii has led to the assumption that cell-mediated immunity is the most important immune component for protection against this pathogen. However, passive immunization with immune serum can protect naïve animals from challenge with virulent C. burnetii, indicating a role for antibody (Ab) in protection. The mechanism of this Ab-mediated protection is unknown. Therefore, we conducted a study to determine whether Fc receptors (FcR) or complement contribute to Ab-mediated immunity (AMI) to C. burnetii. Results: Virulent C. burnetii infects and replicates within human dendritic cells (DC) without inducing their maturation or activation. We investigated the effects of Ab opsonized C. burnetii on human monocyte-derived and murine bone marrow-derived DC. Infection of DC with Ab-opsonized C. burnetii resulted in increased expression of maturation markers and inflammatory cytokine production. Bacteria that had been incubated with naïve serum had minimal effect on DC, similar to virulent C. burnetii alone. The effect of Ab opsonized C. burnetii on DC was FcR dependent as evidenced by a reduced response of DC from FcR knockout (FcR k/o) compared to C57Bl/6 (B6) mice. To address the potential role of FcR in Ab-mediated protection in vivo, we compared the response of passively immunized FcR k/o mice to the B6 controls. Interestingly, we found that FcR are not essential for AMI to C. burnetii in vivo. We subsequently examined the role of complement in AMI by passively immunizing and challenging several different strains of complement-deficient mice and found that AMI to C. burnetii is also complement-independent. Conclusion: Despite our data showing FcR-dependent stimulation of DC in vitro, Ab-mediated immunity to C. burnetii in vivo is FcR-independent. We also found that passive immunity to this pathogen is independent of complement.