��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)² are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1–5). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8–10). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (14–18) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (27–30). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.     

Synergy between Ficolin-2 and Pentraxin 3 Boosts Innate Immune Recognition and Complement Deposition

The long pentraxin 3 (PTX3) is a multifunctional soluble pattern recognition molecule that is crucial in innate immune protection against opportunistic fungal pathogens such as Aspergillus fumigatus. The mechanisms that mediate downstream effects of PTX3 are largely unknown. However, PTX3 interacts with C1q from the classical pathway of the complement. The ficolins are recognition molecules of the lectin complement pathway sharing structural and functional characteristics with C1q. Thus, we investigated whether the ficolins (Ficolin-1, -2, and -3) interact with PTX3 and whether the complexes are able to modulate complement activation on A. fumigatus. Ficolin-2 could be affinity-isolated from human plasma on immobilized PTX3. In binding studies, Ficolin-1 and particularly Ficolin-2 interacted with PTX3 in a calcium-independent manner. Ficolin-2, but not Ficolin-1 and Ficolin-3, bound A. fumigatus directly, but this binding was enhanced by PTX3 and vice versa. Ficolin-2-dependent complement deposition on the surface of A. fumigatus was enhanced by PTX3. A polymorphism in the FCN2 gene causing a T236M amino acid change in the fibrinogen-like binding domain of Ficolin-2, which affects the binding to GlcNAc, reduced Ficolin-2 binding to PTX3 and A. fumigatus significantly. These results demonstrate that PTX3 and Ficolin-2 may recruit each other on pathogens. The effect was alleviated by a common amino acid change in the fibrinogen-like domain of Ficolin-2. Thus, components of the humoral innate immune system, which activate different complement pathways, cooperate and amplify microbial recognition and effector functions.The ficolins are multimeric collagen-like proteins consisting of an N-terminal domain, a collagen-like domain (CD),² and a C-terminal fibrinogen-like (FBG) domain involved in innate immune defense (1, 2). In humans, three types of ficolins have been identified as follows: Ficolin-1 (M-ficolin), Ficolin-2 (L-ficolin), and Ficolin-3 (H-ficolin/Hakata antigen). They function as recognition molecules in the lectin complement pathway along with mannose-binding lectin but with differentiated complement activating capacity (3). Ficolin-2 and Ficolin-3 circulate in the blood with a median concentration of 5 and 25 μg/ml, respectively (4, 5). Ficolin-2 is mainly produced in the liver, whereas Ficolin-3 is synthesized in both the liver and lungs, with the highest expression in the lungs (3). Ficolin-1 is primarily expressed by bone marrow-derived cells and lung epithelial cells (3, 6–8) and has recently been shown to be present in the blood with a median plasma concentration of 60 ng/ml (9). The ficolin genes (FCN1, -2, and -3) are polymorphic, and particularly polymorphisms in FCN2 regulate both the level and function of Ficolin-2 (4, 10, 11). In this respect, a base substitution in exon 8 at position 6359 (C→T) causing a threonine to be replaced by a methionine (T236M) in the FBG domain of Ficolin-2 has been shown to cause decreased binding activity toward GlcNAc (10).Ficolin-1 has been reported to bind to GlcNAc, GalNAc, and sialic acid (8, 12). It may opsonize Staphylococcus aureus via GlcNAc and interact with a smooth-type strain of Salmonella typhimurium through an unknown ligand, the binding of which is not diminished by GlcNAc (8). Ficolin-2 has been shown to recognize specific pathogen-associated molecular patterns, which are typically located in pathogen cell membranes, such as lipoteichoic acid and peptidoglycan in Gram-positive bacteria cell walls, lipopolysaccharide in Gram-negative bacteria cell walls, and 1,3-β-d-glucan in yeast and fungal cell walls (13, 14). The ligand specificity of Ficolin-2 has also been defined as acetyl groups, including those of N-acetylmannosamine, GlcNAc, GalNAc as well as acetyl groups on cysteine, glycine, and choline (15). Ficolin-2 recognizes clinically important pathogens, like S. typhimurium, S. aureus, and Streptococcus pneumoniae (13, 16, 17). Ficolin-3 shows affinity for GlcNAc, GalNAc, and d-fucose and may interact with S. typhimurium, Salmonella minnesota, and Aerococcus viridans (17, 18).The long pentraxin 3 (PTX3) is a soluble pattern recognition molecule mediating innate immune recognition (19). PTX3 is a glycoprotein of 45 kDa, which assembles into an octameric structure through protomer linkage by disulfide bonds (20). PTX3 shares C-terminal structural similarity with the classic short pentraxins, C-reactive protein (CRP), and serum amyloid P component, whereas the N-terminal sequence differs from the other proteins (21). Myeloid cells are a major source of PTX3, but PTX3 has also been shown in vitro to be produced by a variety of cells in response to inflammatory signals (21). During inflammation PTX3 is rapidly up-regulated and released into the surrounding tissue and into the bloodstream. PTX3 interacts with C1q and participates in activation of the classical complement pathway (22, 23). Moreover, it has also been shown that PTX3 binds the complement regulatory factor H and that this interaction regulates the alternative pathway of complement (24).PTX3 can interact with a number of different pathogens, bacteria as well as fungi and viruses. A specific binding has been observed for selected Gram-positive and Gram-negative bacteria, including S. aureus, Pseudomonas aeruginosa, S. typhimurium, Klebsiella pneumoniae, S. pneumoniae, and Neisseria meningitidis (21). PTX3 also binds zymosan and conidia from Aspergillus fumigatus) (25). Furthermore, it has been shown that ptx3 knock-out mice are extremely susceptible to invasive pulmonary aspergillosis. The phenotypic defect can be completely reversed by treatment with recombinant PTX3 (25, 26). These data indicate that PTX3 is important in protection against A. fumigatus, which has become a major cause of morbidity in medical institutions because of the increasing number of immunosuppressed patients (27).Based on the knowledge of the structural and functional similarities between C1q and the ficolins, this study was designed to characterize a possible interaction between the ficolins and PTX3 using A. fumigatus as a model. Based on our data, we propose an important role for previously unlinked collaboration of PTX3 and Ficolin-2, but not Ficolin-1 and Ficolin-3, in the recognition of A. fumigatus and amplification of complement activation. Moreover, our results demonstrate functional consequences of the Ficolin-2 T236M substitution in the interaction between PTX3 and A. fumigatus.