Novel inhibitory effect of galectin-3 on the respiratory burst induced by Staphylococcus aureus in human neutrophils.

Among the responders to microbial invasion, neutrophils represent the earliest and perhaps the most important immune cells that contribute to host defense with the primary role to kill invading microbes using a plethora of stored anti-microbial molecules. One such process is the production of reactive oxygen species (ROS) by the neutrophil enzyme complex NADPH-oxidase, which can be assembled and active either extracellularly or intracellularly in phagosomes (during phagocytosis) and/or granules (in the absence of phagocytosis). One soluble factor modulating the interplay between immune cells and microbes is galectin-3 (gal-3), a carbohydrate-binding protein that regulates a wide variety of neutrophil functions. Gal-3 has been shown to potentiate neutrophil interaction with bacteria, including Staphylococcus aureus, and is also a potent activator of the neutrophil respiratory burst, inducing large amounts of granule-localized ROS in primed cells. Herein, the role of gal-3 in regulating S. aureus phagocytosis and S. aureus-induced intracellular ROS was analyzed by imaging flow cytometry and luminol-based chemiluminescence, respectively. Although gal-3 did not interfere with S. aureus phagocytosis per se, it potently inhibited phagocytosis-induced intracellular ROS production. Using the gal-3 inhibitor GB0139 (TD139) and carbohydrate recognition domain of gal-3 (gal-3C), we found that the gal-3-induced inhibitory effect on ROS production was dependent on the carbohydrate recognition domain of the lectin. In summary, this is the first report of an inhibitory role of gal-3 in regulating phagocytosis-induced ROS production.

Galectin-3 Is a Target for Proteases Involved in the Virulence of Staphylococcus aureus.

Staphylococcus aureus is a major cause of skin and soft tissue infection. The bacterium expresses four major proteases that are emerging as virulence factors: aureolysin (Aur), V8 protease (SspA), staphopain A (ScpA), and staphopain B (SspB). We hypothesized that human galectin-3, a ß-galactoside-binding lectin involved in immune regulation and antimicrobial defense, is a target for these proteases and that proteolysis of galectin-3 is a novel immune evasion mechanism. Indeed, supernatants from laboratory strains and clinical isolates of S. aureus caused galectin-3 degradation. Similar proteolytic capacities were found in Staphylococcus epidermidis isolates but not in Staphylococcus saprophyticus Galectin-3-induced activation of the neutrophil NADPH oxidase was abrogated by bacterium-derived proteolysis of galectin-3, and SspB was identified as the major protease responsible. The impact of galectin-3 and protease expression on S. aureus virulence was studied in a murine skin infection model. In galectin-3+/+ mice, SspB-expressing S. aureus caused larger lesions and resulted in higher bacterial loads than protease-lacking bacteria. No such difference in bacterial load or lesion size was detected in galectin-3-/- mice, which overall showed smaller lesion sizes than the galectin-3+/+ animals. In conclusion, the staphylococcal protease SspB inactivates galectin-3, abrogating its stimulation of oxygen radical production in human neutrophils and increasing tissue damage during skin infection.

Galectin-3 type-C self-association on neutrophil surfaces; The carbohydrate recognition domain regulates cell function.

Galectin-3 is an endogenous ß-galactoside-binding lectin comprising a carbohydrate recognition domain (CRD) linked to a collagen-like N-domain. Both domains are required for galectin-3 to induce cellular effects; a C-terminal fragment of galectin-3, galectin-3C, containing the CRD but lacking the N-domain, binds cell surface glycoconjugates but does not induce cellular effects since cross-linking promoted by the N-domain is thought to be required. Instead, galectin-3C is proposed to antagonize the effects of galectin-3 by competing for binding sites. The aim of this study was to investigate the effects of galectin-3C on galectin-3 interactions with human neutrophils. Recombinant galectin-3C inhibited galectin-3-induced production of reactive oxygen species in primed neutrophils. Surprisingly, this inhibition was not due to competitive inhibition of galectin-3 binding to the cells. In contrast, galectin-3C potentiated galectin-3 binding, in line with emerging evidence that galectin-3 can aggregate not only through the N-domain but also through the CRD. The cell surface interaction between galectin-3C and galectin-3 was corroborated by colocalization of fluorescently labeled galectin-3 and galectin-3C. Galectin-3C can be generated in vivo through cleavage of galectin-3 by proteases. Indeed, in circulation, galectin-3 and galectin-3C were both attached to the cell surface of neutrophils, which displayed great capacity to bind additional galectin-3 and galectin-3C. In conclusion, galectin-3C enhances galectin-3 binding to neutrophils by nonactivating type-C self-association, in parallel to inhibiting neutrophil activation by galectin-3 (induced by type-N self-association). This implicates type-C self-association as a termination system for galectin-3-induced cell activation, with the purpose of avoiding oxidant-dependent tissue damage.

Phenol-Soluble Modulin α Peptide Toxins from Aggressive Staphylococcus aureus Induce Rapid Formation of Neutrophil Extracellular Traps through a Reactive Oxygen Species-Independent Pathway.

Neutrophils have the ability to capture and kill microbes extracellularly through the formation of neutrophil extracellular traps (NETs). These are DNA and protein structures that neutrophils release extracellularly and are believed to function as a defense mechanism against microbes. The classic NET formation process, triggered by, e.g., bacteria, fungi, or by direct stimulation of protein kinase C through phorbol myristate acetate, is an active process that takes several hours and relies on the production of reactive oxygen species (ROS) that are further modified by myeloperoxidase (MPO). We show here that NET-like structures can also be formed by neutrophils after interaction with phenol-soluble modulin α (PSMα) that are cytotoxic membrane-disturbing peptides, secreted from community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The PSMα-induced NETs contained the typical protein markers and were able to capture microbes. The PSMα-induced NET structures were disintegrated upon prolonged exposure to DNase-positive S. aureus but not on exposure to DNase-negative Candida albicans. Opposed to classic NETosis, PSMα-triggered NET formation occurred very rapidly, independently of ROS or MPO, and was also manifest at 4°C. These data indicate that rapid NETs release may result from cytotoxic membrane disturbance by PSMα peptides, a process that may be of importance for CA-MRSA virulence.