Veronica Osla

Mutational Tuning of Galectin-3 Specificity and Biological Function

Galectins are defined by a conserved β-galactoside binding site that has been linked to many of their important functions in e.g. cell adhesion, signaling, and intracellular trafficking. Weak adjacent sites may enhance or decrease affinity for natural β-galactoside-containing glycoconjugates, but little is known about the biological role of this modulation of affinity (fine specificity). We have now produced 10 mutants of human galectin-3, with changes in these adjacent sites that have altered carbohydrate-binding fine specificity but that retain the basic β-galactoside binding activity as shown by glycan-array binding and a solution-based fluorescence anisotropy assay. Each mutant was also tested in two biological assays to provide a correlation between fine specificity and function. Galectin-3 R186S, which has selectively lost affinity for LacNAc, a disaccharide moiety commonly found on glycoprotein glycans, has lost the ability to activate neutrophil leukocytes and intracellular targeting into vesicles. K176L has increased affinity for β-galactosides substituted with GlcNAcβ1–3, as found in poly-N-acetyllactosaminoglycans, and increased potency to activate neutrophil leukocytes even though it has lost other aspects of galectin-3 fine specificity. G182A has altered carbohydrate-binding fine specificity and altered intracellular targeting into vesicles, a possible link to the intracellular galectin-3-mediated anti-apoptotic effect known to be lost by this mutant. Finally, the mutants have helped to define the differences in fine specificity shown by Xenopus, mouse, and human galectin-3 and, as such, the evidence for adaptive change during evolution.

Increased Intracellular Oxygen Radical Production in Neutrophils During Febrile Episodes of Periodic Fever, Aphthous Stomatitis, Pharyngitis, and Cervical Adenitis Syndrome

OBJECTIVE Periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome is an autoinflammatory disease of unknown etiology that primarily affects preschool-aged children. PFAPA syndrome is characterized by recurrent attacks of fever and symptoms of inflammation consistent with the disease acronym. Since autoinflammatory diseases are, by definition, mediated by cells of the innate immune system, the aim of this study was to evaluate the functional features of neutrophils, the most abundant innate immune cell in the circulation, in children with PFAPA syndrome. METHODS Blood polymorphonuclear leukocytes (PMNs), obtained from patients with PFAPA syndrome during both febrile and asymptomatic, afebrile phases of the disease, as well as from healthy children (afebrile controls) and children with fever and abdominal pain (febrile controls), were analyzed for 3 key neutrophil characteristics: 1) apoptosis (measured by annexin V/7-aminoactinomycin D staining), 2) production of reactive oxygen species (ROS) (measured by luminol/isoluminol-amplified chemiluminescence), and 3) priming status (measured as responsiveness to galectin-3 and up-regulation of CD11b). RESULTS Compared to PMNs obtained from patients with PFAPA syndrome during an afebrile interval and those from febrile controls, PMNs obtained from patients during a PFAPA syndrome flare produced elevated levels of intracellular NADPH oxidase-derived ROS, had significantly diminished rates of spontaneous apoptosis, and displayed signatures of priming. In contrast, PMNs from afebrile patients with PFAPA syndrome had a significantly elevated rate of spontaneous apoptosis compared to PMNs from afebrile controls. CONCLUSION These findings demonstrate that 3 key aspects of neutrophil innate immune function, namely, apoptosis, priming, and generation of an intracellular oxidative burst, are altered, most prominently during febrile attacks, in children with PFAPA syndrome.

Cord blood neutrophils display a galectin-3 responsive phenotype accentuated by vaginal delivery

BackgroundTerm neonates are at increased risk of infections due to undeveloped immune mechanisms, and proper neutrophil function is important for perinatal immune defence. Galectin-3, an endogenous β-galactoside-binding lectin, is emerging as an inflammatory mediator and we have previously shown that primed/activated, but not resting, adult neutrophils respond to this lectin by production of reactive oxygen species (ROS). We investigated if galectin-3 is of importance in perinatal immune defence, focusing on plasma levels and neutrophil responsiveness.MethodsNeutrophils were isolated from peripheral blood of healthy adults and cord blood (CB) after elective Caesarean section (CSCB) and vaginal delivery (VDCB). ROS production was measured by chemiluminescence, L-selectin expression by flow cytometry, and interleukin-8 (IL-8) and galectin-3 concentrations by ELISA. Statistical evaluations were performed using the Mann–Whitney test.ResultsIn response to galectin-3, CSCB neutrophils showed a small but clear ROS production not evident in adult cells, signifying that neonatal neutrophils exist in a primed state. IL-8 production was elevated in CSCB cells while L-selectin exposure was equal to adult cells. Comparing CSCB to VDCB neutrophils, the latter showed an extensive galectin-3 responsiveness, indicating that the degree of priming is dependent on mode of delivery. VDCB neutrophils were increasingly prone to shed L-selectin, while the amount of IL-8 was similar to CSCB cells. The endogenous galectin-3 levels were higher in neonatal as compared to adult plasma, unaffected by mode of delivery.ConclusionsNeutrophils enter a pre-primed state already in the fetus. Upon exposure to the inflammatory stimuli that are associated with labor, the neutrophils develop a reactive phenotype with extensive priming features.

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

ABSTRACT 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 β-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 β‐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.

Elevated Mitochondrial Reactive Oxygen Species and Cellular Redox Imbalance in Human NADPH-Oxidase-Deficient Phagocytes

Chronic granulomatous disease (CGD) is caused by mutations in genes that encode the NADPH-oxidase and result in a failure of phagocytic cells to produce reactive oxygen species (ROS) via this enzyme system. Patients with CGD are highly susceptible to infections and often suffer from inflammatory disorders; the latter occurs in the absence of infection and correlates with the spontaneous production of inflammatory cytokines. This clinical feature suggests that NADPH-oxidase-derived ROS are not required for, or may even suppress, inflammatory processes. Experimental evidence, however, implies that ROS are in fact required for inflammatory cytokine production. By using a myeloid cell line devoid of a functional NADPH-oxidase and primary CGD cells, we analyzed intracellular oxidants, signs of oxidative stress, and inflammatory cytokine production. Herein, we demonstrate that phagocytes lacking a functional NADPH-oxidase, namely primary CGD phagocytes and a gp91phox-deficient cell line, display elevated levels of ROS derived from mitochondria. Accordingly, these cells, despite lacking the major source of cellular ROS, display clear signs of oxidative stress, including an induced expression of antioxidants and altered oxidation of cell surface thiols. These observed changes in redox state were not due to abnormalities in mitochondrial mass or membrane integrity. Finally, we demonstrate that increased mitochondrial ROS enhanced phosphorylation of ERK1/2, and induced production of IL8, findings that correlate with previous observations of increased MAPK activation and inflammatory cytokine production in CGD cells. Our data show that elevated baseline levels of mitochondria-derived oxidants lead to the counter-intuitive observation that CGD phagocytes are under oxidative stress and have enhanced MAPK signaling, which may contribute to the elevated basal production of inflammatory cytokines and the sterile inflammatory manifestations in CGD.

Neutrophils from patients with SAPHO syndrome show no signs of aberrant NADPH oxidase-dependent production of intracellular reactive oxygen species

Objective. We aimed to investigate if aberrant intracellular production of NADPH oxidase-derived reactive oxygen species (ROS) in neutrophils is a disease mechanism in the autoinflammatory disease SAPHO syndrome, characterized by synovitis, acne, pustulosis, hyperostosis and osteitis, as has previously been suggested based on a family with SAPHO syndrome-like disease. Methods. Neutrophil function was explored in a cohort of four patients with SAPHO syndrome, two of whom were sampled during both inflammatory and non-inflammatory phase. Intracellular neutrophil ROS production was determined by luminol-amplified chemiluminescence in response to phorbol myristate acetate. Results. Cells from all patients produced normal amounts of ROS, both intra- and extracellularly, when compared with internal controls as well as with a large collection of healthy controls assayed in the laboratory over time (showing an extensive inter-personal variability in a normal population). Further, intracellular production of ROS increased during the inflammatory phase. Neutrophil activation markers were comparable between patients and controls. Conclusion. Dysfunctional generation of intracellular ROS in neutrophils is not a generalizable feature in SAPHO syndrome. Secondly, serum amyloid A appears to be a more sensitive inflammatory marker than CRP during improvement and relapses in SAPHO syndrome.

MPO deficiency confers impaired processing of neutrophil reactive oxygen species in a patient with severe CRMO.

We report a severe case of chronic recurrent multifocal osteomyelitis (CRMO) associated with total myeloperoxidase (MPO) deficiency. The etiology of CRMO is in most cases unknown, and this is to our knowledge the first case associated with MPO-deficiency. Leukocyte MPO-deficiency renders neutrophils unable to process superoxide to secondary reactive oxygen species (ROS). Partial MPO deficiency is seldom associated with pathology but little is known about the effects of total MPO deficiency.

Challenging the opinion that SAPHO syndrome is associated with low intracellular ROS production in neutrophils

Background SAPHO syndrome, characterized by synovitis, acne, pustulosis, hyperostosis, and osteitis, belongs to the autoinflammatory bone disorders, in which dysregulation of innate immunity typically causes inflammation in sterile bone. The mechanisms underlying SAPHO syndrome are unknown, but neutrophil activation is suggested as part of disease pathophysiology. Previously, a patient with SAPHO syndrome-like phenotype was shown to lack production of intracellular NADPH-oxidase-derived reactive oxygen species (ROS) in response to phorbol myristate acetate (PMA; Ferguson et al, Arthritis and Rheumatism, 2008). In absence of phagosome-formation, such ROS are produced in intracellular granules, and are suggested to be part of regulatory signaling associated with hyperinflammatory disease.

Severe chronic non-bacterial osteomyelitis associated with MPO deficiency

We report a severe case of chronic non-bacterial osteomyelitis (CNO) associated with total myeloperoxidase (MPO) deficiency. Chronic non-bacterial osteomyelitis (CNO) is often considered an autoinflammatory disease. In pediatric literature CNO is often referred to as chronic recurrent multifocal ostemyelitis (CRMO). CNO is occasionally associated with extremely rare monogenic disease but in most cases the etiology is unknown. MPO deficiency is characterized by low levels of leukocyte MPO, and in some cases even total lack of the enzyme. The condition is seldom associated with pathology; severe Candida infection occurs in about 5% of cases and there is a slightly increased frequency of minor infections.