Gajendra S. Naika

Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation

Mitochondrial DNA (mtDNA) is a highly potent inflammatory trigger and is reportedly found outside the cells in blood in various pathologies. Platelets are abundant in blood where they promote hemostasis. Although lacking a nucleus, platelets contain functional mitochondria. On activation, platelets produce extracellular vesicles known as microparticles. We hypothesized that activated platelets could also release their mitochondria. We show that activated platelets release respiratory-competent mitochondria, both within membrane-encapsulated microparticles and as free organelles. Extracellular mitochondria are found in platelet concentrates used for transfusion and are present at higher levels in those that induced acute reactions (febrile nonhemolytic reactions, skin manifestations, and cardiovascular events) in transfused patients. We establish that the mitochondrion is an endogenous substrate of secreted phospholipase A2 IIA (sPLA2-IIA), a phospholipase otherwise specific for bacteria, likely reflecting the ancestral proteobacteria origin of mitochondria. The hydrolysis of the mitochondrial membrane by sPLA2-IIA yields inflammatory mediators (ie, lysophospholipids, fatty acids, and mtDNA) that promote leukocyte activation. Two-photon microscopy in live transfused animals revealed that extracellular mitochondria interact with neutrophils in vivo, triggering neutrophil adhesion to the endothelial wall. Our findings identify extracellular mitochondria, produced by platelets, at the midpoint of a potent mechanism leading to inflammatory responses.

Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA

Significance On activation, blood platelets package components from their cytoplasm into microparticles (MPs), tiny vesicles released by cytoplasmic membrane budding and shedding. Given that MPs can impact other cellular lineages on internalization, we aimed to decipher the mechanisms promoting MP internalization by cellular recipients. We modeled MP internalization by neutrophils and identified a predominant lipid, 12(S)-hydroxyeicosatetranoic acid, as a mediator critical for the promotion of MP internalization. MPs were found inside neutrophils from individuals with rheumatoid arthritis, and their presence in neutrophils in the joints of mice treated with arthritogenic serum is dependent on the expression of enzymes implicated in the generation of 12(S)-hydroxyeicosatetranoic acid. These findings reveal a unique molecular mechanism implicated in MP internalization relevant to inflammatory processes. Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

Interfacial Kinetic and Binding Properties of Mammalian Group IVB Phospholipase A2 (cPLA2β) and Comparison with the Other cPLA2 Isoforms

The cytosolic (group IV) phospholipase A2 (cPLA2s) family contains six members. We have prepared recombinant proteins for human α, mouse β, human γ, human δ, human ϵ, and mouse ζ cPLA2s and have studied their interfacial kinetic and binding properties in vitro. Mouse cPLA2β action on phosphatidylcholine vesicles is activated by anionic phosphoinositides and cardiolipin but displays a requirement for Ca2+ only in the presence of cardiolipin. This activation pattern is explained by the effects of anionic phospholipids and Ca2+ on the interfacial binding of mouse cPLA2β and its C2 domain to vesicles. Ca2+-dependent binding of mouse cPLA2β to cardiolipin-containing vesicles requires a patch of basic residues near the Ca2+-binding surface loops of the C2 domain, but binding to phosphoinositide-containing vesicles does not depend on any specific cluster of basic residues. Human cPLA2δ also displays Ca2+- and cardiolipin-enhanced interfacial binding and activity. The lysophospholipase, phospholipase A1, and phospholipase A2 activities of the full set of mammalian cPLA2s were quantified. The relative level of these activities is very different among the isoforms, and human cPLA2δ stands out as having relatively high phospholipase A1 activity. We also tested the susceptibility of all cPLA2 family members to a panel of previously reported inhibitors of human cPLA2α and analogs of these compounds. This led to the discovery of a potent and selective inhibitor of mouse cPLA2β. These in vitro studies help determine the regulation and function of the cPLA2 family members.

Characterization of functional intermediates of endoglucanase from Aspergillus aculeatus during urea and guanidine hydrochloride unfolding.

Low concentrations of urea and GuHCl (2 M) enhanced the activity of endoglucanase (EC 3.1.2.4) from Aspergillus aculeatus by 2.3- and 1.9-fold, respectively. The K(m) values for controls, in the presence of 2 M urea and GuHCl, were found to be 2.4 +/- 0.2 x 10(-8) mol L(-1), 1.4 +/- 0.2 x 10(-8) mol L(-1), and 1.6 +/- 0.2 x 10(-8) mol L(-1), respectively. The dissociation constant (Kd) showed changes in the affinity of the enzyme for the substrate with increases in the Kcat suggesting an increased turnover number in the presence of urea and GuHCl. Fluorescence studies showed changes in the microenvironment of the protein. The increase in the activity of this intermediate state was due to conformational changes accompanied by increased flexibility at the active site.

Secreted PLA2 group X orchestrates innate and adaptive immune responses to inhaled allergen

Phospholipase A2 (PLA2) enzymes regulate the formation of eicosanoids and lysophospholipids that contribute to allergic airway inflammation. Secreted PLA2 group X (sPLA2-X) was recently found to be increased in the airways of asthmatics and is highly expressed in airway epithelial cells and macrophages. In the current study, we show that allergen exposure increases sPLA2-X in humans and in mice, and that global deletion of Pla2g10 results in a marked reduction in airway hyperresponsiveness (AHR), eosinophil and T cell trafficking to the airways, airway occlusion, generation of type-2 cytokines by antigen-stimulated leukocytes, and antigen-specific immunoglobulins. Further, we found that Pla2g10-/- mice had reduced IL-33 levels in BALF, fewer type-2 innate lymphoid cells (ILC2s) in the lung, less IL-33-induced IL-13 expression in mast cells, and a marked reduction in both the number of newly recruited macrophages and the M2 polarization of these macrophages in the lung. These results indicate that sPLA2-X serves as a central regulator of both innate and adaptive immune response to proteolytic allergen.

Effect of cosolvents on the structural stability of endoglucanase from Aspergillus aculeatus.

The effects of cosolvents such as sucrose, glycerol, and sorbitol on endoglucanase have been studied by activity, circular dichroic spectroscopy, fluorescence, and apparent thermal transition temperature measurements. The endoglucanase activity increased by 4-fold at 40% cosolvent concentration under optimum conditions. The endoglucanase lost 50% of its activity when exposed to 90 degrees C for more than 30 min (1 h). In the presence of cosolvents, it maintained its original activity and native conformation as indicated by far UV-CD at 70 degrees C. The app T(m) increased from a control value of 57 degrees C to a value of 66 degrees C in the presence of 40% sucrose. The partial specific volume of endoglucanase was 0.723 mL/g in sodium acetate buffer. The preferential interaction parameters were negative in all cosolvents, and the maximum hydration of the protein was observed in 40% glycerol where the preferential interaction parameter was -0.126 g/g. The thermal stability of endoglucanase increased in the presence of cosolvents.

In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A2

ABSTRACT We have previously shown that secreted phospholipases A2 (sPLA2s) from animal venoms inhibit the in vitro development of Plasmodium falciparum, the agent of malaria. In addition, the inflammatory-type human group IIA (hGIIA) sPLA2 circulates at high levels in the serum of malaria patients. However, the role of the different human sPLA2s in host defense against P. falciparum has not been investigated. We show here that 4 out of 10 human sPLA2s, namely, hGX, hGIIF, hGIII, and hGV, exhibit potent in vitro anti-Plasmodium properties with half-maximal inhibitory concentrations (IC50s) of 2.9 ± 2.4, 10.7 ± 2.1, 16.5 ± 9.7, and 94.2 ± 41.9 nM, respectively. Other human sPLA2s, including hGIIA, are inactive. The inhibition is dependent on sPLA2 catalytic activity and primarily due to hydrolysis of plasma lipoproteins from the parasite culture. Accordingly, purified lipoproteins that have been prehydrolyzed by hGX, hGIIF, hGIII, and hGV are more toxic to P. falciparum than native lipoproteins. However, the total enzymatic activities of human sPLA2s on purified lipoproteins or plasma did not reflect their inhibitory activities on P. falciparum. For instance, hGIIF is 9-fold more toxic than hGV but releases a lower quantity of nonesterified fatty acids (NEFAs). Lipidomic analyses of released NEFAs from lipoproteins demonstrate that sPLA2s with anti-Plasmodium properties are those that release polyunsaturated fatty acids (PUFAs), with hGIIF being the most selective enzyme. NEFAs purified from lipoproteins hydrolyzed by hGIIF were more potent at inhibiting P. falciparum than those from hGV, and PUFA-enriched liposomes hydrolyzed by sPLA2s were highly toxic, demonstrating the critical role of PUFAs. The selectivity of sPLA2s toward low- and high-density (LDL and HDL, respectively) lipoproteins and their ability to directly attack parasitized erythrocytes further explain their anti-Plasmodium activity. Together, our findings indicate that 4 human sPLA2s are active against P. falciparum in vitro and pave the way to future investigations on their in vivo contribution in malaria pathophysiology.

Influence of Ethylenediaminetetraacetic Acid (EDTA) on the Structural Stability of Endoglucanase from Aspergillus aculeatus

The effect of the chelating agent ethylenediaminetetraacetic acid (EDTA) on the structure and function of endoglucanase is studied. In the presence of 2 mM EDTA, endoglucanase showed an enhanced enzymatic activity of 1.5-fold compared to control. No further change in activity was observed with increase in the concentration of EDTA to 5 mM. The K(m) values for control and in the presence of EDTA are 0.060 and 0.044%, respectively, and K(cat) was 1.9 min(-1) in the presence of EDTA. The kinetic parameters indicated a decrease in the K(m) with an increase in the K(cat). Far-ultraviolet circular dichroism (far-UV-CD) results showed a 20% decrease in ellipticity values at 217 nm in the presence of EDTA compared to native enzyme. The apparent T(m) shifted from a control value of 57 ± 1 to 76 ± 1 °C in the presence of EDTA (5 mM). The above results suggested that the enhanced activity in the presence of EDTA is due to an increase in the K(cat) and flexible conformation of the enzyme. The stability of endoglucanase increased in the presence of EDTA.

Study of the Role of Cytosolic Phospholipase A2 Alpha in Eicosanoid Generation and Thymocyte Maturation in the Thymus

The thymus is a primary lymphoid organ, home of maturation and selection of thymocytes for generation of functional T-cells. Multiple factors are involved throughout the different stages of the maturation process to tightly regulate T-cell production. The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses. In this study, we determined the profile of expression of the eicosanoids present in the mouse thymus at different stages of thymocyte development. As the group IVA cytosolic phospholipase A2 (cPLA2α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA2α deficient mice to our investigations. We found that a vast array of eicosanoids is expressed in the thymus, which expression is substantially modulated through thymocyte development. The cPLA2α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA2α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA2α inhibitor pyrrophenone did not impact thymocyte maturation. This study provides information on the eicosanoid repertoire present during thymocyte development and suggests that thymocyte maturation can occur independently of cPLA2α.