The Role of Serum Albumin and Secretory Phospholipase A2 in Sepsis.

Sepsis is caused by a dysregulated host response to an infection that leads to cascading cell death and eventually organ failure. In this study, the role of inflammatory response serum secretory phospholipase A2 (sPLA2) and albumin in sepsis was investigated by determining the activities of the two proteins in serial serum samples collected on different days from patients with sepsis after enrollment in the permissive underfeeding versus standard enteral feeding protocols in an intensive care unit. Serum sPLA2 and albumin showed an inverse relationship with increasing sPLA2 activity and decreasing albumin membrane-binding activity in patients with evolving complications of sepsis. The activities of sPLA2 and albumin returned to normal values more rapidly in the permissive underfeeding group than in the standard enteral feeding group. The inverse sPLA2-albumin activity relationship suggests a complex interplay between these two proteins and a regulatory mechanism underlying cell membrane phospholipid homeostasis in sepsis. The decreased albumin-membrane binding activity in patients' serum was due to its fatty acid-binding sites occupied by pre-bound fatty acids that might alter albumin's structure, binding capacities, and essential functions. The sPLA2-albumin dual serum assays may be useful in determining whether nutritional intervention effectively supports the more rapid recovery of appropriate immune responses in critically ill patients with sepsis.

Up-regulation of IL-1ß and sPLA2-III in the medial prefrontal cortex contributes to orofacial and somatic hyperalgesia induced by malocclusion via glial-neuron crosstalk.

The medial prefrontal cortex (mPFC) has been identified as a key brain region involved in the modulation of chronic pain. Our recent study demonstrated that unilateral anterior crossbite (UAC) developed the comorbidity model of temporomandibular disorders (TMD) and fibromyalgia syndrome (FMS), which was characterized by both orofacial and somatic hyperalgesia. In the present study, UAC rats exhibited significant changes in gene expression in the mPFC. Enrichment analysis revealed that the significantly involved pathways were cytokines-cytokine receptor interaction and immune response. The expression of group III secretory phospholipase A2 (sPLA2-III) was significantly increased in the mPFC of UAC rats. Silencing sPLA2-III expression in the mPFC blocked the orofacial and somatic hyperalgesia. Immunofluorescence showed that sPLA2-III was mainly localized in neurons. The expression of interleukin-1ß (IL-1ß) in the mPFC significantly increased after UAC. Injection of IL-1ß antibody into the mPFC blocked orofacial and somatic hyperalgesia. IL-1ß was mainly localized in microglia cells. Furthermore, injection of IL-1ß antibody significantly reduced the expression of sPLA2-III. These results indicate that neuroinflammatory cascade responses induced by glial-neuron crosstalk in the mPFC may contribute to the development of TMD and FMS comorbidity, and IL-1ß and sPLA2-III are identified as novel potential therapeutic targets for the treatment of chronic pain in the comorbidity of TMD and FMS.

Extracellular vesicles as a hydrolytic platform of secreted phospholipase A2.

Extracellular vesicles (EVs) represent small vesicles secreted from cells, including exosomes (40-150 nm in diameter), which are released via the multivesicular endosomal pathway, and microvesicles and ectosomes (100-1000 nm), which are produced by plasma membrane budding. Broadly, EVs also include vesicles generated from dying cells, such as apoptotic bodies (5-10 µm), as well as exomeres (< 50 nm), which are very small, non-membranous nanoparticles. EVs play important roles in cell-to-cell signaling in various aspects of cancer, immunity, metabolism, and so on by transferring proteins, microRNAs (miRNAs), and metabolites as cargos from donor cells to recipient cells. Although lipids are one of the major components of EVs, they have long been recognized as merely the "wall" that partitions the lumen of the vesicle from the outside. However, it has recently become obvious that lipid composition of EVs influences their properties and functions, that EVs act as a carrier of a variety of lipid mediators, and that lipid mediators are produced in EV membranes by the hydrolytic action of secreted phospholipase A2s (sPLA2s). In this article, we will make an overview of the roles of lipids in EVs, with a particular focus on sPLA2-driven mobilization of lipid mediators from EVs and its biological significance.

Cardiolipin in myocardial ischaemia-reperfusion injury: From molecular mechanisms to clinical strategies.

Myocardial reperfusion injury occurs when blood flow is restored after ischemia, an essential process to salvage ischemic tissue. However, this phenomenon is intricate, characterized by various harmful effects. Tissue damage in ischemia-reperfusion injury arises from various factors, including the production of reactive oxygen species, the sequestration of proinflammatory immune cells in ischemic tissues, the induction of endoplasmic reticulum stress, and the occurrence of postischemic capillary no-reflow. Secretory phospholipase A2 (sPLA2) plays a crucial role in the eicosanoid pathway by releasing free arachidonic acid from membrane phospholipids' sn-2 position. This liberated arachidonic acid serves as a substrate for various eicosanoid biosynthetic enzymes, including cyclooxygenases, lipoxygenases, and cytochromes P450, ultimately resulting in inflammation and an elevated risk of reperfusion injury. Therefore, the activation of sPLA2 directly correlates with the heightened and accelerated damage observed in myocardial ischemia-reperfusion injury (MIRI). Presently, clinical trials are in progress for medications aimed at sPLA2, presenting promising avenues for intervention. Cardiolipin (CL) plays a crucial role in maintaining mitochondrial function, and its alteration is closely linked to mitochondrial dysfunction observed in MIRI. This paper provides a critical analysis of CL modifications concerning mitochondrial dysfunction in MIRI, along with its associated molecular mechanisms. Additionally, it delves into various pharmacological approaches to prevent or alleviate MIRI, whether by directly targeting mitochondrial CL or through indirect means.

PKC-α is involved in the signaling of phagocytosis induced by two snake venom secretory PLA2S in macrophages.

Phagocytosis, an essential process for host defense, requires the coordination of a variety of signaling reactions. MT-II, an enzymatically inactive Lys49 phospholipase A2 (PLA2) homolog, and MT-III, a catalytically-active Asp49 PLA2, are known to activate phagocytosis in macrophages. In this study, the signaling pathways mediating phagocytosis, focusing on protein kinases, were investigated. Macrophages from male Swiss mice peritoneum were obtained 96 h after intraperitoneal thioglycolate injection. Phagocytosis was evaluated using non-opsonized zymosan particles in the presence or absence of specific inhibitors, as well as PKC and PKC-α localization by confocal microscopy. Moreover, protein kinase C (PKC) activity was assessed by γP32 ATP in macrophages stimulated by both PLA2s. Data showed that both sPLA2s increased phagocytosis. Cytochalasin D, staurosporine/H7, wortmannin, and herbimycin, inhibitors of actin polymerization, PKC, phosphoinositide 3-kinase (PI3K), and protein tyrosine kinase (PTK), respectively, significantly reduced phagocytosis induced by both PLA2s. PKC activity was increased in macrophages stimulated by both PLA2s. Actin polymerization and talin were evidenced by immunofluorescence and talin was recruited 5 min after both PLA2s stimulation. PKC and PKC-α localization within the cell were increased after 60 min of MT-II and MT-III stimulation. These data suggest that the effect of both PLA2s depends on actin cytoskeleton rearrangements and the activation of PKC, PI3K, and PTK signaling events required for phagocytosis.

[Identification and expression pattern analysis of a secretory phospholipase A2 gene in Bombyx mori].

Phospholipase A2 (PLA2) is widely distributed in animals, plants, and microorganisms, and it plays an important role in many physiological activities. In a previous study, we have identified a secretory PLA2 in Bombyx mori (BmsPLA2-1-1). In this study, we further identified four new sPLA2 genes (BmsPLA2-1-2, BmsPLA2-2, BmsPLA2-3, and BmsPLA2-4) in B. mori genome. All four genes exhibits the characteristic features of sPLA2, including the sPLA2 domain, metal binding sites, and highly conserved catalytic domain. This study completed the cloning, in vitro expression, and expression pattern analysis of the BmsPLA2-4 gene in B. mori. The full length of BmsPLA2-4 is 585 bp, and the recombinant protein obtained through prokaryotic expression has an estimated size of 25 kDa. qRT-PCR analysis revealed that the expression level of BmsPLA2-4 reached its peak on the first day of the fifth instar larval stage. Tissue expression profiling analysis showed that BmsPLA2-4 had the highest expression level in the midgut, followed by the epidermis and fat body. Western blotting analysis results were consistent with those of qRT-PCR. Furthermore, after infecting fifth instar 1-day-old larvae with Escherichia coli and Staphylococcus aureus, the expression level of the BmsPLA2-4 gene significantly increased in 24 h. The findings of this study provides a theoretical basis and valuable experimental data for future related research.

Role of nitric oxide and signaling pathways modulating the stimulatory effect of snake venom secretory PLA2S on non-opsonized zymosan phagocytosis by macrophages.

The phagocytic activity of macrophages activated with MT-II, a Lys-49 PLA2 homolog, and MT-III, an Asp-49 PLA2, from Bothrops asper snake venom, was investigated in this study using a pharmacological approach. Stimulating thioglycollate-elicited macrophages with both venom components enhanced their ability to phagocytose non-opsonized zymosan particles. MT-II and MT-III-induced phagocytosis was drastically inhibited by pretreating cells with L-NAME, aminoguanidine or L-NIL, cNOS or iNOS inhibitors, or with ODQ (sGC inhibitor) or Rp-cGMPS (PKG inhibitor). These results indicate that the NO/sGC/GMP/PKG pathway plays an essential role in the ß-glucan-mediated phagocytosis induced in macrophages by these venom-secretory PLA2s.

Hawthorn leaf flavonoids alleviate the deterioration of atherosclerosis by inhibiting SCAP-SREBP2-LDLR pathway through sPLA2-â ¡A signaling in macrophages in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Hawthorn leaves are a combination of the dried leaves of the Rosaceae plants, i.e., Crataegus pinnatifida Bge. or Crataegus pinnatifida Bge. var. major N. E. Br., is primarily cultivated in East Asia, North America, and Europe. hawthorn leaf flavonoids (HLF) are the main part of extraction. The HLF have demonstrated potential in preventing hypertension, inflammation, hyperlipidemia, and atherosclerosis. However, the potential pharmacological mechanism behind its anti-atherosclerotic effect has yet to be explored. AIM OF THE STUDY: The in vivo and in vitro effects of HLF on lipid-mediated foam cell formation were investigated, with a specific focus on the levels of secreted phospholipase A2 type IIA (sPLA2-II A) in macrophage cells. MATERIALS AND METHODS: The primary constituents of HLF were analyzed using ultra-high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. In vivo, HLF, at concentrations of 5 mg/kg, 20 mg/kg, and 40 mg/kg, were administered to apolipoprotein E knockout mice (ApoE-/-) fed by high-fat diet (HFD) for 16 weeks. Aorta and serum samples were collected to identify lesion areas and lipids through mass spectrometry analysis to dissect the pathological process. RAW264.7 cells were incubated with oxidized low-density lipoprotein (ox-LDL) alone, or ox-LDL combined with different doses of HLF (100, 50, and 25 µg/ml), or ox-LDL plus 24-h sPLA2-IIA inhibitors, for cell biology analysis. Lipids and inflammatory cytokines were detected using biochemical analyzers and ELISA, while plaque size and collagen content of plaque were assessed by HE and the Masson staining of the aorta. The lipid deposition in macrophages was observed by Oil Red O staining. The expression of sPLA2-IIA and SCAP-SREBP2-LDLR was determined by RT-qPCR and Western blot analysis. RESULTS: The chemical profile of HLF was studied using UPLC-Q-TOF-MS/MS, allowing the tentative identification of 20 compounds, comprising 1 phenolic acid, 9 flavonols and 10 flavones, including isovitexin, vitexin-4″-O-glucoside, quercetin-3-O-robibioside, rutin, vitexin-2″-O-rhamnoside, quercetin, etc. HLF decreased total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and non-high-density lipoprotein cholesterol (non-HDL-C) levels in ApoE-/- mice (P < 0.05), reduced ox-LDL uptake, inhibited level of inflammatory factors, such as IL-6, IL-8, TNF-α, and IL-1ꞵ (P < 0.001), and alleviated aortic plaques with a thicker fibrous cap. HLF effectively attenuated foam cell formation in ox-LDL-treated RAW264.7 macrophages, and reduced levels of intracellular TC, free cholesterol (FC), cholesteryl ester (CE), IL-6, TNF-α, and IL-1ß (P < 0.001). In both in vivo and in vitro experiments, HLF significantly downregulated the expression of sPLA2-IIA, SCAP, SREBP2, LDLR, HMGCR, and LOX-1 (P < 0.05). Furthermore, sPLA2-IIA inhibitor effectively mitigated inflammatory release in RAW264.7 macrophages and regulated SCAP-SREBP2-LDLR signaling pathway by inhibiting sPLA2-IIA secretion (P < 0.05). CONCLUSION: HLF exerted a protective effect against atherosclerosis through inhibiting sPLA2-IIA to diminish SCAP-SREBP2-LDLR signaling pathway, to reduce LDL uptake caused foam cell formation, and to slow down the progression of atherosclerosis in mice.

Screening of insect immune suppressors using a recombinant phospholipase A2 of a lepidopteran insect.

Phospholipase A2 (PLA2 ) catalyzes phospholipids at the sn-2 position to release free fatty acids, including arachidonic acid (AA) or its precursor. The free AA is then oxygenated into different eicosanoids, which mediate the diverse physiological processes in insects. Any inhibition of the PLA2 catalysis would give rise to serious malfunctioning in insect growth and development. An onion moth, Acrolepiopsis sapporensis, encodes four different PLA2 genes (As-PLA2 A-As-PLA2 D), in which As-PLA2 A is dominantly expressed at all developmental stages and in different larval tissues. RNA interference of the As-PLA2 A expression significantly reduced the PLA2 activity of A. sapporensis, which suffered from immunosuppression. A recombinant As-PLA2 A protein was purified from a bacterial expression system, which exhibited a typical Michaelis-Menten kinetics and hence susceptible to a specific inhibitor to sPLA2 and dithiothreitol. A total of 19 bacterial metabolites derived from Xenorhabdus and Photorhabdus were screened against the recombinant As-PLA2 A. Five potent metabolites were highly inhibitory and followed a competitive enzyme inhibition. These five inhibitors suppressed the immune responses of A. sapporensis by inhibiting hemocyte-spreading behavior and phenoloxidase activity. However, an addition of AA could significantly rescue the immunosuppression induced by the selected inhibitors. These studies suggest that the recombinant As-PLA2 A protein can be applied for high-throughput screening of insect immunosuppressive compounds.

High purity of human secreted phospholipase A2 group IIE in Pichia pastoris using basal salts medium comparison with YPD medium.

Secreted phospholipase A2s (sPLA2s) are a group of enzymes with 6-8 disulfide bonds that participate in numerous physiological processes by catalyzing the hydrolysis of phospholipids at the sn-2 position. Due to their high content of disulfide bonds and hydrolytic activity toward cell membranes, obtaining the protein of sPLA2s in the soluble and active form is challenging, which hampers their functional study. In this study, one member of recombinant human sPLA2s, tag-free group IIE (GIIE), was expressed in Pichia pastoris. The protein GIIE was purified from the crude culture supernatant by a two-step chromatography procedure, a combination of cation exchange and size-exclusion chromatography. In the shake flask fermentation, Protein of GIIE with higher purity was successfully obtained, using basal salts medium (BSM) instead of YPD medium. In the large-scale fermentation, each liter of BSM produced a final yield of 1.2 mg pure protein GIIE. This protocol will facilitate further research of GIIE and provide references for the production of other sPLA2 members.

Lysophospholipase D from Thermocrispum limits psoriatic inflammation by hydrolyzing epidermal lysoplasmalogen produced by group IIF secreted phospholipase A2.

Epidermal lipids play important roles in skin homeostasis and diseases. Psoriasis is an inflammatory disease characterized by keratinocyte hyperproliferation and Th17 immune responses. We previously reported that ethanolamine-type lysoplasmalogen (P-LPE), preferentially produced by group IIF secreted PLA2 (sPLA2-IIF/PLA2G2F) that is expressed in the suprabasal epidermis, promotes epidermal hyperplasia in psoriatic inflammation. Herein, we show that forcible degradation of epidermal P-LPE by topical application of recombinant lysophospholipase D (LyPls-PLD) from Thermocrispum, a lysoplasmalogen-specific hydrolase, attenuated epidermal hyperplasia and inflammation in imiquimod-induced and K5.Stat3C-transgenic mouse psoriasis models. In humans, P-LPE levels were elevated in the tape-stripped stratum corneum of patients with psoriasis. Moreover, in primary cultured human epidermal keratinocytes, aberrant cell proliferation and activation by psoriatic cytokines were sPLA2-IIF/P-LPE-dependent and were suppressed by the addition of LyPls-PLD with a decrease in P-LPE. These findings confirm that the sPLA2-IIF/P-LPE axis in the epidermis indeed regulates psoriasis, that P-LPE is a lipid biomarker that predicts the severity of psoriasis, and that pharmacological removal of this bioactive lipid is useful to prevent the disease. Thus, our study may lead to the development of drug discovery and diagnostic techniques based on this pathway.

Lipid clearance and amyloid formation by serum amyloid A: exploring the links between beneficial and pathologic actions of an enigmatic protein.

Serum amyloid A (SAA) is named after a life-threatening disease, yet this small evolutionarily conserved protein must have played a vital role in host defense. Most circulating SAA binds plasma lipoproteins and modulates their metabolism. However, this hardly justifies the rapid and dramatic SAA upregulation in inflammation, which is concomitant with upregulation of secretory phospholipase A2 (sPLA2). We proposed that these proteins synergistically clear cell membrane debris from the sites of injury. The present study uses biochemical and biophysical approaches to further explore the beneficial function of SAA and its potential links to amyloid formation. We show that murine and human SAA1 are powerful detergents that solubilize diverse lipids, including mammalian biomembranes, converting them into lipoprotein-size nanoparticles. These nanoparticles provide ligands for cell receptors, such as scavenger receptor CD36 or heparin/heparan sulfate, act as substrates of sPLA2, and sequester toxic products of sPLA2. Together, these functions enable SAA to rapidly clear unprotected lipids. SAA can also adsorb, without remodeling, to lipoprotein-size nanoparticles such as exosomal liposomes, which are proxies for lipoproteins. SAA in complexes with zwitterionic phospholipids stabilizes α-helices, while SAA in complexes containing anionic lipids or micelle-forming sPLA2 products forms metastable ß-sheet-rich species that readily aggregate to form amyloid. Consequently, the synergy between SAA and sPLA2 extends from the beneficial lipid clearance to the pathologic amyloid formation. Furthermore, we show that lipid composition alters SAA conformation and thereby can influence the metabolic fate of SAA-lipid complexes, including their proamyloidogenic and proatherogenic binding to heparan sulfate.

Early life surfactant protein-D levels in bronchoalveolar lavage fluids of extremely preterm neonates.

Surfactant protein-D (SP-D) is a hydrophilic protein with multiple crucial anti-inflammatory and immunological functions. It might play a role in the development and course of pulmonary infections, acute respiratory distress syndrome, and other respiratory disorders. Only few small neonatal studies have investigated SP-D: we aimed to investigate the links between this protein, measured in the first hours of life in extremely preterm neonates, and clinical outcomes, as well its relationship with pulmonary secretory phospholipase A2 (sPLA2). Bronchoalveolar lavage fluids were obtained within the first 3 h of life. SP-D and sPLA2 were measured with ELISA and radioactive method, respectively; epithelial lining fluid concentrations were estimated with urea ratio. Clinical data were prospectively collected. One hundred extremely preterm neonates were nonconsecutively studied. SP-D was significantly raised with increasing gestational age (24-26 wk: 68 [0-1,694], 27 or 28 wk: 286 [0-1,328], 29 or 30 wk: 1,401 [405-2,429] ng/mL, overall P = 0.03). SP-D was significantly higher in cases with clinical chorioamnionitis with fetal involvement (1,138 [68-3,336]) than in those without clinical chorioamnionitis with fetal involvement (0 [0-900] ng/mL, P < 0.001). SP-D was lower in infants with bronchopulmonary dysplasia (BPD) (251 [0-1,550 ng/mL]) compared with those without bronchopulmonary dysplasia (BPD) or who died before its diagnosis (977 [124-5,534 ng/mL], P = 0.05) and this was also significant upon multivariate analysis [odds ration (OR): 0.997 (0.994-0.999), P = 0.024], particularly in neonates between 27- and 28-wk gestation. SP-D significantly correlated with the duration of hospital stay (ρ = -0.283, P = 0.002), invasive ventilation (ρ = -0.544, P = 0.001), and total sPLA2 activity (ρ = 0.528, P = 0.008). These findings help understanding the role of SP-D early in life and support further investigation about the role of SP-D in developing BPD.NEW & NOTEWORTHY Surfactant protein-D increases with gestational age and is inversely associated with BPD development. These results have been obtained in the first hours of life of extremely preterm neonates with optimal perinatal care.

Skeletal muscle fiber hypercontraction induced by Bothrops asper myotoxic phospholipases A2 ex vivo does not involve a direct action on the contractile apparatus.

Myonecrosis is a frequent clinical manifestation of envenomings by Viperidae snakes, mainly caused by the toxic actions of secreted phospholipase A2 (sPLA2) enzymes and sPLA2-like homologs on skeletal muscle fibers. A hallmark of the necrotic process induced by these myotoxins is the rapid appearance of hypercontracted muscle fibers, attributed to the massive influx of Ca2+ resulting from cell membrane damage. However, the possibility of myotoxins having, in addition, a direct effect on the contractile machinery of skeletal muscle fibers when internalized has not been investigated. This question is here addressed by using an ex vivo model of single-skinned muscle fibers, which lack membranes but retain an intact contractile apparatus. Rabbit psoas skinned fibers were exposed to two types of myotoxins of Bothrops asper venom: Mt-I, a catalytically active Asp49 sPLA2 enzyme, and Mt-II, a Lys49 sPLA2-like protein devoid of phospholipolytic activity. Neither of these myotoxins affected the main parameters of force development in striated muscle sarcomeres of the skinned fibers. Moreover, no microscopical alterations were evidenced after their exposure to Mt-I or Mt-II. In contrast to the lack of effects on skinned muscle fibers, both myotoxins induced a strong hypercontraction in myotubes differentiated from murine C2C12 myoblasts, with drastic morphological alterations that reproduce those described in myonecrotic tissue in vivo. As neither Mt-I nor Mt-II showed direct effects upon the contractile apparatus of skinned fibers, it is concluded that the mechanism of hypercontraction triggered by both myotoxins in patients involves indirect effects, i.e., the large cytosolic Ca2+ increase after sarcolemma permeabilization.

The Roles of sPLA2s in Skin Homeostasis and Disease.

Among the phospholipase A2 (PLA2) family, the secreted PLA2 (sPLA2) family in mammals contains 11 members that exhibit unique tissue or cellular distributions and enzymatic properties. Current studies using knockout and/or transgenic mice for a nearly full set of sPLA2s, in combination with comprehensive lipidomics, have revealed the diverse pathophysiological roles of sPLA2s in various biological events. Individual sPLA2s exert specific functions within tissue microenvironments, likely through the hydrolysis of extracellular phospholipids. Lipids are an essential biological component for skin homeostasis, and disturbance of lipid metabolism by deletion or overexpression of lipid-metabolizing enzymes or lipid-sensing receptors often leads to skin abnormalities that are easily visible on the outside. Over the past decades, our studies using knockout and transgenic mice for various sPLA2s have uncovered several new aspects of these enzymes as modulators of skin homeostasis and disease. This article summarizes the roles of several sPLA2s in skin pathophysiology, providing additional insight into the research fields of sPLA2s, lipids, and skin biology.

Multiple calcium signaling genes play a role in the circadian period of Neurospora crassa.

The Ca2+ signaling genes cpe-1, plc-1, ncs-1, splA2, camk-1, camk-2, camk-3, camk-4, cmd, and cnb-1 are necessary for a normal circadian period length in Neurospora crassa. In addition, the Q10 values ranged between 0.8 and 1.2 for the single mutants lacking cpe-1, splA2, camk-1, camk-2, camk-3, camk-4, and cnb-1, suggesting that the circadian clock exhibits standard temperature compensation. However, the Q10 value for the ∆plc-1 mutant was 1.41 at 25 and 30 °C, 1.53 and 1.40 for the ∆ncs-1 mutant at 20 and 25 °C, and at 20 and 30 °C, respectively, suggesting a partial loss of temperature compensation in these two mutants. Moreover, expression of frq, a regulator of the circadian period, and the blue light receptor wc-1, were increased >2-fold in the Δplc-1, ∆plc-1; ∆cpe-1, and the ∆plc-1; ∆splA2 mutants at 20 °C. The frq mRNA level was increased >2-fold in the Δncs-1 mutant compared to the ras-1bd strain at 20 °C. Therefore, multiple Ca2+ signaling genes regulate the circadian period, by influencing expression of the frq and wc-1 genes that are critical for maintaining the normal circadian period length in N. crassa.

Secreted phospholipase A2 XIIA triggers a mitochondrial damage-induced senescence in chronic obstructive pulmonary disease fibroblasts.

RATIONALE: Lung fibroblast senescence is involved in the pathophysiology of chronic obstructive pulmonary disease (COPD). However, the mechanisms underlining this phenomenon are still poorly understood. Secreted phospholipases (sPLA2, a subclass of phospholipases) are secreted by senescent cells and can in turn induce senescence. However, their role in fibroblasts senescence in COPD is unknown. OBJECTIVES: The aim of this study was to analyze the role of sPLA2 in pulmonary fibroblast senescence. METHODS: Fibroblasts were isolated from patients with COPD and control subjects, and senescence markers and inflammatory profile was analyzed. sPLA2 levels were quantified in serum of COPD and controls. MAIN RESULTS: In comparison with non-smokers and smoker controls, senescent lung COPD fibroblasts exhibited a higher mRNA and protein expression of the sPLA2 isoform XIIA and of syndecan 4 (one of its receptors). sPLA2 XIIA induced in turn senescence of non-senescent pulmonary fibroblasts via a pathway involving consecutively syndecan 4, activation of MAPK and p-serine 727 STAT-3, increased mitochondrial ROS production, and activation of AMPK/p53. This pathway was associated with a specific inflammatory secretome (IL-10, IL-12 and TNFα), globally suggesting occurrence of a mitochondrial damage-induced senescence. COPD fibroblasts were more susceptible to this sPLA2 XIIA effect than cells from controls subjects. sPLA2 XIIA levels were significantly higher in serum from COPD patients as compared to controls. CONCLUSION: sPLA2 XIIA is involved in senescence in COPD and could be a potential target to dampen this process.

Cellular Distribution of Secreted Phospholipase A2 in Lungs of IPF Patients and Its Inhibition in Bleomycin-Induced Pulmonary Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease with a very poor prognosis as it has a 2.5 to 5 years mean survival after proper diagnosis. Even nintedanib and pirfenidone cannot halt the progression, though they slow the progression of IPF. Hence, there is a need to understand the novel pathophysiology. Phospholipase A2 (PLA2) could be the ideal candidate to study in IPF, as they have a role in both inflammation and fibrosis. In the present study, we have shown the expression profile of various secretory Phospholipase A2 (PLA2) isoforms by analyzing publicly available transcriptome data of single cells from the lungs of healthy individuals and IPF patients. Among 11 members of sPLA2, PLA2G2A is found to be increased in the fibroblasts and mesothelial cells while PLA2G5 is found to be increased in the fibroblasts of IPF patients. We identified a subset of fibroblasts expressing high PLA2G2A with moderate expression of PLA2G5 and which are specific to IPF only; we named it as PLA2G2A+ IPF fibroblast. Pathway analysis revealed that these PLA2G2A+ IPF fibroblast have upregulation of both inflammatory and fibrosis-related pathways like the TGF-ß signaling pathway, IL-17 signaling, the arachidonic acid metabolism pathway and ECM-receptor interaction. In addition to this, we found elevated levels of sPLA2-IIA in plasma samples of IPF patients in our cohort. PLA2G3, PLA2G10 and PLA2G12B are found in to be increased in certain epithelial cells of IPF patients. Thus, these findings indicate that these five isoforms have a disease-dominant role along with innate immune roles as these isoforms are found predominantly in structural cells of IPF patients. Further, we have targeted sPLA2 in mice model of bleomycin-induced lung fibrosis by pBPB, a known sPLA2 inhibitor. pBPB treatment attenuated lung fibrosis induced by bleomycin along with a reduction in TGF-ß and deposition of extracellular matrix in lung. Thus, these findings indicate that these sPLA2 isoforms especially PLA2G2A may serve as a therapeutic target in lung fibrosis.

Modulation of immunity by the secreted phospholipase A2 family.

Among the phospholipase A2 (PLA2 ) superfamily, which typically catalyzes the sn-2 hydrolysis of phospholipids to yield fatty acids and lysophospholipids, the secreted PLA2 (sPLA2 ) family contains 11 isoforms in mammals. Individual sPLA2 s have unique enzymatic specificity toward fatty acids and polar heads of phospholipid substrates and display distinct tissue/cellular distributions, suggesting their distinct physiological functions. Recent studies using knockout and/or transgenic mice for a full set of sPLA2 s have revealed their roles in modulation of immunity and related disorders. Application of mass spectrometric lipidomics to these mice has enabled to identify target substrates and products of individual sPLA2 s in given tissue microenvironments. sPLA2 s hydrolyze not only phospholipids in the plasma membrane of activated, damaged or dying mammalian cells, but also extracellular phospholipids such as those in extracellular vesicles, microbe membranes, lipoproteins, surfactants, and dietary phospholipids, thereby exacerbating or ameliorating various diseases. The actions of sPLA2 s are dependent on, or independent of, the generation of fatty acid- or lysophospholipid-derived lipid mediators according to the pathophysiological contexts. In this review, we make an overview of our current understanding of the roles of individual sPLA2 s in various immune responses and associated diseases.

Secretory Phospholipase A2 and Interleukin-6 Levels as Predictive Markers of the Severity and Outcome of Patients with COVID-19 Infections.

Coronavirus disease (COVID-19) has become a global pandemic. COVID-19 patients need immediate diagnosis and rehabilitation, which makes it urgent to identify new protein markers for a prognosis of the severity and outcome of the disease. The aim of this study was to analyze the levels of interleukin-6 (IL-6) and secretory phospholipase (sPLA2) in the blood of patients regarding the severity and outcome of COVID-19 infection. The study included clinical and biochemical data obtained from 158 patients with COVID-19 treated at St. Petersburg City Hospital No. 40. A detailed clinical blood test was performed on all patients, as well as an assessment of IL-6, sPLA2, aspartate aminotransferase (AST), total protein, albumin, lactate dehydrogenase (LDH), APTT, fibrinogen, procalcitonin, D-dimer, C-reactive protein (CRB), ferritin, and glomerular filtration rate (GFR) levels. It was found that the levels of PLA2, IL-6, APTV, AST, CRP, LDH, IL-6, D-dimer, and ferritin, as well as the number of neutrophils, significantly increased in patients with mild to severe COVID-19 infections. The levels of IL-6 were positively correlated with APTT; the levels of AST, LDH, CRP, D-dimer, and ferritin; and the number of neutrophils. The increase in the level of sPLA2 was positively correlated with the levels of CRP, LDH, D-dimer, and ferritin, the number of neutrophils, and APTT, and negatively correlated with the levels of GFR and lymphocytes. High levels of IL-6 and PLA2 significantly increase the risk of a severe course by 13.7 and 2.24 times, and increase the risk of death from COVID-19 infection by 14.82 and 5.32 times, respectively. We have shown that the blood levels of sPLA2 and IL-6 increase in cases which eventually result in death and when patients are transferred to the ICU (as the severity of COVID-19 infection increases), showing that IL-6 and sPLA2 can be considered as early predictors of aggravation of COVID-19 infections.