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1.
FASEB J ; 38(8): e23619, 2024 Apr 30.
Article in English | MEDLINE | ID: mdl-38661031

ABSTRACT

Exosomes, which are small membrane-encapsulated particles derived from all cell types, are emerging as important mechanisms for intercellular communication. In addition, exosomes are currently envisioned as potential carriers for the delivery of drugs to target tissues. The natural population of exosomes is very variable due to the limited amount of cargo components present in these small vesicles. Consequently, common components of exosomes may play a role in their function. We have proposed that membrane phospholipids could be a common denominator in the effect of exosomes on cellular functions. In this regard, we have previously shown that liposomes made of phosphatidylcholine (PC) or phosphatidylserine (PS) induced a robust alteration of macrophage (Mϕ) gene expression. We herewith report that these two phospholipids modulate gene expression in Mϕs by different mechanisms. PS alters cellular responses by the interaction with surface receptors, particularly CD36. In contrast, PC is captured by a receptor-independent process and likely triggers an activity within endocytic vesicles. Despite this difference in the capture mechanisms, both lipids mounted similar gene expression responses. This investigation suggests that multiple mechanisms mediated by membrane phospholipids could be participating in the alteration of cellular functions by exosomes.


Subject(s)
Exosomes , Macrophages , Phosphatidylserines , Macrophages/metabolism , Animals , Mice , Phosphatidylserines/metabolism , Exosomes/metabolism , Phosphatidylcholines/metabolism , Inflammation/metabolism , Phospholipids/metabolism , Mice, Inbred C57BL , CD36 Antigens/metabolism , CD36 Antigens/genetics , Liposomes
2.
Membranes (Basel) ; 13(2)2023 Jan 21.
Article in English | MEDLINE | ID: mdl-36837644

ABSTRACT

Phospholipids are the major components of cellular membranes and cell-derived vesicles such as exosomes. They are also key components of artificial lipid nanoparticles, allowing the encapsulation and transport of various biological or chemical cargos. Both artificial and natural vesicles could be captured by cells delivering important information that could modulate cellular functions. However, the potential contribution of phospholipids within vesicles altering cellular physiology has been largely underestimated. Here, we showed that macrophages exposed to liposomes made exclusively with palmitoyl oleoyl phosphatidylcholine (POPC) in vivo resulted in a dramatic alteration of the transcriptome profile. Differential gene expression analysis indicated that the exposure to POPC liposomes resulted in a change in the expression of 1598 genes. Moreover, 146 genes were upregulated, and 69 genes were downregulated by incubation with POPC liposomes in contrast to palmitoyl oleoyl phosphatidylserine (POPS) exposure. Signaling pathway impact analysis revealed that 24 signaling pathways were significantly modulated after exposure to POPC liposomes, including the activation of the NF-κB pathway. Indeed, the expression of several cytokines (TNF-α, IL-6, and IL-10) and chemokines (Cxcl1 and Cxcl2) were increased. These observations were validated by the exposure of macrophages to POPC liposomes in culture conditions. In addition, the proteomic analysis of peritoneal cells exposed to POPC liposomes performed by mass spectrometry revealed that the expression of 107 proteins was downregulated after POPC exposure, whereas the expression of 12 proteins was significantly upregulated by this treatment, including seven proteins involved in the neutrophil degranulation pathway. This observation was confirmed by flow cytometry analysis showing the rapid recruitment of neutrophils into the peritoneal cavity after POPC exposure. Overall, these findings demonstrate that the presence of phospholipids within artificial and natural vesicles could be responsible for changes in the function of target cells.

3.
Infect Immun ; 89(4)2021 03 17.
Article in English | MEDLINE | ID: mdl-33431705

ABSTRACT

Sepsis is a life-threatening condition that arises from a poorly regulated inflammatory response to pathogenic organisms. Current treatments are limited to antibiotics, fluid resuscitation, and other supportive therapies. New targets for monitoring disease progression and therapeutic interventions are therefore critically needed. We previously reported that lipocalin-2 (Lcn2), a bacteriostatic mediator with potent proapoptotic activities, was robustly induced in sepsis. Other studies showed that Lcn2 was a predictor of mortality in septic patients. However, how Lcn2 is regulated during sepsis is poorly understood. We evaluated how IkBζ, an inducer of Lcn2, was regulated in sepsis using both the cecal ligation and puncture (CLP) and endotoxemia (lipopolysaccharide [LPS]) animal models. We show that Nfkbiz, the gene encoding IkBζ, was rapidly stimulated but, unlike Lcn2, whose expression persists during sepsis, mRNA levels of Nfkbiz decline to near basal levels several hours after its induction. In contrast, we observed that IkBζ expression remained highly elevated in septic animals following CLP but not LPS, indicating the occurrence of a CLP-specific mechanism that extends IkBζ half-life. By using an inhibitor of IkBζ, we determined that the expression of Lcn2 was largely controlled by IkBζ. Altogether, these data indicate that the high IkBζ expression in tissues likely contributes to the elevated expression of Lcn2 in sepsis. Since IkBζ is also capable of promoting or repressing other inflammatory genes, it might exert a central role in sepsis.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Disease Susceptibility , I-kappa B Proteins/metabolism , Sepsis/etiology , Sepsis/metabolism , Shock, Septic/etiology , Shock, Septic/metabolism , Animals , Animals, Outbred Strains , Disease Models, Animal , Lipocalin-2/genetics , Lipocalin-2/metabolism , Lipopolysaccharides/adverse effects , Macrophages/immunology , Macrophages/metabolism , Mice , Sepsis/pathology , Shock, Septic/pathology
4.
Am J Physiol Regul Integr Comp Physiol ; 317(1): R160-R168, 2019 07 01.
Article in English | MEDLINE | ID: mdl-31091156

ABSTRACT

Sepsis is a major clinical challenge, with therapy limited to supportive interventions. Therefore, the search for novel remedial approaches is of great importance. We addressed whether hyperbaric oxygen therapy (HBOT) could improve the outcome of sepsis using an acute experimental mouse model. Sepsis was induced in male CD-1 mice by cecal ligation and puncture (CLP) tailored to result in 80-90% mortality within 72 h of the insult. After CLP, mice were randomized into two groups receiving HBOT or not at different times after the initial insult or subjected to multiple HBOT treatments. HBOT conditions were 98% oxygen pressurized to 2.4 atmospheres for 1 h. HBOT within 1 h after CLP resulted in 52% survival in comparison with mice that did not receive the treatment (13% survival). Multiple HBOT at 1 and 6 h or 1, 6, and 21 h displayed an increase in survival of >50%, but they were not significantly different from a single treatment after 1 h of CLP. Treatments at 6 or 21 h after CLP, excluding the 1 h of treatment, did not show any protective effect. Early HBO treatment did not modify bacterial counts after CLP, but it was associated with decreased expression of TNF-α, IL-6, and IL-10 expression in the liver within 3 h after CLP. The decrease of cytokine expression was reproduced in cultured macrophages after exposure to HBOT. Early HBOT could be of benefit in the treatment of sepsis, and the protective mechanism may be related to a reduction in the systemic inflammatory response.


Subject(s)
Disease Models, Animal , Hyperbaric Oxygenation , Sepsis/therapy , Animals , Cecum/injuries , Cytokines/genetics , Cytokines/metabolism , Gene Expression Regulation , Ligation , Lipopolysaccharides/toxicity , Macrophages/metabolism , Male , Mice , Mitochondria/metabolism , Oxygen Consumption , Punctures
5.
Shock ; 52(6): 604-611, 2019 12.
Article in English | MEDLINE | ID: mdl-30601409

ABSTRACT

The omentum is a large mesenchymal fibro-fatty tissue with remarkable healing capability. It is also rich in immune cells, including macrophages and lymphocytes, within particular structures named milky spots. Clinical observations indicate a high incidence of peritonitis after the removal of the omentum suggesting that it may play a role in sepsis. To test this possibility, male CD-1 mice underwent simultaneous omentectomy and cecal ligation and puncture (CLP), omentectomy-sham operation and CLP alone, and mortality was documented within 72 h post the insults. A significant increase in mortality was observed in mice subjected to omentectomy and CLP in comparison with CLP alone. Mortality was correlated with an increase in cytokine gene expression within the lung after omentectomy and CLP as opposed to CLP alone. However, no differences in bacterial load were observed within the peritoneum or blood between groups. To test the long-term effect of omentectomy, mice were subjected to omentum removal or sham operation, allowed to recover from surgery for 14 or 28 days, and then both were subjected to CLP. In these cases, no differences in mortality were observed between the groups suggesting that the lack of omentum triggers a compensatory mechanism. Finally, omentectomy and sham operation altered the composition of peritoneal immune cells with the disappearance of F4/80 macrophages and the appearance of a new population of F4/80 macrophages within 1 or 14 days post-surgery. The F4/80 positive cells reappeared after 28 days following the procedures. All of these observations suggest that the omentum plays an early role in the outcome from sepsis.


Subject(s)
Macrophages, Peritoneal/metabolism , Omentum/metabolism , Sepsis/metabolism , Animals , Disease Models, Animal , Macrophages, Peritoneal/pathology , Male , Mice , Mice, Inbred ICR , Omentum/pathology , Sepsis/pathology
6.
FASEB J ; 33(2): 2995-3009, 2019 02.
Article in English | MEDLINE | ID: mdl-30325674

ABSTRACT

Extracellular vesicles (ECVs) are heterogeneous membrane-enclosed structures containing proteins, nucleic acids, and lipids that participate in intercellular communication by transferring their contents to recipient cells. Although most of the attention has been directed at the biologic effect of proteins and microRNA, the contribution of phospholipids present in ECVs on cellular activation has not been extensively addressed. We investigated the biologic effect of phosphatidylserine (PS) and phosphatidylcholine (PC), 2 phospholipids highly abundant in ECVs. A transcriptomic analysis revealed that ∼4700 genes were specifically modified by exposing peritoneal macrophages to PS or PC liposomes in vivo. Among them, the expression of several chemokines and cytokines was highly upregulated by PS liposome treatment, translating into a massive neutrophil infiltration of the peritoneum capable of neutralizing a septic polymicrobial insult. Both the l and d stereoisomers of PS induced the same response, suggesting that the effect was related to the negative charge of the phospholipid head. We concluded that an increase in the internal negative charge of the cell triggers a signaling cascade activating an innate immune response capable of controlling infection.-Cauvi, D. M., Hawisher, D., Dores-Silva, P. R., Lizardo, R. E., De Maio, A. Macrophage reprogramming by negatively charged membrane phospholipids controls infection.


Subject(s)
Cellular Reprogramming , Coinfection/prevention & control , Extracellular Vesicles/drug effects , Macrophages, Peritoneal/drug effects , Phosphatidylcholines/pharmacology , Phosphatidylserines/pharmacology , Sepsis/prevention & control , Animals , Cells, Cultured , Coinfection/immunology , Coinfection/metabolism , Coinfection/microbiology , Extracellular Vesicles/immunology , Extracellular Vesicles/metabolism , Female , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/metabolism , Male , Mice , Mice, Inbred ICR , Sepsis/immunology , Sepsis/metabolism , Sepsis/microbiology
7.
Cell Stress Chaperones ; 23(5): 943-953, 2018 09.
Article in English | MEDLINE | ID: mdl-29796787

ABSTRACT

Extracellular vesicles (ECV) reflect physiological or pathological conditions, emerging as potential biomarkers for disease. They can be obtained from a variety of body fluids, particularly urine that is an ideal source because it can be obtained in great quantities, recurrently and with minimal intervention. However, the characterization of urine ECV is challenging because the preparation is usually contaminated with soluble proteins, such as uromodulin (UMOD) or Tamm-Horsfall glycoprotein that forms large extracellular filaments co-sedimenting with ECV. We developed a method to obtain human urine ECV free of UMOD by the addition of ZnSO4 prior to vesicle isolation by differential centrifugation. Treatment with ZnSO4 did not affect the size and concentration of the vesicle preparation and preserved the storage of the samples at low temperatures. We did not observe a variation in the number of vesicles isolated during different times of the day or different days between different donors. The glycoprotein pattern of urine ECV was characterized by binding to concanavalin A (Con A) and mass spectroscopy. Several markers were found, including dipeptidyl peptidase IV (CD26), vacuolar protein sorting factor 4A (VPS4A) and dipeptidase 1 (DPEP1), and galectin 3 binding protein (G3-BP). The levels of VPS4A and DPEP1 were similar in ECV preparations obtained from several donors of both sexes. Con A binding pattern and monosaccharide composition were also comparable between subjects. In summary, our method for the isolation of highly pure ECV derived from human urine is likely to help in the use of these vesicles as potential biomarkers.


Subject(s)
Extracellular Vesicles/chemistry , Urine/cytology , Adult , Biomarkers/urine , Centrifugation , Exosomes/chemistry , Female , Glycoproteins/urine , Humans , Male , Uromodulin/urine
8.
Infect Immun ; 85(12)2017 12.
Article in English | MEDLINE | ID: mdl-28947644

ABSTRACT

Sepsis remains a major health problem at the levels of mortality, morbidity, and economic burden to the health care system, a condition that is aggravated by the development of secondary conditions such as septic shock and multiple-organ failure. Our current understanding of the etiology of human sepsis has advanced, at least in part, due to the use of experimental animal models, particularly the model of cecum ligation and puncture (CLP). Antibiotic treatment has been commonly used in this model to closely mirror the treatment of human septic patients. However, whether their use may obscure the elucidation of the cellular and molecular mechanisms involved in the septic response is questionable. The objective of the present study was to determine the effect of antibiotic treatment in the outcome of a fulminant model of CLP. Various dosing strategies were used for the administration of imipenem, which has broad-spectrum coverage of enteric bacteria. No statistically significant differences in the survival of mice were observed between the different antibiotic dosing strategies and no treatment, suggesting that live bacteria may not be the only factor inducing septic shock. To further investigate this hypothesis, mice were challenged with sterilized or unsterilized cecal contents. We found that exposure of mice to sterilized cecal contents also resulted in a high mortality rate. Therefore, it is possible that bacterial debris, apart from bacterial proliferation, triggers a septic response and contributes to mortality in this model, suggesting that additional factors are involved in the development of septic shock.


Subject(s)
Anti-Bacterial Agents/administration & dosage , Imipenem/administration & dosage , Sepsis/drug therapy , Animals , Disease Models, Animal , Mice , Survival Analysis , Treatment Outcome
9.
Innate Immun ; 22(3): 141-51, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26951856

ABSTRACT

Macrophages (Mφs) play a critical role in the defense against pathogens, orchestrating the inflammatory response during injury and maintaining tissue homeostasis. During these processes, macrophages encounter a variety of environmental conditions that are likely to change their gene expression pattern, which modulates their function. In this study, we found that murine Mφs displayed two different subpopulations characterized by differences in morphologies, expression of surface markers and phagocytic capacity under non-stimulated conditions. These two subpopulations could be recapitulated by changes in the culture conditions. Thus, Mφs grown in suspension in the presence of serum were highly phagocytic, whereas subtraction of serum resulted in rapid attachment and reduced phagocytic activity. The difference in phagocytosis between these subpopulations was correlated with the expression levels of FcγR. These two cell subpopulations also differed in their responses to LPS and the expression of surface markers, including CD14, CD86, scavenger receptor A1, TLR4 and low-density lipoprotein receptor. Moreover, we found that the lipid/cholesterol content in the culture medium mediated the differences between these two cell subpopulations. Thus, we described a mechanism that modulates Mφ function depending on the exposure to lipids within their surrounding microenvironment.


Subject(s)
Cellular Microenvironment , Lipids/immunology , Macrophages/physiology , Animals , B7-2 Antigen/metabolism , Cell Differentiation , Cell Line , Lipopolysaccharide Receptors/metabolism , Lipopolysaccharides/immunology , Mice , Mice, Inbred Strains , Phagocytosis , Receptors, IgG/metabolism , Receptors, Scavenger/metabolism , Toll-Like Receptor 4/metabolism
10.
Innate Immun ; 19(1): 30-41, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22751446

ABSTRACT

Scavenger receptor A (Sra), also known as macrophage scavenger receptor 1 (Msr1), is a surface glycoprotein preferentially present in macrophages that plays a primary role in innate immunity. Previous studies have shown that Sra is a modifier gene for the response to bacterial LPS in mice at the level of IL-10 production, in particular. In the present study, we found that Sra(-/-) mice are more resistant to septic shock induced by cecal ligation and puncture than wild-type C57BL/6 J (B6) mice. In addition, Sra(-/-) mice displayed initial elevated high density lipoprotein (HDL) circulating levels. Naïve peritoneal macrophages (PMs) were isolated from Sra(-/-) mice to understand the possible protective mechanism. Incubation of these cells with LPS was found to modulate TLR4 signaling, leading to a reduction in IL-10 and IL-6 mRNA levels, but not TNF-α expression, at low concentrations of LPS in comparison with PMs isolated from B6 mice. No differences were found in LPS binding between PMs derived from Sra(-/-) or B6 mice. The lack of Sra binding to LPS was confirmed after transfection of Chinese hamster ovary (CHO) cells with the Sra gene. The contribution of Sra to the outcome of sepsis may be a combination of changes in TLR4 signaling pathway and elevated levels of HDL in circulation, but also LPS toxicity.


Subject(s)
Cytokines/metabolism , Macrophages, Peritoneal/immunology , Scavenger Receptors, Class A/metabolism , Shock, Septic/immunology , Toll-Like Receptor 4/metabolism , Animals , CHO Cells , Cricetinae , Lipopolysaccharides/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Scavenger Receptors, Class A/genetics , Shock, Septic/genetics , Signal Transduction/genetics , Signal Transduction/immunology
11.
J Biol Chem ; 287(24): 19804-15, 2012 Jun 08.
Article in English | MEDLINE | ID: mdl-22518839

ABSTRACT

Sepsis is a major health problem in the United States with high incidence and elevated patient care cost. Using an animal model of sepsis, cecum ligation, and puncture, we observed that mice became rapidly hypothermic reaching a threshold temperature of 28 °C within 5-10 h after initiation of the insult, resulting in a reliable predictor of mortality, which occurred within 30-72 h of the initial procedure. We also observed that the inflammatory gene expression in lung and liver developed early within 1-2 h of the insult, reaching maximum levels at 6 h, followed by a decline, approaching basal conditions within 20 h. This decrease in inflammatory gene expression at 20 h after cecal ligation and puncture was not due to resolution of the insult but rather was an immune dysfunction stage that was demonstrated by the inability of the animal to respond to a secondary external inflammatory stimulus. Removal of the injury source, ligated cecum, within 6 h of the initial insult resulted in increased survival, but not after 20 h of cecal ligation and puncture. We concluded that the therapeutic window for resolving sepsis is early after the initial insult and coincides with a stage of hyperinflammation that is followed by a condition of innate immune dysfunction in which reversion of the outcome is no longer possible.


Subject(s)
Gene Expression Regulation/immunology , Immunity, Innate , Sepsis/immunology , Sepsis/therapy , Animals , Humans , Inflammation/immunology , Inflammation/pathology , Male , Mice , Sepsis/economics , Sepsis/epidemiology , Sepsis/pathology , Time Factors , United States/epidemiology
12.
J Surg Res ; 174(2): 344-51, 2012 May 15.
Article in English | MEDLINE | ID: mdl-21474146

ABSTRACT

BACKGROUND: Sepsis is a major health problem in the United States that affects more than three-quarters of a million people every year. Previous studies have shown that scavenger receptor A (Sra), also known as macrophage scavenger receptor 1 (Msr1), is a modifier of interleukin 10 (IL-10) expression after injection of bacterial lipopolysaccharide (LPS). Therefore, we investigated the response to sepsis in Sra knock out mice. MATERIALS AND METHODS: C57BL/6J (B6) (n = 88) and Sra (-/-) mice (n = 88) were subjected to cecal ligation and puncture (CLP) using 18G or 16G needles, sham operation, or non-operated controls. At the end, mice were autopsied for the determination of abnormalities after the procedure. Cytokine gene expression was examined in lung and liver samples by quantitative RT-PCR (qRT-PCR), and circulating cholesterol levels were also measured. RESULTS: Sra (-/-) mice displayed an enlargement of the gallbladder after CLP that was not detected in sham or non-operated mice or in B6 mice (wild-type) after CLP. The enlarged gallbladder resembles a condition of acute acalculous cholecystitis observed in humans. Sra (-/-) mice presented high cholesterol levels in circulation as opposed to wild type B6 mice. Moreover, Sra (-/-) mice exhibited a reduction in IL-10 mRNA levels in lungs compared to wild-type B6 mice after CLP. CONCLUSIONS: The development of acute acalculous cholecystitis may be the combination of pre-existing conditions, such as hypercholesterolemia associated with a defect in Sra (Msr1) and a robust inflammation induced by sepsis.


Subject(s)
Acalculous Cholecystitis/etiology , Scavenger Receptors, Class A/genetics , Sepsis/complications , Acalculous Cholecystitis/metabolism , Animals , Cecum/surgery , Cholesterol/blood , Disease Models, Animal , Interleukin-10/metabolism , Ligation , Lung/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Sepsis/genetics , Sepsis/metabolism
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