Membrane phospholipids activate the inflammatory response in macrophages by various mechanisms.

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.

Regulation of the Nfkbiz Gene and Its Protein Product IkBζ in Animal Models of Sepsis and Endotoxic Shock.

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.

Macrophage reprogramming by negatively charged membrane phospholipids controls infection.

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.

Early hyperbaric oxygen therapy improves survival in a model of severe sepsis.

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.

Urbanization in Sub-Saharan Africa: Declining Rates of Chronic and Recurrent Infection and Their Possible Role in the Origins of Non-communicable Diseases.

BACKGROUND: Non-communicable diseases (NCDs), such as atherosclerosis and cancers, are a leading cause of death worldwide. An important, yet poorly explained epidemiological feature of NCDs is their low incidence in under developed areas of low-income countries and rising rates in urban areas. METHODS: With the goal of better understanding how urbanization increases the incidence of NCDs, we provide an overview of the urbanization process in sub-Saharan Africa, discuss gene expression differences between rural and urban populations, and review the current NCD determinant model. We conclude by identifying research priorities. RESULTS: Declining rates of chronic and recurrent infection are the hallmark of urbanization in sub-Saharan Africa. Gene profiling studies show urbanization results in complex molecular changes, with almost one-third of the peripheral blood leukocyte transcriptome altered. The current NCD determinant model could be improved by including a possible effect from declining rates of infection and expanding the spectrum of diseases that increase with urbanization. CONCLUSIONS: Urbanization in sub-Saharan Africa provides a unique opportunity to investigate the mechanism by which the environment influences disease epidemiology. Research priorities include: (1) studies to define the relationship between infection and risk factors for NCDs, (2) explaining the observed differences in the inflammatory response between rural and urban populations, and (3) identification of animal models that simulate the biological changes that occurs with urbanization. A better understanding of the biological changes that occur with urbanization could lead to new prevention and treatment strategies for some of the most common surgical diseases in high-income countries.

Why Antibiotic Treatment Is Not Enough for Sepsis Resolution: an Evaluation in an Experimental Animal Model.

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.

Pulmonary preconditioning, injury, and inflammation modulate expression of the candidate tumor suppressor gene ECRG4 in lung.

PURPOSE: The human c2orf40 gene encodes a candidate tumor suppressor called Esophageal Cancer-Related Gene-4 (ECRG4) that is a cytokine-like epigenetically-regulated protein that is characteristically downregulated in cancer, injury, inflammation, and infection. Here, we asked whether ECRG4 gene expression is detectable in lung epithelial cells and if its expression changes with inflammation, infection, and/or protective preconditioning. MATERIALS AND METHODS: We used immunoblotting, PCR, and quantitative PCR to measure ECRG4 and either inhalation anesthesia preconditioning, lipopolysaccharide injection, or laparotomy to modulate lung inflammation. RESULTS: Immunoblotting establishes the presence of the full-length 14 kDa ECRG4 peptide in mouse lung. Immunohistochemistry localizes ECRG4 to type l alveolar epithelial cells. Basal ECRG4 mRNA is greater than TNF-α, IL-1ß, and IL-6 but following inflammatory lung injury, TNF-α, IL-1ß, IL-6, and IL-10 are upregulated while ECRG4 gene expression is decreased. Similar findings are observed after an intravenous administration of lipopolysaccharide. In contrast, lung preconditioning with isoflurane anesthesia increases lung ECRG4 gene expression. Over-expression of ECRG4 in human lung epithelial cells in vitro decreases cell proliferation implying that a loss of ECRG4 in vivo would be permissive to cell growth. CONCLUSIONS: This study supports the hypothesis that ECRG4 acts as a sentinel growth inhibitor in lung alveolar epithelial cells. Its downregulation by injury, infection, and inflammation and upregulation by preconditioning supports a role for ECRG4 in regulating the alveolar epithelium response to injury and inflammation. By extension, the findings support a functional consequence to its inhibition by promoter hypermethylation (i.e. lung cancer) and suggest potential benefits to its upregulation.

Period of irreversible therapeutic intervention during sepsis correlates with phase of innate immune dysfunction.

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.

Phosphatidylcholine Liposomes Reprogram Macrophages toward an Inflammatory Phenotype.

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.

The Small GTPase Rab7 Regulates Release of Mitochondria in Extracellular Vesicles in Response to Lysosomal Dysfunction.

Mitochondrial quality control is critical for cardiac homeostasis as these organelles are responsible for generating most of the energy needed to sustain contraction. Dysfunctional mitochondria are normally degraded via intracellular degradation pathways that converge on the lysosome. Here, we identified an alternative mechanism to eliminate mitochondria when lysosomal function is compromised. We show that lysosomal inhibition leads to increased secretion of mitochondria in large extracellular vesicles (EVs). The EVs are produced in multivesicular bodies, and their release is independent of autophagy. Deletion of the small GTPase Rab7 in cells or adult mouse heart leads to increased secretion of EVs containing ubiquitinated cargos, including intact mitochondria. The secreted EVs are captured by macrophages without activating inflammation. Hearts from aged mice or Danon disease patients have increased levels of secreted EVs containing mitochondria indicating activation of vesicular release during cardiac pathophysiology. Overall, these findings establish that mitochondria are eliminated in large EVs through the endosomal pathway when lysosomal degradation is inhibited.

Human HSP70-escort protein 1 (hHep1) interacts with negatively charged lipid bilayers and cell membranes.

Human Hsp70-escort protein 1 (hHep1) is a cochaperone that assists in the function and stability of mitochondrial HSPA9. Similar to HSPA9, hHep1 is located outside the mitochondria and can interact with liposomes. In this study, we further investigated the structural and thermodynamic behavior of interactions between hHep1 and negatively charged liposomes, as well as interactions with cellular membranes. Our results showed that hHep1 interacts peripherally with liposomes formed by phosphatidylserine and cardiolipin and remains partially structured, exhibiting similar affinities for both. In addition, after being added to the cell membrane, recombinant hHep1 was incorporated by cells in a dose-dependent manner. Interestingly, the association of HSPA9 with hHep1 improved the incorporation of these proteins into the lipid bilayer. These results demonstrated that hHep1 can interact with lipids also present in the plasma membrane, indicating roles for this cochaperone outside of mitochondria.

Mitochondria are secreted in extracellular vesicles when lysosomal function is impaired.

Mitochondrial quality control is critical for cardiac homeostasis as these organelles are responsible for generating most of the energy needed to sustain contraction. Dysfunctional mitochondria are normally degraded via intracellular degradation pathways that converge on the lysosome. Here, we identified an alternative mechanism to eliminate mitochondria when lysosomal function is compromised. We show that lysosomal inhibition leads to increased secretion of mitochondria in large extracellular vesicles (EVs). The EVs are produced in multivesicular bodies, and their release is independent of autophagy. Deletion of the small GTPase Rab7 in cells or adult mouse heart leads to increased secretion of EVs containing ubiquitinated cargos, including intact mitochondria. The secreted EVs are captured by macrophages without activating inflammation. Hearts from aged mice or Danon disease patients have increased levels of secreted EVs containing mitochondria indicating activation of vesicular release during cardiac pathophysiology. Overall, these findings establish that mitochondria are eliminated in large EVs through the endosomal pathway when lysosomal degradation is inhibited.

Acute acalculous cholecystitis-like phenotype in scavenger receptor A knock-out mice.

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.

Tumor necrosis factor expression is ameliorated after exposure to an acidic environment.

BACKGROUND: It has been well established that laparoscopic surgery presents several clinical benefits, including reduced pain and a shorter hospital stay. These effects have been associated with a decrease in the inflammatory response. Previous studies have demonstrated that reduced inflammation after laparoscopic surgery is the product of carbon dioxide insufflation, which decreases peritoneal pH. The objective of this study was to investigate the cellular and molecular mechanisms responsible for the reduced response after exposure to acidic environments. MATERIALS AND METHODS: A murine macrophage line (J744) was incubated in culture medium at pH 6.0 or pH 7.4 for 3 h at 37°C. Then, cells were stimulated with lipopolysaccharide (LPS) at pH 7.4, the expression of TNF-α (qRT-PCR or enzyme-linked immunosorbent assay (ELISA) and intracellular pH were measured. In addition, CD14 and Toll-like receptor 4 expression and NF-κB nuclear translocation were analyzed. RESULTS: A significant decrease in LPS-induced TNF-α expression levels was observed in cells pre-incubated at pH 6.0 in comparison with cells at neutral pH conditions. This decrease in TNF-α levels was not associated with a reduction in cell surface expression of CD14 and Toll-like receptor 4. Exposure to an extracellular acidic environment resulted in a reduction of IκB phosphorylation and NF-κB nuclear translocation, secondary to a significant drop in cytosolic pH. CONCLUSIONS: These observations provide a potential mechanism for the reduced expression of TNF-α after exposure to low extracellular pH, which may be related to acidification after CO(2) insufflation during laparoscopic surgery. In addition, extracellular acidic pH environments could emerge as an important regulator of macrophage function.

The interaction of heat shock proteins with cellular membranes: a historical perspective.

The interaction of heat shock proteins (HSP) with cellular membranes has been an enigmatic process, initially observed by morphological studies, inferred during the purification of HSP70s, and confirmed after the detection of these proteins on the surface of cancer cells and their insertion into artificial lipid bilayers. Today, the association of several HSP with lipid membranes is well established. However, the mechanisms for membrane insertion have been elusive. There is conclusive evidence indicating that HSP70s have a great selectivity for negatively charged phospholipids, whereas other HSP have a broader spectrum of lipid specificity. HSP70 also oligomerizes upon membrane insertion, forming ion conductance channels. The functional role of HSP70 lipid interactions appears related to membrane stabilization that may play a role during cell membrane biogenesis. They could also play a role as membrane chaperones as well as during endocytosis, microautophagy, and signal transduction. Moreover, HSP membrane association is a key component in the extracellular export of these proteins. The presence of HSP70 on the surface of cancer cells and its interaction with lysosome membranes have been envisioned as potential therapeutic targets. Thus, the biology and function of HSP membrane association are reaching a new level of excitement. This review is an attempt to preserve the recollection of the pioneering contributions of many investigators that have participated in this endeavor.

Heat shock proteins and the biogenesis of cellular membranes.

The successful function of cells is importantly contributed by lipid membranes that are more than a simple physical barrier. The major components of cellular membranes are lipids, in particular glycerophospholipids, that have the capacity to assemble spontaneously into vesicles containing a lipid bilayer after exposure to an aqueous milieu due to their amphiphilic characteristics. The lipid capacity to form vesicles and encapsulate substrates has been proposed as a fundamental event during the biogenesis of cells. However, the stability of small vesicles is compromised during their expansion into larger and more complex particles. Recent observations by (Cornell et al. Proc Natl Acad Sci U S A 116:17239-17244, 2019) have shown that the insertion of amino acids into rudimentary vesicles could play a stabilizing role that was critical to the formation of early cells. Fatty acids were likely substituted by glycerophospholipids and amino acids replaced by polypeptides during the evolution of protocells. Thus, archaic peptides displaying lipid-binding and membrane-penetrating capacities could have played a key function in the development of current cells. In this regard, heat shock proteins (HSP), particularly the Hsp70 (HSPA) and small HSP (HSPB) families, could have portrayed that role. Indeed, bacterial DnaK is closest in sequence to the earliest members of the Hsp70 family and inserts into lipid membranes spontaneously. Moreover, extensive studies by the Vigh group have shown that, certainly, Hsp70s stabilize membranes. Thus, the ability of ancestral HSP70s and small HSPs to associate with lipids and stabilize membranes could have been a fundamental event in the genesis of cells.

Human heat shock cognate protein (HSC70/HSPA8) interacts with negatively charged phospholipids by a different mechanism than other HSP70s and brings HSP90 into membranes.

Heat shock proteins (HSP) are critical elements for the preservation of cellular homeostasis by participating in an array of biological processes. In addition, HSP play an important role in cellular protection from various environmental stresses. HSP are part of a large family of different molecular mass polypeptides, displaying various expression patterns, subcellular localizations, and diversity functions. An unexpected observation was the detection of HSP on the cell surface. Subsequent studies have demonstrated that HSP have the ability to interact and penetrate lipid bilayers by a process initiated by the recognition of phospholipid heads, followed by conformational changes, membrane insertion, and oligomerization. In the present study, we described the interaction of HSPA8 (HSC70), the constitutive cytosolic member of the HSP70 family, with lipid membranes. HSPA8 showed high selectivity for negatively charged phospholipids, such as phosphatidylserine and cardiolipin, and low affinity for phosphatidylcholine. Membrane insertion was mediated by a spontaneous process driven by increases in entropy and diminished by the presence of ADP or ATP. Finally, HSPA8 was capable of driving into the lipid bilayer HSP90 that does not display any lipid biding capacity by itself. This observation suggests that HSPA8 may act as a membrane chaperone.

New insights on human Hsp70-escort protein 1: Chaperone activity, interaction with liposomes, cellular localizations and HSPA's self-assemblies remodeling.

The 70 kDa heat shock proteins (Hsp70) are prone to self-assembly under thermal stress conditions, forming supramolecular assemblies (SMA), what may have detrimental consequences for cellular viability. In mitochondria, the cochaperone Hsp70-escort protein 1 (Hep1) maintains mitochondrial Hsp70 (mtHsp70) in a soluble and functional state, contributing to preserving proteostasis. Here we investigated the interaction between human Hep1 (hHep1) and HSPA9 (human mtHsp70) or HSPA1A (Hsp70-1A) in monomeric and thermic SMA states to unveil further information about the involved mechanisms. hHep1 was capable of blocking the formation of HSPA SMAs under a thermic treatment and stimulated HSPA ATPase activity in both monomeric and preformed SMA. The interaction of hHep1 with both monomeric and SMA HSPAs displayed a stoichiometric ratio close to 1, suggesting that hHep1 has access to most protomers within the SMA. Interestingly, hHep1 remodeled HSPA9 and HSPA1A SMAs into smaller forms. Furthermore, hHep1 was detected in the mitochondria and nucleus of cells transfected with the respective coding DNA and interacted with liposomes resembling mitochondrial membranes. Altogether, these new features reinforce that hHep1 act as a "chaperone for a chaperone", which may play a critical role in cellular proteostasis.