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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.

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.

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.

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.

BackgroundOlder aged adults and those with pre-existing conditions are at highest risk for severe COVID-19 associated outcomes.MethodsUsing a large dataset of genome-wide RNA-seq profiles derived from human dermal fibroblasts (GSE113957) we investigated whether age affects the expression of pattern recognition receptor (PRR) genes and ACE2, the receptor for SARS-CoV-2.ResultsExtremes of age are associated with increased expression of selected PRR genes, ACE2 and four genes that encode proteins that have been shown to interact with SAR2-CoV-2 proteins.ConclusionsAssessment of PRR expression might provide a strategy for stratifying the risk of severe COVID-19 disease at both the individual and population levels.

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.

Heat shock proteins (HSPs) are ubiquitous polypeptides expressed in all living organisms that participate in several basic cellular processes, including protein folding, from which their denomination as molecular chaperones originated. There are several HSPs, including HSPA5, also known as 78-kDa glucose-regulated protein (GRP78) or binding immunoglobulin protein (BIP) that is an ER resident involved in the folding of polypeptides during their translocation into this compartment prior to the transition to the Golgi network. HSPA5 is detected on the surface of cells or secreted into the extracellular environment. Surface HSPA5 has been proposed to have various roles, such as receptor-mediated signal transduction, a co-receptor for soluble ligands, as well as a participant in tumor survival, proliferation, and resistance. Recently, surface HSPA5 has been reported to be a potential receptor of some viruses, including the novel SARS-CoV-2. In spite of these observations, the association of HSPA5 within the plasma membrane is still unclear. To gain information about this process, we studied the interaction of HSPA5 with liposomes made of different phospholipids. We found that HSPA5 has a high affinity for negatively charged phospholipids, such as palmitoyl-oleoyl phosphoserine (POPS) and cardiolipin (CL). The N-terminal and C-terminal domains of HSPA5 were independently capable of interacting with negatively charged phospholipids, but to a lesser extent than the full-length protein, suggesting that both domains are required for the maximum insertion into membranes. Interestingly, we found that the interaction of HSPA5 with negatively charged liposomes promotes an oligomerization process via intermolecular disulfide bonds in which the N-terminus end of the protein plays a critical role.

Urbanization in low-income countries represents an important inflection point in the epidemiology of disease, with rural populations experiencing high rates of chronic and recurrent infections and urban populations displaying a profile of noncommunicable diseases. To investigate if urbanization alters the expression of genes encoding mitochondrial proteins, we queried gene microarray data from rural and urban populations living in Morocco (GSE17065). The R Bioconductor packages edgeR and limma were used to identify genes with different expression. The experimental design was modeled upon location and sex. Nuclear genes encoding mitochondrial proteins were identified from the MitoCarta2.0 database. Of the 1158 genes listed in the MitoCarta2.0 database, 847 genes (73%) were available for analysis in the Moroccan dataset. The urban-rural comparison with the greatest environmental differences showed that 76.5% of the MitoCarta2.0 genes were differentially expressed, with 97% of the genes having an increased expression in the urban area. Enrichment analysis revealed 367 significantly enriched pathways (adjusted p value

Once thought of as an inert fatty tissue present only to provide insulation for the peritoneal cavity, the omentum is currently recognized as a vibrant immunologic organ with a complex structure uniquely suited for defense against pathogens and injury. The omentum is a source of resident inflammatory and stem cells available to participate in the local control of infection, wound healing, and tissue regeneration. It is intimately connected with the systemic vasculature and communicates with the central nervous system and the hypothalamic pituitary adrenal axis. Furthermore, the omentum has the ability to transit the peritoneal cavity and sequester areas of inflammation and injury. It contains functional, immunologic units commonly referred to as milky spots that contribute to the organs immune response. These milky spots are complex nodules consisting of macrophages and interspersed lymphocytes, which are gateways for the infiltration of inflammatory cells into the peritoneal cavity in response to infection and injury. The omentum contains far greater complexity than is currently conceptualized in clinical practice and investigations directed at unlocking its beneficial potential may reveal new mechanisms underlying its vital functions and the secondary impact of omentectomy for the staging and treatment of a variety of diseases.

The Hsp70 family of heat shock proteins plays a critical function in maintaining cellular homeostasis within various subcellular compartments. The human mitochondrial Hsp70 (HSPA9) has been associated with cellular death, senescence, cancer and neurodegenerative diseases, which is the rational for the name mortalin. It is well documented that mortalin, such as other Hsp70s, is prone to self-aggregation, which is related to mitochondria biogenesis failure. Here, we investigated the assembly, structure and function of thermic aggregates/oligomers of recombinant human mortalin and Hsp70-1A (HSPA1A). Summarily, both Hsp70 thermic aggregates have characteristics of supramolecular assemblies. They display characteristic organized structures and partial ATPase activity, despite their nanometric size. Indeed, we observed that the interaction of these aggregates/oligomers with liposomes is similar to monomeric Hsp70s and, finally, they were non-toxic over neuroblastoma cells. These findings revealed that high molecular mass oligomers of mortalin and Hsp70-1A preserved some of the fundamental functions of these proteins.

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.

Increasing evidence shows that heat shock proteins (hsp) escape the cytosol gaining access to the extracellular environment, acting as signaling agents. Since the majority of these proteins lack the information necessary for their export via the classical secretory pathway, attention has been focused on alternative releasing mechanisms. Crossing the plasma membrane is a major obstacle to the secretion of a cytosolic protein into the extracellular milieu. Several mechanisms have been proposed, including direct interaction with the plasma membrane or their release within extracellular vesicles (ECV). HSPB1 (Hsp27), which belongs to the small hsp family, was detected within the membrane of ECV released from stressed HepG2 cells. To further investigate this finding, we studied the interaction of HSPB1 with lipid membranes using liposomes. We found that HSPB1 interacted with liposomes made of palmitoyl oleoyl phosphatidylserine (POPS), palmitoyl oleoyl phosphatidylcholine (POPC), and palmitoyl oleoyl phosphatidylglycerol (POPG), with different characteristics. Another member of the small hsp family, HSPB5 (αB-crystallin), has also been detected within ECV released from HeLa cells transfected with this gene. This protein was found to interact with liposomes as well, but differently than HSPB1. To address the regions interacting with the membrane, proteoliposomes were digested with proteinase K and the protected domains within the liposomes were identified by mass spectroscopy. We observed that large parts of HSPB1 and HSPB5 were embedded within the liposomes, particularly the alpha-crystallin domain. These observations suggest that the interaction with lipid membranes may be part of the mechanisms of export of these proteins.

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.

BackgroundNon-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.MethodsWith 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.ResultsDeclining 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.ConclusionsUrbanization 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.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to dementia caused by advanced neuronal dysfunction and death. The most significant symptoms of AD are observed at late stages of the disease when interventions are most likely too late to ameliorate the condition. Currently, the predominant theory for AD is the "amyloid hypothesis," which states that abnormally increased levels of amyloid β (Aβ) peptides result in the production of a variety of aggregates that are neurotoxic. The specific mechanisms for Aβ peptide-induced cytotoxicity have not yet been completely elucidated. However, since the majority of Aβ is released into the extracellular milieu, it is reasonable to assume that toxicity begins outside the cells and makes its way inside where it disrupts the basic cellular process resulting in cell death. There is increasing evidence that hsp, particularly Hsp70, are exported into the extracellular milieu by an active export mechanism independent of cell death. Therefore, both Aβ peptides and Hsp70 may coexist in a common environment during pathological conditions. We observed that Hsp70 affected the Aβ assembling process in vitro preventing oligomer formation. Moreover, the presence of Hsp70 reduced the Aβ peptide-induced toxicity of cultured neurons (N2A cells). These results suggest a potential mechanism for the reduction of the detrimental effects of Aβ peptides in AD.

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.

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.

PurposeThe 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 methodsWe used immunoblotting, PCR, and quantitative PCR to measure ECRG4 and either inhalation anesthesia preconditioning, lipopolysaccharide injection, or laparotomy to modulate lung inflammation.ResultsImmunoblotting 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-1β, and IL-6 but following inflammatory lung injury, TNF-α, IL-1β, 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.ConclusionsThis 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.