TREML4 receptor regulates inflammation and innate immune cell death during polymicrobial sepsis.

Sepsis is a biphasic disease characterized by an acute inflammatory response, followed by a prolonged immunosuppressive phase. Therapies aimed at controlling inflammation help to reduce the time patients with sepsis spend in intensive care units, but they do not lead to a reduction in overall mortality. Recently, the focus has been on addressing the immunosuppressive phase, often caused by apoptosis of immune cells. However, molecular triggers of these events are not yet known. Using whole-genome CRISPR screening in mice, we identified a triggering receptor expressed on myeloid cells (TREM) family receptor, TREML4, as a key regulator of inflammation and immune cell death in sepsis. Genetic ablation of Treml4 in mice demonstrated that TREML4 regulates calcium homeostasis, the inflammatory cytokine response, myeloperoxidase activation, the endoplasmic reticulum stress response and apoptotic cell death in innate immune cells, leading to an overall increase in survival rate, both during the acute and chronic phases of polymicrobial sepsis.

Vesiclepedia 2024: an extracellular vesicles and extracellular particles repository.

Vesiclepedia (http://www.microvesicles.org) is a free web-based compendium of DNA, RNA, proteins, lipids and metabolites that are detected or associated with extracellular vesicles (EVs) and extracellular particles (EPs). EVs are membranous vesicles that are secreted ubiquitously by cells from all domains of life from archaea to eukaryotes. In addition to EVs, it was reported recently that EPs like exomeres and supermeres are secreted by some mammalian cells. Both EVs and EPs contain proteins, nucleic acids, lipids and metabolites and has been proposed to be implicated in several key biological functions. Vesiclepedia catalogues proteins, DNA, RNA, lipids and metabolites from both published and unpublished studies. Currently, Vesiclepedia contains data obtained from 3533 EV studies, 50 550 RNA entries, 566 911 protein entries, 3839 lipid entries, 192 metabolite and 167 DNA entries. Quantitative data for 62 822 entries from 47 EV studies is available in Vesiclepedia. The datasets available in Vesiclepedia can be downloaded as tab-delimited files or accessible through the FunRich-based Vesiclepedia plugin.

Bovine milk-derived extracellular vesicles enhance doxorubicin sensitivity in triple negative breast cancer cells by targeting metabolism and STAT signalling.

Metastatic triple-negative breast cancer (TNBC) has a low 5-year survival rate of below 30% with systemic chemotherapy being the most widely used treatment. Bovine milk-derived extracellular vesicles (MEVs) have been previously demonstrated to have anti-cancer attributes. In this study, we isolated bovine MEVs from commercial milk and characterised them according to MISEV guidelines. Bovine MEVs sensitised TNBC cells to doxorubicin, resulting in reduced metabolic potential and cell-viability. Label-free quantitative proteomics of cells treated with MEVs and/or doxorubicin suggested that combinatorial treatment depleted various pro-tumorigenic interferon-inducible gene products and proteins with metabolic function, previously identified as therapeutic targets in TNBC. Combinatorial treatment also led to reduced abundance of various STAT proteins and their downstream oncogenic targets with roles in cell-cycle and apoptosis. Taken together, this study highlights the ability of bovine MEVs to sensitise TNBC cells to standard-of-care therapeutic drug doxorubicin, paving the way for novel treatment regimens.

Engineering Extracellular Vesicles for Cancer Therapy.

Extracellular vesicles (EVs) are lipid bilayer containing nanovesicles that have a predominant role in intercellular communication and cargo delivery. EVs have recently been used as a means for drug delivery and have been depicted to elicit no or minimal immune response in vivo. The stability, biocompatibility and manipulatable tumour homing capabilities of these biological vessels make them an attractive target for the packaging and delivery of drugs and molecules to treat various diseases including cancer. The following chapter will summarise current EV engineering techniques for the purpose of delivering putative drugs and therapeutic molecules for the treatment of cancer. The relevance of EV source will be discussed, as well as the specific modifications required to manufacture them into suitable vehicles for molecular drug delivery. Furthermore, methods of EV cargo encapsulation will be evaluated with emphasis on intercellular coordination to allow for the effective emptying of therapeutic contents into target cells. While EVs possess properties making them naturally suitable nanocarriers for drugs and molecules, many challenges with clinical translation of EV-based platforms remain. These issues need to be addressed in order to harness the true potential of the EV-based therapeutic avenue.

Extracellular Vesicles Contain Putative Cancer Biomarkers.

Emerging evidences have implicated extracellular vesicles (EVs), nanoparticles secreted by cells, in regulating cancer progression. Several seminal studies on EVs have added an additional layer to the previously unanswered questions in understanding the complexity of diseases such as cancer. It has been observed that EV content is highly heterogenous and it likely reflects the dynamic state of the parent cell. Hence, these nano-sized vesicles have been proposed as reservoirs of cancer biomarkers for diagnostic and prognostic purposes. Due to their presence in almost all biological fluids, ability to display membrane, and sometimes cytosolic, cargo of its host cell and increase in their number during disease states has supported the potential utility of EVs as an alternative to current methods of cancer diagnosis. The following chapter will discuss the use of cancer cell-derived EVs as a resource of tumor specific biomarkers for the early diagnosis of disease. In addition, EVs could also be used in personalised medicine as a resource of predictive biomarkers to understand a patient's response to therapy. Overall, EVs could be exploited as a source of cancer biomarkers and could aid in treatment and stratification options to improve patient survival and quality of life.

Extracellular Vesicles Regulate Cancer Metastasis.

Metastatic cancer is a complex disease associated with poor prognosis and accounts for the majority of cancer related deaths. To date, many of the molecular mechanisms driving metastatic disease remain elusive and require further investigation for the development of effective treatment strategies. Recent studies have shown that extracellular vesicles (EVs) can be exploited by tumors to assist in cancer cell growth, proliferation, migration, invasion and metastasis. Cancer cells have proven efficient in educating fibroblasts, within their microenvironment, to secrete EVs as communicative vessels for mediating phenotypic changes in recipient cells. Using this vesicular delivery system, cancer cells can establish a new metastatic niche within distant sites, away from the primary tumor, thus favoring cancer progression. These findings demonstrate the availability of a new route for therapeutic intervention in the inhibition of cancer dissemination. Although, several approaches to target cancer cell secretion of EVs are detailed in the literature, there is still no defined way to currently apply them in clinical settings. Hence, further studies are required to unravel the molecular mechanisms of metastasis - governed by the establishment and release of cancer associated EVs.

Extracellular Vesicles in Metabolism and Metabolic Diseases.

As living organisms constantly need energy to maintain and perform cellular functions, metabolism plays a vital role in producing the required energy to execute these processes. Hence, various metabolic pathways are highly regulated and disruption in critical pathways can result in the onset of multiple disorders such as hypertension, diabetes, obesity, and dyslipidaemia. Extracellular vesicles (EVs) are membrane-bound nanosized vesicles that are known to be secreted by various cell types into their respective extracellular environment. EVs have been implicated in cell-to-cell communication via mediating cellular signaling and can functionally impact recipient cells with the transport of bioactive proteins, nucleic acids, lipids and cellular metabolites. Recently, several studies have highlighted the role of EVs in metabolism. Alterations in the plasma derived EV concentration and their cargo in patients with metabolic disorders have been reported by multiple studies, further proposing EVs as a potential source of disease biomarkers. The following chapter will discuss the functional significance of EVs in metabolic diseases and the processes by which EVs act as cellular messengers to reprogram the metabolic machinery in recipient cells.

BCL-2 family protein BOK is a positive regulator of uridine metabolism in mammals.

BCL-2 family proteins regulate the mitochondrial apoptotic pathway. BOK, a multidomain BCL-2 family protein, is generally believed to be an adaptor protein similar to BAK and BAX, regulating the mitochondrial permeability transition during apoptosis. Here we report that BOK is a positive regulator of a key enzyme involved in uridine biosynthesis; namely, uridine monophosphate synthetase (UMPS). Our data suggest that BOK expression enhances UMPS activity, cell proliferation, and chemosensitivity. Genetic deletion of Bok results in chemoresistance to 5-fluorouracil (5-FU) in different cell lines and in mice. Conversely, cancer cells and primary tissues that acquire resistance to 5-FU down-regulate BOK expression. Furthermore, we also provide evidence for a role for BOK in nucleotide metabolism and cell cycle regulation. Our results have implications in developing BOK as a biomarker for 5-FU resistance and have the potential for the development of BOK-mimetics for sensitizing 5-FU-resistant cancers.

Semiquantitative Proteomics Enables Mapping of Murine Neutrophil Dynamics following Lethal Influenza Virus Infection.

Neutrophils are rapidly deployed innate immune cells, and excessive recruitment is causally associated with influenza-induced pathologic conditions. Despite this, the complete set of influenza lethality-associated neutrophil effector proteins is currently unknown. Whether the expression of these proteins is predetermined during bone marrow (BM) neutrophil maturation or further modulated by tissue compartment transitions has also not been comprehensively characterized at a proteome-wide scale. In this study, we used high-resolution mass spectrometry to map how the proteomes of murine neutrophils change comparatively across BM, blood, and the alveolar airspaces to deploy an influenza lethality-associated response. Following lethal influenza infection, mature neutrophils undergo two infection-dependent and one context-independent compartmental transitions. Translation of type I IFN-stimulated genes is first elevated in the BM, preceding the context-independent downregulation of ribosomal proteins observed in blood neutrophils. Following alveolar airspace infiltration, the bronchoalveolar lavage (BAL) neutrophil proteome is further characterized by a limited increase in type I IFN-stimulated and metal-sequestering proteins as well as a decrease in degranulation-associated proteins. An influenza-selective and dose-dependent increase in antiviral and lipid metabolism-associated proteins was also observed in BAL neutrophils, indicative of a modest capacity for pathogen response tuning. Altogether, our study provides new and comprehensive evidence that the BAL neutrophil proteome is shaped by BM neutrophil maturation as well as subsequent compartmental transitions following lethal influenza infection.

The E3 ubiquitin ligase NEDD4 regulates chemoresistance to 5-fluorouracil in colorectal cancer cells by altering JNK signalling.

Colorectal cancer (CRC) is the second leading cause of cancer deaths. Though chemotherapy is the main treatment option for advanced CRC, patients invariably acquire resistance to chemotherapeutic drugs and fail to respond to the therapy. Although understanding the mechanisms regulating chemoresistance has been a focus of intense research to manage this challenge, the pathways governing resistance to drugs are poorly understood. In this study, we provide evidence for the role of ubiquitin ligase NEDD4 in resistance developed against the most commonly used CRC chemotherapeutic drug 5-fluorouracil (5-FU). A marked reduction in NEDD4 protein abundance was observed in a panel of CRC cell lines and patient-derived xenograft samples that were resistant to 5-FU. Knockout of NEDD4 in CRC cells protected them from 5-FU-mediated apoptosis but not oxaliplatin or irinotecan. Furthermore, NEDD4 depletion in CRC cells reduced proliferation, colony-forming abilities and tumour growth in mice. Follow-up biochemical analysis highlighted the inhibition of the JNK signalling pathway in NEDD4-deficient cells. Treatment with the JNK activator hesperidin in NEDD4 knockout cells sensitised the CRC cells against 5-FU. Overall, we show that NEDD4 regulates cell proliferation, colony formation, tumour growth and 5-FU chemoresistance in CRC cells.

Extracellular Vesicles as Drug Targets and Delivery Vehicles for Cancer Therapy.

Extracellular vesicles (EVs) are particles that are released from cells into the extracellular space both under pathological and normal conditions. It is now well established that cancer cells secrete more EVs compared to non-cancerous cells and that, captivatingly, several proteins that are involved in EV biogenesis and secretion are upregulated in various tumours. Recent studies have revealed that EVs facilitate the interaction between cancer cells and their microenvironment and play a substantial role in the growth of tumours. As EVs are involved in several aspects of cancer progression including angiogenesis, organotropism, pre-metastatic niche formation, fostering of metastasis, and chemoresistance, inhibiting the release of EVs from cancer and the surrounding tumour microenvironment cells has been proposed as an ideal strategy to treat cancer and associated paraneoplastic syndromes. Lately, EVs have shown immense benefits in preclinical settings as a novel drug delivery vehicle. This review provides a brief overview of the role of EVs in various hallmarks of cancer, focusing on (i) strategies to treat cancer by therapeutically targeting the release of tumour-derived EVs and (ii) EVs as valuable drug delivery vehicles. Furthermore, we also outline the drawbacks of the existing anti-cancer treatments and the future prospective of EV-based therapeutics.

Absence of pro-survival A1 has no impact on inflammatory cell survival in vivo during acute lung inflammation and peritonitis.

Inflammation is a natural defence mechanism of the body to protect against pathogens. It is induced by immune cells, such as macrophages and neutrophils, which are rapidly recruited to the site of infection, mediating host defence. The processes for eliminating inflammatory cells after pathogen clearance are critical in preventing sustained inflammation, which can instigate diverse pathologies. During chronic inflammation, the excessive and uncontrollable activity of the immune system can cause extensive tissue damage. New therapies aimed at preventing this over-activity of the immune system could have major clinical benefits. Here, we investigated the role of the pro-survival Bcl-2 family member A1 in the survival of inflammatory cells under normal and inflammatory conditions using murine models of lung and peritoneal inflammation. Despite the robust upregulation of A1 protein levels in wild-type cells upon induction of inflammation, the survival of inflammatory cells was not impacted in A1-deficient mice compared to wild-type controls. These findings indicate that A1 does not play a major role in immune cell homoeostasis during inflammation and therefore does not constitute an attractive therapeutic target for such morbidities.

Inflammation and Cell Death of the Innate and Adaptive Immune System during Sepsis.

Sepsis is a life-threatening medical condition that occurs when the host has an uncontrolled or abnormal immune response to overwhelming infection. It is now widely accepted that sepsis occurs in two concurrent phases, which consist of an initial immune activation phase followed by a chronic immunosuppressive phase, leading to immune cell death. Depending on the severity of the disease and the pathogen involved, the hosts immune system may not fully recover, leading to ongoing complications proceeding the initial infection. As such, sepsis remains one of the leading causes of morbidity and mortality world-wide, with treatment options limited to general treatment in intensive care units (ICU). Lack of specific treatments available for sepsis is mostly due to our limited knowledge of the immuno-physiology associated with the disease. This review will provide a comprehensive overview of the mechanisms and cell types involved in eliciting infection-induced immune activation from both the innate and adaptive immune system during sepsis. In addition, the mechanisms leading to immune cell death following hyperactivation of immune cells will be explored. The evaluation and better understanding of the cellular and systemic responses leading to disease onset could eventuate into the development of much needed therapies to combat this unrelenting disease.

Circulating BiP/Grp78 is a novel prognostic marker for sepsis-mediated immune cell death.

Sepsis remains to be a major contributor to mortality in ICUs, and immune suppression caused by immune cell apoptosis determines the overall patient survival. However, diagnosis of sepsis-induced lymphopenia remains problematic with no accurate prognostic techniques or biomarkers for cell death available. Developing reliable prognostic tools for sepsis-mediated cell death is not only important for identifying patients at increased risk of immune suppression but also to monitor treatment progress of currently trialed immunotherapy strategies. We have previously shown an important role for endoplasmic reticulum stress (ER stress) in inducing sepsis-mediated cell death and here report on the identification of a secreted form of the ER chaperone BiP (immunoglobulin binding protein) as a novel circulating prognostic biomarker for immune cell death and ER stress during sepsis. Using biochemical purification and mass spectrometry coupled with an established in vitro sepsis cell death assay, we identified BiP/Grp78 as a factor secreted by lipopolysaccharide-activated macrophages that is capable of inducing cell death in target cells. Quantitative ELISA analysis showed significantly elevated levels of circulating BiP in mice undergoing polymicrobial sepsis, which was absent in Bim-/- mice that are protected from sepsis-induced lymphopenia. Using blood serum from human sepsis patients, we could detect a significant difference in levels of secreted BiP in sepsis patients compared to nonseptic controls, suggesting that secreted circulating BiP could indeed be used as a prognostic marker that is directly correlative to immune cell death during sepsis.

Oral administration of bovine milk-derived extracellular vesicles induces senescence in the primary tumor but accelerates cancer metastasis.

The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.

TLR4: the fall guy in sepsis?

Sepsis and its impact on human health can be traced back to 1000 BC and continues to be a major health burden today. It causes about 11 million deaths world-wide of which, more than a third are due to neonatal sepsis. There is no effective treatment other than fluid resuscitation therapy and antibiotic treatment that leave patients immunosuppressed and vulnerable to nosocomial infections. Added to that, ageing population and the emergence of antibiotic resistant bacteria pose new challenges. Most of the deleterious effects of sepsis are due to the host response to the systemic infection. In the initial phase of infection, hyper activation of the immune system leads to cytokine storm, which could lead to organ failure and this accounts for about 15% of overall deaths. However, the subsequent immune paralysis phase (mostly attributed to apoptotic death of immune cells) accounts for about 85% of all deaths. Past clinical trials (more than 100 in the last 30 years) all targeted the inflammatory phase with little success, predictably, for inflammation is a necessary process to fight infection. In order to identify the regulators of immune cell death during sepsis, we carried out an unbiased, whole genome CRISPR screening in mice and identified Trigger Receptor Expressed in Myeloid-like 4 (Treml4) as the receptor that controls both the inflammatory phase and the immune suppression phase in sepsis (Nedeva et al. (2020) Nature Immunol, doi: 10.1038/s41590-020-0789-z). Characterising the Treml4 gene knockout mice revealed new insights into the relative roles of TLR4 and TREML4 in inducing the inflammatory cytokine storm during sepsis.

ROCK1 but not LIMK1 or PAK2 is a key regulator of apoptotic membrane blebbing and cell disassembly.

Many cell types are known to undergo a series of morphological changes during the progression of apoptosis, leading to their disassembly into smaller membrane-bound vesicles known as apoptotic bodies (ApoBDs). In particular, the formation of circular bulges called membrane blebs on the surface of apoptotic cells is a key morphological step required for a number of cell types to generate ApoBDs. Although apoptotic membrane blebbing is thought to be regulated by kinases including ROCK1, PAK2 and LIMK1, it is unclear whether these kinases exhibit overlapping roles in the disassembly of apoptotic cells. Utilising both pharmacological and CRISPR/Cas9 gene editing based approaches, we identified ROCK1 but not PAK2 or LIMK1 as a key non-redundant positive regulator of apoptotic membrane blebbing as well as ApoBD formation. Functionally, we have established an experimental system to either inhibit or enhance ApoBD formation and demonstrated the importance of apoptotic cell disassembly in the efficient uptake of apoptotic materials by various phagocytes. Unexpectedly, we also noted that ROCK1 could play a role in regulating the onset of secondary necrosis. Together, these data shed light on both the mechanism and function of cell disassembly during apoptosis.

Sepsis: Inflammation Is a Necessary Evil.

Sepsis is one of the leading causes of deaths world-wide and yet there are no therapies available other than ICU treatment. The patient outcome is determined by a complex interplay between the pro and anti-inflammatory responses of the body i.e., a homeostatic balance between these two competing events to be achieved for the patient's recovery. The initial attempts on drug development mainly focused on controlling inflammation, however, without any tangible outcome. This was despite most deaths occurring during the immune paralysis stage of this biphasic disease. Recently, the focus has been shifting to understand immune paralysis (caused by apoptosis and by anti-inflammatory cytokines) to develop therapeutic drugs. In this review we put forth an argument for a proper understanding of the molecular basis of inflammation as well as apoptosis for developing an effective therapy.

DR5 and caspase-8 are dispensable in ER stress-induced apoptosis.

The endoplasmic reticulum (ER) stress response constitutes cellular reactions triggered by a wide variety of stimuli that disturb folding of proteins, often leading to apoptosis. ER stress-induced apoptotic cell death is thought to be an important contributor to many human pathological conditions. The molecular mechanism of this apoptosis process has been highly controversial with both the receptor and the mitochondrial pathways being implicated. Using knockout mouse models and RNAi-mediated gene silencing in cell lines, our group and others had demonstrated the importance of the mitochondrial apoptotic pathway in ER stress-induced cell death, particularly the role of the pro-apoptotic BH3-only BCL-2 family members, BIM and PUMA. However, a recent report suggested a central role for the death receptor, DR5, activated in a ligand-independent manner, and the initiator caspase, caspase-8, in ER stress-induced cell death. This prompted us to re-visit our previous observations and attempt to reproduce the newly published findings. Here we report that the mitochondrial apoptotic pathway, activated by BH3-only proteins, is essential for ER stress-induced cell death and that, in contrast to the previous report, DR5 as well as caspase-8 are not required for this process.

Isolation of Cardiomyocytes and Cardiofibroblasts for Ex Vivo Analysis.

Heart failure (HF) is a common clinical endpoint to several underlying causes including aging, hypertension, stress, and cardiomyopathy. It is characterized by a significant decline in the cardiac output. Cardiomyocytes are terminally differentiated cells and therefore, apoptotic death due to beta adrenergic (ß-AR) signaling contributes to high attrition rate of these cells. Past treatments of HF offer some survival benefit to patients (e.g., the beta blockers), but at the expense of blocking the compensatory beta-adrenergic signaling in surviving cells. One prerequisite for developing new therapeutics is to be able to grow cardiomyocytes ex vivo, and test their apoptotic response to drugs. Here we describe methods for isolation and culturing of neonatal and adult calcium tolerant cardiomyocytes. Similarly, cardiofibroblasts can also be isolated using the same protocol and subsequently, immortalized with SV40 T-Antigen for ex vivo studies.