Background: Pulmonary arterial hypertension (PAH) is a lethal disease for which the fundamental molecular mechanisms are only partially understood. Existing therapies, which primarily focus on endothelial dysfunction, have limited effects on improving outcomes. Increases in pulmonary vascular resistance in PAH may be attributed to complex lung arterial remodeling which result in obliterative “plexiform” lesions, a pathological hallmark of this disease. Recent studies have shown that endothelial cell (ECs) apoptosis may be a central trigger for PAH, resulting in the emergence of growth-dysregulated and apoptosis-resistant ECs that contribute to the formation of complex neoplastic-like vascular lesions. However, the mechanism which links ECs apoptosis to dysregulated growth is not yet known. Previous studies in our lab have identified increased expression of translationally controlled tumor protein (TCTP) and its gene (TPT1), previously implicated in the transformation of neoplastic cells in cancer, and in blood outgrowth ECs from patients with PAH. Moreover, TCTP expression was found to be elevated in the lungs of patients with PAH, and tightly localized to complex arterial lesions. In addition, it was detected in obliterative intimal lesions of an experimental rat model of severe PAH. Hypothesis: TCTP represents a central molecular mechanism linking ECs apoptosis to the emergence of growth-dysregulated lung vascular cells and occlusive, complex arterial remodelling in PAH. Specific Hypotheses: - Lentiviral overexpression of TCTP in human umbilical vein endothelial cells (HUVECs) and pulmonary artery smooth muscle cells (PASMCs) leads to a hyperproliferative and apoptosis-resistant phenotype. - Overexpression of TCTP will increase its export into apoptotic extracellular vesicles, thereby augmenting cell-cell signalling between ECs and neighbouring SMCs. Purpose: My objective was to examine the effects TCTP overexpression on ECs and SMCs survival in terms of proliferation and apoptosis, and TCTP release on the survival of nontransduced ECs and SMCs. Methods and Results: The effect of TCTP overexpression on ECs growth and survival was studied using in vitro models. TCTP was overexpressed via a lentivirus vector in HUVECs and PASMCs. Compared to non-transfected or null transfected cells, TCTP overexpression led to increases in BrdU incorporation, consistent with hyper-proliferation, and decreases in caspase activity, consistent with apoptosis resistance. As well, TCTP was selectively exported into the conditioned media of apoptotic ECs, but not SMCs, despite similar levels of overexpression. In addition, the level of release was greater in serum starved conditioned media in comparison to the exosome fraction. Finally, our data demonstrates a selective effect of conditioned media (CM) from serum-starved ECs on PASMCs, but not ECs, in terms of an increase in proliferation and a decrease in apoptosis. Conclusions: These support the idea that TCTP overexpression confers an increase in the survival of SMCs and HUVECs. Moreover, TCTP released from apoptotic ECs leads to a growth-dysregulated phenotype within SMCs (but not ECs) and may contribute to the formation of complex lung arterial lesions, leading to arteriolar obliteration in PAH. Finally, an increase in the level of TCTP expression via lentiviral transduction led to an increased TCTP export into the media, but this appeared to be mostly in the soluble portion, and less was associated with exosomes.

Cancer cells require survival strategies to respond to microenviromental changes and out-compete their neighbours. They activate stress response mechanisms under extreme microenvironmental conditions, some of which are controlled by the amino acid-sensitive kinase complex, mechanistic Target of Rapamycin Complex 1 (mTORC1). Exosomes are secreted nanovesicles made inside intracellular endosomal compartments that mediate a specialised and complex form of intercellular signalling that can reprogramme target cells via the action of multiple active cargos. I investigated whether mTORC1 activity might modulate the type of exosome secreted in response to microenvironmental changes. Here I identify a new form of mTORC1-regulated exosome biogenesis and signalling involving recycling multivesicular endosomes (rMVEs), a previously unrecognised site for exosome biogenesis. Reduced activity of a specific form of glutamine-sensitive mTORC1 in HCT116 colorectal cancer cells results in an ‘exosome switch’ in which exosomes are preferentially released from these compartments instead of late endosomes. Importantly, RAB11a is found in association with at least a proportion of rMVEs that generate these alternative exosomes and is loaded on to some of their ILVs, providing a RAB signature of compartmental origin. I provide evidence that this exosome switch is conserved in other cancer cell types. My study also presents a proteomics analysis of extracellular vesicle (EV) preparations from normal and mTORC1-inhibited cells. I demonstrate that EV preparations isolated following exosome switching have enhanced pro-angiogenic properties and novel tumour growth-promoting activities. Activation of the receptor tyrosine kinase c-MET and its downstream mitogen-activated protein kinase (MAPK) ERK via phosphorylation is stimulated by these EVs, providing a potential explanation for their growth-promoting effects. Subsequent studies in the lab have demonstrated that several of these pro-tumorigenic activities are mediated by exosomes. I conclude that stress-induced mTORC1 inhibition allows tumour cells to initiate a novel exosome secretion pathway that potentially mediates a cancer cell survival plan that reverses microenvironmental change and supports tumour adaptation. In the future, blocking this response could improve patient outcome following treatment with mTORC1-inhibitory or anti-angiogenic drugs that have currently met with limited success in the clinic.

The dysfunction of autophagy pathways has been linked to the development and progression of numerous human diseases, in particular neurological disorders and cancer. Investigating these pathological autophagy mechanisms is essential to gain insights into the underlying disease mechanisms, identify novel biomarkers, and develop targeted therapies. In this thesis, I present three manuscripts that investigate the regulatory mechanisms of autophagy machinery in human diseases. In the first manuscript (Chitiprolu et al., 2018), we investigated the mechanism of p62-mediated selective autophagic clearance of RNA stress granules implicated in Amyotrophic Lateral Sclerosis (ALS). Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. By employing mass spectrometry, high resolution imaging and biochemical assays, we demonstrated that the autophagy receptor p62 associates with C9ORF72 to eliminate stress granules by autophagy. This requires p62 to associate with proteins that are symmetrically methylated on arginines. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients. The second manuscript (Guo, Chitiprolu et al., 2014) describes the mechanism by which autophagy degrades retrotransposon RNA from both long and short interspersed elements, thereby preventing new retrotransposon insertions into the genome. By employing quantitative imaging tools, we demonstrated that retrotransposon RNA localizes to RNA granules that are selectively degraded by the autophagy receptors NDP52 and p62. Mice lacking a copy of Atg6/Beclin1, a gene critical for autophagy, also accumulate both retrotransposon RNA and genomic insertions. This suggests a mechanism for the increased tumorigenesis upon autophagy inhibition and therefore a role for autophagy in tempering evolutionary change. Finally, the third manuscript (Guo, Chitiprolu et al., 2017) examines the intersection of autophagy machinery with exosome release and function in cancer metastasis. By employing dynamic light scattering, Nanosight particle tracking, electron microscopy, super-resolution imaging and Western blotting, we robustly quantified exosome identity and purity in multiple cell lines. We demonstrated that exosome production is strongly reduced in cells lacking Atg5 and Atg16L1, but this is independent of Atg7 and canonical autophagy. The effect of Atg5 on exosome production promotes the migration and in vivo metastasis of orthotopic breast cancer cells. These findings delineate autophagy-independent pathways by which autophagy-related genes can contribute to metastasis. Taken together, data presented in the three manuscripts highlight the molecular mechanisms of autophagy core machinery proteins and selective receptors such as Atg5, p62 and NDP52, in the pathogenesis of cancer and neurodegeneration. In these diseases characterized by mutations in autophagy pathways, the mechanisms we uncover provide insights into their causes and serve as potential therapeutic targets.

Extracellular vesicles (exosomes) are the subject of current nephrology proteomics research as they are considered as a promising source of potential biomarkers of kidney disease. This work is focused on discovery of the most appropriate procedure for the urinary exosomes isolation. We have compared already described methods, based on different physicochemical principles of isolation: hydrostatic filtration dialysis (HFD), differential ultracentrifugation, ultrafiltration through a 100 kDa filter, or sample precipitation with Total Exosome Isolation (from urine) kit. Characterization of individual isolated exosomal fractions was performed using SDS-PAGE method (presence of contaminating proteins), western blot analysis (detection of exosomal markers TSG101, alix), nanoparticle tracking analysis (NTA, vesicle size and concentration) or transmission electron microscopy (TEM, vesicles morphology). Due to the presence of contaminating proteins in urine samples, which could distort the results of subsequent proteomic assays, the conditions for the cleavage of undesirable proteins by proteinase K prior to their own isolation were optimized. It has been found that the best yield and purity of the isolated exosomal fractions were provided by a process combining HFD with differential ultracentrifugation...

Rôle du métabolisme post-transcriptionnel des ARNm dans la régulation de la dormance des semences d’Arabidopsis thaliana.Une étude physiologique nous a permis d'identifier l'influence de la température et de l'humidité relative (HR) lors du stockage des graines dormantes d’Arabidopsis. Après une levée de dormance atteinte en 7 semaines avec des cinétiques variables selon les conditions, on observe une induction de la dormance secondaire à faible HR et une perte progressive de la viabilité à forte HR. La levée et l’induction de la dormance sont associées à la régulation de gènes liés aux voies de l'acide abscissique et des gibbérellines. Nous avons étudié la dynamique d’association des ARNm aux polysomes et comparé la transcription et la traduction des graines dormantes et non dormantes au cours de l’imbibition. Nous montrons qu'il n'y a pas de corrélation entre transcriptome et traductome et que la régulation de la germination est principalement liée à la traduction. Ceci suppose un recrutement sélectif et dynamique des ARNm liés aux polysomes dans les graines dormantes et non dormantes. Certaines caractéristiques de la région 5'UTR des transcrits semble impliquées dans la sélection des ARNm traduits pendant la germination. Les phénotypes de mutants d’éléments du catabolisme des ARN montrent que la dormance est également associée à une dégradation sélective des ARNm. Les P-bodies (localisés dans des lignées YFP-DCP1) sont d’ailleurs en quantité plus importante dans les graines non-dormantes. La comparaison des transcriptomes des mutants vcs-8 et xrn4-5 a permis l'identification de plusieurs transcrits dégradés via VCS ou XRN4, dont le rôle sur la dormance a été confirmé par génétique inverse. Certains motifs spécifiques semblent être impliqués dans la sélection de transcrits pour leur dégradation. / Role of mRNA post-transcriptional metabolism in the regulation of Arabidopsis thaliana dormancy.A physiological study allowed us to reveal the effect of temperature and relative humidity (RH) during Arabidopsis seed storage. Seven weeks of after ripening lead to alleviation of dormancy with different kinetics according to the conditions. Longer storage induced an induction of secondary dormancy at low RH and progressive loss of viability at high RH. Dormancy release and induction of secondary dormancy were associated with induction or repression of key genes related to abscissic acid and gibberellins pathways. We have studied the dynamics of mRNAs association with polysomes and compared transcriptome and translatome of dormant and non-dormant seeds. There was no correlation between transcriptome and translatome and germination regulation is largely translational, implying a selective and dynamic recruitment of mRNAs to polysomes in both dormant and non-dormant seeds. Some identified 5'UTR features could play a role in this selective. Dormancy is also associated with mRNA decay as demonstrated by phenotyping mutants altered in mRNA metabolism. Moreover we have shown that P-bodies were more abundant in non-dormant seeds that in dormant ones. Transcriptome analysis of xrn4-5 and vcs-8 mutants allowed us to identify several transcripts degraded via VCS ou XRN4 proteins, having a role in dormancy. This role was confirmed by reverse genetics for some of them. Some specific motifs were identified as involved in mRNA decay selection.

Duas patologias principais estão associadas ao HTLV-1: a leucemia/linfoma de células T do adulto (ATLL) e a mielopatia associada ao HTLV-1/paraparesia espástica tropical (HAM/TSP). O mecanismo pelo qual ocorre o desenvolvimento destas doenças em apenas 1 a 5% dos indivíduos infectados é desconhecido. Sabe-se que diversos fatores estão associados ao desenvolvimento destas patologias, como carga proviral, características celulares, genéticas e imunológicas do hospedeiro. No entanto, tais mecanismos não foram totalmente elucidados e novas abordagens de estudos são necessárias para a compreensão da patogênese viral. Nesse sentido, vários estudos relatam o papel dos exossomos no desenvolvimento de infecções virais como HIV, EBV e HCV através do transporte de componentes incluindo proteínas, RNAm e miRNA. A possibilidade da transferência do conteúdo viral por essas vesículas para células não infectadas na ausência de vírus é um mecanismo intrigante. Dessa forma, esse trabalho teve como objetivo isolar os exossomos diretamente do soro de indivíduos infectados pelo HTLV-1 e comparar o conteúdo dessas vesículas quanto à expressão dos genes virais tax e HBZ entre os grupos assintomático e sintomático. Para tanto, os exossomos foram isolados por precipitação polimérica (Exoquick(TM)) e caracterizados por Western Blot (expressão dos marcadores CD9, CD81, Alix e citocromo c) e Análise de Rastreamento de Nanopartículas (tamanho e concentração). Além disso, o conteúdo dos exossomos foi analisado por PCR em tempo real para quantificação dos genes virais regulatórios tax e HBZ. Por fim, no intuito de estabelecer o papel dos exossomos como biomarcadores para o desenvolvimento da HAM/TSP, os níveis de expressão destes genes foram associados à carga proviral do HTLV-1. Os transcritos de RNAm de tax e HBZ foram detectados apenas em exossomos isolados do soro dos indivíduos sintomáticos, com ausência nos assintomáticos. Além disso, a expressão de tax e HBZ nos exossomos dos indivíduos infectados pelo HTLV-1 foi correlacionada positivamente com a CPV. Dessa forma, os resultados sugerem que os exossomos podem desempenhar importante papel na patogênese da mielopatia, através do transporte de genes virais de uma célula infectada para não-infectada. No entanto, mais estudos que relacionam o papel dos exossomos na infecção pelo HTLV-1 são necessários. / Two major pathologies has been associated with HTLV-1 infection: adult T- cell leukemia/lymphoma (ATLL) and HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM / TSP). The mechanism which is responsible for the appearance of these diseases in only 1 to 5% of the infected individuals is unknown. Is known that several factors has been related to the development of these diseases, such as proviral load and cellular, genetic and immunological characteristics of the host. However, these mechanisms have not been fully elucidated and new approaches are necessary to understand the viral pathogenesis. Thus, several studies have reported the involvement of exosomes in the development of the HIV, HCV, and EBV viral infections carrying components including proteins, mRNA and miRNA. The possibility of transfer of viral content via these vesicles to uninfected cells in the absence of virus is an intriguing mechanism. Therefore, this proposal aimed to isolate exosomes from serum of HTLV-1 infected patients and compare the content of these vesicles regarding viral gene expression of tax and HBZ between asymptomatic and symptomatic group. For this, the exosomes were isolated by polymeric precipitation (Exoquick(TM)) and characterized by Western Blot (expression of markers CD9, CD81, and Alix) and Nanoparticle tracking Analysis (size and concentration). Furthermore, the exosomes content were analyzed by real-time PCR for the quantification of tax and HBZ regulatory viral genes. Finally, to establish the role of exosomes like biomarkers to HAM/TSP development, levels of these genes were associated with proviral load of HTLV-1. Viral mRNA transcripts including tax and HBZ were found only in exosomes isolated from serum of symptomatic individuals and absence in assymptomatic. Furthermore, tax and HBZ expression in exosomes from HTLV-1 infected individuals were positive correlated with proviral load. Thus, the results suggests that exosomes can able to develop important role in myelopathy pathogenesis carrying viral genes of infected cell to uninfected cell. However, more studies that relate the role of exosomes in HTLV-1 are needed

No description available.

Les syndromes myélodysplasiques (SMD) sont dus à une atteinte oligoclonale de la cellule souche hématopoïétique aboutissant à une dysplasie des lignées myéloïdes, des cytopénies sanguines et une évolution fréquente vers la leucémie aiguë. De nombreuses mutations décrites dans des gènes contrôlant la régulation épigénétique sont responsables de la genèse des SMD. Mais des travaux récents montrent également que des anomalies du microenvironnement médullaire, notamment des cellules stromales mésenchymateuses (CSM), peuvent induire et propager un SMD suggérant l’idée d’une communication intercellulaire étroite entre la niche et les cellules hématopoïétiques. L’invalidation du gène Dicer1 (RNASE de type III impliquée dans le processing des microARN) dans les progéniteurs ostéoblastiques murins induit un véritable SMD avec dysmyélopoïèse.Nous avons confirmé la sous-expression de Dicer1 dans les CSM SMD à partir de prélèvements primaires de moelle totale et dans les CSM en expansion. La sous-expression de Dicer1 s’accompagne d’une dérégulation du profil des microARN au sein de CSM SMD mise en évidence par étude transcriptomique des CSM SMD vs CSM témoins. Nous avons découvert une possible cible thérapeutique : le miR-486-5p que nous avons retrouvé constamment surexprimé dans les CSM SMD. Un des moyens pour les CSM d’influer sur les cellules souches hématopoïétiques peut se faire par la sécrétion de vésicules extracellulaires (EVs). Ces EVs sont hétérogènes et peuvent être définies par leur taille. Nous nous sommes plus particulièrement intéressés aux petites vésicules extracellulaires (sEVs) contenant la fraction exosomale qui est connue comme pouvant transporter des microARN, mARN et des protéines entre les cellules. Nous avons retrouvé ce miR-486-5p transporté comme cargo dans les sEVs sécrétées des CSM, des CSM vers les CD34+. De plus, nous montrons dans un modèle de co-incubation (sEVs avec CD34+ de sujets sains), que sur le plan fonctionnel, les sEVs provenant de CSM SMD induisent plus d’apoptose, plus de stress oxydatif ainsi que plus de dommage à l’ADN.Par ailleurs, la surcharge martiale observée chez les patients SMD est également responsable d’un stress oxydatif. Le déférasirox (DFX), un chélaleur de fer, a montré dans le cadre d’études rétrospectives une amélioration de l’érythropoïèse chez des patients SMD. Grâce à un modèle de différenciation érythroïde avec surcharge martiale, nous avons montré que de faibles doses de DFX induisent une meilleure prolifération des progéniteurs érythroïdes (moins d’apoptose et plus de cellules en cycle) via une activation de NF-κB. Cette activation est due à une diminution du niveau de dérivés réactifs de l’oxygène (ROS) en rapport avec une diminution du fer labile et est contrôlée de manière très fine par le niveau de ROS.Enfin, nous avons utilisé les propriétés du microenvironnement médullaire pour établir un modèle murin de SMD humain. En effet, la relative incapacité des cellules souches myélodysplasiques humaines de greffer et de reconstituer une hématopoïèse pathologique dans des souris immunodéprimées suggère que ces cellules souches SMD doivent avoir besoin d’un support extrinsèque du microenvironnement. Nous avons réalisé un modèle de souris humanisées en co-injectant des CSM et des CD34+ en intratibial. Une prise de greffe a été observée chez toutes les souris injectées et avons pu étudier l’évolution clonale au fil des générations dans les différentes sous-populations de progéniteurs myéloïdes (common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte–erythroid progenitor (MEP)). Notre modèle est stable au cours des générations avec persistance du clone fondateur initial.En conclusion, ce travail confirme le rôle prépondérant du microenvironnement médullaire dans la genèse et la physiopathologie des syndromes myélodysplasiques et ouvre la voie à de nouvelles possibilités thérapeutiques. / Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) oligoclonal diseases leading to dysplasia, blood cytopenia and evolution to acute leukemia. Numerous mutations in genes involved in epigenetic regulation are responsible of MDS genesis. But recently, studies show that medullar microenvironment, particularly mesenchymal stromal cells (MSC), could induces and propagates a truly MDS suggesting a narrow communication between HSC and this niche. Dicer1’s (type III RNAse implicating in microRNA processing) invalidation in murine osteoblastic progenitors induces a MDS with sign of dysplasia.In this work, we have confirmed the under expression of Dicer1 in MDS mesenchymal stromal cells from total bone marrow and cultured MSC. Dicer1 down regulation leads to a deregulation of miRNome profile in MDS MSC highlighted by transcriptomic approaches. We found a potential therapeutic target: miR-486-5p which is constantly overexpressed in MDS MSC. Extracellular vesicles (EVs) could be a possible way for MSC to influence HSC fates. Those EVs are heterogeneous are could be characterized by their sight. We mainly focused on small EVs (sEVs) containing the exosomal fraction known to be able to carry miRNA, mRNA and proteins. We found that miR-486-5p is carry from MSC to the HSC. Transcriptomic analyses of HD HSC overexpressing miR-486-5p are ongoing. Moreover, in a co-incubation model (sEVs and healthy donor (HD) HSC), sEVs coming from MDS MSC induced apoptosis, oxidative stress and DNA damages.Moreover, iron overload seen in MDS patients is also able to induce DNA damages and oxidative stress. Deferasirox (DFX), an iron chelator, has shown an erythropoiesis improvement in MDS patients. Using an erythroid differentiation model with iron overload, we have observed that low dose of DFX induce a better proliferation of erythroid progenitors (less apoptosis and more cycling cells) due to NF-κB activation. This activation is due to a decrease of reactive oxygen species level in relation to a decrease of the labile iron pool.Finally, we have used medullar microenvironment properties to establish a murine model of MDS. Indeed, MDS HSC incapacity to reconstitute a pathological hematopoiesis in immunocompromised mice suggests that MDS HSC need an extrinsic support from the microenvironment. We have engineered a MDS patient derived xenograft (PDX) model by intra-tibial co-injection CD34+ cells with MSC. All mice engrafted et we have follow the clonal evolution over mice generation in the different subset of myeloid progenitors. (common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte–erythroid progenitor (MEP)). Our model is stable over generations with persistence of the initial founding clone.In conclusion, this work confirms the preponderant role of the medullary microenvironment in the genesis and physiopathology of myelodysplastic syndromes and opens the way to new therapeutic possibilities.

abstract: Extracellular vesicles (EVs) are membranous particles that are abundantly secreted in the circulation system by most cells and can be found in most biological fluids. Among different EV subtypes, exosomes are small particles (30 – 150 nm) that are generated through the double invagination of the lipid bilayer membrane of cell. Therefore, they mirror the cell membrane proteins and contain proteins, RNAs, and DNAs that can represent the phenotypic state of their cell of origin, hence considered promising biomarker candidates. Importantly, in most pathological conditions, such as cancer and infection, diseased cells secrete more EVs and the disease associated exosomes have shown great potential to serve as biomarkers for early diagnosis, disease staging, and treatment monitoring. However, using EVs as diagnostic or prognostic tools in the clinic is hindered by the lack of a rapid, sensitive, purification-free technique for their isolation and characterization. Developing standardized assays that can translate the emerging academic EV biomarker discoveries to clinically relevant procedures is a bottleneck that have slowed down advancements in medical research. Integrating widely known immunoassays with plasmonic sensors has shown the promise to detect minute amounts of antigen present in biological sample, based on changes of ambient optical refractive index, and achieve ultra-sensitivity. Plasmonic sensors take advantage of the enhanced interaction of electromagnetic radiations with electron clouds of plasmonic materials at the dielectric-metal interface in tunable wavelengths. / Dissertation/Thesis / Doctoral Dissertation Biomedical Engineering 2020

Sepsis is dysregulated host immune response to infection causing life-threatening organ dysfunction. Endothelial cell dysfunction and uncontrolled inflammatory responses are two contributors for sepsis-induced mortality. The crosstalk between endothelial and immune cells plays a critical role in the pathophysiology of sepsis. Therefore, understanding the mechanism of interaction between endothelial and immune cells will provide novel information to develop therapeutic strategies for sepsis. Pathogen associated moleculear patterns (PAMPs) and/or damage associated molecular patterns (DAMPs) produced during sepsis, activate endothelial cells to increase the expression of adhesion molecules, attracting immune cell infiltration into the tissues. Uncontrolled inflammatory responses during the early phase of sepsis contribute to organ failure and lethality. Over 100 clinical trials, targeting inflammatory responses in sepsis, have failed in the past three decades. Thereby, developing novel therapeutic strategies for sepsis are urgent. Heat shock protein A12B (HSPA12B), as one member of HSP70 family, predominately expressed in the endothelial cells, plays important roles in many pathophysiological processes. Currently, we observed endothelial cell specific HSPA12B deficiency (HSPA12B-/-) exacerbates mortality in sepsis induced by cecal ligation puncture (CLP). HSPA12B-/- septic mice exhibits increased expressions of adhesion molecule and infiltrated macrophages in the myocardium and activated macrophages in the peritoneal cavity. In vitro studies show that HSPA12B could be secreted from endothelial cells via exosome. HSPA12B carried by exosomes can be uptaken by macrophages to downregulate macrophage NF-kB activation and pro-inflammatory cytokine production. Trained immunity, induced by β-glucan, causes immune memory in innate immune cells, with an altered response towards another challenge. We have found that mice received β-glucan seven days before CLP sepsis exhibit attenuated mortality with decreased pro-inflammatory responses. We found that β-glucan significantly increased the levels of HSPA12B in endothelial cells and endothelial exosomes. β-glucan induced endothelial exosomes markedly suppress macrophage NF-kB activation and pro-inflammatory responses. The current data suggests that HSPA12B plays a novel role in the regulation of immune and inflammatory responses and that HSPA12B could be an important mediator for the crosstalk between endothelial cells and macrophages during sepsis. β-glucan regulates endothelial cell functions and immune/inflammatory responses, thus improving survival outcome in CLP sepsis.