Genetic and dietary modulators of the inflammatory response in the gastrointestinal tract of the BXD mouse genetic reference population.

peer reviewedInflammatory gut disorders, including inflammatory bowel disease (IBD), can be impacted by dietary, environmental, and genetic factors. While the incidence of IBD is increasing worldwide, we still lack a complete understanding of the gene-by-environment interactions underlying inflammation and IBD. Here, we profiled the colon transcriptome of 52 BXD mouse strains fed with a chow or high-fat diet (HFD) and identified a subset of BXD strains that exhibit an IBD-like transcriptome signature on HFD, indicating that an interplay of genetics and diet can significantly affect intestinal inflammation. Using gene co-expression analyses, we identified modules that are enriched for IBD-dysregulated genes and found that these IBD-related modules share cis-regulatory elements that are responsive to the STAT2, SMAD3, and REL transcription factors. We used module quantitative trait locus analyses to identify genetic loci associated with the expression of these modules. Through a prioritization scheme involving systems genetics in the mouse and integration with external human datasets, we identified Muc4 and Epha6 as the top candidates mediating differences in HFD-driven intestinal inflammation. This work provides insights into the contribution of genetics and diet to IBD risk and identifies two candidate genes, MUC4 and EPHA6, that may mediate IBD susceptibility in humans

Characterization of Blood Immune Cells in Patients With Decompensated Cirrhosis Including ACLF

Background and Aims: Patients with cirrhosis and acute-on-chronic liver failure (ACLF) have immunosuppression, indicated by an increase in circulating immune-deficient monocytes. The aim of this study was to investigate simultaneously the major blood-immune cell subsets in these patients. Material and Methods: Blood taken from 67 patients with decompensated cirrhosis (including 35 critically ill with ACLF in the intensive care unit), and 12 healthy subjects, was assigned to either measurements of clinical blood counts and microarray (genomewide) analysis of RNA expression in whole-blood; microarray (genomewide) analysis of RNA expression in blood neutrophils; or assessment of neutrophil antimicrobial functions. Results: Several features were found in patients with ACLF and not in those without ACLF. Indeed, clinical blood count measurements showed that patients with ACLF were characterized by leukocytosis, neutrophilia, and lymphopenia. Using the CIBERSORT method to deconvolute the whole-blood RNA-expression data, revealed that the hallmark of ACLF was the association of neutrophilia with increased proportions of macrophages M0-like monocytes and decreased proportions of memory lymphocytes (of B-cell, CD4 T-cell lineages), CD8 T cells and natural killer cells. Microarray analysis of neutrophil RNA expression revealed that neutrophils from patients with ACLF had a unique phenotype including induction of glycolysis and granule genes, and downregulation of cell-migration and cell-cycle genes. Moreover, neutrophils from these patients had defective production of the antimicrobial superoxide anion. Conclusions: Genomic analysis revealed that, among patients with decompensated cirrhosis, those with ACLF were characterized by dysregulation of blood immune cells, including increases in neutrophils (that had a unique phenotype) and macrophages M0-like monocytes, and depletion of several lymphocyte subsets (including memory lymphocytes). All these lymphocyte alterations, along with defective neutrophil superoxide anion production, may contribute to immunosuppression in ACLF, suggesting targets for future therapies

J Clin Invest

Arterial cardiovascular events are the leading cause of death in patients with JAK2V617F myeloproliferative neoplasms (MPN). However, their mechanisms are poorly understood. The high prevalence of myocardial infarction without significant coronary stenosis or atherosclerosis in patients with MPN suggests that vascular function is altered. Consequences of JAK2V617F mutation on vascular reactivity are unknown. We observe here increased responses to vasoconstrictors in arteries from Jak2V617F mice, resulting from disturbed endothelial nitric oxide pathway and increased endothelial oxidative stress. This response was reproduced in wild-type mice by circulating microvesicles isolated from patients carrying JAK2V617F and by erythrocyte-derived microvesicles from transgenic mice. Microvesicles of other cellular origins had no effect. This effect was observed ex vivo on isolated aortas, but also in vivo on femoral arteries. Proteomic analysis of microvesicles derived from JAK2V617F erythrocytes identified increased expression of myeloperoxidase as the likely mechanism accounting for microvesicles effect. Myeloperoxidase inhibition in microvesicles derived from JAK2V617F erythrocytes supressed their effect on oxidative stress. Antioxidants, such as simvastatin and N-acetyl-cysteine, improved arterial dysfunction in Jak2V617F mice. In conclusion, JAK2V617F MPN are characterized by exacerbated vasoconstrictor responses resulting from increased endothelial oxidative stress caused by circulating erythrocyte-derived microvesicles. Simvastatin appears as promising therapeutic strategy in this setting

Pathophysiology of cardiovascular events in Bcr/Abl-negative myeloproliferative neoplasms

Les syndromes myéloprolifératifs Bcr/Abl-negatifs (SMP) sont des maladies hématopoïétiques clonales, secondaires dans 80% des cas à la mutation sporadique JAK2V617F. JAK2V617F a été récemment mise en évidence dans les cellules endothéliales. Les deuxièmes mutations en ordre de fréquence sont les mutations de Calréticulin (CALR). Les évènements cardiovasculaires sont la première cause de mortalité des malades atteints de SMP (2/3 artériels et 1/3 veineux). Les évènements veineux sont caractérisés par une fréquence particulièrement élevée de thromboses survenant dans des sites inhabituels, comme les veines splanchniques (veines hépatiques (syndrome de Budd-Chiari) ou veine porte). Les mécanismes responsables de ces évènements cardiovasculaires chez les malades atteints de SMP sont mal compris. Les conséquences phénotypiques et fonctionnelles de la mutation JAK2V617F sur les cellules endothéliales n'ont pas été évaluées. L'objectif global de ce travail est de mieux comprendre la physiopathologie des évènements cardio-vasculaires associés aux SMP, sur le plan artériel, j'ai montré, grâce à des expériences de myographie, que les aortes de souris portant JAK2V617F à la fois dans leurs cellules hématopoïétiques et endothéliales ont une très forte augmentation de la réponse aux agents vasoconstricteurs alors que cet effet n'est pas observé lorsque la mutation est uniquement endothéliale. J'ai ensuite isolé des microvésicules plasmatiques de malades porteurs de JAK2V617F, non traités pour leur SMP, et ai observé que ces microvésicules reproduisent l'effet d'hyperréponse artérielle aux agents vasoconstricteurs. J'ai par la suite montré que seules les microvésicules de globules rouges portant la mutation JAK2V617F étaient responsables de cet effet. J'ai ensuite analysé les mécanismes impliqués et ai déterminé que cette hyperréactivité vasculaire est dépendante de l'endothélium et des NO synthases. De plus, j'ai aussi mis en évidence une forte augmentation du stress oxydant dans l'endothélium des aortes de souris portant la mutation JAK2V617F en comparaison aux souris sauvages, suggérant que les perturbations de la voie du NO résultent d'une génération de stress oxydant induite par les microvésicules de globules rouges. Ces données nous ont poussés à évaluer de nouvelles thérapeutiques, comme les statines, qui sont des molécules anti-cholestérolémiques ayant indépendamment du cholestérol un effet anti-oxydant bénéfique sur la fonction endothélial. J'ai montré que l'utilisation de simvastatine chez les souris portant la mutation JAK2V617F diminue significative cette hyperréactivité vasculaire en comparaison aux souris contrôles. Mes résultats suggèrent que chez les malades atteints de SMP une hyperréponse aux agents vasoconstricteurs induite par les microvésicules circulantes d'origine érythrocytaire pourrait participer aux accidents artériels et que de nouvelles thérapeutiques, comme les statines pourraient être prometteuses. Sur le plan veineux, en analysant une cohorte prospective de 312 patients atteints de thromboses splanchniques, j'ai pu déterminer la prévalence des mutations CALR dans cette population (2%) et identifier un groupe de malades (ceux sans JAK2V617F et ayant une taille de rate >ou= 16cm et des plaquettes > 200G/L) chez lesquels la recherche des mutations CALR doit être effectuée. Ces critères ont une excellente valeur prédictive négative (100%) et permettent d'éviter 96% de tests inutiles. J'ai confirmé ces critères grâce à une collaboration européenne dans une cohorte de validation espagnole comprenant 209 patients. Je me suis aussi attachée à déterminer le rôle de JAK2V617F endothélial dans les conséquences hépatiques des thromboses des veines hépatiques. Nous avons montré que la présence de la mutation JAK2V617F dans l'endothélium n'aggrave pas le développement des lésions hépatiques secondaires à un syndrome de Budd-Chiari, ni en termes de fibrose hépatique ni en termes d'hypertension portale.Classical myeloproliferative neoplasms (MPNs) are clonal hematopoietic diseases. The sporadic mutation JAK2V617F is present in 80% of patients. JAK2V617F has recently been demonstrated in endothelial cells. The second most frequent mutations are Calreticulin mutations (CALR). Cardiovascular events are the leading cause of death in patients with MPNs (2/3 arterial and 1/3 venous). Venous events are characterized by a particularly high frequency of unusual sites involvement, such as splanchnic veins thrombosis (hepatic veins (Budd-Chiari syndrome) or portal vein). Mechanisms responsible for these cardiovascular events in patients with MPNs are poorly understood. The phenotypic and functional consequences of JAK2V617F mutation in endothelial cells have not been evaluated. The overall objective of this work is to better understand the pathophysiology of cardio-vascular events associated with MPNs. On the arterial side, I have shown, through myography experiments, that mouse aorta carrying JAK2V617F both in their hematopoietic and endothelial cells display a very strong increased vasoconstrictive response whereas this effect is not observed when the mutation is only endothelial. I then isolated circulating microvesicles from patients carrying JAK2V617F mutation and observed that these microvesicles reproduce the increased arterial vasoconstrictive response. I subsequently showed that only microvesicles from red blood cells carrying the JAK2V617F mutation were responsible for this effect. I then analysed the mechanisms involved and determined that this vascular hyperreactivity is endothelial-dependent via NO synthases inhibition. In addition, I also found a strong increase in oxidative stress in the aortic endothelium of mouse carrying the JAK2V617F mutation compared to wild-type mice, suggesting that the NO pathway inhibition results from oxidative stress induced by red blood cells microvesicles. These data prompted us to evaluate new therapeutics, such as statins, which are anti-cholesterolemic molecules that have antioxidant effect beneficial to endothelial function. I have shown that the use of simvastatin in mice carrying the JAK2V617F mutation significantly decreases this vascular hyperreactivity compared to control. My results suggest that patients with MPNs may have an increased arterial vasoconstrictive response induced by erythrocytes microvesicles and that new therapies such as statins may be promising. On the venous side, by analysing a prospective cohort of 312 patients with splanchnic thrombosis, I was able to determine the prevalence of CALR mutations in this population (2%) and identify a group of patients (those without JAK2V617F and having a spleen >or= 16cm and platelets> 200G / L) in which the search for CALR mutations must be performed. These criteria have an excellent negative predictive value (100%) and 96% of useless tests could be avoided. I confirmed these criteria through European collaboration in a Spanish validation cohort of 209 patients. I also focused on determining the role of endothelial JAK2V617F in hepatic consequences of hepatic veins thrombosis. We have shown that the presence of the JAK2V617F mutation in the endothelium does not worsen the development of hepatic lesions secondary to Budd-Chiari syndrome, nor in terms of hepatic fibrosis nor in terms of portal hypertension

Leukemia

Thrombosis, both in arterial and venous territories, is the major complication of myeloproliferative neoplasms and is responsible for a high rate of morbidity and mortality. The currently accepted risk factors are an age over 60 years and a history of thrombosis. However, many complex mechanisms contribute to this increased prothrombotic risk, with involvement of all blood cell types, plasmatic factors, and endothelial cells. Besides, some cardiovascular events may originate from arterial vasospasm that could contribute to thrombotic complications. In this review, we discuss recent results obtained in mouse models in the light of data obtained from clinical studies. We emphasize on actors of thrombosis that are currently not targeted with current therapeutics but could be promising targets, i.e, neutrophil extracellular traps and vascular reactivity

Microvésicules de globules rouges et vasospasmes artériels dans les syndromes myéloprolifératifs avec mutation JAK2

International audienc

Prognostic Value of Prospective Longitudinal CRP to Albumin Ratio among Older Outpatients with Cancer

International audienceThe prognostic value of the CRP to albumin ratio (CAR) among older adults with cancer is not known. Six hundred and three older outpatients with cancer and undergoing geriatric assessment before therapeutic decisions were prospectively recruited from the PF-EC cohort study. Serum albumin levels, serum CRP levels and the CAR were prospectively recorded at baseline, and at each consultation thereafter, as follows: 1, 3, 6, 9, 12, 18, 24 and 36 months. Frailty was defined as a G8-index ≤ 14. The primary endpoint was longitudinal variation in the CAR during the study follow-up. Two clusters in the longitudinal trajectories of the CAR were identified, one favourable, with lower values and better overall survival (cluster A), and the second with higher values and less favourable overall survival (cluster B). The median CAR [95% CI] for clusters A and B were respectively: 0.17 [0.04–0.48] and 0.26 [0.04–0.79] at baseline (p = 0.01), and 0.18 [0.02–3.17] and 0.76 [0.03–6.87] during the study follow-up (p < 0.0001). Cluster B was associated with the frailest patients with metastatic disease, mainly driven by a high CRP level at baseline, and low albumin during the study follow-up. Our study results suggest that the most risk-prone patients have a cancer-cachexia trajectory

Liver sinusoidal endothelial cells: Physiology and role in liver diseases

International audienceLiver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells representing the interface between blood cells on the one side and hepatocytes and hepatic stellate cells on the other side. LSECs represent a permeable barrier. Indeed, the association of 'fenestrae', absence of diaphragm and lack of basement membrane make them the most permeable endothelial cells of the mammalian body. They also have the highest endocytosis capacity of human cells. In physiological conditions, LSECs regulate hepatic vascular tone contributing to the maintenance of a low portal pressure despite the major changes in hepatic blood flow occurring during digestion. LSECs maintain hepatic stellate cell quiescence, thus inhibiting intrahepatic vasoconstriction and fibrosis development. In pathological conditions, LSECs play a key role in the initiation and progression of chronic liver diseases. Indeed, they become capillarized and lose their protective properties, and they promote angiogenesis and vasoconstriction. LSECs are implicated in liver regeneration following acute liver injury or partial hepatectomy since they renew from LSECs and/or LSEC progenitors, they sense changes in shear stress resulting from surgery, and they interact with platelets and inflammatory cells. LSECs also play a role in hepatocellular carcinoma development and progression, in ageing, and in liver lesions related to inflammation and infection. This review also presents a detailed analysis of the technical aspects relevant for LSEC analysis including the markers these cells express, the available cell lines and the transgenic mouse models. Finally, this review provides an overview of the strategies available for a specific targeting of LSECs

Expression of enzymes involved in synthesis and metabolism of estradiol in human breast as studied by immunocytochemistry and in situ hybridization

It is well documented that human breast is actively involved in the local formation of estrogens. To determine the site(s) of action of enzymes involved in synthesis and metabolism of the most potent estrogen estradiol (E2), we have studied the expression of the following enzymes: 3ß-hydroxysteroid dehydrogenase (3-HSD), 17ß-HSD types 1, 2, 5, 7 and 12, aromatase, steroid sulfatase (STS) and estrogen sulfotransferase (EST) 1E1 at the cellular level in breast. Both in situ hybridization and immunocytochemistry were used for enzyme localization in normal breast tissues. For immunocytochemistry, we used rabbit antibodies, while in situ hybridization studies were performed using (35S)- labeled cRNA probes. Similar results were obtained with both approaches. All the enzymes (3ß-HSD; 17ß-HSD types 1, 5, 7 and 12; aromatase) involved in the conversion of circulating dehydroepiandrosterone (DHEA) to E2 as well as STS which converts estradiol sulfate (E2-S) to E2 have been found to be expressed in epithelial cells of acini and/or ducts as well as the stromal cells. Moreover, 17ß-HSD type 2 and EST1E1, two enzymes which inactivate E2, have been also localized in the same cell types. The present results indicate the enzymes which play a role in the synthesis and metabolism of E2 are expressed in both epithelial and stromal cells in human breast

Serum Leptin Levels, Nutritional Status, and the Risk of Healthcare-Associated Infections in Hospitalized Older Adults

We aimed to determine whether serum leptin levels are predictive of the occurrence of healthcare-associated infections (HAIs) in hospitalized older patients. In a prospective cohort, 232 patients had available data for leptin and were monitored for HAIs for 3 months. Admission data included comorbidities, invasive procedures, the Mini Nutritional Assessment (MNA), BMI, leptin, albumin and C-reactive protein levels, and CD4 and CD8 T-cell counts. Multivariate logistic regression modelling was used to identify predictors of HAIs. Of the 232 patients (median age: 84.8; females: 72.4%), 89 (38.4%) experienced HAIs. The leptin level was associated with the BMI (p &lt; 0.0001) and MNA (p &lt; 0.0001) categories. Women who experienced HAIs had significantly lower leptin levels than those who did not (5.9 &mu;g/L (2.6&ndash;17.7) and 11.8 (4.6&ndash;26.3), respectively; p = 0.01; odds ratio (OR) (95% confidence interval): 0.67 (0.49&ndash;0.90)); no such association was observed for men. In a multivariate analysis of the women, a lower leptin level was significantly associated with HAIs (OR = 0.70 (0.49&ndash;0.97)), independently of comorbidities, invasive medical procedures, and immune status. However, leptin was not significantly associated with HAIs after adjustments for malnutrition (p = 0.26) or albuminemia (p = 0.15)&mdash;suggesting that in older women, the association between serum leptin levels and subsequent HAIs is mediated by nutritional status