A human postnatal lymphoid progenitor capable of circulating and seeding the thymus

Identification of a thymus-seeding progenitor originating from human bone marrow (BM) constitutes a key milestone in understanding the mechanisms of T cell development and provides new potential for correcting T cell deficiencies. We report the characterization of a novel lymphoid-restricted subset, which is part of the lineage-negative CD34+CD10+ progenitor population and which is distinct from B cell–committed precursors (in view of the absence of CD24 expression). We demonstrate that these Lin−CD34+CD10+CD24− progenitors have a very low myeloid potential but can generate B, T, and natural killer lymphocytes and coexpress recombination activating gene 1, terminal deoxynucleotide transferase, PAX5, interleukin 7 receptor α, and CD3ε. These progenitors are present in the cord blood and in the BM but can also be found in the blood throughout life. Moreover, they belong to the most immature thymocyte population. Collectively, these findings unravel the existence of a postnatal lymphoid-polarized population that is capable of migrating from the BM to the thymus

Asthma and COPD Are Not Risk Factors for ICU Stay and Death in Case of SARS-CoV2 Infection

BACKGROUND: Asthmatics and patients with chronic obstructive pulmonary disease (COPD) have more severe outcomes with viral infections than people without obstructive disease. OBJECTIVE: To evaluate if obstructive diseases are risk factors for intensive care unit (ICU) stay and death due to coronavirus disease 2019 (COVID19). METHODS: We collected data from the electronic medical record from 596 adult patients hospitalized in University Hospital of Liege between March 18 and April 17, 2020, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection. We classified patients into 3 groups according to the underlying respiratory disease, present before the COVID19 pandemic. RESULTS: Among patients requiring hospitalization for COVID19, asthma and COPD accounted for 9.6% and 7.7%, respectively. The proportions of asthmatics, patients with COPD, and patients without obstructive airway disease hospitalized in the ICU were 17.5%, 19.6%, and 14%, respectively. One-third of patients with COPD died during hospitalization, whereas only 7.0% of asthmatics and 13.6% of patients without airway obstruction died due to SARS-CoV2. The multivariate analysis showed that asthma, COPD, inhaled corticosteroid treatment, and oral corticosteroid treatment were not independent risk factors for ICU admission or death. Male gender (odds ratio [OR]: 1.9; 95% confidence interval [CI]: 1.1-3.2) and obesity (OR: 8.5; 95% CI: 5.1-14.1) were predictors of ICU admission, whereas male gender (OR 1.9; 95% CI: 1.1-3.2), older age (OR: 1.9; 95% CI: 1.6-2.3), cardiopathy (OR: 1.8; 95% CI: 1.1-3.1), and immunosuppressive diseases (OR: 3.6; 95% CI: 1.5-8.4) were independent predictors of death. CONCLUSION: Asthma and COPD are not risk factors for ICU admission and death related to SARS-CoV2 infection

LEAFY expression and temporal sequence of floral transition in Sinapis alba L.

The shoot apical meristem (SAM) of Sinapis alba can be switched from vegetative to reproductive fate by exposure of 2-month old plants to a single long day (LD). Floral transition then occurs in good synchrony within a population, and a number of biochemical, cellular, and morphological changes have been described. Our aim is to integrate gene expression patterns into this timing. We report here the analysis of SaLFY, orthologous to the floral meristem identity gene LEAFY of Arabidopsis. Materials and Methods: Plants of Sinapis were grown in 8-h shorts days for two months before being induced to flower by one 22-h LD. Shoot apices were harvested 24, 32, 40, 48, 56 and 80h after start of the LD, and prepared for in situ hybridization (Melzer et al., 1999). Results and Conclusions: As expected, the expression of SaLFY was very strong in flower primordia. More surprisingly, SaLFY was expressed well before the initiation of flowers. First, a strong signal was detected in the tip of young leaf primordia of vegetative plants. Secondly, SaLFY was transiently expressed in the SAM of induced plants, from 32h after the start of the LD, when cell proliferation increased. The signal formed, in transverse sections, a discontinuous ring with activation where last leaves were to be initiated. Interestingly, this activation in the SAM matched in time and space early growth changes previously described during the transition to flowering, namely an increase of leaf primordia growth and an acceleration of last leaf initiation (Bernier, 1997), suggesting that SaLFY may have dual functions in fate specification during the floral transition of the SAM. References: Bernier G 1997. J Exp Bot 48; 1071-1077. Melzer S, Kampmann G, Chandler J, Apel K 1999. Plant J 18; 395-405

Contribution du lipopolysaccharide de Leptospira interrogans à l'échappement des bactéries au système immunitaire inné

La leptospirose est une zoonose négligée ré-émergente causée par la bactérie pathogène Leptospira interrogans. Chez l'homme, l'infection provoque généralement un syndrome grippal, mais la forme grave, également appelée maladie de Weil, peut provoquer une défaillance de plusieurs organes et être potentiellement mortelle. L. interrogans peut infecter tous les vertébrés, qui peuvent présenter des symptômes différents selon les hôtes. Les souris et les rats sont résistants à la forme aiguë de la maladie et peuvent être infectés de manière chronique au niveau rénal, agissant ainsi comme un réservoir naturel. L. interrogans sont des bactéries furtives qui ont déjà été décrites comme évitant certaines réponses immunitaires innées. Les objectifs de ce doctorat étaient d'étudier la contribution de certains motifs moléculaires des leptospires (MAMPs) à l'évasion immunitaire innée des bactéries dans les macrophages de différents hôtes. Plus spécifiquement, nous nous sommes concentrés sur l'étude de la flagelline, du lipopolysaccharide atypique et des lipoprotéines des leptospires, et leur reconnaissance respective par les récepteurs de type Toll (TLRs) et les caspases. Tout d'abord, nous avons terminé un projet sur l'échappement à la reconnaissance par TLR5 de l'endoflagelle des leptospires chez les hôtes humain, murin et bovin (Holzapfel, Bonhomme et al. 2020). Ensuite, nous avons étudié le LPS atypique des leptospires, objet principal de cette thèse. Nous avons démontré dans le modèle de macrophage de souris que ce LPS particulier échappe à la reconnaissance par la voie TLR4-TRIF, avec un rôle de l'antigène O et des lipoprotéines co-purifiées (Bonhomme et al. 2020). Des résultats préliminaires ont aussi montré que ce LPS échappait aussi à la voie de la caspase 11, impliquée dans l'activation de l'inflammasome non-canonique, expliquant ainsi l'absence d'induction de la pyroptose et de la mort cellulaire lors de l'infection par les leptospires. Nous avons ensuite travaillé sur d'autres mécanismes liés au LPS de L. interrogans. En travaillant avec des données obtenues précédemment dans le laboratoire, nous avons étudié la modulation de l'autophagie lors de l'infection et montré que L. interrogans, par la signalisation de son LPS, induisait une légère diminution de l'autophagie. De plus, nous avons montré que ce LPS était responsable de l'activation de l'axe p62/NRF2 dans les cellules, contribuant potentiellement à la diminution de l'autophagie. Enfin, nous avons montré que les leptospires étaient internalisées dans les macrophages humains et murins lors de l'infection mais disparaissaient rapidement. Nous avons étudié le rôle potentiel de TLR2 et TLR4 et montré que les diverses réponses antimicrobiennes induites par ces récepteurs (espèces réactives de l'oxygène, oxyde nitrique) n'étaient pas responsables du contrôle des leptospires intracellulaires. Bien que le mécanisme de disparition intracellulaire des leptospires reste inconnu, nous avons pu exclure de nombreuses voies potentiellement impliquées (phagocytose, autophagie, ROS), clarifiant ainsi la littérature conflictuelle actuelle. En conclusion, nous avons contribué à l'étude des deux facettes de l'interaction hôte-pathogène entre Leptospira interrogans et ses hôtes. Nous avons mis en évidence à la fois des mécanismes d'échappement et de reconnaissance qui pourraient expliquer la furtivité des leptospires.Leptospirosis is a re-emerging neglected zoonosis caused by the pathogenic bacteria Leptospira interrogans. In humans, leptospirosis usually presents as a flu-like syndrome but the severe form, also called Weil's disease, can provoke multi-organ failure and is potentially fatal. Leptospira interrogans can infect all vertebrates, but with different symptoms depending on the hosts. Mice and rats are resistant to the acute form of the disease and can become chronically infected in their kidneys, acting as natural reservoir. L. interrogans are stealth pathogens that have already been described to avoid some innate immune responses. The objectives of this Ph.D were to study the contribution of some leptospiral Microbial Associated Molecular Patterns (MAMPs) to the innate immune escape of the bacteria in macrophages from different hosts. More specifically, we focused on the study of the leptospiral flagellin, atypical lipopolysaccharide and lipoproteins and their respective recognition by Toll-Like Receptors (TLRs) and caspases. First, we finished a project on the escape of TLR5 recognition by live leptospires in human, murine and bovine hosts, due to the periplasmic localization of their endoflagella (Holzapfel, Bonhomme et al. 2020). Then, we studied the atypical leptospiral LPS recognition, the main focus of this thesis. We demonstrated in the mouse macrophage model that the leptospiral LPS escapes the recognition by the murine TLR4-TRIF pathway, with a role of the O antigen and co-purifying lipoproteins (Bonhomme et al. 2020). Preliminary results also showed that the leptospiral LPS escaped the caspase 11 pathway of the non-canonical inflammasome and hence explained the lack of induction of pyroptosis and cell death upon infection with leptospires. We then worked on other mechanisms related to the leptospiral LPS. Working with data previously obtained in the laboratory, we studied the modulation of autophagy upon infection and showed that L. interrogans, through signaling of their LPS, induced a slight diminution of autophagy. Furthermore, we showed that the leptospiral LPS induced the activation of p62/NRF2 axis in the cells, potentially contributing to autophagy dampening. Finally, we also showed that leptospires were internalized upon infection in murine and human macrophages, but disappeared quickly. We investigated the potential role of TLR2 & TLR4 and showed that various antimicrobial responses induced by these receptors (Reactive Oxygen Species, nitric oxide) were not accountable for the control of intracellular leptospires. Although the mechanism of intracellular leptospiral disappearance remains unexplained, we excluded many pathways potentially involved (phagocytosis, autophagy, ROS), hence clarifying the current conflictual literature. In conclusion, we contributed to the study of the two sides of the host-pathogen interaction between Leptospira interrogans and their hosts. We highlighted both escape and recognition mechanisms that could shed light on the stealthiness of the leptospires

Purification of LPS from Leptospira

International audienceLeptospira species are one of the few spirochetes to possess a lipopolysaccharide (LPS) embedded in their outer membrane. Two protocols are currently available to extract and/or purify the leptospiral lipopolysaccharides: the rapid proteinase K method and the classical hot water/phenol extraction. The first method allows to get a quick overview of the LPS O antigen structure, whereas the second method is fitted to study the immunological properties of the leptospiral LPS. These two methods will be detailed in this chapter. Methodologies to assess the quality of the purification, such as the modified silver staining coloration, will also be reviewed. Both advantages and limitations of the different analyses will be described

Potentialités de la microscopie électronique analytique à transmission pour l'étude de l'interface os/implant

International audienc

A cytokinin route to flowering in Arabidopsis

Cytokinins (CKs) are involved in many physiological processes. We observed that the application of N6-benzylaminopurine (BAP) to the roots of hydroponically grown plants of Arabidopsis thaliana promotes flowering in non-inductive short days. The response to BAP treatment does no require FLOWERING LOCUS T (FT), but activates its paralogue TWIN SISTER OF FT (TSF), as well as FD and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) (D'Aloia et al., 2011). We present here complementary data obtained with transgenic plants overexpressing a catalytic CK OXIDASE/DEHYDROGENASE (CKX) in the roots. The high efficiency of BAP in promoting flowering in our experimental system contrasts with the variability that emerges from studies gathered in literature. Many factors, either experimental or inherent to plant material, might explain these discrepancies and we are interested in identifying endogenous regulators that might provide a mechanistic explanation. We are therefore investigating whether the endogenous pathways underlying plant developmental phase changes might regulate the relative contribution of CKs to flowering

Proteomic and phosphoproteomic analyses of chromatin-associated proteins from Arabidopsis thaliana

International audienceThe nucleus is the organelle where basically all DNA-related processes take place in eukaryotes, such as replication, transcription, and splicing as well as epigenetic regulation. The identification and description of the nuclear proteins is one of the requisites toward a comprehensive understanding of the biological functions accomplished in the nucleus. Many of the regulatory mechanisms of protein functions rely on their PTMs among which phosphorylation is probably one of the most important properties affecting enzymatic activity, interaction with other molecules, localization, or stability. So far, the nuclear and subnuclear proteome and phosphoproteome of the model plant Arabidopsis thaliana have been the subject of very few studies. In this work, we developed a purification protocol of Arabidopsis chromatin-associated proteins and performed proteomic and phosphoproteomic analyses identifying a total of 879 proteins of which 198 were phosphoproteins that were mainly involved in chromatin remodeling, transcriptional regulation, and RNA processing. From 230 precisely localized phosphorylation sites (phosphosites), 52 correspond to hitherto unidentified sites. This protocol and data thereby obtained should be a valuable resource for many domains of plant research

Quantitative phosphoproteomic analysis reveals shared and specific targets of Arabidopsis mitogen-activated protein kinases (MAPKs) MPK3, MPK4, and MPK6

In Arabidopsis, mitogen-activated protein kinases MPK3, MPK4, and MPK6 constitute essential relays for a variety of functions including cell division, development and innate immunity. Although some substrates of MPK3, MPK4 and MPK6 have been identified, the picture is still far from complete. To identify substrates of these MAPKs likely involved in cell division, growth and development we compared the phosphoproteomes of wild-type and mpk3, mpk4, and mpk6. To study the function of these MAPKs in innate immunity, we analyzed their phosphoproteomes following microbe-associated molecular pattern (MAMP) treatment. Partially overlapping substrates were retrieved for all three MAPKs, showing target specificity to one, two or all three MAPKs in different biological processes. More precisely, our results illustrate the fact that the entity to be defined as a specific or a shared substrate for MAPKs is not a phosphoprotein but a particular (S/T) P phosphorylation site in a given protein. One hundred fifty-two peptides were identified to be differentially phosphorylated in response to MAMP treatment and/or when compared between genotypes and 70 of them could be classified as putative MAPK targets. Biochemical analysis of a number of putative MAPK substrates by phosphorylation and interaction assays confirmed the global phosphoproteome approach. Our study also expands the set of MAPK substrates to involve other protein kinases, including calcium-dependent (CDPK) and sugar nonfermenting (SnRK) protein kinases