Roles of the STAT5 transcription factor in neutrophil homeostasy and inflammation

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Characterization of pentraxin 3 in the horse and its expression in airways

The long pentraxin 3 (PTX3) plays an important role in host defence and its over-expression may contribute to airway injury. The aim of the present study was therefore to characterize in more detail PTX3 and its expression in the horses’ airway. Six healthy horses and six horses affected by recurrent airway obstruction (R.A.O.) were submitted to a dusty environment challenge. PTX3 DNA and cDNA were cloned and sequenced. PTX3 expression was evaluated by RT-qPCR, Western blotting and immuno-histochemistry in bronchoalveolar lavage fluid (BALF) cells, BALF supernatant and bronchial epithelial cells. An alternative splicing of the second exon of PTX3 occurred, resulting in two forms of the protein: “spliced” (32 kDa) and “full length” (42 kDa). PTX3 was detected in BALF macrophages, neutrophils and bronchial epithelial cells. It was over-expressed in the BALF supernatant from R.A.O.-affected horses in crisis. However, dust was unable to induce PTX3 in BALF cells ex vivo, indicating that dust is an indirect inducer of PTX3. Dust exposure in-vivo induced PTX3 in BALF macrophages but there was no significant difference between healthy and R.A.O.-affected horses. Conversely, PTX3 was over-expressed in the bronchial epithelial cells from R.A.O.-affected horses in crisis. These data indicate a differential regulatory mechanism in inflammatory and bronchial epithelial cells and offer therapeutically interesting perspectives

Comparison of inulin with urea as dilutional markers of bronchoalveolar lavage in healthy and heaves-affected horses.

Solute analysis in bronchoalveolar lavage fluid involves the use of dilutional markers to correct for variable recovery of pulmonary epithelial lining fluid (PELF). Urea is the best characterised endogenous marker, whereas inulin appears to meet the requirements of an exogenous marker. In horses, the use of inulin has never been investigated and the impact of lower airway diseases such as heaves, on PELF recovery is unknown. In this study, five healthy and five heaves-affected horses underwent airway endoscopy and bronchoalveolar lavage. PELF recovery from bronchoalveolar lavage was calculated by the inulin and the urea method. The inulin method was compared to the urea method and differences between healthy and heaves-affected horses were analysed. From a technical and analytical point of view, inulin fulfilled the requirements of a marker of dilution as well as urea. When both healthy and heaves-affected horses groups were pooled together, PELF recovery calculated by the inulin method was significantly higher than by the urea method (6.43+/-4.08% versus 0.789+/-0.299%, P < 0.005). No significant differences were observed between healthy and heaves-affected horses, neither by the inulin nor by the urea method. Inulin did not present major advantages over urea, but the combined use of both markers can improve the standardisation of studies comparing PELF compounds, by providing upper limits (inulin dilution) and lower limits (urea dilution) of PELF recovery.Peer reviewe

Generation of a comprehensive molecular cell atlas of the healthy canine lung

peer reviewedSingle cell RNA sequencing (scRNA-seq) is a powerful transcriptomic technique to analyse cell expression profiles across various tissues or conditions, and to identify new cell subpopulations. It has been extensively used in human and mouse studies, and more recently for identification of cellular subpopulations in the bronchoalveolar lavage fluid of dogs. To date, the molecular state of all cell types in canine lung tissue has not been profiled. Such study will help to determine specific cell markers, often lacking in the canine species, and will also provide the foundation for further comparisons with specific lung diseases at single-cell level, such as canine pulmonary fibrosis or neoplasia. In this context, we had a particular interest in fibroblast subpopulations and their expression profiles. Indeed, molecules expressed by fibroblasts (and by cancer-associated fibroblasts) are of potential interest for further development of early markers of disease and of novel molecular fibroblast-targeting therapies. We performed droplet-based scRNA-seq on fresh healthy lung biopsies from three dogs. Two biopsies were collected from dogs euthanized for unrelated reasons, and one was collected from the tumour-free area of a lung lobe resected for primary adenocarcinoma. Biopsies were systematically collected at the peripheral part of the right caudal lobe. Tissues were dissociated to obtain single-cell suspensions, which were loaded into the Chromium Controller (10x Genomics). Clustering, visualization and gene profiling was achieved using the Seurat package in R. Distinct cell populations were identified based on canonical or literature-described cell markers. A total of 22,424 cells were sequenced. Four main cell compartments were identified and individually investigated: epithelial cells (EPCAM+, 5 subpopulations), immune cells (PTPRC+, 17 subpopulations), endothelial cells (PECAM1+, 5 subpopulations) and mesenchymal cells (EPCAM-/PTPRC-/PECAM1-, 10 subpopulations). Clustering resolution was high enough to consistently discriminate different cell subpopulations within classical cell types such as fibroblasts, smooth muscle cells, lymphocytes or macrophages, for example. Among fibroblasts, cluster analysis highlighted subpopulations already identified in humans such as alveolar or adventitial fibroblasts. Differential gene expression profiles were defined for all 37 cell subpopulations, thus identifying new specific cell markers for all cells of the canine lung. This is the first report of scRNA-seq analysis of canine lung tissue, expanding our knowledge of canine distal lung cell subpopulations. This study provides the foundation for comparisons with specific lung diseases at single-cell level, such as canine pulmonary fibrosis or canine pulmonary neoplasia, for which development of emerging therapies are cruelly required

Morpho-functional Characterisation of Cœlomocytes in the Aquacultivated Sea Cucumber Holothuria Scabra: From Cell Diversity to Transcriptomic Immune Response

Holothuria scabra is one of the most valuable species of sea cucumber owing to its exploitation as a seafood product. This study aims to describe the main molecular and cellular actors in the immunology of the holothuroid H. scabra. First of all, a detailed description of the immune cells – the cœlomocytes – is provided, highlighting five main cell types including phagocytes, small round cells (SRCs), spherulocytes, fusiform cells, and crystal cells, with a further five subtypes identified using transmission electron microscopy. Cœlomocyte aggregates were also described morphologically, yielding two main types, one comprising three successive maturation stages. A comparison of the concentration and proportion of cell populations was carried out between the two main body fluids, namely the hydrovascular fluid of the Polian vesicle (HF) and the perivisceral fluid of the general cavity (PF), and no clear relation could be revealed. Next, the cœlomocyte immune response was studied 24 hours after a lipopolysaccharide (LPS) injection. Firstly, the fluctuation in cell populations was assessed, and despite a high inter-individual variability, it shows a decrease in the phagocyte proportion and an increase in the SRC proportion. Secondly, the differential gene expression of PF cœlomocytes was studied by de novo RNA-sequencing between LPS-injected and control-injected individuals: 945 genes were differentially expressed, including 673 up-regulated and 272 down-regulated in the LPS-injected individuals. Among these genes, 80 had a presumed function in immunity based on their annotation, covering a wide range of immune mechanisms. Overall, this study reveals a complex immune system at both molecular and cellular levels and constitutes a baseline reference on H. scabra immunity, which may be useful for the development of sustainable aquaculture and provides valuable data for comparative immunology

Lead Drives Complex Dynamics of a Conjugative Plasmid in a Bacterial Community.

Plasmids carrying metal resistance genes (MRGs) have been suggested to be key ecological players in the adaptation of metal-impacted microbial communities, making them promising drivers of bio-remediation processes. However, the impact of metals on plasmid-mediated spread of MRGs through selection, plasmid loss, and transfer is far from being fully understood. In the present study, we used two-member bacterial communities to test the impact of lead on the dispersal of the IncP plasmid pKJK5 from a Pseudomonas putida KT2440 plasmid donor and two distinct recipients, Variovorax paradoxus B4 or Delftia acidovorans SPH-1 after 4 and 10 days of mating. Two versions of the plasmid were used, carrying or not carrying the lead resistance pbrTRABCD operon, to assess the importance of fitness benefit and conjugative potential for the dispersal of the plasmid. The spread dynamics of metal resistance conveyed by the conjugative plasmid were dependent on the recipient and the lead concentration: For V. paradoxus, the pbr operon did not facilitate neither lead resistance nor variation in plasmid spread. The growth gain brought by the pbr operon to D. acidovorans SPH-1 and P. putida KT2440 at 1 mM Pb enhanced the spread of the plasmid. At 1.5 mM Pb after 4 days, the proteomics results revealed an oxidative stress response and an increased abundance of pKJK5-encoded conjugation and partitioning proteins, which most likely increased the transfer of the control plasmid to D. acidovorans SPH-1 and ensured plasmid maintenance. As a consequence, we observed an increased spread of pKJK5-gfp. Conversely, the pbr operon reduced the oxidative stress response and impeded the rise of conjugation- and partitioning-associated proteins, which slowed down the spread of the pbr carrying plasmid. Ultimately, when a fitness gain was recorded in the recipient strain, the spread of MRG-carrying plasmids was facilitated through positive selection at an intermediate metal concentration, while a high lead concentration induced oxidative stress with positive impacts on proteins encoding plasmid conjugation and partitioning

Identification of pro-fibrotic macrophage populations by single-cell transcriptomic analysis in West Highland white terriers affected with canine idiopathic pulmonary fibrosis.

Canine idiopathic pulmonary fibrosis (CIPF) affects old dogs from the West Highland white terrier (WHWT) breed and mimics idiopathic pulmonary fibrosis (IPF) in human. The disease results from deposition of fibrotic tissue in the lung parenchyma causing respiratory failure. Recent studies in IPF using single-cell RNA sequencing (scRNA-seq) revealed the presence of profibrotic macrophage populations in the lung, which could be targeted for therapeutic purpose. In dogs, scRNA-seq was recently validated for the detection of cell populations in bronchoalveolar lavage fluid (BALF) from healthy dogs. Here we used the scRNA-seq to characterize disease-related heterogeneity within cell populations of macrophages/monocytes (Ma/Mo) in the BALF from 5 WHWTs affected with CIPF in comparison with 3 healthy WHWTs. Gene set enrichment analysis was also used to assess pro-fibrotic capacities of Ma/Mo populations. Five clusters of Ma/Mo were identified. Gene set enrichment analyses revealed the presence of pro-fibrotic monocytes in higher proportion in CIPF WHWTs than in healthy WHWTs. In addition, monocytes-derived macrophages enriched in pro-fibrotic genes in CIPF compared with healthy WHWTs were also identified. These results suggest the implication of Ma/Mo clusters in CIPF processes, although, further research is needed to understand their role in disease pathogenesis. Overexpressed molecules associated with pulmonary fibrosis processes were also identified that could be used as biomarkers and/or therapeutic targets in the future

MafB-restricted local monocyte proliferation precedes lung interstitial macrophage differentiation.

peer reviewedResident tissue macrophages (RTMs) are differentiated immune cells that populate distinct niches and exert important tissue-supportive functions. RTM maintenance is thought to rely either on differentiation from monocytes or on RTM self-renewal. Here, we used a mouse model of inducible lung interstitial macrophage (IM) niche depletion and refilling to investigate the development of IMs in vivo. Using time-course single-cell RNA-sequencing analyses, bone marrow chimeras and gene targeting, we found that engrafted Ly6C+ classical monocytes proliferated locally in a Csf1 receptor-dependent manner before differentiating into IMs. The transition from monocyte proliferation toward IM subset specification was controlled by the transcription factor MafB, while c-Maf specifically regulated the identity of the CD206+ IM subset. Our data provide evidence that, in the mononuclear phagocyte system, the ability to proliferate is not merely restricted to myeloid progenitor cells and mature RTMs but is also a tightly regulated capability of monocytes developing into RTMs in vivo

University population-based prospective cohort study of SARS-CoV- 2 infection and immunity (SARSSURV-ULiège): a study protocol

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