Biology subjects

Wilhelm, J.

Publications and source records attributed to Wilhelm, J..

29 records · Page 2Linked to original sources

bioRxiv2022

Fgfr2b signaling is essential for the maintenance of the alveolar epithelial type 2 lineage during lung homeostasis in mice

Fibroblast growth factor receptor 2b (Fgfr2b) signaling is essential throughout lung development to form the alveolar epithelial lineage. However, its role in alveolar epithelial type 2 cells (AT2s) homeostasis was recently considered dispensable. SftpcCreERT2; tdTomatoflox/flox mice were used to delete Fgfr2b expression in cells belonging to the AT2 lineage, which contains mature AT2s and a novel SftpcLow lineage-traced population called "injury activated alveolar progenitors" or IAAPs. Upon continuous tamoxifen exposure for either one or two weeks to delete Fgfr2b, a shrinking of the AT2s is observed. Mature AT2s exit the cell cycle, undergo apoptosis and fail to form alveolospheres in vitro. However, the lung morphometry appears normal, suggesting the involvement of compensatory mechanisms. In mutant lungs, IAAPs which escaped Fgfr2b deletion expand, display enhanced alveolosphere formation in vitro and increase drastically their AT2 signature suggesting differentiation towards mature AT2s. Interestingly, a significant increase in AT2s and decrease in IAPPs occurs after a one-week tamoxifen exposure followed by an eight-week chase period. While mature AT2s partially recover their alveolosphere formation capabilities, the IAAPs no longer display this property. Single-cell RNA seq analysis confirms that AT2s and IAAPs represent stable and distinct cell populations and recapitulate some of their characteristics observed in vivo. Our results underscore the essential role played by Fgfr2b signaling in the maintenance of the AT2 lineage in the adult lung and suggest that the IAAPs could represent a new population of AT2 progenitors.

developmental biology↗

bioRxiv2022

Characterization of alveolar epithelial lineage heterogeneity during the late pseudoglandular stage of mouse lung development

The specification, characterization, and fate of alveolar type 1 and type 2 (AT1 and 2) progenitors during embryonic lung development remains mostly elusive. In this paper, we build upon our previously published work on the regulation of airway epithelial progenitors by fibroblast growth factor receptor 2b (Fgfr2b) signalling during early (E12.5) and mid (E14.5) pseudoglandular lung development. Here, we looked at the regulation by Fgfr2b signalling on alveolar progenitors during late pseudoglandular/early canalicular (E14.5-E16.5) development. Using a dominant negative mouse model to conditionally inhibit Fgfr2b ligands at E16.5, we used gene array analyses to characterize a set of potential direct targets of Fgfr2b signalling. By mining published single-cell RNA sequence (scRNAseq) datasets, we showed that these Fgfr2b signature genes narrow on a discreet subset of AT2 cells at E17.5 and in adult lungs. Furthermore, we demonstrated that Fgfr2b signalling is lost in AT2 cells in their transition to AT1 cells during repair after injury. We also used CreERT2-based mouse models to conditionally knock-out the Fgfr2b gene in AT2 and in AT1 progenitors, as well as lineage label these cells. We found, using immunofluorescence, that in wildtype controls AT1 progenitors labeled at E14.5-E15.5 contribute a significant proportion to AT2 cells at E18.5; while AT2 progenitors labeled at the same time contribute significantly to the AT1 lineage. We show, using immunofluorescence and FACS-based analysis, that knocking out of Fgfr2b at E14.5-E15.5 in AT2 progenitors leads to an increase in lineage-labeled AT1 cells at E18.5; while the reverse is true in AT1 progenitors. Furthermore, we demonstrate that increased Fgfr signalling in AT2 progenitors reduces their contribution to the AT1 pool. Taken together, our results suggest that a significant proportion of AT2 and AT1 progenitors are cross-lineage committed during late pseudoglandular development, and that lineage commitment is regulated in part by Fgfr2b signalling. We have characterized a set of direct Fgfr2b targets at E16.5 which are likely involved in alveolar lineage formation. These signature genes concentrate on a subpopulation of AT2 cells later in development, and are downregulated in AT2 cells transitioning to the AT1 lineage during repair after injury in adults. Our findings highlight the extensive heterogeneity of alveolar cells by elucidating the role of Fgfr2b signalling in these cells during early alveolar lineage formation, as well as during repair after injury.

developmental biology↗

bioRxiv2021

Lactate Increases Stemness of CD8+ T Cells to Augment Anti-Tumor Immunity

Nutrients and metabolites play important roles in immune functions. Recent studies show lactate instead of glucose can serve as a primary carbon fuel source for most tissues. The role of lactate in tumor immunity is not well understood with immune suppressive functions reported for lactic acid, the conjugate acid form of lactate. In this study, we report lactate increases the stemness of CD8+ T cells and augments anti-tumor immunity. Subcutaneous administration of lactate but not glucose shows CD8+ T cell-dependent tumor growth inhibition. Single cell transcriptomics analysis revealed lactate treatment increased a subpopulation of stem-like TCF-1-expressing CD8+ T cells, which is further validated by ex vivo culture of CD8+ T cells from mouse splenocytes and human peripheral blood mononuclear cells. The inhibition of histone deacetylase activity by lactate increased acetylation in the histone H3K27 site at the Tcf7 super enhancer locus and increased the gene expression of Tcf7. Adoptive transfer of CD8+ T cells pretreated with lactate in vitro showed potent tumor growth inhibition in vivo. Our results elucidate the immune protective role of lactate in anti-tumor immunity without the masking effect of acid. These results may have broad implications for T cell therapy and the understanding of lactate in immune metabolism.

immunology↗

bioRxiv2021

Variation in root system architecture among the founder parents of two 8-way MAGIC wheat populations for selection in breeding

Root system architecture (RSA) is a target for breeding because of the interest to develop crops with roots that use nutrients and water more effectively. Breeding for RSA requires phenotypic diversity in populations amenable to QTL identification to provide markers for large breeding programs. This study examined the variation for root traits across the parents of two multi-parent advanced generation inter-cross (MAGIC) wheat populations from NIAB and CSIRO for 16 days in an upgraded version of the non-invasive, germination paper-based phenotyping platform, GrowScreen-PaGe. Across all parents, total root length varied up to 1.90 fold, root biomass 2.25 fold and seminal root angle 1.16 fold. The CSIRO parents grew faster, exhibited slightly wider seminal root angle and produced larger root systems compared to NIAB parents. Lateral root lengths, leaf lengths and biomass contrasted most between fastest (Robigus - NIAB and AC Barrie - CSIRO) and slowest growing parents (Rialto - NIAB and G204 Xiaoyan54 - CSIRO). Lengths of lateral and total root, and leaf number and length had moderate to high heritability (0.30-0.67) and repeatability. Lengths of lateral roots and leaves are good targets for enhancing wheat crop establishment, a critical stage for crop productivity.

plant biology↗

bioRxiv2021

Characterization of changes in the hemagglutinin that accompanied the emergence of H3N2/1968 pandemic influenza viruses

The hemagglutinin (HA) of A/H3N2 pandemic influenza viruses (IAVs) of 1968 differed from its inferred avian precursor by eight amino acid substitutions. To determine their phenotypic effects, we studied recombinant variants of A/Hong Kong/1/1968 virus containing either human-type or avian-type amino acids in the corresponding positions of HA. The precursor HA displayed receptor binding profile and high conformational stability typical for duck IAVs. Substitutions Q226L and G228S, in addition to their known effects on receptor specificity and replication, marginally decreased HA stability. Substitutions R62I, D63N, D81N and N193S reduced HA binding avidity. Substitutions R62I, D63N, D81N and A144G promoted virus replication in human airway epithelial cultures. Analysis of HA sequences revealed that substitutions D63N and D81N accompanied by the addition of N-glycans represent common markers of avian H3 HA adaptation to mammals. Our results advance understanding of genotypic and phenotypic changes in IAV HA required for avian-to-human adaptation and pandemic emergence.

microbiology↗

bioRxiv2021

Kinetic and structural characterization of the self-labeling protein tags HaloTag7, SNAP-tag and CLIP-tag

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study on the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rates with certain rhodamine substrates, which are more than two orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rates however are less affected by the structure of the label than those of HaloTag7, which vary over six orders of magnitude for commonly employed substrates. Solving the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.

biochemistry↗

bioRxiv2021

Characterization in mice of the stromal niche maintaining AT2 stem cell self-renewal in homeostasis and disease

Resident mesenchymal cells (rMCs defined as Cd31NegCd45NegEpcamNeg) control the self-renewal and differentiation of alveolar epithelial type 2 (AT2) stem cells in vitro. The identity of these rMCs is still elusive. Among them, Axin2Pos mesenchymal alveolar niche cells (MANCs), which are expressing Fgf7, have been previously described. We propose that an additional population of rMCs, expressing Fgf10 (called rMC-Sca1PosFgf10Pos) are equally important to maintain AT2 stem cell self-renewal. The alveolosphere model, based on the AT2-rMC co-culture in growth factor reduced Matrigel, was used to test the efficiency of different rMC subpopulations isolated by FACS from adult murine lung to sustain the self-renewal and differentiation of AT2 stem cells. We demonstrate that rMC-Sca1PosFgf10Pos cells are efficient to promote the self-renewal and differentiation of AT2 stem cells. Co-staining of adult lung for Fgf10 mRNA and Sftpc protein respectively, indicate that 28% of Fgf10Pos cells are located close to AT2 cells. Co-ISH for Fgf7 and Fgf10 indicate that these two populations do not significantly overlap. Gene arrays comparing rMC-Sca1PosAxin2Pos and rMC-Sca1PosFgf10Pos support that these two cell subsets express differential markers. In addition, rMC function is decreased in diabetic and obese ob/ob mutant compared to WT mice with a much stronger loss of function in males compared to females. In conclusion, rMC-Sca1PosFgf10Pos cells play important role in supporting AT2 stem cells self-renewal and differentiation. This result sheds a new light on the subpopulations of rMCs contributing to the AT2 stem cell niche in homeostasis and in the context of COVID-19 pathogenesis. Key messageO_ST_ABSWhat is already known about the subject?C_ST_ABSResident mesenchymal cells (rMCs defined as Cd31NegCd45NegEpcamNeg) control the self-renewal and differentiation of alveolar epithelial type 2 (AT2) stem cells in vitro. The identity of these rMCs is still elusive. Among them, Axin2Pos mesenchymal alveolar niche cells (MANCs), which are expressing Fgf7, have been previously described. What does this study add?Our study shows that an additional population of rMCs, expressing Fgf10 (called rMC-Sca1PosFgf10Pos) is equally important to maintain AT2 stem cell self-renewal. rMC-Sca1PosFgf10Pos are LipidToxHigh and are located close to AT2s. In addition, rMC-Sca1PosFgf10Pos cells support AT2 stem cell self-renewal and differentiation thereby identifying these cells as bone fide functional lipofibroblasts (LIFs). We have previously reported that LIF can transdifferentiate into activated MYF in the context of bleomycin-induced fibrosis in mice [1] and that activated MYF isolated from the lungs of end stage idiopathic fibrosis human patients can respond to Metformin to undergo transdifferentiation back to the LIF phenotype [2]. We also show that the function of rMCs-Sca1Pos is negatively impacted by gender and obesity, which represent two major aggravating factors for COVID-19 pathogenesis, leading to either death or major complications after infection recovery such as lung fibrosis. How might this impact on clinical practice and future development?By establishing that rMC-Sca1PosFgf10Pos are different from the MANCs, our study opens the way for a new key mesenchymal cell population that should be targeted to either prevent or reverse fibrosis. In addition, as this population maintains the AT2 stem cells self-renewal and differentiation, such targeting will also allow to progressively recover the loss in respiratory function.

developmental biology↗

bioRxiv2020

Identification a novel subset of alveolar type 2 cells expanding following pneumonectomy and enriched in PD-L1

Alveolar type 2 (AT2) cells are heterogeneous cells; where specialized AT2 subpopulations within this lineage exhibit stem cell properties. However, the existence of quiescent, immature cells within the AT2 lineage, which get activated during lung regeneration, is unknown. SftpcCreERT2/+; tdTomatoflox/flox mice were used for the labelling of AT2 cells and labeled subpopulations were analyzed by flow cytometry, qPCR, ATAC-seq, gene arrays, pneumonectomy, and culture of precision-cut lung slides. Human lungs from donor and IPF were also analyzed. In mice, we detected two distinct AT2 subpopulations with low tdTomato level (TomLow) and high tdTomato level (TomHigh). TomLow express lower level of AT2 differentiation markers, Fgfr2b and Etv5, while TomHigh, as bona fide mature AT2 cells, show higher level of Sftpc, Sftpb, Sftpa1, Fgfr2b, and Etv5. ATAC-seq analysis indicates that TomLow and TomHigh constitute two distinct cell populations with specific silencing of Sftpc, Rosa26 and cell cycle gene loci in TomLow. Upon pneumonectomy, TomLow but not TomHigh cells proliferate and upregulate the expression of Fgfr2b, Etv5, Sftpc, Ccnd1 and Ccnd2 compared to sham. TomLow cells overexpress PD-L1, an immune inhibitory membrane receptor ligand, which is used by flow cytometry to differentially isolate these two sub-populations. In the human lung, PD-L1 and HTII-280 antibodies are used by flow cytometry to differentially sort mature AT2 (HTII-280-high, PD-L1-low) as well as an additional subpopulation of epithelial cells characterized by HTII-280-Low and PD-L1-high. We have identified a novel population of AT2 quiescent immature progenitor cells in mouse that proliferate upon pneumonectomy and provided evidence for the existence of such cells in human. Significance of the workThe characterization and mechanism of the proliferation of a novel and relevant pool of AT2 progenitor cells for the repair/regeneration process after injury are critical to improving respiratory function in patients with lung disease.

cell biology↗

bioRxiv2019

Restored alveolar epithelial differentiation and reversed human lung fibrosis upon Notch inhibition

Alveolar epithelial cell type II (AEC2) injury underlies idiopathic pulmonary fibrosis (IPF). Here we show increased Notch1 signaling in AEC2s in human IPF and IPF models, causing enhanced proliferation and de-differentiation of AEC2s. As a result, we observed defective surfactant protein (SP)-B/C processing, elevated alveolar surface tension, repetitive alveolar collapse and development of lung fibrosis. Similar changes were encountered upon pharmacological inhibition of SP-B/C processing in vivo by pepstatin A. Inhibition of Notch signaling in cultured human IPF precision cut lung slices improved surfactant processing capacity of AEC2s and reversed fibrosis. Notch1 therefore offers as novel therapeutic target.\n\nOne sentence summaryNotch1 inhibition restores alveolar epithelial differentiation and surface tension and reverses matrix deposition in lung fibrosis

cell biology↗

bioRxiv2018

Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

Idiopathic pulmonary fibrosis is a fatal, incurable lung disease in which the intricate alveolar network of the human lung is progressively replaced by fibrotic scars, eventually leading to respiratory failure. Myofibroblasts are the effector cells that lead to abnormal deposition of extracellular matrix proteins and therefore mediate fibrotic disease not only in the lung but also in other organs. Emerging literature suggests a correlation between fibrosis and metabolic alterations in IPF. In this study, we show that the first-line antidiabetic drug, metformin, exerts potent antifibrotic effects in the lung by modulating metabolic pathways, inhibiting TGF{beta}1 action, suppressing collagen formation, activating PPAR{gamma} signaling and inducing lipogenic differentiation in lung myofibroblasts derived from human patients. Using genetic lineage tracing in a murine model of lung fibrosis, we show that metformin alters the fate of myofibroblasts and accelerates fibrosis resolution by inducing myofibroblast-tolipofibroblast transdifferentiation. Detailed pathway analysis showed that the reduction of collagen synthesis was largely AMPK-dependent, whereas the transdifferentiation of myo- to lipofibroblasts occurred in a BMP2-PPAR{gamma}-dependent fashion and was largely AMPK-independent. Our data report an unprecedented role for metformin in lung fibrosis, thus warranting further therapeutic evaluation.

cell biology↗

bioRxiv2018

A critical role for miR-142 in alveolar epithelial lineage formation

The development of a functional lung, capable of gas exchange, requires proper alveologenesis. Mechanisms regulating AT1 and AT2 cell maturation are poorly defined. We report the activation of the glucocorticoid pathway in an in vitro alveolar epithelial lineage differentiation assay led to increased AT2 marker Sftpc and decreased miR-142 expression. Using a constitutive KO mouse model, we further demonstrate a relative increase of AT2 and a decrease in AT1 cell number with a global decrease of AT2 gene profile signature in miR-142 KO AT2 cells. Over-expression of miR-142 in alveolar progenitor cells in vivo led to an opposite effect. Examination of the KO lungs at E18.5, revealed enhanced expression miR-142 targets like Apc, Ep300 and Kras associated with increased Ctnnb1 and p-Erk signaling. Pharmacological inhibition of Ep300-Ctnnb1 in vitro prevented an increase in Sftpc expression triggered by loss of miR-142. These results together suggest glucocorticoid-miR-142-p300 signaling axis controls pneumocyte maturation.

developmental biology↗