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Hematopoietic stem/progenitor cells (HSPC) are characterized by their unique capacities of self-renewal and multi-differentiation potential. This second property makes them able to adapt their differentiation profile depending on the local environment they reach. Taking advantage of an animal model of peritonitis,induced by injection of the TLR-2 ligand,zymosan,we sought to study the relationship between bone marrow-derived hematopoietic stem/progenitor cells (BM-HSPCs) and innate lymphoid cells (ILCs) regarding their emergence and differentiation at the site of inflammation. Our results demonstrate that the strength of the inflammatory signals affects the capacity of BM-derived HSPCs to migrate and give rise in situ to ILCs. Both low- and high-dose of zymosan injections trigger the appearance of mature ILCs in the peritoneal cavity where the inflammation occurs. Herein,we show that only in low-dose injected mice,the recovered ILCs are dependent on an in situ differentiation of BM-derived HSPCs and/or ILC2 precursors (ILC2P) wherein high-dose,the stronger inflammatory environment seems to be able to induce the emergence of ILCs independently of BM-derived HSPCs. We suggest that a relationship between HSPCs and ILCs seems to be affected by the strength of the inflammatory stimuli opening new perspectives in the manipulation of these early hematopoietic cells. View Publication

Cellular senescence,which is characterized by an irreversible cell-cycle arrest1 accompanied by a distinctive secretory phenotype2,can be induced through various intracellular and extracellular factors. Senescent cells that express the cell cycle inhibitory protein p16INK4A have been found to actively drive naturally occurring age-related tissue deterioration3,4 and contribute to several diseases associated with ageing,including atherosclerosis5 and osteoarthritis6. Various markers of senescence have been observed in patients with neurodegenerative diseases7-9; however,a role for senescent cells in the aetiology of these pathologies is unknown. Here we show a causal link between the accumulation of senescent cells and cognition-associated neuronal loss. We found that the MAPTP301SPS19 mouse model of tau-dependent neurodegenerative disease10 accumulates p16INK4A-positive senescent astrocytes and microglia. Clearance of these cells as they arise using INK-ATTAC transgenic mice prevents gliosis,hyperphosphorylation of both soluble and insoluble tau leading to neurofibrillary tangle deposition,and degeneration of cortical and hippocampal neurons,thus preserving cognitive function. Pharmacological intervention with a first-generation senolytic modulates tau aggregation. Collectively,these results show that senescent cells have a role in the initiation and progression of tau-mediated disease,and suggest that targeting senescent cells may provide a therapeutic avenue for the treatment of these pathologies. View Publication

As the sole target of broadly neutralizing antibodies (bnAbs) to HIV,the envelope glycoprotein (Env) trimer is the focus of vaccination strategies designed to elicit protective bnAbs in humans. Because HIV Env is densely glycosylated with 75-90 N-glycans per trimer,most bnAbs use or accommodate them in their binding epitope,making the glycosylation of recombinant Env a key aspect of HIV vaccine design. Upon analysis of three HIV strains,we here find that site-specific glycosylation of Env from infectious virus closely matches Envs from corresponding recombinant membrane-bound trimers. However,viral Envs differ significantly from recombinant soluble,cleaved (SOSIP) Env trimers,strongly impacting antigenicity. These results provide a benchmark for virus Env glycosylation needed for the design of soluble Env trimers as part of an overall HIV vaccine strategy. View Publication

Cytokine responses from monocytes and macrophages exposed to bacteria are of particular importance in innate immunity. Focusing on the impact of the immunoregulatory cytokine interleukin (IL)-27 on control of innate immune system responses,we examined human immune responses to bacterial products and bacterial infection by E. coli and S. typhimurium. Since the effect of IL-27 treatment in human myeloid cells infected with bacteria is understudied,we treated human monocytes and macrophages with IL-27 and either LPS,flagellin,or bacteria,to investigate the effect on inflammatory signaling and cytokine responses. We determined that simultaneous stimulation with IL-27 and LPS derived from E. coli or S. typhimurium resulted in enhanced IL-12p40,TNF-$\alpha$,and IL-6 expression compared to that by LPS alone. To elucidate if IL-27 manipulated the cellular response to infection with bacteria,we infected IL-27 treated human macrophages with S. typhimurium. While IL-27 did not affect susceptibility to S. typhimurium infection or S. typhimurium-induced cell death,IL-27 significantly enhanced proinflammatory cytokine production in infected cells. Taken together,we highlight a role for IL-27 in modulating innate immune responses to bacterial infection. View Publication

Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here,we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the $\beta$-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation,there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70{\%} of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting,HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules,UM171 and SR1,stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of $\beta$-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research. View Publication

Neutrophils are short-lived cells that play important roles in both health and disease. Neutrophils and monocytes originate from the granulocyte monocyte progenitor (GMP) in bone marrow; however,unipotent neutrophil progenitors are not well defined. Here,we use cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) methodologies to identify a committed unipotent early-stage neutrophil progenitor (NeP) in adult mouse bone marrow. Importantly,we found a similar unipotent NeP (hNeP) in human bone marrow. Both NeP and hNeP generate only neutrophils. NeP and hNeP both significantly increase tumor growth when transferred into murine cancer models,including a humanized mouse model. hNeP are present in the blood of treatment-naive melanoma patients but not of healthy subjects. hNeP can be readily identified by flow cytometry and could be used as a biomarker for early cancer discovery. Understanding the biology of hNeP should allow the development of new therapeutic targets for neutrophil-related diseases,including cancer. View Publication

Traumatic spinal cord injury results in persistent disability due to disconnection of surviving neural elements. Neural stem cell transplantation has been proposed as a therapeutic option,but optimal cell type and mechanistic aspects remain poorly defined. Here,we describe robust engraftment into lesioned immunodeficient mice of human neuroepithelial stem cells derived from the developing spinal cord and maintained in self-renewing adherent conditions for long periods. Extensive elongation of both graft and host axons occurs. Improved functional recovery after transplantation depends on neural relay function through the grafted neurons,requires the matching of neural identity to the anatomical site of injury,and is accompanied by expression of specific marker proteins. Thus,human neuroepithelial stem cells may provide an anatomically specific relay function for spinal cord injury recovery. View Publication

Enterochromaffin (EC) cells constitute the largest population of intestinal epithelial enteroendocrine (EE) cells. EC cells are proposed to be specialized mechanosensory cells that release serotonin in response to epithelial forces,and thereby regulate intestinal fluid secretion. However,it is unknown whether EE and EC cells are directly mechanosensitive,and if so,what the molecular mechanism of their mechanosensitivity is. Consequently,the role of EE and EC cells in gastrointestinal mechanobiology is unclear. Piezo2 mechanosensitive ion channels are important for some specialized epithelial mechanosensors,and they are expressed in mouse and human EC cells. Here,we use EC and EE cell lineage tracing in multiple mouse models to show that Piezo2 is expressed in a subset of murine EE and EC cells,and it is distributed near serotonin vesicles by superresolution microscopy. Mechanical stimulation of a subset of isolated EE cells leads to a rapid inward ionic current,which is diminished by Piezo2 knockdown and channel inhibitors. In these mechanosensitive EE cells force leads to Piezo2-dependent intracellular Ca2+ increase in isolated cells as well as in EE cells within intestinal organoids,and Piezo2-dependent mechanosensitive serotonin release in EC cells. Conditional knockout of intestinal epithelial Piezo2 results in a significant decrease in mechanically stimulated epithelial secretion. This study shows that a subset of primary EE and EC cells is mechanosensitive,uncovers Piezo2 as their primary mechanotransducer,defines the molecular mechanism of their mechanotransduction and mechanosensitive serotonin release,and establishes the role of epithelial Piezo2 mechanosensitive ion channels in regulation of intestinal physiology. View Publication

Uncoupling of oxidative phosphorylation in epithelial mitochondria results in decreased epithelial barrier function as characterized by increased internalization of non-invasive Escherichia coli and their translocation across the epithelium. We hypothesized that the increased burden of intracellular commensal bacteria would activate the enterocyte,with the potential to promote inflammation. Treatment of human colon-derived epithelial cell lines in vitro with dinitrophenol (DNP) and commensal E. coli (strains F18,HB101) provoked increased production of interleukin (IL-8),which was not observed with conditioned medium from the bacteria,lipopolysaccharide or inert beads. The IL-8 response was inhibited by co-treatment with cytochalasin-D (blocks F-actin rearrangement),chloroquine (blocks phagosome acidification) and a MyD88 inhibitor (blocks TLR signaling),consistent with TLR-signaling mediating IL-8 synthesis subsequent to bacterial internalization. Use of the mitochondria-targeted antioxidant,mitoTEMPO,or U0126 to block ERK1/2 MAPK signalling inhibited DNP+E. coli-evoked IL-8 production. Mutations in the NOD2 (the intracellular sensor of bacteria) or ATG16L1 (autophagy protein) genes are susceptibility traits for Crohn's,and epithelia lacking either protein displayed enhanced IL-8 production in comparison to wild-type cells when exposed to DNP + E coli. Thus,metabolic stress perturbs the normal epithelial-bacterial interaction resulting in increased IL-8 production due to uptake of bacteria into the enterocyte: this potentially pro-inflammatory event is enhanced in cells lacking NOD2 or ATG16L1 that favor increased survival of bacteria within the enterocyte. We speculate that by increasing epithelial permeability and IL-8 production,reduced mitochondria function in the enteric epithelium would contribute to the initiation,pathophysiology,and reactivation of inflammatory disease in the gut. View Publication

YAP,a key effector of Hippo pathway,is activated by its translocation from cytoplasm to nucleus to regulate gene expression and promote tumorigenesis. Although the mechanism by which YAP is suppressed in cytoplasm has been well-studied,how the activated YAP is sequestered in the nucleus remains unknown. Here,we demonstrate that YAP is a nucleocytoplasmic shuttling protein and its nuclear export is controlled by SET1A-mediated mono-methylation of YAP at K342,which disrupts the binding of YAP to CRM1. YAP mimetic methylation knockin mice are more susceptible to colorectal tumorigenesis. Clinically,YAP K342 methylation is reversely correlated with cancer survival. Collectively,our study identifies SET1A-mediated mono-methylation at K342 as an essential regulatory mechanism for regulating YAP activity and tumorigenesis. View Publication

Cattle are an economically important domestic animal species. In vitro 2D cultures of intestinal epithelial cells or epithelial cell lines have been widely used to study cell function and host-pathogen interactions in the bovine intestine. However,these cultures lack the cellular diversity encountered in the intestinal epithelium,and the physiological relevance of monocultures of transformed cell lines is uncertain. Little is also known of the factors that influence cell differentiation and homeostasis in the bovine intestinal epithelium,and few cell-specific markers that can distinguish the different intestinal epithelial cell lineages have been reported. Here we describe a simple and reliable procedure to establish in vitro 3D enteroid,or mini gut" cultures from bovine small intestinal (ileal) crypts. These enteroids contained a continuous central lumen lined with a single layer of polarized enterocytes bound by tight junctions with abundant microvilli on their apical surfaces. Histological and transcriptional analyses suggested that the enteroids comprised a mixed population of intestinal epithelial cell lineages including intestinal stem cells enterocytes Paneth cells goblet cells and enteroendocrine cells. We show that bovine enteroids can be successfully maintained long-term through multiple serial passages without observable changes to their growth characteristics morphology or transcriptome. Furthermore the bovine enteroids can be cryopreserved and viable cultures recovered from frozen stocks. Our data suggest that these 3D bovine enteroid cultures represent a novel physiologically-relevant and tractable in vitro system in which epithelial cell differentiation and function and host-pathogen interactions in the bovine small intestine can be studied." View Publication

OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease1,2 and embryonic lethality during mouse development3. Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus,knock-in mice that express catalytically inactive OTULIN,either constitutively or selectively in endothelial cells,resembled LUBAC-deficient mice4 and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1). Inactivation of OTULIN in adult mice also caused pro-inflammatory cell death. Accordingly,embryonic lethality and adult auto-inflammation were prevented by the combined loss of cell death mediators: caspase 8 for apoptosis and RIPK3 for necroptosis. Unexpectedly,OTULIN mutant mice that lacked caspase 8 and RIPK3 died in the perinatal period,exhibiting enhanced production of type I interferon that was dependent on RIPK1. Collectively,our results indicate that OTULIN and LUBAC function in a linear pathway,and highlight a previously unrecognized interaction between linear ubiquitination,regulators of cell death,and induction of type I interferon. View Publication