Search

Single cell data for"The cellular states and fates of shed intestinal cells" paper. Folder: Count_tables and Objects This folder contains BG subtracted UMI tables and metadata for both the fecal wash UMAP and the fecal/tissue enterocytes combined UMAP 1. BG_subtracted_count_table_all_Fecal_cells_before_filt.txt - BG subtracted UMI table of all cells before Seurat filtration and analysis 2. BG_subtracted_count_table_all_Fecal_cells_after_filt.txt - BG subtracted UMI table of all cells after Seurat filtration and analysis. 3. Metadata_all_Fecal_cells_after_filteration.txt - Metadata for "BG_subtracted_count_table_all_Fecal_cells_after_filt.txt" 4. Combine_all_Fecal_cells_Obj.rds - Seurat Object for BG_subtracted_count_table_all_Fecal_cells_after_filt.txt 5. BG_subtracted_count_table_all_Enterocytes_before_filt.txt - BG subtracted UMI table of all cells before Seurat filtration and analysis 6. BG_subtracted_count_table_all_Enterocytes_after_filt.txt - BG subtracted UMI table of all cells after Seurat filtration and analysis. 7. Metadata_all_Enterocytes_after_filteration.txt - Metadata for "BG_subtracted_count_table_all_Enterocytes_after_filt.txt" 8. Combine_Enterocytes_Obj.rds - Seurat Object for BG_subtracted_count_table_all_Enterocytes_after_filt.txt

SummaryWe recently showed that intravenous infusion of mouse or human, fetal or adult lung cells following conditioning of recipient mice leads to lung chimerism within alveolar and bronchiolar lineages, in distinct ‘patches’ containing both epithelial and endothelial cells. We show here, using R26R-Confetti mice as donors, that these multi-lineage patches are derived from a single lung progenitor. FACS of adult mouse lung cells revealed that the putative patch-forming progenitors co-express the endothelial marker CD31 (PECAM-1) and the epithelial marker CD326 (EPCAM). Transplantation of lung cells from transgenic Cre/lox mice expressing membrane GFP under the VEcad promoter (VEcad-Cre-mTmG), led to GFP+ patches comprising both GFP+ endothelial and epithelial cells in vivo, and in ex-vivo culture of CD326+CD31+ progenitors. Single cell mRNAseq of CD326+CD31+ lung cells revealed a subpopulation expressing canonical epithelial and endothelial genes. Such double positive GFP+NKX2.1+SOX17+ cells were also deteceted by immuno-histologial staining in lungs of VEcad-Cre-nTnG (expressing nulear GFP) mice in proximity to blood vessels. These findings provide new insights on lung progenitros and lung development and suggest a potential novel approach for lung regeneration.

Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81−/− mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection., Lactate is a by-product of glycolysis that can function via its G protein receptor GPR81. Here the authors show that LPS or Salmonella infection enhances glycolytic metabolism in bone marrow neutrophils, resulting in lactate production, which increases endothelial barrier permeability and mobilization of these neutrophils by targeting endothelial GPR81.

Enduring immunity against harmful pathogens depends on the generation of immunological memory. Serum immunoglobulins are constantly secreted by long-lived antibody-producing cells, which provide extended protection from recurrent exposures. These cells originate mainly from germinal center structures, wherein B cells introduce mutations to their immunoglobulin genes followed by affinity-based selection. Generation of high-affinity antibodies relies on physical contacts between T and B cells, a process that facilitates the delivery of fate decision signals. T-B cellular engagements are mediated through interactions between the T cell receptor and its cognate peptide presented on B cell major histocompatibility class II molecules. Here, we describe the cellular and molecular aspects of these cognate T-B interactions, and highlight exceptional cases, especially those arising at intestinal lymphoid organs, at which T cells provide help to B cells in an atypical manner, independent of T cell specificity.

Antibody-mediated long-lasting protection from harmful pathogens depends on collaboration of immune cells within immunological niches. Stoler-Barak et al. introduce an approach that enables the visualization of all the germinal center niches and activated B cells within intact lymph nodes., Germinal centers (GCs) are sites wherein B cells proliferate and mutate their immunoglobulins in the dark zone (DZ), followed by affinity-based selection in the light zone (LZ). Here, we mapped the location of single B cells in the context of intact lymph nodes (LNs) throughout the GC response, and examined the role of BCR affinity in dictating their position. Imaging of entire GC structures and proximal single cells by light-sheet fluorescence microscopy revealed that individual B cells that previously expressed AID are located within the LN cortex, in an area close to the GC LZ. Using in situ photoactivation, we demonstrated that B cells migrate from the LZ toward the GC outskirts, while DZ B cells are confined to the GC. B cells expressing very-low-affinity BCRs formed GCs but were unable to efficiently disperse within the follicles. Our findings reveal that BCR affinity regulates B cell positioning during the GC response.

Long-lasting immunity depends on the generation of protective antibodies through the germinal center (GC) reaction. N6-methyladenosine (m6A) modification of mRNAs by METTL3 activity modulates transcript lifetime primarily through the function of m6A readers; however, the physiological role of this molecular machinery in the GC remains unknown. Here, we show that m6A modifications by METTL3 are required for GC maintenance through the differential functions of m6A readers. Mettl3-deficient GC B cells exhibited reduced cell-cycle progression and decreased expression of proliferation- and oxidative phosphorylation–related genes. The m6A binder, IGF2BP3, was required for stabilization of Myc mRNA and expression of its target genes, whereas the m6A reader, YTHDF2, indirectly regulated the expression of the oxidative phosphorylation gene program. Our findings demonstrate how two independent gene networks that support critical GC functions are modulated by m6A through distinct mRNA binders.

Consecutive exposures to different pathogens are highly prevalent and often alter the host immune response. However, it remains unknown how a secondary bacterial infection affects an ongoing adaptive immune response elicited against primary invading pathogens. We demonstrated that recruitment of Sca-1

Generation of antibodies is crucial for establishing enduring protection from invading pathogens, as well as for maintaining homeostasis with commensal bacteria at mucosal surfaces. Chronic exposure to microbiota- and dietary- derived antigens results in continuous production of antibody producing cells within the Peyer’s patch germinal center structures. Recently, we have shown that B cells responding to gut-derived antigens colonize the subepithelial dome (SED) in Peyer’s patches and rapidly proliferate independently of their relative BCR affinity. To evaluate B cell proliferation within different niches in Peyer’s patches, we applied in vivo EdU incorporation assay as described in this protocol.

In germinal centers, B cells interact with antigen in the light zone and clonally expand in the dark zone. Davidzohn et al. show that BCR-induced Syk degradation in the light zone attenuates signal transduction, impedes plasma cell formation, and promotes B cell transition to the dark zone., Germinal centers (GCs) are sites at which B cells proliferate and mutate their antibody-encoding genes in the dark zone (DZ), followed by affinity-based selection in the light zone (LZ). B cell antigen receptor (BCR) signals induce Syk activation followed by rapid phosphatase-mediated desensitization; however, how degradation events regulate BCR functions in GCs is unclear. Here, we found that Syk degradation restrains plasma cell (PC) formation in GCs and promotes B cell LZ to DZ transition. Using a mouse model defective in Cbl-mediated Syk degradation, we demonstrate that this machinery attenuates BCR signaling intensity by mitigating the Kras/Erk and PI3K/Foxo1 pathways, and restricting the expression of PC transcription factors in GC B cells. Inhibition of Syk degradation perturbed gene expression, specifically in the LZ, and enhanced the generation of PCs without affecting B cell proliferation. These findings reveal how long-lasting attenuation of signal transduction by degradation events regulates cell fate within specialized microanatomical sites., Graphical Abstract

Summary Protective immune responses depend on the formation of immune synapses between T cells and antigen-presenting cells (APCs). The two main LFA-1 ligands, ICAM-1 and ICAM-2, are co-expressed on many cell types, including APCs and blood vessels. Although these molecules were suggested to be key players in immune synapses studied in vitro , their contribution to helper T cell priming in vivo is unclear. Here, we used transgenic mice and intravital imaging to examine the role of dendritic cell (DC) ICAM-1 and ICAM-2 in naive CD4 T cell priming and differentiation in skin-draining lymph nodes. Surprisingly, ICAM deficiency on endogenous CD40-stimulated lymph node DCs did not impair their ability to arrest and prime CD4 lymphocyte activation and differentiation into Th1 and Tfh effectors. Thus, functional T cell receptor (TCR)-specific helper T cell synapses with antigen-presenting DCs and subsequent proliferation and early differentiation into T effectors do not require LFA-1-mediated T cell adhesiveness to DC ICAMs.

The molecular mechanism governing affinity-based B cell selection for germinal center colonization is unclear. Zaretsky et al. show that B cell ICAMs promote efficient B cell selection for clonal expansion by supporting sustained interactions with T follicular helper cells., The germinal center (GC) reaction begins with a diverse and expanded group of B cell clones bearing a wide range of antibody affinities. During GC colonization, B cells engage in long-lasting interactions with T follicular helper (Tfh) cells, a process that depends on antigen uptake and antigen presentation to the Tfh cells. How long-lasting T–B interactions and B cell clonal expansion are regulated by antigen presentation remains unclear. Here, we use in vivo B cell competition models and intravital imaging to examine the adhesive mechanisms governing B cell selection for GC colonization. We find that intercellular adhesion molecule 1 (ICAM-1) and ICAM-2 on B cells are essential for long-lasting cognate Tfh–B cell interactions and efficient selection of low-affinity B cell clones for proliferative clonal expansion. Thus, B cell ICAMs promote efficient antibody immune response by enhancement of T cell help to cognate B cells.

Although aging-regulating pathways were discovered a few decades ago, it is not entirely clear how their activities are orchestrated, to govern lifespan and proteostasis at the organismal level. Here, we utilized the nematode Caenorhabditis elegans to examine whether the alteration of aging, by reducing the activity of the Insulin/IGF signaling (IIS) cascade, affects protein SUMOylation. We found that IIS activity promotes the SUMOylation of the germline protein, CAR-1, thereby shortening lifespan and impairing proteostasis. In contrast, the expression of mutated CAR-1, that cannot be SUMOylated at residue 185, extends lifespan and enhances proteostasis. A mechanistic analysis indicated that CAR-1 mediates its aging-altering functions, at least partially, through the notch-like receptor glp-1. Our findings unveil a novel regulatory axis in which SUMOylation is utilized to integrate the aging-controlling functions of the IIS and of the germline and provide new insights into the roles of SUMOylation in the regulation of organismal aging.

Performance and safety are impaired in employees engaged in shift work. Combat divers who use closed-circuit oxygen diving apparatus undergo part of their training during the night hours. The greatest risk involved in diving with such apparatus is the development of central nervous system oxygen toxicity (CNS-OT). We investigated whether the switch from day-to-night activity may be a risk factor for the development of CNS-OT using a diurnal animal model, the fat sand rat (Psammomys obesus). Animals were kept on a 12:12 light-dark schedule (6 a.m. to 6 p.m. at 500 lx). The study included two groups: (1) Control group: animals were kept awake and active during the day, between 09:00 and 15:00. (2) Experimental group: animals were kept awake and active during the night, between 21:00 and 03:00, when they were exposed to dim light in order to simulate the conditions prevalent during combat diver training. This continued for a period of 3 weeks, 5 days a week. On completion of this phase, 6-sulphatoxymelatonin (6-SMT) levels in urine were determined over a period of 24 h. Animals were then exposed to hyperbaric oxygen (HBO). To investigate the effect of acute melatonin administration, melatonin (50 mg/kg) or its vehicle was administered to the animals in both groups 20 min prior to HBO exposure. After the exposure, the activity of superoxide dismutase, catalase and glutathione peroxidase was measured, as were the levels of neuronal nitric oxide synthase (nNOS) and overall nitrotyrosylation in the cortex and hippocampus. Latency to CNS-OT was significantly reduced after the transition from day-to-night activity. This was associated with alterations in the level of melatonin metabolites secreted in the urine. Acute melatonin administration had no effect on latency to CNS-OT in either of the groups. Nevertheless, the activity of superoxide dismutase and catalase, as well as nitrotyrosine and nNOS levels, were altered in the hippocampus following melatonin administration. On the basis of these results, we suggest that a switch from diurnal to nocturnal activity may represent an additional risk factor for the development of CNS-OT. Utilizing a diurnal animal model may contribute to our understanding of the heightened risk of developing CNS-OT when diving with closed-circuit oxygen apparatus at night.

Background: Structural changes in the human body resulting from aging may affect the response to altered levels of O2 and CO2. An abnormal ventilatory response to a buildup of CO2 in the inspired air due to rebreathing may result in adverse effects, which will impair the individual's ability to function under stress. The purpose of this study was to evaluate the effect of age on the respiratory response to wearing an escape hood at rest and during mild exercise. Methods: Subjects were seven healthy, young adult males (20–30 years) and seven healthy, middle-aged males (45–65 years). Inspired CO2 and O2, breathing pattern (tidal volume [VT] and breathing frequency [F]), and mouth inspiratory and expiratory pressures, were measured at rest and during mild exercise (50 w) while wearing the CAPS 2000 escape hood (Shalon Chemical Industries and Supergum-Rubber and Plastic Technology, Tel Aviv, Israel). Findings: Resting inspired CO2 was higher in the middle-aged group compared with the young group (2.2...

Spatial information from NICHE-seq Immune functions depend on the interactions of heterogeneous cells in a range of microenvironments in the body. Although information regarding immune cell function has been collected using single-cell RNA-sequencing methods, these techniques have traditionally lacked spatial information. Medaglia et al. describe NICHE-seq, a technique that allows the sorting and analysis of cells from within visually selected territories in transgenic mice that express photoactivatable green fluorescent protein. The method successfully identified T and B cell-specific niches in mouse lymph nodes and spleens after virus infection. The approach will allow us to bridge the gap between cellular and spatial information in studies of organs. Science , this issue p. 1622

Findings regarding blood glucose level (BGL) on exposure to hyperbaric oxygen (HBO) are contradictory. We investigated the influence of HBO on BGL, and of BGL on latency to central nervous system oxygen toxicity (CNS-OT). The study was conducted on five groups of rats: Group 1, exposure to oxygen at 2.5 atmospheres absolute (ATA), 90 min/day for 7 days; Group 2, exposure to oxygen once a week from 2 to 6 ATA in increments of 1 ATA/wk, for a period of time calculated as 60% of the latency to CNS-OT (no convulsions); Group 3, exposure to 6 ATA breathing a gas mixture with a pO2of 0.21; Group 4, received 10 U/kg insulin to induce hypoglycemia before exposure to HBO; Group 5, received 33% glucose to induce hyperglycemia before exposure to HBO. Blood samples were drawn before and after exposures for measurement of BGL. No change was observed in BGL after exposure to oxygen at 2.5 ATA, 90 min/day for 7 days. BGL was significantly elevated after exposure to oxygen at 6 ATA until the appearance of convulsions, and following exposure to 4, 5, and 6 ATA without convulsions ( P < 0.01). No change was observed in BGL after exposure to 6 ATA breathing a gas mixture with a pO2of 0.21. Hypoglycemia shortened latency to CNS oxygen toxicity, whereas hyperglycemia had no effect. Our results demonstrate an influence of HBO exposure on elevation of BGL, starting at 4 ATA. This implies that BGL may serve as a marker for the generation of CNS-OT.

The two main LFA-1 ligands ICAM-1 and ICAM-2 are constitutively co-expressed on antigen presenting cells (APCs) and on the lymph node stroma. We find abrogated antigen specific CD4 T cell priming in ICAM-1 and ICAM-2 deficient spleens. Nevertheless, the in vivo contribution of these ligands to lymphocyte activation proliferation and differentiation in lymph nodes has been difficult to dissect in total knockout mice due to their multiple roles in lymphocyte entry and priming in these organs. We therefore constructed lethally irradiated chimeric mice reconstituted with ICAM-1 and 2 double knock out (DKO) APCs in which lymphocyte entry into peripheral lymph nodes was reconstituted. Strikingly, these mice elicited normal antigen specific OT-II T cell activation, proliferation and differentiation into Th1 and Tfh effectors. OT-II T cells could also normally arrest on DCs deficient in ICAM-1 and 2. A compensatory role of VCAM-1 in ICAM deficient DCs was ruled out. Nevertheless, polyclonal T cell proliferation in response to endogenous self-antigens required the presence of ICAM-1 and ICAM-2 on endogenous lymph node DCs. Our results are the first indication that high avidity TCR peptide-MHC-II recognition can bypass integrin-mediated lymphocyte adhesions to DCs under specific settings.

Central nervous system oxygen toxicity (CNS-OT) can occur in humans at pressures above 2 atmospheres absolute (ATA), and above 4.5 ATA in the rat. Pulmonary oxygen toxicity appears at pressures above 0.5 ATA. We hypothesized that exposure to mild HBO following extreme exposure might provide protection against CNS, but not pulmonary oxygen toxicity. We measured the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), and nitrotyrosine and nNOS levels in the brain and lung in the following groups: (1) Sham rats, no pressure exposure (SHAM); (2) Exposure to 6 ATA oxygen for 60% of latency to CNS-OT (60%LT); (3) Exposure to 6 ATA for 60% of latency to CNS-OT, followed by 20 min at 2.5 ATA for recovery (REC); (4) Exposure to 6 ATA for 60% of latency to CNS-OT, followed by 20 min at 2.5 ATA oxygen and a subsequent increase in pressure to 6 ATA until the appearance of convulsions (CONV); (5) Control rats exposed to 6 ATA until the appearance of convulsions (C). SOD and CAT activity were reduced in both brain and lung in the REC group. GPX activity was reduced in the hippocampus in the REC group, but not in the cortex or the lung. nNOS levels were reduced in the hippocampus in the REC group. Contrary to our hypothesis, no difference was observed between the brain and the lung for the factors investigated. We suggest that at 2.5 ATA and above, CNS and pulmonary oxygen toxicity may share similar mechanisms.

Gut-derived antigens trigger immunoglobulin A (IgA) immune responses that are initiated by cognate B cells in Peyer's patches (PPs). These cells colonize the subepithelial domes (SEDs) of the PPs and subsequently infiltrate pre-existing germinal centers (GCs). Here we defined the pre-GC events and the micro-anatomical site at which affinity-based B cell selection occurred in PPs. Using whole-organ imaging, we showed that the affinity of the B cell antigen receptor (BCR) regulated the infiltration of antigen-specific B cells into GCs but not clonal competition in the SED. Follicular helper-like T cells resided in the SED and promoted its B cell colonization, independently of the magnitude of BCR affinity. Imaging and immunoglobulin sequencing indicated that selective clonal expansion ensued during infiltration into GCs. Thus, in contrast to the events in draining lymph nodes and spleen, in PPs, T cells promoted mainly the population expansion of B cells without clonal selection during pre-GC events. These findings have major implications for the design of oral vaccines.

The germinal center (GC) reaction in Peyer′s patches (PP) requires continuous access to antigens, but how this is achieved is not known. Here we show that activated antigen-specific CCR6+CCR1+GL7− B cells make close contact with M cells in the subepithelial dome (SED). Using in situ photoactivation analysis of antigen-specific SED B cells, we find migration of cells towards the GC. Following antigen injection into ligated intestinal loops containing PPs, 40% of antigen-specific SED B cells bind antigen within 2 h, whereas unspecifc cells do not, indicating B cell-receptor involvment. Antigen-loading is not observed in M cell-deficient mice, but is unperturbed in mice depleted of classical dendritic cells (DC). Thus, we report a M cell-B cell antigen-specific transporting pathway in PP that is independent of DC. We propose that this antigen transporting pathway has a critical role in gut IgA responses, and should be taken into account when developing mucosal vaccines., Gut lumen antigens must be continuously sampled by the immune system to maintain proper immune homeostasis. Here the authors show that activated CCR6+CCR1+GL7- gut B cells retrieve lumen antigens from specialized M cells and transfer them across the subepithelial dome in the Peyer’s patch to contribute to the maintenance of gut humoral immunity.

Summary Antibodies secreted within the intestinal tract provide protection from the invasion of microbes into the host tissues. Germinal center (GC) formation in lymph nodes and spleen strictly requires SLAM-associated protein (SAP)-mediated T cell functions; however, it is not known whether this mechanism plays a similar role in mucosal-associated lymphoid tissues. Here, we find that in Peyer’s patches (PPs), SAP-mediated T cell help is required for promoting B cell selection in GCs, but not for clonal diversification. PPs of SAP-deficient mice host chronic GCs that are absent in T cell-deficient mice. GC B cells in SAP-deficient mice express AID and Bcl6 and generate plasma cells in proportion to the GC size. Single-cell IgA sequencing analysis reveals that these mice host few diversified clones that were subjected to mild selection forces. These findings demonstrate that T cell-derived help to B cells in PPs includes SAP-dependent and SAP-independent functions., Graphical Abstract, Highlights • Chronic germinal centers in Peyer’s patches are formed in SAP-deficient mice • SAP-independent germinal centers arise in response to influenza infection • Few highly diversified clones dominate the SAP-independent germinal centers • Germinal center B cells in SAP-deficient mice are subjected to mild selection forces, SAP is required for proper T cell help in germinal centers (GCs). Biram et al. show that SAP-independent GCs are formed within Peyer’s patches. These GCs host highly diversified clones that are subjected to mild selection forces, demonstrating that clonal diversification can be uncoupled from clonal selection in chronic GCs.