Single-cell analysis of lung epithelial cells reveals age and cell population-specific responses to SARS-CoV-2 infection in ciliated cells.

Introduction: The ability of SARS-CoV-2 to evade antiviral immune signaling in the airway contributes to the severity of COVID-19 disease. Additionally, COVID-19 is influenced by age and has more severe presentations in older individuals. This raises questions about innate immune signaling as a function of lung development and age.

Reduced Notch1 Cleavage Promotes the Development of Pulmonary Hypertension.

Clinical trials of Dll4 (Delta-like 4) neutralizing antibodies (Dll4nAbs) in cancer patients are ongoing. Surprisingly, pulmonary hypertension (PH) occurs in 14% to 18% of patients treated with Dll4nAbs, but the mechanisms have not been studied. Here, PH progression was measured in mice treated with Dll4nAbs.

Histone H2A.X phosphorylation and Caspase-Initiated Chromatin Condensation in late-stage erythropoiesis.

Background: Condensation of chromatin prior to enucleation is an essential component of terminal erythroid maturation, and defects in this process are associated with inefficient erythropoiesis and anemia. However, the mechanisms involved in this phenomenon are not well understood. Here, we describe a potential role for the histone variant H2A.

β2M Signals Monocytes Through Non-Canonical TGFβ Receptor Signal Transduction.

Rationale: Circulating monocytes can have proinflammatory or proreparative phenotypes. The endogenous signaling molecules and pathways that regulate monocyte polarization in vivo are poorly understood. We have shown that platelet-derived β2M (β-2 microglobulin) and TGF-β (transforming growth factor β) have opposing effects on monocytes by inducing inflammatory and reparative phenotypes, respectively, but each bind and signal through the same receptor.

Lung megakaryocytes are immune modulatory cells.

Although platelets are the cellular mediators of thrombosis, they are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered bone marrow-resident (BM-resident) cells.

Codanin-1 mutations engineered in human erythroid cells demonstrate role of CDAN1 in terminal erythroid maturation.

The generation of a functional erythrocyte from a committed progenitor requires significant changes in gene expression during hemoglobin accumulation, rapid cell division, and nuclear condensation. Congenital dyserythropoietic anemia type I (CDA-I) is an autosomal recessive disease that presents with erythroid hyperplasia in the bone marrow. Erythroblasts in patients with CDA-I are frequently binucleate and have chromatin bridging and defective chromatin condensation.

Hyperacetylated chromatin domains mark cell type-specific genes and suggest distinct modes of enhancer function.

Stratification of enhancers by signal strength in ChIP-seq assays has resulted in the establishment of super-enhancers as a widespread and useful tool for identifying cell type-specific, highly expressed genes and associated pathways. We examine a distinct method of stratification that focuses on peak breadth, termed hyperacetylated chromatin domains (HCDs), which classifies broad regions exhibiting histone modifications associated with gene activation. We find that this analysis serves to identify genes that are both more highly expressed and more closely aligned to cell identity than super-enhancer analysis does using multiple data sets.

The histone methyltransferase Setd8 alters the chromatin landscape and regulates the expression of key transcription factors during erythroid differentiation.

Background: SETD8 is the sole methyltransferase capable of mono-methylating histone H4, lysine 20. SETD8 and H4K20me1 play a role in a number of essential biologic processes, including cell cycle progression, establishment of higher order chromatin structure, and transcriptional regulation. SETD8 is highly expressed in erythroid cells and erythroid deletion of Setd8 is embryonic lethal by embryonic day 11.

Disruption of normal patterns of FOXF1 expression in a lethal disorder of lung development.

Background: Alveolar capillary dysplasia with misalignment of the pulmonary veins (ACDMPV) is a lethal disorder of lung development. ACDMPV is associated with haploinsufficiency of the transcription factor , which plays an important role in the development of the lung and intestine. CNVs upstream of the FOXF1 gene have also been associated with an ACDMPV phenotype, but mechanism(s) by which these deletions disrupt lung development are not well understood.

IFNγ induces epigenetic programming of human T-bet B cells and promotes TLR7/8 and IL-21 induced differentiation.

Although B cells expressing the IFNγR or the IFNγ-inducible transcription factor T-bet promote autoimmunity in Systemic Lupus Erythematosus (SLE)-prone mouse models, the role for IFNγ signaling in human antibody responses is unknown. We show that elevated levels of IFNγ in SLE patients correlate with expansion of the T-bet expressing IgDCD27CD11cCXCR5 (DN2) pre-antibody secreting cell (pre-ASC) subset. We demonstrate that naïve B cells form T-bet pre-ASCs following stimulation with either Th1 cells or with IFNγ, IL-2, anti-Ig and TLR7/8 ligand and that IL-21 dependent ASC formation is significantly enhanced by IFNγ or IFNγ-producing T cells.

Human erythroblasts with c-Kit activating mutations have reduced cell culture costs and remain capable of terminal maturation.

A major barrier to the in vitro production of red blood cells for transfusion therapy is the cost of culture components, with cytokines making up greater than half of the culture costs. Cell culture cytokines also represent a major expense for in vitro studies of human erythropoiesis. HUDEP-2 cells are an E6/E7 immortalized erythroblast line used for the in vitro study of human erythropoiesis.

Aged marrow macrophages expand platelet-biased hematopoietic stem cells via Interleukin1B.

The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function, though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mφs) directs HSC platelet-bias. Mφs from the marrow of aged mice and humans exhibited an activated phenotype, with increased expression of inflammatory signals.

A novel in vitro model of primary human pediatric lung epithelial cells.

Background: Current in vitro human lung epithelial cell models derived from adult tissues may not accurately represent all attributes that define homeostatic and disease mechanisms relevant to the pediatric lung.

Methods: We report methods for growing and differentiating primary Pediatric Human Lung Epithelial (PHLE) cells from organ donor infant lung tissues. We use immunohistochemistry, flow cytometry, quantitative RT-PCR, and single cell RNA sequencing (scRNAseq) analysis to characterize the cellular and transcriptional heterogeneity of PHLE cells.

Platelet-derived β2M regulates monocyte inflammatory responses.

β-2 Microglobulin (β2M) is a molecular chaperone for the major histocompatibility class I (MHC I) complex, hemochromatosis factor protein (HFE), and the neonatal Fc receptor (FcRn), but β2M may also have less understood chaperone-independent functions. Elevated plasma β2M has a direct role in neurocognitive decline and is a risk factor for adverse cardiovascular events. β2M mRNA is present in platelets at very high levels, and β2M is part of the activated platelet releasate.

regulates age-dependent differences in murine platelet function.

Platelets are essential for hemostasis; however, several studies have identified age-dependent differences in platelet function. To better understand the origins of fetal platelet function, we have evaluated the contribution of the fetal-specific RNA binding protein in the megakaryocyte/platelet lineage. Because activated fetal platelets have very low levels of P-selectin, we hypothesized that the expression of platelet P-selectin is part of a fetal-specific hematopoietic program conferred by Lin28b.

EVI1 overexpression reprograms hematopoiesis via upregulation of Spi1 transcription.

Inv(3q26) and t(3:3)(q21;q26) are specific to poor-prognosis myeloid malignancies, and result in marked overexpression of EVI1, a zinc-finger transcription factor and myeloid-specific oncoprotein. Despite extensive study, the mechanism by which EVI1 contributes to myeloid malignancy remains unclear. Here we describe a new mouse model that mimics the transcriptional effects of 3q26 rearrangement.

Kit ligand has a critical role in mouse yolk sac and aorta-gonad-mesonephros hematopoiesis.

Few studies report on the requirement for hematopoietic niche factors in the mammalian embryo. Here, we comprehensively analyze the requirement for Kit ligand (Kitl) in the yolk sac and aorta-gonad-mesonephros (AGM) niche. In-depth analysis of loss-of-function and transgenic reporter mouse models show that Kitl-deficient embryos harbor decreased numbers of yolk sac erythro-myeloid progenitor (EMP) cells, resulting from a proliferation defect following their initial emergence.

Microfluidic assay of the deformability of primitive erythroblasts.

Primitive erythroblasts (precursors of red blood cells) enter vascular circulation during the embryonic period and mature while circulating. As a result, primitive erythroblasts constantly experience significant hemodynamic shear stress. Shear-induced deformation of primitive erythroblasts however, is poorly studied.

Analysis of Erythropoiesis Using Imaging Flow Cytometry.

Erythroid maturation has been classically defined based on the remarkable changes visualized through microscopy. These involve the decrease in cell size, nuclear condensation and organelle loss, and include the final unique asymmetric division creating the short-lived nucleated pyrenocyte and the enucleate reticulocyte that matures into the red blood cell. Understanding the regulation of these processes has been challenging due to the difficulty in obtaining sufficient numbers of cells, particularly of rare intermediates, to study by microscopy.

Functional Analysis of Erythroid Progenitors by Colony-Forming Assays.

The capacity of erythroid-lineage progenitors to form colonies of maturing red blood cells in semisolid media has provided a functional assay for these progenitors and has greatly contributed to our understanding of erythropoiesis. Studies since the 1970s have led to the development of a model of the erythron, whereby the earliest erythroid-committed progenitor, the immature burst-forming unit erythroid (BFU-E), gives rise sequentially to late-stage BFU-E and to colony-forming units erythroid (CFU-E). CFU-E give rise, in turn, to maturing erythroblast precursors that hemoglobinize.

Circulating primitive erythroblasts establish a functional, protein 4.1R-dependent cytoskeletal network prior to enucleating.

Hematopoietic ontogeny is characterized by distinct primitive and definitive erythroid lineages. Definitive erythroblasts mature and enucleate extravascularly and form a unique membrane skeleton, composed of spectrin, 4.1R-complex, and ankyrinR-complex components, to survive the vicissitudes of the adult circulation.