Optimization of single-cell plate sorting for high throughput sequencing applications.

Single cell sequencing has recently been applied to many immunological studies. Flow cytometric index sorting isolates cells for single cell sequencing with protein level data linked to sequences. However, successful sequencing of index sorted samples requires careful optimization of several sort parameters, including nozzle size, flow rate, threshold rate, and yield calculations. In this study, considerations and optimization data for each of these variables are presented. Our analysis focused on index sorting, but the findings can be applied to any plate sorting protocol. Minimization of flow rates and use of the 70 µm nozzle improved cell yields. Improvements in total read counts after sequencing were obtained by decreasing the threshold rate, or the number of cells processed per second. In addition, this technique provided linked protein and gene expression analysis of the cytokine interferon (IFN)γ, demonstrating that on a single cell basis IFNγ+ cells tend to express IFNG mRNA, and IFNγ- cells do not. Through rigorous optimization and quality control, we have identified parameters important to plate sorting and recommend the use of the 70 µm nozzle and low flow and threshold rates for analysis of rare populations of human lymphocytes.

Endothelial Hypoxia-Inducible Factor-2α Is Required for the Maintenance of Airway Microvasculature.

BACKGROUND: Hypoxia-inducible factors (HIFs), especially HIF-1α and HIF-2α, are key mediators of the adaptive response to hypoxic stress and play essential roles in maintaining lung homeostasis. Human and animal genetics studies confirm that abnormal HIF correlates with pulmonary vascular pathology and chronic lung diseases, but it remains unclear whether endothelial cell HIF production is essential for microvascular health. The large airway has an ideal circulatory bed for evaluating histological changes and physiology in genetically modified rodents. METHODS: The tracheal microvasculature of mice, with conditionally deleted or overexpressed HIF-1α or HIF-2α, was evaluated for anatomy, perfusion, and permeability. Angiogenic signaling studies assessed vascular changes attributable to dysregulated HIF expression. An orthotopic tracheal transplantation model further evaluated the contribution of individual HIF isoforms in airway endothelial cells. RESULTS: The genetic deletion of Hif-2α but not Hif-1α caused tracheal endothelial cell apoptosis, diminished pericyte coverage, reduced vascular perfusion, defective barrier function, overlying epithelial abnormalities, and subepithelial fibrotic remodeling. HIF-2α promoted microvascular integrity in airways through endothelial angiopoietin-1/TIE2 signaling and Notch activity. In functional tracheal transplants, HIF-2α deficiency in airway donors accelerated graft microvascular loss, whereas HIF-2α or angiopoietin-1 overexpression prolonged transplant microvascular perfusion. Augmented endothelial HIF-2α in transplant donors promoted airway microvascular integrity and diminished alloimmune inflammation. CONCLUSIONS: Our findings reveal that the constitutive expression of endothelial HIF-2α is required for airway microvascular health.

NF-κB/MAPK activation underlies ACVR1-mediated inflammation in human heterotopic ossification.

BACKGROUND: Inflammation helps regulate normal growth and tissue repair. Although bone morphogenetic proteins (BMPs) and inflammation are known contributors to abnormal bone formation, how these pathways interact in ossification remains unclear. METHODS: We examined this potential link in patients with fibrodysplasia ossificans progressiva (FOP), a genetic condition of progressive heterotopic ossification caused by activating mutations in the Activin A type I receptor (ACVR1/ALK2). FOP patients show exquisite sensitivity to trauma, suggesting that BMP pathway activation may alter immune responses. We studied primary blood, monocyte, and macrophage samples from control and FOP subjects using multiplex cytokine, gene expression, and protein analyses; examined CD14+ primary monocyte and macrophage responses to TLR ligands; and assayed BMP, TGF-ß activated kinase 1 (TAK1), and NF-κB pathways. RESULTS: FOP subjects at baseline without clinically evident heterotopic ossification showed increased serum IL-3, IL-7, IL-8, and IL-10. CD14+ primary monocytes treated with the TLR4 activator LPS showed increased CCL5, CCR7, and CXCL10; abnormal cytokine/chemokine secretion; and prolonged activation of the NF-κB pathway. FOP macrophages derived from primary monocytes also showed abnormal cytokine/chemokine secretion, increased TGF-ß production, and p38MAPK activation. Surprisingly, SMAD phosphorylation was not significantly changed in the FOP monocytes/macrophages. CONCLUSIONS: Abnormal ACVR1 activity causes a proinflammatory state via increased NF-κB and p38MAPK activity. Similar changes may contribute to other types of heterotopic ossification, such as in scleroderma and dermatomyositis; after trauma; or with recombinant BMP-induced bone fusion. Our findings suggest that chronic antiinflammatory treatment may be useful for heterotopic ossification.

Loss of Iroquois homeobox transcription factors 3 and 5 in osteoblasts disrupts cranial mineralization.

Cranial malformations are a significant cause of perinatal morbidity and mortality. Iroquois homeobox transcription factors (IRX) are expressed early in bone tissue formation and facilitate patterning and mineralization of the skeleton. Mice lacking Irx5 appear grossly normal, suggesting that redundancy within the Iroquois family. However, global loss of both Irx3 and Irx5 in mice leads to significant skeletal malformations and embryonic lethality from cardiac defects. Here, we study the bone-specific functions of Irx3 and Irx5 using Osx-Cre to drive osteoblast lineage-specific deletion of Irx3 in Irx5(-/-) mice. Although we found that the Osx-Cre transgene alone could also affect craniofacial mineralization, newborn Irx3 (flox/flox) /Irx5(-/-)/Osx-Cre (+) mice displayed additional mineralization defects in parietal, interparietal, and frontal bones with enlarged sutures and reduced calvarial expression of osteogenic genes. Newborn endochondral long bones were largely unaffected, but we observed marked reductions in 3-4-week old bone mineral content of Irx3 (flox/flox) /Irx5(-/-)/Osx-Cre (+) mice. Our findings indicate that IRX3 and IRX5 can work together to regulate mineralization of specific cranial bones. Our results also provide insight into the causes of the skeletal changes and mineralization defects seen in Hamamy syndrome patients carrying mutations in IRX5.

Increased Gs Signaling in Osteoblasts Reduces Bone Marrow and Whole-Body Adiposity in Male Mice.

Bone is increasingly recognized as an endocrine organ that can regulate systemic hormones and metabolism through secreted factors. Although bone loss and increased adiposity appear to be linked clinically, whether conditions of increased bone formation can also change systemic metabolism remains unclear. In this study, we examined how increased osteogenesis affects metabolism by using an engineered G protein-coupled receptor, Rs1, to activate Gs signaling in osteoblastic cells in ColI(2.3)(+)/Rs1(+) transgenic mice. We previously showed that these mice have dramatically increased bone formation resembling fibrous dysplasia of the bone. We found that total body fat was significantly reduced starting at 3 weeks of age. Furthermore, ColI(2.3)(+)/Rs1(+) mice showed reduced O2 consumption and respiratory quotient measures without effects on food intake and energy expenditure. The mice had significantly decreased serum triacylglycerides, leptin, and adiponectin. Resting glucose and insulin levels were unchanged; however, glucose and insulin tolerance tests revealed increased sensitivity to insulin. The mice showed resistance to fat accumulation from a high-fat diet. Furthermore, ColI(2.3)(+)/Rs1(+) mouse bones had dramatically reduced mature adipocyte differentiation, increased Wingless/Int-1 (Wnt) signaling, and higher osteoblastic glucose utilization than controls. These findings suggest that osteoblasts can influence both local and peripheral adiposity in conditions of increased bone formation and suggest a role for osteoblasts in the regulation of whole-body adiposity and metabolic homeostasis.

Hematopoietic stem cell fate decisions are regulated by Wnt antagonists: comparisons and current controversies.

Wingless and int (Wnt) proteins are secreted proteins that are important for regulating hematopoietic stem cell self-renewal and differentiation in the bone marrow microenvironment in mice. The mechanisms by which Wnt signaling regulates these hematopoietic cell fate decisions are not fully understood. Secreted Wnt antagonists, which are expressed in bone and bone marrow stromal cells, either bind to Wnt ligands directly or block Wnt receptors and co-receptors to halt Wnt-mediated signal transduction in both osteolineage and hematopoietic cell types. Secreted frizzled related proteins-1 and -2, Wnt inhibitory factor-1, Dickkopf-1, and Sclerostin are Wnt antagonists that influence hematopoietic cell fate decisions in the bone marrow niche. In this review, we compare and contrast the roles of these Wnt antagonists and their effects on hematopoietic development in mice, and also discuss the clinical significance of targeting Wnt antagonists within the context of hematopoietic disease.

Absence of sclerostin adversely affects B-cell survival.

Increased osteoblast activity in sclerostin-knockout (Sost(-/-)) mice results in generalized hyperostosis and bones with small bone marrow cavities resulting from hyperactive mineralizing osteoblast populations. Hematopoietic cell fate decisions are dependent on their local microenvironment, which contains osteoblast and stromal cell populations that support both hematopoietic stem cell quiescence and facilitate B-cell development. In this study, we investigated whether high bone mass environments affect B-cell development via the utilization of Sost(-/-) mice, a model of sclerosteosis. We found the bone marrow of Sost(-/-) mice to be specifically depleted of B cells because of elevated apoptosis at all B-cell developmental stages. In contrast, B-cell function in the spleen was normal. Sost expression analysis confirmed that Sost is primarily expressed in osteocytes and is not expressed in any hematopoietic lineage, which indicated that the B-cell defects in Sost(-/-) mice are non-cell autonomous, and this was confirmed by transplantation of wild-type (WT) bone marrow into lethally irradiated Sost(-/-) recipients. WT→Sost(-/-) chimeras displayed a reduction in B cells, whereas reciprocal Sost(-/-) →WT chimeras did not, supporting the idea that the Sost(-/-) bone environment cannot fully support normal B-cell development. Expression of the pre-B-cell growth stimulating factor, Cxcl12, was significantly lower in bone marrow stromal cells of Sost(-/-) mice, whereas the Wnt target genes Lef-1 and Ccnd1 remained unchanged in B cells. Taken together, these results demonstrate a novel role for Sost in the regulation of bone marrow environments that support B cells.