Neutrophils Derived from Genetically Modified Human Induced Pluripotent Stem Cells Circulate and Phagocytose Bacteria In Vivo

Abstract Bacterial and fungal infections are a major cause of morbidity and mortality in neutropenic patients. Donor‐derived neutrophil transfusions have been used for prophylaxis or treatment for infection in neutropenic patients. However, the short half‐life and the limited availability of large numbers of donor‐derived neutrophils for transfusion remain a significant hurdle in the implementation of neutrophil transfusion therapy. Here, we investigate the in vitro and in vivo activity of neutrophils generated from human induced pluripotent stem cells (iPSC), a potentially unlimited resource to produce neutrophils for transfusion. Phenotypic analysis of iPSC‐derived neutrophils reveal reactive oxygen species production at similar or slightly higher than normal peripheral blood neutrophils, but have an ∼50%–70% reduced Escherichia coli phagocytosis and phorbol 12‐myristate 13‐acetate induced formation of neutrophil extracellular traps (NET). Signaling of granulocytic precursors identified impaired AKT activation, but not ERK or STAT3, in agonist‐stimulated iPSC‐derived neutrophils. Expression of a constitutively activated AKT in iPSC‐derived neutrophils restores most phagocytic activity and NET formation. In a model of bacterial induced peritonitis in immunodeficient mice, iPSC‐derived neutrophils, with or without corrected AKT activation, migrate similarly to the peritoneal fluid as peripheral blood neutrophils, whereas the expression of activated AKT significantly improves their phagocytic activity in vivo. Stem Cells Translational Medicine 2019;8:557–56

TRAF6 Mediates Basal Activation of NF-κB Necessary for Hematopoietic Stem Cell Homeostasis

Summary: Basal nuclear factor κB (NF-κB) activation is required for hematopoietic stem cell (HSC) homeostasis in the absence of inflammation; however, the upstream mediators of basal NF-κB signaling are less well understood. Here, we describe TRAF6 as an essential regulator of HSC homeostasis through basal activation of NF-κB. Hematopoietic-specific deletion of Traf6 resulted in impaired HSC self-renewal and fitness. Gene expression, RNA splicing, and molecular analyses of Traf6-deficient hematopoietic stem/progenitor cells (HSPCs) revealed changes in adaptive immune signaling, innate immune signaling, and NF-κB signaling, indicating that signaling via TRAF6 in the absence of cytokine stimulation and/or infection is required for HSC function. In addition, we established that loss of IκB kinase beta (IKKβ)-mediated NF-κB activation is responsible for the major hematopoietic defects observed in Traf6-deficient HSPC as deletion of IKKβ similarly resulted in impaired HSC self-renewal and fitness. Taken together, TRAF6 is required for HSC homeostasis by maintaining a minimal threshold level of IKKβ/NF-κB signaling. : Fang et al. identify TRAF6 as an essential regulator of hematopoietic stem cell (HSC) self-renewal and quiescence. TRAF6 preserves HSC homeostasis by maintaining a minimal threshold level of NF-κB signaling in the absence of inflammation. Keywords: TRAF6, hematopoietic stem cell, NF-kB, innate immune signaling, toll-like receptor, IKKbet

Disorganization and degeneration of liver sympathetic innervations in nonalcoholic fatty liver disease revealed by 3D imaging

Hepatic nerves have a complex role in synchronizing liver metabolism. Here, we used three-dimensional (3D) immunoimaging to explore the integrity of the hepatic nervous system in experimental and human nonalcoholic fatty liver disease (NAFLD). We demonstrate parallel signs of mild degeneration and axonal sprouting of sympathetic innervations in early stages of experimental NAFLD and a collapse of sympathetic arborization in steatohepatitis. Human fatty livers display a similar pattern of sympathetic nerve degeneration, correlating with the severity of NAFLD pathology. We show that chronic sympathetic hyperexcitation is a key factor in the axonal degeneration, here genetically phenocopied in mice deficient of the Rac-1 activator Vav3. In experimental steatohepatitis, 3D imaging reveals a severe portal vein contraction, spatially correlated with the extension of the remaining nerves around the portal vein, enlightening a potential intrahepatic neuronal mechanism of portal hypertension. These fundamental alterations in liver innervation and vasculature uncover previously unidentified neuronal components in NAFLD pathomechanisms.Funding Agencies|Swedish Research CouncilSwedish Research CouncilEuropean Commission [04X-2887]; Knut and Alice Wallenberg FoundationKnut &amp; Alice Wallenberg Foundation; Sven Mattssons Foundation; Ahlen-Foundation; NovoNordisk postdoctoral Fellowship</p

Factors affecting availability for detection: An example using radio-collared Northern Bobwhite (Colinus virginianus)

Avian monitoring strategies are usually linked to bird singing or calling behavior. Individual availability for detection can change as a result of conspecific factors affecting bird behavior, though the magnitude of these effects is difficult to quantify. We evaluated behavioral and temporal factors affecting Northern Bobwhite (Colinus virginianus) breeding season individual availability for detection during three common survey times (3 min, 5 min, 10 min). We conducted 10-minute surveys associated with radio-collared male Northern Bobwhites on Peabody Wildlife Management Area, Kentucky, from 2010-2011. We homed to within 50 m of radio-collared males and recorded number of distinct Northern Bobwhite whistles (singing rate) per 1-minute interval, number of other males calling during the survey, minutes-since-sunrise, and day-of-season. We also recorded the number of minutes during a 10-minute survey that radio-collared male Northern Bobwhites called. We used logistic regression to estimate availability of radio-collared individuals for 3-minute, 5-minute, and 10-minute surveys. We also modeled number of minutes during 10-minute surveys that radio-collared Northern Bobwhites called, and we modeled singing rate. Individual availability for detection of radio-collared individuals during a 10-minute survey increased by 100% when at least 1 other Northern Bobwhite called during a survey (6.5% to 13.1%) and by 626% when 6 other Northern Bobwhites were calling (6.5% to 47.6%). Individual availability was 30% greater for 10-minute surveys than 5-minute surveys or 55% greater for 10-minute surveys than 3-minute surveys. Northern Bobwhite called most (2.8 ± 0.66 minutes/10-min survey) and at a greater rate (11.8 ± 1.3 calls/10-min period) when at least 5 other Northern Bobwhites called. Practitioners risk biasing population estimates low if individual availability is unaccounted for because species with low populations will not be stimulated by other calling males, are less likely to call, call less frequently, and call fewer times per minute, reducing their individual availability and likelihood to be counted on a survey even when they are present