A20 and the noncanonical NF-κB pathway are key regulators of neutrophil recruitment during fetal ontogeny.

Newborns are at high risk of developing neonatal sepsis, particularly if born prematurely. This has been linked to divergent requirements the immune system has to fulfill during intrauterine compared with extrauterine life. By transcriptomic analysis of fetal and adult neutrophils, we shed new light on the molecular mechanisms of neutrophil maturation and functional adaption during fetal ontogeny. We identified an accumulation of differentially regulated genes within the noncanonical NF-κB signaling pathway accompanied by constitutive nuclear localization of RelB and increased surface expression of TNF receptor type II in fetal neutrophils, as well as elevated levels of lymphotoxin α in fetal serum. Furthermore, we found strong upregulation of the negative inflammatory regulator A20 (Tnfaip3) in fetal neutrophils, which was accompanied by pronounced downregulation of the canonical NF-κB pathway. Functionally, overexpressing A20 in Hoxb8 cells led to reduced adhesion of these neutrophil-like cells in a flow chamber system. Conversely, mice with a neutrophil-specific A20 deletion displayed increased inflammation in vivo. Taken together, we have uncovered constitutive activation of the noncanonical NF-κB pathway with concomitant upregulation of A20 in fetal neutrophils. This offers perfect adaption of neutrophil function during intrauterine fetal life but also restricts appropriate immune responses particularly in prematurely born infants.

Priming anti-tumor immunity by radiotherapy: Dying tumor cell-derived DAMPs trigger endothelial cell activation and recruitment of myeloid cells.

The major goal of radiotherapy is the induction of tumor cell death. Additionally, radiotherapy can function as in situ cancer vaccination by exposing tumor antigens and providing adjuvants for anti-tumor immune priming. In this regard, the mode of tumor cell death and the repertoire of released damage-associated molecular patterns (DAMPs) are crucial. However, optimal dosing and fractionation of radiotherapy remain controversial. Here, we examined the initial steps of anti-tumor immune priming by different radiation regimens (20 Gy, 4 × 2 Gy, 2 Gy, 0 Gy) with cell lines of triple-negative breast cancer in vitro and in vivo. Previously, we have shown that especially high single doses (20 Gy) induce a delayed type of primary necrosis with characteristics of mitotic catastrophe and plasma membrane disintegration. Now, we provide evidence that protein DAMPs released by these dying cells stimulate sequential recruitment of neutrophils and monocytes in vivo. Key players in this regard appear to be endothelial cells revealing a distinct state of activation upon exposure to supernatants of irradiated tumor cells as characterized by high surface expression of adhesion molecules and production of a discrete cytokine/chemokine pattern. Furthermore, irradiated tumor cell-derived protein DAMPs enforced differentiation and maturation of dendritic cells as hallmarked by upregulation of co-stimulatory molecules and improved T cell-priming. Consistently, a recurring pattern was observed: The strongest effects were detected with 20 Gy-irradiated cells. Obviously, the initial steps of radiotherapy-induced anti-tumor immune priming are preferentially triggered by high single doses - at least in models of triple-negative breast cancer.

Circadian Expression of Migratory Factors Establishes Lineage-Specific Signatures that Guide the Homing of Leukocyte Subsets to Tissues.

The number of leukocytes present in circulation varies throughout the day, reflecting bone marrow output and emigration from blood into tissues. Using an organism-wide circadian screening approach, we detected oscillations in pro-migratory factors that were distinct for specific vascular beds and individual leukocyte subsets. This rhythmic molecular signature governed time-of-day-dependent homing behavior of leukocyte subsets to specific organs. Ablation of BMAL1, a transcription factor central to circadian clock function, in endothelial cells or leukocyte subsets demonstrated that rhythmic recruitment is dependent on both microenvironmental and cell-autonomous oscillations. These oscillatory patterns defined leukocyte trafficking in both homeostasis and inflammation and determined detectable tumor burden in blood cancer models. Rhythms in the expression of pro-migratory factors and migration capacities were preserved in human primary leukocytes. The definition of spatial and temporal expression profiles of pro-migratory factors guiding leukocyte migration patterns to organs provides a resource for the further study of the impact of circadian rhythms in immunity.

A Novel ex vivo Mouse Mesometrium Culture Model for Investigating Angiogenesis in Microvascular Networks.

BACKGROUND: The development of models that incorporate intact microvascular networks enables the investigation of multicellular dynamics during angiogenesis. Our laboratory introduced the rat mesentery culture model as such a tool, which would be enhanced with mouse tissue. Since mouse mesentery is avascular, an alternative is mouse mesometrium, the connective tissue of uterine horns. The study's objective was to demonstrate that mouse mesometrium contains microvascular networks that can be cultured to investigate multicellular dynamics during angiogenesis. METHODS: Harvested mesometrium tissues from C57Bl/6 female mice were cultured in media with serum for up to 7 days. PECAM, NG2, αSMA, and LYVE-1 labeling identified endothelial cells, pericytes, smooth muscle cells, and lymphatic endothelial cells, respectively. RESULTS: These cells comprised microvascular networks with arterioles, venules, and capillaries. Compared to day 0, capillary sprouts per vascular length were increased by 3 and 5 days in culture (day 0, 0.08 ± 0.01; day 3, 3.19 ± 0.78; day 5, 2.49 ± 0.05 sprouts/mm; p < 0.05). Time-lapse imaging of cultured tissues from FlkEGFP mice showcases the use of the model for lineage studies. The impact is supported by the identification of endothelial cell jumping from one sprout to another. CONCLUSION: These results introduce a novel culture model for investigating multicellular dynamics during angiogenesis in real-time ex vivo microvascular networks.

Rac GTPase Activating Protein ARHGAP25 Regulates Leukocyte Transendothelial Migration in Mice.

ARHGAP25 is a Rac-specific GTPase-activating protein that is expressed primarily in hematopoietic cells. The involvement of ARHGAP25 in regulating the recruitment of leukocytes to inflammatory sites was investigated in genetically modified mice. Using intravital microscopy, we show that Arhgap25 deficiency affects all steps of leukocyte recruitment with a predominant enhancement of transendothelial migration of neutrophilic granulocytes. Increased transmigration of Arhgap25-deficient leukocytes is demonstrated in inflamed cremaster muscle venules, in a peritonitis model, and in an in vitro chemotaxis assay. Using bone marrow chimeric mice lacking ARHGAP25 in the hematopoietic compartment, we show that enhanced migration in the absence of ARHGAP25 is due to defective leukocyte function. In search for potential mechanisms of ARHGAP25-regulated migration of neutrophils, we detected an increase in the amount of active, GTP-bound Rac and Rac-dependent cytoskeletal changes in the absence of ARHGAP25, suggesting a critical role of ARHGAP25 in counterbalancing the Rac-activating effect of nucleotide exchange factors. Taken together, using Arhgap25-deficient mice, we identified ARHGAP25 as a relevant negative regulator of leukocyte transendothelial migration.

Extracellular MRP8/14 is a regulator of ß2 integrin-dependent neutrophil slow rolling and adhesion.

Myeloid-related proteins (MRPs) 8 and 14 are cytosolic proteins secreted from myeloid cells as proinflammatory mediators. Currently, the functional role of circulating extracellular MRP8/14 is unclear. Our present study identifies extracellular MRP8/14 as an autocrine player in the leukocyte adhesion cascade. We show that E-selectin-PSGL-1 interaction during neutrophil rolling triggers Mrp8/14 secretion. Released MRP8/14 in turn activates a TLR4-mediated, Rap1-GTPase-dependent pathway of rapid ß2 integrin activation in neutrophils. This extracellular activation loop reduces leukocyte rolling velocity and stimulates adhesion. Thus, we identify Mrp8/14 and TLR4 as important modulators of the leukocyte recruitment cascade during inflammation in vivo.

Endothelial cell-derived chemerin promotes dendritic cell transmigration.

ChemR23 is a chemotactic receptor expressed by APCs, such as dendritic cells, macrophages, and NK cells. Chemerin, the ChemR23 ligand, was detected by immunohistochemistry, to be associated with inflamed endothelial cells in autoimmune diseases, such as lupus erythematosus, psoriasis, and rheumatoid arthritis. This study reports that blood and lymphatic murine endothelial cells produce chemerin following retinoic acid stimulation. Conversely, proinflammatory cytokines, such as TNF-α, IFN-γ, and LPS, or calcitriol, are not effective. Retinoic acid-stimulated endothelial cells promoted dendritic cell adhesion under shear stress conditions and transmigration in a ChemR23-dependent manner. Activated endothelial cells upregulated the expression of the atypical chemotactic receptor CCRL2/ACKR5, a nonsignaling receptor able to bind and present chemerin to ChemR23(+) dendritic cells. Accordingly, activated endothelial cells expressed chemerin on the plasma membrane and promoted in a more efficient manner chemerin-dependent transmigration of dendritic cells. Finally, chemerin stimulation of myeloid dendritic cells induced the high-affinity binding of VCAM-1/CD106 Fc chimeric protein and promoted VCAM-1-dependent arrest to immobilized ligands under shear stress conditions. In conclusion, this study reports that retinoic acid-activated endothelial cells can promote myeloid and plasmacytoid dendritic cell transmigration across endothelial cell monolayers through the endogenous production of chemerin, the upregulation of CCRL2, and the activation of dendritic cell ß1 integrin affinity.

Neutrophil and endothelial adhesive function during human fetal ontogeny.

Attenuation of the immune response contributes to the high rate of neonatal infections, particularly in premature infants. Whereas our knowledge of innate immune functions in mature neonates is growing, little is known about the ontogeny of neutrophil recruitment. We investigated neutrophils and ECs in the course of gestation with respect to rolling and adhesive functions. With the use of microflow chambers, we demonstrate that the neutrophil's ability to roll and adhere directly correlates with gestational age. These adhesion-related abilities are very rare in extremely premature infants (<30 weeks of gestation), which may correlate with our observation of markedly reduced expression of PSGL-1 and Mac-1 on neutrophils in preterm infants. In parallel, the capacity of HUVECs to mediate neutrophil adhesion under flow increases with gestational age. In addition, HUVECs from extremely premature infants exerting the lowest ability to recruit adult neutrophils show a diminished up-regulation of E-selectin and ICAM-1. Finally, by following neutrophil function postnatally, we show that maturation of PMN recruitment proceeds equivalently during extra- and intrauterine development. Thus, PMN recruitment and EC adhesion-related functions are ontogenetically regulated in the fetus, which might contribute significantly to the high risk of life-threatening infections in premature infants.