Neutrophil recruitment is an important early step in controlling tissue infections or injury. Here, we report that this influx depends on both tissue-resident mast cells and macrophages. Mice with mast cell deficiency recruit reduced numbers of neutrophils in the first few hours of intraperitoneal lipopolysaccharide (LPS) stimulation. Conversely, in mice with clodronate-ablated macrophages, neutrophils extravasate, but have limited ability to reach the peritoneal fluid. Tissue macrophages synthesize neutrophil chemoattractants CXCL1/CXCL2 (CXC chemokine ligands 1/2) in response to LPS. Mast cells also produce these chemokines of which a proportion are preformed in granules. Release of the granules and new CXCL1/CXCL2 synthesis is Toll-like receptor 4–dependent. Both in vivo studies with blocking monoclonal antibodies and in vitro chemotaxis experiments show the neutrophil response to mast cells and macrophages to be CXCL1/CXCL2-dependent. The data are in keeping with the model that mast cells, optimally positioned in close proximity to the vasculature, initiate an early phase of neutrophil recruitment by releasing the chemoattractants CXCL1/CXCL2. Having arrived within the stimulated tissue, neutrophils penetrate further in a macrophage-dependent manner. Therefore, we demonstrate a positive role for mast cells in tissue inflammation and define how this comes about with contribution from a second tissue cell, the macrophage.

The decline in hematopoietic function seen during aging involves a progressive reduction in the immune response and an increased incidence of myeloid malignancy, and has been linked to aging of hematopoietic stem cells (HSCs). The molecular mechanisms underlying HSC aging remain unclear. Here we demonstrate that elevated activity of the small RhoGTPase Cdc42 in aged HSCs is causally linked to HSC aging and correlates with a loss of polarity in aged HSCs. Pharmacological inhibition of Cdc42 activity functionally rejuvenates aged HSCs, increases the percentage of polarized cells in an aged HSC population, and restores the level and spatial distribution of histone H4 lysine 16 acetylation to a status similar to that seen in young HSCs. Our data therefore suggest a mechanistic role for Cdc42 activity in HSC biology and epigenetic regulation, and identify Cdc42 activity as a pharmacological target for ameliorating stem cell aging.

Imaging the unforeseen fate of neutrophils: Inflammation that results from insults such as ischemia and reperfusion or trauma in the absence of microorganisms is known as “sterile inflammation.” Neutrophils are recruited in vast numbers during sterile inflammation and have been thought to play a detrimental role. Wang et al. used intravital microscopy to show that neutrophils actually perform helpful tasks such as removing and regenerating thermally damaged blood vessels in the liver (see the Perspective by Garner and de Visser). Moreover, neutrophils neither die nor are phagocytosed. Instead, they return to the circulation in a process called “reverse transmigration,” making a pit stop in the lungs, before ending their lives where they began—in the bone marrow. Thus, a reconsideration of the use of anti-neutrophil therapies after injury may be warranted. Science , this issue p. 111 ; see also p. 42

Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunocompromised patients. Macrophages and neutrophils are known to kill conidia, whereas hyphae are killed mainly by neutrophils. Since hyphae are too large to be engulfed, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs) consisting of nuclear DNA decorated with fungicidal proteins. However, until now NET formation in response to A. fumigatus has only been demonstrated in vitro, the importance of neutrophils for their production in vivo is unclear and the molecular mechanisms of the fungus to defend against NET formation are unknown. Here, we show that human neutrophils produce NETs in vitro when encountering A. fumigatus. In time-lapse movies NET production was a highly dynamic process which, however, was only exhibited by a sub-population of cells. NETosis was maximal against hyphae, but reduced against resting and swollen conidia. In a newly developed mouse model we could then demonstrate the existence and measure the kinetics of NET formation in vivo by 2-photon microscopy of Aspergillus-infected lungs. We also observed the enormous dynamics of neutrophils within the lung and their ability to interact with and phagocytose fungal elements in situ. Furthermore, systemic neutrophil depletion in mice almost completely inhibited NET formation in lungs, thus directly linking the immigration of neutrophils with NET formation in vivo. By using fungal mutants and purified proteins we demonstrate that hydrophobin RodA, a surface protein making conidia immunologically inert, led to reduced NET formation of neutrophils encountering Aspergillus fungal elements. NET-dependent killing of Aspergillus-hyphae could be demonstrated at later time-points, but was only moderate. Thus, these data establish that NET formation occurs in vivo during host defence against A. fumigatus, but suggest that it does not play a major role in killing this fungus. Instead, NETs may have a fungistatic effect and may prevent further spreading. The fungus Aspergillus fumigatus grows on decaying organic matter and produces large numbers of spores, called conidia, which are constantly inhaled by humans. This is harmless, because we have a functioning defence system of immune cells called neutrophil granulocytes, but people with too few or non-functioning neutrophils can die of Aspergillus infections. Neutrophils invade the lung, engulf/phagocytose and thereby kill conidia. Dying neutrophils can also throw their nuclear DNA on hyphal elements as NETs (Neutrophil Extracellular Traps) that are decorated with antimicrobial proteins. Thus, larger fungal amounts, including tissue-invading hyphae, can still be controlled. However, until today the formation of NETs has not been demonstrated in Aspergillus-infected lungs, the role of neutrophils for this process was unknown and whether the fungus has anti-NET defence strategies on its own was not clear. We demonstrate here the existence of NETs in Aspergillus-infected lungs, show that neutrophils produce these structures and that they phagocytose fungal elements within the lung tissue. Furthermore, we show that Aspergillus camouflages its spores by means of the surface protein hydrophobin RodA, which is able to strongly prevent NET formation by neutrophils. These studies shed new light on the dynamics and molecular mechanisms of this key process of host-pathogen interaction. Although these data establish that NET formation occurs in vivo during host defence against A. fumigatus, we suggest that NET formation does not play a major role in killing this fungus.