Acquisition of microbes by the neonate, which begins immediately during birth, is influenced by gestational age and mother’s microbiota and modified by exposure to antibiotics1. In neonates, prolonged duration of antibiotic therapy is associated with increased risk of sepsis after 4 days of life, known as late-onset sepsis (LOS)2, a disorder critically controlled by neutrophils3, but a role for the microbiota in regulating neutrophil behavior in the neonate has not been described. We exposed pregnant mouse dams to antibiotics in drinking water to limit transfer of maternal microbes to the neonates. Antibiotic exposure of dams decreased the total number of microbes in the intestine, altered the structure of intestinal microbiota and changed the pattern of microbial colonization. These changes were associated with decreased numbers of circulating and bone marrow neutrophils and granulocyte/macrophage restricted progenitor cells in the bone marrow. Antibiotic-exposure of dams attenuated the postnatal granulocytosis by reducing the number of interleukin (IL) 17-producing cells in intestine and consequent production of granulocyte colony stimulating factor (G-CSF). Relative granulocytopenia contributed to increased susceptibility of antibiotic-exposed neonatal mice to Escherichia coli K1 and Klebsiella pneumoniae sepsis, which could be partially reversed by administration of G-CSF. Restoration of normal microbiota, through TLR4- and MYD88-dependent mechanism, induced accumulation of IL17-producing type 3 innate lymphoid cells (ILC) in the intestine, promoted granulocytosis, and restored the IL17-dependent resistance to sepsis. Specific depletion of ILCs prevented the IL17- and G-CSF-dependent granulocytosis and resistance to sepsis. These data support a role for the intestinal microbiota in regulation of granulocytosis and host resistance to sepsis in the neonates.

The circadian system is as an important regulator of immune function. Human inflammatory lung diseases frequently show time-of-day variation in symptom severity and lung function, but the mechanisms and cell types that are underlying these effects remain unclear. We show that pulmonary antibacterial responses are modulated by a circadian clock within epithelial club (Clara) cells. These drive circadian neutrophil recruitment to the lung via the chemokine CXCL5. Genetic ablation of the clock gene Bmal1 (also called Arntl or MOP3) in bronchiolar cells disrupts rhythmic Cxcl5 expression, resulting in exaggerated inflammatory responses to lipopolysaccharide and bacterial infection. Adrenalectomy blocks rhythmic inflammatory responses and the circadian regulation of CXCL5, suggesting a key role for the adrenal axis in driving CXCL5 expression and pulmonary neutrophil recruitment. Glucocorticoid receptor occupancy at the Cxcl5 locus shows circadian oscillations, but this is disrupted in mice with bronchiole-specific ablation of Bmal1, leading to enhanced CXCL5 expression despite normal corticosteroid secretion. In clock-gene disrupted mice the synthetic glucocorticoid dexamethasone loses anti-inflammatory efficacy. We now define a regulatory mechanism that links the circadian clock and glucocorticoid hormones to control both time-of-day variation and also the magnitude of pulmonary inflammation and responses to bacterial infection.

Summary The chemokine sink hypothesis pertaining to erythrocyte Duffy Antigen Receptor for Chemokines (DARC) during inflammation has received considerable attention, but lacks direct in vivo evidence. Here we demonstrate, using mice with a targeted deletion in CXCL5, that CXCL5 bound erythrocyte DARC and impaired its chemokine scavenging in blood. CXCL5 increased the plasma concentrations of CXCL1 and CXCL2 in part through inhibiting chemokine scavenging, impairing chemokine gradients and desensitizing CXCR2, which led to decreased neutrophil influx to the lung, increased lung bacterial burden and mortality in an Escherichia coli pneumonia model. In contrast, CXCL5 exerted a predominant role in mediating neutrophil influx to the lung during inflammation after LPS inhalation. Platelets and lung resident cells were the sources of homeostatic CXCL5 in blood and inflammatory CXCL5 in the lung respectively. This study presents a paradigm whereby platelets and red cells alter chemokine scavenging and neutrophil-chemokine interaction during inflammation.

Neutrophils are essential for maintaining innate immune surveillance under normal conditions, but also represent a major contributor to tissue damage during inflammation. Neutrophil homeostasis is therefore tightly regulated. Cxcr2 plays a critical role in neutrophil homeostasis, as Cxcr2 -/-mice demonstrate mild neutrophilia and severe neutrophil hyperplasia in the bone marrow. The mechanisms underlying these phenotypes, however, are unclear.

Sister chromatid separation depends on the release of cohesion by the activity of Esp1, a member of the caspase family [1, 2]. In budding yeast, Esp1p is kept inactive by its association with Pds1p, until the onset of anaphase, when Pds1p is ubiquitinated by the APC/Cdc20 complex [3--5] and subsequently degraded by the 26S proteasome. Pds1 is not an essential gene in budding yeast, but is required for cell cycle arrest prior to anaphase in response to the disruption of spindle structures [6, 7]. Thus, Pds1 mutant yeast cells display precocious sister chromatid separation in the presence of nocodazole [6]. Mammalian orthologs of yeast Esp1 and Pds1, separin and securin, have been identified [8], and, as anticipated, a nondegradable mutant form of securin inhibits sister separation when added to mitotic Xenopus egg extracts [8]. Securin was also independently identified as PTTG (pituitary tumor transforming gene), a gene overexpressed in pituitary tumors [9]. The relationship between its overexpression in tumors and its control of sister chromatid cohesion remains ill defined. To explore securin function in mammals, we took a targeted gene disruption approach in mice. Here, we report that securin is neither essential for cell viability nor required for spindle checkpoint function, and mice lacking securin are viable and apparently normal, but mouse embryonic fibroblasts lacking securin grow abnormally in culture.

Lad is an SH2 domain-containing adaptor protein that binds MEK kinase 2 (MEKK2), a mitogen-activated protein kinase (MAPK) kinase kinase for the extracellular signal-regulated kinase 5 (ERK5) and JNK pathways. Lad and MEKK2 are in a complex in resting cells. Antisense knockdown of Lad expression and targeted gene disruption of MEKK2 expression results in loss of epidermal growth factor (EGF) and stress stimuli-induced activation of ERK5. Activation of MEKK2 and the ERK5 pathway by EGF and stress stimuli is dependent on Src kinase activity. The Lad-binding motif is encoded within amino acids 228 to 282 in the N terminus of MEKK2, and expression of this motif blocks Lad-MEKK2 interaction, resulting in inhibition of Src-dependent activation of MEKK2 and ERK5. JNK activation by EGF is similarly inhibited by loss of Lad or MEKK2 expression and by blocking the interaction of MEKK2 and Lad. Our studies demonstrate that Src kinase activity is required for ERK5 activation in response to EGF, MEKK2 expression is required for ERK5 activation by Src, Lad and MEKK2 association is required for Src activation of ERK5, and EGF and Src stimulation of ERK5-regulated MEF2-dependent promoter activity requires a functional Lad-MEKK2 signaling complex.Extracellular signal-regulated kinase 5 (ERK5)/big mitogenactivated kinase 1 is a member of the mitogen-activated protein kinase (MAPK) family. Efforts to define the ERK5 signaling module have led to the identification of MEK5 as a specific ERK5 kinase (61). Sequence comparisons indicate that MEK5 is most related to the ERK1/2 kinases MEK1 and MEK2 (42). However, MEK5 is not significantly phosphorylated by the MEK1/2 kinase Raf-1 or MEKK1, nor does MEK5 phosphorylate ERK1/2 or JNK (13), indicating that MEK5/ ERK5 represents a distinct MAPK signaling cascade. The ERK5 pathway has been implicated in stress response and growth factor-promoted cell growth and survival (1,20). In neurons, blocking ERK5 activation diminished retrograde survival responses initiated by neurotrophin stimulation of axon terminals (56). A recent study showed that expression of an activated form of MEK5 resulted in serial assembly of sarcomeres in cardiomyocytes and eccentric cardiac hypertrophy in transgenic mice, suggesting a role for the MEK5/ERK5 module in mediating cytokine signaling associated with cardiac hypertrophy (36). ERK5 activates the MADS box transcription factors MEF2A, -C, and -D (19, 21, 28) and the Ets-domain transcription factor Sap1a (18). One consequence of activation of the MEF2 proteins is to induce c-jun expression that is essential for cell growth and cell cycle progression (7,19,21,23,28). Additionally, ERK5 regulates muscle cell differentiation via a mechanism probably involving MEF2 proteins and myocyte-specific activators such as MyoD (12).We and others have recently demonstrated that the two highly homologous MEK kinases, MEKK2 and -3, specifically interact with MEK5 and activate the ERK5 pathway (8, 49). Significantly, we found that MEKK2 is much more potent than MEKK3 in ERK5 activa...

Platelets and neutrophils contribute to the development of acute lung injury (ALI). However, the mechanism by which platelets make this contribution is incompletely understood. We investigated whether the two most abundant platelet chemokines, CXCL7, which induces neutrophil chemotaxis and activation, and CXCL4, which does neither, mediate ALI through complementary pathogenic pathways. To examine the role of platelet-derived chemokines in the pathogenesis of ALI using Cxcl7 2/2 and Cxcl4 2/2 knockout mice and mice that express human CXCL7 or CXCL4, we measured levels of chemokines in these mice. ALI was then induced by acid aspiration, and the severity of injury was evaluated by histology and by the presence of neutrophils and protein in the bronchoalveolar lavage fluid. Pulmonary vascular permeability was studied in vivo by measuring extravasation of fluorescently labeled dextran. Murine CXCL7, both recombinant and native protein released from platelets, can be N-terminally processed by cathepsin G to yield a biologically active CXCL7 fragment. Although Cxcl7 2/2 mice are protected from lung injury through the preservation of endothelial/epithelial barrier function combined with impaired neutrophils transmigration, Cxcl4 2/2 mice are protected through improved barrier function without affecting neutrophils transmigration to the airways. Sensitivity to ALI is restored by transgenic expression of CXCL7 or CXCL4. Platelet-derived CXCL7 and CXCL4 contribute to the pathogenesis of ALI through complementary effects on neutrophil chemotaxis and through activation and vascular permeability.

Annual epidemics and unexpected pandemics of influenza are threats to human health. Lung immune and inflammatory responses, such as those induced by respiratory infection influenza virus, determine the outcome of pulmonary pathogenesis. Platelet-derived chemokine (C-X-C motif) ligand 4 (CXCL4) has an immunoregulatory role in inflammatory diseases. Here we show that CXCL4 is associated with pulmonary influenza infection and has a critical role in protecting mice from fatal H1N1 virus respiratory infection. CXCL4 knockout resulted in diminished viral clearance from the lung and decreased lung inflammation during early infection but more severe lung pathology relative to wild-type mice during late infection. Additionally, CXCL4 deficiency decreased leukocyte accumulation in the infected lung with markedly decreased neutrophil infiltration into the lung during early infection and extensive leukocyte, especially lymphocyte accumulation at the late infection stage. Loss of CXCL4 did not affect the activation of adaptive immune T and B lymphocytes during the late stage of lung infection. Further study revealed that CXCL4 deficiency inhibited neutrophil recruitment to the infected mouse lung. Thus the above results identify CXCL4 as a vital immunoregulatory chemokine essential for protecting mice against influenza A virus infection, especially as it affects the development of lung injury and neutrophil mobilization to the inflamed lung.