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.