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The microbiota regulates neutrophil homeostasis and host resistance to Escherichia coli K1 sepsis in neonatal mice.

Deshmukh HS, Liu Y, Menkiti OR, Mei J, Dai N, O'Leary CE, Oliver PM, Kolls JK, Weiser JN, Worthen GS - Nat. Med. (2014)

Bottom Line: Antibiotic exposure of dams decreased the total number and composition of microbes in the intestine of the neonates.Antibiotic exposure of dams reduced the number of interleukin-17 (IL-17)-producing cells in the intestine and production of granulocyte colony-stimulating factor (G-CSF).Granulocytopenia was associated with impaired host defense and increased susceptibility to Escherichia coli K1 and Klebsiella pneumoniae sepsis in antibiotic-treated neonates, which could be partially reversed by administration of G-CSF.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Neonatology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. [2] Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

ABSTRACT
Neonatal colonization by microbes, which begins immediately after birth, is influenced by gestational age and the mother's microbiota and is modified by exposure to antibiotics. In neonates, prolonged duration of antibiotic therapy is associated with increased risk of late-onset sepsis (LOS), a disorder controlled by neutrophils. A role for the microbiota in regulating neutrophil development and susceptibility to sepsis in the neonate remains unclear. 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 and composition of microbes in the intestine of the neonates. This was associated with decreased numbers of circulating and bone marrow neutrophils and granulocyte/macrophage-restricted progenitor cells in the bone marrow of antibiotic-treated and germ-free neonates. Antibiotic exposure of dams reduced the number of interleukin-17 (IL-17)-producing cells in the intestine and production of granulocyte colony-stimulating factor (G-CSF). Granulocytopenia was associated with impaired host defense and increased susceptibility to Escherichia coli K1 and Klebsiella pneumoniae sepsis in antibiotic-treated neonates, which could be partially reversed by administration of G-CSF. Transfer of a normal microbiota into antibiotic-treated neonates induced IL-17 production by group 3 innate lymphoid cells (ILCs) in the intestine, increasing plasma G-CSF levels and neutrophil numbers in a Toll-like receptor 4 (TLR4)- and myeloid differentiation factor 88 (MyD88)-dependent manner and restored IL-17-dependent resistance to sepsis. Specific depletion of ILCs prevented IL-17- and G-CSF-dependent granulocytosis and resistance to sepsis. These data support a role for the intestinal microbiota in regulation of granulocytosis, neutrophil homeostasis and host resistance to sepsis in neonates.

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Microbiota regulates postnatal granulocytosis and controls host resistance to E. coli(a) 5 day-old sex-matched neonatal mice exposed to combination of 3 (3ABX) or 5 antibiotics (5ABX) or no antibiotics (No ABX) were examined for susceptibility after inoculation with E. coli via intraperitoneal route. (b) 5 day-old sex-matched neonatal mice exposed to combination of 3 (3ABX) or (c) 5 antibiotics (5ABX) or no antibiotics (No ABX) were examined for circulating neutrophils or (d) plasma G-CSF levels 4h after inoculation with E. coli. (e) 5 day-old sex-matched neonatal mice exposed to combination of 3 (3ABX) or (f) 5 antibiotics (5ABX) or no antibiotics (No ABX) were treated with G-CSF and examined for susceptibility after inoculation with E. coli via intraperitoneal route. * Significantly different from neonatal mice not exposed to antibiotics (No ABX), ** significantly different from ABX-exposed neonatal mice. (g) Transfer of intestinal contents from postnatal day 3 old control (no antibiotic-exposed) mice to sex-matched neonatal mice exposed to combination of 5 antibiotics (5ABX) or (h) 3 antibiotics (3ABX) via oral gavage on postnatal day 3 and assessment of susceptibility to E. coli 48 h following transfer (postnatal day 5). (i) Transfer of intestinal contents from postnatal day 3 old control (no antibiotic-exposed) mice to sex-matched neonatal mice exposed to combination of 5 antibiotics (ABX) via oral gavage on postnatal day 3 and assessment of circulating or (j) bone marrow neutrophils 48 h following transfer (postnatal day 5). Data are representative of three independent experiments with 12 mice per group. Results are shown as the means ± s.e.m. * Significantly different from control (No ABX) neonatal mice, ** significantly different from ABX-exposed neonatal mice.
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Figure 2: Microbiota regulates postnatal granulocytosis and controls host resistance to E. coli(a) 5 day-old sex-matched neonatal mice exposed to combination of 3 (3ABX) or 5 antibiotics (5ABX) or no antibiotics (No ABX) were examined for susceptibility after inoculation with E. coli via intraperitoneal route. (b) 5 day-old sex-matched neonatal mice exposed to combination of 3 (3ABX) or (c) 5 antibiotics (5ABX) or no antibiotics (No ABX) were examined for circulating neutrophils or (d) plasma G-CSF levels 4h after inoculation with E. coli. (e) 5 day-old sex-matched neonatal mice exposed to combination of 3 (3ABX) or (f) 5 antibiotics (5ABX) or no antibiotics (No ABX) were treated with G-CSF and examined for susceptibility after inoculation with E. coli via intraperitoneal route. * Significantly different from neonatal mice not exposed to antibiotics (No ABX), ** significantly different from ABX-exposed neonatal mice. (g) Transfer of intestinal contents from postnatal day 3 old control (no antibiotic-exposed) mice to sex-matched neonatal mice exposed to combination of 5 antibiotics (5ABX) or (h) 3 antibiotics (3ABX) via oral gavage on postnatal day 3 and assessment of susceptibility to E. coli 48 h following transfer (postnatal day 5). (i) Transfer of intestinal contents from postnatal day 3 old control (no antibiotic-exposed) mice to sex-matched neonatal mice exposed to combination of 5 antibiotics (ABX) via oral gavage on postnatal day 3 and assessment of circulating or (j) bone marrow neutrophils 48 h following transfer (postnatal day 5). Data are representative of three independent experiments with 12 mice per group. Results are shown as the means ± s.e.m. * Significantly different from control (No ABX) neonatal mice, ** significantly different from ABX-exposed neonatal mice.

Mentions: Neutrophils are essential in controlling infection due to E. coli serotype K1, an important cause of LOS and meningitis in human neonates15 and the leading cause of death in preterm infants16. Neutropenia is an important risk factor in fatal neonatal sepsis3. In order to test whether antibiotic exposure modified host defense against infection, neonatal mice (postnatal day 3 or 5 or 7 or 14 old) were exposed to the combination of 5 antibiotics or the more clinically relevant combination of ampicillin, gentamicin and vancomycin through their Dams beginning 5 days before birth and continuing until day 14. Antibiotic-exposed or age-matched (no antibiotic-exposed) controls, were inoculated intraperitoneally with 104 CFU g−1 of E. coli K1 or 106 CFU g−1 of Klebsiella pneumoniae, another important gram negative pathogen in neonates. Neonatal mice exposed to the 5-antibiotic combination or to the 3-antibiotic combination demonstrated marked susceptibility to E. coli K1 as compared to controls (median survival 8 h and 10 h respectively vs. >72 h) (Fig. 2a, Fig. S2a-b), with increased bacteria in blood, spleen and peritoneal fluid, indicating bacteremia and decreased neutrophil recruitment in the peritoneal fluid as compared to age-matched controls (Fig. S2c-e). Similarly, antibiotic-exposed postnatal day 5 old neonatal mice demonstrated marked susceptibility to K. pneumoniae as compared to controls (median survival 14 h vs. >72 h) (Fig. S2f).


The microbiota regulates neutrophil homeostasis and host resistance to Escherichia coli K1 sepsis in neonatal mice.

Deshmukh HS, Liu Y, Menkiti OR, Mei J, Dai N, O'Leary CE, Oliver PM, Kolls JK, Weiser JN, Worthen GS - Nat. Med. (2014)

Microbiota regulates postnatal granulocytosis and controls host resistance to E. coli(a) 5 day-old sex-matched neonatal mice exposed to combination of 3 (3ABX) or 5 antibiotics (5ABX) or no antibiotics (No ABX) were examined for susceptibility after inoculation with E. coli via intraperitoneal route. (b) 5 day-old sex-matched neonatal mice exposed to combination of 3 (3ABX) or (c) 5 antibiotics (5ABX) or no antibiotics (No ABX) were examined for circulating neutrophils or (d) plasma G-CSF levels 4h after inoculation with E. coli. (e) 5 day-old sex-matched neonatal mice exposed to combination of 3 (3ABX) or (f) 5 antibiotics (5ABX) or no antibiotics (No ABX) were treated with G-CSF and examined for susceptibility after inoculation with E. coli via intraperitoneal route. * Significantly different from neonatal mice not exposed to antibiotics (No ABX), ** significantly different from ABX-exposed neonatal mice. (g) Transfer of intestinal contents from postnatal day 3 old control (no antibiotic-exposed) mice to sex-matched neonatal mice exposed to combination of 5 antibiotics (5ABX) or (h) 3 antibiotics (3ABX) via oral gavage on postnatal day 3 and assessment of susceptibility to E. coli 48 h following transfer (postnatal day 5). (i) Transfer of intestinal contents from postnatal day 3 old control (no antibiotic-exposed) mice to sex-matched neonatal mice exposed to combination of 5 antibiotics (ABX) via oral gavage on postnatal day 3 and assessment of circulating or (j) bone marrow neutrophils 48 h following transfer (postnatal day 5). Data are representative of three independent experiments with 12 mice per group. Results are shown as the means ± s.e.m. * Significantly different from control (No ABX) neonatal mice, ** significantly different from ABX-exposed neonatal mice.
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Figure 2: Microbiota regulates postnatal granulocytosis and controls host resistance to E. coli(a) 5 day-old sex-matched neonatal mice exposed to combination of 3 (3ABX) or 5 antibiotics (5ABX) or no antibiotics (No ABX) were examined for susceptibility after inoculation with E. coli via intraperitoneal route. (b) 5 day-old sex-matched neonatal mice exposed to combination of 3 (3ABX) or (c) 5 antibiotics (5ABX) or no antibiotics (No ABX) were examined for circulating neutrophils or (d) plasma G-CSF levels 4h after inoculation with E. coli. (e) 5 day-old sex-matched neonatal mice exposed to combination of 3 (3ABX) or (f) 5 antibiotics (5ABX) or no antibiotics (No ABX) were treated with G-CSF and examined for susceptibility after inoculation with E. coli via intraperitoneal route. * Significantly different from neonatal mice not exposed to antibiotics (No ABX), ** significantly different from ABX-exposed neonatal mice. (g) Transfer of intestinal contents from postnatal day 3 old control (no antibiotic-exposed) mice to sex-matched neonatal mice exposed to combination of 5 antibiotics (5ABX) or (h) 3 antibiotics (3ABX) via oral gavage on postnatal day 3 and assessment of susceptibility to E. coli 48 h following transfer (postnatal day 5). (i) Transfer of intestinal contents from postnatal day 3 old control (no antibiotic-exposed) mice to sex-matched neonatal mice exposed to combination of 5 antibiotics (ABX) via oral gavage on postnatal day 3 and assessment of circulating or (j) bone marrow neutrophils 48 h following transfer (postnatal day 5). Data are representative of three independent experiments with 12 mice per group. Results are shown as the means ± s.e.m. * Significantly different from control (No ABX) neonatal mice, ** significantly different from ABX-exposed neonatal mice.
Mentions: Neutrophils are essential in controlling infection due to E. coli serotype K1, an important cause of LOS and meningitis in human neonates15 and the leading cause of death in preterm infants16. Neutropenia is an important risk factor in fatal neonatal sepsis3. In order to test whether antibiotic exposure modified host defense against infection, neonatal mice (postnatal day 3 or 5 or 7 or 14 old) were exposed to the combination of 5 antibiotics or the more clinically relevant combination of ampicillin, gentamicin and vancomycin through their Dams beginning 5 days before birth and continuing until day 14. Antibiotic-exposed or age-matched (no antibiotic-exposed) controls, were inoculated intraperitoneally with 104 CFU g−1 of E. coli K1 or 106 CFU g−1 of Klebsiella pneumoniae, another important gram negative pathogen in neonates. Neonatal mice exposed to the 5-antibiotic combination or to the 3-antibiotic combination demonstrated marked susceptibility to E. coli K1 as compared to controls (median survival 8 h and 10 h respectively vs. >72 h) (Fig. 2a, Fig. S2a-b), with increased bacteria in blood, spleen and peritoneal fluid, indicating bacteremia and decreased neutrophil recruitment in the peritoneal fluid as compared to age-matched controls (Fig. S2c-e). Similarly, antibiotic-exposed postnatal day 5 old neonatal mice demonstrated marked susceptibility to K. pneumoniae as compared to controls (median survival 14 h vs. >72 h) (Fig. S2f).

Bottom Line: Antibiotic exposure of dams decreased the total number and composition of microbes in the intestine of the neonates.Antibiotic exposure of dams reduced the number of interleukin-17 (IL-17)-producing cells in the intestine and production of granulocyte colony-stimulating factor (G-CSF).Granulocytopenia was associated with impaired host defense and increased susceptibility to Escherichia coli K1 and Klebsiella pneumoniae sepsis in antibiotic-treated neonates, which could be partially reversed by administration of G-CSF.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Neonatology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. [2] Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

ABSTRACT
Neonatal colonization by microbes, which begins immediately after birth, is influenced by gestational age and the mother's microbiota and is modified by exposure to antibiotics. In neonates, prolonged duration of antibiotic therapy is associated with increased risk of late-onset sepsis (LOS), a disorder controlled by neutrophils. A role for the microbiota in regulating neutrophil development and susceptibility to sepsis in the neonate remains unclear. 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 and composition of microbes in the intestine of the neonates. This was associated with decreased numbers of circulating and bone marrow neutrophils and granulocyte/macrophage-restricted progenitor cells in the bone marrow of antibiotic-treated and germ-free neonates. Antibiotic exposure of dams reduced the number of interleukin-17 (IL-17)-producing cells in the intestine and production of granulocyte colony-stimulating factor (G-CSF). Granulocytopenia was associated with impaired host defense and increased susceptibility to Escherichia coli K1 and Klebsiella pneumoniae sepsis in antibiotic-treated neonates, which could be partially reversed by administration of G-CSF. Transfer of a normal microbiota into antibiotic-treated neonates induced IL-17 production by group 3 innate lymphoid cells (ILCs) in the intestine, increasing plasma G-CSF levels and neutrophil numbers in a Toll-like receptor 4 (TLR4)- and myeloid differentiation factor 88 (MyD88)-dependent manner and restored IL-17-dependent resistance to sepsis. Specific depletion of ILCs prevented IL-17- and G-CSF-dependent granulocytosis and resistance to sepsis. These data support a role for the intestinal microbiota in regulation of granulocytosis, neutrophil homeostasis and host resistance to sepsis in neonates.

Show MeSH
Related in: MedlinePlus