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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-derived signals regulate postnatal granulocytosis via TLR4 and MyD88 dependent pathway(a and c) Neonatal mice deficient in MyD88 (Myd88−/−) or TLR4 (Tlr4−/−) and age and sex matched wild type (WT) littermates exposed or not exposed to combination of 5 antibiotics (ABX) were examined for number of circulating neutrophils or (b and d) bone marrow neutrophils. (e) Administration of LPS (10 ng) to neonatal mice exposed to combination of 5 antibiotics (ABX) or no antibiotics (No ABX) via oral gavage and assessment of IL17 transcripts in small intestine or (f) plasma G-CSF (g) circulating neutrophils or (h) bone marrow neutrophils 48 h following gavage. Data are representative of three independent experiments with 8-10 mice per group. Results are shown as the means ± s.e.m.
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Figure 4: Microbiota-derived signals regulate postnatal granulocytosis via TLR4 and MyD88 dependent pathway(a and c) Neonatal mice deficient in MyD88 (Myd88−/−) or TLR4 (Tlr4−/−) and age and sex matched wild type (WT) littermates exposed or not exposed to combination of 5 antibiotics (ABX) were examined for number of circulating neutrophils or (b and d) bone marrow neutrophils. (e) Administration of LPS (10 ng) to neonatal mice exposed to combination of 5 antibiotics (ABX) or no antibiotics (No ABX) via oral gavage and assessment of IL17 transcripts in small intestine or (f) plasma G-CSF (g) circulating neutrophils or (h) bone marrow neutrophils 48 h following gavage. Data are representative of three independent experiments with 8-10 mice per group. Results are shown as the means ± s.e.m.

Mentions: Since both commensal and pathogenic bacteria can be recognized by a number of pattern recognition receptors (PRR)29, we hypothesized that signals derived from intestinal microbiota could regulate postnatal granulocytosis through signaling via TLR and NOD family members. We examined the postnatal granulocytic response of mice deficient in TLR2 (Tlr2−/−), TLR4 (Tlr4−/−), NOD2 (Nod2−/−) and MYD88 (MyD88−/−), the latter of which encodes a critical adaptor molecule, which regulates signaling through multiple TLRs30. The number of circulating and bone marrow neutrophils in neonatal Tlr4−/− and MyD88−/− mice (Fig. 4a-d), but not Tlr2−/− and Nod2−/− mice (Fig. S4a-d) was lower as compared to age- and strain-matched controls, consistent with previous observations that commensal detection by PRR and subsequent signaling via MYD88 promotes systemic immune responses31. Tlr4−/− mice have distinctly different microbiota from WT mice32, therefore to control for potential differences in the microbiota, we blocked TLR4 signaling in WT neonatal mice with a neutralizing antibody. Treatment of neonatal mice with anti-TLR4 neutralizing antibody resulted in decreased number of circulating and bone marrow neutrophils compared to neonatal mice treated with isotype-control antibody (Fig. S4e-f). Nevertheless, Tlr4−/− mice or mice treated with anti-TLR4 neutralizing antibody or MyD88−/− mice failed to completely recapitulate the phenotype observed in antibiotic-treated or Il17ra−/− neonatal mice, suggesting that TLR4 and MYD88 signaling pathways do not completely account for postnatal granulocytosis and alternate PRR pathways are involved in regulating postnatal granulocytosis. TIR-domain-containing adapter-inducing interferon-β (TRIF), which relays signals from TLR4 independent of MYD8833 is critical in neutrophil homeostasis34. Therefore TRIF, or members of the NOD like receptor (NLR) family, such as NLR635 which recognize microbial patterns and mediate intestinal homeostasis36 could be additional microbial sensor in regulating postnatal granulocytosis. Furthermore, levels of short-chain fatty acids (SCFA), gut microbiota-derived fermentation products, which have a critical role in intestinal inflammation and host resistance are decreased in the intestinal contents of antibiotic-exposed mice37. Therefore SCFA could play a potential role in regulating postnatal granulocytosis.


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-derived signals regulate postnatal granulocytosis via TLR4 and MyD88 dependent pathway(a and c) Neonatal mice deficient in MyD88 (Myd88−/−) or TLR4 (Tlr4−/−) and age and sex matched wild type (WT) littermates exposed or not exposed to combination of 5 antibiotics (ABX) were examined for number of circulating neutrophils or (b and d) bone marrow neutrophils. (e) Administration of LPS (10 ng) to neonatal mice exposed to combination of 5 antibiotics (ABX) or no antibiotics (No ABX) via oral gavage and assessment of IL17 transcripts in small intestine or (f) plasma G-CSF (g) circulating neutrophils or (h) bone marrow neutrophils 48 h following gavage. Data are representative of three independent experiments with 8-10 mice per group. Results are shown as the means ± s.e.m.
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Figure 4: Microbiota-derived signals regulate postnatal granulocytosis via TLR4 and MyD88 dependent pathway(a and c) Neonatal mice deficient in MyD88 (Myd88−/−) or TLR4 (Tlr4−/−) and age and sex matched wild type (WT) littermates exposed or not exposed to combination of 5 antibiotics (ABX) were examined for number of circulating neutrophils or (b and d) bone marrow neutrophils. (e) Administration of LPS (10 ng) to neonatal mice exposed to combination of 5 antibiotics (ABX) or no antibiotics (No ABX) via oral gavage and assessment of IL17 transcripts in small intestine or (f) plasma G-CSF (g) circulating neutrophils or (h) bone marrow neutrophils 48 h following gavage. Data are representative of three independent experiments with 8-10 mice per group. Results are shown as the means ± s.e.m.
Mentions: Since both commensal and pathogenic bacteria can be recognized by a number of pattern recognition receptors (PRR)29, we hypothesized that signals derived from intestinal microbiota could regulate postnatal granulocytosis through signaling via TLR and NOD family members. We examined the postnatal granulocytic response of mice deficient in TLR2 (Tlr2−/−), TLR4 (Tlr4−/−), NOD2 (Nod2−/−) and MYD88 (MyD88−/−), the latter of which encodes a critical adaptor molecule, which regulates signaling through multiple TLRs30. The number of circulating and bone marrow neutrophils in neonatal Tlr4−/− and MyD88−/− mice (Fig. 4a-d), but not Tlr2−/− and Nod2−/− mice (Fig. S4a-d) was lower as compared to age- and strain-matched controls, consistent with previous observations that commensal detection by PRR and subsequent signaling via MYD88 promotes systemic immune responses31. Tlr4−/− mice have distinctly different microbiota from WT mice32, therefore to control for potential differences in the microbiota, we blocked TLR4 signaling in WT neonatal mice with a neutralizing antibody. Treatment of neonatal mice with anti-TLR4 neutralizing antibody resulted in decreased number of circulating and bone marrow neutrophils compared to neonatal mice treated with isotype-control antibody (Fig. S4e-f). Nevertheless, Tlr4−/− mice or mice treated with anti-TLR4 neutralizing antibody or MyD88−/− mice failed to completely recapitulate the phenotype observed in antibiotic-treated or Il17ra−/− neonatal mice, suggesting that TLR4 and MYD88 signaling pathways do not completely account for postnatal granulocytosis and alternate PRR pathways are involved in regulating postnatal granulocytosis. TIR-domain-containing adapter-inducing interferon-β (TRIF), which relays signals from TLR4 independent of MYD8833 is critical in neutrophil homeostasis34. Therefore TRIF, or members of the NOD like receptor (NLR) family, such as NLR635 which recognize microbial patterns and mediate intestinal homeostasis36 could be additional microbial sensor in regulating postnatal granulocytosis. Furthermore, levels of short-chain fatty acids (SCFA), gut microbiota-derived fermentation products, which have a critical role in intestinal inflammation and host resistance are decreased in the intestinal contents of antibiotic-exposed mice37. Therefore SCFA could play a potential role in regulating postnatal granulocytosis.

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