Limits...
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.

Show MeSH

Related in: MedlinePlus

Microbiota-derived signals regulate postnatal granulocytosis via IL17 and G-CSF dependent pathway(a) Age-matched germ free (GF) or conventionalized (CNV) mice or neonatal mice exposed to combination of 5 antibiotics (ABX) or (b) age- and sex-matched neonatal mice exposed to combination of 5 antibiotics (ABX) or no antibiotics (No ABX) were examined for IL17A transcripts in the intestine. (c) Transfer of intestinal contents from age- and sex-matched control (no antibiotic-exposed) mice to neonatal mice exposed to combination of 5 antibiotics (ABX) via oral gavage and assessment of IL17A transcripts 48 h following transfer. (d) Neonatal mice deficient in receptor for IL17A (Il17ra−/−) 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. (e) Treatment with neutralizing antibody against IL17A daily on postnatal day 0-3 and transfer of intestinal contents from postnatal day 3 control (no antibiotic-exposed) mice to neonatal mice exposed to combination of 5 antibiotics (ABX) via oral gavage on postnatal day 3 and assessment of circulating or (f) bone marrow neutrophils 48 h following transfer (postnatal day 5). (g) Age- and sex-matched neonatal mice exposed to combination of 5 antibiotics (ABX) or no antibiotics (No ABX) were examined for frequency of IL17+ cells in small intestine lamina propria. (h) 3 day-old neonatal mice were examined for IL17+ cells in small intestine lamina propria. Flow cytometry plots are gated on live IL17+ cells and frequency of each distinct population of cells is indicated. Representative histograms from 5 separate experiments. (i) Treatment of neonatal Rag1−/− mice with anti-CD90.2 antibody or isotype control antibody (5 μg g-1 body weight) daily on postnatal day 0-3 and assessment of circulating and (j) bone marrow neutrophils on postnatal day 3. Data are representative of three independent experiments with 8-12 mice per group. Results are shown as the means ± s.e.m.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4016187&req=5

Figure 3: Microbiota-derived signals regulate postnatal granulocytosis via IL17 and G-CSF dependent pathway(a) Age-matched germ free (GF) or conventionalized (CNV) mice or neonatal mice exposed to combination of 5 antibiotics (ABX) or (b) age- and sex-matched neonatal mice exposed to combination of 5 antibiotics (ABX) or no antibiotics (No ABX) were examined for IL17A transcripts in the intestine. (c) Transfer of intestinal contents from age- and sex-matched control (no antibiotic-exposed) mice to neonatal mice exposed to combination of 5 antibiotics (ABX) via oral gavage and assessment of IL17A transcripts 48 h following transfer. (d) Neonatal mice deficient in receptor for IL17A (Il17ra−/−) 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. (e) Treatment with neutralizing antibody against IL17A daily on postnatal day 0-3 and transfer of intestinal contents from postnatal day 3 control (no antibiotic-exposed) mice to neonatal mice exposed to combination of 5 antibiotics (ABX) via oral gavage on postnatal day 3 and assessment of circulating or (f) bone marrow neutrophils 48 h following transfer (postnatal day 5). (g) Age- and sex-matched neonatal mice exposed to combination of 5 antibiotics (ABX) or no antibiotics (No ABX) were examined for frequency of IL17+ cells in small intestine lamina propria. (h) 3 day-old neonatal mice were examined for IL17+ cells in small intestine lamina propria. Flow cytometry plots are gated on live IL17+ cells and frequency of each distinct population of cells is indicated. Representative histograms from 5 separate experiments. (i) Treatment of neonatal Rag1−/− mice with anti-CD90.2 antibody or isotype control antibody (5 μg g-1 body weight) daily on postnatal day 0-3 and assessment of circulating and (j) bone marrow neutrophils on postnatal day 3. Data are representative of three independent experiments with 8-12 mice per group. Results are shown as the means ± s.e.m.

Mentions: Regulation of G-CSF by IL17A is a key mechanism controlling granulocytosis20 and neutrophil recruitment during bacterial infection21. Transcript levels of IL17A and were significantly lower in intestine but not lung of GF mice (Fig. 3a, S3a) and antibiotic-exposed neonatal mice (Fig. 3b) suggesting a role for IL17A in regulation of postnatal granulocytosis. Furthermore, transfer of normal microbiota restored IL17A levels in the intestine of antibiotic-exposed neonatal mice, though not to the same extent as age-matched controls (Fig. 3c). To determine the importance of IL17 signaling in postnatal granulocytosis, we utilized mice deficient in IL17A receptor (Il17ra−/−) or blocked IL17A signaling with a neutralizing antibody. The number of circulating neutrophils was significantly lower in Il17ra−/− neonatal mice (Fig. 3d). Interestingly, the number of circulating neutrophils in neonatal Il17ra−/− mice was no higher than antibiotic-exposed age- and strain-matched controls. Treatment of neonatal mice with anti-IL17A blocking antibody resulted in lower neutrophil numbers than neonatal mice treated with isotype-control antibody (Fig. S3b), and in addition, blocked the increase in circulating and bone marrow neutrophils (Fig. 3e-f) and plasma G-CSF (Fig. S3c) in antibiotic-exposed neonatal mice after transfer of normal intestinal microbiota. These data confirm that intestinal microbiota regulates postnatal granulocytosis via IL17A.


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 IL17 and G-CSF dependent pathway(a) Age-matched germ free (GF) or conventionalized (CNV) mice or neonatal mice exposed to combination of 5 antibiotics (ABX) or (b) age- and sex-matched neonatal mice exposed to combination of 5 antibiotics (ABX) or no antibiotics (No ABX) were examined for IL17A transcripts in the intestine. (c) Transfer of intestinal contents from age- and sex-matched control (no antibiotic-exposed) mice to neonatal mice exposed to combination of 5 antibiotics (ABX) via oral gavage and assessment of IL17A transcripts 48 h following transfer. (d) Neonatal mice deficient in receptor for IL17A (Il17ra−/−) 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. (e) Treatment with neutralizing antibody against IL17A daily on postnatal day 0-3 and transfer of intestinal contents from postnatal day 3 control (no antibiotic-exposed) mice to neonatal mice exposed to combination of 5 antibiotics (ABX) via oral gavage on postnatal day 3 and assessment of circulating or (f) bone marrow neutrophils 48 h following transfer (postnatal day 5). (g) Age- and sex-matched neonatal mice exposed to combination of 5 antibiotics (ABX) or no antibiotics (No ABX) were examined for frequency of IL17+ cells in small intestine lamina propria. (h) 3 day-old neonatal mice were examined for IL17+ cells in small intestine lamina propria. Flow cytometry plots are gated on live IL17+ cells and frequency of each distinct population of cells is indicated. Representative histograms from 5 separate experiments. (i) Treatment of neonatal Rag1−/− mice with anti-CD90.2 antibody or isotype control antibody (5 μg g-1 body weight) daily on postnatal day 0-3 and assessment of circulating and (j) bone marrow neutrophils on postnatal day 3. Data are representative of three independent experiments with 8-12 mice per group. Results are shown as the means ± s.e.m.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016187&req=5

Figure 3: Microbiota-derived signals regulate postnatal granulocytosis via IL17 and G-CSF dependent pathway(a) Age-matched germ free (GF) or conventionalized (CNV) mice or neonatal mice exposed to combination of 5 antibiotics (ABX) or (b) age- and sex-matched neonatal mice exposed to combination of 5 antibiotics (ABX) or no antibiotics (No ABX) were examined for IL17A transcripts in the intestine. (c) Transfer of intestinal contents from age- and sex-matched control (no antibiotic-exposed) mice to neonatal mice exposed to combination of 5 antibiotics (ABX) via oral gavage and assessment of IL17A transcripts 48 h following transfer. (d) Neonatal mice deficient in receptor for IL17A (Il17ra−/−) 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. (e) Treatment with neutralizing antibody against IL17A daily on postnatal day 0-3 and transfer of intestinal contents from postnatal day 3 control (no antibiotic-exposed) mice to neonatal mice exposed to combination of 5 antibiotics (ABX) via oral gavage on postnatal day 3 and assessment of circulating or (f) bone marrow neutrophils 48 h following transfer (postnatal day 5). (g) Age- and sex-matched neonatal mice exposed to combination of 5 antibiotics (ABX) or no antibiotics (No ABX) were examined for frequency of IL17+ cells in small intestine lamina propria. (h) 3 day-old neonatal mice were examined for IL17+ cells in small intestine lamina propria. Flow cytometry plots are gated on live IL17+ cells and frequency of each distinct population of cells is indicated. Representative histograms from 5 separate experiments. (i) Treatment of neonatal Rag1−/− mice with anti-CD90.2 antibody or isotype control antibody (5 μg g-1 body weight) daily on postnatal day 0-3 and assessment of circulating and (j) bone marrow neutrophils on postnatal day 3. Data are representative of three independent experiments with 8-12 mice per group. Results are shown as the means ± s.e.m.
Mentions: Regulation of G-CSF by IL17A is a key mechanism controlling granulocytosis20 and neutrophil recruitment during bacterial infection21. Transcript levels of IL17A and were significantly lower in intestine but not lung of GF mice (Fig. 3a, S3a) and antibiotic-exposed neonatal mice (Fig. 3b) suggesting a role for IL17A in regulation of postnatal granulocytosis. Furthermore, transfer of normal microbiota restored IL17A levels in the intestine of antibiotic-exposed neonatal mice, though not to the same extent as age-matched controls (Fig. 3c). To determine the importance of IL17 signaling in postnatal granulocytosis, we utilized mice deficient in IL17A receptor (Il17ra−/−) or blocked IL17A signaling with a neutralizing antibody. The number of circulating neutrophils was significantly lower in Il17ra−/− neonatal mice (Fig. 3d). Interestingly, the number of circulating neutrophils in neonatal Il17ra−/− mice was no higher than antibiotic-exposed age- and strain-matched controls. Treatment of neonatal mice with anti-IL17A blocking antibody resulted in lower neutrophil numbers than neonatal mice treated with isotype-control antibody (Fig. S3b), and in addition, blocked the increase in circulating and bone marrow neutrophils (Fig. 3e-f) and plasma G-CSF (Fig. S3c) in antibiotic-exposed neonatal mice after transfer of normal intestinal microbiota. These data confirm that intestinal microbiota regulates postnatal granulocytosis via IL17A.

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