The microbiota regulates neutrophil homeostasis and host resistance to Escherichia coli sepsis in neonates
Methods: We exposed pregnant dams to antibiotics in drinking water beginning 5 days before delivery and measured bone marrow (BM) and peripheral neutrophils and IL17 producing cells in the gut and determined composition of gut microbiota in their neonates at PN 3-14d by 16S rDNA sequencing. We inoculated pups (PN 3d) with Escherichia coli K1 (104 CFU/g) via i.p. route. We transferred the intestinal contents from no antibiotic-exposed (control) mice (PN 3d) to age-matched antibiotic-exposed mice by gavage, to reconstitute the microbiota in antibiotic-exposed mice. We interrogated the role of IL17 or toll like receptor (TLR) 4 by using gene targeted neonatal mice (PN 3d) (Il17ra-/- or Tlr4-/-) or by using anti-IL17A or anti-TLR4 neutralizing antibody.
Results: Perinatal antibiotic exposure altered the pattern of microbial colonization, abolished the early appearance of Gammaproteobacteria in the gut and increased the susceptibility of neonatal mice to E. coli as compared to controls (median survival 8h vs. >72h). These changes were associated with decreased number of circulating and BM neutrophils, reduced number of IL17 producing cells in the gut. Transfer of normal intestinal microbiota restored the circulating and BM neutrophil counts, IL17 producing cells and restored the host resistance to E. coli (median survival 36h). The numbers of circulating and BM neutrophils were significantly lower in neonatal Il17ra-/- or Tlr4-/-mice. Pretreatment of antibiotic-exposed mice (PN 3d) with anti-IL17A or anti-TLR4 neutralizing antibody blocked the increase in circulating and BM neutrophil counts in antibiotic-exposed neonatal mice after transfer of normal microbiota.
Conclusion: Microbiota are critical in regulating postnatal granulocytosis and host resistance to E. coli via TLR4 and IL17A-dependent mechanisms raising the possibility that therapies to ameliorate antibiotic-induced microbiota dysbiosis might improve neonatal mortality.