502 - Neonatal Vancomycin Treatment Expands Yolk-sac-Derived Colonic Macrophage Population

Poster Number: 502
Publication Number: 502.425

Ellen M. Schill, Washington University in St. Louis, Saint Louis, MO, United States; Vini John, Washington University in St. Louis School of Medicine, Saint Louis, MO, United States; Shreya Gaddipati, Washington University in St. Louis, St.Louis, MO, United States; Alexandria N. Floyd, Washington University in St. Louis School of Medicine, Saint Charles, MO, United States; Keely G. McDonald, Washington University in St. Louis School of Medicine, St. Louis, MO, United States; Rodney D. Newberry, Washington University School of Medicine, Saint Louis, MO, United States

Background: Antibiotics are some of the most prescribed medications in neonatal intensive care units due to the risk of serious bacterial infections in premature infants. Early antibiotic treatment increases an infant’s risk of late-onset sepsis and necrotizing enterocolitis.  Early life antibiotics may alter intestinal homeostasis by disrupting development of neuroimmune interactions. Vancomycin treatment decreases enteric neuron density and slows intestinal transit time in neonatal mice. In adult mice, broad spectrum antibiotics cause slowed intestinal transit time and neuronal disruption by decreasing the number of intestinal macrophages (IMφ).  IMφ are tissue resident macrophages that are initially yolk-sac-derived but are replaced by bone marrow-derived cells in the neonatal period. However, the subset of macrophages that interact with the enteric nervous system (ENS) are long-lived yolk-sac-derived cells. 

Objective: To determine how vancomycin treatment alters IMφ number and phenotype in neonatal mice

Design/Methods: Wildtype mice were fed vancomycin or water daily from postnatal day (P) 1 through P10. Mice were treated per litter.  Mice were sacrificed on P11.  Small intestine and colon were separately washed with EDTA to remove the epithelial layer and then digested into single cell suspensions.  Cells were analyzed via flow cytometry. 

Results: Vancomycin-exposed mice had fewer total and hematopoietic (CD45+) cells in the small intestine (Figure 1A-B).  In the colon, there was a significant increase in total and hematopoietic cells in the vancomycin-exposed mice (Figure 1C-D). There was no difference in the number of IMφ (CD45+ CX3CR1+ CD11b+ F4/80+, Figure 2A) in the small intestine of control versus vancomycin-exposed mice. There was an increase in the number of IMφ in the colon of vancomycin-exposed mice (Figure 2B-C). Colonic macrophages were further characterized as yolk-sac-derived (identified as F4/80HI CD11bLO) or bone marrow-derived (F4/80LO CD11bHI, Figure 2D-E).  There was an increase in both yolk-sac and bone marrow-derived macrophages in the vancomycin-exposed mice compared to control (Figure 2F-G). The magnitude of increase was different in yolk-sac (26x higher in vancomycin-exposed) compared to bone marrow-derived (10x higher in vancomycin-exposed).Conclusion(s): Vancomycin treatment increases the number of yolk-sac-derived, and to a lesser extent, bone marrow-derived macrophages in neonatal mice. As a subset of yolk-sac-derived macrophages preferentially associate with the ENS, these changes could disrupt development of neuron-macrophage interactions. 
Ellen Merrick Schill CVEMS CV 01062022.pdf
Figure 2: Vancomycin exposure expands colonic macrophage populations(A) Gating strategy to identify intestinal macrophages. Cells were gated on FSC-A/SSC-A, FSC-A/FSC-H (not shown), CD45+, CX3CR1+, CD11b+, F480+. Number of intestinal macrophages in the small intestine (B) was unchanged between vancomycin-exposed and control animals. Vancomycin-exposed mice had significantly more macrophages in the colon compared to controls (C). (D) and (E) show identification of yolk-sac-derived and bone marrow-derived macrophages by F480, CD11b staining. Representative plots shown from control (D) and vancomycin-exposed (E) colons. Number of yolk-sac-derived macrophages was significantly increased in vancomycin-exposed colons (F). Number of bone marrow-derived macrophages was also increased (G) in colons of vancomycin-exposed animals but did not reach statistical significance).