340 - Enteral Probiotics Alter Inflammatory Signaling and Lung Remodeling in a Neonatal Sepsis–Associated Lung Injury Model

Publication Number: 1324.340

John J. Morelli, Children's Mercy Hospitals and Clinics, Kansas City, MO, United States; Heather Menden, Children's Mercy Hospitals and Clinics, Kansas City, MO, United States; Sherry M. Mabry, Children's Mercy, Kansas City, MO, United States; Venkatesh Sampath, Children's Mercy Kansas City, kansas city, KS, United States

Background: In premature infants, sepsis-induced acute lung injury (ALI) is a known risk factor for the development of bronchopulmonary dysplasia (BPD), a chronic lung disease marked by impaired alveolar and vascular growth. The gut-lung axis, linking intestinal microbiota to pulmonary immune homeostasis, has emerged as a potential target to mitigate systemic inflammation. Data suggest probiotics reduce late-onset sepsis and necrotizing enterocolitis in premature neonates; however, their impact on pulmonary inflammation modulation remains uncertain. We hypothesized that enteral probiotics would attenuate Toll-like receptor (TLR)-induced inflammation and tissue remodeling in a murine model of neonatal sepsis-associated ALI and BPD.

Objective: To determine whether early, multi-strain probiotic exposure attenuates systemic endotoxin lipopolysaccharide (LPS)-induced a) pro-inflammatory TLR signaling and ALI, and b) subsequent chronic alveolar remodeling in a murine model of neonatal sepsis-associated lung injury.

Design/Methods: C57BL6/J pups received enteral UltraFlora® Baby Probiotic (0.05 mL of 10⁸ CFU/mL) or vehicle. For the acute group, mice received probiotic or vehicle on postnatal days (P) 4-6, followed by intraperitoneal LPS (2 mg/kg) or saline at P6, then euthanized on P7. Lung samples underwent RT-PCR for inflammatory (Kc, Il-6, Il-1β) cytokine expression, as well as western blot analysis to evaluate lung p-p65 (NF-κB component), p-p38 (MAPK), ICAM1, iNOS, and cleaved caspase-3 (apoptosis) protein expression. In a separate BPD-model group, probiotic or vehicle was administered to mice on P4-7, and P10, followed by LPS or saline on P5 and P7, then euthanized on P14. Inflation-fixed P14 lungs were analyzed for alveolar remodeling using radial alveolar counts (RAC). ANOVA with Bonferroni corrections were performed for statistical analysis.

Results: With acute LPS exposure, probiotics appeared to exaggerate LPS-induced lung Il-1β and Kc expression, with Il-6 significantly increased. Despite elevated transcript levels, western blot revealed reduced phosphorylation of NF-κB (p65) and p38 MAPK with probiotic exposure as well as reduced iNOS and ICAM-1, suggesting suppression of pro-inflammatory TLR4 signaling. In the chronic model, probiotic treatment rescued LPS induced chronic alveolar remodeling as measured by higher RAC values.

Conclusion(s): Acute findings showed divergent transcriptional and post-translational responses to probiotic exposure, whereas the chronic model demonstrated partial recovery. Further investigation into the complex relationships between probiotics and sepsis-induced ALI is warranted.

Enteral probiotic administration effects on RNA and protein expression in an acute lung injury model in neonatal mice
C57BL6/J pups received enteral UltraFlora® Baby Probiotic (Metagenics, Aliso Viejo, CA; 0.05 mL of 10⁸ CFU/mL; Bifidobacterium animalis ssp. lactis and Lactobacillus rhamnosus GG) or vehicle once daily on postnatal days (P) 4-6, followed by intraperitoneal lipopolysaccharide (LPS; 2 mg/kg) or saline at P6; pups were euthanized at P7. (A) Graphical timeline of the acute lung probiotic experiments. (B) Experimental group allocation for the acute lung experiments. Group sizes were n = 3 for control + vehicle (Ctrl), n = 5 for LPS + vehicle (LPS Veh), and n = 5 for LPS + probiotics (LPS Pro). No mouse deaths occurred. (C) Mouse lungs were harvested after the acute lung experiments, and qRT-PCR was performed for inflammatory markers Kc, Il-6, and Il-1β. *p < 0.05. (D) Whole-lung homogenates were immunoblotted for phospho-p65 ((p)P65), phospho-p38 ((p)P38), ICAM1, iNOS, cleaved caspase-3 (CC3), and actin.

Probiotics improved radial alveolar counts and reduced chronic alveolar remodeling in a murine model of sepsis-associated bronchopulmonary dysplasia
C57BL6/J pups were administered UltraFlora® Baby Probiotic (Metagenics, Aliso Viejo, CA; 0.05 mL of 10⁸ CFU/mL; Bifidobacterium animalis ssp. lactis and Lactobacillus rhamnosus GG) or vehicle on postnatal days (P) 4-7 and P10. Lipopolysaccharide (LPS; 2 mg/kg) or saline was given intraperitoneally on P5 and P7, and pups were euthanized at P14. Inflation-fixed lungs collected at P14 were analyzed for alveolar remodeling by radial alveolar counts (RAC). (A) Graphical timeline of the chronic lung probiotic experiments. (B) Experimental group allocation for the chronic lung experiments. Group sizes were n = 5 for control + vehicle, n = 8 for LPS + vehicle, and n = 9 for LPS + probiotics. No mouse deaths occurred. (C) H&E-stained inflated lungs were used to quantify RACs from mice from histologic images with (D) corresponding graphical RAC quantifications. *p < 0.05.