Effect of antibiotic gut microbiota disruption on LPS-induced acute lung inflammation

Abstract

Background: An increasing body of evidence is indicating that the gut microbiota modulates pulmonary inflammatory responses. This so-called gut-lung axis might be of importance in a whole spectrum of inflammatory pulmonary diseases such as acute respiratory distress syndrome, chronic obstructive pulmonary disease and pneumonia. Here, we investigate the effect of antibiotic disruption of gut microbiota on immune responses in the lung after a intranasal challenge with lipopolysaccharide (LPS).

Methods/results: C57Bl/6 mice were treated for two weeks with broad-spectrum antibiotics supplemented to their drinking water. Afterwards, mice and untreated control mice were inoculated intranasally with LPS. Mice were sacrificed 2 and 6 hours post-challenge, after which bronchoalveolar lavage fluid (BALF) and lung tissues were taken. Gut microbiota analysis showed that antibiotic-treated mice had a pronounced reduction in numbers and diversity of bacteria. A modest, but time consistent, significant increase of interleukin (IL)-6 release was seen in BALF of antibiotic treated mice. Release of tumor necrosis factor alpha (TNFα), however, was not statistically different between groups.

Conclusion: Antibiotic induced microbiota disruption is associated with alterations in host responses during LPS-induced lung inflammation. Further studies are required to determine the clinical relevance of the gut-lung axis in pulmonary infection and inflammation.

Fig 1. Experimental design, microbiota disruption and pulmonary cytokine production during LPS-induced inflammation in control and antibiotic pre-treated mice.

A) Schematic overview of performed experiments. Wildtype C57BL/6 mice were treated with two weeks of antibiotics (ABX: ampicillin, neomycin, metronidazole and vancomycin) in their drinking water after which 1 or 10 μg lipopolysaccharide (LPS) from either K. pneumoniae or E. coli, or saline was administered intranasally. Mice were sacrificed 2 or 6 hours after LPS challenge. B) Gut microbiome diversity as measured by Shannon α-diversity of antibiotic (ABX) treated (yellow) and control (grey) mice (left panel). Group averaged relative abundance profiles on Family level between antibiotic treated and control mice (right panel). Both analysis were executed using a rarefaction depth of 20000 reads (n = 12 No ABX, n = 4 Yes ABX mice. Note: The rest of ABX treated mice (n = 12 total) tested did not reach 20000 reads and were therefore excluded from Fig 1B). C) Interleukin (IL)-6 and tumor necrosis factor (TNF)-α were measured in broncho-alveolar lavage fluid of antibiotic pre-treated mice (yellow) or control (grey) sacrificed 2 or 6 hours after intranasal inoculation with 10 μg LPS from K. pneumoniae. Additional control groups are saline inoculated mice with or without antibiotic pre-treatment (white and black bars, respectively). Dashed line represents detection limit. Data are presented as bar plots showing the mean and standard deviation of the mean (n = 6–8 mice/group). One way ANOVA with post-hoc Tukey’s test. na: not applicable, ns: non-significant; *p<0.05, **p<0.01, ***p<0.001,****P<0.0001.