Treatment with a neutrophil elastase inhibitor and ofloxacin reduces P. aeruginosa burden in a mouse model of chronic suppurative otitis media

K M Khomtchouk,P L Bollyky,I Koliesnik,A Xia,P L Santa Maria,S Massa,L I Joseph,B B Khomtchouk,D Pletzer,A Kouhi

doi:10.1038/s41522-021-00200-z

Abstract

Chronic suppurative otitis media (CSOM) is a widespread, debilitating problem with poorly understood immunology. Here, we assess the host response to middle ear infection over the course of a month post-infection in a mouse model of CSOM and in human subjects with the disease. Using multiparameter flow cytometry and a binomial generalized linear machine learning model, we identified Ly6G, a surface marker of mature neutrophils, as the most informative factor of host response driving disease in the CSOM mouse model. Consistent with this, neutrophils were the most abundant cell type in infected mice and Ly6G expression tracked with the course of infection. Moreover, neutrophil-specific immunomodulatory treatment using the neutrophil elastase inhibitor GW 311616A significantly reduces bacterial burden relative to ofloxacin-only treated animals in this model. The levels of dsDNA in middle ear effusion samples are elevated in both humans and mice with CSOM and decreased during treatment, suggesting that dsDNA may serve as a molecular biomarker of treatment response. Together these data strongly implicate neutrophils in the ineffective immune response to P. aeruginosa infection in CSOM and suggest that immunomodulatory strategies may benefit drug-tolerant infections for chronic biofilm-mediated disease.

Highlights

Pseudomonas aeruginosa is a World Health Organization priority pathogen due to its ability to evolve resistance to all available antibiotics[1]
Using our chronic suppurative otitis media (CSOM) mouse model, we examined neutrophil numbers and maturity in more detail over time in samples collected on days 1, 14, and 28 d.p.i. (Fig. 3a)
Patients with conditions other than CSOM had dsDNA levels below the limit of detection. These data indicate that human CSOM is associated with elevated dsDNA levels in the middle ear and support the relevance of our animal model to human disease. These results strongly implicate neutrophils in the ineffective immune response to P. aeruginosa infection observed in CSOM

Summary

INTRODUCTION

Pseudomonas aeruginosa is a World Health Organization priority pathogen due to its ability to evolve resistance to all available antibiotics[1]. Host biofilms are stronger and more developed in the host versus in a petri dish, and NETs can add to the integrity of the aggregate It has previously been demonstrated in CSOM that neutrophil recruitment is increased to the infection site, and neutrophils in the middle ear promote bacterial biofilm stability through their contribution of dsDNA to the biofilm matrix[19]. Neutrophil immunomodulation has been reported for other inflammation-mediated pathology such as atherosclerosis[27] These lines of evidence suggest that adjunctive inhibition of elastase and other hostdirected therapeutics might help counter the growing problem of ineffective antibiotics and promote clearance of P. aeruginosa biofilm infection. We observed that neutrophils became phenotypically altered in Ly6G expression, experiencing a significant reduction in the LyGhi subset by 14 d.p.i. (Fig. 3b)

RESULTS

DISCUSSION

METHODS