Frequent and excessive use of
antibiotics primes patients to
Clostridioides difficile infection (CDI), which leads to fatal
pseudomembranous colitis, with limited treatment options. In earlier reports, we used a
drug repurposing strategy and identified
amoxapine (an
antidepressant),
doxapram (a breathing stimulant), and
trifluoperazine (an
antipsychotic), which provided significant protection to mice against lethal
infections with several pathogens, including C. difficile However, the mechanisms of action of these drugs were not known. Here, we provide evidence that all three drugs offered protection against experimental CDI by reducing bacterial burden and toxin levels, although the drugs were neither bacteriostatic nor bactericidal in nature and had minimal impact on the composition of the microbiota.
Drug-mediated protection was dependent on the presence of the microbiota, implicating its role in evoking host defenses that promoted protective immunity. By utilizing transcriptome sequencing (
RNA-seq), we identified that each
drug increased expression of several innate immune response-related genes, including those involved in the recruitment of neutrophils, the production of
interleukin 33 (IL-33), and the
IL-22 signaling pathway. The
RNA-seq data on selected genes were confirmed by quantitative real-time PCR (qRT-PCR) and
protein assays. Focusing on
amoxapine, which had the best anti-CDI outcome, we demonstrated that neutralization of
IL-33 or depletion of neutrophils resulted in loss of
drug efficacy. Overall, our lead drugs promote disease alleviation and survival in the murine model through activation of
IL-33 and by clearing the pathogen through host defense mechanisms that critically include an early influx of neutrophils.IMPORTANCEClostridioides difficile is a spore-forming anaerobic bacterium and the leading cause of
antibiotic-associated colitis. With few therapeutic options and high rates of disease recurrence, the need to develop new treatment options is urgent. Prior studies utilizing a repurposing approach identified three nonantibiotic Food and Drug Administration-approved drugs,
amoxapine,
doxapram, and
trifluoperazine, with efficacy against a broad range of human pathogens; however, the protective mechanisms remained unknown. Here, we identified mechanisms leading to
drug efficacy in a murine model of lethal C. difficile
infection (CDI), advancing our understanding of the role of these drugs in
infectious disease pathogenesis that center on host immune responses to C. difficile Overall, these studies highlight the crucial involvement of innate immune responses, as well as the importance of
immunomodulation as a potential therapeutic option to combat CDI.