Milk Exosomes Protect Human Microbiota Associated-Mice Against Clostridioides difficile Infection

Abstract

ObjectivesExosomes are natural nanoparticles that facilitate cell-to-cell communication in animals and bacteria. Milk contains ∼1012 exosomes/mL. Evidence suggests that milk exosomes (MEs) contribute to maintaining a tight gut barrier function, bind toxins secreted by gut pathogens, and alter the growth of gut bacteria. Clostridioides difficile infection is a leading cause of health care-associated diarrhea in North America. Our objective of this study is to test if milk exosomes alter the severity of C. difficile infection. MethodsC57BL/6 mice stably colonized with a gut microbiome from a healthy elderly human (HMAmice) were fed ME- and RNA-sufficient (ERS) diet or ME- and RNA-depleted (ERD) diet for four weeks starting at age of three weeks (N = 20 in ERS, 17 in ERD). The content of ME and miRNA cargos in ERD is 15% and 1% of that in ERS. After four weeks, mice were treated with a combination of six antibiotics and challenged with 105C. difficile spores. Disease progression was monitored over time through measurement of weight loss, changes in behavior and stool consistency, and mortality. C. difficile colonization and toxin activity over time were assessed by colony forming units (CFU) and relative toxin units (RTU) in feces. A subset of mice was euthanized on day 3 to collect tissue samples for measurement of tissue damage and inflammation. Mann-Whitney test was used for statistical analysis and P < 0.05 was considered statistically significant. ResultsMice fed ERD diet exhibited more severe disease than mice fed ERS diet. Mice fed ERD diet had an increased mortality rate (58%) compared to mice fed ERS diet (25%, P = 0.0481) as well as 3-fold increase in weight loss two days following C. difficile infection compared to mice on an ERS diet (3.07 ± 3.77% (ERD) vs 1.08 ± 5.92%, P = 0.0488), The level of CFU and toxin activity was also higher in ERD-fed mice than that in ERS-fed mice. Finally, myeloperoxidase (MPO) activity assays (marker of neutrophil infiltration) and IL17a qRT-PCR provided evidence of higher immune activation in ERD-fed mice compared to ERS-fed mice. ConclusionsMEs decreased the severity of C. difficile infection in juvenile HMAmice. Funding SourcesNIH P20GM104320, NIFA 2016-67001-25301 and 2020-67017-30834, USDA W-4002.