DOI: 10.62425/jlasp.1978421 ISSN: 2791-8645

Fecal Extracellular Vesicles from Laboratory Mice Reprogram Neuroinflammatory Responses in Microglia

Sinan Gönüllü, Hande Pelin Özdemir, Sefa Küçükler, Selçuk Özdemir
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This study aims to examine the impact of fecal extracellular vesicles (EVs) derived from laboratory mice on lipopolysaccharide (LPS)-induced neuroinflammatory responses in BV2 microglia cells. EVs were isolated using size exclusion chromatography (SEC) and characterized through transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot techniques. TEM analysis demonstrated characteristic vesicular morphology, NTA confirmed particle distribution within the nanometer size range, and Western blot analysis indicated flotillin positivity while calnexin was absent as a negative control, thereby validating EV purity. 3-(4,5-dimetiltiyazol-2-il)-2,5-difeniltetrazolyum bromür (MTT) assay for dose optimization in BV2 microglia cells revealed that a concentration of 10 µg/mL was cytotoxic; therefore, 2.5 µg/mL and 5 µg/mL were selected as safe doses for further investigations. The effects of EV administration on LPS-induced inflammatory responses were assessed using RT-qPCR and ELISA. In addition, the expression levels of neuroinflammation- and microglial activation-related markers, including neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba1), and triggering receptor expressed on myeloid cells 2 (TREM2), were also analyzed. The findings indicated that EV treatment markedly inhibited the production of pro-inflammatory mediators, including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). In addition, fecal EV treatment attenuated the LPS-induced elevation of the anti-inflammatory mediators interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1). Furthermore, changes in the expression of neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba1), and triggering receptor expressed on myeloid cells 2 (TREM2) supported the modulation of microglial activation and neuroinflammatory processes by EVs. In conclusion, fecal EVs may modulate neuroinflammatory responses by influencing microglial inflammation and could serve as functional communication mediators within the microbiota-gut-brain axis.

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