Effects of a Mycobacterium smegmatis-related Strain on the Inflammatory Signaling of BV-2 Murine Microglia Assessed via the NanoString Platform

Sanders, William Jacob

Undergraduate Honors Thesis

Effects of a Mycobacterium smegmatis-related Strain on the Inflammatory Signaling of BV-2 Murine Microglia Assessed via the NanoString Platform Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/6682x526j

Abstract

Individuals residing in high-income, industrialized countries are facing higher rates of inflammatory disease and stress-linked psychiatric disorders for which inflammation is a risk factor. The “Old Friends” Hypothesis asserts that this trend exists in part due to reduced exposure to commensal microorganisms (Rook et al., 2013). Mycobacterium vaccae NCTC 11659, a strain of soil-derived and non-pathogenic bacterium, has been shown to attenuate inflammation and stress-linked behavior in murine studies (Reber et al., 2016). Analyzing the effects of related bacterial strains on immune tissue provides information on the yet undiscovered molecular mechanisms at play and can help determine which species have the strongest immunoregulatory effects.

This experiment investigates the effects of a yet unnamed species of Mycobacterium, closely related to M. vaccae and M. smegmatis, on BV-2 murine microglia. Microglia, which reside in the central nervous system, are not typically exposed to bacteria and must be able to defend neural tissue when necessary. Due to their functional niche, the BV-2 cells are expected to exhibit a pro-inflammatory response after bacterial contact, along with possible strain-specific responses (Loane & Byrnes, 2010). Cultured BV-2 cells were divided into 12 samples (three replicates of four conditions) and each condition received doses of an inflammatory agent (lipopolysaccharide, LPS), the novel mycobacterial strain, and/or growth media. Extraction of mRNA using Qiagen RNeasy kits permitted NanoString transcript sequencing, where relative levels of mRNA transcripts are measured and compared. Although one replicate in the LPS + M. smegmatis-related strain group could not be included in statistical analysis, the data do not indicate any strong immunomodulatory effects from treatment with the novel strain, and instead suggest that it generates a mild inflammatory phenotype in microglia.

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