One cell, two cell, female cell, male cell: Sex Differences in Immune Cell Activation
Creators: Friedman, Timothy N.
Publisher: name: University of Alberta Library
Year: 2023

<p>The overarching idea of this thesis stemmed from previous work in our lab, where it was noticed that females and males exhibited different disease trajectories in a model of Multiple Sclerosis. Combined with our observation of different outcome metrics for peripheral nociception suggested that something interesting was happening in the female peripheral nervous that may not be happening in males, or to the same extent. This idea is also relevant to clinical MS and other autoimmune diseases, where women not only have different incidence rates than men but also exhibit subtleties in disability and chronic pain. It is well established that there are sex differences in immune cell activation or pathways in immune activation, so we aimed to investigate if these differences were present specifically in the peripheral nervous system structures responsible for pain signal generation.<br>
To begin my investigation, I took a general approach and aimed to look from an overhead view by RNA sequencing male and female DRGs to identify major differences. This encompassed most of Chapter 2, where I analyzed bulk RNA and miR sequencing data from male and female DRGs. I discovered that the female DRGs had an incredible amount of both gene and miR dysregulation, focusing on genes related to the innate immune system and phagocytic roles. I discovered that miR-21a-5p, a microRNA previously known to be upregulated in CNS tissues in MS and EAE, was also upregulated in the DRG of both sexes. I identified a candidate gene for follow-up investigation (TLR7) as it was highly upregulated in the female DRGs and is well described in phagocytic innate immune cells.<br>
Chapter 3 took a reductionist approach by focusing on the innate immune cells indicated in the first chapter. I stimulated male and female cells with TNF-alpha, one of the cytokines responsible for activating immune cells, as it was more prevalent in the female disease course in EAE and interesting in this system. I did not find anything that contradicted the previously described M1-like phenotype, but there were some interesting subtleties. Females had a much higher mitotic capability, as well as higher motility and sensitivity for activation. I found some differences in cytokine profiles, particularly in the levels of CCL12, CCL22, and IL-16 as standout candidates for sex differential effects, which may have knock-on effects on inflammatory pain. This suggests that this cytokine profile could potentially help resolve inflammatory pain.<br>
In Chapter 4, I continued the reductionist approach and shifted the in vitro system from bone marrow-derived macrophages to dorsal ganglion neurons. By culturing neurons in different types of inflammatory conditioned media, we investigated the plastic properties that these medias could impart on the neurons. Interestingly, I found that the conditions exhibiting high amounts of growth also contained neurons with highly spontaneous electrical activity immediately after plating, which then became quiescent over time. I further explored the link between excitability and plasticity by modulating excitability using pharmacokinetic tools and discovered that exogenous silencing of neurons prevented later outgrowth.<br>
This thesis aimed to investigate sex differences in the innate immune system and their potential implications in peripheral pain signal generation in Multiple Sclerosis. Through a combination of bioinformatics, in vitro cell culture, and pharmacological approaches, I discovered unique effects of the innate immune system on each sex of neurons. My findings suggest that understanding the molecular mechanisms of these pathways may provide valuable information to guide pharmacological therapeutics that could benefit one or both sexes. Furthermore, my work highlights the importance of considering sex differences in autoimmune diseases such as Multiple Sclerosis, as they not only have different incidence rates between the sexes but also exhibit subtleties in disability and chronic pain. Ultimately, my research provides insight into the role of the innate immune system in sex-specific pain signaling and identifies potential targets for future therapeutic interventions.</p>