DYSFUNCTION OF BONE-MARROW-DERIVED MACROPHAGES IN GDX KNOCKOUT MICE

Macrophages are cells of the immune system responsible for clearing up the organism, removing damaged and dead cells, pathogens and other foreign substances. They remove and destroy these compounds by a process called phagocytosis, in which the macrophages recognize what is to be destroyed, engulf it and digest it in the lysosome. The phagocytosis mechanism requires special cytoskeleton configurations formed by actin branches that allow the engulfment of substances into the cells. The cytoskeleton structures formed by actin branching are also known to be necessary during the macrophage migration towards its target. Our group has shown that the small cytosolic GdX protein has an important role in the actin filament branching. Therefore, the aim of this study is to analyze the migration ability and the phagocytosis potential of wild-type (WT) and GdX knockout macrophages (KO). Bone marrow cells from mice were isolated and cultured in presence of Macrophage Colony-Stimulating Factor (MCSF) to induce cell differentiation into macrophages. The bone marrow derived macrophages were then subjected to phagocytosis and migration assays. The results from the phagocytosis assay indicated no significant difference in the percentage of cells capable of internalizing the beads or in the amount of engulfed beads between WT and KO macrophages. However, the results from the migration assay suggested a decrease in the migration ability in GdX KO cells when compared to WT macrophages. Therefore,our results imply that the lack of GdX may cause macrophage dysfunction.M.S. in Cellular and Molecular Biology, May 201

Understanding the Role of Lipids Derived from the Gut Microbes in a Mouse Model of Obesity-Induced Peripheral Pain

The increase in obesity has been accompanied by a rise in the prevalence of painful peripheral neuropathy. Recently, studies have suggested a role for gut microbiome in the development of some peripheral pain, including chemotherapy- induced pain and fibromyalgia. In the present dissertation, we showed that modulation of gut microbiome in obese mice alleviated neuropathic indices, concurrent with changes in immune cell profile within the peripheral nerve system. We demonstrated that fecal transplantation from lean to obese mice decreased obesity-induced pain and restored nerve density in the skin. These improvements were accompanied by changes in peripheral nerve system gene expression, calcium signaling and inflammatory cells. Our results suggested that circulating butyrate, a metabolite secreted by gut microbiome upon fiber digestion, may be involved in pain improvement by acting directly on peripheral nerve system cells. We also observed that increasing circulating butyrate correlated with mechanical pain improvement and decreased inflammatory markers in the PNS of obese mice.Because obesity is a state of chronic low-grade inflammation, we hypothesized that butyrate could modulate inflammation levels to improve neuropathy in obese mice. Using a caspase-1 biosensor murine model to monitor inflammation, we observed caspase-1 activation in metabolic and neural tissues, including heart, brown adipose tissue and brain of lean, obese controls and obese mice subjected to butyrate treatment. Our data highlighted that butyrate acts on a tissue-specific manner and rescue some caspase 1-dependent inflammatory responses dysregulated in obesity settings

Fecal transplantation and butyrate improve neuropathic pain, modify immune cell profile, and gene expression in the PNS of obese mice

Obesity affects over 2 billion people worldwide and is accompanied by peripheral neuropathy (PN) and an associated poorer quality of life. Despite high prevalence, the molecular mechanisms underlying the painful manifestations of PN are poorly understood, and therapies are restricted to use of painkillers or other drugs that do not address the underlying disease. Studies have demonstrated that the gut microbiome is linked to metabolic health and its alteration is associated with many diseases, including obesity. Pathologic changes to the gut microbiome have recently been linked to somatosensory pain, but any relationships between gut microbiome and PN in obesity have yet to be explored. Our data show that mice fed a Western diet developed indices of PN that were attenuated by concurrent fecal microbiome transplantation (FMT). In addition, we observed changes in expression of genes involved in lipid metabolism and calcium handling in cells of the peripheral nerve system (PNS). FMT also induced changes in the immune cell populations of the PNS. There was a correlation between an increase in the circulating short-chain fatty acid butyrate and pain improvement following FMT. Additionally, butyrate modulated gene expression and immune cells in the PNS. Circulating butyrate was also negatively correlated with distal pain in 29 participants with varied body mass index. Our data suggest that the metabolite butyrate, secreted by the gut microbiome, underlies some of the effects of FMT. Targeting the gut microbiome, butyrate, and its consequences may represent novel viable approaches to prevent or relieve obesity-associated neuropathies

CEBPβ regulation of endogenous IGF-1 in adult sensory neurons can be mobilized to overcome diabetes-induced deficits in bioenergetics and axonal outgrowth

Aberrant insulin-like growth factor 1 (IGF-1) signaling has been proposed as a contributing factor to the development of neurodegenerative disorders including diabetic neuropathy, and delivery of exogenous IGF-1 has been explored as a treatment for Alzheimer's disease and amyotrophic lateral sclerosis. However, the role of autocrine/paracrine IGF-1 in neuroprotection has not been well established. We therefore used in vitro cell culture systems and animal models of diabetic neuropathy to characterize endogenous IGF-1 in sensory neurons and determine the factors regulating IGF-1 expression and/or affecting neuronal health. Single-cell RNA sequencing (scRNA-Seq) and in situ hybridization analyses revealed high expression of endogenous IGF-1 in non-peptidergic neurons and satellite glial cells (SGCs) of dorsal root ganglia (DRG). Brain cortex and DRG had higher IGF-1 gene expression than sciatic nerve. Bidirectional transport of IGF-1 along sensory nerves was observed. Despite no difference in IGF-1 receptor levels, IGF-1 gene expression was significantly (P < 0.05) reduced in liver and DRG from streptozotocin (STZ)-induced type 1 diabetic rats, Zucker diabetic fatty (ZDF) rats, mice on a high-fat/ high-sugar diet and db/db type 2 diabetic mice. Hyperglycemia suppressed IGF-1 gene expression in cultured DRG neurons and this was reversed by exogenous IGF-1 or the aldose reductase inhibitor sorbinil. Transcription factors, such as NFAT1 and CEBPβ, were also less enriched at the IGF-1 promoter in DRG from diabetic rats vs control rats. CEBPβ overexpression promoted neurite outgrowth and mitochondrial respiration, both of which were blunted by knocking down or blocking IGF-1. Suppression of endogenous IGF-1 in diabetes may contribute to neuropathy and its upregulation at the transcriptional level by CEBPβ can be a promising therapeutic approach