The role of neurons and glia in ethanol-induced innate immune signaling

The innate immune system is an unexpected and unique addition to signaling pathways in the brain. The brain functions as a largely sterile environment, void of infiltrating infectious agents due to the blood brain barrier. However, in recent years it has been discovered that multiple components of the innate immune system, including Toll-like receptors (TLRs), pro-inflammatory cytokines, and transcription factors such as NFκB that regulate innate immune genes, are upregulated in post-mortem human alcoholic brain. This has further been replicated both in rodent models of alcohol consumption as well as in brain slice cultures. However, it is still unknown how different cell types in brain contribute to this response and how innate immune signaling molecules function as communication mediators between cells. In this dissertation, we determine the role of neurons and glia (microglia and astrocytes) in ethanol-induced innate immune system by utilizing specific cell lines: SH-SY5Y neurons, BV2 microglia, and U373 astrocytes. In Chapter 2, we treat SH-SY5Y neurons and BV2 microglia with either ethanol, the TLR3 agonist Poly(I:C), or the TLR4-agonist LPS, and discover that ethanol induces a broad and highly sensitive response to ethanol in SH-SY5Y neurons. In Chapter 3, we use a co-culture model of BV2 microglia and SH-SY5Y to determine how co-culture impacts ethanol-induced innate immune signaling between these two cell types. We discover that co-culture modifies multiple innate immune genes in both cell types, as well as increasing ethanol-induced IL-4/IL-13 signaling, suggesting a novel microglial-neuronal signaling pathway. In Chapter 4, we discover that ethanol induces interferons in SH-SY5Y neurons and U373 astrocytes, but not BV2 microglia, indicating interferons as a neuronal and astrocytic-specific response to ethanol. We further determined using conditioned media experiments that astrocyte-induced TRAIL, an interferon response gene, induces interferons in SH-SY5Y neurons. This suggests novel TRAIL-IFN signaling pathways between astrocytes and neurons. Overall, these results suggest that neurons have a unique involvement in ethanol-induced innate immune signaling, and that innate immune signaling molecules function as cell-to-cell signaling mediators in brain. In addition, these results indicate that future therapeutic strategies may be utilized to target both specific cell type and cell-to-cell signaling responses.Doctor of Philosoph

CD4+ Regulatory and Effector/Memory T Cell Subsets Profile Motor Dysfunction in Parkinson’s Disease

Animal models and clinical studies have linked the innate and adaptive immune system to the pathology of Parkinson’s disease (PD). Despite such progress, the specific immune responses that influence disease progression have eluded investigators. Herein, we assessed relationships between T cell phenotype and function with PD progression. Peripheral blood lymphocytes from two separate cohorts, a discovery cohort and a validation cohort, totaling 113 PD patients and 96 age- and environment-matched caregivers were examined by flow cytometric analysis and T cell proliferation assays. Increased effector/memory T cells (Tem), defined as CD45RO+ and FAS+ CD4+ T cells and decreased CD31+ and α4β7+ CD4+ T cells were associated with progressive Unified Parkinson’s Disease Rating Scale III scores. However, no associations were seen between immune biomarkers and increased age or disease duration. Impaired abilities of regulatory T cells (Treg) from PD patients to suppress effector T cell function was observed. These data support the concept that chronic immune stimulation, notably Tem activation and Treg dysfunction is linked to PD pathobiology and disease severity, but not disease duration. The association of T cell phenotypes with motor symptoms provides fresh avenues for novel biomarkers and therapeutic designs

Tumor necrosis factor-alpha levels in HIV-1 seropositive injecting drug users.

TNF-alpha is a highly pleiotropic cytokine and plays an important role in regulating HIV-1 replication. It may compromise the integrity of the blood-brain-barrier and, thus, may contribute to the neurotoxicity of HIV-1-infection. Both intravenous drug abuse (IDU) and HIV infection can increase TNF-alpha activity, but little information is available on the effects of a combination of these factors on TNF-alpha. We investigated plasma TNF-alpha levels and mRNA in the peripheral monocytes of 166 men and women in three groups: HIV-1-positive IDUs, HIV-1-negative IDUs, and HIV-negative non-IDU control participants. HIV-1-positive IDUs had higher TNF-alpha levels than HIV-1-negative IDUs who, in turn, had higher levels than controls. TNF-alpha mRNA expression in peripheral monocytes was significantly increased in both HIV-1-positive and negative IDUs compared to controls. These findings show that the effects of HIV infection and intravenous drug use may be additive in increasing TNF-alpha levels. Given the multiple effects of TNF-alpha in HIV infection, additional investigation of its role is needed

Role of the IKKβ/NF-κB pathway in alcoholism

Pro-inflammatory neuroimmune signaling pathways are implicated in the acute and chronic effects of alcohol exposure. Genetic association studies in humans, gene expression microarray studies in postmortem brains of alcoholics, transcriptome meta-analysis in rodents, and drinking models in mice support the role of neuroimmune signaling in alcohol abuse disorder. Nuclear factor kappa-B (NF-κB) is a ubiquitously expressed transcription factor that controls the expression of genes important for innate- and adaptive-immune responses, cell proliferation/death, and inflammation. More specifically, the NF-κB canonical pathway is responsible for the expression of pro-inflammatory genes. The inhibitory kappa-B kinase (IKK) complex, composed of IKKα, IKKβ, and IKKγ represents a point of convergence for many extracellular signals and regulates the NF-κB canonical pathway by targeting the inhibitor of NF-κB (IκB) for degradation. NF-κB is disinhibited, translocates to the nucleus, and acts as a transcription factor for numerous pro-inflammatory chemokines and cytokines. However, IKKβ is the only member of the IKK complex that specifically mediates this pathway. As such, I hypothesized that inhibiting IKKβ/NF-κB pathway would limit/decrease voluntary ethanol consumption. This was studied by determining the brain region and cell type-specificity of all the IKK isoforms. It was observed that all IKKs were primarily expressed in neurons and ubiquitously expressed throughout the brain regions studied [prefrontal cortex (PFC), nucleus accumbens (NAc), amygdala (AMY), and ventral tegmental area (VTA)]. Subsequently, the effects of inhibiting/knocking down IKKβ were investigated both systemically and centrally to determine the effects on voluntary ethanol drinking. It was observed that both antagonizing IKKβ peripherally and genetically knocking it down centrally in the NAc and central amygdala (CeA) reduced voluntary ethanol consumption and preference in two bottle choice (2BC) ethanol drinking paradigms. Lastly, ethanol-responsive microRNAs were explored in the PFC, NAc, and AMY. Several differentially expressed MicroRNAs were discovered that were either predicted/validated to target genes of the IKKβ/NF-κB pathway. One candidate, let-7g, was manipulated in vivo to determine its effect on voluntary ethanol drinking behaviors, however, no significant phenotypes were observed. These results demonstrate that blocking IKKβ decreases voluntary ethanol consumption and indicate its role as a potential therapeutic target for alcohol abuse.Cellular and Molecular Biolog

HLA-II-Dependent Neuroimmune Changes in Group A Streptococcal Necrotizing Fasciitis

Streptococcus pyogenes (Group A Streptococcus, GAS) bacteria cause a spectrum of human diseases ranging from self-limiting pharyngitis and mild, uncomplicated skin infections (impetigo, erysipelas, and cellulitis) to highly morbid and rapidly invasive, life-threatening infections such as streptococcal toxic shock syndrome and necrotizing fasciitis (NF). HLA class II allelic polymorphisms are linked with differential outcomes and severity of GAS infections. The dysregulated immune response and peripheral cytokine storm elicited due to invasive GAS infections increase the risk for toxic shock and multiple organ failure in genetically susceptible individuals. We hypothesized that, while the host immune mediators regulate the immune responses against peripheral GAS infections, these interactions may simultaneously trigger neuropathology and, in some cases, induce persistent alterations in the glial phenotypes. Here, we studied the consequences of peripheral GAS skin infection on the brain in an HLA-II transgenic mouse model of GAS NF with and without treatment with an antibiotic, clindamycin (CLN). Mice expressing the human HLA-II DR3 (DR3) or the HLA-II DR4 (DR4) allele were divided into three groups: (i) uninfected controls, (ii) subcutaneously infected with a clinical GAS strain isolated from a patient with GAS NF, and (iii) GAS-infected with CLN treatment (10 mg/kg/5 days, intraperitoneal). The groups were monitored for 15 days post-infection. Skin GAS burden and lesion area, splenic and hippocampal mRNA levels of inflammatory markers, and immunohistochemical changes in hippocampal GFAP and Iba-1 immunoreactivity were assessed. Skin GAS burden and hippocampal mRNA levels of the inflammatory markers S100A8/A9, IL-1β, IL-33, inflammasome-related caspase-1 (Casp1), and NLRP6 were elevated in infected DR3 but not DR4 mice. The levels of these markers were significantly reduced following CLN treatment in DR3 mice. Although GAS was not detectable in the brain, astrocyte (GFAP) and microglia (Iba-1) activation were evident from increased GFAP and Iba-1 mRNA levels in DR3 and DR4 mice. However, CLN treatment significantly reduced GFAP mRNA levels in DR3 mice, not DR4 mice. Our data suggest a skin–brain axis during GAS NF, demonstrating that peripherally induced pathological conditions regulate neuroimmune changes and gliotic events in the brai

Acute neuroinflammation elicited by TLR-3 systemic activation combined with early life stress induces working memory impairments in male adolescent mice

Toll-like Receptors (TLRs) are implicated with the pathogenesis of cognitive impairment induced by inflammation. Early life stress is associated with altered trajectories of neuroimmune signaling with implications for cognitive development. However, effects of TLR-3 activation on early life stress-related cognitive outcomes are understudied. We investigated the effects of maternal separation (MS) during postnatal development and a viral immune challenge during adolescence on working memory performance. BALB/c mice exposed to MS were separated from their dams daily for 180-min from postnatal day (PND) 2 to 15. At PND 45, animals were challenged with a single i.p. injection of either Poly (I:C) or sterile saline, and then subjected to a spatial working memory test in a Y-maze apparatus. Gene expression was determined by qPCR. Protein levels of oxidative stress markers were also assessed. A single peripheral administration of a TLR-3 agonist was able to induce working memory impairments in adolescent mice exposed to MS. At a molecular level, exposure to MS was associated with lower mRNA levels of Tlr3 in the medial prefrontal cortex (mPFC). However, when MS animals were exposed to Poly (I:C), a more robust activation of Tlr3, Il6 and Nfkb1 gene transcription was observed in these mice compared with control animals. These modifications did not result in oxidative stress. Finally, higher mRNA levels of Nfkb1 in the mPFC were correlated with lower working memory performance, suggesting that altered NF-\u3baB signaling might be related with poor cognitive functioning. These results have implications for how ELS affects neuroimmune signaling in the mPFC

Life Sci

Aims:Growing evidence suggests that Gulf War Illness (GWI) is the result of underlying neuroimmune dysfunction. For example, previously we found that several GWI-relevant organophosphate acetylcholinesterase inhibitors produce heightened neuroinflammatory responses following subchronic exposure to stress hormone as a mimic of high physiological stress. The goal of the current study was to evaluate the potential for the \u3b2-adrenergic receptor inhibitor and anti-inflammatory drug, propranolol, to treat neuroinflammation in a novel long-term mouse model of GWI.Main methods:Adult male C57BL/6J mice received a subchronic exposure to corticosterone (CORT) at levels mimicking high physiological stress followed by exposure to the sarin surrogate, diisopropyl fluorophosphate (DFP). These mice were then re-exposed to CORT every other week for a total of five weeks, followed by a systemic immune challenge with lipopolysaccharide (LPS). Animals receiving the propranolol treatment were given a single dose (20 mg/kg, i.p.) either four or 11 days prior to the LPS challenge. The potential anti-neuroinflammatory effects of propranolol were interrogated by analysis of cytokine mRNA expression.Key findings:We found that our long-term GWI model produces a primed neuroinflammatory response to subsequent immune challenge that is dependent upon GWI-relevant organophosphate exposure. Propranolol treatment abrogated the elaboration of inflammatory cytokine mRNA expression in the brain instigated in our model, having no treatment effects in non-DFP exposed groups.Significance:Our results indicate that propranolol may be a promising therapy for GWI with the potential to treat the underlying neuroinflammation associated with the illness.CC999999/ImCDC/Intramural CDC HHSUnited States/2022-04-28T00:00:00Z34563566PMC90470581148

Expression and Differential Responsiveness of Central Nervous System Glial Cell Populations to the Acute Phase Protein Serum Amyloid A

Acute-phase response is a systemic reaction to environmental/inflammatory insults and involves hepatic production of acute-phase proteins, including serum amyloid A (SAA). Extrahepatically, SAA immunoreactivity is found in axonal myelin sheaths of cortex in Alzheimer's disease and multiple sclerosis (MS), although its cellular origin is unclear. We examined the responses of cultured rat cortical astrocytes, microglia and oligodendrocyte precursor cells (OPCs) to master pro-inflammatory cytokine tumour necrosis factor (TNF)-\u3b1 and lipopolysaccaride (LPS). TNF-\u3b1 time-dependently increased Saa1 (but not Saa3) mRNA expression in purified microglia, enriched astrocytes, and OPCs (as did LPS for microglia and astrocytes). Astrocytes depleted of microglia were markedly less responsive to TNF-\u3b1 and LPS, even after re-addition of microglia. Microglia and enriched astrocytes showed complementary Saa1 expression profiles following TNF-\u3b1 or LPS challenge, being higher in microglia with TNF-\u3b1 and higher in astrocytes with LPS. Recombinant human apo-SAA stimulated production of both inflammatory mediators and its own mRNA in microglia and enriched, but not microglia-depleted astrocytes. Co-ultramicronized palmitoylethanolamide/luteolin, an established anti-inflammatory/neuroprotective agent, reduced Saa1 expression in OPCs subjected to TNF-\u3b1 treatment. These last data, together with past findings suggest that co-ultramicronized palmitoylethanolamide/luteolin may be a novel approach in the treatment of inflammatory demyelinating disorders like MS

Involvement of MicroRNA in Microglia-Mediated Immune Response

MicroRNAs (miRNAs) are an abundant class of small noncoding RNA molecules that play an important role in the regulation of gene expression at the posttranscriptional level. Due to their ability to simultaneously modulate the fate of different genes, these molecules are particularly well suited to act as key regulators during immune cell differentiation and activation, and their dysfunction can contribute to pathological conditions associated with neuroinflammation. Recent studies have addressed the role of miRNAs in the differentiation of progenitor cells into microglia and in the activation process, aiming at clarifying the origin of adult microglia cells and the contribution of the central nervous system (CNS) environment to microglia phenotype, in health and disease. Altered expression of several miRNAs has been associated with Alzheimer’s disease, multiple sclerosis, and ischemic injury, hence strongly advocating the use of these small molecules as disease markers and new therapeutic targets. This review summarizes the recent advances in the field of miRNA-mediated regulation of microglia development and activation. We discuss the role of specific miRNAs in the maintenance and switching of microglia activation states and illustrate the potential of this class of nucleic acids both as biomarkers of inflammation and new therapeutic tools for the modulation of microglia behavior in the CNS

DISCOVERY OF NATURAL PRODUCT ANALOGS AGAINST ETHANOL-INDUCED CYTOTOXICITY IN HIPPOCAMPAL SLICE CULTURES

An estimated 13.9% of Americans currently meet criteria for an alcohol (ethanol; EtOH) use disorder (AUD). While there are 4 medications approved by the Food and Drug Administration (FDA) to treat AUD, these treatments have demonstrated poor clinical efficacy. Our ongoing research program encompasses a multi-tiered screening of a natural product library and validation process to provide novel information about the mechanisms underlying EtOH-induced changes in neurobiology and to identify novel chemical scaffolds to be exploited in the development of pharmacological treatments for AUD in a rodent organotypic hippocampal slice culture model. Initial screens of several natural product compounds identified 3 compounds which attenuate 48 h EtOH-induced cytotoxicity in vitro. As analogs of natural products can be developed to have enhanced therapeutic potential over parental structures, Study 1 sought to extend on prior findings via the screening of several natural product analogs for their ability to attenuate EtOH-induced cytoxicity. Nine natural produce analogs demonstrated potent cytoprotective effects against EtOH-induced toxicity at 48 h. Several reports suggest EtOH-induced neurotoxicity may be secondary to the induction of persistent neuroimmune activation, and isoflavonoids have been shown to have effects on neuroimmune signaling. Thus, Study 2 compared the effects of compound 9b, an isoflavonoid analog identified in Study 1, to daidzein (DZ), a prototypical isoflavonoid, in the same 48 h model, with the addition of a neuroimmune component. Specifically, culture media was collected to assess for the release of the neuroimmune mediators HMGB1, TNF-α, IL-6, and IL-10 via ELISA. Compound 9b and DZ protected against EtOH-induced cytotoxicity at 48 h. EtOH exposure significantly increased secretion of HMGB1 and IL-6 into culture media at 48h. Compound 9b and DZ attenuated these increases at all concentrations tested. These results suggest potential neuroimmune modulating properties of isoflavonoids which may contribute to their neuroprotective effects against EtOH in vitro. These findings highlight the potential applications DZ and the novel isoflavonoid analog 9b for use in the treatment of AUD