Moderate Prenatal Alcohol Exposure Increases Toll-like Receptor Activity in Umbilical Cord Blood at Birth: A Pilot Study.

The prevalence of prenatal alcohol exposure (PAE) is increasing, with evidence suggesting that PAE is linked to an increased risk of infections. PAE is hypothesized to affect the innate immune system, which identifies pathogens through pattern recognition receptors, of which toll-like receptors (TLRs) are key components. We hypothesized that light-to-moderate PAE would impair immune responses, as measured by a heightened response in cytokine levels following TLR stimulation. Umbilical cord samples (10 controls and 8 PAE) from a subset of the Ethanol, Neurodevelopment, Infant and Child Health Study-2 cohort were included. Peripheral blood mononuclear cells (PMBCs) were stimulated with one agonist (TLR2, TLR3, TLR4, or TLR9). TLR2 agonist stimulation significantly increased pro-inflammatory interleukin-1-beta in the PAE group after 24 h. Pro- and anti-inflammatory cytokines were increased following stimulation with the TLR2 agonists. Stimulation with TLR3 or TLR9 agonists displayed minimal impact overall, but there were significant increases in the percent change of the control compared to PAE after 24 h. The results of this pilot investigation support further work into the impact on TLR2 and TLR4 response following PAE to delineate if alterations in levels of pro- and anti-inflammatory cytokines have clinical significance that could be used in patient management and/or attention to follow-up.

Maternal alcohol drinking patterns predict offspring neurobehavioral outcomes.

The timing, rate, and quantity of gestational alcohol consumption, collectively referred to here as Maternal Drinking Patterns (MDPs), are of known importance to fetal developmental outcomes. However, few studies have directly evaluated the impact of MDPs on offspring behavior. To do so, we used specialized equipment to record the precise amount and timing of alcohol consumption in pregnant dams, and then characterized MDPs using Principle Component Analysis (PCA). We next tested offspring on behaviors we have previously identified as impacted by prenatal alcohol exposure, and evaluated them where possible in the context of MDPs. Male alcohol exposed mice exhibited longer latencies to fall on the rotarod compared to their controls, which we attribute to a delayed decrease in body weight-gain. This effect was mediated by MDPs within the first 15 min of alcohol access (i.e. alcohol frontloading), where the highest performing male offspring came from dams exhibiting the highest rate of alcohol frontloading. Female alcohol exposed mice displayed reduced locomotor activity in the open field compared to controls, which was mediated by MDPs encompassing the entire drinking session. Surprisingly, total gestational alcohol exposure alone was not associated with any behavioral outcomes. Finally, we observed allodynia in alcohol exposed mice that developed more quickly in males compared to females, and which was not observed in controls. To our knowledge, this report represents the highest resolution assessment of alcohol drinking throughout gestation in mice, and one of few to have identified relationships between specific alcohol MDPs and neurobehavioral outcomes in offspring.

Prenatal alcohol exposure promotes NLRP3 inflammasome-dependent immune actions following morphine treatment and paradoxically prolongs nerve injury-induced pathological pain in female mice.

BACKGROUND: Neuroimmune dysregulation from prenatal alcohol exposure (PAE) may contribute to neurological deficits associated with fetal alcohol spectrum disorders (FASD). PAE is a risk factor for developing peripheral immune and spinal glial sensitization and release of the proinflammatory cytokine IL-1ß, which lead to neuropathic pain (allodynia) from minor nerve injury. Although morphine acts on µ-opioid receptors, it also activates immune receptors, TLR4, and the NLRP3 inflammasome that induces IL-1ß. We hypothesized that PAE induces NLRP3 sensitization by morphine following nerve injury in adult mice. METHODS: We used an established moderate PAE paradigm, in which adult PAE and non-PAE control female mice were exposed to a minor sciatic nerve injury, and subsequent allodynia was measured using the von Frey fiber test. In control mice with standard sciatic damage or PAE mice with minor sciatic damage, the effects of the NLRP3 inhibitor, MCC950, were examined during chronic allodynia. Additionally, minor nerve-injured mice were treated with morphine, with or without MCC950. In vitro studies examined the TLR4-NLRP3-dependent proinflammatory response of peripheral macrophages to morphine and/or lipopolysaccharide, with or without MCC950. RESULTS: Mice with standard sciatic damage or PAE mice with minor sciatic damage developed robust allodynia. Blocking NLRP3 activation fully reversed allodynia in both control and PAE mice. Morphine paradoxically prolonged allodynia in PAE mice, while control mice with minor nerve injury remained stably non-allodynic. Allodynia resolved sooner in nerve-injured PAE mice without morphine treatment than in morphine-treated mice. MCC950 treatment significantly shortened allodynia in morphine-treated PAE mice. Morphine potentiated IL-1ß release from TLR4-activated PAE immune cells, while MCC950 treatment greatly reduced it. CONCLUSIONS: In female mice, PAE prolongs allodynia following morphine treatment through NLRP3 activation. TLR4-activated PAE immune cells showed enhanced IL-1ß release with morphine via NLRP3 actions. Similar studies are needed to examine the adverse impact of morphine in males with PAE. These results are predictive of adverse responses to opioid pain therapeutics in individuals with FASD.

Biomass smoke inhalation promotes neuroinflammatory and metabolomic temporal changes in the hippocampus of female mice.

Smoke from wildland fires has been shown to produce neuroinflammation in preclinical models, characterized by neural infiltrations of neutrophils and monocytes, as well as altered neurovascular endothelial phenotypes. To address the longevity of such outcomes, the present study examined the temporal dynamics of neuroinflammation and metabolomics after inhalation exposures from biomass-derived smoke. 2-month-old female C57BL/6 J mice were exposed to wood smoke every other day for 2 weeks at an average exposure concentration of 0.5 mg/m3. Subsequent serial euthanasia occurred at 1-, 3-, 7-, 14-, and 28-day post-exposure. Flow cytometry of right hemispheres revealed two endothelial populations of CD31Hi and CD31Med expressors, with wood smoke inhalation causing an increased proportion of CD31Hi. These populations of CD31Hi and CD31Med were associated with an anti-inflammatory and pro-inflammatory response, respectively, and their inflammatory profiles were largely resolved by the 28-day mark. However, activated microglial populations (CD11b+/CD45low) remained higher in wood smoke-exposed mice than controls at day 28. Infiltrating neutrophil populations decreased to levels below controls by day 28. However, the MHC-II expression of the peripheral immune infiltrate remained high, and the population of neutrophils retained an increased expression of CD45, Ly6C, and MHC-II. Utilizing an unbiased approach examining the metabolomic alterations, we observed notable hippocampal perturbations in neurotransmitter and signaling molecules, such as glutamate, quinolinic acid, and 5-α-dihydroprogesterone. Utilizing a targeted panel designed to explore the aging-associated NAD+ metabolic pathway, wood smoke exposure drove fluctuations and compensations across the 28-day time course, ending with decreased hippocampal NAD+ abundance on day 28. Summarily, these results indicate a highly dynamic neuroinflammatory environment, with potential resolution extending past 28 days, the implications of which may include long-term behavioral changes, systemic and neurological sequalae directly associated with wildfire smoke exposure.

Prenatal alcohol exposure alters mRNA expression for stress peptides, glucocorticoid receptor function and immune factors in acutely stressed neonatal brain.

Background: The amygdala, hippocampus and hypothalamus are critical stress regulatory areas that undergo functional maturation for stress responding initially established during gestational and early postnatal brain development. Fetal alcohol spectrum disorder (FASD), a consequence of prenatal alcohol exposure (PAE), results in cognitive, mood and behavioral disorders. Prenatal alcohol exposure negatively impacts components of the brain stress response system, including stress-associated brain neuropeptides and glucocorticoid receptors in the amygdala, hippocampus and hypothalamus. While PAE generates a unique brain cytokine expression pattern, little is known about the role of Toll-like receptor 4 (TLR4) and related proinflammatory signaling factors, as well as anti-inflammatory cytokines in PAE brain stress-responsive regions. We hypothesized that PAE sensitizes the early brain stress response system resulting in dysregulated neuroendocrine and neuroimmune activation. Methods: A single, 4-h exposure of maternal separation stress in male and female postnatal day 10 (PND10) C57Bl/6 offspring was utilized. Offspring were from either prenatal control exposure (saccharin) or a limited access (4 h) drinking-in-the-dark model of PAE. Immediately after stress on PND10, the hippocampus, amygdala and hypothalamus were collected, and mRNA expression was analyzed for stress-associated factors (CRH and AVP), glucocorticoid receptor signaling regulators (GAS5, FKBP51 and FKBP52), astrocyte and microglial activation, and factors associated with TLR4 activation including proinflammatory interleukin-1ß (IL-1ß), along with additional pro- and anti-inflammatory cytokines. Select protein expression analysis of CRH, FKBP and factors associated with the TLR4 signaling cascade from male and female amygdala was conducted. Results: The female amygdala revealed increased mRNA expression in stress-associated factors, glucocorticoid receptor signaling regulators and all of the factors critical in the TLR4 activation cascade, while the hypothalamus revealed blunted mRNA expression of all of these factors in PAE following stress. Conversely, far fewer mRNA changes were observed in males, notably in the hippocampus and hypothalamus, but not the amygdala. Statistically significant increases in CRH protein, and a strong trend in increased IL-1ß were observed in male offspring with PAE independent of stressor exposure. Conclusion: Prenatal alcohol exposure creates stress-related factors and TLR-4 neuroimmune pathway sensitization observed predominantly in females, that is unmasked in early postnatal life by a stress challenge.

Neuroinflammatory and Metabolomic Temporal Dynamics Following Wood Smoke Inhalation.

Smoke from wildland fires has been shown to produce neuroinflammation in preclinical models, characterized by neural infiltrations of neutrophils and monocytes, as well as altered neurovascular endothelial phenotypes. To address the longevity of such outcomes, the present study examined the neuroinflammatory and metabolomic temporal dynamics after inhalation exposures from biomass-derived smoke. 2-month-old female C57BL/6J mice were exposed to wood smoke every other day for two weeks at an average exposure concentration of 0.5mg/m 3 . Subsequent serial euthanasia occurred at 1-, 3-, 7-, 14-, and 28-days post-exposure. Flow cytometry of right hemispheres revealed two endothelial populations of PECAM (CD31), high and medium expressors, with wood smoke inhalation causing an increased proportion of PECAM Hi . These populations of PECAM Hi and PECAM Med were associated with an anti-inflammatory and pro-inflammatory response, respectively, and their inflammatory profiles were largely resolved by the 28-day mark. However, activated microglial populations (CD11b + /CD45 low ) remained higher in wood smoke-exposed mice than controls at day 28. Infiltrating neutrophil populations decreased to levels below controls by day 28. However, the MHC-II expression of the peripheral immune infiltrate remained high, and the population of neutrophils retained an increased expression of CD45, Ly6C, and MHC-II. Utilizing an unbiased approach examining the metabolomic alterations, we observed notable hippocampal perturbations in neurotransmitter and signaling molecules like glutamate, quinolinic acid, and 5-α-dihydroprogesterone. Utilizing a targeted panel designed to explore the aging-associated NAD + metabolic pathway, wood smoke exposure drove fluctuations and compensations across the 28-day time course, ending with decreased hippocampal NAD + abundance at day 28. Summarily, these results indicate a highly dynamic neuroinflammatory environment, with potential resolution extending past 28 days, the implications of which may include long-term behavioral changes, systemic and neurological sequalae directly associated wtith wildfire smoke exposure.

Prenatal alcohol exposure dysregulates spinal and circulating immune cell circular RNA expression in adult female rats with chronic sciatic neuropathy.

Alcohol consumption during pregnancy is associated with Fetal Alcohol Spectrum Disorders (FASD) that results in a continuum of central nervous system (CNS) deficits. Emerging evidence from both preclinical and clinical studies indicate that the biological vulnerability to chronic CNS disease in FASD populations is driven by aberrant neuroimmune actions. Our prior studies suggest that, following minor nerve injury, prenatal alcohol exposure (PAE) is a risk factor for developing adult-onset chronic pathological touch sensitivity or allodynia. Allodynia in PAE rats occurs concurrently with heightened proinflammatory peripheral and spinal glial-immune activation. However, minor nerve-injured control rats remain non-allodynic, and corresponding proinflammatory factors are unaltered. A comprehensive molecular understanding of the mechanism(s) that underlie PAE-induced proinflammatory bias during adulthood remains elusive. Non-coding circular RNAs (circRNAs) are emerging as novel modulators of gene expression. Here, we hypothesized that PAE induces dysregulation of circRNAs that are linked to immune function under basal and nerve-injured conditions during adulthood. Utilizing a microarray platform, we carried out the first systematic profiling of circRNAs in adult PAE rats, prior to and after minor nerve injury. The results demonstrate a unique circRNA profile in adult PAE rats without injury; 18 circRNAs in blood and 32 spinal circRNAs were differentially regulated. Following minor nerve injury, more than 100 differentially regulated spinal circRNAs were observed in allodynic PAE rats. Bioinformatic analysis identified that the parental genes of these circRNAs are linked to the NF-κB complex, a central transcription factor for pain-relevant proinflammatory cytokines. Quantitative real-time PCR was employed to measure levels of selected circRNAs and linear mRNA isoforms. We have validated that circVopp1 was significantly downregulated in blood leukocytes in PAE rats, concurrent with downregulation of Vopp1 mRNA levels. Spinal circVopp1 levels were upregulated in PAE rats, regardless of nerve injury. Additionally, PAE downregulated levels of circItch and circRps6ka3, which are linked to immune regulation. These results demonstrate that PAE exerts long-lasting dysregulation of circRNA expression in blood leukocytes and the spinal cord. Moreover, the spinal circRNA expression profile following peripheral nerve injury is differentially modulated by PAE, potentially contributing to PAE-induced neuroimmune dysregulation.

The maternal-placental-fetal interface: Adaptations of the HPA axis and immune mediators following maternal stress and prenatal alcohol exposure.

This review addresses underlying physiological, cellular, and molecular factors that alter the developing fetal brain stress circuits and responses of the hypothalamic-pituitary-adrenal (HPA) axis caused by maternal stress and prenatal alcohol exposure (PAE). An emphasis is placed on the contribution of the placenta following maternal stress separately, and as a co-occurrence with PAE. Altered fetal HPA axis ultimately results in dysregulation of the brain stress-response system long after birth and possibly lifelong. Additional consideration of the role of placentally-derived endocrine and sex hormones, as well as a brief discussion of epigenetic mechanisms of altered placental expression of genes encoding the glucocorticoid receptor and the enzymes 11ß-HSD that rapidly convert glucocorticoids into its active or inactive forms are reviewed. Data highlighting the strong, reciprocal interactions between the neuroimmune and neuroendocrine systems during fetal development that are impacted by maternal stress and PAE are considered, emphasizing the role of the placenta as a key contributor to the dysregulation of these systems. In view of the maternal-placental-fetal interface, important physiological, cellular, and molecular factors underlying later life dysregulated stress responses are additionally considered. Literature from animal models of PAE and maternal stress is reviewed that support clinical observations of the effect of maternal stress and alcohol exposure during fetal development on later-life adult stress responses and associated mood dysregulation. An appreciation of dysregulated stress responses in individuals with fetal alcohol spectrum disorders (FASD) are addressed given the greater prevalence of adult dysregulated stress responses and a greater co-occurrence of mood disorders in individuals diagnosed with FASD.

Effects of prenatal opioid and alcohol exposures on immune and serotonin factors in human placenta.

PURPOSE: Opioids and alcohol impact critical serotonin (5-HT) function in the developing placenta and fetus through the actions of immune proinflammatory factors. Yet, possible convergent effects of opioids and alcohol on human placental toll-like receptor 4 (TLR4) activation and subsequent 5-HT homeostasis remain entirely unknown. The purpose of this study was to examine the effect of prenatal exposure to opioids with or without prenatal alcohol exposure (PAE) on the expression of key placental immune and serotonin signaling factors in human placental tissue obtained from a well-characterized prospective cohort. METHODS: Data were collected from a subset of participants enrolled in the prospective pre-birth Ethanol, Neurodevelopment, Infant, and Child Health (ENRICH-1) cohort. Women were recruited and classified into four study groups: 1) PAE (n = 20); 2) those taking medications for opioid use disorder (MOUD; n = 28), 3) concurrent PAE and MOUD (n = 20); and 4) controls (HC; n = 20) based on prospective, repeated self-report, and biomarker analysis. Placenta samples underwent tissue processing to identify mRNA for TLR4, nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), serotonin transporter (SERT), tryptophan hydroxylase (TPH1), indoleamine 2,3-Dioxygenase 1 (IDO) as well as protein concentrations of TLR4, IL-1ß, TNF-α, SERT. To consider the association between study group and mRNA/protein expression of our targets, multivariable regression models were developed with inclusion of a priori selected covariates. RESULTS: There was a significant negative association between PAE and SERT mRNA (ß = -0.01; p < 0.01) and a positive association with TPH1 mRNA expression (ß = 0.78; p < 0.05). In addition, there was a negative association between MOUD and TNF-α protein expression (ß = -0.12; p < 0.05). CONCLUSIONS: This study provides the first evidence that PAE may inhibit SERT expression while simultaneously promoting increased TPH1 protein expression in human placenta. This may result in increased 5-HT in fetal circulation known to affect neurodevelopment. Our data suggest that opioids and alcohol may disturb the bidirectional, dynamic interaction between the placental immune and serotonin system. Given the implication for brain development and health across the life-span further investigation of these critical mechanisms in well-defined cohorts is required.

Neuroinflammatory and Neurometabolomic Consequences From Inhaled Wildfire Smoke-Derived Particulate Matter in the Western United States.

Utilizing a mobile laboratory located >300 km away from wildfire smoke (WFS) sources, this study examined the systemic immune response profile, with a focus on neuroinflammatory and neurometabolomic consequences, resulting from inhalation exposure to naturally occurring wildfires in California, Arizona, and Washington in 2020. After a 20-day (4 h/day) exposure period in a mobile laboratory stationed in New Mexico, WFS-derived particulate matter (WFPM) inhalation resulted in significant neuroinflammation while immune activity in the peripheral (lung, bone marrow) appeared to be resolved in C57BL/6 mice. Importantly, WFPM exposure increased cerebrovascular endothelial cell activation and expression of adhesion molecules (VCAM-1 and ICAM-1) in addition to increased glial activation and peripheral immune cell infiltration into the brain. Flow cytometry analysis revealed proinflammatory phenotypes of microglia and peripheral immune subsets in the brain of WFPM-exposed mice. Interestingly, endothelial cell neuroimmune activity was differentially associated with levels of PECAM-1 expression, suggesting that subsets of cerebrovascular endothelial cells were transitioning to resolution of inflammation following the 20-day exposure. Neurometabolites related to protection against aging, such as NAD+ and taurine, were decreased by WFPM exposure. Additionally, increased pathological amyloid-beta protein accumulation, a hallmark of neurodegeneration, was observed. Neuroinflammation, together with decreased levels of key neurometabolites, reflect a cluster of outcomes with important implications in priming inflammaging and aging-related neurodegenerative phenotypes.

SPARC coordinates extracellular matrix remodeling and efficient recruitment to and migration of antigen-specific T cells in the brain following infection.

Central nervous system (CNS) injury and infection can result in profound tissue remodeling in the brain, the mechanism and purpose of which is poorly understood. Infection with the protozoan parasite Toxoplasma gondii causes chronic infection and inflammation in the brain parenchyma. Control of parasite replication requires the continuous presence of IFNγ-producing T cells to keep T. gondii in its slowly replicating cyst form. During infection, a network of extracellular matrix fibers, revealed using multiphoton microscopy, forms in the brain. The origin and composition of these structures are unknown but the fibers have been observed to act as a substrate for migrating T cells. In this study, we show a critical regulator of extracellular matrix (ECM) remodeling, Secreted Protein, Acidic, Rich in Cysteine (SPARC), is upregulated in the brain during the early phases of infection in the frontal cortex. In the absence of SPARC, a reduced and disordered fibrous network, increased parasite burden, and reduced antigen-specific T cell entry into the brain points to a role for SPARC in T cell recruitment to and migration within the brain. We also report SPARC can directly bind to CCR7 ligands CCL19 and CCL21 but not CXCL10, and enhance migration toward a chemokine gradient. Measurement of T cell behavior points to tissue remodeling being important for access of immune cells to the brain and facilitating cellular locomotion. Together, these data identify SPARC as an important regulatory component of immune cell trafficking and access to the inflamed CNS.

The LFA-1 antagonist BIRT377 reverses neuropathic pain in prenatal alcohol-exposed female rats via actions on peripheral and central neuroimmune function in discrete pain-relevant tissue regions.

Previous reports show that moderate prenatal alcohol exposure (PAE) poses a risk factor for developing neuropathic pain following adult-onset peripheral nerve injury in male rats. Recently, evidence suggests that immune-related mechanisms underlying neuropathic pain in females are different compared to males despite the fact that both sexes develop neuropathy of similar magnitude and duration following chronic constriction injury (CCI) of the sciatic nerve. Data suggest that the actions of peripheral T cells play a greater role in mediating neuropathy in females. The goal of the current study is to identify specificity of immune cell and cytokine changes between PAE and non-PAE neuropathic females by utilizing a well-characterized rodent model of sciatic nerve damage, in an effort to unmask unique signatures of immune-related factors underlying the risk of neuropathy from PAE. Cytokines typically associated with myeloid cell actions such as interleukin (IL)-1ß, tumor necrosis factor (TNF), IL-6, IL-4 and IL-10 as well as the neutrophil chemoattractant CXCL1, are examined. In addition, transcription factors and cytokines associated with various differentiated T cell subtypes are examined (anti-inflammatory FOXP3, proinflammatory IL-17A, IL-21, ROR-γt, interferon (IFN)-γ and T-bet). Lymphocyte function associated antigen 1 (LFA-1) is an adhesion molecule expressed on peripheral immune cells including T cells, and regulates T cell activation and extravasation into inflamed tissue regions. A potential therapeutic approach was explored with the goal of controlling proinflammatory responses in neuroanatomical regions critical for CCI-induced allodynia by blocking LFA-1 actions using BIRT377. The data show profound development of hindpaw allodynia in adult non-PAE control females following standard CCI, but not following minor CCI, while minor CCI generated allodynia in PAE females. The data also show substantial increases in T cell-associated proinflammatory cytokine mRNA and proteins, along with evidence of augmented myeloid/glial activation (mRNA) and induction of myeloid/glial-related proinflammatory cytokines, CCL2, IL-1ß and TNF in discrete regions along the pain pathway (damaged sciatic nerve, dorsal root ganglia; DRG, and spinal cord). Interestingly, the characteristic anti-inflammatory IL-10 protein response to nerve damage is blunted in neuropathic PAE females. Moreover, T cell profiles are predominantly proinflammatory in neuropathic Sac and PAE females, augmented levels of Th17-specific proinflammatory cytokines IL-17A and IL-21, as well as the Th1-specific factor, T-bet, are observed. Similarly, the expression of RORγt, a critical transcription factor for Th17 cells, is detected in the spinal cord of neuropathic females. Blocking peripheral LFA-1 actions with intravenous (i.v.) BIRT377 reverses allodynia in Sac and PAE rats, dampens myeloid (IL-1ß, TNF, CXCL1)- and T cell-associated proinflammatory factors (IL-17A and RORγt) and spinal glial activation. Moreover, i.v. BIRT377 treatment reverses the blunted IL-10 response to CCI observed only in neuropathic PAE rats and elevates FOXP3 in pain-reversed Sac rats. Unexpectedly, intrathecal BIRT377 treatment is unable to alter allodynia in either Sac or PAE neuropathic females. Together, these data provide evidence that: 1) fully differentiated proinflammatory Th17 cells recruited at the sciatic nerve, DRGs and lumbar spinal cord may interact with the local environment to shape the immune responses underlying neuropathy in female rats, and, 2) PAE primes peripheral and spinal immune responses in adult females. PAE is a risk factor in females for developing peripheral neuropathy after minor nerve injury.

Ischemic preconditioning induces cortical microglial proliferation and a transcriptomic program of robust cell cycle activation.

Ischemic preconditioning (IPC) is an experimental phenomenon in which a subthreshold ischemic insult applied to the brain reduces damage caused by a subsequent more severe ischemic episode. Identifying key molecular and cellular mediators of IPC will provide critical information needed to develop novel therapies for stroke. Here we report that the transcriptomic response of acutely isolated preconditioned cortical microglia is dominated by marked upregulation of genes involved in cell cycle activation and cellular proliferation. Notably, this transcriptional response occurs in the absence of cortical infarction. We employed ex vivo flow cytometry, immunofluorescent microscopy, and quantitative stereology methods on brain tissue to evaluate microglia proliferation following IPC. Using cellular colocalization of microglial (Iba1) and proliferation (Ki67 and BrdU) markers, we observed a localized increase in the number of microglia and proliferating microglia within the preconditioned hemicortex at 72, but not 24, hours post-IPC. Our quantification demonstrated that the IPC-induced increase in total microglia was due entirely to proliferation. Furthermore, microglia in the preconditioned hemisphere had altered morphology and increased soma volumes, indicative of an activated phenotype. Using transgenic mouse models with either fractalkine receptor (CX3CR1)-haploinsufficiency or systemic type I interferon signaling loss, we determined that microglial proliferation after IPC is dependent on fractalkine signaling but independent of type I interferon signaling. These findings suggest there are multiple distinct targetable signaling pathways in microglia, including CX3CR1-dependent proliferation that may be involved in IPC-mediated protection.

LFA-1 antagonist (BIRT377) similarly reverses peripheral neuropathic pain in male and female mice with underlying sex divergent peripheral immune proinflammatory phenotypes.

AIM: The majority of preclinical studies investigating aberrant glial-neuroimmune actions underlying neuropathic pain have focused on male rodent models. Recently, studies have shown peripheral immune cells play a more prominent role than glial cells in mediating pathological pain in females. Here, we compared the onset and duration of allodynia in males and females, and the anti-allodynic action of a potentially novel therapeutic drug (BIRT377) that not only antagonizes the action of lymphocyte function-associated antigen-1 (LFA-1) to reduce cell migration in the periphery, but may also directly alter the cellular inflammatory bias. METHODS: Male and female mice were subjected to peripheral nerve injury chronic constriction injury (CCI) applying two methods, using either 4-0 or 5-0 chromic gut suture material, to examine potential sex differences in the onset, magnitude and duration of allodynia. Hindpaw sensitivity before and after CCI and application of intravenous BIRT377 was assessed. Peripheral and spinal tissues were analyzed for protein (multiplex electrochemiluminescence technology) and mRNA expression (quantitative real-time PCR). The phenotype of peripheral T cells was determined using flow cytometry. RESULTS: Sex differences in proinflammatory CCL2 and IL-1ß and the anti-inflammatory IL-10 were observed from a set of cytokines analyzed. A profound proinflammatory T cell (Th17) response in the periphery and spinal cord was also observed in neuropathic females. BIRT377 reversed pain, reduced IL-1ß and TNF, and increased IL-10 and transforming growth factor (TGF)-ß1, also an anti-inflammatory cytokine, in both sexes. However, female-derived T cell cytokines are transcriptionally regulated by BIRT377, as demonstrated by reducing proinflammatory IL-17A production with concurrent increases in IL-10, TGF-ß1 and the anti-inflammatory regulatory T cell-related factor, FOXP3. CONCLUSION: This study supports that divergent peripheral immune and neuroimmune responses during neuropathy exists between males and females. Moreover, the modulatory actions of BIRT377 on T cells during neuropathy are predominantly specific to females. These data highlight the necessity of including both sexes for studying drug efficacy and mechanisms of action in preclinical studies and clinical trials.

CXCR2 Blockade Mitigates Neural Cell Injury Following Preclinical Chorioamnionitis.

Minimizing central nervous system (CNS) injury from preterm birth depends upon identification of the critical pathways that underlie essential neurodevelopmental and CNS pathophysiology. While chorioamnionitis (CHORIO), is a leading cause of preterm birth, the precise mechanism linking prenatal brain injury and long-term CNS injury is unknown. The chemokine (C-X-C motif) ligand 1 (CXCL1) and its cognate receptor, CXCR2, are implicated in a variety of uterine and neuropathologies, however, their role in CNS injury associated with preterm birth is poorly defined. To evaluate the putative efficacy of CXCR2 blockade in neural repair secondary to CHORIO, we tested the hypothesis that transient postnatal CXCR2 antagonism would reduce neutrophil activation and mitigate cerebral microstructural injury in rats. To this end, a laparotomy was performed on embryonic day 18 (E18) in Sprague Dawley rats, with uterine arteries transiently occluded for 60 min, and lipopolysaccharide (LPS, 4 µg/sac) injected into each amniotic sac. SB225002, a CXCR2 antagonist (3 mg/kg), was administered intraperitoneally from postnatal day 1 (P1)-P5. Brains were collected on P7 and P21 and analyzed with western blot, immunohistochemistry and ex vivo diffusion tensor imaging (DTI). Results demonstrate that transient CXCR2 blockade reduced cerebral neutrophil activation (myeloperoxidase expression/MPO) and mitigated connexin43 expression, indicative of reduced neuroinflammation at P7 (p < 0.05 for all). CXCR2 blockade also reduced alpha II-spectrin calpain-mediated cleavage, improved pNF/NF ratio, and minimized Iba1 and GFAP expression consistent with improved neuronal and axonal health and reduced gliosis at P21. Importantly, DTI revealed diffuse white matter injury and decreased microstructural integrity following CHORIO as indicated by lower fractional anisotropy (FA) and elevated radial diffusivity (RD) in major white matter tracts (p < 0.05). Early postnatal CXCR2 blockade also reduced microstructural abnormalities in white matter and hippocampus at P21 (p < 0.05). Together, these data indicate that transient postnatal blockade of CXCR2 ameliorates perinatal abnormalities in inflammatory signaling, and facilitates neural repair following CHORIO. Further characterization of neuroinflammatory signaling, specifically via CXCL1/CXCR2 through the placental-fetal-brain axis, may clarify stratification of brain injury following preterm birth, and improve use of targeted interventions in this highly vulnerable patient population.

Targeting the ß2-integrin LFA-1, reduces adverse neuroimmune actions in neuropathic susceptibility caused by prenatal alcohol exposure.

Recently, moderate prenatal alcohol exposure (PAE) was shown to be a risk factor for peripheral neuropathy following minor nerve injury. This effect coincides with elevated spinal cord astrocyte activation and ex vivo immune cell reactivity assessed by proinflammatory cytokine interleukin (IL) -1ß protein expression. Additionally, the ß2-integrin adhesion molecule, lymphocyte function-associated antigen-1 (LFA-1), a factor that influences the expression of the proinflammatory/anti-inflammatory cytokine network is upregulated. Here, we examine whether PAE increases the proinflammatory immune environment at specific anatomical sites critical in the pain pathway of chronic sciatic neuropathy; the damaged sciatic nerve (SCN), the dorsal root ganglia (DRG), and the spinal cord. Additionally, we examine whether inhibiting LFA-1 or IL-1ß actions in the spinal cord (intrathecal; i.t., route) could alleviate chronic neuropathic pain and reduce spinal and DRG glial activation markers, proinflammatory cytokines, and elevate anti-inflammatory cytokines. Results show that blocking the actions of spinal LFA-1 using BIRT-377 abolishes allodynia in PAE rats with sciatic neuropathy (CCI) of a 10 or 28-day duration. This effect is observed (utilizing immunohistochemistry; IHC, with microscopy analysis and protein quantification) in parallel with reduced spinal glial activation, IL-1ß and TNFα expression. DRG from PAE rats with neuropathy reveal significant increases in satellite glial activation and IL-1ß, while IL-10 immunoreactivity is reduced by half in PAE rats under basal and neuropathic conditions. Further, blocking spinal IL-1ß with i.t. IL-1RA transiently abolishes allodynia in PAE rats, suggesting that IL-1ß is in part, necessary for the susceptibility of adult-onset peripheral neuropathy caused by PAE. Chemokine mRNA analyses from SCN, DRG and spinal cord reveal that increased CCL2 occurs following CCI injury regardless of PAE and BIRT-377 treatment. These data demonstrate that PAE creates dysregulated proinflammatory IL-1ß and TNFα /IL-10 responses to minor injury in the sciatic-DRG-spinal pain pathway. PAE creates a risk for developing peripheral neuropathies, and LFA-1 may be a novel therapeutic target for controlling dysregulated neuroimmune actions as a consequence of PAE.

Chorioamnionitis in Rats Precipitates Extended Postnatal Inflammatory Lymphocyte Hyperreactivity.

Preterm birth is an important cause of perinatal brain injury (PBI). Neurological injury in extremely preterm infants often begins in utero with chorioamnionitis (CHORIO) or inflammation/infection of the placenta and concomitant placental insufficiency. Studies in humans have shown dysregulated inflammatory signaling throughout the placental-fetal brain axis and altered peripheral immune responses in children born preterm with cerebral palsy (CP). We hypothesized that peripheral immune responses would be altered in our well-established rat model of CP. Specifically, we proposed that isolated peripheral blood mononuclear cells (PBMCs) would be hyperresponsive to a second hit of inflammation throughout an extended postnatal time course. Pregnant Sprague-Dawley dams underwent a laparotomy on embryonic day 18 (E18) with occlusion of the uterine arteries (for 60 min) followed by intra-amniotic injection of lipopolysaccharide (LPS, 4 µg/sac) to induce injury in utero. Shams underwent laparotomy only, with equivalent duration of anesthesia. Laparotomies were then closed, and the rat pups were born at E22. PBMCs were isolated from pups on postnatal day 7 (P7) and P21, and subsequently stimulated in vitro with LPS for 3 or 24 h. A secreted inflammatory profile analysis of conditioned media was performed using multiplex electrochemiluminescent immunoassays, and the composition of inflammatory cells was assayed with flow cytometry (FC). Results indicate that CHORIO PBMCs challenged with LPS are hyperreactive and secrete significantly more tumor necrosis factor α (TNFα) and C-X-C chemokine ligand 1 at P7. FC confirmed increased intracellular TNFα in CHORIO pups at P7 following LPS stimulation, in addition to increased numbers of CD11b/c immunopositive myeloid cells. Notably, TNFα secretion was sustained until P21, with increased interleukin 6, concomitant with increased expression of integrin ß1, suggesting both sustained peripheral immune hyperreactivity and a heightened activation state. Taken together, these data indicate that in utero injury primes the immune system and augments enhanced inflammatory signaling. The insidious effects of primed peripheral immune cells may compound PBI secondary to CHORIO and/or placental insufficiency, and thereby render the brain susceptible to future chronic neurological disease. Further understanding of inflammatory mechanisms in PBI may yield clinically important biomarkers and facilitate individualized repair strategies and treatments.

Lifelong Impacts of Moderate Prenatal Alcohol Exposure on Neuroimmune Function.

In utero alcohol exposure is emerging as a major risk factor for lifelong aberrant neuroimmune function. Fetal alcohol spectrum disorder encompasses a range of behavioral and physiological sequelae that may occur throughout life and includes cognitive developmental disabilities as well as disease susceptibility related to aberrant immune and neuroimmune actions. Emerging data from clinical studies and findings from animal models support that very low to moderate levels of fetal alcohol exposure may reprogram the developing central nervous system leading to altered neuroimmune and neuroglial signaling during adulthood. In this review, we will focus on the consequences of low to moderate prenatal alcohol exposure (PAE) on neuroimmune interactions during early life and at different stages of adulthood. Data discussed here will include recent studies suggesting that while abnormal immune function is generally minimal under basal conditions, following pathogenic stimuli or trauma, significant alterations in the neuroimmune axis occur. Evidence from published reports will be discussed with a focus on observations that PAE may bias later-life peripheral immune responses toward a proinflammatory phenotype. The propensity for proinflammatory responses to challenges in adulthood may ultimately shape neuron-glial-immune processes suspected to underlie various neuropathological outcomes including chronic pain and cognitive impairment.

Aging Exacerbates Neuroinflammatory Outcomes Induced by Acute Ozone Exposure.

The role of environmental stressors, particularly exposure to air pollution, in the development of neurodegenerative disease remains underappreciated. We examined the neurological effects of acute ozone (O3) exposure in aged mice, where increased blood-brain barrier (BBB) permeability may confer vulnerability to neuroinflammatory outcomes. C57BL/6 male mice, aged 8-10 weeks or 12-18 months were exposed to either filtered air or 1.0 ppm O3 for 4 h; animals received a single IP injection of sodium fluorescein (FSCN) 20 h postexposure. One-hour post-FSCN injection, animals were transcardially perfused for immunohistochemical analysis of BBB permeability. ß-amyloid protein expression was assessed via ELISA. Flow cytometric characterization of infiltrating immune cells, including neutrophils, macrophages, and microglia populations was performed 20 h post-O3 exposure. Flow cytometry analysis of brains revealed increased microglia "activation" and presentation of CD11b, F4/80, and MHCII in aged animals relative to younger ones; these age-induced differences were potentiated by acute O3 exposure. Cortical and limbic regions in aged brains had increased reactive microgliosis and ß-amyloid protein expression after O3 insult. The aged cerebellum was particularly vulnerable to acute O3 exposure with increased populations of infiltrating neutrophils, peripheral macrophages/monocytes, and Ly6C+ inflammatory monocytes after insult, which were not significantly increased in the young cerebellum. O3 exposure increased the penetration of FSCN beyond the BBB, the infiltration of peripheral immune cells, and reactive gliosis of microglia. Thus, the aged BBB is vulnerable to insult and becomes highly penetrable in response to O3 exposure, leading to greater neuroinflammatory outcomes.

Preclinical chorioamnionitis dysregulates CXCL1/CXCR2 signaling throughout the placental-fetal-brain axis.

In the United States, perinatal brain injury (PBI) is a major cause of infant mortality and childhood disability. For a large proportion of infants with PBI, central nervous system (CNS) injury begins in utero with inflammation (chorioamnionitis/CHORIO) and/or hypoxia-ischemia. While studies show CHORIO contributes to preterm CNS injury and is also a common independent risk factor for brain injury in term infants, the molecular mechanisms mediating inflammation in the placental-fetal-brain axis that result in PBI remain a gap in knowledge. The chemokine (C-X-C motif) ligand 1 (CXCL1), and its cognate receptor, CXCR2, have been clinically implicated in CHORIO and in mature CNS injury, although their specific role in PBI pathophysiology is poorly defined. Given CXCL1/CXCR2 signaling is essential to neural cell development and neutrophil recruitment, a key pathological hallmark of CHORIO, we hypothesized CHORIO would upregulate CXCL1/CXCR2 expression in the placenta and fetal circulation, concomitant with increased CXCL1/CXCR2 signaling in the developing brain, immune cell activation, neutrophilia, and microstructural PBI. On embryonic day 18 (E18), a laparotomy was performed in pregnant Sprague Dawley rats to induce CHORIO. Specifically, uterine arteries were occluded for 60min to induce placental transient systemic hypoxia-ischemia (TSHI), followed by intra-amniotic injection of lipopolysaccharide (LPS). Pups were born at E22. Placentae, serum and brain were collected along an extended time course from E19 to postnatal day (P)15 and analyzed using multiplex electrochemiluminescence (MECI), Western blot, qPCR, flow cytometry (FC) and diffusion tensor imaging (DTI). Results demonstrate that compared to sham, CHORIO increases placental CXCL1 and CXCR2 mRNA levels, concomitant with increased CXCR2+ neutrophils. Interestingly, pup serum CXCL1 expression in CHORIO parallels this increase, with sustained elevation through P15. Analyses of CHORIO brains reveal similarly increased CXCL1/CXCR2 expression through P7, together with increased neutrophilia, microgliosis and peripheral macrophages. Similar to the placenta, cerebral neutrophilia was defined by increased CXCR2 surface expression and elevated myeloperoxidase expression (MPO), consistent with immune cell activation. Evaluation of microstructural brain injury at P15 with DTI reveals aberrant microstructural integrity in the callosal and capsular white matter, with reduced fractional anisotropy in superficial and deep layers of overlying cortex. In summary, using an established model of CHORIO that exhibits mature CNS deficits mimicking those of preterm survivors, we show CHORIO induces injury throughout the placental-fetal-brain axis with a CXCL1/CXCR2 inflammatory signature, neutrophilia, and microstructural abnormalities. These data are concomitant with abnormal cerebral CXCL1/CXCR2 expression, and support temporal aberrations in CXCL1/CXCR2 and neutrophil dynamics in the placental-fetal-brain axis following CHORIO. These investigations define novel targets for directed therapies for infants at high risk for PBI.