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.

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.

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.

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.

Prenatal alcohol exposure is a risk factor for adult neuropathic pain via aberrant neuroimmune function.

BACKGROUND: Clinical studies show that prenatal alcohol exposure (PAE) results in effects that persist into adulthood. Experimental animal models of moderate PAE demonstrate that young adults with PAE display potentiated sensitivity to light touch, clinically termed allodynia, following sciatic nerve chronic constriction injury (CCI) that coincides with heightened spinal glial, spinal macrophage, and peripheral immune responses. However, basal touch sensitivity and corresponding glial and leukocyte activation are unaltered. Therefore, the current study explored whether the enduring pathological consequences of moderate PAE on sensory processing are unmasked only following secondary neural insult. METHODS: In middle-aged (1 year) Long Evans rats that underwent either prenatal saccharin exposure (control) or moderate PAE, we modified the well-characterized model of sciatic neuropathy, CCI, to study the effects of PAE on neuro-immune responses in adult offspring. Standard CCI manipulation required 4 chromic gut sutures, while a mild version applied a single suture loosely ligated around one sciatic nerve. Spinal glial immunoreactivity was examined using immunohistochemistry. The characterization and functional responses of leukocyte populations were studied using flow cytometry and cell stimulation assays followed by quantification of the proinflammatory cytokines interleukin-1beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α). Data were statistically analyzed by ANOVA and unpaired t tests. RESULTS: The current report demonstrates that mild CCI generates robust allodynia only in PAE rats, while the pathological effects of PAE following the application of a standard CCI are revealed by enhanced allodynia and elevated spinal glial activation. Additionally, mild CCI increases spinal astrocyte activation but not microglia, suggesting astrocytes play a larger role in PAE-induced susceptibility to aberrant sensory processing. Leukocyte populations from PAE are altered under basal conditions (i.e., prior to secondary insult), as the distribution of leukocyte populations in lymphoid organs and other regions are different from those of controls. Lastly, following in vitro leukocyte stimulation, only PAE augments the immune response to antigen stimulation as assessed by heightened production of TNF-α and IL-1ß. CONCLUSIONS: These studies demonstrate PAE may prime spinal astrocytes and peripheral leukocytes that contribute to enduring susceptibility to adult-onset neuropathic pain that is not apparent until a secondary insult later in life.

Prenatal alcohol exposure potentiates chronic neuropathic pain, spinal glial and immune cell activation and alters sciatic nerve and DRG cytokine levels.

A growing body of evidence indicates that prenatal alcohol exposure (PAE) may predispose individuals to secondary medical disabilities later in life. Animal models of PAE reveal neuroimmune sequelae such as elevated brain astrocyte and microglial activation with corresponding region-specific changes in immune signaling molecules such as cytokines and chemokines. The aim of this study was to evaluate the effects of moderate PAE on the development and maintenance of allodynia induced by chronic constriction injury (CCI) of the sciatic nerve in adult male rat offspring. Because CCI allodynia requires the actions of glial cytokines, we analyzed lumbar spinal cord glial and immune cell surface markers indicative of their activation levels, as well as sciatic nerve and dorsal root ganglia (DRG) cytokines in PAE offspring in adulthood. While PAE did not alter basal sensory thresholds before or after sham manipulations, PAE significantly potentiated adult onset and maintenance of allodynia. Microscopic analysis revealed exaggerated astrocyte and microglial activation, while flow cytometry data demonstrated increased proportions of immune cells with cell surface major histocompatibility complex II (MHCII) and ß-integrin adhesion molecules, which are indicative of PAE-induced immune cell activation. Sciatic nerves from CCI rats revealed that PAE potentiated the proinflammatory cytokines interleukin (IL)-1ß, IL-6 and tumor necrosis factor-alpha (TNFα) protein levels with a simultaneous robust suppression of the anti-inflammatory cytokine, IL-10. A profound reduction in IL-10 expression in the DRG of PAE neuropathic rats was also observed. Taken together, our results provide novel insights into the vulnerability that PAE produces for adult-onset central nervous system (CNS) pathological conditions from peripheral nerve injury.

Chronic Sciatic Neuropathy in Rat Reduces Voluntary Wheel-Running Activity With Concurrent Chronic Mechanical Allodynia.

BACKGROUND: Animal models of peripheral neuropathy produced by a number of manipulations are assessed for the presence of pathologic pain states such as allodynia. Although stimulus-induced behavioral assays are frequently used and important to examine allodynia (ie, sensitivity to light mechanical touch; von Frey fiber test), other measures of behavior that reflect overall function are not only complementary to stimulus-induced responsive measures, but are also critical to gain a complete understanding of the effects of the pain model on quality of life, a clinically relevant aspect of pain on general function. Voluntary wheel-running activity in rodent models of inflammatory and muscle pain is emerging as a reliable index of general function that extends beyond stimulus-induced behavioral assays. Clinically, reports of increased pain intensity occur at night, a period typically characterized with reduced activity during the diurnal cycle. We therefore examined in rats whether alterations in wheel-running activity were more robust during the inactive phase compared with the active phase of their diurnal cycle in a widely used rodent model of chronic peripheral neuropathic pain, the sciatic nerve chronic constriction injury (CCI) model. METHODS: In adult male Sprague Dawley rats, baseline (BL) hindpaw threshold responses to light mechanical touch were assessed using the von Frey test before measuring BL activity levels using freely accessible running wheels (1 hour/day for 7 sequential days) to quantify the distance traveled. Running wheel activity BL values are expressed as total distance traveled (m). The overall experimental design was after BL measures, rats underwent either sham or CCI surgery followed by repeated behavioral reassessment of hindpaw thresholds and wheel-running activity levels for up to 18 days after surgery. Specifically, separate groups of rats were assessed for wheel-running activity levels (1 hour total/trial) during the onset (within first 2 hours) of either the (1) inactive (n = 8/group) or (2) active (n = 8/group) phase of the diurnal cycle. An additional group of CCI-treated rats (n = 8/group) was exposed to a locked running wheel to control for the potential effects of wheel-running exercise on allodynia. The 1-hour running wheel trial period was further examined at discrete 20-minute intervals to identify possible pattern differences in activity during the first, middle, and last portions of the 1-hour trial. The effect of neuropathy on activity levels was assessed by measuring the change from their respective BLs to distance traveled in the running wheels. RESULTS: Although wheel-running distances between groups were not different at BL from rats examined during either the inactive phase of the diurnal cycle or active phase of the diurnal cycle, sciatic nerve CCI reduced running wheel activity levels compared with sham-operated controls during the inactive phase. In addition, compared with sham controls, bilateral low-threshold mechanical allodynia was observed at all time points after surgical induction of neuropathy in rats with free-wheel and locked-wheel access. Allodynia in CCI compared with shams was replicated in rats whose running wheel activity was examined during the active phase of the diurnal cycle. Conversely, no significant reduction in wheel-running activity was observed in CCI-treated rats compared with sham controls at any time point when activity levels were examined during the active diurnal phase. Finally, running wheel activity patterns within the 1-hour trial period during the inactive phase of the diurnal cycle were relatively consistent throughout each 20-minute phase. CONCLUSIONS: Compared with nonneuropathic sham controls, a profound and stable reduction of running wheel activity was observed in CCI rats during the inactive phase of the diurnal cycle. A concurrent robust allodynia persisted in all rats regardless of when wheel-running activity was examined or whether they ran on wheels, suggesting that acute wheel-running activity does not alter chronic low-intensity mechanical allodynia as measured using the von Frey fiber test. Overall, these data support that acute wheel-running exercise with limited repeated exposures does not itself alter allodynia and offers a behavioral assay complementary to stimulus-induced measures of neuropathic pain.

Chitinase dependent control of protozoan cyst burden in the brain.

Chronic infections represent a continuous battle between the host's immune system and pathogen replication. Many protozoan parasites have evolved a cyst lifecycle stage that provides it with increased protection from environmental degradation as well as endogenous host mechanisms of attack. In the case of Toxoplasma gondii, these cysts are predominantly found in the immune protected brain making clearance of the parasite more difficult and resulting in a lifelong infection. Currently, little is known about the nature of the immune response stimulated by the presence of these cysts or how they are able to propagate. Here we establish a novel chitinase-dependent mechanism of cyst control in the infected brain. Despite a dominant Th1 immune response during Toxoplasma infection there exists a population of alternatively activated macrophages (AAMØ) in the infected CNS. These cells are capable of cyst lysis via the production of AMCase as revealed by live imaging, and this chitinase is necessary for protective immunity within the CNS. These data demonstrate chitinase activity in the brain in response to a protozoan pathogen and provide a novel mechanism to facilitate cyst clearance during chronic infections.

CCR7-dependent immunity during acute Toxoplasma gondii infection.

The chemokine receptor CCR7 is a well-established homing receptor for dendritic cells and T cells. Interactions with its ligands, CCL19 and CCL21, facilitate priming of immune responses in lymphoid tissue, yet CCR7-independent immune responses can be generated in the presence of sufficient antigen. In these studies, we investigated the role of CCR7 signaling in the generation of protective immune responses to the intracellular protozoan parasite Toxoplasma gondii. The results demonstrated a significant increase in the expression of CCL19, CCL21, and CCR7 in peripheral and central nervous system (CNS) tissues over the course of infection. Unexpectedly, despite the presence of abundant antigen, CCR7 was an absolute requirement for protective immunity to T. gondii, as CCR7(-/-) mice succumbed to the parasite early in the acute phase of infection. Although serum levels of interleukin 12 (IL-12), IL-6, tumor necrosis factor alpha (TNF-alpha), and IL-10 remained unchanged, there was a significant decrease in CCL2/monocyte chemoattractant protein 1 (MCP-1) and inflammatory monocyte recruitment to the site of infection. In addition, CCR7(-/-) mice failed to produce sufficient gamma interferon (IFN-gamma), a critical Th1-associated effector cytokine required to control parasite replication. As a result, there was increased parasite dissemination and a significant increase in parasite burden in the lungs, livers, and brains of infected mice. Adoptive-transfer experiments revealed that expression of CCR7 on the T-cell compartment alone is sufficient to enable T-cell priming, increase IFN-gamma production, and allow the survival of CCR7(-/-) mice. These data demonstrate an absolute requirement for T-cell expression of CCR7 for the generation of protective immune responses to Toxoplasma infection.