Distinct trans-placental effects of maternal immune activation by TLR3 and TLR7 agonists: implications for schizophrenia risk

Exposure to infection in utero predisposes towards psychiatric diseases such as autism, depression and schizophrenia in later life. The mechanisms involved are typically studied by administering mimetics of double-stranded (ds) virus or bacterial infection to pregnant rats or mice. The effect of single-stranded (ss) virus mimetics has been largely ignored, despite evidence linking prenatal ss virus exposure with psychiatric disease. Understanding the effects of gestational ss virus exposure has become even more important with recent events. In this study, in pregnant mice, we compare directly the effects, on the maternal blood, placenta and the embryonic brain, of maternal administration of ds-virus mimetic poly I:C (to activate Toll-like receptor 3, TLR3) and ss-virus mimetic resiquimod (to activate TLR7/8). We find that, 4 h after the administration, both poly I:C and resiquimod elevated the levels of IL-6, TNFα, and chemokines including CCL2 and CCL5, in maternal plasma. Both agents also increased placental mRNA levels of IL-6 and IL-10, but only resiquimod increased placental TNFα mRNA. In foetal brain, poly I:C produced no detectable immune-response-related increases, whereas pronounced increases in cytokine (e.g. Il-6, Tnfα) and chemokine (e.g. Ccl2, Ccl5) expression were observed with maternal resiquimod administration. The data show substantial differences between the effect of maternal exposure to a TLR7/8 activator as compared to a TLR3 activator. There are significant implications for future modelling of diseases where maternal ss virus exposure contributes to environmental disease risk in offspring

Neuroinflammation: exploring brain cell responses and leukocyte infiltration

In recent years, the relationship between inflammation and psychiatric disorders has gained substantial research interest. Multiple studies have demonstrated that psychiatric disorders are associated with a dysregulation of the immune response, and changes in inflammatory factors are being explored as possible disease biomarkers (Gibney and Drexhage, 2013, Yuan et al., 2019). The Cavanagh group has studied the influence of peripheral inflammation on central nervous system (CNS) responses and behaviour for a number of years using animal models including the Aldara model of psoriasis-like skin inflammation. The active ingredient, imiquimod (IMQ), is a synthetic toll-like receptor-7 (TLR-7) agonist. Topical application of Aldara triggers a strong pro-inflammatory response in the periphery and the brain characterised by increased cytokine and chemokine transcription, immune cell entry into the brain and a decrease in motivated behaviour (McColl et al., 2016, Nerurkar et al., 2017). After initially regarding this as a model of peripheral inflammation, mass spectrometry analysis revealed that IMQ enters the brain rapidly after topical application, raising questions about whether this stimulus can act on neural cells directly (Nerurkar et al., 2017). This thesis explores whether neural cells can respond to IMQ directly and how immune cells enter the brain parenchyma in response to Aldara treatment. To investigate the reaction of neural cells to IMQ, we treated newly optimised primary mouse brain cell cultures in vitro. Results showed that primary brain cells (neurons, microglia, astrocytes and oligodendrocytes) upregulate the same pro-inflammatory cytokines and chemokines involved in the IMQ response in vivo. We also examined the cellular source of some of these factors and found astrocytes and oligodendrocytes to be majorly involved in their production. As interferons (IFNs) are the main mediators of the TLR-7 response, we also examined the influence of type I and type II IFNs on brain cells and found that they too trigger chemokine and cytokine transcription, albeit to different extents. The second part of this thesis focuses on leukocyte entry into the CNS within the Aldara model. Tracer dye experiments showed mild signs of blood-brain barrier (BBB) integrity loss, although genes involved in upholding BBB integrity were not downregulated. Ifnar1 knock out (KO) experiments highlighted the importance of type I IFNs (IFN-α and IFN-β) for T cell and monocyte recruitment following Aldara treatment. Inflammatory chemokine receptor (iCCR) KOs revealed iCCR importance in monocyte recruitment to the CNS, with CCR2 being the main iCCR expressed on infiltrating monocytes. Notably, both KO experiments showed a small monocyte population that continued to enter the CNS, despite the absence of either Ifnar1 or iCCRs. Overall, this thesis demonstrates that IMQ can act on neural cells directly and trigger a strong pro-inflammatory response in the absence of peripheral immune cell influence. In vivo, Aldara treatment causes mild BBB disruption and immune cell recruitment is reliant on type I IFNs as well as iCCRs, albeit to different extents across different leukocyte subsets. Further experiments are needed to understand immune cell entry mechanisms and examine the cellular sources of inflammatory mediators in the CNS. Overall, the Aldara model produces symptoms of mild encephalitis which makes it an easy, well controlled alternative to virus-based encephalitis models

Stress, immune system and the brain

Stress encompasses the psychological perception of pressure from the environment, and the body’s physiological response to it. The sources of stress have evolved over time, from predation and natural disasters, to things like interpersonal conflicts and economic insecurities. While in the past, stressors evoked a very acute physical ‘fight or flight’ response, these events are rare in today’s terms. In contrast, the stressors we experience in the modern world are arguably more trivial – they are not often immediately life threatening – however they are more persistent, manifesting as a chronic, low level source of anxiety in our daily lives. The natural stress response involves multiple systems and is designed to provide short-term beneficial effects to the individual to help see them through a threatening situation. It is thought this response is mediated largely through glucocorticoid (GC) production and will rapidly normalize following the stressful event. In the event of chronic exposure to stress, some of these short-term physiological changes fail to return to ‘normality’, and as a result, the nature of our homeostasis is altered. This chapter will focus on the changes to the immune system and brain mediated through exposure to stress, with particular emphasis on the detrimental effects of chronic stress

Differential effects of toll-like receptor activation and differential mediation by MAP kinases of immune responses in microglial cells

Microglial activation is believed to play a role in many psychiatric and neurodegenerative diseases. Based largely on evidence from other cell types, it is widely thought that MAP kinase (ERK, JNK and p38) signalling pathways contribute strongly to microglial activation following immune stimuli acting on toll-like receptor (TLR) 3 or TLR4. We report here that exposure of SimA9 mouse microglial cell line to immune mimetics stimulating TLR4 (lipopolysaccharide—LPS) or TLR7/8 (resiquimod/R848), results in marked MAP kinase activation, followed by induction of nitric oxide synthase, and various cytokines/chemokines. However, in contrast to TLR4 or TLR7/8 stimulation, very few effects of TLR3 stimulation by poly-inosine/cytidine (polyI:C) were detected. Induction of chemokines/cytokines at the mRNA level by LPS and resiquimod were, in general, only marginally affected by MAP kinase inhibition, and expression of TNF, Ccl2 and Ccl5 mRNAs, along with nitrite production, were enhanced by p38 inhibition in a stimulus-specific manner. Selective JNK inhibition enhanced Ccl2 and Ccl5 release. Many distinct responses to stimulation of TLR4 and TLR7 were observed, with JNK mediating TNF protein induction by the latter but not the former, and suppressing Ccl5 release by the former but not the latter. These data reveal complex modulation by MAP kinases of microglial responses to immune challenge, including a dampening of some responses. They demonstrate that abnormal levels of JNK or p38 signalling in microglial cells will perturb their profile of cytokine and chemokine release, potentially contributing to abnormal inflammatory patterns in CNS disease states

Dissociation between iron accumulation and ferritin upregulation in the aged substantia nigra:Attenuation by dietary restriction

Despite regulation, brain iron increases with aging and may enhance aging processes including neuroinflammation. Increases in magnetic resonance imaging transverse relaxation rates, R2 and R2*, in the brain have been observed during aging. We show R2 and R2* correlate well with iron content via direct correlation to semi-quantitative synchrotron-based X-ray fluorescence iron mapping, with age-associated R2 and R2* increases reflecting iron accumulation. Iron accumulation was concomitant with increased ferritin immunoreactivity in basal ganglia regions except in the substantia nigra (SN). The unexpected dissociation of iron accumulation from ferritin-upregulation in the SN suggests iron dyshomeostasis in the SN. Occurring alongside microgliosis and astrogliosis, iron dyshomeotasis may contribute to the particular vulnerability of the SN. Dietary restriction (DR) has long been touted to ameliorate brain aging and we show DR attenuated age-related in vivo R2 increases in the SN over ages 7 – 19 months, concomitant with normal iron-induction of ferritin expression and decreased microgliosis. Iron is known to induce microgliosis and conversely, microgliosis can induce iron accumulation, which of these may be the initial pathological aging event warrants further investigation. We suggest iron chelation therapies and anti-inflammatory treatments may be putative ‘anti-brain aging’ therapies and combining these strategies may be synergistic

Establishing mixed neuronal and glial cell cultures from embryonic mouse brains to study infection and innate immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods