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- Uncovering the microglia response during neonatal Group B Streptococcus meningitisPublication . Soares, Joana; Lorga, Inês; Bravo, Joana; Summavielle, Teresa; Nova, Manuel Vila; Bonifácio Andrade, ElvaGroup B Streptococcus (GBS) remains the most common bacterial cause of meningitis in neonates. Microglia, the brain resident immune cells, have a critical role in the development of neural circuits. However, the role of GBS infection on microglia activation and neurological sequelae remains poorly characterised. Here, we aimed to evaluate whether GBS induces changes in microglia profile during the acute phase of infection, using a mouse model that mimics key steps of GBS pathophysiology in humans. Female C57BL/6 mice were intra-vaginally inoculated with GBS during gestation, and CFU analysis was performed on postnatal days (P) 1, 3 and 5. Bacterial colonisation was found at all ages, peaking at P3. When analysing the status of microglia by flow cytometry in the whole brain of male pups at P3, an overall activation was observed in the infected group. Mainly, we found a significant increase in microglia frequency, as well as the mean fluorescence intensities (MFIs) of CD45, CD11b and F4/80. Additionally, we also analysed some microglial receptors that are important neuro-immune regulators with relevant functions during development. We observed increased CX3CR1 expression in microglia, whereas Sirp and CD200r were not altered. Moreover, analysing the cortex and hippocampus, relevant regions for cognition, we found similar numbers in Iba1+ cells, a known microglia marker, in the hippocampus of infected pups. In contrast, a significant decrease was observed in the cortex, suggesting altered migration of these cells. Furthermore, microglia phagocytosis was increased in the cortex of infected pups but not in the hippocampus. Interestingly, quantification of neurons revealed a significant decrease in the hippocampus of infected pups while being increased in the cortex, compared with age-match controls. Altogether, our results show that GBS meningitis alters the neonatal microglia profile. Further studies will be necessary to better understand the microglia inflammatory state after GBS infection.
- Development of bioluminescent Group B streptococcal strains for longitudinal infection studiesPublication . Lorga, Inês; Geraldo, Rafaela; Soares, Joana; Oliveira, Liliana; Firon, Arnaud; Bonifácio Andrade, ElvaGroup B Streptococcus (GBS) remains the leading bacterial cause of invasive neonatal disease, resulting in substantial morbidity and mortality. New therapeutic approaches beyond antibacterial treatment to prevent neonatal disease outcomes are urgent. One significant limitation in studying GBS disease and progression is the lack of non-invasive technologies for longitudinal studies. Here, we develop and compare three bioluminescent GBS strains for in vivo pathogenic analysis. Bioluminescence is based on the luxABCDE operon on a replicative vector (luxGBS-CC17), and the red-shifted firefly luciferase on a replicative vector (fflucGBS-CC17) or integrated in the genome (glucGBS-CC17). We show that luxGBS-CC17 is suitable for in vitro analysis but does not produce a significant bioluminescent signal in infected pups. In contrast, the fflucGBS-CC17 results in a strong bioluminescent signal proportional to the organ colonisation level. However, the stability of the replicative vector depends on the route of infection, especially when pups acquire the bacteria from infected vaginal mucosa. Stable chromosomal integration of luciferase in glucGBS-CC17 leads to significant bioluminescence in both haematological and vertical infection models associated with high systemic colonisation. These strains will allow the preclinical evaluation of treatment efficacy against GBS invasive disease using wholemouse bioluminescence imaging.
- The impact of psychostimulants on central and peripheral neuro-immune regulation: a scoping review of cytokine profiles and their implications for addictionPublication . Bravo, Joana; Magalhães, Catarina; Andrade, Elva B.; Magalhães, Ana; Summavielle, TeresaIt is now well-accepted that psychostimulants act on glial cells causing neuroinflammation and adding to the neurotoxic effects of such substances. Neuroinflammation can be described as an inflammatory response, within the CNS, mediated through several cytokines, reactive oxygen species, chemokines and other inflammatory markers. These inflammatory players, in particular cytokines, play important roles. Several studies have demonstrated that psychostimulants impact on cytokine production and release, both centrally and at the peripheral level. Nevertheless, the available data is often contradictory. Because understanding how cytokines are modulated by psychoactive substances seems crucial to perspective successful therapeutic interventions, here, we conducted a scoping review of the available literature. We have focused on how different psychostimulants impact on the cytokine profile. Publications were grouped according to the substance addressed (methamphetamine, cocaine, methylphenidate, MDMA or other amphetamines), the type of exposure and period of evaluation (acute, short- or long-term exposure, withdrawal, and reinstatement). Studies were further divided in those addressing central cytokines, circulating (peripheral) levels, or both. Our analysis showed that the classical pro-inflammatory cytokines TNF-α, IL-6, and IL-1β were those more investigated. The majority of studies have reported increased levels of these cytokines in the central nervous system after acute or repeated drug. However, studies investigating cytokine levels during withdrawal or reinstatement have shown higher variability in their findings. Although we have identified fewer studies addressing circulating cytokines in humans, the available data suggest that the results may be more robust in animal models than in patients with problematic drug use. As a major conclusion, an extensive use of arrays for relevant cytokines should be considered to better determine which cytokines, upon the classical ones, may be involved in the progression from episodic use to the development of addiction. A concerted effort is still necessary to address the link between peripheral and central immune players, including from a longitudinal perspective. Until there, the identification of new biomarkers and therapeutic targets to envision personalized immune-based therapeutics will continue to be unlikely.
- Neuron–microglia contact-dependent mechanisms attenuate methamphetamine-induced microglia reactivity and enhance neuronal plasticityPublication . Bravo, Joana; Ribeiro, Inês; Terceiro, Ana Filipa; Andrade, Elva B.; Portugal, Camila Cabral; Lopes, Igor M.; Azevedo, Maria M.; Sousa, Mafalda; Lopes, Cátia D. F.; Lobo, Andrea C.; Canedo, Teresa; Relvas, João Bettencourt; Summavielle, TeresaExposure to methamphetamine (Meth) has been classically associated with damage to neuronal terminals. However, it is now becoming clear that addiction may also result from the interplay between glial cells and neurons. Recently, we demonstrated that binge Meth administration promotes microgliosis and microglia pro-inflammation via astrocytic glutamate release in a TNF/IP3R2-Ca2+-dependent manner. Here, we investigated the contribution of neuronal cells to this process. As the crosstalk between microglia and neurons may occur by contact-dependent and/or contact-independent mechanisms, we developed co-cultures of primary neurons and microglia in microfluidic devices to investigate how their interaction affects Meth-induced microglia activation. Our results show that neurons exposed to Meth do not activate microglia in a cell-autonomous way but require astrocyte mediation. Importantly, we found that neurons can partially prevent Meth-induced microglia activation via astrocytes, which seems to be achieved by increasing arginase 1 expression and strengthening the CD200/CD200r pathway. We also observed an increase in synaptic individual area, as determined by co-localization of pre- and post-synaptic markers. The present study provides evidence that contact-dependent mechanisms between neurons and microglia can attenuate pro-inflammatory events such as Meth-induced microglia activation.
- A mouse model reproducing the pathophysiology of neonatal group B streptococcal infectionPublication . Bonifácio Andrade, Elva; Magalhães, Ana; Puga, Ana; Costa, Madalena; Bravo, Joana; Portugal, Camila Cabral; Ribeiro, Adília; Correia-Neves, Margarida; Faustino, Augusto; Firon, Arnaud; Trieu-Cuot, Patrick; Summavielle, Teresa; Ferreira, PaulaGroup B streptococcal (GBS) meningitis remains a devastating disease. The absence of an animal model reproducing the natural infectious process has limited our understanding of the disease and, consequently, delayed the development of effective treatments. We describe here a mouse model in which bacteria are transmitted to the offspring from vaginally colonised pregnant females, the natural route of infection. We show that GBS strain BM110, belonging to the CC17 clonal complex, is more virulent in this vertical transmission model than the isogenic mutant BM110∆cylE, which is deprived of hemolysin/cytolysin. Pups exposed to the more virulent strain exhibit higher mortality rates and lung inflammation than those exposed to the attenuated strain. Moreover, pups that survive to BM110 infection present neurological developmental disability, revealed by impaired learning performance and memory in adulthood. The use of this new mouse model, that reproduces key steps of GBS infection in newborns, will promote a better understanding of the physiopathology of GBS-induced meningitis.
- Regulation of CD47 expression by interferon-gamma under chronic Methamphetamine exposure TitlePublication . Rodrigues, João; Bravo, Joana; Bonifácio Andrade, Elva; Canedo, Teresa; Azevedo, Maria; Summavielle, TeresaExposure to methamphetamine (Meth), a highly addictive widely used psychostimulant, is classically associated with damage to neuronal terminals, but its neurotoxicity can also be mediated via activation of the neuroinflammatory response. Microglia, the resident immune cells of the brain, become highly activated and increase the release of proinflammatory mediators upon exposure to Meth. However, their role in Meth-associated neurotoxicity is still not sufficiently understood. Data from our lab shows that, in the hippocampus, chronic Meth administration leads to microglia homeostasis dysregulation, synapse dysregulation, and downregulation of cluster-differentiation 47 protein (CD47). The crosstalk between CD47 and its receptor, signal regulatory protein α (SIRPα), is an important “don’t eat me signal” that inhibits phagocytosis. CD47 has been shown to protect synapses from excessive microglia-mediated pruning during development and neurodegeneration. Of note, in cancer cells CD47 expression is modulated by interferon-gamma (IFN-γ). Consistently, after chronic Meth, we observed a significant decrease of meningeal T cells, and a decrease in the production of IFN-y by these cells. Here we aim to clarify if IFNγ is regulating CD47 in the brain after chronic Meth administration, and consequently regulating synaptic pruning, using IFNyKO mice and wild-type mice injected with recombinant IFNy via stereotaxic surgery. Preliminary results indicate that IFNy/CD47 does not modulate microglia morphology and number after chronic Meth in the hippocampus. Currently we are evaluating synapses and phagocytosis, and we further expect to clarify the impact of IFNγ /CD47 in the chronic Meth conditioning and in memory.