Correlation between IL1β expression level and morphological parameters proves the usefulness of morphology measures to predict the degree of activation of microglial cells

It is well known that microglial cells undergo an important change in morphology upon activation, so that form and function are intimately related. Upon activation, microglia cell body enlarges, its ramifications shortens and become thicker. In parallel, a variety of cytokines and inflammatory mediators such as IL1β are released. However the activation process is not all-or-nothing. Rather, cells in subtle activation states or in a deactivation process can occur, so intermediate not obvious phenotypes may appear. Thus, we aimed to correlate the expression level of a well-defined marker of activation, IL1β, with different morphological parameters. To do so, we used an intracerebroventricular injection of neuraminidase to produce an acute inflammation in rats. Brain sections were double-stained with IBA1 to have an image of the whole cell and its ramifications, and with IL1β to assess the level of activation. Images were captured from septofimbria (close to the injection site) and from the hypothalamus. A ratio of IL1β-positive pixels to IBA1-positive pixels was used to estimate the level of IL1β expression for each cell. Single microglial cell images were processed with ImageJ software to obtain outlined and filled shapes, which were used to obtain (by means of FracLac plug in) the following morphological parameters: fractal dimension, lacunarity, area, density and perimeter. All parameters showed a significant correlation with the level of expression of IL1β. This occurred in cells sampled from the two brain areas studied. Density, lacunarity and perimeter resulted as the best predictor parameters of activation, that is, those with a better correlation with the level of expression of IL1β. Area, an extensively used parameter to assess microglial activation, presented the least significant correlation. Thus, objectively measured morphological parameters correlate with the level of expression of IL1β, and could therefore be used as predictors of the activation level of microglial cells.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

FGF2, but not EGF, Induces multiciliated ependymal cells to dedifferentiate and adopt radial glial features in vitro

Multiciliated ependymal cells form an epithelium lining most of the ventricular cavities of vertebrates brain. Although considered postmitotic and completely differentiated, ependymal cells maintain some phenotypic characteristics of neural stem cells. Thereby, under specific conditions they behave as neural stem cells, developing radial glia characteristics, and undergoing asymmetric division. Our group is searching for factors that promote dedifferentiation of ependymal cells in vitro. We developed a simple method to obtain pure cultures of non-adherent multiciliated ependymal cells from adult rats. These cultures were used to investigate the effect of FGF2 on the differentiation state and the aggregation of ependymal cells. Thus, FGF2 treated ependymal cells lose cilia and hence mobility, and after 7 days they aggregate to form irregular spheres (diameter ≥ 20-30 μm). Such changes were not observed when EFG was used instead of FGF2. To assess the specificity of FGF2 action on cell aggregation, the FGF receptor inhibitor PD166866 and an anti-FGF2 neutralizing antibody were used. In both conditions the aggregative effect of FGF2 was abolished. No cell proliferation was observed during sphere formation, at least in such experimental conditions. Spheres were analyzed by immunocytochemistry using radial glia markers. They were positive for GFAP, vimentin, BLBP and GLAST. These data suggest that FGF2 promotes the identity loss in multiciliated ependymal cells in vitro, which are transformed into cells with radial glia features.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Neuraminidase-activated microglia compromise the viability of ependymocytes

Neuraminidase (NA) is a sialidase present in the envelope/wall of some virus/bacteria responsible for brain infections, such as flu, mumps or meningitis. The intracerebroventricular injection of NA in the rat brain provokes ependymal detachment and death, and an acute inflammatory process. Although inflammation reverses, ependymal lining is not regenerated. Complement system activation within the CSF contributes to ependymal damage, but is not the only cause (Granados-Duran et al, 2016). Here we aimed to investigate if microglial activation might also play a role. For this purpose we used pure isolated ependymocytes (Grondona et al, 2013) and ventricular wall explants, which were co-cultured with microglial cells, both in basal conditions and with agents that induce microglial activation: NA, LPS, or Pam3CSK4 (synthetic lipopeptide). The viability of the ependymal cells was assessed by trypan blue exclusion. The viability of isolated ependymocytes was reduced when NA or LPS were added to the culture, compared to controls without additives. In the absence of microglia, NA or LPS did not compromise viability significantly, indicating that microglia was involved in ependymocytes death. The addition of NA to cultured explants reduced ependymocytes viability only when microglial cells were present in the culture; a similar reduction was observed when LPS or Pam3CSK4 were added. Conversely, explants cultured in the absence of microglia did not suffer a significant decrease in ependymocytes viability upon NA addition to the medium. We hypothesized that cytokines released by activated microglia, such as IL1β or TNFα, could mediate ependymocytes death. RT-PCR performed in RNA obtained from pure ependymocytes confirmed the presence of IL1β and TNFα receptors in ependymal cells. Nevertheless further experiments are required to confirm this hypothesis. We conclude that microglia activated by NA mediates, at least in part, ependymal cell death, what might be relevant for neuroinflammatory diseases mediated by NA bearing virus/bacteria.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Neuraminidase-induced neuroinflammation is largely dependent on microglial TLR4 receptor

The sialidase neuraminidase (NA) cleaves terminal sialic acid from glycoproteins and glycolipids. Among its various locations, it is present in the envelope/membrane of some bacteria/viruses (e.g. influenza virus), where it is involved in infectiveness and dispersion. The injection of NA within the brain lateral ventricle represents a model of acute sterile inflammation. The relevance of the toll-like receptors TLR2 and TLR4 (particularly those in microglial cells) in such process was investigated using mouse strains deficient in these receptors. In septofimbria and hypothalamus, IBA1-positive and IL-1β-positive cell counts increased after NA injection in wild type (WT) mice. In TLR4-/- mice such increases were largely abolished, while only slightly affected in TLR2-/- mice. Similarly, the NA-induced expression of IL-1β, TNFα and IL-6 (evaluated by qPCR) was completely blocked in TLR4-/- mice, and only partially reduced in TLR2-/- mice. Microglia was isolated from the three mouse strains and exposed to NA or to specific TLR2 and TLR4 agonists (Pam3CSK4 and LPS respectively) in vitro. NA induced a cytokine response (IL-1β, TNFα and IL-6) in WT microglia, but was unable to do so in TLR4-/- microglia; TLR2 deficiency partially affected the NA-induced microglia response. To investigate if such response of microglial cells to NA was dependent on the sialidase activity of the enzyme, WT microglia was exposed in vitro to NA previously inactivated with heat, or inhibited with two different sialidase inhibitors (oseltamivir phosphate and N-acetyl-2,3-dehydro-2-deoxyneuraminic acid). In all cases, NA- induced microglia activation was dependent on the intact sialidase activity of NA. Therefore, we conclude that NA is able to directly activate microglial cells, mostly through TLR4 receptor and due to its sialidase activity. Accordingly, the inflammatory reaction induced by NA in vivo is partially dependent on TLR2, while TLR4 plays a crucial role.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Long-term priming of hypothalamic microglia is associated with energy balance disturbances under diet-induced obesity

Exposure of microglia to an inflammatory environment may lead to their priming and exacerbated response to future inflammatory stimuli. Here we aimed to explore hypothalamic microglia priming and its consequences on energy balance regulation. A model of intracerebroventricular administration of neuraminidase (NA, which is present in various pathogens such as influenza virus) was used to induce acute neuroinflammation. Evidences of primed microglia were observed 3 months after NA injection, namely (1) a heightened response of microglia located in the hypothalamic arcuate nucleus after an in vivo inflammatory challenge (high fat diet [HFD] feeding for 10 days), and (2) an enhanced response of microglia isolated from NA-treated mice and challenged in vitro to LPS. On the other hand, the consequences of a previous NA-induced neuroinflammation were further evaluated in an alternative inflammatory and hypercaloric scenario, such as the obesity generated by continued HDF feeding. Compared with sham-injected mice, NA-treated mice showed increased food intake and, surprisingly, reduced body weight. Besides, NA-treated mice had enhanced microgliosis (evidenced by increased number and reactive morphology of microglia) and a reduced population of POMC neurons in the basal hypothalamus. Thus, a single acute neuroinflammatory event may elicit a sustained state of priming in microglial cells, and in particular those located in the hypothalamus, with consequences in hypothalamic cytoarchitecture and its regulatory function upon nutritional challenges.Ministerio de Economía, Industria yCompetitividad, Gobierno de España, Grant/Award Number: SAF2017-83645; Ministeriode Educación y Formación Profesional; Funding for open access charge: Universidadde Málaga/CBU

Morphological traits of surveillant/activated microglia during an experimentally induced acute neuroinflammatory process

Poster en CongresoSeveral studies show that morphological changes of microglia over the course of inflammation are tightly coupled to function. However the progressive transformation into activated microglia is poorly characterized. AIMS: This study aimed to establish a spatiotemporal correlation between quantifiable morphological parameters of microglia and the spread of an acute ventricular inflammatory process. METHODS: Inflammation was induced by a single injection of the enzyme neuraminidase within the lateral ventricle of rats. Animals were sacrificed 2, 4 and 12 hours after injection. Coronal slices were immunostained with Iba1 to label microglia and with IL1β to delimit the spread of inflammation. Digital images were obtained by scanning the labelled sections. Single microglia images were randomly selected from periventricular areas of caudate putamen, hippocampus and hypothalamus. FracLac for ImageJ software was used to measure the following morphological parameters: fractal dimension, lacunarity, area, perimeter and density. RESULTS: Significant differences were found in fractal dimension, lacunarity, perimeter and density of microglia cells of neuraminidase injected rats compared to sham animals. However no differences were found in the parameter “area”. In hipoccampus there was a delay in the significant change of the measured parameters. These morphological changes correlated with IL1β-expression in the same areas. CONCLUSIONS: Ventricular inflammation induced by neuraminidase provokes quantifiable morphological changes in microglia restricted to areas labelled with IL1β. Morphological parameters of microglia such as fractal dimension, lacunarity, perimeter and density are sensitive and valuable tools to quantify activation. However, the extensively used parameter “area” did not change upon microglia activation.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Grant: Junta de Andalucía (Spain) P11-CVI-07637 Ibima, Andalucía Tech, UM

Partial inhibition of CSF1R signaling reverses long-term microglial priming.

Microglial cells are main actors in acute neuroinflammation, during which they activate to later return to a basal resting state. Sometimes they retain immune memory of previous neuroinflammatory events, turning into primed microglia, which develop exacerbated responses to new stimuli. Brain can be depleted of microglia by treatment with the CSF1R inhibitor PLX5622. Treatment termination allows for microglia regeneration, new cells presenting a resting state. Here we aimed to explore if treatment with lower doses of PLX5622 can reverse microglial priming. We induced microglial priming in mice by provoking acute neuroinflammation by icv administration of neuraminidase. After 3 weeks, when neuroinflammation is largely solved, mice were treated with a daily dose of PLX5622 for 12 days. Then, microglial repopulation was allowed for 7 weeks. Finally, a second stimulus was applied (ip LPS) to induce inflammatory activation of primed microglia, and animals were sacrificed 12 hours later. Brains were collected to analyze microglial cell number and activation by morphological analysis, and expression level of key genes by qPCR; these parameters were evaluated in two regions: the periventricular area of the hypothalamus and the hippocampus. In hypothalamic paraventricular nucleus the number of microglial cells was the same regardless the treatment; however, it was slightly reduced in the dentate gyrus of the hippocampus of PLX5622 treated mice. Morphological analysis of microglial cells was carried out by fractal, sholl and skeleton analysis. All of them pointed that microglia sampled from NA injected mice had a more activated profile (less ramified cells), which was reversed by PLX5622 treatment. Besides, expression of pro-inflammatory related genes (IL1β, IL6, TNFα, NLRP3, TLR4) pointed to the same direction. Thus, our results suggest that PLX5622 used at low doses reverses microglial priming, while does not fully deplete microglial population.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Microbial neuraminidase induces TLR4-dependent long-term immune priming in the brain

Innate immune memory explains the plasticity of immune responses after repeated immune stimulation, leading to either enhanced or suppressed immune responses. This process has been extensively reported in peripheral immune cells and also, although modestly, in the brain. Here we explored two relevant aspects of brain immune priming: its persistence over time and its dependence on TLR receptors. For this purpose, we used an experimental paradigm consisting in applying two inflammatory stimuli three months apart. Wild type, toll-like receptor (TLR) 4 and TLR2 mutant strains were used. The priming stimulus was the intracerebroventricular injection of neuraminidase (an enzyme that is present in various pathogens able to provoke brain infections), which triggers an acute inflammatory process in the brain. The second stimulus was the intraperitoneal injection of lipopolysaccharide (a TLR4 ligand) or Pam3CSK4 (a TLR2 ligand). One day after the second inflammatory challenge the immune response in the brain was examined. In wild type mice, microglial and astroglial density, as well as the expression of 4 out of 5 pro-inflammatory genes studied (TNFα, IL1β, Gal-3, and NLRP3), were increased in mice that received the double stimulus compared to those exposed only to the second one, which were initially injected with saline instead of neuraminidase. Such enhanced response suggests immune training in the brain, which lasts at least 3 months. On the other hand, TLR2 mutants under the same experimental design displayed an enhanced immune response quite similar to that of wild type mice. However, in TLR4 mutant mice the response after the second immune challenge was largely dampened, indicating the pivotal role of this receptor in the establishment of immune priming. Our results demonstrate that neuraminidase-induced inflammation primes an enhanced immune response in the brain to a subsequent immune challenge, immune training that endures and that is largely dependent on TLR4 receptor

Primed microglia after acute neuroinflammation may drive an enhanced stress response.

Microglial cells become activated during acute neuroinflammation and usually they return to their basal surveillant state in a few days. However, sometimes microglia evolve towards a primed state characterized by an exacerbated response to new stimuli, which may jeopardize brain functions. Here we aimed to explore microglial priming in the hypothalamus and its consequences on the neuroendocrine regulation of the stress response. To induce priming we used a model of acute ventricular neuroinflammation by intracerebroventricular (ICV) injection of the enzyme neuraminidase (NA). Three months later, an acute stressor (consisting in forced swimming) was applied to investigate the activation of the hypothalamic-pituitary-adrenal axis and the stress response elicited, as well as the inflammatory activation of hypothalamic microglial cells. Stressed rats previously injected with NA had increased plasma levels of corticosterone compared to control rats that were equally stressed but had been ICV injected with saline. Also, qPCR studies revealed that NA-treated rats presented an increased expression of the microglial marker IBA1 and of the inflammasome protein NLRP3. Concomitantly, the morphological analysis of hypothalamic microglial cells showed a morphological bias towards a slightly activated state in microglia of NA injected rats compared to those of saline injected controls. Furthermore, in the open field test NA-treated rats displayed increased locomotor activity. These results suggest that prior neuroinflammatory episodes might result in subtle but persistent changes in microglial cells that could determine the response to future challenges such as stressful events.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Neuraminidase-induced neuroinflammation causes anxiety and microgliosis in the amygdala

An intracerebroventricular (ICV) injection of neuraminidase (NA) within the lateral ventricles originates an acute event of neuroinflammation, which is solved to a great extent after two weeks. Recently, neurological problems or behavioral alterations have been associated with neuroinflammation. Although the majority of them fade along with inflammation resolution, the possibility of long-term sequelae should be taken into consideration. Thus, we aimed to explore if NA-induced neuroinflammation provokes behavioral or neurological disturbances at medium (2 weeks) and long (10 weeks) term. Initially, rats were ICV injected with NA or saline. Two or 10 weeks later they were made to perform a series of neurological tests and behavioral evaluations (open field test). The neuroinflammation status of the brain was studied by immunohistochemistry and qPCR. While no neurological alterations were found, the open field test revealed an increased anxiety state 2 weeks after NA administration, which was not observed after 10 weeks. In accordance with this behavioral findings, an overexpression of the molecular pattern receptor TLR4 was revealed by qPCR in hypothalamic tissue in NA treated animals after 2 weeks of ICV, but not after 10 weeks. Moreover, histological studies showed a microgliosis in the amygdala of NA injected rats 2 weeks post-ICV, as well as a slightly activated state evidenced by morphometric parameters of these cells. These histological findings were not present 10 weeks after the ICV injection. These results suggest that NA-induced neuroinflammation might cause anxiety, with no neurological manifestations, in the medium term, along with a mild microglial activation in amygdala. Such symptoms seem to revert, as they were not detected 10 weeks after NA administration.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech