Interleukin-15 alters hippocampal synaptic transmission and impairs episodic memory formation in mice.

Cytokines are potent immunomodulators exerting pleiotropic effects in the central nervous system (CNS). They influence neuronal functions and circuit activities with effects on memory processes and behaviors. Here, we unravel a neuromodulatory activity of interleukin-15 (IL-15) in mouse brain. Acute exposure of hippocampal slices to IL-15 enhances gamma-aminobutyricacid (GABA) release and reduces glutamatergic currents, while chronic treatment with IL-15 increases the frequency of hippocampal miniature inhibitory synaptic transmission and impairs memory formation in the novel object recognition (NOR) test. Moreover, we describe that serotonin is involved in mediating the hippocampal effects of IL-15, because a selective 5-HT3A receptor antagonist prevents the effects on inhibitory neurotransmission and ameliorates mice performance in the NOR test. These findings provide new insights into the modulatory activities of cytokines in the CNS, with implications on behavior.

Microglial crosstalk with astrocytes and immune cells in amyotrophic lateral sclerosis.

In recent years, biomedical research efforts aimed to unravel the mechanisms involved in motor neuron death that occurs in amyotrophic lateral sclerosis (ALS). While the main causes of disease progression were first sought in the motor neurons, more recent studies highlight the gliocentric theory demonstrating the pivotal role of microglia and astrocyte, but also of infiltrating immune cells, in the pathological processes that take place in the central nervous system microenvironment. From this point of view, microglia-astrocytes-lymphocytes crosstalk is fundamental to shape the microenvironment toward a pro-inflammatory one, enhancing neuronal damage. In this review, we dissect the current state-of-the-art knowledge of the microglial dialogue with other cell populations as one of the principal hallmarks of ALS progression. Particularly, we deeply investigate the microglia crosstalk with astrocytes and immune cells reporting in vitro and in vivo studies related to ALS mouse models and human patients. At last, we highlight the current experimental therapeutic approaches that aim to modulate microglial phenotype to revert the microenvironment, thus counteracting ALS progression.

CCRL2 Expression by Specialized Lung Capillary Endothelial Cells Controls NK-cell Homing in Lung Cancer.

Patterns of receptors for chemotactic factors regulate the homing of leukocytes to tissues. Here we report that the CCRL2/chemerin/CMKLR1 axis represents a selective pathway for the homing of natural killer (NK) cells to the lung. C-C motif chemokine receptor-like 2 (CCRL2) is a nonsignaling seven-transmembrane domain receptor able to control lung tumor growth. CCRL2 constitutive or conditional endothelial cell targeted ablation, or deletion of its ligand chemerin, were found to promote tumor progression in a Kras/p53Flox lung cancer cell model. This phenotype was dependent on the reduced recruitment of CD27- CD11b+ mature NK cells. Other chemotactic receptors identified in lung-infiltrating NK cells by single-cell RNA sequencing (scRNA-seq), such as Cxcr3, Cx3cr1, and S1pr5, were found to be dispensable in the regulation of NK-cell infiltration of the lung and lung tumor growth. scRNA-seq identified CCRL2 as the hallmark of general alveolar lung capillary endothelial cells. CCRL2 expression was epigenetically regulated in lung endothelium and it was upregulated by the demethylating agent 5-aza-2'-deoxycytidine (5-Aza). In vivo administration of low doses of 5-Aza induced CCRL2 upregulation, increased recruitment of NK cells, and reduced lung tumor growth. These results identify CCRL2 as an NK-cell lung homing molecule that has the potential to be exploited to promote NK cell-mediated lung immune surveillance.

Natural killer cells and innate lymphoid cells 1 tune anxiety-like behavior and memory in mice via interferon-γ and acetylcholine.

The mechanisms of communication between the brain and the immune cells are still largely unclear. Here, we characterize the populations of resident natural killer (NK) cells and innate lymphoid cells (ILC) 1 in the meningeal dura layer of adult mice. We describe that ILC1/NK cell-derived interferon-γ and acetylcholine can contribute to the modulation of brain homeostatic functions, shaping synaptic neuronal transmission and neurotransmitter levels with effects on mice behavior. In detail, the interferon-γ plays a role in the formation of non-spatial memory, tuning the frequency of GABAergic neurotransmission on cortical pyramidal neurons, while the acetylcholine is a mediator involved in the modulation of brain circuitries that regulate anxiety-like behavior. These findings disclose mechanisms of immune-to-brain communication that modulate brain functions under physiological conditions.

Environmental enrichment counteracts the effects of glioma in primary visual cortex.

Experience-dependent neuronal changes and brain plasticity occur throughout life as animals adapt to their environment. Structural, morphological, and cellular modifications promoted by exposure to environmental enrichment (EE) have been reported to improve neuronal functions, increase hippocampal neurogenesis, ameliorate memory tasks and cognitive performance, and have beneficial effects on several brain diseases, including cancer. We specifically addressed the role of the EE in counteracting neuronal dysfunction in mice bearing glioma in the primary visual cortex. By recording spontaneous and evoked currents with patch clamp techniques in acute slices obtained from standard and enriched-housed mice, we found that the presence of glioma globally reduced the excitatory and inhibitory transmissions in the peritumoral area. The exposure to an enriched environment counteracts the tumor-mediated depression of both excitatory and inhibitory neuronal activities, with a more pronounced impact on evoked transmission. The effect of EE on glioma was also associated with reduced tumor cell proliferation. These results elucidate the impact of EE on excitatory and inhibitory neurotransmission of the primary visual cortex in control and glioma-bearing mice.

Dialogue among Lymphocytes and Microglia in Glioblastoma Microenvironment.

Microglia and lymphocytes are fundamental constituents of the glioblastoma microenvironment. In this review, we summarize the current state-of-the-art knowledge of the microglial role played in promoting the development and aggressive hallmarks of this deadly brain tumor. Particularly, we report in vitro and in vivo studies related to glioblastoma models and human patients to outline the symbiotic bidirectional interaction between microglia, lymphocytes, and tumor cells that develops during tumor progression. Furthermore, we highlight the current experimental therapeutic approaches that aim to shape these interplays, such as adeno-associated virus (AAV) delivery and CAR-T and -NK cell infusion, and to modulate the tumor microenvironment in an anti-tumoral way, thus counteracting glioblastoma growth.

Blocking immune cell infiltration of the central nervous system to tame Neuroinflammation in Amyotrophic lateral sclerosis.

Neuroinflammation is one of the main hallmarks of amyotrophic lateral sclerosis (ALS). Recently, peripheral immune cells were discovered as pivotal players that promptly participate in this process, speeding up neurodegeneration during progression of the disease. In particular, infiltrating T cells and natural killer cells release inflammatory cytokines that switch glial cells toward a pro-inflammatory/detrimental phenotype, and directly attack motor neurons with specific ligand-receptor signals. Here, we assessed the presence of lymphocytes in the spinal cord of sporadic ALS patients. Furthermore, we demonstrate that blocking the extravasation of immune cells in the central nervous system using Natalizumab (NAT), an antibody for the α4 integrin, reduces the level of interferon-γ in the spinal cord of ALS mouse models, such as the hSOD1G93A and TDP43A315T mice, modifying microglia and astrocytes phenotype, increasing motor neuron number and prolonging the survival time. Taken together, our results establish a central role for the immune cells as drivers of inflammation in ALS.

Short-chain fatty acids promote the effect of environmental signals on the gut microbiome and metabolome in mice.

Gut microorganisms and the products of their metabolism thoroughly affect host brain development, function and behavior. Since alterations of brain plasticity and cognition have been demonstrated upon motor, sensorial and social enrichment of the housing conditions, we hypothesized that gut microbiota and metabolome could be altered by environmental stimuli, providing part of the missing link among environmental signals and brain effects. In this preliminary study, metagenomic and metabolomic analyses of mice housed in different environmental conditions, standard and enriched, identify environment-specific microbial communities and metabolic profiles. We show that mice housed in an enriched environment have distinctive microbiota composition with a reduction in gut bacterial richness and biodiversity and are characterized by a metabolomic fingerprint with the increase of formate and acetate and the decrease of bile salts. We demonstrate that mice treated with a mixture of formate and acetate recapitulate some of the brain plasticity effects modulated by environmental enrichment, such as hippocampal neurogenesis, neurotrophin production, short-term plasticity and cognitive behaviors, that can be further exploited to decipher the mechanisms involved in experience-dependent brain plasticity.

Muscle Damage in Dystrophic mdx Mice Is Influenced by the Activity of Ca2+-Activated KCa3.1 Channels.

Duchenne muscular dystrophy (DMD) is an X-linked disease, caused by a mutant dystrophin gene, leading to muscle membrane instability, followed by muscle inflammation, infiltration of pro-inflammatory macrophages and fibrosis. The calcium-activated potassium channel type 3.1 (KCa3.1) plays key roles in controlling both macrophage phenotype and fibroblast proliferation, two critical contributors to muscle damage. In this work, we demonstrate that pharmacological blockade of the channel in the mdx mouse model during the early degenerative phase favors the acquisition of an anti-inflammatory phenotype by tissue macrophages and reduces collagen deposition in muscles, with a concomitant reduction of muscle damage. As already observed with other treatments, no improvement in muscle performance was observed in vivo. In conclusion, this work supports the idea that KCa3.1 channels play a contributing role in controlling damage-causing cells in DMD. A more complete understanding of their function could lead to the identification of novel therapeutic approaches.

Microglia control glutamatergic synapses in the adult mouse hippocampus.

Microglia cells are active players in regulating synaptic development and plasticity in the brain. However, how they influence the normal functioning of synapses is largely unknown. In this study, we characterized the effects of pharmacological microglia depletion, achieved by administration of PLX5622, on hippocampal CA3-CA1 synapses of adult wild type mice. Following microglial depletion, we observed a reduction of spontaneous and evoked glutamatergic activity associated with a decrease of dendritic spine density. We also observed the appearance of immature synaptic features and higher levels of plasticity. Microglia depleted mice showed a deficit in the acquisition of the Novel Object Recognition task. These events were accompanied by hippocampal astrogliosis, although in the absence ofneuroinflammatory condition. PLX-induced synaptic changes were absent in Cx3cr1-/- mice, highlighting the role of CX3CL1/CX3CR1 axis in microglia control of synaptic functioning. Remarkably, microglia repopulation after PLX5622 withdrawal was associated with the recovery of hippocampal synapses and learning functions. Altogether, these data demonstrate that microglia contribute to normal synaptic functioning in the adult brain and that their removal induces reversible changes in organization and activity of glutamatergic synapses.

Microglia modulate hippocampal synaptic transmission and sleep duration along the light/dark cycle.

Microglia, the brain's resident macrophages, actively contribute to the homeostasis of cerebral parenchyma by sensing neuronal activity and supporting synaptic remodeling and plasticity. While several studies demonstrated different roles for astrocytes in sleep, the contribution of microglia in the regulation of sleep/wake cycle and in the modulation of synaptic activity in the different day phases has not been deeply investigated. Using light as a zeitgeber cue, we studied the effects of microglial depletion with the colony stimulating factor-1 receptor antagonist PLX5622 on the sleep/wake cycle and on hippocampal synaptic transmission in male mice. Our data demonstrate that almost complete microglial depletion increases the duration of NREM sleep and reduces the hippocampal excitatory neurotransmission. The fractalkine receptor CX3CR1 plays a relevant role in these effects, because cx3cr1GFP/GFP mice recapitulate what found in PLX5622-treated mice. Furthermore, during the light phase, microglia express lower levels of cx3cr1 and a reduction of cx3cr1 expression is also observed when cultured microglial cells are stimulated by ATP, a purinergic molecule released during sleep. Our findings suggest that microglia participate in the regulation of sleep, adapting their cx3cr1 expression in response to the light/dark phase, and modulating synaptic activity in a phase-dependent manner.

Microglial Potassium Channels: From Homeostasis to Neurodegeneration.

The growing interest in the role of microglia in the progression of many neurodegenerative diseases is developing in an ever-expedited manner, in part thanks to emergent new tools for studying the morphological and functional features of the CNS. The discovery of specific biomarkers of the microglia phenotype could find application in a wide range of human diseases, and creates opportunities for the discovery and development of tailored therapeutic interventions. Among these, recent studies highlight the pivotal role of the potassium channels in regulating microglial functions in physiological and pathological conditions such as Alzheimer's Disease, Parkinson's Disease, and Amyotrophic Lateral Sclerosis. In this review, we summarize the current knowledge of the involvement of the microglial potassium channels in several neurodegenerative diseases and their role as modulators of microglial homeostasis and dysfunction in CNS disorders.

Enriched Environment Cues Suggest a New Strategy to Counteract Glioma: Engineered rAAV2-IL-15 Microglia Modulate the Tumor Microenvironment.

Several types of cancer grow differently depending on the environmental stimuli they receive. In glioma, exposure to an enriched environment (EE) increases the overall survival rate of tumor-bearing mice, acting on the cells that participate to define the tumor microenvironment. In particular, environmental cues increase the microglial production of interleukin (IL)-15 which promotes a pro-inflammatory (antitumor) phenotype of microglia and the cytotoxic activity of natural killer (NK) cells, counteracting glioma growth, thus representing a virtuous mechanism of interaction between NK cells and microglia. To mimic the effect of EE on glioma, we investigated the potential of creating engineered microglia as the source of IL-15 in glioma. We demonstrated that microglia modified with recombinant adeno-associated virus serotype 2 (rAAV2) carrying IL-15 (rAAV2-IL-15), to force the production of IL-15, are able to increase the NK cells viability in coculture. Furthermore, the intranasal delivery of rAAV2-IL-15 microglia triggered the interplay with NK cells in vivo, enhancing NK cell recruitment and pro-inflammatory microglial phenotype in tumor mass of glioma-bearing mice, and ultimately counteracted tumor growth. This approach has a high potential for clinical translatability, highlighting the therapeutic efficacy of forced IL-15 production in microglia: the delivery of engineered rAAV2-IL-15 microglia to boost the immune response paves the way to design a new perspective therapy for glioma patients.

The feeding behaviour of Amyotrophic Lateral Sclerosis mouse models is modulated by the Ca2+ -activated KCa 3.1 channels.

BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) patients exhibit dysfunctional energy metabolism and weight loss, which is negatively correlated with survival, together with neuroinflammation. However, the possible contribution of neuroinflammation to deregulations of feeding behaviour in ALS has not been studied in detail. We here investigated if microglial KCa 3.1 is linked to hypothalamic neuroinflammation and affects feeding behaviours in ALS mouse models. EXPERIMENTAL APPROACH: hSOD1G93A and TDP43A315T mice were treated daily with 120 mg·kg-1 of TRAM-34 or vehicle by intraperitoneal injection from the presymptomatic until the disease onset phase. Body weight and food intake were measured weekly. The later by weighing food provided minus that left in the cage. RT-PCR and immunofluorescence analysis were used to characterize microglia phenotype and the main populations of melanocortin neurons in the hypothalamus of hSOD1G93A and age-matched non-tg mice. The cannabinoid-opioid interactions in feeding behaviour of hSOD1G93A mice were studied using an inverse agonist and an antagonist of the cannabinoid receptor CB1 (rimonabant) and µ-opioid receptors (naloxone), respectively. KEY RESULTS: We found that treatment of hSOD1G93A mice with the KCa 3.1 inhibitor TRAM-34 (i), attenuates the pro-inflammatory phenotype of hypothalamic microglia, (ii) increases food intake and promotes weight gain, (iii) increases the number of healthy pro-opiomelanocortin (POMC) neurons and (iv), changes the expression of cannabinoid receptors involved in energy homeostasis. CONCLUSION AND IMPLICATIONS: Using ALS mouse models, we describe defects in the hypothalamic melanocortin system that affect appetite control. These results reveal a new regulatory role for KCa 3.1 to counteract weight loss in ALS.

Microglial-glucocorticoid receptor depletion alters the response of hippocampal microglia and neurons in a chronic unpredictable mild stress paradigm in female mice.

Chronic psychological stress is one of the most important triggers and environmental risk factors for neuropsychiatric disorders. Chronic stress can influence all organs via the secretion of stress hormones, including glucocorticoids by the adrenal glands, which coordinate the stress response across the body. In the brain, glucocorticoid receptors (GR) are expressed by various cell types including microglia, which are its resident immune cells regulating stress-induced inflammatory processes. To study the roles of microglial GR under normal homeostatic conditions and following chronic stress, we generated a mouse model in which the GR gene is depleted in microglia specifically at adulthood to prevent developmental confounds. We first confirmed that microglia were depleted in GR in our model in males and females among the cingulate cortex and the hippocampus, both stress-sensitive brain regions. Then, cohorts of microglial-GR depleted and wild-type (WT) adult female mice were housed for 3 weeks in a standard or stressful condition, using a chronic unpredictable mild stress (CUMS) paradigm. CUMS induced stress-related behavior in both microglial-GR depleted and WT animals as demonstrated by a decrease of both saccharine preference and progressive ratio breakpoint. Nevertheless, the hippocampal microglial and neural mechanisms underlying the adaptation to stress occurred differently between the two genotypes. Upon CUMS exposure, microglial morphology was altered in the WT controls, without any apparent effect in microglial-GR depleted mice. Furthermore, in the standard environment condition, GR depleted-microglia showed increased expression of pro-inflammatory genes, and genes involved in microglial homeostatic functions (such as Trem2, Cx3cr1 and Mertk). On the contrary, in CUMS condition, GR depleted-microglia showed reduced expression levels of pro-inflammatory genes and increased neuroprotective as well as anti-inflammatory genes compared to WT-microglia. Moreover, in microglial-GR depleted mice, but not in WT mice, CUMS led to a significant reduction of CA1 long-term potentiation and paired-pulse ratio. Lastly, differences in adult hippocampal neurogenesis were observed between the genotypes during normal homeostatic conditions, with microglial-GR deficiency increasing the formation of newborn neurons in the dentate gyrus subgranular zone independently from stress exposure. Together, these findings indicate that, although the deletion of microglial GR did not prevent the animal's ability to respond to stress, it contributed to modulating hippocampal functions in both standard and stressful conditions, notably by shaping the microglial response to chronic stress.

Chimeric Stimuli-Responsive Liposomes as Nanocarriers for the Delivery of the Anti-Glioma Agent TRAM-34.

Nanocarriers are delivery platforms of drugs, peptides, nucleic acids and other therapeutic molecules that are indicated for severe human diseases. Gliomas are the most frequent type of brain tumor, with glioblastoma being the most common and malignant type. The current state of glioma treatment requires innovative approaches that will lead to efficient and safe therapies. Advanced nanosystems and stimuli-responsive materials are available and well-studied technologies that may contribute to this effort. The present study deals with the development of functional chimeric nanocarriers composed of a phospholipid and a diblock copolymer, for the incorporation, delivery and pH-responsive release of the antiglioma agent TRAM-34 inside glioblastoma cells. Nanocarrier analysis included light scattering, protein incubation and electron microscopy, and fluorescence anisotropy and thermal analysis techniques were also applied. Biological assays were carried out in order to evaluate the nanocarrier nanotoxicity in vitro and in vivo, as well as to evaluate antiglioma activity. The nanosystems were able to successfully manifest functional properties under pH conditions, and their biocompatibility and cellular internalization were also evident. The chimeric nanoplatforms presented herein have shown promise for biomedical applications so far and should be further studied in terms of their ability to deliver TRAM-34 and other therapeutic molecules to glioblastoma cells.

Histone-deacetylase 8 drives the immune response and the growth of glioma.

Many epigenetic modifications occur in glioma, in particular the histone-deacetylase class proteins play a pivotal role in glioma development, driving the proliferation rate and the invasiveness of tumor cells, and modulating the tumor microenvironment. In this study, we evaluated the role of the histone deacetylase HDAC8 in the regulation of the immune response in glioma and tumor growth. We found that inhibition of HDAC8 by the specific inhibitor PCI-34051 reduces tumor volume in glioma mouse models. We reported that HDAC8 modulates the viability and the migration of human and murine glioma cells. Interestingly, HDAC8 inhibition increases the acetylation of alpha-tubulin, suggesting this epigenetic modification controls glioma migration. Furthermore, we identify HDAC8 as a key molecule that supports a poorly immunogenic tumor microenvironment, modulating microglial phenotype and regulating the gene transcription of NKG2D ligands that trigger the Natural Killer cell-mediated cytotoxicity of tumor cells. Altogether, these results identify HDAC8 as a key actor in glioma growth and tumor microenvironment, and pave the way to a better knowledge of the molecular mechanisms of immune escape in glioma.

Synthesis and use of an amphiphilic dendrimer for siRNA delivery into primary immune cells.

Using siRNAs to genetically manipulate immune cells is important to both basic immunological studies and therapeutic applications. However, siRNA delivery is challenging because primary immune cells are often sensitive to the delivery materials and generate immune responses. We have recently developed an amphiphilic dendrimer that is able to deliver siRNA to a variety of cells, including primary immune cells. We provide here a protocol for the synthesis of this dendrimer, as well as siRNA delivery to immune cells such as primary T and B cells, natural killer cells, macrophages, and primary microglia. The dendrimer synthesis entails straightforward click coupling followed by an amidation reaction, and the siRNA delivery protocol requires simple mixing of the siRNA and dendrimer in buffer, with subsequent application to the primary immune cells to achieve effective and functional siRNA delivery. This dendrimer-mediated siRNA delivery largely outperforms the standard electroporation technique, opening a new avenue for functional and therapeutic studies of the immune system. The whole protocol encompasses the dendrimer synthesis, which takes 10 days; the primary immune cell preparation, which takes 3-10 d, depending on the tissue source and cell type; the dendrimer-mediated siRNA delivery; and subsequent functional assays, which take an additional 3-6 d.

Natural killer cells modulate motor neuron-immune cell cross talk in models of Amyotrophic Lateral Sclerosis.

In amyotrophic lateral sclerosis (ALS), immune cells and glia contribute to motor neuron (MN) degeneration. We report the presence of NK cells in post-mortem ALS motor cortex and spinal cord tissues, and the expression of NKG2D ligands on MNs. Using a mouse model of familial-ALS, hSOD1G93A, we demonstrate NK cell accumulation in the motor cortex and spinal cord, with an early CCL2-dependent peak. NK cell depletion reduces the pace of MN degeneration, delays motor impairment and increases survival. This is confirmed in another ALS mouse model, TDP43A315T. NK cells are neurotoxic to hSOD1G93A MNs which express NKG2D ligands, while IFNγ produced by NK cells instructs microglia toward an inflammatory phenotype, and impairs FOXP3+/Treg cell infiltration in the spinal cord of hSOD1G93A mice. Together, these data suggest a role of NK cells in determining the onset and progression of MN degeneration in ALS, and in modulating Treg recruitment and microglia phenotype.

Role of Glia in the Regulation of Sleep in Health and Disease.

Sleep is a naturally occurring physiological state that is required to sustain physical and mental health. Traditionally viewed as strictly regulated by top-down control mechanisms, sleep is now known to also originate locally. Glial cells are emerging as important contributors to the regulation of sleep-wake cycles, locally and among dedicated neural circuits. A few pioneering studies revealed that astrocytes and microglia may influence sleep pressure, duration as well as intensity, but the precise involvement of these two glial cells in the regulation of sleep remains to be fully addressed, across contexts of health and disease. In this overview article, we will first summarize the literature pertaining to the role of astrocytes and microglia in the regulation of sleep under normal physiological conditions. Afterward, we will discuss the beneficial and deleterious consequences of glia-mediated neuroinflammation, whether it is acute, or chronic and associated with brain diseases, on the regulation of sleep. Sleep disturbances are a main comorbidity in neurodegenerative diseases, and in several brain diseases that include pain, epilepsy, and cancer. Identifying the relationships between glia-mediated neuroinflammation, sleep-wake rhythm disruption and brain diseases may have important implications for the treatment of several disorders. © 2020 American Physiological Society. Compr Physiol 10:687-712, 2020.