Serodolin, a ß-arrestin-biased ligand of 5-HT7 receptor, attenuates pain-related behaviors.

G protein­coupled receptors (GPCRs) are involved in regulation of manifold physiological processes through coupling to heterotrimeric G proteins upon ligand stimulation. Classical therapeutically active drugs simultaneously initiate several downstream signaling pathways, whereas biased ligands, which stabilize subsets of receptor conformations, elicit more selective signaling. This concept of functional selectivity of a ligand has emerged as an interesting property for the development of new therapeutic molecules. Biased ligands are expected to have superior efficacy and/or reduced side effects by regulating biological functions of GPCRs in a more precise way. In the last decade, 5-HT7 receptor (5-HT7R) has become a promising target for the treatment of neuropsychiatric disorders, sleep and circadian rhythm disorders, and pathological pain. In this study, we showed that Serodolin is unique among a number of agonists and antagonists tested: it behaves as an antagonist/inverse agonist on Gs signaling while inducing ERK activation through a ß-arrestin­dependent signaling mechanism that requires c-SRC activation. Moreover, we showed that Serodolin clearly decreases hyperalgesia and pain sensation in response to inflammatory, thermal, and mechanical stimulation. This antinociceptive effect could not be observed in 5-HT7R knockout (KO) mice and was fully blocked by administration of SB269-970, a specific 5-HT7R antagonist, demonstrating the specificity of action of Serodolin. Physiological effects of 5-HT7R stimulation have been classically shown to result from Gs-dependent adenylyl cyclase activation. In this study, using a ß-arrestin­biased agonist, we provided insight into the molecular mechanism triggered by 5-HT7R and revealed its therapeutic potential in the modulation of pain response.

LINGO family receptors are differentially expressed in the mouse brain and form native multimeric complexes.

Leucine-rich repeat and immunoglobin-domain containing (LRRIG) proteins that are commonly involved in protein-protein interactions play important roles in nervous system development and maintenance. LINGO-1, one of this family members, is characterized as a negative regulator of neuronal survival, axonal regeneration, and oligodendrocyte precursor cell (OPC) differentiation into mature myelinating oligodendrocytes. Three LINGO-1 homologs named LINGO-2, LINGO-3, and LINGO-4 have been described. However, their relative expression and functions remain unexplored. Here, we show by in situ hybridization and quantitative polymerase chain reaction that the transcripts of LINGO homologs are differentially expressed in the central nervous system. The immunostaining of brain slices confirmed this observation and showed the co-expression of LINGO-1 with its homologs. Using BRET (bioluminescence resonance energy transfer) analysis, we demonstrate that LINGO proteins can physically interact with each of the other ones with comparable affinities and thus form the oligomeric states. Furthermore, co-immunoprecipitation experiments indicate that LINGO proteins form heterocomplexes in both heterologous systems and cortical neurons. Since LINGO-1 is a promising target for the treatment of demyelinating diseases, its ability to form heteromeric complexes reveals a new level of complexity in its functioning and opens the way for new strategies to achieve diverse and nuanced LINGO-1 regulation.

LentiRILES, a miRNA-ON sensor system for monitoring the functionality of miRNA in cancer biology and therapy.

A major unresolved challenge in miRNA biology is the capacity to monitor the spatiotemporal activity of miRNAs expressed in animal disease models. We recently reported that the miRNA-ON monitoring system called RILES (RNAi-inducible expression Luciferase system) implanted in lentivirus expression system (LentiRILES) offers unique opportunity to decipher the kinetics of miRNA activity in vitro, in relation with their intracellular trafficking in glioblastoma cells. In this study, we describe in detail the method for the production of LentiRILES stable cell lines and employed it in several applications in the field of miRNA biology and therapy. We show that LentiRILES is a robust, highly specific and sensitive miRNA sensor system that can be used in vitro as a single-cell miRNA monitoring method, cell-based screening platform for miRNA therapeutics and as a tool to analyse the structure-function relationship of the miRNA duplex. Furthermore, we report the kinetics of miRNA activity upon the intracranial delivery of miRNA mimics in an orthotopic animal model of glioblastoma. This information is exploited to evaluate the tumour suppressive function of miRNA-200c as locoregional therapeutic modality to treat glioblastoma. Our data provide evidence that LentiRILES is a robust system, well suited to resolve the activity of endogenous and exogenously expressed miRNAs from basic research to gene and cell therapy.

Hippocampal interleukin-33 mediates neuroinflammation-induced cognitive impairments.

BACKGROUND: Interleukin (IL)-33 is expressed in a healthy brain and plays a pivotal role in several neuropathologies, as protective or contributing to the development of cerebral diseases associated with cognitive impairments. However, the role of IL-33 in the brain is poorly understood, raising the question of its involvement in immunoregulatory mechanisms. METHODS: We administered recombinant IL-33 (rmIL-33) by intra-hippocampal injection to C57BL/6 J (WT) and IL-1αß deficient mice. Chronic minocycline administration was performed and cognitive functions were examined trough spatial habituation test. Hippocampal inflammatory responses were investigated by RT-qPCR. The microglia activation was assessed using immunohistological staining and fluorescence-activated cell sorting (FACS). RESULTS: We showed that IL-33 administration in mice led to a spatial memory performance defect associated with an increase of inflammatory markers in the hippocampus while minocycline administration limited the inflammatory response. Quantitative assessment of glial cell activation in situ demonstrated an increase of proximal intersections per radius in each part of the hippocampus. Moreover, rmIL-33 significantly promoted the outgrowth of microglial processes. Fluorescence-activated cell sorting analysis on isolated microglia, revealed overexpression of IL-1ß, 48 h post-rmIL-33 administration. This microglial reactivity was closely related to the onset of cognitive disturbance. Finally, we demonstrated that IL-1αß deficient mice were resistant to cognitive disorders after intra-hippocampal IL-33 injection. CONCLUSION: Thus, hippocampal IL-33 induced an inflammatory state, including IL-1ß overexpression by microglia cells, being causative of the cognitive impairment. These results highlight the pathological role for IL-33 in the central nervous system, independently of a specific neuropathological model.

IL-33 receptor ST2 regulates the cognitive impairments associated with experimental cerebral malaria.

Cerebral malaria (CM) is associated with a high mortality rate and long-term neurocognitive impairment in survivors. The murine model of experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA (PbA)-infection reproduces several of these features. We reported recently increased levels of IL-33 protein in brain undergoing ECM and the involvement of IL-33/ST2 pathway in ECM development. Here we show that PbA-infection induced early short term and spatial memory defects, prior to blood brain barrier (BBB) disruption, in wild-type mice, while ST2-deficient mice did not develop cognitive defects. PbA-induced neuroinflammation was reduced in ST2-deficient mice with low Ifng, Tnfa, Il1b, Il6, CXCL9, CXCL10 and Cd8a expression, associated with an absence of neurogenesis defects in hippocampus. PbA-infection triggered a dramatic increase of IL-33 expression by oligodendrocytes, through ST2 pathway. In vitro, IL-33/ST2 pathway induced microglia expression of IL-1ß which in turn stimulated IL-33 expression by oligodendrocytes. These results highlight the IL-33/ST2 pathway ability to orchestrate microglia and oligodendrocytes responses at an early stage of PbA-infection, with an amplification loop between IL-1ß and IL-33, responsible for an exacerbated neuroinflammation context and associated neurological and cognitive defects.

IL-33-mediated protection against experimental cerebral malaria is linked to induction of type 2 innate lymphoid cells, M2 macrophages and regulatory T cells.

Cerebral malaria (CM) is a complex parasitic disease caused by Plasmodium sp. Failure to establish an appropriate balance between pro- and anti-inflammatory immune responses is believed to contribute to the development of cerebral pathology. Using the blood-stage PbA (Plasmodium berghei ANKA) model of infection, we show here that administration of the pro-Th2 cytokine, IL-33, prevents the development of experimental cerebral malaria (ECM) in C57BL/6 mice and reduces the production of inflammatory mediators IFN-γ, IL-12 and TNF-α. IL-33 drives the expansion of type-2 innate lymphoid cells (ILC2) that produce Type-2 cytokines (IL-4, IL-5 and IL-13), leading to the polarization of the anti-inflammatory M2 macrophages, which in turn expand Foxp3 regulatory T cells (Tregs). PbA-infected mice adoptively transferred with ILC2 have elevated frequency of M2 and Tregs and are protected from ECM. Importantly, IL-33-treated mice deleted of Tregs (DEREG mice) are no longer able to resist ECM. Our data therefore provide evidence that IL-33 can prevent the development of ECM by orchestrating a protective immune response via ILC2, M2 macrophages and Tregs.

Spinal cord oligodendrocyte-derived alarmin IL-33 mediates neuropathic pain.

Neuropathic pain from injury to the peripheral and CNS represents a major health care issue. We have investigated the role of IL-33/IL-33 receptor (ST2) signaling in experimental models of neuropathic pain in mice. Chronic constriction injury (CCI) of the sciatic nerve induced IL-33 production in the spinal cord. IL-33/citrine reporter mice revealed that oligodendrocytes are the main cells expressing IL-33 within the spinal cord together with a minor expression by neurons, microglia. and astrocytes. CCI-induced mechanical hyperalgesia was reduced in IL-33R (ST2)(-/ -) mice compared with wild-type (WT) mice. Intrathecal treatment of WT mice with soluble IL-33 receptor (IL-33 decoy receptor) markedly reduced CCI-induced hyperalgesia. Consistent with these observations, intrathecal injection of IL-33 enhanced CCI hyperalgesia and induced hyperalgesia in naive mice. IL-33-mediated hyperalgesia during CCI was dependent on a reciprocal relationship with TNF-α and IL-1ß. IL-33-induced hyperalgesia was markedly attenuated by inhibitors of PI3K, mammalian target of rapamycin, MAPKs (p38, ERK, and JNK), NF-κB, and also by the inhibitors of glial cells (microglia and astrocytes). Furthermore, targeting these signaling pathways and cells inhibited IL-33-induced TNF-α and IL-1ß production in the spinal cord. Our study, therefore, reveals an important role of oligodendrocyte-derived IL-33 in neuropathic pain.

Critical role of IL-33 receptor ST2 in experimental cerebral malaria development.

Cerebral malaria, a severe complication of Plasmodium falciparum infection, can be modeled in murine Plasmodium berghei ANKA (PbA) infection. PbA-induced experimental cerebral malaria (ECM) is CD8(+) T-cell mediated, and influenced by TH 1/TH 2 balance. Here, we show that IL-33 expression is increased in brain undergoing ECM and we address the role of the IL-33/ST2 pathway in ECM development. ST2-deficient mice were resistant to PbA-induced neuropathology. They survived >20 days with no ECM neurological sign and a preserved cerebral microcirculation, while WT mice succumbed within 10 days with ECM, brain vascular leakage, distinct microvascular pathology obstruction, and hemorrhages. Parasitemia and brain parasite load were similar in ST2-deficient and WT mice. Protection was accompanied by reduced brain sequestration of activated CD4(+) T cells and perforin(+) CD8(+) T cells. While IFN-γ and T-cell-attracting chemokines CXCL9 and CXCL10 were not affected in the absence of functional ST2 pathway, the local expression of ICAM-1, CXCR3, and LT-α, crucial for ECM development, was strongly reduced, and this may explain the diminished pathogenic T-cell recruitment and resistance to ECM. Therefore, IL-33 is induced in PbA sporozoite infection, and the pathogenic T-cell responses with local microvascular pathology are dependent on IL-33/ST2 signaling, identifying IL-33 as a new actor in ECM development.

ß-N-Methyl-Amino-L-Alanine cyanotoxin promotes modification of undifferentiated cells population and disrupts the inflammatory status in primary cultures of neural stem cells.

ß-N-Methyl-Amino-L-Alanine (BMAA) produced by 95% of cyanobacteria is in constant augmentation with cyanobacteria worldwide proliferation due to global warming and eutrophication. Previously, it has been shown that this contaminant induced neurological disorders, notably by acting as a developmental toxin. However, very few studies focus on the impact of BMAA on neuroglial cells, like astrocytes and microglial cells, in a developmental context. In the present study, we investigated whether BMAA disturbs neurogenesis from mice subventricular zone (SVZ) cells and whether this neurotoxin induces neuroinflammation. We show that BMAA at 100 µM disturbs the population of undifferentiated cells (B1 and C cells) and promotes their proliferation. Further, BMAA affects the organization of neuroblasts, indicating that SVZ function could be impaired. BMAA affects neuroinflammatory processes by increasing the release of proinflammatory cytokines IL-1ß, IL-6 and TNFα. Our study adds to evidence that BMAA may disturb the central nervous system homeostasis by targeting glial cells. We highlighted that BMAA may impair SVZ niches and drives astrocytes and microglial cells into a proinflammatory status, with an ameboid shape for microglia.

T Lymphocyte Serotonin 5-HT7 Receptor Is Dysregulated in Natalizumab-Treated Multiple Sclerosis Patients.

Serotonin (5-HT) is known as a potent immune cell modulator in autoimmune diseases and should be protective in the pathogenesis of multiple sclerosis (MS). Nevertheless, there is limited knowledge about receptors involved in 5-HT effects as well as induced mechanisms. Among 5-HT receptors, the 5-HT7 receptor is able to activate naïve T cells and influence the inflammatory response; however, its involvement in the disease has never been studied so far. In this study, we collected blood sample from three groups: acute relapsing MS patients (ARMS), natalizumab-treated MS patients (NTZ), and control subjects. We investigated the 5-HT7 expression on circulating lymphocytes and evaluated the effects of its activation on cytokine production with peripheral blood mononuclear cell (PBMC) cultures. We found a significant increase in the 5-HT7 surface expression on T lymphocytes and on the different CD4+ T cell subsets exclusively in NTZ-treated patients. We also showed that the selective agonist 5-carboxamidotryptamine (5-CT)-induced 5-HT7R activation significantly promotes the production of IL-10, a potent immunosuppressive cytokine in PBMCs. This study provides for the first time a dysregulation of 5-HT7 expression in NTZ-MS patients and its ability to promote IL-10 release, suggesting its protective role. These findings strengthen the evidence that 5-HT7 may play a role in the immuno-protective mechanisms of NTZ in MS disease and could be considered as an interesting therapeutic target in MS.

Innate myeloid cell TNFR1 mediates first line defence against primary Mycobacterium tuberculosis infection.

TNF is crucial for controlling Mycobacterium tuberculosis infection and understanding how will help immunomodulating the host response. Here we assessed the contribution of TNFR1 pathway from innate myeloid versus T cells. We first established the prominent role of TNFR1 in haematopoietic cells for controlling M. tuberculosis in TNFR1 KO chimera mice. Further, absence of TNFR1 specifically on myeloid cells (M-TNFR1 KO) recapitulated the uncontrolled M. tuberculosis infection seen in fully TNFR1 deficient mice, with increased bacterial burden, exacerbated lung inflammation, and rapid death. Pulmonary IL-12p40 over-expression was attributed to a prominent CD11b(+) Gr1(high) cell population in infected M-TNFR1 KO mice. By contrast, absence of TNFR1 on T-cells did not compromise the control of M. tuberculosis infection over 6-months. Thus, the protective TNF/TNFR1 pathway essential for controlling primary M. tuberculosis infection depends on innate macrophage and neutrophil myeloid cells, while TNFR1 pathway in T cells is dispensable.

Role of IL-1ß in experimental cystic fibrosis upon P. aeruginosa infection.

Cystic fibrosis is associated with increased inflammatory responses to pathogen challenge. Here we revisited the role of IL-1ß in lung pathology using the experimental F508del-CFTR murine model on C57BL/6 genetic background (Cftr(tm1eur) or d/d), on double deficient for d/d and type 1 interleukin-1 receptor (d/d X IL-1R1-/-), and antibody neutralization. At steady state, young adult d/d mice did not show any signs of spontaneous lung inflammation. However, IL-1R1 deficiency conferred partial protection to repeated P. aeruginosa endotoxins/LPS lung instillation in d/d mice, as 50% of d/d mice succumbed to inflammation, whereas all d/d x IL-1R1-/- double mutants survived with lower initial weight loss and less pulmonary collagen and mucus production, suggesting that the absence of IL-1R1 signaling is protective in d/d mice in LPS-induced lung damage. Using P. aeruginosa acute lung infection we found heightened neutrophil recruitment in d/d mice with higher epithelial damage, increased bacterial load in BALF, and augmented IL-1ß and TNF-α in parenchyma as compared to WT mice. Thus, F508del-CFTR mice show enhanced IL-1ß signaling in response to P. aeruginosa. IL-1ß antibody neutralization had no effect on lung homeostasis in either d/d or WT mice, however P. aeruginosa induced lung inflammation and bacterial load were diminished by IL-1ß antibody neutralization. In conclusion, enhanced susceptibility to P. aeruginosa in d/d mice correlates with an excessive inflammation and with increased IL-1ß production and reduced bacterial clearance. Further, we show that neutralization of IL-1ß in d/d mice through the double mutation d/d x IL-1R1-/- and in WT via antibody neutralization attenuates inflammation. This supports the notion that intervention in the IL-1R1/IL-1ß pathway may be detrimental in CF patients.