Aline Dumas

CXCL1 can be regulated by IL-6 and promotes granulocyte adhesion to brain capillaries during bacterial toxin exposure and encephalomyelitis

BackgroundGranulocytes generally exert protective roles in the central nervous system (CNS), but recent studies suggest that they can be detrimental in experimental autoimmune encephalomyelitis (EAE), the most common model of multiple sclerosis. While the cytokines and adhesion molecules involved in granulocyte adhesion to the brain vasculature have started to be elucidated, the required chemokines remain undetermined.MethodsCXCR2 ligand expression was examined in the CNS of mice suffering from EAE or exposed to bacterial toxins by quantitative RT-PCR and in situ hybridization. CXCL1 expression was analyzed in IL-6-treated endothelial cell cultures by quantitative RT-PCR and ELISA. Granulocytes were counted in the brain vasculature after treatment with a neutralizing anti-CXCL1 antibody using stereological techniques.ResultsCXCL1 was the most highly expressed ligand of the granulocyte receptor CXCR2 in the CNS of mice subjected to EAE or infused with lipopolysaccharide (LPS) or pertussis toxin (PTX), the latter being commonly used to induce EAE. IL-6 upregulated CXCL1 expression in brain endothelial cells by acting transcriptionally and mediated the stimulatory effect of PTX on CXCL1 expression. The anti-CXCL1 antibody reduced granulocyte adhesion to brain capillaries in the three conditions under study. Importantly, it attenuated EAE severity when given daily for a week during the effector phase of the disease.ConclusionsThis study identifies CXCL1 not only as a key regulator of granulocyte recruitment into the CNS, but also as a new potential target for the treatment of neuroinflammatory diseases such as multiple sclerosis.

The Inflammasome Pyrin Contributes to Pertussis Toxin-Induced IL-1β Synthesis, Neutrophil Intravascular Crawling and Autoimmune Encephalomyelitis

Microbial agents can aggravate inflammatory diseases, such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). An example is pertussis toxin (PTX), a bacterial virulence factor commonly used as an adjuvant to promote EAE, but whose mechanism of action is unclear. We have reported that PTX triggers an IL-6-mediated signaling cascade that increases the number of leukocytes that patrol the vasculature by crawling on its luminal surface. In the present study, we examined this response in mice lacking either TLR4 or inflammasome components and using enzymatically active and inactive forms of PTX. Our results indicate that PTX, through its ADP-ribosyltransferase activity, induces two series of events upstream of IL-6: 1) the activation of TLR4 signaling in myeloid cells, leading to pro-IL-1β synthesis; and 2) the formation of a pyrin-dependent inflammasome that cleaves pro-IL-1β into its active form. In turn, IL-1β stimulates nearby stromal cells to secrete IL-6, which is known to induce vascular changes required for leukocyte adhesion. Without pyrin, PTX does not induce neutrophil adhesion to cerebral capillaries and is less effective at inducing EAE in transgenic mice with encephalitogenic T lymphocytes. This study identifies the first microbial molecule that activates pyrin, a mechanism by which infections may influence MS and a potential therapeutic target for immune disorders.

GPR84 deficiency reduces microgliosis, but accelerates dendritic degeneration and cognitive decline in a mouse model of Alzheimer's disease.

Microglia surrounds the amyloid plaques that form in the brains of patients with Alzheimers disease (AD), but their role is controversial. Under inflammatory conditions, these cells can express GPR84, an orphan receptor whose pathophysiological role is unknown. Here, we report that GPR84 is upregulated in microglia of APP/PS1 transgenic mice, a model of AD. Without GPR84, these mice display both accelerated cognitive decline and a reduced number of microglia, especially in areas surrounding plaques. The lack of GPR84 affects neither plaque formation nor hippocampal neurogenesis, but promotes dendritic degeneration. Furthermore, GPR84 does not influence the clinical progression of other diseases in which its expression has been reported, i.e., experimental autoimmune encephalomyelitis (EAE) and endotoxic shock. We conclude that GPR84 plays a beneficial role in amyloid pathology by acting as a sensor for a yet unknown ligand that promotes microglia recruitment, a response affecting dendritic degeneration and required to prevent further cognitive decline.

Interleukin-36γ is expressed by neutrophils and can activate microglia, but has no role in experimental autoimmune encephalomyelitis

BackgroundExperimental autoimmune encephalomyelitis (EAE) is a model of inflammatory demyelinating diseases mediated by different types of leukocytes. How these cells communicate with each other to orchestrate autoimmune attacks is not fully understood, especially in the case of neutrophils, whose importance in EAE is newly established. The present study aimed to determine the expression pattern and role of different components of the IL-36 signaling pathway (IL-36α, IL-36β, IL-36γ, IL-36R) in EAE.MethodsEAE was induced by either active immunization with myelin peptide, passive transfer of myelin-reactive T cells or injection of pertussis toxin to transgenic 2D2 mice. The molecules of interest were analyzed using a combination of techniques, including quantitative real-time PCR (qRT-PCR), flow cytometry, Western blotting, in situ hybridization, and immunohistochemistry. Microglial cultures were treated with recombinant IL-36γ and analyzed using DNA microarrays. Different mouse strains were subjected to clinical evaluation and flow cytometric analysis in order to compare their susceptibility to EAE.ResultsOur observations indicate that both IL-36γ and IL-36R are strongly upregulated in nervous and hematopoietic tissues in different forms of EAE. IL-36γ is specifically expressed by neutrophils, while IL-36R is expressed by different immune cells, including microglia and other myeloid cells. In culture, microglia respond to recombinant IL-36γ by expressing molecules involved in neutrophil recruitment, such as Csf3, IL-1β, and Cxcl2. However, mice deficient in either IL-36γ or IL-36R develop similar clinical and histopathological signs of EAE compared to wild-type controls.ConclusionThis study identifies IL-36γ as a neutrophil-related cytokine that can potentially activate microglia, but that is only correlative and not contributory in EAE.

The pronociceptive effect of proteinase-activated receptor-4 stimulation in rat knee joints is dependent on mast cell activation.

&NA; Proteinase‐activated receptor‐4 (PAR4) is a G‐protein‐coupled receptor activated by serine proteinases released during tissue repair and inflammation. We have previously shown that PAR4 activation sensitises articular primary afferents leading to joint pain. This study examined whether mast cells contribute to this PAR4‐induced sensitisation and consequent heightened pain behaviour. The expression of PAR4 on synovial mast cells was confirmed with immunofluorescent staining of rat knee joint sections. Electrophysiological recordings were made from joint primary afferents in male Wistar rats during both nonnoxious and noxious rotations of the knee. Afferent firing rate was recorded for 15 minutes after close intra‐arterial injection of 10−9 to 10−5 mol of the PAR4 activating peptide, AYPGKF‐NH2, or the inactive peptide, YAPGKF‐NH2 (100‐μl bolus). Rats were either naive or pretreated with the mast cell stabilise, cromolyn (20 mg/kg). Mechanical withdrawal thresholds were determined using a dynamic planter aesthesiometer and weight bearing determined using an incapacitance tester. These behavioural measurements were taken before and after intra‐articular AYPGKF‐NH2, or the inactive peptide, YAPGKF‐NH2 (100 μg). Local administration of AYPGKF‐NH2 caused a significant increase in joint primary afferent firing rate and pain behaviour compared with the control peptide YAPGKF‐NH2. These effects were blocked by pretreatment with cromolyn. These data reveal that PAR4 is expressed on synovial mast cells and the activation of PAR4 has a pronociceptive effect that is dependent on mast cell activation. Proteinase‐activated receptor‐4 is expressed on synovial mast cells, and the activation of Proteinase‐activated receptor‐4 has a pronociceptive effect that is dependent on mast cell activation.

Oncostatin M decreases interleukin-1 β secretion by human synovial fibroblasts and attenuates an acute inflammatory reaction in vivo

Oncostatin M (OSM) is a pleiotropic cytokine of the IL‐6 family and displays both pro‐inflammatory and anti‐inflammatory activities. We studied the impact of OSM on the gene activation profile of human synovial cells, which play a central role in the progression of inflammatory responses in joints. In synovial cells stimulated with lipopolysaccharide and recombinant human granulocyte‐macrophage colony‐stimulating factor, recombinant human OSM and native OSM secreted by human granulocytes both reduced the gene expression and secretion of IL‐1β and CXCL8, but increased that of IL‐6 and CCL2. This impact on synovial cell activation was not obtained using IL‐6 or leukaemia inhibitory factor. Signal transducer and activator of transcription‐1 appeared to mediate the effects of OSM on stimulated human synovial fibroblasts. In the murine dorsal air pouch model of inflammation, OSM reduced the expression of the pro‐inflammatory cytokines IL‐1β and TNF‐α in lining tissues, and their presence in the cavity. These results as a whole suggest an anti‐inflammatory role for OSM, guiding inflammatory processes towards resolution.

Impact of Anti-Inflammatory Agents on the Gene Expression Profile of Stimulated Human Neutrophils: Unraveling Endogenous Resolution Pathways

Adenosine, prostaglandin E2, or increased intracellular cyclic AMP concentration each elicit potent anti-inflammatory events in human neutrophils by inhibiting functions such as phagocytosis, superoxide production, adhesion and cytokine release. However, the endogenous molecular pathways mediating these actions are poorly understood. In the present study, we examined their impact on the gene expression profile of stimulated neutrophils. Purified blood neutrophils from healthy donors were stimulated with a cocktail of inflammatory agonists in the presence of at least one of the following anti-inflammatory agents: adenosine A2A receptor agonist CGS 21680, prostaglandin E2, cyclic-AMP-elevating compounds forskolin and RO 20-1724. Total RNA was analyzed using gene chips and real-time PCR. Genes encoding transcription factors, enzymes and regulatory proteins, as well as secreted cytokines/chemokines showed differential expression. We identified 15 genes for which the anti-inflammatory agents altered mRNA levels. The agents affected the expression profile in remarkably similar fashion, suggesting a central mechanism limiting cell activation. We have identified a set of genes that may be part of important resolution pathways that interfere with cell activation. Identification of these pathways will improve understanding of the capacity of tissues to terminate inflammatory responses and contribute to the development of therapeutic strategies based on endogenous resolution.

Several Classical Mouse Inbred Strains, Including DBA/2, NOD/Lt, FVB/N, and SJL/J, Carry a Putative Loss-of-Function Allele of Gpr84

G protein-coupled receptor 84 (GPR84) is a 7-transmembrane protein expressed on myeloid cells that can bind to medium-chain free fatty acids in vitro. Here, we report the discovery of a 2-bp frameshift deletion in the second exon of the Gpr84 gene in several classical mouse inbred strains. This deletion generates a premature stop codon predicted to result in a truncated protein lacking the transmembrane domains 4-7. We sequenced Gpr84 exon 2 from 58 strains representing different groups in the mouse family tree and found that 14 strains are homozygous for the deletion. Some of these strains are DBA/1J, DBA/2J, FVB/NJ, LG/J, MRL/MpJ, NOD/LtJ, and SJL/J. However, the deletion was not found in any of the wild-derived inbred strains analyzed. Haplotype analysis suggested that the deletion originates from a unique mutation event that occurred more than 100 years ago, preceding the development of the first inbred strain (DBA), from a Mus musculus domesticus source. As GPR84 ostensibly plays a role in the biology of myeloid cells, it could be relevant 1) to consider the existence of this Gpr84 nonsense mutation in several mouse strains when choosing a mouse model to study immune processes and 2) to consider reevaluating data obtained using such strains.

MicroRNA-223 protects neurons from degeneration in Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease characterized by demyelination and neurodegeneration in the brain, spinal cord and optic nerve. Neuronal degeneration and death underlie progressive forms of MS and cognitive dysfunction. Neuronal damage is triggered by numerous harmful factors in the brain that engage diverse signalling cascades in neurons thus therapeutic approaches to protect neurons will need to focus on agents that can target broad biological processes. To target the broad spectrum of signaling events that mediate neurodegeneration in MS we have focused on non-coding small microRNAs (miRNAs). microRNAs are epigenetic regulators of protein expression, targeting messenger RNAs (mRNAs) and inhibiting their translation. Dysregulation of miRNAs has been described in many neurodegenerative diseases including MS. In this study we identified two miRNAs, miR-223-3p and miR-27a-3p, that were upregulated in neurons in the experimental autoimmune encephalomyelitis (EAE) mouse model of CNS inflammation and in active MS lesions. Overexpression of miR-27a-3p or miR-223-3p protected dissociated cortical neurons from degeneration in response to peripheral blood mononuclear cell conditioned media (PBMC-CM). Introduction of miR-223-3p in vivo in mouse retinal ganglion cells (RGCs) protected RGC axons from degeneration in the EAE model. By in silico analysis we found that mRNAs in the glutamate receptor (GluR) pathway are enriched in miR-27a-3p and miR-223-3p targets. Antagonism of the GluR pathway protected neurons from PBMC-CM-dependent degeneration. Our results suggest that miR-223-3p and miR-27a-3p are upregulated in response to inflammation to mediate a compensatory neuroprotective gene expression program that desensitizes neurons to glutamate by downregulating mRNAs involved in GluR signalling.

ICAM1+ neutrophils promote chronic inflammation via ASPRV1 in B cell–dependent autoimmune encephalomyelitis

Neutrophils contribute to demyelinating autoimmune diseases, yet their phenotype and functions have been elusive to date. Here, we demonstrate that ICAM1 surface expression distinguishes extra- from intravascular neutrophils in the mouse CNS during experimental autoimmune encephalomyelitis (EAE). Transcriptomic analysis of these 2 subpopulations indicated that neutrophils, once extravasated, acquire macrophage-like properties, including the potential for immunostimulation and MHC class II-mediated antigen presentation. In corroboration, super-resolution (3D stimulated emission-depletion [STED]) microscopy revealed neutrophils forming synapses with T and B cells in situ. Further, neutrophils specifically express the aspartic retroviral-like protease ASPRV1, which increases in the CNS during EAE and severe cases of multiple sclerosis. Without ASPRV1, mice immunized with a new B cell-dependent myelin antigen (but not with the traditional myelin oligodendrocyte glycoprotein peptide) develop a chronic phase of EAE that is less severe and even completely fades in many individuals. Therefore, ICAM1+ macrophage-like neutrophils can play both shared and nonredundant roles in autoimmune demyelination, among them perpetuating inflammation via ASPRV1.