Effect of experimental stroke on meningeal gene expression and the influence of mast cells on these gene changes
ABSTRACT: Stroke is a leading cause of adult disability and death. Inflammation plays an important role in stroke pathology, but the factors which promote brain inflammation in this setting remain to be fully defined. Here we investigate the meninges, the membranes that envelop the brain, for a potential role in modulating immune cell trafficking to the brain. We also investigate the potential of mast cells (MCs) to modulate this response as MCs are often considered as 'first responders' playing a critical role in the initiation and development of inflammation in many disease settings. We find that stroke increases expression of inflammatory and immune response genes in the meninges in mice consistent with a potential role in modulating immune cell trafficking. Moreover, genetic and cell transfer approaches identify MCs as important modulators of this response. Three categories of male mice were used: wild-type (WT) mice, mast cell-deficient (KO) mice, and mast cell-engrafted mice (EN), which are mast cell-deficient mice repaired of their mast cell deficiency by engraftment of mast cells i.v. 8-10 weeks prior to experimentation. The mouse strain was WBB6F1-Kit+/+ (wild-type ) and WBB6F1-KitW/W-v (mast cell-deficient ). Each mouse category was subdivided into two groups, naïve (N) and stroke (S), with n=3 per group. The stroke model was 30 minute filament occlusion of the middle cerebral artery. The dura were removed from the mouse brains at 2d post-stroke and from aged-matched naïve mice for microarray analysis. Dura were not pooled but run on separate arrays.
Project description:The meninges are generally considered relatively inert tissues that house the CSF and provide protection for the brain and spinal cord. However, our previous studies using Kit mutant (Kit W/Wv) mast cell-deficient mice demonstrated that mast cells residing in the dura mater and pia mater exacerbate the severity of experimental autoimmune encephalomyelitis (EAE), the rodent model of the CNS demyelinating disease, multiple sclerosis. These data suggest that the meninges are sites of active immune responses in disease. Gene expression profiles of meningeal tissue from wild type and mast cell deficient mice prior to and at day 6 post-EAE induction were found highly distinct. Increases in both mast cell- and neutrophil-associated transcripts were among the notable disease-related changes observed in wild type mice. Kinetic analyses show that meningeal mast cells are activated within 24 hours of disease induction to express multiple mediators including IL-1b and TNF as well as the neutrophil chemoattractant, CXCL2, an observation corresponding with an influx of neutrophils to the meninges. Neutrophil recruitment as well as the disease-related loss of BBB integrity is dependent on mast cell-derived TNF. These data provide unequivocal evidence that the meninges are sites of early inflammatory events in EAE. Mast cells residing within these tissues promote disease by orchestrating an early and efficient immune cell co-localization resulting in a robust local inflammatory response and a breach of the proximal BBB. We hypothesize that these events reflect an aberrant manifestation of the normal immune surveillance role of the meninges in infection settings. Immunized WT and Kit W/Wv mice were sacrificed on Day 6 post-immunization and perfused with PBS as were naïve littermate control mice. The dura mater was immediately removed from the calvarium of the skull and pooled (10 mice/group, 4 groups). RNA was isolated using SV Total RNA Isolation System (Promega). Each pool was analyzed in technical triplicates. Briefly, cRNA was synthesized and amplified/labeled using the Affymetrix Express Kit, then fragmented and hybridized to the The GeneChip® Mouse Genome 430 2.0 Array in accordance to the Affymetrix GeneChip expression analysis technical manual (Affymetrix, Santa Clara, CA). After hybridization, arrays were washed and stained with Affymetrix fluidics protocol FS450_0001 and scanned with a 7G Affymetrix GeneChip Scanner. Image data were analyzed with Affymetrix Expression Console™ software and normalized with Robust Multichip Analysis (RMA; www.bioconductor.org/) to determine signal log ratios (CITE: Gentleman, R.C., Carey, V.J., Bates, D.M., Bolstad, B., Dettling, M., Dudoit, S., Ellis, B., Gautier, L., Ge, Y., Gentry, J., et al. (2004). Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5, R80.). The mean fold change was calculated from 3 independent technical replicates for each of the four experimental conditions and assessed by a non-parametric rank product test (CITE: Hong, F., Breitling, R., McEntee, C.W., Wittner, B.S., Nemhauser, J.L., and Chory, J. (2006). RankProd: a bioconductor package for detecting differentially expressed genes in meta-analysis. Bioinformatics 22, 2825-2827). Heat maps were generated with Genesis (Cite: Sturn, A., Quackenbush, J. and Trajanoski, Z. (2002) Genesis: cluster analysis of microarray data. Bioinformatics, 18, 207-208).
Project description:Expression of cell cycle related-genes after early cerebral ischemia in a mouse thrombotic stroke model: a transcriptomic coexpression analysis. Overall design: Cerebral ischemia was perfomed on Swiss male mice. Middle cerebral artery occlusion was performed by FeCl3 application on the intact dura mater. Mice were sacrified at 6 hours post-occlusion. Five independent experiments were performed.
Project description:Among the diversity of antibacterial immune effectors, mast cells (MCs) are emerging. Here, we show an unknown anti-bacterial mechanism of MCs against Coxiella burnetii, an intracellular bacterium responsible of Q fever. C. burnetii interaction with MCs induced the formation of extracellular actin filaments, named cytonemes. The formation of cytonemes was related to C. burnetii virulence. They mediated capture and destruction of entrapped-bacteria and their microbicidal activity was based on antimicrobial peptides including neutrophil elastase and cathelicidin. We provided evidence that MC cytoneme microbicidal activity including cytoneme formation depended on the cooperation of CD36, a scavenger receptor with Toll-like receptor-4 involved in C. burnetii recognition. Taken together, our results identified cytonemes as a non-previously reported antibacterial mechanism of MCs and suggested a protective role of MCs during C. burnetii infection. Overall design: The human mast cell line HMC-1.2 was cultured in Iscove's Modified Dulbecco's Medium (IMDM, Life Technologies) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 50 µg/ml streptomycin (Life Technologies) at 37°C.
Project description:Sleep deprivation (SD) performed before stroke induces an ischemic tolerance state as observed in other forms of preconditioning. As the mechanisms underlying this effect are not well understood we used DNA oligonucleotide microarray analysis, to identify the genes and the gene-pathways underlying SD preconditioning. Gene expression was analyzed 3 days after stroke surgery in four experimental groups: i) SD performed before focal cerebral ischemia induction; ii) SD performed before Sham surgery; iii) IS without SD; and iv) Sham surgery without SD. SD was performed by gentle handling during the last 6h of the light period and ischemia was induced immediately after. Stroke induced a massive alteration in gene expression both in sleep deprived and non-sleep deprived animals. However, compared to animals that underwent ischemia alone, SD induced a general reduction in transcriptional changes with a reduction in the upregulation of genes involved in cell cycle regulation and immune response. Moreover an upregulation of a new neuroendocrine pathway which included melanin concentrating hormone, glycoprotein hormones-ë±-polypeptide and hypocretin was observed exclusively in rats sleep deprived before stroke. Our data indicate that SD before stroke reprogrammed the signaling response to injury. The inhibition of cell cycle regulation and inflammation are neuroprotective mechanisms reported also for other forms of preconditioning treatment whereas the implication of the neuroendocrine function is novel and has never been described before. These results therefore provide new insights into neuroprotective mechanisms involved in ischemic tolerance mechanisms.
Project description:RABGEF1, a guanine nucleotide exchange factor for Rab5 GTPase, can negatively regulate mast cell activation in vitro. In addition, RabGEF1-deficient mice develop morbidity and severe skin inflammation associated with marked increases in skin mast cell numbers (Tam et al., Nat Immunol, 5(8):844-52, 2004). These findings suggest that over-reactive skin mast cells may contribute to the observed skin pathology in vivo. In order to identify the cell type(s) which contribute to skin inflammation when RabGEF1 is absent, we attempted to delete Rabgef1 gene specifically in three major skin cell types, namely mast cells, myeloid cells and keratinocytes; thus, Rabgef1 floxed (fl/fl) mice were crossed with mice expressing the Cre-recombinase under the influence of the promoter of Mcpt5, LysZ or K14, respectively. Unexpectedly, Mcpt5-Cre;Rabgef1fl/fl and Lysz-Cre;Rabgef1fl/fl mice appeared normal without phenotypic abnormalities. However, K14-Cre;Rabgef1fl/fl mice were normal at birth but rapidly developed morbidity and skin inflammation after 2-3 days, and died between 1 and 8 weeks of age. Skin (epidermis + dermis) was sampled from the center of the back behind shoulders, for 2 conditional knock-out mice (Rabgef1 lox/lox K14-Cre, hereafter called Rabgef1K-KO) and 2 control mice (Rabgef1 lox/lox). Total RNA was isolated from mouse skin using Trizol. Gene expression analysis was performed using Affymetrix Mouse Genome 430 2.0 arrays, with two replicates per genotype. Microarray data were analysed using Bioconductor for R.
Project description:Mast cell (MC) activation contributes considerably to immune responses, such as host protection and allergy. Cell surface immunoreceptors expressed on MCs play an important role in MC activation. Although various immunoreceptors on MCs have been identified, the regulatory mechanism of MC activation is not fully understood. To understand the regulatory mechanisms of MC activation, we used gene expression analyses of human MCs to identify a novel immunoreceptor expressed on MCs. We found that Tek, which encodes Tie2, was preferentially expressed in the MCs of humans. Overall design: Total RNA of peripheral blood (PB)-derived cultured MCs and other PR-derived cells were extracted by using an RNeasy Mini kit (Qiagen, Hilden, Germany), and a Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA) was used to remove ribosomal RNA. RNA-seq was performed according to the protocol described in the SOLiD Total RNA-Seq Kit (Life Technologies, Carlsbad, CA). The library was subjected to emulsion PCR (SOLiD™ EZ Bead™ Emulsifier kit, Life Technologies) to generate clonal DNA fragments on beads, followed by bead enrichment (SOLiD™ EZ Bead™ Enrichment kit, Life Technologies). Enriched template beads were sequenced on a SOLiD 5500xl sequencer as single-end, 75-bp reads (Life Technologies). The SOLiD 5500xl output reads were aligned against the human genome reference sequence (hg19) by using LifeScope version 2.5.1 (Life Technologies) to generate BAM files, and subsequent data analysis was performed in Avadis NGS (Strand Scientific Intelligence Inc., San Francisco, CA).
Project description:Interleukin-33 (IL-33) is elevated in afflicted tissues of patients with mast cell-dependent chronic allergic diseases. Based on its acute effects on mouse mast cells (MCs), IL-33 is thought to play a role in the pathogenesis of allergic disease through MC activation. However, the manifestations of chronic IL-33 exposure on human MC function, which best reflect the conditions associated with chronic allergic disease, are unknown. We now find that long-term exposure of human and mouse MCs to IL-33 results in a substantial reduction of MC activation in response to antigen. This reduction required >72 h exposure to IL-33 for onset and 1-2 wk for reversion following IL-33 removal. This hypo-responsive phenotype was determined to be a consequence of MyD88-dependent attenuation of signaling processes necessary for MC activation including antigen-mediated calcium mobilization and cytoskeletal reorganization; potentially as a consequence of down-regulation of the expression of PLCg1 and Hck. These findings suggest that IL-33 may play a protective, rather than a causative role in MC activation under chronic conditions and, furthermore, reveal regulated plasticity in the MC activation phenotype. The ability to down-regulate MC activation in this manner may provide alternative approaches for treatment of MC-driven disease. Overall design: Mouse bone marrow-derived mast cells treated with IL3 or IL3+IL33. 6 replicates each.
Project description:The Forkhead Box f1 (Foxf1) transcriptional factor (previously known as HFH-8 or Freac-1) is expressed in endothelial and smooth muscle cells in the embryonic and adult lung. To assess effects of Foxf1 during lung injury, we used CCl4 injury model. Foxf1+/- mice developed severe airway obstruction and bronchial edema, associated with increased numbers of pulmonary mast cells and increased mast cell degranulation following injury. Pulmonary inflammation in Foxf1+/- mice was associated with diminished expression of Foxf1, increased mast cell tryptase and increased expression of CXCL12, the latter being essential for mast cell migration and chemotaxis. Foxf1 haploinsufficiency caused pulmonary mastocytosis and enhanced pulmonary inflammation following chemically-induced lung injury, indicating an important role for Foxf1 in the pathogenesis of pulmonary inflammatory responses. Keywords: Influence of genetic modification on the pulmonary inflamation Foxf1+/- mice in which the Foxf1 allele was disrupted by an in-frame insertion of a nuclear localizing -galactosidase (-Gal) gene were bred for ten generations into the Black Swiss mouse genetic background. Carbon tetrachloride (CCl4; Sigma, St Louis, MO) was dissolved in mineral oil at a 1:20 ratio v/v and a single intraperitoneal (i.p.) injection of CCl4 (0.5 l of CCl4/ 1g of body weight) was administered to male Foxf1+/- mice or their wild type (WT) littermates as described.
Project description:Interleukin-33 (IL-33) is elevated in afflicted tissues of patients with mast cell-dependent chronic allergic diseases. Based on its acute effects on mouse mast cells (MCs), IL-33 is thought to play a role in the pathogenesis of allergic disease through MC activation. However, the manifestations of chronic IL-33 exposure on human MC function, which best reflect the conditions associated with chronic allergic disease, are unknown. We now find that long-term exposure of human and mouse MCs to IL-33 results in a substantial reduction of MC activation in response to antigen. This reduction required >72 h exposure to IL-33 for onset and 1-2 wk for reversion following IL-33 removal. This hypo-responsive phenotype was determined to be a consequence of MyD88-dependent attenuation of signaling processes necessary for MC activation including antigen-mediated calcium mobilization and cytoskeletal reorganization; potentially as a consequence of down-regulation of the expression of PLCg1 and Hck. These findings suggest that IL-33 may play a protective, rather than a causative role in MC activation under chronic conditions and, furthermore, reveal regulated plasticity in the MC activation phenotype. The ability to down-regulate MC activation in this manner may provide alternative approaches for treatment of MC-driven disease. Mouse bone marrow-derived mast cells treated with IL3 or IL3+IL33. 6 replicates each.