Transcriptional profiles of macrophages in resolving inflammation
ABSTRACT: We have performed a comprehensive transcriptional analysis of specific monocyte and macrophage (MØ) subsets during an acute self-resolving inflammatory insult. Following initial induction of acute inflammation, tissue resident (Resident) MØ are rapidly ‘cleared’ from the inflammatory foci, only becoming recoverable as inflammation resolves. Monocytes are recruited to the inflammatory lesion where they differentiate into MØ. We term these monocyte-derived MØ ‘inflammation-associated’ to distinguish them from Resident MØ which are present throughout the inflammatory response and can renew during the resolution of inflammation by proliferation. Comparative analysis of the Mo and MØ populations (both ‘inflammation-associated’ and Resident MØ) identifies select genes expressed in subsets of ‘inflammation-associated’ and Resident MØ that play important roles in the resolution of inflammation and/or for immunity, including molecules involved in antigen presentation, cell cycle and others associated with ‘immaturity’ and MØ activation. Overall design: We purified monocyte and macrophage populations from the peritoneal cavity of C57BL/6 mice 4, 18, 72 and 168 hours after the induction of inflammation with intraperitoneal administration of zymosan (2x10^7 particles). We also purified tissue resident macrophages and Ly-6B+ bone marrow monocytes from naive mice as reference populations.
Project description:We have performed a comprehensive transcriptional analysis of specific monocyte and macrophage (MØ) subsets during an acute self-resolving inflammatory insult. Following initial induction of acute inflammation, tissue resident (Resident) MØ are rapidly ‘cleared’ from the inflammatory foci, only becoming recoverable as inflammation resolves. Monocytes are recruited to the inflammatory lesion where they differentiate into MØ. We term these monocyte-derived MØ ‘inflammation-associated’ to distinguish them from Resident MØ which are present throughout the inflammatory response and can renew during the resolution of inflammation by proliferation. Comparative analysis of the Mo and MØ populations (both ‘inflammation-associated’ and Resident MØ) identifies select genes expressed in subsets of ‘inflammation-associated’ and Resident MØ that play important roles in the resolution of inflammation and/or for immunity, including molecules involved in antigen presentation, cell cycle and others associated with ‘immaturity’ and MØ activation. We purified monocyte and macrophage populations from the peritoneal cavity of C57BL/6 mice 4, 18, 72 and 168 hours after the induction of inflammation with intraperitoneal administration of zymosan (2x10^7 particles). We also purified tissue resident macrophages and Ly-6B+ bone marrow monocytes from naive mice as reference populations.
Project description:Human and mouse monocytes can be divided into 2 different sub-populations, using CD14-CD16 and Ly6C-CX3CR1 respectively. We investigated the pig monocytes sub-populations and found that all porcine monocyte express CD16 and CD172α but can be divided into 2 subpopulation using CD14 and the scavenger receptor CD163. The CD14hi-CD163low population resemble to the inflammatory monocytes whereas the CD14low-CD163hi display more a resident monocyte type. Pig monocyte can be differentiated into macrophages when cultured with rhCSF-1 and show an increase in size, granularity and autofluorescence, and express the common macrophage markers CD14, CD16 and CD172α. Gene expression in these 2 sub-populations was profiled using the newly-developed and annotated pig whole genome snowball microarray, showing a distinct pattern between inflammatory and resident monocytes but this difference would be more a maturation process instead of two separate subsets. Furthermore, the expression of certain genes such as CD36, CLEC4E or TREM-1 proved to share the same pattern as human monocytes, quite different from mouse monocytes. These results emphasize the potential role of the pigs as a model for human inflammatory disease and will improved our knowledge on the mononuclear phagocyte system development. Porcine PBMCs were isolated from the blood of three seperate pigs, FACS sorted on expression of CD14 and CD163 and RNA isolated from each sample, a total of 6 microarrays were hybridised
Project description:LPS treatment is considered as the paradigmatic stimulus for acquisition of the classical macrophage polarization/activation state. However, this definition does not take into account the influence that other macrophage differentiating/priming factors might have on the LPS response. In spite of the pathophysiological relevance of the response of M-CSF-dependent anti-inflammatory human macrophages (M-MØ) to endogenous and pathogenic stimuli, detailed analysis of the gene signature of activated anti-inflammatory M-CSF-dependent macrophages remained to be determined. We have now compared the early transcriptional signatures of human GM-MØ and M-MØ in response to macrophage-activating agents and identified a unique gene set acquired upon activation of anti-inflammatory M-MØ. Overall design: Macrophages were differentiated from peripheral blood monocytes from 3 healthy donors with M-CSF or GM-CSF to generate anti-inflammatory (M-MØ) or pro-inflammatory (GM-MØ) macrophages, respectively. M-MØ and GM-MØ were stimulated with 10ng/ml LPS for 4h.
Project description:Dunster2014 - WBC Interactions (Model1)
This is a sub-model of a three-step
inflammatory response modelling study. The model includes distinct
populations of white blood cells namely, macrophages and active and
apoptotic neutrophil populations. Neutrophil apoptosis rate is
predicted to be crucial for the qualitative nature of the
This model is described in the article:
The resolution of
inflammation: a mathematical model of neutrophil and macrophage
Dunster JL, Byrne HM, King JR.
Bull. Math. Biol. 2014 Aug; 76(8):
There is growing interest in inflammation due to its
involvement in many diverse medical conditions, including
Alzheimer's disease, cancer, arthritis and asthma. The
traditional view that resolution of inflammation is a passive
process is now being superceded by an alternative hypothesis
whereby its resolution is an active, anti-inflammatory process
that can be manipulated therapeutically. This shift in mindset
has stimulated a resurgence of interest in the biological
mechanisms by which inflammation resolves. The
anti-inflammatory processes central to the resolution of
inflammation revolve around macrophages and are closely related
to pro-inflammatory processes mediated by neutrophils and their
ability to damage healthy tissue. We develop a spatially
averaged model of inflammation centring on its resolution,
accounting for populations of neutrophils and macrophages and
incorporating both pro- and anti-inflammatory processes. Our
ordinary differential equation model exhibits two outcomes that
we relate to healthy and unhealthy states. We use bifurcation
analysis to investigate how variation in the system parameters
affects its outcome. We find that therapeutic manipulation of
the rate of macrophage phagocytosis can aid in resolving
inflammation but success is critically dependent on the rate of
neutrophil apoptosis. Indeed our model predicts that an
effective treatment protocol would take a dual approach,
targeting macrophage phagocytosis alongside neutrophil
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Project description:The interplay between the inflammatory infiltrate and tissue resident cell populations (e.g. epithelial cells, fibroblasts and macrophages) invokes fibrogenesis. However, the temporal and mechanistic contributions of these cell populations to fibrosis remain poorly defined. To address this issue, liver inflammation, ductular reaction (DR) and fibrosis were induced in C57BL/6 mice by thioacetamide (TAA) administration for up to 12 weeks. TAA treatment induced two phases of liver fibrosis. A rapid peri-central inflammatory infiltrate enriched in F4/80+ monocytes co-localized with SMA+ myofibroblasts resulted in early collagen deposition, marking the start of an initial fibrotic phase (1-6 weeks). An expansion of bone marrow derived macrophages proceeded a second phase, characterized by accelerated progression of fibrosis (> 6 weeks) followed the migration of the DR from the portal tracts to the centrilobular site of injury, in association an increase in DR/macrophage interactions. Although CCL2 mRNA was rapidly induced in response to TAA, CCL2 deficiency only partially abrogated fibrosis. In contrast, CSF-1R blockade diminished CCR2neg (Ly6Clo) monocytes, attenuated the DR and significantly reduced fibrosis, illustrating the critical role of CSF-1 dependent monocyte/macrophage differentiation and linking the two phases of injury. We demonstrate that in response to liver injury, CSF-1 drives early monocyte mediated myofibroblast activation and collagen deposition, subsequent macrophage differentiation and their association with the advancing DR, the formation of fibrotic septa and the progression of liver fibrosis to cirrhosis. Overall design: Single colour, Illumina MouseRef-8 v2.0 Beadarrays.
Project description:Tissue resident macrophages are notoriously heterogeneous, exhibiting discrete phenotypes as a consequence of tissue- and micro-anatomical niche-specific functions, but the molecular basis for this is not understood. We resolved a restricted transcriptional profile for the self-renewing population of peritoneal resident macrophages, which is expressed during homeostasis and inflammation and distinct from other MØ. Prominent within this profile was the expression of Gata6. This study represents a characterisation of the role of Gata6 in peritoneal resident macrophage phenotype. We used microarrays to determine the patterns of gene expression in peritoneal resident MØ in the absence of GATA-6 against wild type. Conditional 'floxed' Gata6 deficient sex-matched mice between 7 weeks old were compared against wild type
Project description:PPARγ is known for its anti-inflammatory actions in macrophages. However, which macrophage populations express PPARγ in vivo and how it regulates tissue homeostasis in the steady state and during inflammation is not completely understood. We show that lung and spleen macrophages constitutively expressed PPARγ, while other macrophage populations did not. Recruitment of monocytes to sites of inflammation was associated with induction of PPARγ as they differentiated to macrophages. Its absence in these macrophages led to failed resolution of inflammation, characterized by persistent, low-level recruitment of leukocytes. Conversely, PPARγ agonists supported an earlier cessation in leukocyte recruitment during resolution of acute inflammation and likewise suppressed monocyte recruitment to chronically inflamed atherosclerotic vessels. In the steady state, PPARγ deficiency in macrophages had no obvious impact in the spleen but profoundly altered cellular lipid homeostasis in lung macrophages. Reminiscent of pulmonary alveolar proteinosis, LysM-Cre x PPARγflox/flox mice displayed mild leukocytic inflammation in the steady-state lung and succumbed faster to mortality upon infection with S. pneumoniae. Surprisingly, this mortality was not due to overly exuberant inflammation, but instead to impaired bacterial clearance. Thus, in addition to its anti-inflammatory role in promoting resolution of inflammation, PPARγ sustains functionality in lung macrophages and thereby has a pivotal role in supporting pulmonary host defense. The two major subsets of monocytes (Ly-6C+ and Ly-6Clo) from 12-week old C57Bl/6 mice were sorted and the RNA extracted and hybridized to Affymetrix GeneChip® 430 2.0 arrays. We pooled leukocytes from 5 mice for each sort and sorted 3 to 4 separate times for 3 to 4 biological replicates.
Project description:This study aims to characterize the transcriptional profile of Granulocyte-macrophage colony-stimulating factor induced macrophages (GM-MØ) and M-CSF macrophages (M-MØ) and to investigate in situ a subset of genes and their products specific to each phenotype in human atherosclerosis plaques 18 RNG/MRC two-colour oligonucleotide microarrays (Le Brigand et al. 2006) were used to generate global mRNA expression profiles for GM-CSF-induced, M-CSF-induced, and peritoneal macrophages. The microarray experiments were conducted either as a common reference (peritoneal-like macrophages) design or using a direct design by hybridizing Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced human monocyte-derived macrophage (GM-MØ) and CSF-induced human monocyte-derived macrophages(M-MØ) on the same arrays
Project description:Human monocytes are a heterogeneous cell population consisting of three subsets: classical CD14++CD16-, intermediate CD14++CD16+ and nonclassical CD14+CD16++ monocytes. Intermediate monocytes contribute to inflammation, and their circulating cell counts are increased in many chronic inflammatory diseases, even though the underlying pathways are poorly characterized. We tested in how far epigenetic mechanisms regulate human monocytes heterogeneity in chronic kidney disease, which is a proinflammatory condition of substantial epidemiological importance. By applying next-generation Methyl-Sequencing (Methyl-Seq) we characterized genome-wide DNA methylation within the three monocyte subsets and analyzed the impact of uremia on DNA methylation in differentiating monocytes. We found that each monocyte subset displays a unique phenotype with regards to DNA methylation. Genes with differentially methylated promoter regions in intermediate monocytes were linked to distinct immunological processes, which is in line with results from recent gene expression analyses. In vitro, uremia induced a dysregulation of DNA methylation in differentiating monocytes which affected several transcription factors important for monocyte differentiation (e.g. FLT3, HDAC1, MNT) and led to enhanced generation of intermediate monocytes. As a potential mediator, the uremic toxin and DNA methylation inhibitor S-Adenosylhomocysteine induced shifts in monocyte subsets in vitro, and associated with monocyte subset counts in vivo. In summary, our data support the concept of monocyte trichotomy and the distinct role of intermediate monocytes in human immunity. The shift in monocyte subsets which occurs in chronic kidney disease, a proinflammatory condition of substantial epidemiological impact, may be induced by the accumulation of specific uremic toxins that mediate epigenetic dysregulation. Overall design: Seven healthy donors were recruited for DNA methylation analysis of the three monocyte subsets; DNA methylation analysis of differentiating monocytes was performed in 5 independent experiments
Project description:Mutations in methyl-CpG-binding protein 2 (MeCP2), a major epigenetic regulator, are the predominant cause of Rett syndrome, an X-linked neurodevelopmental disorder. We previously found that Mecp2-null microglia are functionally impaired, and that engraftment of wild-type monocytes into the brain of Mecp2-deficient mice attenuates pathology. In this study we show that Mecp2 is expressed in macrophage and monocyte populations throughout the body, and is indispensable for their transcriptional regulation in multiple contexts. We demonstrate that Mecp2-null mice progressively lose or are chronically deficient in several macrophage populations and resident monocytes. Postnatal re-expression of Mecp2 driven by a tamoxifen-inducible CX3CR1 promoter significantly increased the lifespan of otherwise Mecp2-null mice, suggesting that epigenetic regulation of macrophage function by Mecp2 significantly contributes to pathology. RNA-Seq of acutely isolated microglia and peritoneal macrophages (to our knowledge, the first cell-specific RNA-Seq analysis comparing Mecp2-null and wild type cells of any kind) revealed significantly increased transcription of glucocorticoid- and hypoxia-signaling genes in Mecp2-null cells compared to that in their wild-type counterparts, suggesting that Mecp2 functions as a repressor of these pathways. Furthermore, in-vitro and in vivo validation studies demonstrated that the absence of Mecp2 is associated with cell-intrinsic dysfunction of signaling underlying inflammatory activation, suggesting that Mecp2 is important for regulation of specific macrophage gene-expression programs in response to stimuli and stressors. Our findings demonstrate a fundamental role for Mecp2 in the regulation of macrophage functions, which may provide a link to pathologies in Rett syndrome across multiple organs. Mecp2-null microglia and resident peritoneal macrophages from 10-12 week old mice were acutely isolated via AutoMACS, total RNA collected, and analyzed via RNA-Seq to compare for transcriptional differences in microglia and macrophages in the absence of Mecp2.