Highly dynamic molecular signature of macrophage subsets during sterile inflammation, resolution and tissue repair
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ABSTRACT: Tibialis anterior muscle was damaged by cardiotoxin injection and macrophage subsets were isolated and analyzed by gene expression analysis. We used microarray to obtain global gene expression data of muscle-derived tissue macrophage subsets. Tissue macrophages were collected from regenerating muscle samples, Gr1+/Cx3cr1low and Gr1-/Cx3cr1high macrophage subsets were sorted. The global gene expression patterns of distinct macrophage subsets were analyzed on Affymetrix microarrays.
Project description:Muscle injury was elicited by cardiotoxin injection into the tibialis anterior muscle. Macrophages were isolated 2 days post-injury from the regenerating muscle. We used microarray to obtain global gene expression data of muscle-derived tissue macrophage subsets. Tissue macrophages were collected from regenerating muscle samples of three animals, Ly6C+ F4/80low and Ly6C- F4/80high macrophage subsets were sorted. The global gene expression patterns of distinct macrophage subsets were analyzed on Affymetrix microarrays.
Project description:Muscle injury was elicited by cardiotoxin injection into the tibialis anterior muscle. Macrophages were isolated 2 days post-injury from the regenerating muscle. We used microarray to obtain global gene expression data of muscle-derived tissue macrophage subsets. Tissue macrophages were collected from regenerating muscle samples of three animals, Ly6C+ F4/80low and Ly6C- F4/80high macrophage subsets were sorted. The global gene expression patterns of distinct macrophage subsets were analyzed on Affymetrix microarrays.
Project description:Molecular profiling of infiltrating monocyte-derived macrophages versus resident kupffer cells following acute liver injury The liver has a remarkable capacity to regenerate after injury; yet, the role of macrophages (MF) in this process remains controversial mainly due to difficulties in distinguishing between different MF-subsets. Here, we utilized a murine model of acute liver injury caused by overdose of acetaminophen (APAP) and defined three distinct MF subsets that populate the liver following injury. Accordingly, resident Kupffer cells (KC) were significantly reduced upon APAP-challenge and started recovering by self-renewal at resolution phase without contribution of circulating Ly6Chi monocytes. The latter were recruited in a CCR2 and M-CSF mediated pathway at the necro-inflammatory phase and differentiated into ephemeral Ly6Clo MF subset at resolution phase. Moreover, their inducible ablation resulted in impaired recovery. Microarray based molecular profiling uncovered high similarity between steady state KC and those recovered at the resolution phase. In contrast, KC and monocyte-derived MF displayed distinct pro-restorative genetic signature at the resolution phase. Finally, we show that infiltrating monocytes acquire a pro-restorative polarization manifested by unique expression of pro-angiogenesis mediators and genes involved with inhibition of neutrophil activity and recruitment and promotion of their clearance. Collectively, our results present a novel phenotypic, ontogenic and molecular definition of liver-MF compartment following acute injury. 11 Samples (arrays) were performed. We generated pairwise comparison between all the different macrophages stages, using Partek Genomics Suite. Genes with p?5%[FDR] and a fold-change difference of ?2 or <-2 were selected.
Project description:Splenic red pulp macrophages (RPM) degrade senescent erythrocytes and recycle heme-associated iron. The transcription factor Spic is selectively expressed by RPM and is required for their development, but the physiologic stimulus inducing Spic is unknown. Here, we report that Spic also regulated the development of F4/80+VCAM+ bone marrow macrophages (BMM) and that Spic expression in BMM and RPM development was induced by heme, a metabolite of erythrocyte degradation. Pathologic hemolysis induced loss of RPM and BMM due to excess heme but induced Spic in monocytes to generate new RPM and BMM. Spic expression in monocytes was constitutively inhibited by the transcriptional repressor Bach1. Heme induced proteasome-dependent BACH1 degradation and rapid Spic derepression. Further, cysteine-proline dipeptide motifs in BACH1 that mediate heme-dependent degradation were necessary for Spic induction by heme. These findings are the first example of metabolite-driven differentiation of a tissue-resident macrophage subset and provide new insight into iron homeostasis. Global gene expression pattern of Spic+ monocytes, Spic- monocytes, and Spic high red pulp macrophages were compared by sorting these cells from Spic(igfp/+) splenocytes and performing microarray-based gene expression profiling. Splenocytes were prepared from Spic(igfp/+) mice and were first negatively selected for CD4, CD8, and B220 by MACS (Miltenyi Biotech) purification using the respective microbeads. Negatively selected splenocytes were then stained with anti-CD11b and anti-Ly6c and sorted for Spic+ monocytes (CD11b+Ly6c+Spic+) and Spic- monocytes (CD11b+Ly6c+Spic-). Purified RPM were obtained by staining splenocytes with anti-F4/80 and sorting for F4/80 hi Spic-EGFP hi cells.
Project description:We have found the existence of two independent populations contributing to the skin-resident macrophage pool based on their different origin. We have analyzed their gene profile by deep-sequencing (RNA-Seq). Analysis of RNA-Seq data revealed a differential expression signature between both subsets of skin macrophages for 744 of 17741 genes compiled (198 of them showing similar normalized expression levels across replicates). We have further characterized their specialized functions related to their different gene profiles. Examination of gene profile of 2 different macrophage subsets coexisting in skin under steady state.
Project description:Satellite cells are resident skeletal muscle stem cells responsible for muscle maintenance and repair. In resting muscle, satellite cells are maintained in a quiescent state. Satellite cell activation induces the myogenic commitment factor, MyoD, and cell cycle entry to facilitate transition to a population of proliferating myoblasts that eventually exit the cycle and regenerate muscle tissue. The molecular mechanism involved in the transition of a quiescent satellite cell to a transit-amplifying myoblast is poorly understood. We used microarrays to detail the global program of gene expression of in vivo satellite cell activation through muscle injury and identified RNA post-transcriptional regulation as a key component of satellite cell activation. Wild type or Sdc4-/- satellite cells were FACS isolated from resting muscle or from muscle 12h and 48h following barium chloride-induced muscle injury. 5000 cell equivalents of RNA was labeled and hybridized to MOE430v2 GeneChips (Affymetrix) and scanned as per manufacturers protocol. Probeset intensities were GCRMA normalized for further analysis including UPGMA hierarchical clustering, analysis of variance (ANOVA), and fold change.
Project description:Six-weeks old (C57Bl6, Cx3cr1gfp/+) mice were intraperitonealy infected with a low number (1.104) of L. monocytogenes (EGDe strain) in exponential growth phase (bacteria were grown in BHI at 108/ml, and diluted 10.000x in PBS immediately before injection). Group of three mice were euthanized, before infection. Peritoneal cells were recovered by peritoneal lavage. Cells from individual mice were stained with antibodies to CD11b (PECy7), Gr1 (APC), NK1.1, B220 and CD3 (PE), and F4/80 (biotin-conjugated followed by streptavidinpacific blue) for sorting. Gr1- monocytes were purified as NK1.1- CD3- B220- CD11b+ F4/80low Gr1-, gfphigh; Gr1+ monocytes were purified as NK1.1- CD3- B220- CD11b+ F4/80low Gr1+, gfpint; and polymorphonuclear cells were purified as NK1.1- CD3- B220- CD11b+ F4/80- Gr1high, gfp-. 1.103 cells from each mice, time point, and phenotype were purified by facs sorting according to their phenotype. Samples were kept at 4°C before and during the sort. Cells were directly sorted in the SuperAmp Lysis Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) using a FACS Aria cell-sorter (BD biosciences).
Project description:We compared arginase-1+ macrophages (macrophages were defined by flow cytometry as CD45hi CD11b+ Ly6G-) with arginase-1- brain macrophages following traumatic brain injury (TBI) by isolating these cells from YARG transgenic mice, which express YFP under the arginase-1 promoter. Both cell populations were isolated from YARG brain tissues one day following TBI. We also examined the expression profile of peripheral blood monocytes (monocytes were defined by flow cytometry as CD11bhi F4/80+) from injured YARG mice and from normal YARG mice. Peripheral blood samples were compared to TBI brain macrophages to assess gene expression changes before and after infiltration into the brain. TBI macrophage subsets were identified by using a reporter mouse strain (YARG) that expresses eYFP from an IRES inserted at the 3' end of the gene for arginase-1 (Arg1), a hallmark of alternatively activated (M2) macrophages. One day after TBI, 21±1.5% of ipsilateral brain macrophages expressed relatively high levels of Arg1 as detected by YFP. Gene expression analysis of Arg1+ and Arg1- brain macrophage populations revealed that these populations were distinct from either classically activated (M1) macrophages or M2 macrophages, with features of both. The Arg1+ cells differed from Arg1- cells in multiple aspects, most notably in their chemokine repertoires. Thus, the macrophage response to TBI involves recruitment of at least two major macrophage subsets. Overall, our data indicate that the macrophage response to TBI is heterogeneous and unique. Four groups (Arg1- brain macrophages post-TBI, Arg1+ brain macrophages post-TBI, normal blood monocytes, blood monocytes post-TBI) were analyzed. Four replicates of each group were analyzed for a total of 16 samples (only 3 replicates of the blood monocyte groups are included in this submission).
Project description:We have employed genome-wide transcriptome analysis to characterise the changes in expression that occur between resting and regenerating muscle, and the influence p38-alpha has on these expression profiles.