Dataset Information

Alterations in the membrane-associated proteome of alveolar macrophages in sarcoidosis

ABSTRACT: Here we have performed qualitative profiling of the membrane-associated proteome of alveolar macrophages in sarcoidosis patients and healthy individuals with the aim to identify specific proteins and pathways involved in sarcoidosis pathology.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lung, Macrophage

DISEASE(S): Pulmonary Sarcoidosis

SUBMITTER: Hanna Kjellin

LAB HEAD: Janne Lehtiö

PROVIDER: PXD000752 | Pride | 2016-12-23

REPOSITORIES: Pride

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			Action	DRS
	100524_sarcoidosis_MAP_exp1_fr19-54.msf	Msf
	100524_sarcoidosis_MAP_exp1_fr19-54.pep.xml	Pepxml
	100524_sarcoidosis_MAP_exp1_fr19-54.prot.xml	Xml
	100524_sarcoidosis_MAP_exp1_fr19.raw	Raw
	100524_sarcoidosis_MAP_exp1_fr20.raw	Raw

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Publications

Alterations in the membrane-associated proteome fraction of alveolar macrophages in sarcoidosis.

Kjellin Hanna H Silva Ernesto E Branca Rui Mamede RM Eklund Anders A Jakobsson Per-Johan PJ Grunewald Johan J Lehtiö Janne J Wheelock Åsa M ÅM

Sarcoidosis, vasculitis, and diffuse lung diseases : official journal of WASOG 20160329 1

<h4>Background</h4>Alveolar macrophages are implicated in the pathogenesis of lung sarcoidosis. Their interaction with T-cells leads to an inflammatory response that may either resolve within 2 years, or become chronic with an increased risk to develop lung fibrosis.<h4>Objective</h4>To perform quantitative profiling of the membrane-associated proteome of alveolar macrophages in sarcoidosis patients and healthy individuals to identify specific proteins and pathways involved in sarcoidosis pathol ...[more]

PMID: 27055832

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Project description:Rationale: A well-validated animal model of sarcoidosis can help advance our understanding of the pathobiology of this complex disease. We have developed a multiwall carbon nanotube (MWCNT) based murine model that shows marked histological and inflammatory signal similarities to human sarcoidosis. In this study, we compared the alveolar immune cell transcriptional signatures of our murine model with human sarcoidosis to comprehensively assess overlapping molecular programs. Methods: Total RNA was extracted from alveolar macrophages of 6 MWCNT mice and 6 control mice and hybridized to Illumina Mouse WG-6 v2 microarrays. Transcriptional profiling of alveolar immune cells in 15 sarcoidosis patients and 12 healthy humans was previously performed using Affymetrix Human GeneChip U133A 2.0 microarrays. Rigorous statistical methods were used to identify differentially expressed genes. To better elucidate gene expression pathways, integrated network and gene set enrichment analysis (GSEA) were applied. To confirm differential expression of select network genes, we performed qPCR studies using the ABI Prism 7300 Detection. Results: We identified over 1000 genes that were differentially expressed between the control and the MWCNT mice. Gene Ontology functional analysis showed over-representation of processes primarily involved in immunity and inflammation in MCWNT mice. Applying a more comprehensive pathway analysis using GSEA to both mouse and human samples revealed upregulation of 92 gene sets in MWCNT mice and 142 gene sets in sarcoidosis patients. Commonly activated pathways in both MWCNT mice and sarcoidosis included adaptive immunity, T-cell signaling, IL-12/IL-17 signaling and oxidative phosphorylation. Differences in selective gene set enrichment between MWCNT mice and sarcoidosis patients were also observed, among them IL-27 signaling, NK-mediated cytotoxicity and proteasome pathways. We applied network analysis to differentially expressed genes common between the MWCNT model and human sarcoidosis to identify key drivers of disease and putative therapeutic targets. Conclusions: Our MWCNT murine model has an alveolar macrophage signature that varies significantly from control mice. Application of an unbiased transcriptomic approach revealed substantial functional similarities between a murine MWCNT model and human sarcoidosis particularly with respect to activation of immune-specific pathways.

Project description:Introduction: Sarcoidosis is a multisystem immuno-inflammatory disorder of unknown etiology that most commonly involves the lungs. We hypothesized that an unbiased approach to identify pathways activated in alveolar macrophagesâa key immuno-inflammatory cell in the lungâcan shed light on the pathogenesis of this complex disease. Methods: We recruited 15 patients with various stages of sarcoidosis and 12 healthy controls. All subjects underwent bronchoscopy with lavage. For each subject, total RNA was extracted from bronchoalveolar (BAL) cells and hybridized to an Affymetrix GeneChip Human Genome U133A 2.0 microarray. Rigorous statistical methods were applied to identify differential gene expression between subjects with sarcoidosis vs. controls. To better elucidate pathways differentially activated between these groups, we applied gene set enrichment analysis (GSEA) to the transcriptional profiles of BAL cells. We used false discovery rate (FDR) < 0.01 to designate significant enrichment. Results: Sarcoid patients were either non-smokers or ex-smokers, all had lung involvement and only 2 were on systemic prednisone. Healthy controls were all non-smokers. Comparison of BAL cell gene expression between sarcoidosis and healthy subjects revealed over 1200 differentially expressed genes at an FDR cutoff < 0.01. Several previously described immune mediators, such as interferon gamma, were up-regulated in the sarcoidosis subjects. Since genes often exert their influence through functionally coherent modules, we performed GSEA based on global expression profiles of alveolar macrophages between the subject groups. We identified more than 200 gene sets enriched in patients with sarcoidosis whereas very few pathways were over-represented in the healthy controls. Many of the sarcoidosis-associated pathways mapped to inflammatory and immune-related processes including T-cell signaling, graft vs. host disease, IL-12, IL-23, and IL-17 pathways, and oxidative phosphorylation. However, we also found and confirmed significant alteration in Proteasome-related pathways. Conclusions: BAL cells in sarcoidosis are characterized by enrichment of distinct transcriptional networks involved in immuno-inflammatory and proteasomal processes. Our findings add to the growing evidence implicating airspace resident cells in the pathogenesis of sarcoidoisis and identify specific pathways whose activation may modulate disease progression. Total RNA from BAL cells of 15 subjects with sarcoidosis and 12 healthy controls was hybridized to 27 Affymetrix Genechip Human U133A 2.0 microarrays

Project description:Introduction: Sarcoidosis is a multisystem immuno-inflammatory disorder of unknown etiology that most commonly involves the lungs. We hypothesized that an unbiased approach to identify pathways activated in alveolar macrophages—a key immuno-inflammatory cell in the lung—can shed light on the pathogenesis of this complex disease. Methods: We recruited 15 patients with various stages of sarcoidosis and 12 healthy controls. All subjects underwent bronchoscopy with lavage. For each subject, total RNA was extracted from bronchoalveolar (BAL) cells and hybridized to an Affymetrix GeneChip Human Genome U133A 2.0 microarray. Rigorous statistical methods were applied to identify differential gene expression between subjects with sarcoidosis vs. controls. To better elucidate pathways differentially activated between these groups, we applied gene set enrichment analysis (GSEA) to the transcriptional profiles of BAL cells. We used false discovery rate (FDR) < 0.01 to designate significant enrichment. Results: Sarcoid patients were either non-smokers or ex-smokers, all had lung involvement and only 2 were on systemic prednisone. Healthy controls were all non-smokers. Comparison of BAL cell gene expression between sarcoidosis and healthy subjects revealed over 1200 differentially expressed genes at an FDR cutoff < 0.01. Several previously described immune mediators, such as interferon gamma, were up-regulated in the sarcoidosis subjects. Since genes often exert their influence through functionally coherent modules, we performed GSEA based on global expression profiles of alveolar macrophages between the subject groups. We identified more than 200 gene sets enriched in patients with sarcoidosis whereas very few pathways were over-represented in the healthy controls. Many of the sarcoidosis-associated pathways mapped to inflammatory and immune-related processes including T-cell signaling, graft vs. host disease, IL-12, IL-23, and IL-17 pathways, and oxidative phosphorylation. However, we also found and confirmed significant alteration in Proteasome-related pathways. Conclusions: BAL cells in sarcoidosis are characterized by enrichment of distinct transcriptional networks involved in immuno-inflammatory and proteasomal processes. Our findings add to the growing evidence implicating airspace resident cells in the pathogenesis of sarcoidoisis and identify specific pathways whose activation may modulate disease progression.

Project description:Cardiac involvement is an important determinant of mortality amongst sarcoidosis patients. While granulomatous inflammation is a hallmark finding in cardiac sarcoidosis, the precise immune cell populations that comprise the granuloma remain unresolved. Furthermore, it is unclear how the cellular and transcriptomic landscape of cardiac sarcoidosis differs from other inflammatory heart diseases. We leveraged spatial transcriptomics (GeoMx DSP) and single nucleus RNA sequencing (snRNAseq) to elucidate the cellular and transcriptional landscape of cardiac sarcoidosis. Using GeoMX DSP technology, we compared the transcriptomal profile of CD68+ rich immune cell infiltrates in human cardiac sarcoidosis, giant cell myocarditis, and lymphocytic myocarditis. We performed snRNAseq of human cardiac sarcoidosis to identify immune cell types and examined their transcriptomic landscape and regulation. Using multi-channel immunofluorescence staining, we validated immune cell populations identified by snRNAseq, determined their spatial relationship, and devised an immunostaining approach to distinguish cardiac sarcoidosis from other inflammatory heart diseases. Despite overlapping histological features, spatial transcriptomics identified transcriptional signatures and associated pathways that robustly differentiated cardiac sarcoidosis from giant cell myocarditis and lymphocytic myocarditis. snRNAseq revealed the presence of diverse populations of myeloid cells in cardiac sarcoidosis with distinct molecular features. We identified GPNMB as a novel marker of multinucleated giant cells and predicted that the MITF family of transcription factors regulated this cell type. We also detected additional macrophage populations in cardiac sarcoidosis including HLA-DR+ macrophages, SYTL3+ macrophages and CD163+ resident macrophages. HLA-DR+ macrophages were found immediately adjacent to GPMMB+ giant cells, a distinct feature compared with other inflammatory cardiac diseases. SYTL3+ macrophages were located scattered throughout the granuloma and CD163+ macrophages, CD1c+ dendritic cells, non-classical monocytes, and T-cells were located at the periphery and outside of the granuloma. Finally, we demonstrate mTOR pathway activation is associated with proliferation and is selectively found in HLA-DR+ and SYLT3+ macrophages. In this study, we identified diverse populations of immune cells with distinct molecular signatures that comprise the sarcoid granuloma. These findings provide new insights into the pathology of cardiac sarcoidosis and highlight opportunities to improve diagnostic testing.