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Neutrophil gene expression in response to Mycobacterium abscessus

ABSTRACT: The response of human neutrophils to the emerging pathogen Mycobacterium abscessus has not been described. However, M. abscessus infections are frequently associated with neutrophil-rich abscesses. To better understand the reponse of neutrophils to M. abscessus we performed gene expression analysis using Affymetrix HG-U133A Plus 2.0 microarrays. Human neutrophils from healthy donors were stimulated with isogenic rough and smooth morphotypes of M. abscessus. Staphylococcus aureus was used as a control. Gene expression was compared to neutrophils left unstimulated. Neutrophils from four individual donors were isolated on separate days and stimulated with freshly prepared bacteria. Neutrophils (stimulated and control) were left for 2 hours before total RNA was isolated, and biotinylated cRNA was prepared by standard methods. Analysis indicates that M. abscessus morphotypes induce a limited number of genes, when compared to S. aureus, which are enriched in genes for cytokines and chemokines, including neutrophil-specific chemokines. These data suggest that neutrophils have a limited response to M. abscessus, which may contribute to neutrophil-rich abscess formation.!Series_overall_design = Human neutrophils from healthy donors were exposed to rough Mab (ATCC 19977T), smooth Mab (ATCC 19977T) and S. aureus (CF clinical strain) for two hours; control cells were exposed to saline.

ORGANISM(S): Homo sapiens

PROVIDER: GSE39889 | GEO | 2013/01/10

SECONDARY ACCESSION(S): PRJNA171967

REPOSITORIES: GEO

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Project description:Members of the Mycobacterium (M.) abscessus complex (MABC) are rapidly growing mycobacteria showing smooth and/or rough colony morphotype. While not as virulent as M. tuberculosis, they can cause soft tissue infection and fatal pulmonary disease, especially in patients with cystic fibrosis. Diagnosing MABC pulmonary disease is challenging since the isolation of M. abscessus from respiratory samples is in itself not diagnostic and the clinical features are often non-specific. Immunologic assays, which could aid in the understanding and diagnosis of the disease, are not available. In this study eight rough and six smooth colony morphotype isolates were collected from seven clinical MABC strains and the M. abscessus reference strain ATCC19977, as six strains showed both morphotypes simultaneously and two strains only showed a rough morphotype. Clinical isolates were submitted to whole genome sequencing. Quantitative proteomic analysis was performed on bacterial lysates and the culture supernatant of all 14 isolates. Supernatant proteins present in all isolates were compared in a BLAST search against other clinically significant mycobacterial species to determine species-specific proteins of MABC. In silico B- and T-cell epitope prediction was performed for species-specific proteins. All clinical strains were found to be M. abscessus ssp. abscessus. Six of seven rough colony clinical isolates contained genetic changes in the MAB_4099c gene, which is a likely genetic basis for the rough morphotype. Proteomic analysis detected 3 137 different proteins in total of which 79 proteins were found in the culture supernatants of all isolates. BLAST analyses of these 79 proteins identified 12 of those exclusively encoded by all members of MABC plus M. immunogenum. In silico prediction of epitopes predicted B- and T-cell epitopes in all these 12 species-specific proteins, rendering them promising candidates for future studies on immune pathogenesis and immune diagnostic tools for MABC disease.

Project description:Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens whose siderophores would normally be chelated by 24p3. Specific granule deficiency (SGD) is a rare congenital disorder characterized by complete absence of proteins in secondary granules. Neutrophils from SGD patients, who are prone to bacterial infections, lack normal functions but the potential role of 24p3 in neutrophil dysfunction in SGD is not known. Here we show that neutrophils from 24p3-deficient mice are defective in many neutrophil functions. Specifically, neutrophils in 24p3-deficient mice do not extravasate to sites of infection and are defective for chemotaxis. A transcriptome analysis revealed that genes that control cytoskeletal reorganization are selectively suppressed in 24p3-deficient neutrophils. Additionally, small regulatory RNAs (miRNAs) that control upstream regulators of cytoskeletal proteins are also increased in 24p3-deficient neutrophils. Further, 24p3-deficient neutrophils failed to phagocytose bacteria, which may account for the enhanced sensitivity of 24p3-deficient mice to both intracellular (Listeria monocytogenes) and extracellular (Candida albicans, Staphylococcus aureus) pathogens. Interestingly, Listeria does not secrete siderophores and additionally, the siderophore secreted by Candida is not sequestered by 24p3. Therefore, the heightened sensitivity of 24p3-deficient mice to these pathogens is not due to sequestration of siderophores limiting iron availability, but is a consequence of impaired neutrophil function. Key words: Lipocalin, 24p3, neutrophils, cell motility, chemotaxis, MIRNA-362-3p To address the role of lipocalin 2 in unstimulated and fMLP-stimulated neutrophils derived from mouse bone marrow, we performed micorarray analysis of gene expression in unstimulated wild type (N=3), unstimulated lcn2 knockout (N=3), fMLP-stimulated wild type (N=2) and fMLP-stimulated lcn2 knockout (N=2) neutrophils. Upon stimulation, neutrophils were treated by fMLP at 10 micromolar for 20 minutes at 37 centigrade.

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Neutrophil gene expression in response to Mycobacterium abscessus

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