Broad transcriptional firing represses bactericidal activity in human airway neutrophils [Transcriptional_Blockage_CFASN_Neutrophils]
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE167066 | GEO | 2021/02/19
REPOSITORIES: GEO
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Project description:Neutrophils are often considered terminally differentiated and poised for bacterial killing. In chronic diseases such as cystic fibrosis (CF), an unexplained paradox pits massive neutrophil presence against prolonged bacteria infections. Here, we show that neutrophils recruited to CF airways in vivo and in an in vitro transmigration model display rapid and broad transcriptional firing, leading to an upregulation of survival and anabolic genes, and a downregulation of antimicrobial genes. Newly transcribed RNAs are mirrored by the appearance of corresponding proteins, confirming active translation in these cells. Remarkably, treatment by the RNA polymerase II and III inhibitor alpha-amanitin restored expression of key antimicrobial genes, and increased bactericidal capacity of CF airway neutrophils in vitro and in short-term sputum cultures ex vivo. Broadly, our findings show that neutrophil plasticity is regulated at the terminal organ via RNA and protein synthesis, leading to adaptations that profoundly impact their canonical functions (i.e., bacterial clearance).
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Project description:In this model, naïve blood neutrophils are migrated through a small airway epithelium grown at air-liquid interface toward CF airway fluid supernatant (CFASN, which corresponds to sputum sequentially centrifuged to remove cells and bacteria) or leukotriene B4 (LTB4, transmigration control), placed apically.
2021-02-19 | GSE167069 | GEO
Project description:Neutrophils are highly abundant in the gingival tissues where they play an essential role in immune homeostasis by preventing microbial invasion. Here, we show that the oral periodontal pathogen Porphyromonas gingivalis utilizes its cysteine proteases (gingipains) to disengage phagosomal antimicrobial capacity without inducing neutrophil apoptosis. Arginine gingipains are a sub-family of trypsin-like proteases produced by P. gingivalis that cleave several host proteins at arginine residues. We found that RgpB action is not limited to the extracellular or plasma membrane-associated host proteins but can proteolytically modify several intracellular proteins in neutrophils. We used 2D-DIGE coupled with mass spectrometry to identify neutrophil proteins susceptible to RgpB-mediated cleavage and determined the impact of these modifications on neutrophil antimicrobial capacity and inflammatory responses. RgpB cleaved several cytoskeletal and cytoplasmic proteins, including metabolic enzymes and antimicrobial proteins such as neutrophil elastase, myeloperoxidase, and proteinase 3 within neutrophil granules. RgpB also directly primed neutrophils, increasing integrin expression and inducing broad transcriptional changes, consistent with proinflammatory reprogramming. However, despite their primed status and augmented inflammatory capacity, RgpB-treated neutrophils were conducive to intracellular bacterial survival due to the reduced activity of granule proteins and oxidative burst. Thus, our data show a previously unknown role for P. gingivalis proteases in the systematic inactivation of neutrophil microbicidal capacity via proteolysis of intracellular proteins.
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Project description:Injury to the epithelium is integral to the pathogenesis of many inflammatory lung diseases, and epithelial repair is a critical determinant of clinical outcome. However, the signaling pathways regulating such repair are incompletely understood. Herein, we used in vitro and in vivo models to define these pathways. Human neutrophils were induced to transmigrate across monolayers of human lung epithelial cells in the physiologic basolateral-to-apical direction to a gradient of fMLP. Control wells were treated with fMLP alone. Indicated wells were pretreated with an anti-CD47 blocking antibody, which exacerbates epithelial injury. This allowed study of the neutrophil contribution not only to the initial epithelial injury, but also to its repair, as manifest by restoration of transepithelial resistance (TER) and re-epithelialization of the denuded epithelium. Microarray analysis of epithelial gene expression revealed that neutrophil transmigration activated β-catenin signaling, and this was verified by real time PCR, nuclear translocation of β-catenin, and TOPFlash reporter activity. This pathway represents a potential therapeutic target to accelerate physiologic recovery in inflammatory lung diseases. Total of 20 samples: Calu-3 cells that were untreated, treated with fMLP, treated with fMLP and neutrophil transmigration, and treated with anti-CD47 antibodies, fMLP, and neutrophil transmigration; cDNA was pooled from n=3 experiments; No biological replicates
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Project description:Streptococcus pneumoniae is the dominant cause of community-acquired pneumonia world-wide. Invasion of the pleural space is common and results in increased mortality. We set out to determine the bacterial and host factors that influence invasion of the pleural space. In a murine model of pneumococcal infection, we isolated neutrophil-dominated samples of bronchoalveolar and pleural fluid containing bacteria 48 hours after infection. Using dual RNA-seq, we characterised bacterial and host transcripts that were differentially regulated between these compartments and bacteria in broth and resting neutrophils respectively. Pleural and lung samples showed upregulation of genes involved in positive regulation of neutrophil extravasation but down-regulation of genes mediating bacterial killing. Compared to the lung samples, cells within the pleural space showed marked upregulation of many genes induced by type I interferons, cytokines implicated in preventing bacterial transmigration across epithelial barriers. Differences in the bacterial transcripts between the infected samples and bacteria grown in broth showed upregulation of genes in the bacteriocin locus, the pneumococcal surface adhesin PsaA, and the glycopeptide resistance gene, vanZ; the gene encoding the ClpP protease was downregulated in infection. 169 intergenic putative small bacterial RNAs were also identified, of which 43 (25.4%) had been previously described. 42 of the small RNAs were upregulated in pleura compared to broth, including many previously identified as important in virulence. Our results have identified key host and bacterial responses to invasion of the pleural space that can be potentially exploited to develop alternative antimicrobial strategies for prevention and treatment of pneumococcal pleural disease.
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Project description:Staphylococcus aureus is one of the first and most prevalent pathogens cultured from the airways of cystic fibrosis (CF) patients, which can persist there for extended periods. Airway infections in CF patients are characterized by a strong inflammatory response of highly recruited neutrophils. One killing mechanism of neutrophils is the formation of neutrophil extracellular traps (NETs), which capture and eradicate bacteria by extracellular fibers of neutrophil chromatin decorated with antimicrobial granule proteins. S. aureus secretes nuclease, which can degrade NETs. We hypothesized, that S. aureus adapts to the airways of CF patients during persistent infection by escaping from NET-mediated killing via an increase of nuclease activity. Sputum samples of CF patients with chronic S. aureus infection were visualized by confocal microscopy after immuno-fluorescence staining for NET-specific markers, S. aureus bacteria and overall DNA structures. Nuclease activity was analyzed in sequential isogenic long persisting S. aureus isolates, as confirmed by whole genome sequencing, from an individual CF patient using a FRET-based nuclease activity assay. Additionally, some of these isolates were selected and analyzed by qRT-PCR to determine the expression of nuc1 and regulators of interest. NET-killing assays were performed with clinical S. aureus isolates to evaluate killing and bacterial survival depending on nuclease activity. To confirm the role of nuclease during NET-mediated killing, a clinical isolate with low nuclease activity was transformed with a nuclease expression vector (pCM28nuc). Furthermore, two sputa from an individual CF patient were subjected to RNA-sequence analysis to evaluate the activity of nuclease in vivo. In sputa, S. aureus was associated to extracellular DNA structures. Nuclease activity in clinical S. aureus isolates increased in a time-and phenotype-dependent manner. In the clinical isolates, the expression of nuc1 was inversely correlated to the activity of agr and was independent of saeS. NET-mediated killing was significantly higher in S. aureus isolates with low compared to isolates with high nuclease activity. Importantly, transformation of the clinical isolate with low nuclease activity with pCM28nuc conferred protection against NET-mediated killing confirming the beneficial role of nuclease for protection against NETs. Also, nuclease expression in in vivo sputa was high, which underlines the important role of nuclease within the highly inflamed CF airways. In conclusion, our data show that S. aureus adapts to the neutrophil-rich environment of CF airways with increasing nuclease expression most likely to avoid NET-killing during long-term persistence.
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Project description:Neutrophils are the most prominent cell in the immune system, accounting for 70% of circulating leukocytes, and acting as the first line of defense against various invading pathogens. For example, neutrophils engage in phagocytosis, degranulation, and neutrophil extracellular trap formation to respond to pathogens. Pseudomonas aeruginosa is a Gram-negative bacterium that readily forms biofilms (i.e., high-density bacterial clusters attached to surfaces) to provide protection from the host immune system, antibiotic treatment, and antimicrobial agents. Neutrophils are essential to clear P. aeruginosa biofilms and infections through the actions of antimicrobial proteins and peptides. In this study, we evaluated proteome remodeling of the host following exposure of neutrophils (differentiated from HL-60 cells) to P. aeruginosa biofilms.
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Project description:Neutrophils rapidly respond to inflammation and infection, but to which degree their functional trajectories after mobilization from the bone marrow can be shaped within the circulation remains vague. Phenotypic changes of circulating neutrophils caused by systemic inflammation are thought to result from several factors, including a “left shift” of the neutrophil compartment towards younger bone marrow-derived subsets. However, experimental limitations have so far hampered neutrophil research in human disease. Here, using innovative fixation and single-cell-based toolsets, we profile the human and murine neutrophil transcriptome and proteome during steady state and bacterial infection. We find that peripheral priming of circulating neutrophils leads to plastic shifts dominated by conserved upregulation of antimicrobial genes across neutrophil substates, facilitating pathogen containment. We show the TLR4/NF-κB-signaling dependent upregulation of canonical neutrophil activation markers like CD177/NB-1 during acute inflammation, resulting in functional shifts in vivo. Importantly, blocking de novo RNA synthesis in circulating neutrophils abrogates these plastic shifts and prevents the adaptation of antibacterial neutrophil programs by upregulation of distinct effector molecules upon infection. In summary, these data underline transcriptional plasticity as a relevant mechanism of functional neutrophil reprogramming during acute infection to foster bacterial containment within the circulation. This adds to the understanding of competing modes of adaptation to inflammatory challenges by the neutrophil compartment.
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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 regulating miRNA expression profiling in neutrophils derived from mouse bone marrow, we performed microarray analysis of miRNAs in wild type (N=2) and lcn2 knockout (N=2) neutrophils.
2013-01-31 | E-GEOD-43890 | biostudies-arrayexpress
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.
2013-01-31 | E-GEOD-43889 | biostudies-arrayexpress