Heterogeneity in macrophage phagocytosis of Staphylococcus aureus strains: high-throughput scanning cytometry-based analysis
Alveolar macrophages (AMs) can phagocytose unopsonized pathogens such as S. aureus via innate immune receptors, such as scavenger receptors (SRs). Cytoskeletal events and signaling pathways involved in phagocytosis of unopsonized bacteria likely govern the fate of ingested pathogens, but are poorly...
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| description | Alveolar macrophages (AMs) can phagocytose unopsonized pathogens such as S. aureus via innate immune receptors, such as scavenger receptors (SRs). Cytoskeletal events and signaling pathways involved in phagocytosis of unopsonized bacteria likely govern the fate of ingested pathogens, but are poorly characterized. We have developed a high-throughput scanning cytometry-based assay to quantify phagocytosis of S. aureus by adherent human blood-derived AM-like macrophages in a 96-well microplate format. Differential fluorescent labeling of internalized vs. bound bacteria or beads allowed automated image analysis of collapsed confocal stack images acquired by scanning cytometry, and quantification of total particles bound and percent of particles internalized. We compared the effects of the classic SR blocker polyinosinic acid, the cytoskeletal inhibitors cytochalasin D and nocodazole, and the signaling inhibitors staurosporine, Gö 6976, JNK Inhibitor I and KN-93, on phagocytosis of a panel of live unopsonized S. aureus strains, (Wood, Seattle 1945 (ATCC 25923), and RN6390), as well as a commercial killed Wood strain, heat-killed Wood strain and latex beads. Our results revealed failure of the SR inhibitor polyinosinic acid to block binding of any live S. aureus strains, suggesting that SR-mediated uptake of a commercial killed fluorescent bacterial particle does not accurately model interaction with viable bacteria. We also observed heterogeneity in the effects of cytoskeletal and signaling inhibitors on internalization of different S. aureus strains. The data suggest that uptake of unopsonized live S. aureus by human macrophages is not mediated by SRs, and that the cellular mechanical and signaling processes that mediate S. aureus phagocytosis vary. The findings also demonstrate the potential utility of high-throughput scanning cytometry techniques to study phagocytosis of S. aureus and other organisms in greater detail. |
| doi_str_mv | 10.1371/journal.pone.0006209 |
| format | Article |
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Cytoskeletal events and signaling pathways involved in phagocytosis of unopsonized bacteria likely govern the fate of ingested pathogens, but are poorly characterized. We have developed a high-throughput scanning cytometry-based assay to quantify phagocytosis of S. aureus by adherent human blood-derived AM-like macrophages in a 96-well microplate format. Differential fluorescent labeling of internalized vs. bound bacteria or beads allowed automated image analysis of collapsed confocal stack images acquired by scanning cytometry, and quantification of total particles bound and percent of particles internalized. We compared the effects of the classic SR blocker polyinosinic acid, the cytoskeletal inhibitors cytochalasin D and nocodazole, and the signaling inhibitors staurosporine, Gö 6976, JNK Inhibitor I and KN-93, on phagocytosis of a panel of live unopsonized S. aureus strains, (Wood, Seattle 1945 (ATCC 25923), and RN6390), as well as a commercial killed Wood strain, heat-killed Wood strain and latex beads. Our results revealed failure of the SR inhibitor polyinosinic acid to block binding of any live S. aureus strains, suggesting that SR-mediated uptake of a commercial killed fluorescent bacterial particle does not accurately model interaction with viable bacteria. We also observed heterogeneity in the effects of cytoskeletal and signaling inhibitors on internalization of different S. aureus strains. The data suggest that uptake of unopsonized live S. aureus by human macrophages is not mediated by SRs, and that the cellular mechanical and signaling processes that mediate S. aureus phagocytosis vary. The findings also demonstrate the potential utility of high-throughput scanning cytometry techniques to study phagocytosis of S. aureus and other organisms in greater detail.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0006209</identifier><identifier>PMID: 19593389</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Acids ; Alveoli ; Animals ; Bacteria ; Beads ; Blood ; Blood & organ donations ; Cytochalasin D ; Cytometry ; Cytoskeleton ; Environmental health ; Female ; Flow Cytometry - methods ; Fluorescence ; Heterogeneity ; Humans ; Image acquisition ; Image analysis ; Image processing ; Immunology/Innate Immunity ; Infectious Diseases/Bacterial Infections ; Inhibitors ; Internalization ; JNK protein ; Kinases ; Latex ; Latex beads ; Ligands ; Macrophages ; Macrophages, Alveolar - microbiology ; Mice ; Mice, Inbred BALB C ; Microbiology/Innate Immunity ; Nocodazole ; Particulates ; Pathogens ; Phagocytosis ; Phosphorylation ; Physiology ; Public health ; Receptors ; Scanning ; Scavenger receptors ; Signaling ; Staphylococcus ; Staphylococcus aureus ; Staphylococcus aureus - immunology ; Staurosporine ; Strains (organisms) ; Wood</subject><ispartof>PloS one, 2009-07, Vol.4 (7), p.e6209-e6209</ispartof><rights>COPYRIGHT 2009 Public Library of Science</rights><rights>2009 DeLoid et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>DeLoid et al. 2009</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c693t-445b68a47ef3c4de3479839d716cbbf364ec8d764438af937904609885e9bd9a3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703801/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703801/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79342,79343</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19593389$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>DeLoid, Glen M</creatorcontrib><creatorcontrib>Sulahian, Timothy H</creatorcontrib><creatorcontrib>Imrich, Amy</creatorcontrib><creatorcontrib>Kobzik, Lester</creatorcontrib><title>Heterogeneity in macrophage phagocytosis of Staphylococcus aureus strains: high-throughput scanning cytometry-based analysis</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Alveolar macrophages (AMs) can phagocytose unopsonized pathogens such as S. aureus via innate immune receptors, such as scavenger receptors (SRs). Cytoskeletal events and signaling pathways involved in phagocytosis of unopsonized bacteria likely govern the fate of ingested pathogens, but are poorly characterized. We have developed a high-throughput scanning cytometry-based assay to quantify phagocytosis of S. aureus by adherent human blood-derived AM-like macrophages in a 96-well microplate format. Differential fluorescent labeling of internalized vs. bound bacteria or beads allowed automated image analysis of collapsed confocal stack images acquired by scanning cytometry, and quantification of total particles bound and percent of particles internalized. We compared the effects of the classic SR blocker polyinosinic acid, the cytoskeletal inhibitors cytochalasin D and nocodazole, and the signaling inhibitors staurosporine, Gö 6976, JNK Inhibitor I and KN-93, on phagocytosis of a panel of live unopsonized S. aureus strains, (Wood, Seattle 1945 (ATCC 25923), and RN6390), as well as a commercial killed Wood strain, heat-killed Wood strain and latex beads. Our results revealed failure of the SR inhibitor polyinosinic acid to block binding of any live S. aureus strains, suggesting that SR-mediated uptake of a commercial killed fluorescent bacterial particle does not accurately model interaction with viable bacteria. We also observed heterogeneity in the effects of cytoskeletal and signaling inhibitors on internalization of different S. aureus strains. The data suggest that uptake of unopsonized live S. aureus by human macrophages is not mediated by SRs, and that the cellular mechanical and signaling processes that mediate S. aureus phagocytosis vary. The findings also demonstrate the potential utility of high-throughput scanning cytometry techniques to study phagocytosis of S. aureus and other organisms in greater detail.</description><subject>Acids</subject><subject>Alveoli</subject><subject>Animals</subject><subject>Bacteria</subject><subject>Beads</subject><subject>Blood</subject><subject>Blood & organ donations</subject><subject>Cytochalasin D</subject><subject>Cytometry</subject><subject>Cytoskeleton</subject><subject>Environmental health</subject><subject>Female</subject><subject>Flow Cytometry - methods</subject><subject>Fluorescence</subject><subject>Heterogeneity</subject><subject>Humans</subject><subject>Image acquisition</subject><subject>Image analysis</subject><subject>Image processing</subject><subject>Immunology/Innate Immunity</subject><subject>Infectious Diseases/Bacterial Infections</subject><subject>Inhibitors</subject><subject>Internalization</subject><subject>JNK protein</subject><subject>Kinases</subject><subject>Latex</subject><subject>Latex beads</subject><subject>Ligands</subject><subject>Macrophages</subject><subject>Macrophages, Alveolar - 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Cytoskeletal events and signaling pathways involved in phagocytosis of unopsonized bacteria likely govern the fate of ingested pathogens, but are poorly characterized. We have developed a high-throughput scanning cytometry-based assay to quantify phagocytosis of S. aureus by adherent human blood-derived AM-like macrophages in a 96-well microplate format. Differential fluorescent labeling of internalized vs. bound bacteria or beads allowed automated image analysis of collapsed confocal stack images acquired by scanning cytometry, and quantification of total particles bound and percent of particles internalized. We compared the effects of the classic SR blocker polyinosinic acid, the cytoskeletal inhibitors cytochalasin D and nocodazole, and the signaling inhibitors staurosporine, Gö 6976, JNK Inhibitor I and KN-93, on phagocytosis of a panel of live unopsonized S. aureus strains, (Wood, Seattle 1945 (ATCC 25923), and RN6390), as well as a commercial killed Wood strain, heat-killed Wood strain and latex beads. Our results revealed failure of the SR inhibitor polyinosinic acid to block binding of any live S. aureus strains, suggesting that SR-mediated uptake of a commercial killed fluorescent bacterial particle does not accurately model interaction with viable bacteria. We also observed heterogeneity in the effects of cytoskeletal and signaling inhibitors on internalization of different S. aureus strains. The data suggest that uptake of unopsonized live S. aureus by human macrophages is not mediated by SRs, and that the cellular mechanical and signaling processes that mediate S. aureus phagocytosis vary. The findings also demonstrate the potential utility of high-throughput scanning cytometry techniques to study phagocytosis of S. aureus and other organisms in greater detail.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>19593389</pmid><doi>10.1371/journal.pone.0006209</doi><tpages>e6209</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Acids Alveoli Animals Bacteria Beads Blood Blood & organ donations Cytochalasin D Cytometry Cytoskeleton Environmental health Female Flow Cytometry - methods Fluorescence Heterogeneity Humans Image acquisition Image analysis Image processing Immunology/Innate Immunity Infectious Diseases/Bacterial Infections Inhibitors Internalization JNK protein Kinases Latex Latex beads Ligands Macrophages Macrophages, Alveolar - microbiology Mice Mice, Inbred BALB C Microbiology/Innate Immunity Nocodazole Particulates Pathogens Phagocytosis Phosphorylation Physiology Public health Receptors Scanning Scavenger receptors Signaling Staphylococcus Staphylococcus aureus Staphylococcus aureus - immunology Staurosporine Strains (organisms) Wood |
| title | Heterogeneity in macrophage phagocytosis of Staphylococcus aureus strains: high-throughput scanning cytometry-based analysis |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-11T21%3A57%3A31IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Heterogeneity%20in%20macrophage%20phagocytosis%20of%20Staphylococcus%20aureus%20strains:%20high-throughput%20scanning%20cytometry-based%20analysis&rft.jtitle=PloS%20one&rft.au=DeLoid,%20Glen%20M&rft.date=2009-07-10&rft.volume=4&rft.issue=7&rft.spage=e6209&rft.epage=e6209&rft.pages=e6209-e6209&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0006209&rft_dat=%3Cgale_plos_%3EA472964501%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1290696876&rft_id=info:pmid/19593389&rft_galeid=A472964501&rft_doaj_id=oai_doaj_org_article_a88c0bce98ea460a9ffbe7b7eb07a563&rfr_iscdi=true |