Name: alveolar macrophage phagocytosis and bacteria clearance in mice
Uploaded: 2019-03-02T14:00:00
Description: Watch this Scientific Journal Video about Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice at JoVE.com

Dette arbejde støttes af grant R01HL116826 til X. Zhao.

Denne protokol følger retningslinjerne i den institutionelle Animal Care og brug udvalg (IACUC) af østlige Virginia Medical School.
1. fluorescerende perler fagocytose
<ol><li>Aflive mus (C57BL/6J, 6 uger gamle, kvindelige) af CO2 kvælning som pr IACUC protokoller for den etiske eutanasi af dyr.</li>
	<li>Lå musen mave-up på en dissektion bord dækket med papirservietter. Indkredse sine poter med dens lemmer spread-eagle og krog en streng under dens forreste tænder til at tr...

<ol>
	<li>Hussell, T., Bell, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages:+plasticity+in+a+tissue-specific+context.">Alveolar macrophages: plasticity in a tissue-specific context.</a> Nature Reviews Immunology. 14, 81-93 (2014).</li><li>Belchamber, K. B. R., Donnelly, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+Dysfunction+in+Respiratory+Disease.">Macrophage Dysfunction in Respiratory Disease.</a> Results and Problems in Cell Differentiation. 62, 299-313 (2017).</li><li>Broug-Holub, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages+are+required+for+protective+pulmonary+defenses+in+murine+Klebsiella+pneumonia:+elimination+of+alveolar+macrophages+increases+neutrophil+recruitment+but+decreases+bacterial+clearance+and+survival.">Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival.</a> Infection and Immunity. 65, 1139-1146 (1997).</li><li>Knapp, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages+have+a+protective+antiinflammatory+role+during+murine+pneumococcal+pneumonia.">Alveolar macrophages have a protective antiinflammatory role during murine pneumococcal pneumonia.</a> American Journal of Respiratory and Critical Care Medicine. 167, 171-179 (2003).</li><li>Bhatia, M., Zemans, R. L., Jeyaseelan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+chemokines+in+the+pathogenesis+of+acute+lung+injury.">Role of chemokines in the pathogenesis of acute lung injury.</a> American Journal of Respiratory Cell and Molecular Biology. 46, 566-572 (2012).</li><li>Marriott, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1beta+regulates+CXCL8+release+and+influences+disease+outcome+in+response+to+Streptococcus+pneumoniae,+defining+intercellular+cooperation+between+pulmonary+epithelial+cells+and+macrophages.">Interleukin-1beta regulates CXCL8 release and influences disease outcome in response to Streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages.</a> Infection and Immunity. 80, 1140-1149 (2012).</li><li>Greenlee-Wacker, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clearance+of+apoptotic+neutrophils+and+resolution+of+inflammation.">Clearance of apoptotic neutrophils and resolution of inflammation.</a> Immunological Reviews. 273, 357-370 (2016).</li><li>Haslett, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte+apoptosis+and+its+role+in+the+resolution+and+control+of+lung+inflammation.">Granulocyte apoptosis and its role in the resolution and control of lung inflammation.</a> American Journal of Respiratory and Critical Care Medicine. 160, 5-11 (1999).</li><li>Cox, G., Crossley, J., Xing, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+engulfment+of+apoptotic+neutrophils+contributes+to+the+resolution+of+acute+pulmonary+inflammation+in+vivo.">Macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary inflammation in vivo.</a> American Journal of Respiratory Cell and Molecular Biology. 12, 232-237 (1995).</li><li>Groves, E., Dart, A. E., Covarelli, V., Caron, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+phagocytic+uptake+in+mammalian+cells.">Molecular mechanisms of phagocytic uptake in mammalian cells.</a> Cellular and Molecular Life Sciences. 65, 1957-1976 (2008).</li><li>Mosser, D. M., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Opsonic+Phagocytosis+via+Fc&#947;+Receptors+and+complement+receptors+on+macrophages.">Measuring Opsonic Phagocytosis via Fc&#947; Receptors and complement receptors on macrophages.</a> Current Protocols in Immunology. , (2011).</li><li>He, J. Q., Wiesmann, C., van Lookeren Campagne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+of+macrophage+complement+receptor+CRIg+in+immune+clearance+and+inflammation.">A role of macrophage complement receptor CRIg in immune clearance and inflammation.</a> Molecular Immunology. 45, 4041-4047 (2008).</li><li>Nagre, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Macrophage+Complement+Receptor+CRIg+by+TRIM72+Polarizes+Innate+Immunity+of+the+Lung.">Inhibition of Macrophage Complement Receptor CRIg by TRIM72 Polarizes Innate Immunity of the Lung.</a> American Journal of Respiratory Cell and Molecular Biology. 58 (6), 756-766 (2018).</li><li>Miksa, M., Komura, H., Wu, R., Shah, K. G., Wang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Method+to+Determine+the+Engulfment+of+Apoptotic+Cells+by+Macrophages+using+pHrodo+Succinimidyl+Ester.">A Novel Method to Determine the Engulfment of Apoptotic Cells by Macrophages using pHrodo Succinimidyl Ester.</a> Journal of Immunological Methods. 342 (1-2), 71-77 (2009).</li><li>Su, H., Chen, H., Jen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+exercise+enhances+phagocytosis+by+murine+bronchoalveolar+macrophages.">Severe exercise enhances phagocytosis by murine bronchoalveolar macrophages.</a> Journal of Leukocyte Biology. 69, 75-80 (2001).</li><li>Amiel, E., Lovewell, R. R., O'Toole, G. A., Hogan, D. A., Berwin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa.+evasion+of+phagocytosis+is+mediated+by+loss+of+swimming+motility+and+is+independent+of+flagellum+expression.">Pseudomonas aeruginosa. evasion of phagocytosis is mediated by loss of swimming motility and is independent of flagellum expression.</a> Infection and Immunity. 78, 2937-2945 (2010).</li><li>Giannoni, E., Sawa, T., Allen, L., Wiener-Kronish, J., Hawgood, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactant+Proteins+A+and+D+Enhance+Pulmonary+Clearance+of+Pseudomonas+aeruginosa.">Surfactant Proteins A and D Enhance Pulmonary Clearance of Pseudomonas aeruginosa.</a> American Journal of Respiratory Cell and Molecular Biology. 34, 704-710 (2006).</li></ol>

Mens de udfører en gas udveksling funktion, konfronterer lungen vedvarende fremmede partikler, patogener og allergener. AMs giver den første linje i forsvaret i kraft af deres hovedfunktion, nemlig fagocytose. AMs også koordinere med andre immunceller i ødelægger patogener og i løsningen af betændelse. Her, beskrevet vi metoder til konkret vurdering fagocytose af AMs isoleret fra mus lunge. Protokollen præsenteret i dette manuskript forklarer en detaljeret undersøgelse af fagocytose både in vivo og in vitro, so...

AMs er de store hjemmehørende fagocytter i alveolerne på den hvilende fase og en af de store aktører i medfødte immun svar gennem anerkendelse og internalisering af inhaleret patogener og fremmede partikler1,2. Det er blevet rapporteret, at AMs er afgørende for den hurtig clearance af mange pulmonal patogener som P. aeruginosa og Klebsiella lungebetændelse3,4, så en mangel i AM fagocytose resulterer ofte i luftvejene infektioner, såsom akut lungebetændelse, som forårsager højere dødelighed og sygelighed satser.
AMs også indlede medfødte inflammatoriske respons i lungen ved produktion af cytokiner og kemokiner såsom TNF-α og IL-1β, hvilke krydstale med andre celler af alveolær miljøet at producere kemokiner og rekruttere inflammatoriske neutrofiler, monocytter, og Adaptive immunceller i lunge5. For eksempel, hjælper IL-1β produceret af AMs til prime frigivelsen af den neutrofile chemokine CXCL8 fra epitelceller6. Derudover bidrage AMs til fagocytose af apoptotiske polymorfnukleære leukocytter (PMNs), hvilket fører til vedvarende udsivning af intracellulære enzymer fra PMNs manglende omkringliggende væv, resulterer i vævsskader og langvarig inflammation 7 , 8 , 9.
Fagocytose af AMs er medieret af en direkte anerkendelse af patogen-associeret molekylære mønstre på patogen overfladen af mønster anerkendelse receptorer (PRRs) af AMs eller ved binding af opsonized patogener med immun effektor receptorer af AMs 10. for sidstnævnte, AMs kan genkende mål opsonized med immunoglobulin (IgG) gennem deres Fcγ receptorer (FcγR) eller patogener belagt med supplement fragmenter, C3b og C3bi, gennem deres supplement receptorer (CR)11. Blandt supplement receptorer, CR af immunoglobulin superfamilien (CRIg) er selektivt udtrykt i væv makrofager12, og en nylig har konstateret fremhævet rollen, som CRIg i AM fagocytose i forbindelse med P. aeruginosa lungebetændelse 13.
Mange oprindelige undersøgelser bruge metoder til at vurdere makrofag fagocytose for at beskrive de molekylære mekanismer af makrofag funktion14,15. Metoder som in vivo fagocytose kræver imidlertid en præcis kvantificering af fagocytose. Her, vi opsummere en detaljeret metode til in vitro- og in vivo fagocytose ved hjælp af fluorescein isothiocyanat (FITC)-glas perler og P. aeruginosa grøn fluorescerende proteiner (NGL), henholdsvis. Derudover forklarer vi metode til skelne blandt PRR-, CR- og FcγR-medieret fagocytose. Endelig vil rapportere vi en metode til at karakterisere bakteriel clearance i mus med hensyn til P. aeruginosa lungebetændelse.

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			<td height="21" style="height:21px;width:216px;">18-G Needle</td>
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			<td style="width:272px;">Molecular Biology grade</td>
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			<td height="21" style="height:21px;width:216px;">2,7-diaminofluorene (DAF)</td>
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			<td>Molecular Biology grade</td>
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			<td height="21" style="height:21px;width:216px;">70% Ethanol</td>
			<td style="width:179px;">Decon Labs Inc.</td>
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			<td>Analytical grade</td>
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			<td height="21" style="height:21px;width:216px;">96-well plate</td>
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			<td height="21" style="height:21px;width:216px;">ACK lysis buffer</td>
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			<td>Molecular Biology grade</td>
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			<td height="42" style="height:42px;width:216px;">Alexa fluor-488 Zymosan-A-bioparticle</td>
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			<td>Molecular Biology grade</td>
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			<td>Biochemical reagent</td>
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			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
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			<td style="width:143px;">A78300101 Issue 11</td>
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			<td height="21" style="height:21px;width:216px;">DMEM Cell Culture Media</td>
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			<td>Cell Biology grade</td>
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			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:179px;">Atlanta Biologicals</td>
			<td style="width:143px;">S11550</td>
			<td>Cell Biology grade</td>
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			<td height="21" style="height:21px;width:216px;">Flow Cytometer</td>
			<td style="width:179px;">BD Biosciences</td>
			<td style="width:143px;">FACSCalibur</td>
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			<td>Suitable for laboratory animal dissection</td>
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			<td height="21" style="height:21px;width:216px;">FITC-carboxylated latex beads</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">L4530</td>
			<td>Cell Biology grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GFP-P. aeruginosa</td>
			<td style="width:179px;">ATCC</td>
			<td>101045GFP</td>
			<td>Suitable for&#160; cell infection assays</td>
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			<td height="21" style="height:21px;width:216px;">Glass bottom dish</td>
			<td style="width:179px;">MatTek Corp.</td>
			<td>P35G-0.170-14-C</td>
			<td>Cell Biology grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High-Pressure Syringe</td>
			<td style="width:179px;">Penn-Century</td>
			<td>FMJ-250</td>
			<td>Suitable for laboratory animal use</td>
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			<td height="21" style="height:21px;width:216px;">Homogenizer</td>
			<td style="width:179px;">Omni International</td>
			<td>TH-01</td>
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			<td height="21" style="height:21px;width:216px;">Hydrogen peroxide</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">H1009</td>
			<td>Analytical grade</td>
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			<td height="42" style="height:42px;width:216px;">Inverted Fluorescence Microscope</td>
			<td style="width:179px;">Olympus</td>
			<td style="width:143px;">IX73</td>
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			<td height="21" style="height:21px;width:216px;">Ketamine Hydrochloride</td>
			<td style="width:179px;">Hospira</td>
			<td style="width:143px;">CA-2904</td>
			<td>Pharmaceutical grade</td>
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			<td height="21" style="height:21px;width:216px;">Shandon Kwik-Diff Stains</td>
			<td style="width:179px;">Thermofisher Scientific</td>
			<td style="width:143px;">9990700</td>
			<td>Cell Biology grade</td>
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			<td height="21" style="height:21px;width:216px;">LB Agar</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:143px;">BP1425</td>
			<td>Molecular Biology grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB Broth</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:143px;">BP1427</td>
			<td>Molecular Biology grade</td>
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			<td height="21" style="height:21px;width:216px;">MicroSprayer Aerosolizer</td>
			<td style="width:179px;">Penn-Century</td>
			<td style="width:143px;">IA-1C</td>
			<td>Suitable for laboratory animal use</td>
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			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">P6148</td>
			<td>Reagent grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:179px;">Gibco</td>
			<td style="width:143px;">20012-027</td>
			<td>Cell Biology grade</td>
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			<td height="21" style="height:21px;">rabbit anti-SRBC-IgG&#160;</td>
			<td style="width:179px;">MP Biomedicals</td>
			<td style="width:143px;">55806</td>
			<td>Suitable for immuno-assays</td>
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			<td height="21" style="height:21px;">rabbit anti-SRBC-IgM&#160;</td>
			<td style="width:179px;">Cedarline Laboratories</td>
			<td style="width:143px;">CL9000-M</td>
			<td>Suitable for immuno-assays</td>
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			<td height="21" style="height:21px;">Scissors</td>
			<td style="width:179px;">Miltex</td>
			<td style="width:143px;">5-2</td>
			<td>Suitable for laboratory animal dissection</td>
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			<td height="21" style="height:21px;">Small Animal Laryngoscope</td>
			<td style="width:179px;">Penn-Century</td>
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			<td>Suitable for laboratory animal use</td>
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			<td height="21" style="height:21px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td style="width:179px;">BioRad</td>
			<td style="width:143px;">1610301</td>
			<td>Analytical grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Spring Scissors (Med)</td>
			<td style="width:179px;">Fine Science Tools</td>
			<td style="width:143px;">15012-12</td>
			<td>Suitable for laboratory animal dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring Scissors (Small)</td>
			<td style="width:179px;">Fine Science Tools</td>
			<td style="width:143px;">91500-09</td>
			<td>Suitable for laboratory animal dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sheep red blood cells (SRBCs)&#160;</td>
			<td style="width:179px;">MP Biomedicals</td>
			<td style="width:143px;">55876</td>
			<td>Washed, preserved SRBCs</td>
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			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">U5378</td>
			<td>Molecular Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine&#160;</td>
			<td style="width:179px;">Akorn Animal Health</td>
			<td style="width:143px;">59399-110-20</td>
			<td>Pharmaceutical grade</td>
		</tr>
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alveolar macrophage phagocytosis and bacteria clearance in mice

Alveolær makrofager (AMs) bevogte den alveolære rum af lungen. Fagocytose af AMs spiller en afgørende rolle i forsvaret mod invaderende patogener, fjernelse af døde celler eller fremmede partikler, og i beslutningsforslaget af inflammatoriske respons og væv remodellering, processer der er medieret af forskellige overflade receptorer af AMs. Her rapporterer vi metoder til analyse af AMs ved hjælp af in vitro- og in vivo assays og eksperimenterende strategier til at skelne mellem den mønster anerkendelse receptor-, supplere receptor- og Fc gamma receptor-medieret fagocyterende funktion fagocytose. Endelig vil diskutere vi en metode til at etablere og karakterisere en P. aeruginosa lungebetændelse model i mus at vurdere bakteriel clearance in vivo. Disse assays repræsenterer de mest almindelige metoder til at evaluere AM funktioner og kan også bruges til at studere makrofag funktion og bakteriel clearance i andre organer.

Vi uropført eksperiment for at analysere fagocytose af musen primære AMs. Hele alle analyser sammenlignede vi AMs isoleret fra WT og TRIM72KO mus. Som vist i figur 1A, afslørede Fluorescens mikroskopi at fagocytose af FITC-glasperler med musen primære AMs opstår efter 1 h inkubation. Figur 1 B viser analysen af fagocytose ved flowcytometri. Kvantificering af fagocytose målt ved m...

Watch this Scientific Journal Video about Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice at JoVE.com

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolær makrofag fagocytose og bakterier Clearance i mus

Her rapporterer vi fælles metoder til at analysere funktionen fagocyterende murine alveolær makrofager og bakteriel clearance fra lungen. Disse metoder studere in vitro-fagocytose af fluorescein isothiocyanat perler og in vivo fagocytose af Pseudomonas aeruginosa grøn fluorescerende proteiner. Vi beskriver også en metode til at rydde P. aeruginosa i mus.

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

Pseudomonas aeruginosa er et BSL-2 niveau patogen. Husk at følge alle BSL-2-niveau sikkerhedspraksis, når du håndterer denne organisme. Her vil vi demonstrere primær celleisolering, in vitro fagocytose, og fagocytiske vej analyse, og in vivo fagocytose og bakteriel clearance vurdering i mus.Vellykket intratracheal levering kræver praksis at mestre. Sørg for, at mikrosprøjten er korrekt indlæst, nålen er mellem de to vokalfolder, og stemplets hastighed styres korrekt. Begynd med at fastgøre musen i liggende position med lemmerne spredt på et dissektionsbræt dækket med papirhåndklæder og koble en snor under fortænderne for at trække hovedet tilbage, så luftrøret er placeret lige og i niveau.Desinficere musen med 70% ethanol og bruge regelmæssige kraftvæceser til at trække op huden i midten af kroppen. Brug kirurgisk saks til at skære huden fra maven til toppen af halsen. Brug den stumpe ende af standard kirurgisk saks til omhyggeligt dissekere halsen muskel og bindevæv.Åbn bugvæggen under brystkassen. Skær mellemgulvet, og skær den nederste del af brystkassen til delvist udsætte lungerne. Brug foråret saks til at udsætte luftrøret og bruge de virpint til at gribe en brusk ring.Brug mikro saks til omhyggeligt at gøre en ca 1,5-millimeter snit på ventrale ansigt af luftrøret. Streng forsigtigt en kort længde af sutur tråd under luftrøret og indsætte en 18 gauge kanyle i luftrøret. Når kanylen er på plads, skal du forsigtigt trække lungen ud tre gange med en milliliter frisk PBS pr. vask, forsigtigt trække væsken ind i sprøjten, før den trækkes tilbage i lungerne igen tre gange i træk.Efter hver lavage overføres den opsamlede bronchoalveolar lavagevæske eller BALF til et konisk rør på 15 milliliter til centrifugering af den samlede høstede væske. Pelletsuspendrer pelletet op igen i en milliliter frisk PBS med endnu en centrifugering og opsaltning af de vaskede alveolær makrofager i to milliliter af Dulbeccos Modified Eagles Medium eller DMEM suppleret med 10 % ikke-varmeinaktiveret føtalt kvægserum. Overfør derefter den primære alveolær makrofager til en glasbundet petriskål til deres to-dages kultur ved 37 grader Celsius i en celleinkubator.Efter 48 timer vaskes kulturen med en milliliter PBS, før kulturen fodres med to milliliter frisk medium. Dernæst tilsættes 50 to-mikrometer diameter carboxylated latex FITC-konjugerede perler pr celle til kulturen for en times inkubation ved 37 grader Celsius i en celle inkubator. Ved slutningen af inkubationen vaskes pladen grundigt fem gange med en milliliter frisk PBS pr. vask for at fjerne de ekstracellulære perler og tilfældigt billede 100 celler for at tælle cellerne, der indeholder intracellulære perler.For en opsonization assay, inkubere to gange 10 til den ottende får røde blodlegemer eller SRBCs med 50 mikroliter af kanin anti-SRBC immunglobulin M eller IgM i 30 minutter ved stuetemperatur. Derefter inkuberes de opsoniserede SRBCs med 50 mikroliter C5-mangelfuldt humant serum i 30 minutter ved 37 grader Celsius for at fikse C3b- og C3b-inhibitoren til at komplementere fragmenter på IgM-belagte SRBC'er. Dernæst aspirere medierne og tilføje 100 mikroliter af en gang 10 til de syv opsonized SRBCs per milliliter medium til hver brønd af en 96-brønd plade, der indeholder en gange 10 til den fjerde overnight-dyrkede murine makrofager per brønd.Inkuber SRBC-musemakrofaget coculture i en time ved 37 grader Celsius, og fjern derefter de ubundne SRBCs ved at vaske med 100 mikroliter ammoniumchlorid kaliumlysisbuffer i et minut. Når de ubundne SRBCs er blevet fjernet, skylles med 100 mikroliter medier. Lyse de resterende celler med 0,1% natrium dodecyli sulfat og behandle lysater med 50 mikroliter af 2, 7-diaminofluorene suppleret med 3% hydrogenperoxid og seks molar urea.Derefter måle absorbans af hæmoglobin-katalyseret fluorein blå dannelse på et spektrofotometer på 620 nanometer. Brug en standardkurve ved 620 nanometerabsorbanceværdier med kendt antal SRBCs til at bestemme antallet af opsoniserede SRBCs, der er fagocytized. For at vurdere mønstergenkendelse receptor-medieret fagocytose, vask to-dages dyrket mus primær alveolær makrofager med en milliliter PBS og behandle cellerne med 500 mikroliter frisk medium indeholdende 100 Alexa fluor-488 konjugerede Zymosan-A-biopartikler.Efter en time ved 37 grader Celsius, standse fagocytose med 500 mikroliter af iskold PBS og vaske cellerne i udstrakt grad fem gange med en milliliter PBS per vask. Efter den sidste vask, fastgør cellerne med 4% paraformaldehyd i 10 minutter ved stuetemperatur og vask cellerne fem gange mere som påvist. Efter den sidste vask dækkes cellerne med 500 mikroliter frisk PBS til billeddannelse under differensinterferenskontrast og lysstofkanal ved 488 nanometer for at kvantificere antallet af Zymosan-A-biopartikelholdige alveolær makrofager.For i vivo alveolær makrofag fagocytose evaluering, bekræfte det passende niveau af sedation ved manglende reaktion på tå knivspids i en bedøvet mus og placere musen på et fladt bord med en plastiktråd under de øverste fortænder. Placer musen i en semi-liggende talerstol på 45 graders position med den ventrale overflade vender opad og bruge buede kraftanger til at trække sig ud og undertrykke tungen. Indsæt derefter en mikrosprøjt mellem vokalfolderne for intratracheally at administrere 50 mikroliter på fem gange 10 til de seks kolonidannende enheder af P.aeruginosa GFP i lungerne på det bedøvede dyr.For at bekræfte, at mikrosprøjtensnålen er i luftrøret, skal du forsigtigt flytte sprøjten og observere stemmefolderne på hver side af nålen. En time efter infektionen, indsamle lavage væske som netop påvist og pellet alveolær makrofager ved centrifugering. Opslænt en gange 10 til de tredje celler i 100 mikroliter frisk PBS til cytocentrifuge på et glas dias.Derefter differentieret pletten cytospun dias for alveolær makrofager, neutrofiler, og lymfocytter, i henhold til standard histokemiske protokoller. Tilfældigt vælge 100 alveolær makrofager til at kvantificere den procentdel af celler, fagocytserede bakterier. I det første in vivo-bakterierydningsforsøg injiceres intratrachealt en sublethalisk dosis på ca. 2,5 til fem gange 10 af de femte kolonidannende enheder pr. milliliter P.aeruginosa i bedøvet vildtype og mutantmus og måles hvert dyrs kropsvægt hver dag i seks dage.I det andet forsøg injiceres et nyt sæt vilde typer og mutantmus med en dødelig tre gange 10 til de syv kolonidannende enheder pr. milliliterdosis P.aeruginosa, og dødeligheden registreres inden for to dage efter en injektion, indsamle hele lungevævet på dødstidspunktet eller to dage efter injektionen for kvantificering af lungebakterielle byrde ved maksimal infektion. Efter høst lungerne skæres lungeprøverne i små stykker på is og homogenisere lungefragmenterne ved hjælp af en justeret elektrisk homogenisatorindstilling. Derefter plade 100 mikroliter af homogenat på Pseudomonas isolering agar plader i 10-fold seriel fortyndinger.Fluorescensmikroskopi afslører, at muse primære alveolær makrofag fagocytose af FITC latex perler opstår efter en times inkubation, med TRIM72 knockout makrofager viser en betydeligt højere fagocytiske evne. Omvendt resulterer overekspression af TRIM72, et protein med ukendt funktion i murine makrofagceller, i et mere end femdobling af komplementfagycytose. Alexa fluor-488 konjugerede Zymosan-A partikler, dog indtages i lige stor mængde ved primær alveolær makrofager isoleret fra enten vilde type eller knockout mus.Differential farvning af BALF høstet fra vilde type og knockout dyr afslører et tilsvarende antal makrofager, men en højere fagocytisk evne til makrofager høstet fra knockout mus. Yderligere, TRIM72 knockout mus demonstrere en hurtigere genopretning af deres kropsvægt, bevare deres overlevelse, og udviser en lavere bakteriel byrde end vilde type dyr efter intratracheal P.aeruginosa administration. Ved hjælp af denne teknik, andre fagocytiske processer, der er vigtige for lungebetændelse såsom neutrofil fagocytose og det relative bidrag af andre fagocytter i bakterier clearance kan analyseres.I kombination med farmakologiske hæmmere, adaptive overførsel, og transgene dyr, denne teknik kan hjælpe forskere til at udforske den molekylære komponent af specifikke type fagocytose for fagocytter af interesse.

alveolar-macrophage-phagocytosis-and-bacteria-clearance-in-mice

Research

JoVE Journal

Immunology and Infection

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Bone Marrow-afledt Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

High Throughput Fluorometrisk Teknik for Vurdering af makrofag Fagocytose og actinpolymerisation

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Makrofag Kolesterol Udtømmelse og dens indflydelse på fagocytose af<em&gt; Cryptococcus neoformans</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

Phagocytosis Assay for Apoptotiske Celler i<em&gt; Drosophila</em&gt; Embryoer

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolation og In Vitro kultur af Murine og menneskelige alveolær makrofager

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

Ved hjælp af forbedret grønne fluorescens Protein-udtryk for Escherichia Coli at vurdere musen Peritoneal makrofag fagocytose

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

Kørsler til uorganiske Polyphosphate i bakterier

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing bronchoalveolar lavage (BAL) fluid. The BAL fluid is rich in cells and can thus provide a 'snapshot' of the pulmonary immune milieu. CD4 T cells are the best characterized HIV reservoirs, while there is strong evidence to suggest that tissue macrophages, including alveolar macrophages (AMs), also serve as viral reservoirs. However, much is still unknown about the role of AMs in the context of HIV reservoir establishment and maintenance. Therefore, developing a protocol for processing BAL fluid to obtain cells that may be used in virological and immunological assays to characterize and evaluate the cell populations and subsets within the lung is relevant for understanding the role of the lungs as HIV reservoirs. Herein, we describe such a protocol, employing standard techniques such as simple centrifugation and flow cytometry. The CD4 T cells and AMs may then be used for subsequent applications, including immunophenotyping and HIV DNA and RNA quantification.

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

We describe a method for processing bronchoalveolar lavage fluid and matched peripheral blood from chronically HIV-infected individuals on antiretroviral therapy to assess pulmonary HIV reservoirs. These methods result in the acquisition of highly pure CD4 T cells and alveolar macrophages that may subsequently be used for immunophenotyping and HIV DNA/RNA quantifications by ultrasensitive polymerase chain reaction.

Behandling af Bronkoalveolær lavage væske og matchet blod for alveolær makrofag og CD4+ T-celle Immunophenotyping og hiv reservoir Assessment

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing ...

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

In vivo-vurdering af alveolær makrofag-Efferocytose efter ozoneksponering

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Måling af fagocytose af Aspergillus fumigatus Conidia af humane leukocytter ved hjælp af strømnings cytometri

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...