Name: measuring bacterial load and immune responses in mice infected with listeria monocytogenes
Uploaded: 2011-08-09T13:00:00
Duration: 12 min 50 s
Description: Watch this Scientific Journal Video about Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes at JoVE.com

De auteurs willen graag Anna Walduck, Christina Cheers, Stuart Berzins, Dale Godfrey, Yifan Zhan en Jonathan Wilksch bedanken voor het advies en reagentia. Dit werk werd gefinancierd door de Juvenile Diabetes Research Foundation (1-2008-602) en de Australische National Health en Medical Research Council (575.552). NW wordt ondersteund door een Australische Postgraduate Award. OW wordt ondersteund door een RD Wright Fellowship van de Australische National Health Medical Research Council.

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multigenic+control+of+Listeria+monocytogenes+susceptibility+in+mice.">Multigenic control of Listeria monocytogenes susceptibility in mice.</a> Nat Genet. 27, 259-260 (2001).</li><li>Boyartchuk, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+host+resistance+locus+sst1+controls+innate+immunity+to+Listeria+monocytogenes+infection+in+immunodeficient+mice.">The host resistance locus sst1 controls innate immunity to Listeria monocytogenes infection in immunodeficient mice.</a> J Immunol. 173, 5112-5120 (2004).</li><li>Pan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ipr1+gene+mediates+innate+immunity+to+tuberculosis.">Ipr1 gene mediates innate immunity to tuberculosis.</a> Nature. 434, 767-772 (2005).</li><li>Garifulin, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irf3+polymorphism+alters+induction+of+interferon+beta+in+response+to+Listeria+monocytogenes+infection.">Irf3 polymorphism alters induction of interferon beta in response to Listeria monocytogenes infection.</a> PLoS Genet. 3, 1587-1597 (2007).</li><li>Hof, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+of+different+strains+of+Listeria+monocytogenes+serovar+1/2a.">Virulence of different strains of Listeria monocytogenes serovar 1/2a.</a> Med Microbiol Immunol (Berl. 173, 207-218 (1984).</li><li>Wollert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extending+the+host+range+of+Listeria+monocytogenes+by+rational+protein+design.">Extending the host range of Listeria monocytogenes by rational protein design.</a> Cell. 129, 891-902 (2007).</li><li>Lecuit, 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+transgenic+model+for+listeriosis:+role+of+internalin+in+crossing+the+intestinal+barrier.">A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier.</a> Science. 292, 1722-1725 (2001).</li><li>Marco, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetration+of+Listeria+monocytogenes+in+mice+infected+by+the+oral+route.">Penetration of Listeria monocytogenes in mice infected by the oral route.</a> Microb Pathog. 23, 255-263 (1997).</li><li>Busch, D. H., Vijh, S., Pamer, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+model+for+infection+with+Listeria+monocytogenes.">Animal model for infection with Listeria monocytogenes.</a> Curr Protoc Immunol. Chapter. 19, 19-19 (2001).</li><li>Neuenhahn, M., Schiemann, M., Busch, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DCs+in+mouse+models+of+intracellular+bacterial+infection.">DCs in mouse models of intracellular bacterial infection.</a> Methods Mol Biol. 595, 319-329 (2010).</li><li>Conlan, J. W., North, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+are+essential+for+early+anti-Listeria+defense+in+the+liver,+but+not+in+the+spleen+or+peritoneal+cavity,+as+revealed+by+a+granulocyte-depleting+monoclonal+antibody.">Neutrophils are essential for early anti-Listeria defense in the liver, but not in the spleen or peritoneal cavity, as revealed by a granulocyte-depleting monoclonal antibody.</a> J Exp Med. 179, 259-268 (1994).</li><li>Czuprynski, C. J., Brown, J. F., Wagner, R. D., Steinberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+antigranulocyte+monoclonal+antibody+RB6-8C5+prevents+expression+of+acquired+resistance+to+Listeria+monocytogenes+infection+in+previously+immunized+mice.">Administration of antigranulocyte monoclonal antibody RB6-8C5 prevents expression of acquired resistance to Listeria monocytogenes infection in previously immunized mice.</a> Infect Immun. 62, 5161-5163 (1994).</li></ol>

Geen belangenconflicten verklaard.

Listeria is one of the most widely used organisms to characterize host immune responses to intracellular bacteria6. The protocol presented here enables one to measure bacterial load and immune cell responses within the same tissue of a given mouse. This dual measurement of a particular tissue for each infected mouse provides for more robust comparisons within and between mouse cohorts (either representing different mouse strains or time points post-infection). While Listeria infection by oral administration could also be used to study immune responses in mice, infection by intravenous injection is often used because: 1) it ensures rapid and effective delivery to the bloodstream; 2) it results in a synchronized and consistent systemic infection; and 3) the Mus species harbors a mutation in the gene encoding the E-cadherin receptor, which limits Listeria infection by oral administration (this mutation affects Listeria's ability to bind the mouse E-cadherin receptor and to efficiently cross the epithelial lining of the gastrointestinal tract)16-18.
There are a few critical steps in this protocol. First, the Listeria inoculum stock should be generated from a fresh overnight HBA culture to ensure viability and virulence. Second, it is important to accurately determine the CFU concentration of the inoculum before and after injecting all mice to ensure that the CFU concentration does not differ greatly between the first and last mouse injected. Third, injection of Listeria into the tail vein must be consistent for all mice. Lastly, it is necessary to perfuse the liver to deplete non-resident leukocytes to ensure accurate measurement of immune cells within the liver and not leukocytes passing through in the peripheral blood. All of these steps, if not performed successfully, can result in unwanted variability for bacterial load and/or immune responses between individual mice infected with Listeria. 
Two limitations of this protocol are the investigator's skill for infecting mice by intravenous injection and the detection of bacterial load in tissues. If one person is injecting a large number of mice, Listeria viability (i.e. CFU concentration) may reduce over time once the frozen inoculum is thawed (e.g. &gt;2 hours between injecting first and last mouse). It is up to the investigator to determine how many mice she/he can inject before the inoculum is compromised. Another limitation of this method is that the Listeria load cannot be accurately measured below 100CFU/organ due to the relatively small amounts of cultured tissue homogenate (e.g. Figure 3 indicates that 100CFU/organ is the detection limit). To more accurately measure lower values for CFU/organ, a larger amount of the tissue homogenate can be cultured (up to 0.5mL per plate, multiple plates can be used) so a greater proportion of the tissue is sampled for detection of Listeria. If a Stomacher is not available, then alternative methods to homogenate the tissue, such as using a tissue homogenizer, can be used instead for steps 7.1-7.2. On a practical note, if space in the 37&deg;C incubator is limited for culturing tissue samples from infected mice, then HBA culture plates can be incubated at room temperature for 2-3 days (the colonies will grow slower at room temperature). However, room temperature should not be used when growing cultures for preparation of frozen Listeria stocks.
This protocol provides a basic approach for characterizing Listeria infection in mice and can be used with other Listeria strains besides EGD. In addition to the liver and spleen, single-cell suspensions from lymph nodes can also be generated. In either instance, these single-cell suspensions can be used for a variety of analyses, including measuring immune cell subsets, and in vitro stimulation of sorted immune cells. Once the basic techniques are mastered this protocol can also be modified to isolate Listeria-specific T cells19, characterize dendritic cells20, or perform immune cell depletion at certain time-points after infection21,22 to more thoroughly characterize the immune response in mice infected with Listeria.

<table border="1"><tbody><tr><th> Naam van het serum </th><th> Vennootschap </th><th> Catalogus nummer </th><th> Reacties </th></tr><tr><td> Paard bloed agar base No.2 </td><td> Oxoid </td><td> CM0271 </td><td> De voorbereiding van agar basis voor HBA platen </td></tr><tr><td> Paard bloed (gedefibrineerd) </td><td> Oxoid </td><td> HB1000 </td><td> Voeg 5-10% agar-agar base </td></tr><tr><td> Brain hart infusie (BHI) bouillon (10 ml) </td><td> Oxoid </td><td> TM456 </td><td></td></tr><tr><td> Brain hart infusie uitgedroogd media </td><td> Oxoid </td><td> CM1135 </td><td></td></tr><tr><td> 96-well vlakke bodemplaat </td><td> BD Biosciences </td><td> 353072 </td><td></td></tr><tr><td> 70 um zeef cel </td><td> BD Biosciences </td><td> 352350 </td><td></td></tr><tr><td> Petrischaaltje </td><td> BD Biosciences </td><td> 351007 </td><td></td></tr><tr><td> Stomacher 80 Biomaster lab systeem </td><td> Seward </td><td></td><td></td></tr><tr><td> Plastic zakken Stomacher </td><td> Sarstedt </td><td> 86 9924 530 </td><td></td></tr><tr><td> Bovine serum albumine </td><td> Sigma </td><td> A3912 </td><td></td></tr><tr><td> FACS buffer </td><td></td><td></td><td> 0,1% (w / v) runder serum albumine in PBS </td></tr><tr><td> Isotone Percoll (33,75% Percoll in PBS) </td><td> GE Healthcare </td><td> 17-0891-01 </td><td> 33.75mL Percoll, 3.75mL 10x PBS, 62.5mL 1x PBS maakt 100 ml isotone Percoll </td></tr><tr><td> TAC Buffer </td><td></td><td></td><td> 17mm Tris, 140mm ammoniumchloride in gedestilleerd water </td></tr><tr><td> FCS / EDTA-buffer </td><td></td><td></td><td> Foetaal kalf serum met 10mm EDTA </td></tr><tr><td> FACS / EDTA-buffer </td><td></td><td></td><td> FACS buffer + 5mM EDTA </td></tr><tr><td> Trypan blauw </td><td> Sigma </td><td> T6146 </td><td> 0,4% (w / v) in PBS, filter gesteriliseerd </td></tr><tr><td> 2 ml Cryovial </td><td> Greiner Bio Een </td><td> 121263 </td><td></td></tr><tr><td> 27 gauge/1mL insulinespuit </td><td> Terumo Medical Products </td><td> SS10M2713 </td><td></td></tr><tr><td> Naald </td><td> Terumo Medical Products </td><td> NN-2516R (25G 5/8in) NN-2613R (26G 1/2in) </td><td></td></tr><tr><td> Injectiespuit </td><td> Terumo Medical Products </td><td> SS-01t (1 ml), SS-053 (5 ml), SS-10S (10 ml) </td><td></td></tr></tbody></table>

measuring bacterial load and immune responses in mice infected with listeria monocytogenes

Listeria monocytogenes (Listeria) is een Gram-positieve facultatief intracellulaire pathogeen 1. Muizen studies meestal intraveneuze injectie van Listeria in dienst, wat resulteert in de systemische infectie 2. Na injectie, Listeria verspreidt snel naar de milt en lever als gevolg van opname door CD8α + dendritische cellen en Kupffercellen 3,4. Eenmaal gefagocyteerd, diverse bacteriële eiwitten kunnen Listeria naar het fagosoom ontsnappen, in het cytosol te overleven, en de naburige cellen infecteren 5. Tijdens de eerste drie dagen van de infectie, de verschillende aangeboren immuun cellen (bijvoorbeeld monocyten, neutrofielen, NK-cellen, dendritische cellen) bemiddelen bactericide mechanismen die Listeria proliferatie te minimaliseren. CD8 + T-cellen worden vervolgens gerekruteerd en verantwoordelijk voor de uiteindelijke klaring van Listeria van de gastheer, meestal binnen 10 dagen na infectie 6. Succesvolle klaring van Listeria van geïnfecteerde muizen afhankelijk van de juiste begin van de gastheer immuunreacties 6. Er is een breed scala aan gevoeligheden onder inteelt muizenstammen 7,8. Over het algemeen, muizen met een verhoogde gevoeligheid voor Listeria infecties zijn minder in staat om bacteriële proliferatie controle, waaruit blijkt toegenomen bacteriële belasting en / of vertraagde klaring ten opzichte van resistente muizen. Genetische studies, waaronder linkage analyses en knock-out muis stammen, hebben geïdentificeerd verschillende genen die sequentievariatie gastheer reacties treft om Listeria besmetting 6,8-14. Bepaling en vergelijking van de infectie kinetiek tussen de verschillende muizenstammen is dan ook een belangrijke methode voor het identificeren van gastheer genetische factoren die tot immuunreacties bij te dragen tegen Listeria. Vergelijking van de gastheer reacties op de verschillende stammen Listeria is ook een effectieve manier om bacteriële virulentie factoren die kunnen dienen als potentiële doelwitten voor antibiotica of vaccins ontwerp te identificeren. We beschrijven hier een eenvoudige methode voor het meten van bacteriële belasting (kolonievormende eenheden [CFU] per weefsel) en het voorbereiden van eencellige suspensies van de lever en milt voor FACS-analyse van immuunresponsen bij Listeria-geïnfecteerde muizen. Deze methode is vooral nuttig voor de initiële karakterisering van Listeria-infectie in nieuwe muizenstammen, evenals de vergelijking van de immuunrespons tussen verschillende muizenstammen die besmet zijn met Listeria. We gebruiken de Listeria monocytogenes EGD stam 15 die, wanneer gekweekt op bloed-agar, een karakteristieke stralenkrans rond elke kolonie als gevolg van β-hemolyse 1 (figuur 1) vertoont. Bacteriële belasting en immuunreacties kan worden bepaald op welk tijdstip-punt na de infectie door het kweken van weefsel homogenaat op bloed agar platen en het voorbereiden van weefsel celsuspensies voor FACS analyse met behulp van de hieronder beschreven protocollen. Wij merken dat individuen die immuungecompromitteerd zijn of zwanger bent niet mag Listeria hanteren, en de desbetreffende institutionele bioveiligheid commissie en dier moet het management worden geraadpleegd voordat het werk begint.

Watch this Scientific Journal Video about Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes at JoVE.com

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is een modelorganisme voor het bestuderen van immuunreacties en de genetische gevoeligheid voor bacteriën intracellulaire bij muizen. Deze methode stelt je in staat om bacteriële lading te meten en single-cell suspensies van de lever en de milt van de muizen voor het genereren van FACS-analyse van veranderingen in het immuunsysteem van cellen als gevolg van Listeria-infectie te bepalen.

Het meten van bacteriën en immuunresponsen bij muizen geïnfecteerd met Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

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Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

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