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

Författarna vill tacka Anna Walduck, Christina Cheers, Stuart Berzins, Dale Godfrey, Yifan Zhan och Jonathan Wilksch för råd och reagenser. Detta arbete har finansierats av Juvenile Diabetes Research Foundation (1-2008-602) och den australiska nationella hälso-och medicinska forskningsrådet (575.552). NV stöds av en australisk Forskarutbildning Award. OW stöds av en RD Wright stipendium från Australian National Health Medical Research Council.

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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>

Inga intressekonflikter deklareras.

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> Reagensets namn </th><th> Företag </th><th> Katalognummer </th><th> Kommentarer </th></tr><tr><td> Häst blodagar bas nr 2 </td><td> Oxoid </td><td> CM0271 </td><td> Beredning av agar bas för HBA plattor </td></tr><tr><td> Häst blod (defibrinerat) </td><td> Oxoid </td><td> HB1000 </td><td> Tillsätt 5-10% agar bas </td></tr><tr><td> Brain hjärta infusion (BHI) buljong (10 ml) </td><td> Oxoid </td><td> TM456 </td><td></td></tr><tr><td> Brain hjärta infusion uttorkad medier </td><td> Oxoid </td><td> CM1135 </td><td></td></tr><tr><td> 96-bra plan bottenplatta </td><td> BD Biosciences </td><td> 353.072 </td><td></td></tr><tr><td> 70 ìm cell sil </td><td> BD Biosciences </td><td> 352.350 </td><td></td></tr><tr><td> Små petriskål </td><td> BD Biosciences </td><td> 351.007 </td><td></td></tr><tr><td> Stomacher 80 biomaster lab-systemet </td><td> Seward </td><td></td><td></td></tr><tr><td> Plast Stomacher påsar </td><td> Sarstedt </td><td> 86 9924 530 </td><td></td></tr><tr><td> Bovint serumalbumin </td><td> Sigma </td><td> A3912 </td><td></td></tr><tr><td> FACS buffert </td><td></td><td></td><td> 0,1% (w / v) bovint serumalbumin i PBS </td></tr><tr><td> Isoton Percoll (33,75% Percoll i PBS) </td><td> GE Healthcare </td><td> 17-0891-01 </td><td> 33.75mL Percoll, 3.75mL 10x PBS, gör 62.5mL 1x PBS 100mL isoton Percoll </td></tr><tr><td> TAC Buffer </td><td></td><td></td><td> 17mm Tris, 140mm ammoniumklorid i destillerat vatten </td></tr><tr><td> FCS / EDTA-buffert </td><td></td><td></td><td> Fostrets kalvserum med 10mm EDTA </td></tr><tr><td> FACS / EDTA-buffert </td><td></td><td></td><td> FACS buffert + 5mm EDTA </td></tr><tr><td> Trypan blå </td><td> Sigma </td><td> T6146 </td><td> 0,4% (w / v) i PBS, filter steriliserat </td></tr><tr><td> 2ml Cryovial </td><td> Greiner Bio En </td><td> 121.263 </td><td></td></tr><tr><td> 27 gauge/1mL spruta </td><td> Terumo Läkemedelsverket </td><td> SS10M2713 </td><td></td></tr><tr><td> Needle </td><td> Terumo Läkemedelsverket </td><td> NN-2516R (25G 5/8in) NN-2613R (26G 1/2in) </td><td></td></tr><tr><td> Spruta </td><td> Terumo Läkemedelsverket </td><td> SS-01t (1 ml), SS-053 (5 ml), SS-10S (10mL) </td><td></td></tr></tbody></table>

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

Listeria monocytogenes (Listeria) är en Gram-positiv fakultativt intracellulär patogen 1. Mus studier vanligtvis använder intravenös injektion av Listeria, vilket resulterar i systemisk infektion 2. Efter injektionen sprider Listeria snabbt till mjälten och levern på grund av upptag av CD8α + dendritiska celler och Kupfferceller 3,4. När fagocyteras, olika bakteriella proteiner att Listeria att undkomma phagosome, överleva i cytosolen, och infektera angränsande celler 5. Under de tre första dagarna av infektion, olika medfödda immunförsvaret celler (t ex monocyter, neutrofiler, NK-celler, dendritiska celler) medla bakteriedödande mekanismer som minimerar Listeria spridning. CD8 + T celler senare rekryteras och ansvarig för den slutliga godkännande av Listeria från värden, vanligtvis inom 10 dagar efter infektion 6. Framgångsrik clearance av Listeria från infekterade möss beror på lämpliga uppkomsten av värd immunsvar 6. Det finns ett brett spektrum av känslighet hos inavlade musstammar 7,8. Generellt möss med ökad mottaglighet för Listeria infektion är mindre möjlighet att kontrollera bakteriell spridning, visar ökad bakteriell belastning och / eller fördröjd clearance jämfört med resistenta möss. Genetiska studier, inklusive koppling analyser och knockout stammar mus, har identifierat olika gener som sekvensen variation påverkar värd svar på Listeria infektion 6,8-14. Fastställande och jämförelse av infektion kinetik mellan olika musstammar är därför en viktig metod för att identifiera värd genetiska faktorer som bidrar till att immunsvaret mot Listeria. Jämförelse av värden svar på olika Listeria stammar är också ett effektivt sätt att identifiera bakteriella virulensfaktorer som kan fungera som potentiella mål för behandling med antibiotika eller vaccin design. Vi beskriver här en enkel metod för att mäta bakteriehalten (kolonibildande enheter [CFU] per vävnad) och förbereda encelliga suspensioner av levern och mjälten för FACS analys av immunsvar hos Listeria-infekterade möss. Den här metoden är särskilt användbart för första karakterisering av Listeria infektion i stammar roman mus, samt jämförande av immunsvaret mellan olika musstammar infekterade med Listeria. Vi använder Listeria monocytogenes EGD stam 15 som när den odlade på blodagar, uppvisar en karakteristisk gloria zon runt varje koloni på grund av β-hemolys 1 (Figur 1). Bakteriehalten och immunsvar kan fastställas när som helst-punkt efter infektion genom odling av vävnad homogenatet på tallrikar blodagar och förbereda vävnad suspensioner cell för FACS-analys med de protokoll som beskrivs nedan. Vi vill påpeka att personer som har nedsatt immunförsvar och gravida ska inte hantera Listeria, och de relevanta institutionella biosäkerhet kommittén och djur fastighetsförvaltning bör konsulteras innan arbetet påbörjas.

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 är en modell organism för att studera immunsvaret och genetisk känslighet för intracellulära bakterier i möss. Denna metod gör en att mäta bakteriehalten och generera encelliga suspensioner av levern och mjälten från möss för FACS-analys för att fastställa förändringar i immunceller grund av Listeria infektion.

Att mäta bakteriehalten och immunsvar hos möss som blivit infekterade med 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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