Development of this protocol was supported by an operating grant from CIHR (MOP97807) to SED.

No conflicts of interest to disclose.

Hier beschreiben wir ein Protokoll, wie eine grundlegende experimentelle Infektion durchzuführen mit der EGD Stamm von L. monocytogenes 25 bei männlichen oder weiblichen C57BL / 6J - Mäuse. Dieses Protokoll wurde für die Zwecke der Untersuchung der Wirkung eines neuartigen niedermolekularen IS001 auf IFN-γ Produktion von angeborenen und adaptiven Lymphozyten in vivo 31 eingerichtet. Bakterien-Clearance und das Überleben nach der Infektion Durch die Überwachung wurden Erkenn...

IFN-γ ist ein Zytokin , das für die Vermittlung von Immunität gegen intrazelluläre Pathogene und zur Steuerung des Tumorwachstums 5 entscheidend. Die Bedeutung dieses Zytokin in bakteriellen Resistenz ist in der Beobachtung offensichtlich , die mit Mutationen in der IFN-γ - Signalweg sind sehr anfällig für eine Infektion mit Mykobakterien und Salmonellen 6 Menschen. In ähnlicher Weise defizienten Mäusen entweder IFN-γ oder die IFN-γ - Rezeptor zeigen Defekte im Widerstand gegen Mykobakterien 7-9 und andere intrazelluläre Pathogene einschließlich L. monocytogenes 10,11, Leishmania major 12, S. typhimurium 13 und bestimmte Viren 11. Neben der Bekämpfung von Krankheitserregern, IFN-γ spielt eine entscheidende Rolle bei der Wirtsverteidigung gegen Tumoren 14. Obwohl höhere Produktion von IFN-γ im Rahmen einer Infektion oder Krebs nützlich ist, verlängerte Produktion dieses Zytokins wurde t verknüpfto der Entwicklung von systemischer Autoimmunität 15-17 und der Beschleunigung von Diabetes Typ I in dem nicht-fettleibigen diabetischen Maus - Modell 18. Die Hauptquellen von IFN-γ sind : NK - Zellen, NKT - Zellen, γδ - T - Zellen, T - Helfer 1 (Th1) -Zellen, und zytotoxischen T - Lymphozyten (CTL) 5,19,20. IFN-γ erhöht sowohl angeborenen und erworbenen Immunität durch: (1) Hochregulieren Haupthistokompatibilitätskomplex (MHC) Klasse I und II-Expression, (2) die Expression von co-stimulatorischen Molekülen auf Antigen-präsentierenden Zellen zu erhöhen, (3) Verbesserung der Makrophagen Phagozytose und die Produktion von pro-inflammatorischen Zytokinen und mikrobizide Faktoren (beispielsweise Stickstoffmonoxid und reaktive Sauerstoffspezies), (4) die Förderung der Differenzierung von naiven CD4 + T - Zellen in Th1 - Effektorzellen, (5) Förderung der Antikörperklasse Umschaltung auf Immunglobulin ( Ig) 2a und IgG3 (in der Maus), (6) Induzierung der Produktion von Chemokinen Immunzellen-Stellen von i zu rekrutierennfection und (7) Verbesserung der NK - Zellen und CTL - Antworten 5,19. Aufgrund der entscheidenden Bedeutung der IFN-γ in der Wirtsantwort auf Pathogene und Tumore, rekombinantes IFN-γ wurde als Behandlung für verschiedene Infektionen und Malignitäten (in 19 überprüft) getestet. Da jedoch die systemische Verabreichung von IFN-γ oder Th1 Zytokin Förderung Interleukin-12 (IL-12) mit Nebenwirkungen verbunden ist und dosisabhängige Toxizität 19,21 besteht ein Interesse alternative Strategien bei der Entwicklung von IFN-γ - Produktion zu erhöhen , indem Immunzellen. Entwicklung neuer Biologika und kleinen Molekülen erfordert in vivo Screening - Tools zu testen , ob solche Mittel während einer Immunantwort IFN-γ Produktion zu erhöhen , und ob dies in einer sinnvollen biologischen Wirkungen wie Erhöhung der das Überleben der Tiere. Experimentelle Infektion von Mäusen mit dem grampositiven Bakterium L. monocytogenes ist ein Instrumental - Modell seitEntschlüsselung der Rolle von IFN-γ in Wirtsimmunität gegen intrazelluläre Erreger 1,22. Infektion von Mäusen mit dem Pathogen intravenös oder intraperitoneal (ip) führt zur schnellen Verbreitung der Bakterien an der Milz und der Leber, wo sie von Makrophagen und Hepatozyten mit peak bakterielle Belastungen in der Milz internalisiert zwischen 3 und 4 Tage auftretenden post- Infektion 1,3,22. Produktion von IFN-γ durch NK - Zellen ist wichtig für die Makrophagenaktivierung und frühen Widerstand gegen den Erreger 3; jedoch bei hohen Infektionsdosen, die Produktion von IFN-γ kann auch auf Erreger - Clearance 23 schädlich sein. NKT - Zellen sind auch eine Quelle von IFN-γ in der Milz und Leber während der frühen Kontrolle von Pathogenen 2,24 und diese Produktion wurde 2 gezeigt , IFN-γ - Produktion durch andere Zelltypen , einschließlich NK - Zellen verstärken. Auf der anderen Seite, die später wirkenden adaptive T - Lymphozyten, CD8 + T - Zellen in partidere sind wichtig für das Spiel des Erregers zu vermitteln und den Schutz gegen eine erneute Infektion 1,4,22 bereitstellt. Diese Infektionsmodell wurde für eine Reihe von Gründen für Forscher attraktiv war ( beschrieben in 1). Erstens, eine Infektion mit dem Pathogen ist in hohem Maße reproduzierbar und induziert eine starke Th1 und zellulären Immunantwort. Zweitens, während subletalen Infektion wird bakterielle Belastung in der Leber und der Milz konzentriert, wo es kann leicht gemessen werden. Drittens kann der Erreger sicher unter Biosafety Level 2 (BSL2) Bedingungen behandelt werden. Viertens der Organismus und die Immunantwort, dass es weitgehend charakterisiert worden erzeugt. Schließlich wurde eine Vielzahl von Mutanten und genetisch veränderte Stämme entwickelt, die für die Verwendung zur Verfügung stehen. Beschrieben ist hier ein Basisprotokoll für die Impfung von C57BL / 6J - Mäuse mit dem EGD Stamm von L. monocytogenes und 25 für IFN Messung - γ reAntworten von NK, NKT und adaptive Lymphozyten nach der Infektion. Beschrieben ist , wie bakterielle Belastung in der Milz und Leber nach subletalen Infektion zu messen , und die Überlebensstudien nach der Infektion durchgeführt werden mit einer modifizierten LD 50 -Dosis des Pathogens. Schließlich werden repräsentative Daten gezeigt, wie kann dieses Protokoll verwendet werden , um die Wirkung neuer Therapien auf IFN-γ - Antworten und das Überleben der Mäuse zum Screening von L. monocytogenes Infektion.

<table><tbody><tr><td>Brain Heart Infusion Broth, Modified</td><td>BD</td><td>299070</td><td>any brand should be appropriate</td></tr><tr><td>Agar</td><td>BD</td><td>214010</td><td>any brand should be appropriate</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td>any brand should be appropriate</td></tr><tr><td>1x PBS</td><td>Sigma</td><td>D8537</td><td>any brand should be appropriate</td></tr><tr><td>TissueLyser II</td><td>Qiagen</td><td>85300</td><td>any brand should be appropriate</td></tr><tr><td>Ammonium Chloride (NH&lt;sub&gt;3&lt;/sub&gt;Cl)</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;EDTA</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>22400089</td><td>any brand should be appropriate</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gibco</td><td>12483</td><td>Before use, heat-inactivate at 56 &amp;deg;C for 30 min</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030</td><td>any brand should be appropriate</td></tr><tr><td>Non-essential amino acids</td><td>Gibco</td><td>11140</td><td>any brand should be appropriate</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140</td><td>any brand should be appropriate</td></tr><tr><td>GolgiStop Protein Transport Inhibitor (containing Monensin)</td><td>BD</td><td>554724</td><td>Use 4 &amp;mu;l in 6 ml cell culture</td></tr><tr><td>16% Paraformaldehye</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Dilute to 4% PFA in ddH&lt;sub&gt;2&lt;/sub&gt;O or 1x PBS</td></tr><tr><td>10x Perm/Wash buffer</td><td>BD</td><td>554723</td><td>Dilute 1x in ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Fc block, Anti-Mouse CD16/CD32 Purified</td><td>eBioscience</td><td>14-0161</td><td>Dilute 1:50</td></tr><tr><td>Fixable Viability Dye eFluor 506</td><td>eBioscience</td><td>65-0866</td><td>Dilute 1:1,000 (we have also used viability dyes from Molecular Probes)</td></tr><tr><td>anti-Mouse CD4-PE-Cy5 (GK1.5)</td><td>eBioscience</td><td>15-0041</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>anti-Mouse CD8-FITC (53-6.7)</td><td>eBioscience</td><td>11-0081</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>PBS57/mCD1d tetramer-APC</td><td>NIH Tetramer Core Facility</td><td>N/A</td><td>Obtained as a gift from the facility</td></tr><tr><td>anti-Mouse TCR&amp;beta;-PE-Cy7 (H56-597)</td><td>eBioscience</td><td>25-5961</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>anti-Mouse NKp46-APC-eFluor780 (29A1.4)</td><td>eBioscience</td><td>47-3351</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>anti-Mouse CD45 PE-Cyanine7 (30-F11)</td><td>eBioscience</td><td>25-0451</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>anti-Mouse IFN gamma-PE (XMG1.2)</td><td>eBioscience</td><td>12-7311</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>OneComp eBeads</td><td>eBioscience</td><td>01-1111</td><td>Use one drop per stain</td></tr><tr><td>Mouse IFN gamma ELISA kit</td><td>eBioscience</td><td>88-7314</td><td>Used for measuring the interferon gamma in the culture supernatant</td></tr><tr><td>50 ml vented tubes for culture</td><td></td><td></td><td>Used for culturing the bacteria, any brand should be appropriate</td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>bacterial petri dishes</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>2 ml cyrovials</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>UV spectrometer</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>safety engineered needles</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>C57BL/6J</td><td>Jackson laboratories</td><td>Stock#000664</td><td>Order for arrival at 7 weeks</td></tr><tr><td>Bleach</td><td></td><td></td><td>For decontamination</td></tr><tr><td>70% Ethanol</td><td></td><td></td><td>For decontamination</td></tr><tr><td>Glass beads</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Centrifuge</td><td></td><td></td><td>rotor, buckets, bucket covers.</td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Sterile Glycerol</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Pipette Tips</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Pipette</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Surgical instruments</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>70 micron strainers</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>3 ml syringe</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Pipette gun</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Filtration Units</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Trypan Blue</td><td></td><td></td><td>Dilute 1 to 9 in ddH&lt;sub&gt;2&lt;/sub&gt;O, any brand should be appropriate</td></tr><tr><td>Hemocytometer</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Round bottomed plates</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>FACs tubes</td><td>Fisher Scientific</td><td>14-959-5</td><td></td></tr><tr><td>BD LSR II</td><td>BD</td><td></td><td>Any flow cytometer could be used for acquisition that has an appropriate laser configuration and filter set to discriminate the fluorochormes</td></tr><tr><td>Flowjo software</td><td>Treestar</td><td></td><td>Used for data analysis. Other types of data analysis software will also be appropriate</td></tr><tr><td>Multichannel pipettor (0 - 300 &amp;micro;l)</td><td>Eppendorf</td><td></td><td>Used for washing cells and adding antibodies during flow cytometry staining</td></tr><tr><td>Acetic Acid</td><td></td><td></td><td>Used for washing glass beads, any brand should be appropriate</td></tr><tr><td>Microbank Bacterial Preservation System</td><td>Pro-lab Diagnostics</td><td></td><td>Used as an alternative to glycerol stocks for long-term storage of bacteria</td></tr></tbody></table>

experimental infection with listeria monocytogenes as a model for studying host interferon-&#947; responses

L. monocytogenes ist ein gram-positive Bakterien , die eine Ursache der durch Lebensmittel übertragenen Krankheiten beim Menschen ist. Experimentelle Infektion von Mäusen mit diesem Erreger hat sich auf die Rolle der angeborenen und adaptiven Immunzellen und spezifische Zytokine in Wirtsimmunität gegen intrazelluläre Erreger sehr informativ gewesen. Produktion von IFN-γ durch angeborene Zellen während der Infektion mit subletalen L. monocytogenes ist wichtig für Makrophagen und frühe Kontrolle des Krankheitserregers 1-3 aktiviert. Zusätzlich kann durch adaptive Speicher Lymphozyten IFN-γ - Produktion ist wichtig zur Grundierung , die Aktivierung von angeborenen Zellen nach Reinfektion 4. Die L. monocytogenes - Infektionsmodell dient somit als großes Werkzeug für die Untersuchung , ob neue Therapien , die IFN-γ Produktion haben einen Einfluss auf die IFN-γ - Antworten in vivo und haben produktive biologische Effekte wie die Erhöhung der bakteriellen Clearance oder die Verbesserung der das Überleben der Mäuse zu erhöhen sind so konzipiert, vonInfektion. Beschrieben ist hier ein Basisprotokoll für den Umgang mit der EGD Stamm von L. monocytogenes intraperitoneale Infektionen von C57BL / 6J - Mäusen zu führen und IFN-γ - Produktion durch NK - Zellen, NKT - Zellen und adaptive Lymphozyten mittels Durchflusszytometrie zu messen. Außerdem werden Verfahren beschrieben: (1) wachsen, und die Bakterien zur Inokulation herzustellen, (2) Messung der Keimbelastung in der Milz und in der Leber, und (3) das Überleben der Tiere zu messen Endpunkten. Repräsentative Daten sind vorgesehen , um auch veranschaulichen , wie dieses Infektionsmodell verwendet werden kann , um die Wirkung von spezifischen Mitteln auf IFN-γ Reaktionen auf L. monocytogenes und das Überleben der Mäuse aus dieser Infektion zu testen.

Abbildung 3 zeigt eine typische Durchflusszytometrie Färbung von IFN-γ in Milz- NK und NKT - Zellen bei 24 Stunden nach der Infektion mit 10 5 CFU des Erregers. Diese Figur zeigt auch die Gating - Strategie für die Färbung Tafel in Tabelle 2 beschrieben. Figur 4 zeigt einige repräsentative Daten , die in einem Experiment erhalten wurden , in denen männliche Mäuse mit dem PPAR &amp; agr; Antagonist IS001 oder Vehikelkont...

Watch this Scientific Journal Video about Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-Î³ Responses at JoVE.com

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

Dieses Protokoll beschreibt , wie C57BL / 6J - Mäuse mit dem Stamm EGD von Listeria monocytogenes (L. monocytogenes) und zur Messung der Interferon-γ (IFN-γ) Antworten , die durch natürliche Killer (NK) Zellen, natürliche Killer - T (NKT) -Zellen zu inokulieren, und adaptive T-Lymphozyten nach der Infektion. Dieses Protokoll beschreibt auch , wie Überlebensstudien bei Mäusen mit einer modifizierten LD 50 Dosis des Erregers nach der Infektion zu führen.

Experimentelle Infektion mit<em&gt; Listeria monocytogenes</em&gt; Als Modell für die Untersuchung Host-Interferon-γ Antworten

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

experimental-infection-with-listeria-monocytogenes-as-model-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

13.5K Aufrufe.  University of Toronto.  Dieses Protokoll beschreibt , wie C57BL / 6J - Mäuse mit dem Stamm EGD von Listeria monocytogenes (L. monocytogenes) und zur Messung der Interferon-γ (IFN-γ) Antworten , die durch natürliche Killer (NK) Zellen, natürliche Killer - T (NKT) -Zellen zu inokulieren, und adaptive T-Lymphozyten nach der Infektion. Dieses Protokoll beschreibt auch , wie Überlebensstudien bei Mäusen mit einer modifizierten LD 50 Dosis des Erregers nach der Infektion zu...

Serie: Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses

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

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

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

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Mess-bakterielle Belastung und Immunreaktionen in Mäuse Infizierte mit Listeria monocytogenes</em

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

Forschung

Immunologie und Infektion

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Introducing Schubspannung in der Studie der bakteriellen Adhäsion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

Die<em&gt; Citrobacter rodentium</em&gt; Mouse Model: Studieren Erreger und Wirt Beiträge zur Infectious Colitis

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

Mündliche Übertragung von<em&gt; Listeria monocytogenes</em&gt; In Mäusen über die Aufnahme von kontaminierten Speisen

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Imaging InlC Sekretion to Cellular-Infektion durch den bakteriellen Erreger untersuchen<em&gt; Listeria monocytogenes</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.

Listeria monocytogenes Infektion des Gehirns

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Verwenden eine bakterielle Erreger, Sonde für zelluläre und organismische Ebene Host Antworten

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

Bildgebung der In Situ Interferon Gamma Produktion in der Maus Milz nach Listeria monocytogenes Infektion

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

Assay for the Identification of Interferon-Gamma Secretion in Murine Lymphoid Organs

Source: Mazet, J. M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59819">Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection.</a> J. Vis. Exp. (2019).
This video demonstrates an imaging method to visualize in situ interferon-gamma (IFN-&#947;) secretion in the mouse spleen. Following bacterial infection and the induction of IFN-&#947; accumulation inside T cells and natural killer cells, the spleen is harvested from the mouse. Upon obtaining tissue sections, immunostaining is performed to visualize IFN-&#947; inside the cells.

Assay zur Identifizierung der Interferon-gamma-Sekretion in lymphatischen Organen der Maus

Source: Mazet, J. M., et al. Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Assays and Functional Profiling

Assessing Bacterial Colonization in a Mouse Brain following Listeria monocytogenes Infection

Using forceps, open the skull to expose the brain and place a spatula between the underside of the brain and the base of the skull to carefully transfer the brain into a 15 milliliter tube containing 5 milliliters of 4 degrees Celsius PBS.
In a biosafety cabinet, insert the tip and run a tissue homogenizer for 10 seconds in sequential 5% bleach, sterile water, 75% ethanol, and sterile water solutions in separate 15 milliliter conical tubes. When the tip has been thoroughly sterilized, homogenize the infected brain in its tube.
Prepare tenfold serial dilutions of each organ homogenate in sterile PBS and plate the dilutions on brain heart infusion agar plates. Then, incubate the homogenate dilution cultures at 37 degrees Celsius overnight, and count the number of colonies on each plate to determine the total number of bacteria per organ or milliliter of blood.

Experimentelle Infektion mit

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Mess-bakterielle Belastung und Immunreaktionen in Mäuse Infizierte mit Listeria monocytogenes

Introducing Schubspannung in der Studie der bakteriellen Adhäsion

Die

Mündliche Übertragung von

Imaging InlC Sekretion to Cellular-Infektion durch den bakteriellen Erreger untersuchen

Listeria monocytogenes Infektion des Gehirns

Verwenden eine bakterielle Erreger, Sonde für zelluläre und organismische Ebene Host Antworten

Bildgebung der In Situ Interferon Gamma Produktion in der Maus Milz nach Listeria monocytogenes Infektion

Assay zur Identifizierung der Interferon-gamma-Sekretion in lymphatischen Organen der Maus

Beurteilung der bakteriellen Besiedlung in einem Mäusegehirn nach einer Infektion mit Listeria monocytogenes