Name: experimental infection with listeria monocytogenes as a model for studying host interferon-&#947; responses
Uploaded: 2016-11-16T13:00:00
Description: Watch this Scientific Journal Video about Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-Î³ Responses at JoVE.com

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

Safety Statement
This protocol describes infection of mice with live L. monocytogenes. The pathogen is handled safely under BSL2 conditions by trained personnel who are not immunocompromised. Immunocompromised people include pregnant women, the elderly and individuals who are HIV-infected or have chronic conditions that require treatment with immunosuppressive therapy. Personnel should don a protective lab coat or gown, gloves, mask, and eye protection while handling infected samples. T...

Here we describe a protocol of how to carry out a basic experimental infection with the EGD strain of L. monocytogenes25 in male or female C57BL/6J mice. This protocol was set up for the purpose of studying the effect of a novel small molecule IS001 on IFN-&#947; production by innate and adaptive lymphocytes in vivo31. By monitoring bacterial clearance and survival post-infection, insights were gained into how these changes in IFN-&#947; impacted the host&#39;s ability to control t...

IFN-&#947; is a cytokine that is crucial for mediating immunity against intracellular pathogens and for controlling tumor growth5. The importance of this cytokine in bacterial resistance is evident in the observation that humans with mutations in the IFN-&#947; signaling pathway are highly susceptible to infection with mycobacteria and salmonellae6. Similarly, mice deficient in either IFN-&#947; or the IFN-&#947; receptor exhibit defects in resistance to mycobacteria7-9 and other intracellular pathogens including L. monocytogenes10,11, Leishmania major12, Salmonella typhimurium13, and certain viruses11. In addition to combatting pathogens, IFN-&#947; plays a crucial role in host-defense against tumors14. Though higher production of IFN-&#947; is beneficial in the context of infection or cancer, prolonged production of this cytokine has been linked to the development of systemic autoimmunity15-17 and the acceleration of type I diabetes in the non-obese diabetic mouse model18.
The major sources of IFN-&#947; include NK cells, NKT cells, &#947;&#948; T cells, T helper 1 (Th1) cells, and cytotoxic T lymphocytes (CTL)5,19,20. IFN-&#947; enhances both innate and adaptive immunity by: (1) up-regulating major histocompatibility complex (MHC) class I and II expression, (2) increasing the expression of co-stimulatory molecules on antigen presenting cells, (3) enhancing macrophage phagocytosis and the production of pro-inflammatory cytokines and microbicidal factors (e.g., nitric oxide and reactive oxygen species), (4) promoting the differentiation of na&#239;ve CD4+ T cells into Th1 effector cells, (5) promoting antibody class switching to immunoglobulin (Ig)2a and IgG3 (in mouse), (6) inducing the production of chemokines to recruit immune cells to sites of infection, and (7) enhancing NK cell and CTL responses5,19. Given the crucial importance of IFN-&#947; in the host response to pathogens and tumors, recombinant IFN-&#947; has been tested as a treatment for various infections and malignancies (reviewed in19). However, because systemic administration of IFN-&#947; or the Th1 promoting cytokine interleukin-12 (IL-12) is associated with side effects and dose-related toxicity19,21, there is interest in developing alternative strategies to increase IFN-&#947; production by immune cells. Development of new biologics and small molecules requires in vivo screening tools to test whether such agents increase IFN-&#947; production during an immune response and whether this translates into meaningful biological effects such as increases in animal survival.
Experimental infection of mice with the gram-positive bacterium L. monocytogenes has been an instrumental model for deciphering the role of IFN-&#947; in host-immunity against intracellular pathogens1,22. Infection of mice with the pathogen intravenously or intraperitoneally (i.p.) leads to the rapid dissemination of the bacteria to the spleen and liver, where they become internalized by resident macrophages and hepatocytes with peak bacterial loads in the spleen occurring between 3 and 4 days post-infection1,3,22. Production of IFN-&#947; by NK cells is important for macrophage activation and early resistance against the pathogen3; however at high infectious doses, production of IFN-&#947; can also be detrimental to pathogen clearance23. NKT cells are also a source of IFN-&#947; in the spleen and liver during early control of pathogens2,24 and this production has been shown to amplify IFN-&#947; production by other cell types including NK cells2. On the other hand, later-acting adaptive T lymphocytes, CD8+ T cells in particular, are important for mediating the clearance of the pathogen and providing protection against re-infection1,4,22.
This infection model has been attractive to researchers for a number of reasons (reviewed in1). First, infection with the pathogen is highly reproducible and induces a strong Th1 and cellular immune response. Secondly, during sublethal infection, bacterial load is concentrated in the liver and spleen where it can be easily measured. Thirdly, the pathogen can be safely handled under Biosafety Level 2 (BSL2) conditions. Fourthly, the organism and the immune response that it generates have been extensively characterized. Finally, a variety of mutant and genetically-modified strains have been developed that are available for use.
Described here is a basic protocol for inoculation of C57BL/6J mice with the EGD strain of L. monocytogenes25 and for measuring IFN-&#947; responses by NK, NKT, and adaptive lymphocytes post-infection. Also described is how to measure bacterial load in the spleen and liver after sublethal infection and to carry out survival studies after infection with a modified LD50 dose of the pathogen. Finally, representative data are shown of how this protocol can be used to screen the effect of new treatments on IFN-&#947; responses and mouse survival from L. monocytogenes infection.

<table border="0" cellpadding="0" cellspacing="0" width="923">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Brain Heart Infusion Broth, Modified</td>
			<td style="width:191px;">BD</td>
			<td style="width:107px;">299070</td>
			<td style="width:273px;">any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>BD</td>
			<td style="width:107px;">214010</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Triton X-100</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:107px;">X100</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1xPBS</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:107px;">D8537</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">TissueLyser II</td>
			<td style="width:191px;">Qiagen</td>
			<td style="width:107px;">85300</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Ammonium Chloride (NH3Cl)</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">KHCO3</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Na2EDTA</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">RPMI 1640</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:107px;">22400089</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Fetal Bovine Serum&#160;</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:107px;">12483</td>
			<td>Before use, heat-inactivate at 56 &#176;C for 30 min</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">L-glutamine</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:107px;">25030</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Non-essential amino acids</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:107px;">11140</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Penicillin/Streptomycin</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:107px;">15140</td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">GolgiStop Protein Transport Inhibitor (containing Monensin)</td>
			<td style="width:191px;">BD</td>
			<td style="width:107px;">554724</td>
			<td>Use 4 &#956;l in 6 ml cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">16% Paraformaledehye</td>
			<td style="width:191px;">Electron Microscopy Sciences</td>
			<td style="width:107px;">15710</td>
			<td>Dilute to 4% PFA in ddH20 or 1xPBS</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:353px;">10 x Perm/Wash buffer</td>
			<td style="width:191px;">BD</td>
			<td style="width:107px;">554723</td>
			<td>Dilute 10x in ddH20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Fc block, Anti-Mouse CD16/CD32 Purified</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">14-0161</td>
			<td>Dilute 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Fixable Viability Dye eFluor 506</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">65-0866</td>
			<td>Dilute 1:1000 (we have also used viability dyes from Molecular Probes)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">anti-Mouse CD4-PE-Cy5 (GK1.5)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">15-0041</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">anti-Mouse CD8-FITC (53-6.7)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">11-0081</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">PBS57/mCD1d tetramer-APC</td>
			<td style="width:191px;">NIH Tetramer Core Facility</td>
			<td style="width:107px;">N/A</td>
			<td>Obtained as a gift from the facility</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Mouse TCR&#946;-PE-Cy7 (H56-597)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">25-5961</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Mouse NKp46-APC-eFluor780 (29A1.4)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">47-3351</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">anti-Mouse CD45 PE-Cyanine7 (30-F11)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">25-0451</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">anti-Mouse IFN gamma-PE (XMG1.2)</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">12-7311</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">OneComp eBeads</td>
			<td style="width:191px;">eBioscience</td>
			<td style="width:107px;">01-1111</td>
			<td>Manufacturer recommends a certain test size; however this should be titrated before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Mouse IFN gamma ELISA kit</td>
			<td style="width:191px;">eBioscience</td>
			<td>88-7314</td>
			<td>Used for measuring the interferon gamma in the culture supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">50 mL vented tubes for culture</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>Used for culturing the bacteria, any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1.5 ml microcentrifuge tubes</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">bacterial petri dishes</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">2 ml cyrovials</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">UV spectrometer</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">safety engineered needles</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">C57BL6/J</td>
			<td style="width:191px;">Jackson laboratories</td>
			<td style="width:107px;">Stock#000664</td>
			<td>Order for arrival at 7 wks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Bleach</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>For decontamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">70% Ethanol</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>For decontamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Glass beads</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Centrifuge</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>rotor, buckets, bucket covers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Microcentrifuge</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Sterile Glycerol</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Pipette Tips</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Pipette</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Surgical instruments</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">70 micron strainers</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">3 ml syringe</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Pipette gun</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Filtration Units</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Trypan Blue</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>Dilute 1 to 9 in ddH20, any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Hemocytometer</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Round bottomed plates</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">FACs tubes</td>
			<td style="width:191px;">BD</td>
			<td style="width:107px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">BD LSR II</td>
			<td style="width:191px;">BD</td>
			<td style="width:107px;"></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 height="21">
			<td height="21" style="height:21px;width:353px;">Flowjo software</td>
			<td style="width:191px;">Treestar</td>
			<td style="width:107px;"></td>
			<td>Used for data analysis. Other types of data analysis software will also be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Multichannel pipettor (0-300 &#181;l)</td>
			<td style="width:191px;">Eppendorf</td>
			<td style="width:107px;"></td>
			<td>Used for washing cells and adding antibodies during flow cytometry staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Acetic Acid</td>
			<td style="width:191px;"></td>
			<td style="width:107px;"></td>
			<td>Used for washing glass beads, any brand should be appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Microbank Bacterial Preservation System</td>
			<td style="width:191px;">Pro-lab Diagnositics</td>
			<td style="width:107px;"></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 is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Figure 3 presents some typical flow cytometry staining of IFN-&#947; in splenic NK and NKT cells at 24 hr post-infection with 105 CFU of the pathogen. This figure also illustrates the gating strategy for the staining panel described in Table 2. Figure 4 shows some representative data that were obtained in one experiment where male mice were treated with the PPAR&#945; antagonist IS001 or vehicle control, infected with 105<...

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 &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

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

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 ...

The overall goals of this protocol are to cultivate and infect C57 blacksix mice with the EGG strain of listeria monocytogenes and to measure the bacterial load, interferon gamma responses, and survival and response to infection by this pathogen. This protocol can help researchers screen for agents or genes that have an impact on interferon gamma responses and on animal survival after bacterial infection. The main advantage of this technique is that it is relatively simple to set up in the laboratory.Even for those with little experience in cultivating bacteria. Also demonstrating the procedure will be Jennifer Ahn, a graduate student from my laboratory. Begin by dipping a sterile pipette tip into room temperature thawed glycerol stock of the L monocytogenes EGG strain.And immediately streak the tip back and forth across a section of a brain heart infusion or BHI plate. This is the primary streak. Turn the plate 90 degrees and drag a fresh pipette tip through the first streak, spreading the stock to the next quarter of the plate.This is the secondary streak. Then, turn and spread the secondary streak into a third quarter of the plate to make the tertiary streak. Now, turn the plate upside down at 37 degrees celsius, for an overnight incubation.Single uniform colonies should be visible in the last set of streaks between 16 and 24 hours. When the colonies appear, dispense 10 milliliters of sterile BHI broth into a sterile vented 50 milliliter tube and use a sterile pipette tip to transfer one L monocytogenes colony from the plate into the broth. Culture the inoculated broth in an orbital shaking incubator overnight at 37 degrees celsius and 225 rotations per minute, until the OD600 equals one.For preparation of the experimental L monocytogenes infection inoculum, in a biosafety cabinet, inoculate a tube of BHI medium with 100 microliters of the overnight culture. After another incubation at 37 degrees celsius, with shaking until the desired OD600 is reached, transfer the culture contents into a sterile centrifuge tube for centrifugation. And use a vacuum attached to a trap flask containing bleach to aspirate the supernatant.Wash the pellet two times with PBS under the same centrifuge conditions. After the second wash, resuspend the bacteria in PBS and dilute at the appropriate concentration. To deliver the CFU of interest to each mouse in a 200 microliter volume.Next, mix the suspension thoroughly with a sterile pipette tip to ensure that the bacteria are homogeneously distributed. Then draw 200 microliters of inoculum into a one milliliter safety engineered syringe fitted with a 25 gauge needle. Now, scruff a mouse with the less dominant hand by the loose skin around the shoulders.And inject all 200 microliters of the inoculum in the lower quadrant of the abdomen, just lateral to the midline to avoid the bladder, using a new needle and syringe for each animal. To measure the peak infection bacterial load, on day three post infection, restrain the limbs of an experimental animal in the supine position on a dissecting board. And disinfect the skin with 70 percent ethanol.Using sterile tough cut scissors, make an incision from the chest to the groin. Then make an incision from the mid groin toward each knee and from the mid chest toward each elbow. Blunt, dissect, and reflect back the skin, pinning it open with 25 gauge needles.Disinfect the muscle layer with more 70 percent ethanol. Then using sterile fine scissors, make a midline incision in the peritoneal wall. Use forceps to grasp the xiphoid process.Make incisions in the peritoneal wall starting at the xiphoid process and moving laterally on each side. Following the ribcage to just below the diaphragm. This will reveal the liver.Next, use sterile scissors to cut an approximately 100 milligram piece out of one lobe. Place the tissue in a preweighed 1.5 milliliter micro centrifuge tube containing 0.2 to 0.3 grams of 1.5 to 2 millimeter acid washed sterile glass beads. And use the forceps to gently push aside the organs on the left side of the peritoneal cavity to visualize the spleen.Use the forceps to gently grasp the spleen, cutting away the surrounding connective tissue to release the organ from the peritoneal cavity. Place the spleen in a different tube than the liver, and transfer the tissues to the laboratory in a leak proof container containing ice. Shake the tubes while using a bead mill homogenizer for three minutes at a 30 hertz frequency.Then, set up a tenfold dilution series of the homogenates in 0.1 percent trident X 100 in PBS. Next, use a sterile spreader to spread 100 microliters of each diluted homogenate onto a BHI agar plate. And transfer the plates to a 37 degrees celsius incubator for an overnight incubation.Keep the plates that contain up to 300 colonies per plate. Count the colonies on each plate. To measure CD4 in CT8 t cell interferon gamma responses, harvest the spleens from mice infected with two tenths into the fourths CFU of the pathogen as just demonstrated.Process the spleens into a single red blood cell life suspension. And resuspend the pellet at four times ten to the six cells per milliliter concentration in complete RPMI medium containing FCS. Next, seed one milliliter of cells per well into a 24 well plate with an equal number in volume of thawed, heat killed L monocytogenes and place the cold cultures into a 37 degrees celsius incubator.After 20 hours, add 0.66 microliters per milliliter of protein transport inhibitor to the wells. And return the plate to the incubator. Four hours later, in a biosafety cabinet, collect 500 microliters of culture supernatant and freeze the samples at negative 80 degrees celsius for the later measurement of interferon gamma by Eliza.Then transfer the cells into a single sterile 15 milliliter tube, washing the wells with PBS and pooling the wash with the collected cells for flow cytometric staining and analysis. The peak of bacterial load occurs in the spleen two to three days post infection. And is reduced dramatically by the addition of a PPAR Alpha antagonist prior to infection.At seven days post infection, the restimulation of spleen CD4 positive and CD8 positive t cells x vivo with heat killed pathogen elicits a positive interferon gamma response from CD4 positive and CD8 positive cells as detected by flow citometry. Treatment with the PPAR alpha antagonist prior to infection boosts the interferon gamma responses by CD4 and CD8 cells. Further, PPAR alpha antagonist treatment increases most survival after infection with the LD50 dose of the pathogen, illustrating the facility of this model for investigating the impact of new drugs that modulate interferon gamma responses on animal survival from infection.Once mastered, all the procedures in this protocol can generate the desired data in several weeks, if performed properly. The technique can be further adapted by infecting mice with strains of listeria monocytogenes that express model antigens and using MAC class one and two tetramery agents specific for these antigens. This will allow one to measure T cell expansion post infection.After its development, this technique became an instrumental model for deciphering the role of interferon gamma in host immunity to intracellular pathogens. After watching this video, you should have a good understanding of how to cultivate and infect C57 blacksix mice with listeria monocytogenes. And to measure the backtrail load and interferon gamma responses to infection.Don't forget that working with listeria monocytogenes can be hazardous to immuno compromised individuals such as pregnant women, the very young or elderly. Therefore the pathogen should only be handled by immuno competent individuals under biosafety level two conditions.

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Research

JoVE Journal

Immunology and Infection

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.

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

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.

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.

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.

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

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

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.

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

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

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.