JHN is supported by the Swiss National Foundation (SNSF 310030_146290).

Mice were bred and kept under specific pathogen-free (SPF) conditions in the animal facility of Department of Biomedicine, University of Basel (Basel, Switzerland). All mouse experiments were performed in accordance with the Swiss Federal and Cantonal regulations (animal protocol number 2816 [Canton of Basel-Stadt]).
1. Preparation of LPS Solution
<ol>
	<li>Open the stock of LPS purified from Escherichia coli 0111:B4 under sterile conditions and reconstitute it in water to the concentration of 5 mg/mL.</li>
	<li>Dilute the LPS stock with the sterile phosphate buffered saline (PBS) to the working concentration of 0.2 &#181;g/&#181;L.</li>
</ol>
2. Intraperitoneal Injection of LPS to Mice
<ol>
	<li>Aspirate the LPS working solution in a 0.5 mL syringe with a 30 G needle in a laminar air flow.</li>
	<li>Weigh female C57Bl/6 mice (six to 10 weeks of age) and calculate the amount of LPS that will be injected: 10 &#181;L (2 &#181;g)/g body weight. 
	NOTE: For example, if a mouse weighs 20 g, then 20 g x 10 &#181;L = 200 &#181;L LPS working solution needs to be injected.</li>
	<li>Handle the mouse gently but firmly and restrain the animal in one hand. Ensure that the animal is able to breathe normally but not to twist and turn during the restraint.</li>
	<li>Tilt the mouse nose slightly towards the floor to expose the abdomen for injection. Locate the midline of the abdomen and the injection place in the low abdomen left or right from the midline. Inject i.p. PBS instead of LPS into control mice.</li>
	<li>With the free hand, inject the appropriate volume (the volume calculated in step 2.2) of the LPS working solution. Make sure that the needle has entered the peritoneal cavity as otherwise, mis-injections into the abdominal muscle layer may occur. Still, do not go too deep with the needle into the peritoneal cavity to avoid injuring inner organs located in that area.</li>
	<li>Return the animal carefully to the cage with up to 5 mice per cage.</li>
</ol>
3. Monitor the Mice
<ol>
	<li>Monitor the animals at the time of injection and every 2 h after the injection for 8 h for the occurrence and severity of endotoxemia. Therefore, use a score sheet (Table 1) that has been adapted from a previously published manuscript 24.</li>
	<li>Score the LPS injected mice for the following parameters listed in Table 1
	<ol>
		<li>Check for the appearance of the fur which is normally smooth. If the fur appears ruffled, spiky or puffy fur which indicates discomfort and/or pain, score them as 1, 2, or 3.</li>
		<li>Check for the activity of the mouse. 
		NOTE: Mice normally move freely around the whole cage eating, drinking, climbing, but suppressed or restricted activity could be a sign of pain.</li>
		<li>Check for the level of consciousness. 
		NOTE: Mice are very curious, and they normally move around the cage investigating the surrounding. Lack of or reduced movement can suggest pain and discomfort.</li>
		<li>Check the eyes. 
		NOTE: Fully open eyes are expected in healthy mice, but partially or fully closed eyes, possibly with secretion, can be an indication of pain.</li>
		<li>Check for the respiration rate. 
		NOTE: Healthy mice usually have a normal and rapid respiration rate, a reduced respiration rate could be an indication of discomfort).</li>
		<li>Check for the respiration quality. 
		NOTE: Labored breathing with or without gasping is a sign of pain.</li>
	</ol>
	</li>
</ol>
4. Tissue Sampling for Ribonucleic Acid (RNA) Extraction and Homogenization
<ol>
	<li>Prepare the collection/homogenization tubes: Add 1 mL single-step RNA isolation reagent to 2 mL tubes prefilled with 1.4 mm ceramic spheres.</li>
	<li>At the appropriate time points (before (0 h) and 2 h, 4 h and 8 h after LPS injection) euthanize the mouse with anesthetic overdose (isoflurane) followed by exsanguination and proceed with the dissection.</li>
	<li>Cut the skin and the muscle layer exposing the abdominal cavity with the inner organs with a pair of scissors.</li>
	<li>Carefully dissect the spleen, liver, and colon, clean the organs from fat and keep them in PBS on ice.</li>
	<li>Cut out a small piece (about 0.5 cm long) from each organ using scissors or scalpel. 
	NOTE: It is important to always take the piece from approximately the same part of the organ in each mouse (same lobe of liver, proximal or distal part of the colon).</li>
	<li>Shortly dry the tissue on a piece of paper tissue and place it in the collection/homogenization tube making sure that it is completely immersed in RNA isolation reagent.</li>
	<li>Homogenize the tissue in a high-speed benchtop homogenizer. For soft tissues like spleen, liver, and colon use 1 cycle of homogenization at speed 6.00 for 30 s.</li>
	<li>Incubate the samples for 5 min at room temperature.</li>
	<li>Carefully place the tube in the liquid nitrogen to snap freeze the tissue lysate. Store the snap frozen lysate at -80 &#176;C until the RNA isolation.</li>
</ol>
5. RNA Extraction from Tissue
NOTE: RNA isolation reagents, chloroform, and alcohols are considered as hazardous material. Perform RNA extraction under a chemical hood. Use certified RNase-free reagents and equipment to maintain an RNase-free environment.
<ol>
	<li>Phase separation
	<ol>
		<li>Thaw the frozen tissue lysate on ice.</li>
		<li>Centrifuge the sample on 1,000 x g for 5 min at 4 &#176;C to pellet the remaining tissue particles that might be left.</li>
		<li>Transfer the supernatant to a new nuclease-free 1.5 mL tube.</li>
		<li>Add 200 &#181;L ice-cold chloroform per 1 mL RNA isolation Reagent and shake vigorously by hand for 10-15 s.</li>
		<li>Incubate for 2-3 min at room temperature.</li>
		<li>Centrifuge at 12, 000 x g for 15 min at 4 &#176;C. 
		NOTE: The sample will now contain 3 phases: lower red phenol-chloroform phase, the interphase and the upper colorless aqueous phase. The RNA is present in the upper aqueous phase.</li>
		<li>Collect the upper aqueous phase ( 50% of total volume) and transfer into a new nuclease-free 1.5 mL tube.</li>
	</ol>
	</li>
	<li>RNA precipitation
	<ol>
		<li>Add 0.5 mL ice-cold isopropanol per 1 mL RNA isolation reagent and mix gently by inverting the tube 5-6 times.</li>
		<li>Incubate for 10-15 min at room temperature.</li>
		<li>Centrifuge at 12,000 x g for 10 min at 4 &#176;C. 
		NOTE: RNA pellet should be visible at this stage.</li>
	</ol>
	</li>
	<li>Washing of RNA pellet
	<ol>
		<li>Completely remove the supernatant containing the isopropanol without disturbing the pellet.</li>
		<li>Wash the pellet with 1 mL ice-cold 75% ethanol per 1 mL RNA isolation reagent used for the initial lysis.</li>
		<li>Vortex the sample briefly.</li>
		<li>Centrifuge the sample at 7,500 (or 12,000) x g for 5 min at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Dissolving RNA
	<ol>
		<li>Completely remove the ethanol in the supernatant without disturbing the pellet.</li>
		<li>Leave the opened tube under the chemical hood for 3-5 min to air-dry the RNA pellet at room temperature (do not over-dry as in that case it will be difficult to dissolve RNA).</li>
		<li>Dissolve the RNA pellet in 20-50 &#181;L nuclease-free water by pipetting carefully up and down.</li>
		<li>Incubate the sample at 55 &#176;C for 10-15 min to further improve the solution of the RNA. 
		NOTE: At this stage, RNA can be stored at -80 &#176;C till further analysis.</li>
	</ol>
	</li>
</ol>
6. Digestion of Remaining DNA
<ol>
	<li>Measure the RNA concentration with a spectrophotometer by pipetting an aliquot (2 &#181;l) into the spectrophotometer and adjust to the recommended concentration.</li>
	<li>Start the digestion of contaminating DNA with max. 10 &#181;g RNA in 50 &#181;L nuclease-free water.</li>
	<li>Add 0.1 volume of 10x DNase I Buffer and 1 &#181;L rDNase I per sample and mix gently by pipetting up and down.</li>
	<li>Incubate at 37 &#176;C for 20-30 min.</li>
	<li>Vortex DNase Inactivation Reagent and add 0.1 volume to the sample mixing well with the pipette.</li>
	<li>Incubate for 5 min at room temperature mixing occasionally by hand.</li>
	<li>Centrifuge at 10,000 x g for 1.5 min.</li>
	<li>Transfer the supernatant containing DNA-free RNA into the new nuclease-free tube.</li>
	<li>Measure the RNA concentration and quality by spectrophotometer.</li>
</ol>
7. Reverse Transcription
<ol>
	<li>Use a commercially available Deoxyribonucleic acid cDNA reverse transcription (RT) kit for reverse transcription. 
	NOTE: Here, up to 2 &#181;g of RNA can be reversed transcribed.</li>
	<li>Calculate the volume, which contains 2 &#181;g RNA. Pipette 2 &#181;g RNA in a new PCR tube.</li>
	<li>Add nuclease-free water to the sample in the PCR tube to the final volume of 10 &#181;L.</li>
	<li>Prepare 2x Master Mix by mixing the following components listed in Table 2</li>
	<li>Add 10 &#181;L 2x Master Mix to 10 &#181;l RNA to obtain 1x Master Mix in the total volume of 20 &#181;L.</li>
	<li>Close the polymerase chain reaction (PCR) tube and spin down the sample briefly.</li>
	<li>Place the samples in a PCR Thermocycler and set the settings listed in Table 3.</li>
	<li>Store the generated cDNA at -20 &#176;C for further analysis.</li>
</ol>
8. Quantitative PCR (qPCR)
<ol>
	<li>Perform the qPCR reaction with a commercially available kit.</li>
	<li>Self-design and test the intron-spanning primers, before usage, with different concentrations of template cDNA. Analyze the efficacy of the primer by creating a standard curve and use the primers with high efficacy and correct melting curves. 
	NOTE: The sequences of primers used in this experiment are listed in Table 2.</li>
	<li>Prepare the quantitative polymerase chain reaction (qPCR) reaction in the 384-well plate with the reaction setup described in Table 4.</li>
	<li>Keep all solutions and the plate on ice using aluminum foil to prevent the plate getting wet when the reaction mixture is prepared.</li>
	<li>Perform all the reactions in triplicates.</li>
	<li>Carry out the PCR reaction on a thermocycler using the setting listed in Table 5.</li>
	<li>Calculate the average Ct value for all reactions from the triplicates. Normalize all target genes to the expression level of glycerinealdehyde-3-phosphate-dehydrogenase (Gapdh) housekeeping gene by calculating 2^(-&#916;Ct). 
	NOTE: All the target genes with the average Ct &#62;35 were considered to be under the detection limit.</li>
</ol>

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The authors have nothing to disclose.

This protocol mimics immunological processes that occur after the invasion by microbial-derived products. Critical steps within the protocol are the selection of the mouse line, the hygiene status of the mice, the dose of LPS, the monitoring of the animals for the occurrence of endotoxemia, and the time point of experiment termination. Most importantly, the genetic background of the mouse strain has to be considered. Different mouse strains have different susceptibility to LPS. For example, the C3H/HeJ and C57BL/10ScCr m...

An erratum was issued for: <a href="https://www.jove.com/video/57610/injections-lipopolysaccharide-into-mice-to-mimic-entrance-microbial">Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach</a>
One of the authors&#39; names was corrected from:
Katarina Raduolovic
to:
Katarina Radulovic

An erratum was issued for: <a href="https://www.jove.com/video/57610/injections-lipopolysaccharide-into-mice-to-mimic-entrance-microbial">Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach</a>.&#160;An author name was updated.

Erratum: Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

The human intestine is colonized with a large consortium of microorganisms that forms the microbiota, who has developed a mutually beneficial relationship with the host during the evolution. In this relationship, the host provides a secure niche for the microbiota, whereas the microbiota provides vitamins, nutrient digestion and protection from pathogens to the host, where the microbiota resides1. When this beneficial relationship between the host and the microbiota is disturbed, diseases can develop, such as inflammatory bowel disease (IBD). IBD is a multifactorial chronic inflammatory disease of the intestine caused by genetic and environmental factors that occur in two major forms, Crohn&#39;s disease (CD) and ulcerative colitis (UC). Despite similarities between the two IBD forms, they are characterized by certain differences in the location and nature of inflammatory modifications. CD is a relapsing transmural inflammatory disorder that can potentially extend to any part of the gastrointestinal tract, while UC is non-transmural and is restricted to the colon. Furthermore, mutations in Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a pattern recognition receptor (PRR) that recognizes muramyl dipeptide (MDP), a component of the cell wall of most Gram-positive and - negative bacteria, is associated with CD2. Furthermore, Escherichia coli (E. coli), Listeria and Streptococci and their products were all found within macrophages in CD patients that have entered the host after a barrier breach3. When bacteria or their products enter the host during the development of CD, the immune system develops a response leading to the production of circulating anti-bacterial antibodies4. Maybe, the most convincing evidence for the role of the microbiota in the pathogenesis of IBD stems from mouse models. When animals are treated with antibiotics, or when mice are kept in germ-free (GF) conditions, the severity of the disease is reduced in most colitis models, such as in IL-10-/- mice that do not develop colitis in GF facilities5,6. Furthermore, colitis also disturbs the composition of the microbiota, which is characterized by an imbalanced composition and reduced richness called dysbiosis7. The consequence of IBD can be an increased intestinal permeability that can lead to the entrance of microbes and microbial-derived products into the host.
In animals, the application of Dextran Sodium Sulfate (DSS) induces an intestinal epithelial breach leading to an increased permeability of the epithelial barrier8. Portal LPS concentrations are elevated in animals with DSS colitis9. Interestingly, animals lacking the C type lectin receptor specific intracellular adhesion molecule-3 grabbing nonintegrin homolog-related 1 (SIGN-R1) are protected from DSS colitis and LPS-induced endotoxemia10. To further disseminate into the host, bacteria or bacteria derived products have to pass the vascular barrier11, the peritoneal cavity, in which the small and large intestine is located, the mesenteric lymph nodes and/or the liver12. To reduce the complexity of this system, a defined bacterial-derived compound was used. LPS, which causes endotoxemia after intraperitoneal (i.p.) or intravenous (i.v.) injection13 was injected into mice, to study the expression of the interleukins Il6 and Ilb and the cytokine Tnfa in response to LPS.
LPS is a pathogen-associated molecular pattern (PAMP) expressed as a cell-wall component of Gram-negative bacteria, that consists of lipid A (the main PAMP in the structure of LPS), a core oligosaccharide and an O side chain14. Toll-like receptor 4 (TLR4) expressed by dendritic cells, macrophages, and epithelial cells recognizes LPS15, that requires co-receptors for appropriate binding. The acute phase protein LPS-binding protein (LBP) binds LPS to form a complex that transfers LPS to the cluster of differentiation 14 (CD14), a glycosylphosphatidylinositol-anchored membrane protein. CD14 further shuttles LPS to Lymphocyte antigen 96 or also known as MD-2, which is associated with the extracellular domain of TLR4. The binding of LPS to MD-2 facilitates the dimerization of TLR4/MD-2 to induce conformational changes to recruit intracellular adaptor molecules to activate the downstream signaling pathway14, which includes the myeloid differentiation primary response gene 88 (MyD88)-dependent pathway and the TIR domain-containing adaptor-inducing interferon-&#946; (TRIF)-dependent pathway16. The recognition of LPS by TLR4 then activates the NF-&#954;B pathway and induces the expression of proinflammatory cytokines, such as TNF&#945;, IL-6, and IL-1&#946;17.
In particular, when LPS is injected into animals, the concentration of LPS given to the animals, the genetic background of the animal and the diet has to be considered. High concentrations of LPS leads to a septic shock, characterized by hypotension and multiple organ failures, and finally to death18. Mice are less sensitive to LPS compared to humans, where LPS concentrations between 2-4 ng/kg body weight (BW) are able to induce a cytokine storm19. For mice, the lethal dose (LD50), which induces death in half of the mice ranges from 10-25 mg/kg BW20 depending on the mouse strain used. For the commonly used mouse strains, C57Bl/6 and BALB/c, the lethal dose 50% (LD50) is 10 mg/kg BW. In contrast, the strains C3H/HeJ and C57BL/10ScCr are protected from LPS induced endotoxemia, which is due to mutations in Tlr421. Consequently, Tlr4-deficient mice are hyporesponsive to injections with LPS22. Other genetically modified mouse lines, such as PARP1-/- mice23 are resistant to LPS-induced toxic shock.
The mouse model described here uses a sublethal dose of LPS administered systemically to mimic the consequences of LPS dissemination after a barrier breach of the body`s surfaces. The chosen LPS concentration (2 mg/kg BW) did not induce mortality in C56Bl/6 mice, but the induced release of pro-inflammatory cytokines.

<table border="0" cellpadding="0" cellspacing="0" width="896">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">DreamTaq Green PCR Master Mix (2x)</td>
			<td style="width:303px;">Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">K1081</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High Capacity cDNA Reverse Transcription Kit,</td>
			<td style="width:303px;">Applied Biosystems, Foster City, CA, USA</td>
			<td>4368813</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase-Free DNase Set,</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LPS Escherichia coli O111:B4</td>
			<td>Invivogen, San Diego, CA, USA.</td>
			<td>tlrl-eblps</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Omnican 50 Single-use insulin syringe</td>
			<td>B. Braun Melsungen, Melsungen, Germany</td>
			<td>9151125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bioanalyzer 2100</td>
			<td>Agilent Technologie, Santa Clara, USA</td>
			<td style="width:119px;">not applicable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge 5430</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td style="width:119px;">not applicable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge Mikro 220R</td>
			<td>Hettich, Kirchlengern, Germany</td>
			<td style="width:119px;">not applicable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection tools</td>
			<td>Aesculap, Tuttlingen, Germany</td>
			<td style="width:119px;">not applicable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast-Prep-24 5G Sample Preparation System</td>
			<td>M.P. Biomedicals, Santa Ana, CA, USA</td>
			<td style="width:119px;">not applicable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop ND-1000</td>
			<td>NanoDrop Products, Wilmington, DE, USA</td>
			<td style="width:119px;">not applicable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRI Reagent</td>
			<td>Zymo Research, Irvine, CA, USA</td>
			<td>R2050-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

injections of lipopolysaccharide into mice to mimic entrance of microbial-derived products after intestinal barrier breach

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the entrance of bacteria or bacteria-derived products into the host, accessing the host circulation and inner organs leading to the uncontrolled inflammation as observed in patients with inflammatory bowel disease (IBD), that are characterized by an increased intestinal epithelial permeability.
To mimic the entrance of bacterial-derived compounds into the host, an endotoxemia model has been adopted in which lipopolysaccharide (LPS), a component of the outer cell wall of Gram-negative bacteria, were injected into mice. In this study, a sublethal dose of LPS was intraperitoneally injected and the mice were subsequently monitored for 8 h using a disease score. Furthermore, the expression levels of the inflammatory cytokines Il6, Il1b, and Tnfa were analyzed in the spleen, liver and colon by qPCR at different time points post LPS injection. This model could be useful for the studies involving investigation of immune responses after the invasion of microorganisms or bacterial-derived products caused by a barrier breach of body surfaces.

To mimic the consequences for the host after the entrance of bacteria or bacterial-derived products that occurs after intestinal barrier breach, LPS was injected into C57Bl/6 mice in a sublethal dose (2 &#181;g/g body weight). Every single mouse was monitored and scored for the occurrence of endotoxemia with parameters listed in score sheet that includes, the appearance of the mice, the activity of the animals, the condition of eyes, and the respiration rate and quality (Table 1</...

Watch this Scientific Journal Video about Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach at JoVE.com

Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach | Immunology and Infection | Video

Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

Here a protocol to mimic the entrance of bacterial-derived compounds after intestinal barrier breach is presented. A low sublethal dose of lipopolysaccharide was injected systemically into mice, which were monitored for 24 h post-injection. The expression of pro-inflammatory cytokines was determined at several time points in spleen, liver, and colon.

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the ...

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19.3K Views.  University of Basel. Here a protocol to mimic the entrance of bacterial-derived compounds after intestinal barrier breach is presented. A low sublethal dose of lipopolysaccharide was injected systemically into mice, which were monitored for 24 h post-injection. The expression of pro-inflammatory cytokines was determined at several time points in spleen, liver, and colon.

Video: Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic Poly(I:C)

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric disorders. Numerous studies have demonstrated that MIA model is able to show face, construct, and predictive validity that are relevant to autism and schizophrenia. To model MIA, investigators often use viral mimic polyinosinic:polycytidylic acid (poly(I:C)) to activate the immune system in pregnant rodents. Generally, the offspring from immune activated dam exhibit behavioral abnormalities and physiological alterations that are associated with autism and schizophrenia. However, poly(I:C) injection with different dosages and at different time points could lead to different outcomes by perturbing brain development at different stages. Here we provide a detailed method of inducing MIA by intraperitoneal (i.p.) injection of 20 mg/kg poly(I:C) at mid-gestational embryonic 12.5 days (E12.5). This method has been shown to induce acute inflammatory response in the maternal-placental-fetal axis, which ultimately results in the brain perturbations and behavioral phenotypes that are associated with autism and schizophrenia.

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C

Maternal immune activation (MIA) is a model for an environmental risk factor of autism and schizophrenia. The goal of this article is to provide a step-by-step procedure of how to induce MIA in the pregnant mice in order to enhance the reproducibility of this model.

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) face and is frequently associated with a poor clinical outcome. While, for instance, GvHD manifestations of the skin are usually responsive to established immune-suppressive therapies and are, hence, not taking a fatal course, the presence and the intensity of intestinal GvHD, especially of the mid-to-lower parts of the gut, strongly influence the outcome and overall survival of patients with acute GvHD. Therapeutic options are essentially limited to the classic immune-suppressive agents yielding only moderate disease-mitigating effects. Hence, detailed knowledge about the tissue-resident immune cascade, changes in the intestinal microbiota, and the stromal response prior, upon, and after intestinal GvHD onset are urgently needed to understand the events and mechanisms underlying its pathogenesis and to develop innovative therapeutic options. Murine models of GvHD are frequently employed to identify and functionally assess molecules and pathways putatively driving intestinal GvHD. However, means to specifically monitor and evaluate intestinal inflammation over time are essentially lacking since established scores to assess and grade acute GvHD are routinely comprised of various parameters which rather reflect&#160;systemic GvHD manifestations. The detailed evaluation of intestinal GvHD has been restricted to studies using euthanized mice, thereby essentially excluding longitudinal (i.e., kinetic) analyses of the colonic compartment under a given experimental condition (e.g., antibody-mediated blockade of a proinflammatory cytokine) in live mice (i.e., in vivo). The mini-endoscopic in situ assessment of the distal colon of allo-HCT-treated mice described here allows a) a detailed macroscopic evaluation of different aspects of intestinal inflammation and b) the option to collect tissue samples for downstream analyses at various time points over the course of the observation period. Overall, the mini-endoscopic approach provides a major advance in preclinical noninvasive monitoring and assessment of intestinal GvHD.

Here, we present a protocol that describes allogeneic hematopoietic stem cell transplantation and allows repetitive mini-endoscopic evaluations of the distal colon in situ for the presence, characteristics, and severity of colonic inflammation within live mice suffering from intestinal graft-versus-host disease.

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell ...

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn&#39;s-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn&#39;s-associated intestinal fibrosis.

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has ...

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates the isolated patient&#8217;s blood mononuclear cells (e.g., lymphocytes and monocytes) through an apheresis machine, co-cultures the patient&#8217;s blood mononuclear cells with adherent cord blood-derived stem cells (CB-SC) in the SCE device, and then returns these &#8220;educated&#8221; immune cells to the patient&#8217;s blood. Exosomes are nano-sized extracellular vesicles between 30&#8210;150 nm existing in all biofluid and cell culture media. To further explore molecular mechanisms underlying SCE therapy and determine the actions of exosomes released from CB-SC, we investigate which cells phagocytize these exosomes during the treatment with CB-SC. By co-culturing Dio-labeled CB-SC-derived exosomes with human peripheral blood mononuclear cells (PBMC), we found that CB-SC-derived exosomes were predominantly taken up by human CD14-positive monocytes, leading to the differentiation of monocytes into type 2 macrophages (M2), with spindle-like morphology and expression of M2-associated surface molecular markers. Here, we present a protocol for the isolation and characterization of CB-SC-derived exosomes and the protocol for the co-culture of CB-SC-derived exosomes with human monocytes and the monitoring of M2 differentiation.

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell CB-SC-Derived Exosomes

Exosome application is an emerging tool for drug development and regenerative medicine. We establish an exosome isolation protocol with high purity to isolate exosomes from novel identified stem cells called CB-SC for mechanistic studies. We also coculture CB-SC-derived exosomes with human monocytes, leading to their differentiation into phenotypically distinct macrophages.

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates ...