Name: adoptive transfer of il-33-stimulated macrophages into bleomycin-induced mouse models to study their effect on idiopathic pulmonary fibrosis in vivo
Uploaded: 2023-05-05T13:30:00
Description: Watch this Scientific Journal Video about Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo at JoVE.c

The authors acknowledge the Special Topic of Laboratory Management of Jiangnan University: Construction of Digital Slice Library Based on Pathological Specimens (JDSYS202223) and the National Natural Science Foundation of China (81800065).

All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals. All the animal experiments were approved by the Experimental Animal Welfare Ethics committee of Jiangnan University (JN No. 20211130m1720615[501]).
NOTE: In total, 10 male C57BL/6 mice aged 6-8 weeks old and weighing 20-25 g were used in this study. The three experimental groups in the study included three recipient mice each, and one host mouse was used for the IM isolation.
...

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This study provides an effective method to deplete, isolate, culture, and transfer macrophages, which can help in studying the mechanisms of pulmonary fibrosis in mice. There are many methods for mouse macrophage depletion, such as tracheal administration, tail vein injection, and nasal inhalation11. This study optimized the nasal inhalation method, which is simple to operate and can effectively deplete pulmonary macrophages8,9. After the ...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64742">Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo</a>. The Protocol section was updated.
Step 2.1 of the Protocol was updated from:
Anesthetize the host mouse with 3% isoflurane, and then euthanize it by cervical dislocation. Disinfect the skin of the euthanized mouse with 75% alcohol and iodine. Use scissors to cut through the skin and expose the cardiopulmonary tissue
to:
Anesthetize the host mouse&#160;with an intraperitoneal injection of Ketamine (120 mg/kg) and Xylazine (16 mg/kg). Confirm the depth of anesthesia via loss of the toe pinch reflex. Apply veterinary ointment to both eyes to prevent dryness under anesthesia. Then, disinfect the skin of the anesthetized mouse with 75% alcohol and iodine. Use scissors to cut through the skin and expose the cardiopulmonary tissue

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64742">Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo</a>. The Protocol section was updated.

Erratum: Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo

Idiopathic pulmonary fibrosis (IPF) is a diffuse pulmonary inflammatory disease caused by many factors1. In the cytokine microenvironment of the Th1 and Th2 immune response, macrophages can be polarized into classically activated macrophages (M1) and alternatively activated macrophages (M2). Lipopolysaccharides (LPS) or the cytokine IFN- &#947; induce M1 macrophages to polarize and produce pro-inflammatory cytokines, including iNOS, IL-1, IL-6, TNF-&#945;, and IL-12. In contrast, the type II cytokines IL-4 and IL-13 drive the polarization of M2 macrophages, which can produce different fibroblast growth-promoting factors, such as TGF-&#946; and PDGF, that promote pulmonary fibrosis2. The pathological process of IPF is accompanied by macrophage activation and infiltration. IPF mediates injury repair, inflammation, and fibrosis through the release of cytokines3. As only limited therapeutic options are available, exploring the molecular pathological mechanisms of IPF holds great significance for developing new strategies for IPF prevention and treatment. Previous studies by our group and other researchers4,5 have confirmed the increased release of IL-33 in IPF patients and in mouse models with bleomycin (BLM)-induced IPF. IL-33 is released by the epithelial and endothelial cells during fibrosis and is involved in macrophage activation, resulting in the abnormal proliferation of fibroblasts, leukocyte infiltration, and the eventual loss of lung function5. The current protocol describes the adoptive transfer of IL-33-stimulated interstitial macrophages (IMs) into the alveoli as a means to study IPF development in mouse models. Here, IMs were isolated from the lung tissue of host mice, cultured in vitro, stimulated with IL-33 for 24 h, and then adoptively transferred into the alveoli of recipient mice by tracheal injection. The direct collection of stimulated mouse macrophages and their adoptive transfer into the recipient alveoli was found to aggravate the degree of pulmonary fibrosis and can more clearly illustrate the influence of stimulating factors on fibrosis compared to the previous studies6. The technique described in this paper can enable researchers to explore the function of macrophages stimulated by potential cytokines in the development of IPF.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;DMEM</td>
			<td>Life technologies Biotechnology,USA</td>
			<td>1508012</td>
			<td></td>
		</tr>
		<tr>
			<td>Arterial indwelling needle</td>
			<td>B Braun Melsingen AG,Germany</td>
			<td>21G15G8393</td>
			<td></td>
		</tr>
		<tr>
			<td>BD&#160;Accuri&#160;C6 Plus</td>
			<td>Becton Dickinson,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleomycin</td>
			<td>Biotang, USA</td>
			<td>Ab9465</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide incubator</td>
			<td>Thermo Forma, USA</td>
			<td>Thermo Forma370</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>R&#38;D Systems,USA</td>
			<td>1124F</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>R&#38;D Systems,USA</td>
			<td>N418</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>Thermo Forma, USA</td>
			<td>174926</td>
			<td></td>
		</tr>
		<tr>
			<td>Clodronate liposomes&#160;</td>
			<td>Clodronate liposomes,Netherlands</td>
			<td>CI-150-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Sigma-Aldrich, USA</td>
			<td>10103578001</td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>R&#38;D Systems,USA</td>
			<td>521204</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Cell Strainer</td>
			<td>Becton,Dickinson and Company, USA</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Life technologies,USA</td>
			<td>1047571</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin Eosin&#160;</td>
			<td>Nanjing Jiancheng Technology,China</td>
			<td>06-570</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 PCR detection system</td>
			<td>Roche, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Murine recombinant factor IL-33</td>
			<td>Peprotech, USA</td>
			<td>210-33</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon microscope</td>
			<td>Nikon Corporation, Japan</td>
			<td>941185</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin, streptomycin</td>
			<td>Life technologies,USA</td>
			<td>877113</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer (PBS)</td>
			<td>Guangdong Huankai Microbial Technology ,China</td>
			<td>1535882</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>Beyotime Biotechnology Company,China</td>
			<td>C3702</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Isolater</td>
			<td>Vazyme company,China</td>
			<td>R401-01-AA</td>
			<td>Total RNA extraction reagent</td>
		</tr>
		<tr>
			<td>RWD Inhalation Anesthesia Machine</td>
			<td>Shenzhen Rayward Life Technology ,China</td>
			<td>R500</td>
			<td></td>
		</tr>
		<tr>
			<td>Semi-automatic paraffin slicer</td>
			<td>Leica, Germany</td>
			<td>LeicaRM2245</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Premix Ex Taq</td>
			<td>Takara, Japan</td>
			<td>410800</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25%</td>
			<td>Life Technologies, USA</td>
			<td>1627172</td>
			<td></td>
		</tr>
	</tbody>
</table>

adoptive transfer of il-33-stimulated macrophages into bleomycin-induced mouse models to study their effect on idiopathic pulmonary fibrosis in vivo

The inflammatory response caused by early lung injury is one of the important causes of the development of idiopathic pulmonary fibrosis (IPF), which is accompanied by the activation of inflammatory cells such as macrophages and neutrophils, as well as the release of inflammatory factors including TNF-&#945;, IL-1&#946;, and IL-6. Early inflammation caused by activated pulmonary interstitial macrophages (IMs) in response to IL-33 stimulation is known to play a vital role in the pathological process of IPF. This protocol describes the adoptive transfer of IMs stimulated by IL-33 into the lungs of mice to study IPF development. It involves the isolation and culture of primary IMs from host mouse lungs, followed by the adoptive transfer of stimulated IMs into the alveoli of bleomycin (BLM)-induced IPF recipient mice (which have been previously depleted of alveolar macrophages by treatment with clodronate liposomes), and the pathological evaluation of those mice. The representative results show that the adoptive transfer of IL-33-stimulated macrophages aggravates pulmonary fibrosis in mice, suggesting that the establishment of the macrophage adoptive transfer experiment is a good technical means to study IPF pathology.

The protocol used here is summarized in the flowchart in Figure 1. The inhalation of clodronate liposomes through the nose (Figure 2) was used to deplete the pulmonary macrophages of adult C57BL/6 mice, and this produced a good recipient mouse model. Pulmonary IMs were isolated from another untreated (host) mouse (Figure 3A,B) and cultured in vitro. The isolated macrophages were stimulated with IL-33 for 24...

Watch this Scientific Journal Video about Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo at JoVE.c

Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo

This protocol describes the isolation of pulmonary interstitial macrophages (IMs) and their adoptive transfer after IL-33 stimulation of the lung alveoli in a mouse model, which can facilitate the in vivo study of idiopathic pulmonary fibrosis (IPF).

Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo

The inflammatory response caused by early lung injury is one of the important causes of the development of idiopathic pulmonary fibrosis (IPF), which is ...

The protocol describes the isolation of pulmonary IMS and adoptive transfer after IL-33 determination in a mouse model, which can facilitate the in vivo study of idiopathic pulmonary fibrosis. The technique enables the researchers to explore the function of macrophages, simulated by traditional cytokines in developing IPF. To begin, take out the vial of clodronate liposomes from the refrigerator.Allow it to warm for 30 minutes at room temperature and invert several times to ensure uniform mixing. Aspirate 60 microliters of clodronate liposomes using a pipette with a sterile suction tip. Administer the control and drug drop-wise into the nasal cavity of the anesthetized mouse.After each drop, ensure the mouse inhales the drug completely and breathes evenly. After the depletion of alveolar macrophages in the recipient mouse, begin by disinfecting the skin of the anesthetized host mouse with 75%alcohol and iodine. Use scissors to cut through the skin and expose the cardiopulmonary tissue.Aspirate 10 milliliters of PBS in a syringe equipped with a 20-gauge needle, and insert the tip of the needle into the right atrium of the mouse. Then cut the inferior vena cava of the mouse with surgical scissors and manually perfuse the mouse with PBS at a constant speed of 10 to 20 milliliters per minute until the lung tissue turns white. Further, excise the lung tissue and transfer it into ice cold PBS in a culture dish.Cut the lung tissue into fragments and add 15 milliliters of DMEM medium containing 1%collagenase A to isolate the macrophages, then incubate it at 100 RPM for 30 minutes on a 37 degree Celsius shaking table. Aspirate the lung tissue suspension approximately 20 times through a 10-milliliter syringe to make it fine. Filter the suspension through a 40-micrometer cell strainer.Centrifuge the filtrate at 400 G for 10 minutes. After discarding the supernatant, lyse the red blood with three milliliters of cell lysis buffer on ice for two to three minutes. Centrifuge at 150 G for five minutes and resuspend the cell pellet in 10 milliliters of DMEM.Then count the cells using a hemocytometer. Seed the cells into a 10-centimeter adherent cell culture dish and incubate for one hour at 37 degrees Celsius and 5%carbon dioxide to adhere the lung interstitial macrophages to the dishes. After one hour, aspirate the supernatant and floating cells.Add 10 milliliters of the fresh, complete culture medium and incubate for over eight hours or overnight. To stimulate the isolated interstitial macrophages, replace the spent culture medium with a complete medium interleukins 33. Also prepare a control plate by adding PBS instead of interleukins 33.Incubate the plates for 24 hours at 37 degrees Celsius and 5%carbon dioxide. To dissociate the pulmonary interstitial macrophages, treat them with one milliliter of 0.25%trypsin for five minutes. Then add three milliliters of fresh culture complete medium to quench the digestion and centrifuge the cell suspension to harvest the pulmonary macrophages.After counting the cells, resuspend the cells in PBS to a final concentration of five times 10 to the fifth cells per 50 microliters. To begin the transfer of interstitial macrophages, fix the limbs of the anesthetized recipient mouse on a vertical plate with medical tape and gently pull the mouse's tongue to one side, using a cotton swab. After turning the intubation lamp on, shine it on the throat of the mouse to see the trachea, which should be close to the base of the tongue.Insert the indwelling needle cannula into the trachea using the 22-gauge intubation lamp and 0.4 millimeter diameter guide wire. To finish the intubation, remove the guide wire and push the cannula into the mouse trachea. Now inject 50 microliters of the cell suspension into the recipient mouse through the trachea when the cannula is seen to enter the trachea.After 24 hours, administer bleomycin via the trachea to induce idiopathic pulmonary fibrosis. Also administer an equal volume of saline to the control group. After 21 days, determine the degree of severity of pulmonary fibrosis by evaluating the expression of markers for fibrosis and the Ashcroft scores.The HE staining showed that the adoptive transfer of interleukins 33 stimulated macrophages exacerbated the degree of lung tissue destruction and increased fibroblast aggregation in the bleomycin-stimulated mice, compared to control. The increase in the Ashcroft score also illustrates the degree of pulmonary fibrosis. The expression levels of mRNA of alpha SMA and fibronectin in lung tissues of the recipient mouse containing adoptively transferred interleukins 33-stimulated interstitial macrophages and treated with bleomycin were higher, compared with those of the BLM-treated wild-type mice.500 microliters of air should be immediately entered into the lungs with a one-millimeter syringe to ensure that the cell suspension in the cannula can completely enter the lung tissue.

adoptive-transfer-il-33-stimulated-macrophages-into-bleomycin-induced

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Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

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To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

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 ...

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles (MPs) are one such type of vesicles, ranging in diameter from 0.1 &#956;m to 1 &#956;m, and typically shed from the plasma membrane of eukaryotic cells undergoing activation or apoptosis. Here we describe the generation of T lymphocyte&#8211;derived microparticles (LMPs) from apoptotic CEM T cells stimulated with actinomycin D. LMPs are isolated through a multistep differential centrifugation process and characterized using flow cytometry. This protocol also presents an in situ cell death detection method for demonstrating the proapoptotic effect of LMPs on bronchial epithelial cells derived from mouse primary respiratory bronchial tissue explants. Methods described herein provide a reproducible procedure for isolating abundant quantities of LMPs from apoptotic lymphocytes in vitro. LMPs derived in this manner can be used to evaluate the characteristics of various disease models, and for pharmacology and toxicology testing. Given that the airway epithelium offers a protective physical and functional barrier between the external environment and underlying tissue, use of bronchial tissue explants rather than immortalized epithelial cell lines provides an effective model for investigations requiring airway tract tissue.

Cell membrane&#8211;shed microparticles (MPs) are active biological vesicles that can be isolated and their pathophysiological effects investigated in various models. Here we describe a method for generating MPs derived from T lymphocytes (LMPs) and for demonstrating their proapoptotic effect on airway epithelial cells.

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...