This work was partially supported by the German Research Foundation (DFG) 2886 PANDORA Project-No.B3; SCHA 2040/1-1; MU 4240/2-1; CRC1181(C03); TRR241(B04), H2020-FETOPEN-2018-2020 Project 861878, and by the Volkswagen-Stiftung (Grant 97744) to MH.

All procedures involving animals were conducted according to the institutional guidelines on animal welfare and approved by the animal welfare commission of the Friedrich-Alexander-University Erlangen-Nuremberg (FAU) (permit number: 55.2.2-2532-2-1217). Female C57Bl/6 mice, aged 7-9 weeks were used for the present study. The mice were obtained from commercial sources (see Table of Materials) and kept in specific pathogen-free conditions with 12 h day/night cycles.
1. Induction of murine ocular surface inflammation
<ol>
	<li>Perform immunogen preparation for immunization.
	<ol>
		<li>Prepare the immunogen freshly on the day of immunization, mixing ovalbumin (OVA, 50 mg/mL) and pertussis toxin (100 &#181;g/mL) in saline and the adjuvant aluminum hydroxide (40 mg/mL) (see Table of Materials) in a 1:1 proportion. 
		CAUTION: Perform the opening, reconstitution, and immunogen preparation of pertussis toxin in a laminar safety cabinet. Wear protective clothing and avoid any contact with the skin.</li>
		<li>Incubate the immunogen and adjuvant mixture at room temperature for 30 min and load 100 &#181;L into a 1 mL syringe.</li>
		<li>Perform intraperitoneal injection of the immunogen solution in a non-anesthetized mouse.
		<ol>
			<li>Hold the mouse softly by the tail while grasping the cage grid. Hold firmly the skin of the back and the neck region between the thumb and index finger and fix the tail and lower limbs between the ring and little finger against the palm of the hand.</li>
			<li>Keep the fixed mouse with its head downward.</li>
			<li>Inject 100 &#181;L of the prepared immunogen solution in the right or left quadrant of the lower abdominal cavity.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform ocular surface challenge 2 weeks after immunization.
	<ol>
		<li>Anesthetize the mouse with isoflurane (2.5%).</li>
		<li>Apply 5 &#181;L of OVA (50 mg/mL) or saline (0.9 % NaCl) per eye on both eyes and wait until the drop gets absorbed by the eye. This takes ~5 min.</li>
		<li>Repeat the procedure 1x daily for 7 days. 
		&#8203;NOTE: The topical administration of medications can be done in the same way.</li>
	</ol>
	</li>
</ol>
2. Collection of ocular exudates
<ol>
	<li>Recover the ocular exudates formed during the challenge phase by applying 50 &#181;L of sterile saline to the eye immediately after the challenge.</li>
	<li>To obtain a single-cell suspension, treat the collected ocular discharge with recombinant MNase (2 x 106 gel U/mL, see Table of Materials) containing the cofactor calcium (5 mM) at 37 &#176;C for 20 min.</li>
	<li>Centrifuge at 400 x g for 7 min at room temperature.</li>
	<li>Isolate the supernatant and measure the cytokines and chemokines using multiplexed ELISA according to the manufacturer&#39;s instructions (see Table of Materials). 
	&#8203;NOTE: The obtained supernatant can be used for protein analysis and the pellet for functional assays such as immunophenotyping, phagocytosis, degranulation, and gene expression.</li>
</ol>
3. Excision of ocular surface tissues
<ol>
	<li>Dissect the eyelids and eye globe following the steps below.
	<ol>
		<li>Euthanize the mouse by CO2 asphyxiation and cervical dislocation.</li>
		<li>Place the mouse on an even surface.</li>
		<li>Disinfect the orbital area around the eye with a swab impregnated with 70% ethanol.</li>
		<li>Make an incision between the ear and the retro-orbital sinus and along the surface above the squamous bone vertically, extending the incision horizontally below the lower eyelid along the maxillary bone and above the upper eyelid along the frontal bone. This forms an incision around the eye (Figure 1).</li>
		<li>Carefully hold the dissected tissue around the eye using curved forceps and pull the tissue and eyeball out.</li>
		<li>Place the excised organs in sterile PBS.</li>
		<li>Trim excess facial muscle tissue around the excised upper eyelids using a scalpel and under the stereomicroscope.</li>
	</ol>
	</li>
	<li>Collect the conjunctiva following the steps below.
	<ol>
		<li>Place the upper eyelid on a dry Petri dish under the stereomicroscope.</li>
		<li>Using fine tweezers and a scalpel, peel the whitish-mucous layer very gently out of the inner surface of the eyelid.</li>
	</ol>
	</li>
	<li>Next, dissect the cornea following the steps below.
	<ol>
		<li>Place the eye globe on a new dry Petri dish on top of dry ice for 3 min.</li>
		<li>Take the Petri dish onto the bench surface in a stable position.</li>
		<li>Make a small incision at the border of the cornea next to the limbus using fine sharp scissors.</li>
		<li>Prolong the incision with the scalpel around the eye globe, separating the sclera from the cornea.</li>
		<li>Remove remnants of the iris and the lens from the backside of the cornea by generously flushing with saline solution.</li>
	</ol>
	</li>
</ol>
4. Documentation of Meibomian gland (MG) obstruction
<ol>
	<li>To assess the MG and its orifices, place excised eyelids in an upright position under the stereomicroscope (Figure 2).</li>
	<li>Capture images with white light epi-illumination according to the camera&#39;s exposure time and ISO settings. 
	NOTE: Morphometric analysis of these images provides reliable quantification of the plug size at the outlet of the glands. The quantification can be performed by outlining with the wand tool the eye plugs and executing the &#34;Analyze Particles&#34; command of the Image J software (see Table of Materials).</li>
</ol>
5. Transillumination of eyelids (Meibomian gland morphology)
<ol>
	<li>To assess the MG area, place the excised eyelids in a horizontal position and turn on the backlight of the stereomicroscope equipped with an infrared camera (Figure 3).</li>
	<li>Capture images by adjusting the exposure time according to the camera&#39;s (see Table of Materials) ISO. 
	NOTE: Infrared imaging of these transilluminated eyelids under the stereomicroscope can help measure the shape and size of each acini. Morphometric analysis of these images provides reliable quantification of the size and number of MG. The quantification can be performed by outlining the MG with the wand tool and executing the &#34;Analyze Particles&#34; command of the Image J software.</li>
</ol>

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The authors have no conflicts of interest to disclose.

The oily secretion of the Meibomian glands is of great importance for a healthy eye22. However, the obstruction of these sebaceous glands by aggregated neutrophil extracellular traps (aggNETs) that line up as parallel strands located on the tarsal plates of both eyelids can disrupt the tear film23. This disruption results in Meibomian gland dysfunction (MGD)1 and accelerated tear evaporation and conditions the damage of the ocular surface<sup class="...

Every blink of an eye replenishes the smooth tear film dispersed over the cornea. The ocular surface epithelia facilitate the distribution and correct orientation of the tear film on the ocular surface. Mucins are provided by the cornea and conjunctiva epithelial cells to help position the aqueous part of the tear film coming from the lacrimal glands on the eyes&#39; surface. Finally, MG secretes lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film1,2,3. In this fashion, the coordinated functions of all the ocular organs protect the ocular surface from invading pathogens or injury and support crystal clear vision without any pain or discomfort.
In a healthy ocular surface, the ocular flowing discharge or eye rheum sweeps away dust, dead epithelial cells, bacteria, mucus, and immune cells. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin and incorporate these components in the eye rheum. AggNETs resolve inflammation by the proteolytic degradation of pro-inflammatory cytokines and chemokines4. However, when they become dysfunctional, these aberrant aggNETs drive the pathogenesis of diseases such as vascular occlusions in COVID-195, gallstones6, and sialolithiasis7. Similarly, aggNETs on the ocular surface play a protective role and contribute to resolving inflammation of the highly exposed surface8. Either an exaggerated formation or lack of aggNETs in the ocular surface can impair the tear film stability and/or cause corneal wounds, cicatrizing conjunctivitis, and dry eye disease. For example, the obstruction of MG is a leading cause of dry eye disease9. AggNETs are also known to plug the flow of lipid secretion from the ducts of MG and cause Meibomian gland dysfunction (MGD). The congestion of MG orifices by aggNETs causes a lack of fatty fluid enveloping the ocular surface and retrograde bottled-up fluid, resulting in dysfunction of the gland function and acinar damage. This dysfunction can result in tear film evaporation, fibrosis of the margins on the eyelids, eye inflammation, and detrimental damage to the MG10,11.
Several animal models have been developed over the years to imitate the pathological process of MGD in humans. For example, C57BL/6 mice aged 1 year have helped study age-related effects on dry eye disease (DED) and MGD, reflecting the ocular disease pathology in patients aged 50 years and older12,13,14. Furthermore, rabbits are appropriate models for investigating the effects of pharmacological interventions. Therefore, inducing MGD in rabbits has been reported by either the topical administration of epinephrine or the systemic introduction of 13-cis-retinoic acid (isotretinoin)15,16,17,18,19.
Although these animal models were adequate for determining the different factors contributing to the pathophysiology of MGD, they were restricted in their utilization. For instance, the murine model of age-related MGD was ideal for deciphering elements in older adults only, and hence, rabbits appeared to be the most suitable animal model to study ocular surface diseases, as they enable the investigation of multiple pathophysiological mechanisms. However, due to the lack of comprehensive analytical tools to detect proteins at the ocular surface and because many parts of the rabbit genome are unannotated, they are limited for investigations20,21.
In addition, these animal models used to investigate the pathogenesis of dry eye disease did not provide adequate details to analyze the immunological arm of the disorder that instigates the inflammation of the ocular surface. Accordingly, the murine model of MGD developed by Reyes et al. showed an association between allergic eye disease in mice and MGD in humans and highlighted the immune etiology responsible for obstructive MGD21. This model associates allergic eye disease with a TH17 response that recruits neutrophils to the conjunctiva and eyelid, causing MGD and chronic ocular inflammation21. The induction of MGD and ocular inflammation in this murine model is a valuable tool for investigating upstream events during the development of local inflammation driven by an ongoing immune response21. The current protocol describes the ocular surface inflammation accompanied by obstructive MGD. In this method, mice are immunized and, after 2 weeks, challenged on the ocular surface with the immunogen for 7 days. Furthermore, the steps to isolate ocular exudate and the associated ocular organs during acute inflammation and the dissection of the cornea, conjunctiva, and eyelids are described.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x PBS</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminium Hydroxide</td>
			<td>Imject alum Adjuvant</td>
			<td>77161</td>
			<td>40 mg/ mL 
			Final Concentration: in vivo: 1 mg/ 100 &#181;L</td>
		</tr>
		<tr>
			<td>C57Bl/6 mice, aged 7&#8211;9 weeks</td>
			<td>Charles River Laboratories&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium</td>
			<td>Carl roth</td>
			<td>CN93.1</td>
			<td>1 M 
			Final Concentration: 5 mM</td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>FST by Dumont SWITZERLAND</td>
			<td>5/45 11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine sharp scissor</td>
			<td>FST Stainless steel, Germany</td>
			<td>15001-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar safety cabinet</td>
			<td>Herasafe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophotography Camera</td>
			<td>Canon</td>
			<td>EOS6D</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophotography Camera (without IR filter)</td>
			<td>Nikon</td>
			<td>D5300</td>
			<td></td>
		</tr>
		<tr>
			<td>Mnase</td>
			<td>New England biolabs</td>
			<td>M0247S</td>
			<td>2 x 106 gel U/mL</td>
		</tr>
		<tr>
			<td>Multi-analyte flow assay kit (Custom mouse 13-plex panel)</td>
			<td>Biolegend</td>
			<td>CLPX-200421AM-UERLAN</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0,9% (Saline)</td>
			<td>B.Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ovalbumin (OVA)</td>
			<td>Endofit, Invivogen</td>
			<td>9006-59-1</td>
			<td>10 mg/200 &#181;L in saline</td>
		</tr>
		<tr>
			<td>Pertussis toxin&#160;</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>PHZ1174</td>
			<td>50 &#181;g/ 500 &#181;L in saline 
			Final Concentration: in vivo: 100 &#181;g/ 100 &#181;L</td>
		</tr>
		<tr>
			<td>Petridish</td>
			<td>Greiner bio-one</td>
			<td>628160</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Feather disposable scalpel</td>
			<td>No. 21&#160;</td>
			<td>Final Concentration: in vivo:&#160; 300 ng/ 100 &#181;L</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Zaiss</td>
			<td>Stemi508</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (corneal/iris washing)</td>
			<td>BD Microlane</td>
			<td>27 G x 3/4 - Nr.20 0,4 x 19 mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (i.p immunization)</td>
			<td>BD Microlane</td>
			<td>24 G1&#34;-Nr 17, 055* 25 mm</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of ocular surface inflammation and collection of involved tissues

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

The present protocol describes the sequential steps for establishing a murine model of ocular surface inflammation. The protocols aim to show how to apply therapeutics locally, obtain ocular exudates, and excise associated accessory organs such as healthy and inflamed eyelids (Figure 2), the cornea, and the conjunctiva. Attention must be paid when the upper eyelids are dissected for the isolation of the conjunctiva, and it must be stored in 1x PBS during the dissection of the cornea. This wi...

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Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD).

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular ...

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Research

JoVE Journal

Immunology and Infection

2.1K Views.  Friedrich-Alexander-University Erlangen-Nürnberg and Universitätsklinikum Erlangen. Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD). 

Video: Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

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 ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.