The authors wish to thank Edith Janssen for the critical review of the manuscript and Navin Rao and Jennifer Towne for their support of the publication of this manuscript.

All animal experiments were conducted in accordance within the policies of the Institutional Animal Care and Use Committee (IACUC) of Janssen R&#38;D.
1. Cryogenic Freezer Mill-based Pulverization Method
<ol>
	<li>At termination of the CIA study, sacrifice mice with 1.5% isoflurane followed by cervical dislocation.</li>
	<li>On the day of tissue processing, pre-chill all instruments (spatulas, grinding vials, etc.) on dry ice for a minimum of 10 min. Wear thermal gloves to avoid freeze damage.</li>
	<li>Cut hind paws with a scissor at the fur line as depicted in Figure 1A.</li>
	<li>Transfer each paw into one 1.5 mL microcentrifuge tube, snap freeze in liquid nitrogen immediately, and store frozen paws at -80 &#176;C. Do not keep samples frozen for more than one week.</li>
	<li>Fill freezer mill with liquid nitrogen as shown in Figure 1B and let it equilibrate for at least 10 min.</li>
	<li>Keep unprocessed sample on dry ice and avoid freeze-thaw cycles.</li>
	<li>Transfer one hind paw into pre-chilled large polycarbonate grinding vial with a bottom steel plug, insert the pre-chilled steel impactor and close the polycarbonate grinding vial with the pre-chilled top steel plug (Figure 1C).</li>
	<li>Transfer large polycarbonate grinding vials with the samples into the freezer mill pre-filled with liquid and close the lid with the rubber clasp found at the front of the instrument.</li>
	<li>Let samples cool in liquid nitrogen for 1 minute.</li>
	<li>Set the freezer mill to the 1 minute program with 10 cycles per second and press the start button. Wait for the freezer mill to complete its cycle. 
	NOTE: The machine makes a drumming sound as the steel impactor moves back and forth. Use ear plugs to avoid hearing loss.</li>
	<li>Open the lid of the freezer mill, take out the large polycarbonate grinding vial and place it in an extractor as depicted in Figure 1D. Remove the top steel plug by placing downward pressure on the black handle until the steel plug slides out of the polycarbonate tube.</li>
	<li>Transfer the opened large polycarbonate grinding vial onto dry ice. Remove the steel impactor with pre-chilled forceps.</li>
	<li>Transfer the frozen powder into pre-chilled 50 mL conical tube.</li>
	<li>Weight 30-50 mg of frozen powder into a tared frozen 1.5 mL microcentrifuge tube for RNA extraction and 100-200 mg of frozen powder for protein extraction. 
	NOTE: The frozen powder should look like white or light pink fine sand as depicted in Figure 1E.</li>
	<li>Store pulverized samples at -80 &#176;C and proceed to RNA and protein isolations within 24 h.</li>
</ol>
2. RNA Extraction
<ol>
	<li>Add 10 &#181;L of &#946;-mercaptoethanol (&#946;-ME) per 1 mL of RLT buffer.</li>
	<li>Calculate the volume of RLT Buffer corresponding to a ratio of 23.3 mL/mg of tissue and add the appropriate volume of RLT Buffer with &#946;-ME to the tube with frozen powder. This ratio was empirically determined to be ideal for mouse paws.</li>
	<li>Vortex the sample at 3,000 RPM for 10 s.</li>
	<li>Mix well by pipetting up-and-down 10 times vigorously with a 1 mL pipettor.</li>
	<li>Vortex the sample again at 3,000 RPM for 20 s.</li>
	<li>Centrifuge the tube at 13,000 x g for 2 min.</li>
	<li>Transfer 700 &#181;L of the supernatants to a fresh tube and add 700 &#181;L of 70% ethanol. 
	NOTE: If &#60;700 &#181;L is in the tube, it is acceptable to proceed, still adding equivalent volume of 70% ethanol.</li>
	<li>Load 700 &#181;L of the sample onto a RNA purification column.</li>
	<li>Centrifuge the tube at &#8805;10,000 x g for 30 s.</li>
	<li>Discard the flow-through and load the remaining part of the sample to the RNeasy column.</li>
	<li>Centrifuge the tube at &#8805;10,000 x g for 30 s.</li>
	<li>Discard the flow through and wash the column by adding 700 &#181;L of RW1 buffer with centrifugation of &#8805;10,000 x g for 30 s.&#160;If performing option DNASE digestion, 350 &#181;L of RW1 is added prior to DNASE treatment. &#160;And an additional 350 &#181;L of RW1 is added following DNASE treatment.</li>
	<li>Optional) Perform a DNase digestion step follow manufacturer&#39;s instructions.</li>
	<li>Wash the tube twice by adding 500 &#181;L of RPE buffer followed by centrifugation at &#8805;10,000 x g for 30 s. Discard flow through each time.</li>
	<li>Dry the column by centrifugation at &#8805;10,000 x g for 2 min.</li>
	<li>Elute RNA by adding 50 &#181;L water with centrifugation at &#8805;10,000 x g for 2 min. Collect flow through and transfer into a new 1.5 mL tube.</li>
	<li>Determine the quantity of RNA using the researcher&#39;s method of choice and store at -80 &#176;C before further analysis.</li>
</ol>
3. Protein Extraction
<ol>
	<li>Dilute the 10x cell lysis stock solution in 1x cell lysis stock solution using cell culture grade water.</li>
	<li>Reconstitute the Protease Inhibitor Cocktail Set I with 1 mL of water to make a 100x protease inhibitor stock.</li>
	<li>Add 100 &#181;L of protease inhibitor to 9.9 mL of 1x Cell lysis to make a 1x final stock solution.</li>
	<li>Add 4 &#181;L of ice cold 1x Cell Lysis Buffer per mg tissue powder (e.g., 800 &#181;L of lysis buffer to 200 mg of tissue powder).</li>
	<li>Add one 5 mm stainless steel bead to the tube.</li>
	<li>Vortex the tube at 3,000 RPM for 60 s. Transfer the tube to wet ice and continue to the next sample.</li>
	<li>Place all sample tubes in a box and shake the centrifuge box on a rocker at 1,000 RPM, 4 &#176;C for 1 h.&#160; 
	NOTE:&#160;Researchers may find that placing the box vertical can facilitate better mixing with the bead.</li>
	<li>Centrifuge the tubes at 10,000 x g and 4 &#176;C for 15 min.</li>
	<li>Transfer the supernatants into a fresh tube and avoid the fat layer on the top.</li>
	<li>Determine the total protein concentration. 10 to 30-fold dilutions of protein lysate are ideal for a BCA test.</li>
	<li>Aliquot the sample into 1.5 mL microcentrifuge tubes and store at -80 &#176;C before further analysis.</li>
</ol>

<ol>
	<li>Firestein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+mechanisms+in+the+pathogenesis+of+rheumatoid+arthritis.">Immunologic mechanisms in the pathogenesis of rheumatoid arthritis.</a> Journal of Clinical Rheumatology. 11, S39-S44 (2005).</li><li>McInnes, I. B., Schett, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+of+rheumatoid+arthritis.">The pathogenesis of rheumatoid arthritis.</a> New England Journal of Medicine. 365, 2205-2219 (2011).</li><li>Ahlqvist, E., Hultqvist, M., Holmdahl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+value+of+animal+models+in+predicting+genetic+susceptibility+to+complex+diseases+such+as+rheumatoid+arthritis.">The value of animal models in predicting genetic susceptibility to complex diseases such as rheumatoid arthritis.</a> Arthritis Research and Therapy. 11, 226 (2009).</li><li>David, C. S., Taneja, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+major+histocompatibility+complex+genes+in+murine+collagen-induced+arthritis:+a+model+for+human+rheumatoid+arthritis.">Role of major histocompatibility complex genes in murine collagen-induced arthritis: a model for human rheumatoid arthritis.</a> American Journal of the Medical Sciences. 327, 180-187 (2004).</li><li>Tarkowski, A., Holmdahl, R., Klareskog, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheumatoid+factors+in+mice.">Rheumatoid factors in mice.</a> Monographs in Allergy. 26, 214-229 (1989).</li><li>Schurgers, E., Billiau, A., Matthys, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-induced+arthritis+as+an+animal+model+for+rheumatoid+arthritis:+focus+on+interferon-gamma.">Collagen-induced arthritis as an animal model for rheumatoid arthritis: focus on interferon-gamma.</a> Journal of Interferon &amp; Cytokine Research. 31, 917-926 (2011).</li><li>Joosten, L. A., Helsen, M. M., van de Loo, F. A., van den Berg, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticytokine+treatment+of+established+type+II+collagen-induced+arthritis+in+DBA/1+mice.+A+comparative+study+using+anti-TNF+alpha+anti-IL-1+alpha/beta,+and+IL-1Ra.">Anticytokine treatment of established type II collagen-induced arthritis in DBA/1 mice. A comparative study using anti-TNF alpha anti-IL-1 alpha/beta, and IL-1Ra.</a> Arthritis &amp; Rheumatology. 39, 797-809 (1996).</li><li>Asquith, D. L., Miller, A. M., McInnes, I. B., Liew, F. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+rheumatoid+arthritis.">Animal models of rheumatoid arthritis.</a> European Journal of Immunology. 39, 2040-2044 (2009).</li><li>Williams, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-induced+arthritis+as+a+model+for+rheumatoid+arthritis.">Collagen-induced arthritis as a model for rheumatoid arthritis.</a> Methods in Molecular Medicine. 98, 207-216 (2004).</li><li>Shen, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+Blockade+of+TNF-alpha+and+IL-17A+Alleviates+Progression+of+Collagen-Induced+Arthritis+without+Causing+Serious+Infections+in+Mice.">Combined Blockade of TNF-alpha and IL-17A Alleviates Progression of Collagen-Induced Arthritis without Causing Serious Infections in Mice.</a> Journal of Immunology. 202, 2017-2026 (2019).</li></ol>

The authors BJ, SH, ML, RM, ML, TO and FS are employees of Janssen Research &#38; Development Inc and are shareholders in the parent company, Johnson &amp; Johnson, Inc.

Although there is strong scientific rationale to evaluate molecular pathways in synovial tissues, many reports on immune profiling of murine CIA model were focused on peripheral blood, while protein and RNA analysis data of inflamed paws are rather limited. There are several possible reasons for this bias: murine ankle joints are no larger than ~2 cm; the affected areas consist of skin, bone and connective tissues which are often difficult to homogenize using traditional methods like tissue grinders, pestle and mortar, s...

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease with persistent symmetric synovitis in joints and extra articular involvement of organs such as the skin, heart, lungs, and eyes1. Although the systemic manifestations of the immune response are evident in human patients, one of the hallmarks of RA pathology is infiltration of immune cells in synovial tissue and proliferation of synovial fibroblast cells2.
Similar to human RA, mouse collagen induced arthritis (CIA) model elicits strong tissue inflammation with active immune responses in synovial tissues and systemic compartments. The susceptibility of different mouse strains to CIA model links to Major Histocompatibility Complex (MHC) haplotype and antigen specific T cell and B cell interactions3,4. In addition, many pathogenic pathways in human RA, including autoantibody production, immune complex deposition, myeloid cell activation, polyarticular manifestations and pannus formation with synovial immune infiltration, are also evident in this model5,6. Investigators have employed this well-established CIA model to investigate effects of anti-inflammatory cytokine treatments7. Many biologics approved for autoimmune or inflammatory diseases, such as anti-TNF&#945; and anti-IL-6, are found to be efficacious in the CIA model8,9.
Profiling the interactions of the immune system in synovial tissue is crucial to elucidate molecular mechanisms associated with the pathogenesis of RA. In the human clinical setting, a common practice is to perform needle synovial biopsies under the guidance of ultrasound imaging. In the preclinical settings, the smaller architecture of the murine joints makes biopsy procedures much more difficult if not impossible. Recently, we demonstrated the utilization of the murine CIA model to evaluate combinations of drugs to impact disparate end-points and resolve disease in a combinatorial approach10. A cryogenic freezer mill-based pulverization method was employed to process inflamed murine paws into homogeneous fine powders and established downstream processes to extract RNA and proteins. This method protects RNA and protein from enzymatic and chemical degrative processes and enables us to apply multiple analytical methods to a single homogenized sample source.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 mm stainless steel bead</td>
			<td>Qiagen</td>
			<td>69989</td>
			<td></td>
		</tr>
		<tr>
			<td>beta-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td>Sample reducing agent that inhibits RNASE enzymes</td>
		</tr>
		<tr>
			<td>Bioanalyzer Kit</td>
			<td>Agilent</td>
			<td>5067-1511</td>
			<td>RNA qualification kit</td>
		</tr>
		<tr>
			<td>b-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Grade Water</td>
			<td>Corning</td>
			<td>25-055-CI</td>
			<td>Water</td>
		</tr>
		<tr>
			<td>Cell lysis stock solution</td>
			<td>Cell Signaling</td>
			<td>9803</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Tube</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td>Microfuge tubes</td>
		</tr>
		<tr>
			<td>Eppendorf tube centrifuge box</td>
			<td>Nalgene</td>
			<td>5055</td>
			<td>Box for holding eppendorf tubes in horizontal tube arrangement</td>
		</tr>
		<tr>
			<td>Everlast 247 Variable Speed Rocker</td>
			<td>Benchmark Scientific</td>
			<td>BR5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer Mill</td>
			<td>Spex Sample Prep</td>
			<td>6875</td>
			<td>Freezer/Mill for processing paws into pulverized powder</td>
		</tr>
		<tr>
			<td>Grinding Vial</td>
			<td>Spex Sample Prep</td>
			<td>6801</td>
			<td>Polycarbonate vial for processing paws into pulverized powder</td>
		</tr>
		<tr>
			<td>Pierce BCA kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td>Kit for Total Protein Quantification</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail set 1</td>
			<td>Calbiochem</td>
			<td>539131</td>
			<td>Protease Inhibitors</td>
		</tr>
		<tr>
			<td>Protein BCA Kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantigene Kit</td>
			<td>Thermofisher</td>
			<td>QP1013</td>
			<td>bDNA analysis Kit</td>
		</tr>
		<tr>
			<td>Refrigerated microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td>Centrifugation</td>
		</tr>
		<tr>
			<td>RLT Buffer</td>
			<td>Qiagen</td>
			<td>79216</td>
			<td>RNA extraction buffer</td>
		</tr>
		<tr>
			<td>RNeasy mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>including RNeasy column, RLT Buffer and RW1 Buffer</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Benchmark Scientific</td>
			<td>BR5000</td>
			<td>Rocker/Shaker</td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>VWR</td>
			<td>10806-412</td>
			<td>Spatula for powder transfer</td>
		</tr>
		<tr>
			<td>Stainless Steel Bead</td>
			<td>Qiagen</td>
			<td>69989</td>
			<td>Bead for mixing during protein extraction</td>
		</tr>
		<tr>
			<td>Tube Extractor</td>
			<td>Spex Sample Prep</td>
			<td>6884</td>
			<td>Extractor for removing the top of grinding vial</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>VWR</td>
			<td>10153-838</td>
			<td>Sample mixing</td>
		</tr>
	</tbody>
</table>

a cryo-pulverization protocol for processing mouse paws to evaluate molecular pathways of tissue inflammation in a collagen induced arthritis model

Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of arthritis research, many studies are focused on inflamed joints that are composed of a complex mixture of bone, cartilage, muscle, stromal cells and immune cells. Here, we established a reliable and robust mechanical method to disrupt inflamed mouse paws into homogeneous pulverized samples in a cryogenically controlled environment. Protein and RNA lysates were processed to enable proteomic and transcriptional endpoints and molecular characterization of relevant disease pathways in local tissue.

Here, we show a representative gel image visualization of RNA extracted from front paws of CIA mice in Figure 2A. The 28S rRNA and the 18S rRNA band indicate all samples have sufficient amount of intact RNA. Next, we show a representative scatter plot of total protein concentrations based on protein BCA analysis in Figure 2B. Total protein concentrations from na&#239;ve mice, CIA mice or CIA mice...

Watch this Scientific Journal Video about A Cryo-pulverization Protocol for Processing Mouse Paws to Evaluate Molecular Pathways of Tissue Inflammation in a Collagen Induced Arthritis Model at JoVE.co

A Cryo-pulverization Protocol for Processing Mouse Paws to Evaluate Molecular Pathways of Tissue Inflammation in a Collagen Induced Arthritis Model

A cryogenic pulverization method to process murine paws using a liquid nitrogen freezer mill was developed to improve the yield and quality of RNA or protein extracted from the tissues and enable the analysis of molecular profiles associated with inflammatory responses.

Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of ...

a-cryo-pulverization-protocol-for-processing-mouse-paws-to-evaluate

Research

JoVE Journal

Immunology and Infection

8.0K Views.  Janssen Research & Development, Inc.. A cryogenic pulverization method to process murine paws using a liquid nitrogen freezer mill was developed to improve the yield and quality of RNA or protein extracted from the tissues and enable the analysis of molecular profiles associated with inflammatory responses.

Video: A Cryo-pulverization Protocol for Processing Mouse Paws to Evaluate Molecular Pathways of Tissue Inflammation in a Collagen Induced Arthritis Model

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

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 ...

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 ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...