We thank Ms. Karla Gorena for technical assistance in flow cytometry. L.S. is supported by the China Scholarship Council and Hunan Provincial Innovation Foundation for Postgraduate (CX201713068). H.H.A. is supported by the Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Jouf University, Sakaka, Saudi Arabia. X.D.L. is supported by the UT Health San Antonio School of Medicine Startup Fund and the Max and Minnei Voelcker Fund.

Experimental procedures described here were approved by the Institutional Animal Care and Use Committee of University of Texas Health San Antonio.
1. Dot blot to show the presence of dsRNA structures in HDM total RNA
<ol>
	<li>Total RNA isolation from allergens, insects, and non-insect allergens
	<ol>
		<li>Put HDM, insects, or non-insect animals collected alive or obtained commercially into 50 mL tubes, and quickly freeze with liquid-N2. Then store at -70 &#176;C for subsequent total RNA isolation. 
		NOTE: In this experiment, HDM, insect, and non-insect animals were selected because they are known to be common sources of allergens. Further, an immunostimulatory function of their RNAs remain unclear.</li>
		<li>Transfer a proper amount (equivalent to 100 &#956;L in volume or less) of HDM, insects or non-insect animals stored at -70 &#176;C into a 2 mL tube containing beads (1.4 mm ceramic spheres), then freeze tubes in a liquid-N2 container for ~10 min.</li>
		<li>For the total RNA isolation, add 1 mL of guanidinium thiocyanate-based RNA isolation reagent24 to each tube, then break the insect and non-insect small animals with a high-energy cell disrupter at the maximum speed for 45 s and chill on ice. Repeat this step twice.</li>
		<li>Transfer the solution from step 1.1.3 into a new 1.5 mL tube and add 200 &#956;L of chloroform to each tube and vortex. Centrifuge tubes at 14,000 x g for 14 min at 4 &#176;C.</li>
		<li>Once centrifugation is completed, transfer the upper aqueous phase (200 &#956;L) into a new 1.5 mL tube containing 500 &#956;L of isopropanol to precipitate RNA pellet. Do not disturb the interphase. The recommended volume ratio of the upper phase versus isopropanol is 1:2.5 ratio.</li>
		<li>Mix by gentle vortexing, then centrifuge tubes at 14,000 x g for 14 min at 4 &#176;C.</li>
		<li>Aspirate the supernatant with caution then wash the RNA pellet with 500 &#956;L of 75% ethanol and centrifuge at 7,500 x g for 10 min at 4 &#176;C. Remove all liquid with caution, air-dry the pellet and dissolve the RNA pellet with 20-50 &#956;L of RNase-free H2O.</li>
		<li>Measure the RNA concentration with a spectrophotometer using the following parameters:
		<ol>
			<li>Open the associated software and select the type of nucleic acids to measure. Change the sample type to RNA.</li>
			<li>Perform the blank measurement with 1-2 &#956;L of RNase-free H2O. Wipe off the RNase-free H2O. Now, the instrument is ready for the measurement.</li>
			<li>Load 1-2 &#956;L of the RNA sample and measure the RNA concentration (&#956;g/&#956;L). 
			NOTE: The ratio of the absorbance at 260 and 280 nm (A260/280) at ~2.0 (1.9-2.2) is generally accepted as &#8220;pure&#8221; for RNA. If not processed immediately, store RNA samples at -70 &#730;C and avoid the freeze-thaw cycles to keep the RNA intact.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Detection of dsRNA structure in the total RNA using dsRNA specific J2 antibody
	<ol>
		<li>Prepare two 20 &#956;L of RNA samples (200 ng/&#956;L). One with RNase-III treatment (1 &#956;L for 1 &#956;g RNA, incubate at 37 &#176;C for 60 min), and the other without RNase-III treatment. 
		NOTE: RNase III is used here to specifically degrade dsRNA, but not single-stranded RNA25.</li>
		<li>Use a pencil to draw grids where RNA samples will be blotted on the membrane.</li>
		<li>Spot 2 &#956;L of the 200 ng/&#956;L of the RNA sample onto the positively charged nylon membrane.</li>
		<li>Crosslink the samples to the membrane at 1,200 microjoules x 100 in a UV crosslinker. Repeat steps 1.2.3 and 1.2.4 two more times in the sample spot place. This will result in total 0.8&#160;&#956;g per blot. 
		NOTE: Do not spot more than 2 &#956;L of RNA sample on the membrane at a time.</li>
		<li>Block non-specific binding with 5% milk in TBS-T for 1 h with shaking at room temperature. Remove the blocking solution from step 1.2.5 and add the anti-dsRNA J2 antibody at the 1:1,000 dilution in 1% milk in TBS-T and incubate overnight with shaking at 4 &#176;C.</li>
		<li>Wash the membrane with TBS-T for 5 min and repeat this step for 3 times. Add the secondary antibody (Alkaline phosphatase-conjugated Anti-Mouse IgG diluted in 1% milk 1:5,000) and incubate for 1 h on a shaker at room temperature. Wash the membrane with TBS-T for 5 min and repeat this step for 3x.</li>
		<li>Add the substrate (BCIP/NBT) and incubate for 5-15 min until a desired signal is visible.</li>
		<li>Stop the reaction by rinsing the membrane with ddH2O.</li>
		<li>Dry the membrane on tissue papers and take a photograph using a smartphone (a representative result is shown in Figure 1).</li>
	</ol>
	</li>
</ol>
2. RT-qPCR to measure the ability of HDM total RNA in stimulating lung ISGs expression
<ol>
	<li>RNA isolation from mice lung tissues 
	NOTE: Mice (female, 8-12 weeks old, C57BL/6J) were maintained under specific pathogen-free conditions.
	<ol>
		<li>Briefly anesthetize the animal with isoflurane and administer via the intratracheal instillation with 5 &#956;g (diluted in 80 &#956;L PBS) of HDM RNAs treated with or without RNase III.</li>
		<li>After 16-18 h post HDM RNA treatment, sacrifice the mouse by CO2 inhalation for a few minutes. Then, place the mouse on a platform and pin limbs with needles.</li>
		<li>Disinfect the mouse with 70% ethanol then cut the skin starting from abdomen to the neck with a sterilized scissor.</li>
		<li>Fix the skin with needles and cut the ribs to expose the lungs. Remove the whole lungs and wash them with cold PBS. Place the lungs on tissue papers and excise one small piece of each lung-lobe into a 2 mL tube containing beads (200-300 &#956;L in volume, 1.4 mm ceramic spheres). 
		NOTE: The purpose of using ceramic beads is to grind whole lung tissues</li>
		<li>Freeze the lung samples by placing tubes into a liquid-N2 container for ~10 min.</li>
		<li>Add 500 &#956;L of guanidinium thiocyanate-based RNA isolation reagent to each tube and break the lung tissues with a homogenizer for 45 s. Chill on ice between each step. Repeat this step twice.</li>
		<li>Follow the steps 1.1.4- 1.1.7 for lung RNA isolation.</li>
		<li>Air-dry the pellet and dissolve the RNA pellet with proper amount of RNase-free H2O (~20-30 &#956;L).</li>
		<li>Measure the RNA concentration as described in step 1.1.8.</li>
	</ol>
	</li>
	<li>RT-qPCR to determine the ability of HDM RNA in stimulating lung gene expression.
	<ol>
		<li>Using 100 ng/&#956;L of RNA extracted from lung tissues as the template, perform the cDNA synthesis according to the referenced protocol26.</li>
		<li>Set up an RT-qPCR reaction at 10 &#956;L/well for a 384-well plate using cDNA generated above and the gene-specific primer pairs (Table 1 and Table 2).</li>
		<li>Seal the wells tightly with a transparent adhesive film and vortex the plate for 30 s. Spin the plate at 1,000 x g for 30 s to collect samples at the bottom of the wells.</li>
		<li>Load the plate onto a RT-qPCR machine and start to run the RT-qPCR reaction using the thermal cycler protocol (Table 3).</li>
		<li>Export the results into a spreadsheet file or analyze the data using the software provided by the manufacture after the program is completed (a representative result is shown in Figure 2).</li>
	</ol>
	</li>
</ol>
3. FACS analysis to determine the effects of HDM RNA on the infiltration of eosinophils in BAL and lung
<ol>
	<li>BAL fluid collection for FACS analysis
	<ol>
		<li>Euthanize mice (female, 8-12 weeks old, C57BL/6J) that were treated with HDM allergen extracts (according to the experimental design shown in Figure 3B) by CO2 inhalation.</li>
		<li>Place the mouse on a platform and pin limbs with needles.</li>
		<li>Disinfect the mouse with 70% ethanol. Use scissors to cut the skin from the upper area of the abdomen to the neck.</li>
		<li>Gently, pull the salivary glands and the sternohyoid muscle carefully apart using the forceps to expose the trachea. Place a nylon string (~10 cm) under the trachea using forceps.</li>
		<li>Make an incision in the trachea (~2 mm under the larynx) just enough to insert a cannula. Do not cut through the trachea. Knot the string around trachea and cannula.</li>
		<li>Load the syringe with 1 mL of PBS+EDTA and attach it to the end of the cannula. Inject 1 mL of PBS+EDTA into the lung and completely aspirate the solution. Detach the syringe from the cannula carefully and transfer&#160;the solution into a 15 mL tube on ice.</li>
		<li>Reload the syringe with the fresh PBS+EDTA and repeat this step 2x.</li>
		<li>Centrifuge the tube containing the pooled BAL obtained in step 3.1.7 to pellet the cells at 500 x g for 7 min at 4 &#176;C. Record the volume of BAL fluid then transfer the supernatant to two 1.5 mL tubes without disturbing the pellet. 
		NOTE: The supernatant of BAL can be stored at -70 &#176;C for future analysis e.g., ELISA.</li>
		<li>In case there are RBCs present in the pellet due to severe lung inflammation, after removing the supernatant, add 500 &#956;L of RBC lysis buffer and mix well by resuspension. Transfer the solution into a new 1.5 mL tube and centrifuge for 7 min at the speed of 500 x g at 4 &#176;C.</li>
		<li>Remove the supernatant and resuspend the pellet in 150 &#956;L of FACS buffer.</li>
		<li>Transfer the 150 &#956;L of the resuspended sample into 96-well plate and centrifuge the plate for 7 min at the speed of 500 x g at 4 &#176;C.</li>
		<li>Quickly, invert the plate on tissue papers to collect the cells residing at the bottom of the wells.</li>
		<li>Stain the cells with antibodies in FACS buffer in the presence of 2.4G2 blocking antibody (2.5 &#956;g / 100 &#956;L). Incubate the plate at room temperature for 30 min in a dark place.</li>
		<li>After staining, centrifuge the plate to pellet the cells at 500 x g for 7 min at 4 &#176;C.</li>
		<li>Remove the staining solution by inverting the plate on tissue paper then wash by resuspending with 100 &#956;L of FACS buffer. Next, centrifuge the plate again at 500 x g for 7 min at 4 &#176;C and remove the FACS buffer by inverting the plate on tissue paper.</li>
		<li>Resuspend samples into 150 &#956;L of FACS buffer and transfer samples to the FACS tubes containing 350 &#956;L of FACS buffer. Add 25 &#956;L of counting beads to each sample. Samples are now ready for flow cytometry analysis. 
		NOTE: Various cell types in BAL fluid were labeled with antibodies as indicated. Counting beads were added before the FACS run. Flow cytometry data were analyzed using a commercially available software. Refer to Figure 3 and Table 4 for gating strategy.</li>
	</ol>
	</li>
	<li>Lung tissue digestion for the FACS analysis
	<ol>
		<li>Follow steps 3.1.1 - 3.1.3.</li>
		<li>Cut the skin starting from abdomen to the neck with a sterilized scissor. Fix the skin with needles and cut the ribs to expose the lungs.</li>
		<li>Remove the whole lungs and wash them with cold PBS. Place samples in the 1.5 mL tube containing 50 &#956;L of lung digestion solution.</li>
		<li>Mince the lung tissues into small pieces with a curved scissor. Transfer the lung tissues into a 6-well plate, then add 8 mL of lung digestion solution. Place the plate on a shaker in 37 &#176;C incubator for 45 min.</li>
		<li>After incubation, use the top of 1.5 mL tube to grind the lung tissues. Place a 70 &#181;m strainers on a new 6-well plate and apply the sample through 0.22 &#181;m filter.</li>
		<li>Transfer the filtered solution into a 15 mL tube, then centrifuge the tubes at 500 x g for 7 min at 4 &#176;C. Aspirate the supernatant and resuspend the pellet in 1 mL of RBC lysis buffer and leave it on ice for 3 min.</li>
		<li>Transfer the sample into 1.5 mL tube and centrifuge at 500 x g for 7 min at 4 &#176;C. Repeat 2x</li>
		<li>Wash the lung cells 2x with 1 mL FACS buffer. Aspirate the supernatant and resuspend the pellet in 1 mL of FACS buffer, and then transfer 100 &#956;L of the sample into 96 well plate.</li>
		<li>Centrifuge the plate for 7 min at the speed of 500 x g at 4 &#176;C. Follow the steps described in BAL fluid collection for FACS analysis (3.1.13 to 3.1.16) to stain the cells of digested lung tissue samples. 
		NOTE: Eosinophils in the lungs were labeled with antibodies as indicated, then mixed with counting beads for further FACS analysis. Flow cytometry data were analyzed using associated software. Refer to Figure 3 for evaluating HDM RNA-induced immune responses.</li>
	</ol>
	</li>
</ol>
4. Statistical analysis
<ol>
	<li>Perform statistical analysis using a commercially available software.</li>
	<li>Determine the p values by unpaired two-tailed Student t test for the comparison of two groups.</li>
	<li>Calculate the absolute numbers of eosinophils based on reference beads (top panel) using the formula</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/61183/61183equ01.jpg" />
<ol start="4">
	<li>Determine the p values by two-way ANOVA and Sidak&#8217;s multiple comparisons test for the comparison of more than two groups.</li>
	<li>Consider a p value smaller than 0.05 as statistically significant. The p values are indicated on plots as *p &#60;0.05, **p&#60;0.01, ***p&#60;0.001, and ****p&#60;0.0001. 
	NOTE: All buffer recipes are provided in Table 5.</li>
</ol>

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We have nothing to disclose.

The current protocol describes how to evaluate the immunostimulatory properties of allergen-associated microbial RNA and their impacts on the development of eosinophilic lung inflammation in a mouse model of allergic asthma. Although long dsRNAs are known as the replication intermediates of many viruses that can potently activate interferon responses in mammalian cells, their presences in HDM allergens have been unknown until our recent work15. The combination of RNA dot blot, RT-qPCR and FACS ana...

Based on the hygiene hypothesis originally proposed by Strachan1, early childhood exposure to environmental microbial factors such as endotoxin can protect against the development of allergic disorders2,3. During microbial infections, e.g., viral infections, the innate immune detection of foreign nucleic acids (RNA/DNA) triggers host defense responses4,5,6. However, the existence and prevalence of immunogenic nucleic acids such as long double-stranded RNA (dsRNA) species in house dust mites (HDM) or other insect allergens remain unknown. This protocol was designed to determine whether HDM or insect and non-insect allergens contain long dsRNA species that can activate a protective immune response to counteract the development of severe eosinophilic lung inflammation in a mouse model of allergic asthma. Here, we provide three simple and fast methods to evaluate the structural determinants in HDM total RNA that are required for regulating allergen-induced eosinophilic lung inflammation.
The mucosal immune system is the largest immune organ in the body and serves as the first line of host defense against both microbial infections and allergic insults7,8. The long dsRNA, the replication intermediate of many viruses, is known to function as a pathogen-associated molecular pattern (PAMP) to potently stimulate innate responses via Toll like receptor 3 (TLR3) to induce the expression of interferon stimulated genes (ISGs)9,10,11,12,13,14. We have recently shown that HDM total RNA contained dsRNA structures, which upregulated the expression of ISGs and reduced severe eosinophilic lung inflammation when administered via the intratracheal instillation in a murine model of allergic asthma induced by HDM extracts15. The severity of lung inflammations is determined by analyzing the immune cell types in bronchoalveolar lavage (BAL) and lung tissue via flow cytometry16,17,18,19,20.
This protocol includes three assays: 1) rapid detection of dsRNA structures with RNA dot blot using a mouse monoclonal antibody J2 which specifically binds to the dsRNA (&#8805;40bp) in a sequence-independent manner; 2) quick evaluation for in vivo effects of immunostimulatory RNA in mouse lungs by measuring the induction of ISGs using RT-qPCR; 3) accurate quantification of eosinophils in BAL and lung in the context of HDM-induced lung inflammation using flow cytometry analysis.
The above assays can be used to study not only allergic lung diseases, but also respiratory bacterial and viral infections. For example, the dsRNA specific J2 antibody can also be used in other applications such as immunoaffinity chromatography, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and immunostaining21,22,23. In addition, several applications downstream of BAL fluid collection can be utilized for quantifying soluble contents such as cytokines and chemokines using ELISA, and transcriptional profiling of cells in the airways (e.g., alveolar macrophages). Although there are a variety of protocols available in the literature to evaluate lung conditions, most of these protocols often focus on the target validation. The procedures described here can be applied to identify components in environmental allergens that are important for regulating the development of allergic diseases.

<table>
	<tbody>
		<tr>
			<td>0.40 &#181;m Falcon Cell Strainer</td>
			<td>Thermo Fisher Scientific</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringes</td>
			<td>Henke Sass Wolf</td>
			<td>5010.200V0</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Tube</td>
			<td>TH.Geyer</td>
			<td>7696702</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Tube</td>
			<td>TH.Geyer</td>
			<td>7696705</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Decon Labs</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute Counting Beads</td>
			<td>Life Technologies Europe B.V.</td>
			<td>C36950</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK-RBC lysing buffer</td>
			<td>Lonza</td>
			<td>10-548E</td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Hybond-N+ Membrane</td>
			<td>GE Healthcare</td>
			<td>RPN203B</td>
			<td></td>
		</tr>
		<tr>
			<td>Ant</td>
			<td>San Antonio</td>
			<td>Note: Locally collected</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody dilution buffer</td>
			<td></td>
			<td>(see Table 5 for recipe)</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD11b V450 Rat (clone M1/70)</td>
			<td>BD Bioscience</td>
			<td>560456</td>
			<td>1 to 200 dilution</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD11c PE-Cy7 (clone N418)</td>
			<td>BioLegend</td>
			<td>117317</td>
			<td>1 to 200 dilution</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD19 Alexa Flour 647 (clone 1D3)</td>
			<td>eBioscience</td>
			<td>15-0193-81</td>
			<td>1 to 200 dilution</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD3e APC (clone 145-2C11)</td>
			<td>Invitrogen</td>
			<td>15-0031-81</td>
			<td>1 to 200 dilution</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD45 APC-Cy7 (clone: 30-F11)</td>
			<td>BioLegend</td>
			<td>103130</td>
			<td>1 to 200 dilution</td>
		</tr>
		<tr>
			<td>Anti-Mouse Fixable Viabillity Dye eFluor 506</td>
			<td>Invitrogen</td>
			<td>65-0866-14</td>
			<td>1 to 200 dilution</td>
		</tr>
		<tr>
			<td>Anti-Mouse IgG (H+L), AP Conjugate</td>
			<td>Promega</td>
			<td>S3721</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Ly-6G FITC (clone RB6-8C5)</td>
			<td>Invitrogen</td>
			<td>11-5931-82</td>
			<td>1 to 200 dilution</td>
		</tr>
		<tr>
			<td>Anti-Mouse MHC II APC-eFluor 780 (clone M5/114.15.2)</td>
			<td>eBioscience</td>
			<td>47-5321-80</td>
			<td>1 to 200 dilution</td>
		</tr>
		<tr>
			<td>Anti-Mouse Siglec-F PE (clone E50-2440)</td>
			<td>BD Pharmingen</td>
			<td>552126</td>
			<td>1 to 200 dilution</td>
		</tr>
		<tr>
			<td>BCIP/NBT substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>PI34042</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking Buffer</td>
			<td></td>
			<td>(see Table 5 for recipe)</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannual, 20G X 1.5&#8221;</td>
			<td>CADENCE SCIENCE</td>
			<td>9920</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75004030</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX384 Touch Real-Time PCR Detection System</td>
			<td>Bio-Rad Laboratories</td>
			<td>1855485</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Thermo Fisher Scientific</td>
			<td>C298-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cockroach</td>
			<td>Greer Laboratories</td>
			<td>B26</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>01-1234-42</td>
			<td></td>
		</tr>
		<tr>
			<td>D. farinae</td>
			<td>Greer Laboratories</td>
			<td>B81</td>
			<td></td>
		</tr>
		<tr>
			<td>D. pteronyssinus</td>
			<td>Greer Laboratories</td>
			<td>B82</td>
			<td></td>
		</tr>
		<tr>
			<td>Denville Cell Culture Plates with lid, 96 well cell culture plate</td>
			<td>Thomas Scientific</td>
			<td>1156F03</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Dry Bath - Four Blocks</td>
			<td>Universal Medical, Inc.</td>
			<td>BSH1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Earthworm</td>
			<td>San Antonio</td>
			<td>Note: Locally collected</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E6511</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS buffer</td>
			<td></td>
			<td>(see recipe in Table 5)</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Polypropylene Tubes, 5 mL</td>
			<td>STEMCELLTM TECHNOLOGIES</td>
			<td>38056</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer (BD FACS Celesta)</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fly</td>
			<td>Greer Laboratories</td>
			<td>B8</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Hausser Scientific</td>
			<td>3110</td>
			<td></td>
		</tr>
		<tr>
			<td>HT-DNA</td>
			<td>Sigma</td>
			<td>D6898</td>
			<td></td>
		</tr>
		<tr>
			<td>In Vivo MAb anti-mouse CD16/CD32 (clone: 2.4G2)</td>
			<td>Bio X Cell</td>
			<td>BE0307</td>
			<td></td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad Laboratories</td>
			<td>1708891</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott Labs</td>
			<td>sc-363629Rx</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP2618500</td>
			<td></td>
		</tr>
		<tr>
			<td>J2 anti-dsRNA monoclonal antibody</td>
			<td>SCICONS</td>
			<td>10010200</td>
			<td></td>
		</tr>
		<tr>
			<td>Lung digestion solution</td>
			<td></td>
			<td>(see recipe in Table 5)</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing Matrix D</td>
			<td>MP Biomedicals</td>
			<td>116913050-CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing Matrix D, 2 mL tube</td>
			<td>MP Biomedicals</td>
			<td>SKU:116913100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice (female, 8-12 weeks old, C57BL/6J)</td>
			<td>Jackson Laboratory</td>
			<td>#000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube 1.5 mL</td>
			<td>Sigma-Aldrich</td>
			<td>30120.094</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>CK30</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-BeadBeater</td>
			<td>Homogenizers</td>
			<td>SKU:BS:607</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Beadbeater-16</td>
			<td>Biospec</td>
			<td>607</td>
			<td></td>
		</tr>
		<tr>
			<td>Mosquito</td>
			<td>Greer Laboratories</td>
			<td>B55</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000C</td>
			<td>Thermo Scientific Spectophotometer Medex Supply</td>
			<td>TSCND2000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle, 21 G x 1 1/2 in</td>
			<td>BD Biosciences</td>
			<td>305167</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat milk</td>
			<td>Bio-Rad Laboratories</td>
			<td>1706404</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon string</td>
			<td>Dynarex</td>
			<td>3243</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered Saline (PBS)</td>
			<td>Lonza</td>
			<td>BE17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase III</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM2290</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase T1</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM2283</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-6802</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker or Small laboratory mixer</td>
			<td>Boekel Scientific</td>
			<td>201100</td>
			<td></td>
		</tr>
		<tr>
			<td>SPHERO AccuCount Fluorescent</td>
			<td>Spherotech</td>
			<td>ACFP-70-5</td>
			<td>1 to 10 dilution</td>
		</tr>
		<tr>
			<td>Spider</td>
			<td>San Antonio</td>
			<td>Note: Locally collected</td>
			<td></td>
		</tr>
		<tr>
			<td>TBS</td>
			<td></td>
			<td>(see recipe in Table 5)</td>
			<td></td>
		</tr>
		<tr>
			<td>TBS-T</td>
			<td></td>
			<td>(see recipe in Table 5)</td>
			<td></td>
		</tr>
		<tr>
			<td>Total cell medium</td>
			<td></td>
			<td>(see recipe in Table 5)</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Stratalinker 2400 UV</td>
			<td>LabX</td>
			<td>20447</td>
			<td></td>
		</tr>
		<tr>
			<td>Wasp</td>
			<td>San Antonio</td>
			<td>Note: Locally collected</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification and characterization of immunogenic rna species in hdm allergens that modulate eosinophilic lung inflammation

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses and microbial substances that induce innate immune responses. These allergen-associated microbial components play an important role in regulating the development of type 2 inflammatory conditions such as allergic asthma. However, the underlying mechanisms remain largely undefined. The protocol presented here determines the structural characteristics and in vivo activity of allergen-associated immunostimulatory RNA. Specifically, common allergens are examined for the presence of double-stranded RNA (dsRNA) species that can stimulate IFN responses in lungs and restrain&#160;the development of severe lung eosinophilia in a mouse model of HDM-induced allergic asthma. Here, we have included the following three assays: Dot blot to show the dsRNA structures in total RNA isolated from allergens including HDM species, RT-qPCR to measure the activities of HDM RNA in interferon stimulating genes (ISGs) expression in mouse lungs and FACS analysis to determine the effects of HDM RNA on the number of eosinophils in BAL and lung, respectively.

The presence of long dsRNA structures in HDM, insects and non-insect small animals was examined by dot blot using a dsRNA-specific mouse monoclonal antibody J2 (&#8805; 40bp). RNase III was used to digest dsRNA into 12&#8211;15 bp dsRNA fragments, which were undetectable by J2 (Figure 1).
The ability of HDM total RNA to stimulate an innate immune response in mouse lungs in a dose-dependent manner&#160;was analyzed by RT-qPCR (Figure 2...

Watch this Scientific Journal Video about Identification and Characterization of Immunogenic RNA Species in HDM Allergens that Modulate Eosinophilic Lung Inflammation at JoVE.com

Identification and Characterization of Immunogenic RNA Species in HDM Allergens that Modulate Eosinophilic Lung Inflammation

Environmental allergens such as house dust mites (HDM) often contain microbial substances that activate innate immune responses to regulate allergic inflammation. The protocol presented here demonstrates the identification of dsRNA species in HDM allergens and characterization of their immunogenic activities in modulating eosinophilic lung inflammation.

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses ...

identification-characterization-immunogenic-rna-species-hdm-allergens

Research

JoVE Journal

Immunology and Infection

5.9K Views.  University of Texas Health San Antonio. Environmental allergens such as house dust mites (HDM) often contain microbial substances that activate innate immune responses to regulate allergic inflammation. The protocol presented here demonstrates the identification of dsRNA species in HDM allergens and characterization of their immunogenic activities in modulating eosinophilic lung inflammation.

Video: Identification and Characterization of Immunogenic RNA Species in HDM Allergens that Modulate Eosinophilic Lung Inflammation

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

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 ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

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.

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...