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

Summary

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.

Abstract

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 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.

Introduction

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 disorders^2,3. During microbial infections, e.g., viral infections, the innate immune detection of foreign nucleic acids (RNA/DNA) triggers host defense responses^4,5,6. 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 insults^7,8. 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 cytometry^{16,17,18,19,20}.

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 (≥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 immunostaining^21,22,23. 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.

Protocol

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

1. Total RNA isolation from allergens, insects, and non-insect allergens
   1. 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 °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.
   2. Transfer a proper amount (equivalent to 100 μL in volume or less) of HDM, insects or non-insect animals stored at -70 °C into a 2 mL tube containing beads (1.4 mm ceramic spheres), then freeze tubes in a liquid-N₂ container for ~10 min.
   3. 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.
   4. Transfer the solution from step 1.1.3 into a new 1.5 mL tube and add 200 μL of chloroform to each tube and vortex. Centrifuge tubes at 14,000 x g for 14 min at 4 °C.
   5. Once centrifugation is completed, transfer the upper aqueous phase (200 μL) into a new 1.5 mL tube containing 500 μL 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.
   6. Mix by gentle vortexing, then centrifuge tubes at 14,000 x g for 14 min at 4 °C.
   7. Aspirate the supernatant with caution then wash the RNA pellet with 500 μL of 75% ethanol and centrifuge at 7,500 x g for 10 min at 4 °C. Remove all liquid with caution, air-dry the pellet and dissolve the RNA pellet with 20-50 μL of RNase-free H₂O.
   8. Measure the RNA concentration with a spectrophotometer using the following parameters:
      1. Open the associated software and select the type of nucleic acids to measure. Change the sample type to RNA.
      2. Perform the blank measurement with 1-2 μL of RNase-free H₂O. Wipe off the RNase-free H₂O. Now, the instrument is ready for the measurement.
      3. Load 1-2 μL of the RNA sample and measure the RNA concentration (μg/μL).
         NOTE: The ratio of the absorbance at 260 and 280 nm (A260/280) at ~2.0 (1.9-2.2) is generally accepted as “pure” for RNA. If not processed immediately, store RNA samples at -70 ˚C and avoid the freeze-thaw cycles to keep the RNA intact.
2. Detection of dsRNA structure in the total RNA using dsRNA specific J2 antibody
   1. Prepare two 20 μL of RNA samples (200 ng/μL). One with RNase-III treatment (1 μL for 1 μg RNA, incubate at 37 °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.
   2. Use a pencil to draw grids where RNA samples will be blotted on the membrane.
   3. Spot 2 μL of the 200 ng/μL of the RNA sample onto the positively charged nylon membrane.
   4. 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 μg per blot.
      NOTE: Do not spot more than 2 μL of RNA sample on the membrane at a time.
   5. 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 °C.
   6. 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.
   7. Add the substrate (BCIP/NBT) and incubate for 5-15 min until a desired signal is visible.
   8. Stop the reaction by rinsing the membrane with ddH₂O.
   9. Dry the membrane on tissue papers and take a photograph using a smartphone (a representative result is shown in Figure 1).

2. RT-qPCR to measure the ability of HDM total RNA in stimulating lung ISGs expression

1. RNA isolation from mice lung tissues
   NOTE: Mice (female, 8-12 weeks old, C57BL/6J) were maintained under specific pathogen-free conditions.
   1. Briefly anesthetize the animal with isoflurane and administer via the intratracheal instillation with 5 μg (diluted in 80 μL PBS) of HDM RNAs treated with or without RNase III.
   2. 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.
   3. Disinfect the mouse with 70% ethanol then cut the skin starting from abdomen to the neck with a sterilized scissor.
   4. 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 μL in volume, 1.4 mm ceramic spheres).
      NOTE: The purpose of using ceramic beads is to grind whole lung tissues
   5. Freeze the lung samples by placing tubes into a liquid-N₂ container for ~10 min.
   6. Add 500 μL 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.
   7. Follow the steps 1.1.4- 1.1.7 for lung RNA isolation.
   8. Air-dry the pellet and dissolve the RNA pellet with proper amount of RNase-free H₂O (~20-30 μL).
   9. Measure the RNA concentration as described in step 1.1.8.
2. RT-qPCR to determine the ability of HDM RNA in stimulating lung gene expression.
   1. Using 100 ng/μL of RNA extracted from lung tissues as the template, perform the cDNA synthesis according to the referenced protocol²⁶.
   2. Set up an RT-qPCR reaction at 10 μL/well for a 384-well plate using cDNA generated above and the gene-specific primer pairs (Table 1 and Table 2).
   3. 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.
   4. Load the plate onto a RT-qPCR machine and start to run the RT-qPCR reaction using the thermal cycler protocol (Table 3).
   5. 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).

3. FACS analysis to determine the effects of HDM RNA on the infiltration of eosinophils in BAL and lung

1. BAL fluid collection for FACS analysis
   1. 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.
   2. Place the mouse on a platform and pin limbs with needles.
   3. Disinfect the mouse with 70% ethanol. Use scissors to cut the skin from the upper area of the abdomen to the neck.
   4. 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.
   5. 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.
   6. 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 the solution into a 15 mL tube on ice.
   7. Reload the syringe with the fresh PBS+EDTA and repeat this step 2x.
   8. 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 °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 °C for future analysis e.g., ELISA.
   9. In case there are RBCs present in the pellet due to severe lung inflammation, after removing the supernatant, add 500 μL 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 °C.
   10. Remove the supernatant and resuspend the pellet in 150 μL of FACS buffer.
   11. Transfer the 150 μL of the resuspended sample into 96-well plate and centrifuge the plate for 7 min at the speed of 500 x g at 4 °C.
   12. Quickly, invert the plate on tissue papers to collect the cells residing at the bottom of the wells.
   13. Stain the cells with antibodies in FACS buffer in the presence of 2.4G2 blocking antibody (2.5 μg / 100 μL). Incubate the plate at room temperature for 30 min in a dark place.
   14. After staining, centrifuge the plate to pellet the cells at 500 x g for 7 min at 4 °C.
   15. Remove the staining solution by inverting the plate on tissue paper then wash by resuspending with 100 μL of FACS buffer. Next, centrifuge the plate again at 500 x g for 7 min at 4 °C and remove the FACS buffer by inverting the plate on tissue paper.
   16. Resuspend samples into 150 μL of FACS buffer and transfer samples to the FACS tubes containing 350 μL of FACS buffer. Add 25 μL 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.
2. Lung tissue digestion for the FACS analysis
   1. Follow steps 3.1.1 - 3.1.3.
   2. 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.
   3. Remove the whole lungs and wash them with cold PBS. Place samples in the 1.5 mL tube containing 50 μL of lung digestion solution.
   4. 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 °C incubator for 45 min.
   5. After incubation, use the top of 1.5 mL tube to grind the lung tissues. Place a 70 µm strainers on a new 6-well plate and apply the sample through 0.22 µm filter.
   6. Transfer the filtered solution into a 15 mL tube, then centrifuge the tubes at 500 x g for 7 min at 4 °C. Aspirate the supernatant and resuspend the pellet in 1 mL of RBC lysis buffer and leave it on ice for 3 min.
   7. Transfer the sample into 1.5 mL tube and centrifuge at 500 x g for 7 min at 4 °C. Repeat 2x
   8. 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 μL of the sample into 96 well plate.
   9. Centrifuge the plate for 7 min at the speed of 500 x g at 4 °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.

4. Statistical analysis

1. Perform statistical analysis using a commercially available software.
2. Determine the p values by unpaired two-tailed Student t test for the comparison of two groups.
3. Calculate the absolute numbers of eosinophils based on reference beads (top panel) using the formula

4. Determine the p values by two-way ANOVA and Sidak’s multiple comparisons test for the comparison of more than two groups.
5. Consider a p value smaller than 0.05 as statistically significant. The p values are indicated on plots as *p <0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
   NOTE: All buffer recipes are provided in Table 5.

Results

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 (≥ 40bp). RNase III was used to digest dsRNA into 12–15 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 was analyzed by RT-qPCR (Figure 2, upper). The RNa...

Discussion

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...

Materials

List of materials used in this article

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