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 (HD....

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 allerg....

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