House Dust Mite‑Induced Asthma Models: Choosing Between Der p and Der f—Standardized Protocols for In‑vitro Cellular and In‑vivo Murine Experiments

For studies on glucocorticoid‑resistant severe asthma, Dermatophagoides pteronyssinus (Der p, house dust mite) is the preferred inducer. For investigations of airway fibrosis and remodeling, Dermatophagoides farinae (Der f, storage mite) should be adopted. A 1:1 mixture of both mites recapitulates the clinical scenario of dual‑mite sensitization. Inappropriate mite selection may lead to inconsistent phenotypic readouts and biased conclusions. This manuscript elaborates the full in‑vitro/in‑vivo experimental workflows, biological distinctions between Der p and Der f, and critical troubleshooting notes to facilitate successful model establishment.

1 Rationale for House Dust Mite (HDM) Models: Superior Clinical Relevance versus OVA Models

Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f) represent the major indoor allergens affecting 85% of patients with allergic asthma worldwide. HDM‑extract‑based experimental models concurrently activate innate immunity as well as adaptive Th2/Th17 immune responses, recapitulating key pathological hallmarks of human asthma including eosinophilic‑neutrophilic mixed inflammation, airway remodeling, epithelial senescence, and glucocorticoid resistance. These features confer notable advantages over the artificial ovalbumin (OVA) model.

Mechanistic overview:

• ① Chitin exoskeleton → triggers innate immunity via TLR2/CLR pattern‑recognition receptors;
• ② Fecal pellets plus lipopolysaccharide (LPS) → exert intrinsic natural adjuvant activity without exogenous supplements;
• ③ Immunodominant epitopes from Der p/Der f → drive adaptive Th2‑type immune responses.

The synergistic actions of above components enable sensitization in the absence of aluminium‑based adjuvants, which underpin the superior clinical resemblance of HDM‑induced asthma models.

2 Applicable Scenarios for Three Major Experimental Models

3 In‑vitro Cellular Model Using Primary Human Nasal Epithelial Cells (HNEpC)

3.1 Key Reagents

• Cells: Primary human nasal epithelial cells (HNEpC, PromoCell Cat. No. C‑1260)
• Stimulants: Der p extract (abs47039044); Der f extract (abs47039045). For combined group: final concentration of 15 μg/mL Der p plus 15 μg/mL Der f.
• Culture medium: Complete airway epithelial cell medium supplemented with penicillin‑streptomycin
• Assay kits: LDH cytotoxicity kit (abs580236), PrestoBlue cell viability assay, inflammation‑targeted ELISA kits, qPCR primers for autophagy / UPR / MMP / senescence‑associated genes.

3.2 Experimental Workflow

1. Cell seeding and cultivation: Seed HNEpC and culture until reaching ~80% confluence (approximately 3 days); ensure intact cell morphology and minimal floating dead cells.

2. HDM stimulation: Assign experimental groups: blank control, Der p, Der f, combined HDM groups. Apply a unified final HDM concentration of 30 μg/mL for 24 h stimulation.

3. Sample collection:

• Cell culture supernatant: measure IL‑1β, IL‑6, IL‑8, CXCL1, CCL2, VEGF and IL‑10 by ELISA.
• Cell pellets: perform qPCR for autophagy‑related genes (ATG3/5/7, Beclin1), UPR‑related genes (ATF6, CHOP, IRE1), MMP family genes (MMP2/9/12), and senescence‑related genes (p21/p16/p27).
• Cellular functional readouts: LDH assay (cytotoxicity), PrestoBlue assay (cell viability), β‑galactosidase staining (cellular senescence).

3.3 Criteria for Valid In‑vitro Model

All of the following endpoints should be satisfied:

1. Decreased cell viability and elevated LDH release (epithelial barrier damage);
2. Elevated pro‑inflammatory mediators (IL‑1β, IL‑6, CXCL8, CXCL1) accompanied by reduced IL‑10;
3. Significant transcriptional up‑regulation of genes linked to autophagy, UPR, MMPs and cellular senescence;
4. Increased proportion of β‑galactosidase‑positive senescent epithelial cells.

4 In‑vivo Murine Asthma Models: 36‑day Comprehensive Protocol versus 5‑week Accelerated Protocol

4.1 Basic Reagents and Animals

• Animals: 5‑6‑week‑old female BALB/c mice (female mice exhibit stable inflammatory responses).
• Challenge reagents: Der p or Der f extracts mixed with aluminium hydroxide adjuvant.
• Administration route: Intraperitoneal sensitization followed by intranasal challenge (classical two‑phase protocol).
• Specimens for analysis: Bronchoalveolar lavage fluid (BALF), lung tissues, serum.

4.2 36‑day Comprehensive Protocol (Recommended for Airway Remodeling Studies)

4.3 Terminal Detection Endpoints

Interpretation of pulmonary‑function parameters: Rrs (total airway resistance) and Rn (central‑airway resistance) indicate airway obstruction; G (tissue damping) reflects inflammatory viscoelastic alterations; H (tissue elastance) serves as the core marker for pulmonary fibrosis and tissue stiffness, which represents a key phenotypic feature prominent in Der f‑challenged animals.

4.4 5‑week Accelerated Protocol (For Preliminary Probiotic / Drug Screening)

Omit intra‑peritoneal sensitization. Perform intranasal HDM challenge (25 μg per mouse) for 5 consecutive days per week, for a total of 5 weeks.

For intervention groups, intragastric administration of test agents commences 7 days prior to model establishment and continues throughout the experimental period.

Terminal readouts include BALF inflammation, histological scoring, serum IgE and gut microbiota profiling (ideal for gut‑lung axis investigations).

4.5 Validation Criteria for Valid In‑vivo HDM Asthma Model

All four dimensions should be fulfilled:

1. Airway hyperresponsiveness: significantly elevated Rrs, Rn, G and H relative to vehicle controls;
2. BALF profiles: increased total cell count, elevated eosinophils, neutrophils and lymphocytes, with reduced macrophage proportion;
3. Histopathology: robust peribronchial inflammatory infiltration, PAS‑positive mucus overproduction, up‑regulated remodeling‑associated proteins (fibronectin, MMPs);
4. Molecular signatures: elevated pulmonary IL‑13 / IL‑33, increased serum total IgE and HDM‑specific IgG1.

5 Biological Differences between Der p and Der f: Selection Decision‑Tree

According to the parallel‑control study reported by Wang et al. (2020), despite shared capacity for asthma induction, Der p and Der f produce distinct pathological phenotypes that heavily influence experimental conclusions.

5.1 Core Phenotypic Differences

5.2 Experimental‑model Selection Decision‑Tree

Selection mnemonic: Use Der p for glucocorticoid‑resistance studies; choose Der f for fibrotic‑remodeling assays; adopt 1:1 mixture for dual‑sensitization scenarios; either mite is acceptable for in‑vitro cellular assays.

6 Troubleshooting: Six Quality‑control Principles for HDM‑induced Asthma Models

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