Physiologic Response to Chronic House Dust Mite Exposure in Mice is Dependent on Lot Characteristics

Capsule Summary

Physiological responses to HDM extracts are variable. To increase the transparency and reproducibility of published work, lot numbers, concentration of extract components, and methods for dose normalization need to be thoroughly reported.

To the Editor:

Multiple models of allergic airway inflammation using house dust mite (HDM) extract have been developed to study experimental asthma in mice. Acute exposure models develop airway hyper-responsiveness (AHR), T helper type 2 (T2)-mediated airway inflammation, and mucus secretion (1, 2), but airway remodeling with peribronchial collagen deposition requires chronic exposures (3–5). Presently there are no standard exposure methodologies, as each model varies with respect to the species, duration, and/or dose of HDM used and different exposure protocols affect phenotypic outcomes (4). In addition, phenotypic outcomes depend on the HDM extract source (6). Lack of reproducible physiological responses to allergen extracts has long been known and is well documented in mice and humans after cutaneous exposure (7, 8), but has been less well detailed in models of allergen inhalation.

The National Institutes of Health stresses the importance of scientific rigor and transparency for enhancing reproducibility in research. Among several key areas, they highlight authentication of biological and/or chemical resources. While the majority of recently published manuscripts (Pubmed; 2016–2018) using murine chronic HDM exposure cite the HDM source, fewer than 11% provide the extract lot number or describe its components in detail; and almost 40% of papers did not cite the species of HDM extract (see Table E1; Online Repository). Furthermore, HDM dose normalization in these studies can be based on protein content, antigen content, or dry extract weight. As HDM extracts are prepared from live mites, and the allergen and endotoxin content can differ between preparations, we hypothesized that the response to extracts may differ amongst lots even when prepared from the same commercial supplier and used in the same exposure protocol.

As proof of concept, we started with two lots of HDM (HDM0 and HDM1) with similar concentrations of total protein, Der P1, and endotoxin units (Greer Laboratories). Respiratory mechanics were evaluated after either acute or chronic administration of HDM. Mechanics parameters were then fit to two different mathematical models of the lung; the Single Compartment Model, and the Constant Phase Model, to determine total respiratory system and compartmentalized responses, respectively (see supplemental methods). When administered acutely (intranasal 100µg 1x/wk for 3wk), both lots increased respiratory system resistance (R_rs) and elastance (E_rs) in response to methacholine (MCh) challenge (Figure 1A). Interestingly, after chronic exposure (oropharyngeal aspiration 100µg 3x/wk for 5wk), HDM0 challenge resulted in AHR to MCh, while HDM1 did not (Figure 1B). Due to the striking differences in AHR, we evaluated respiratory mechanics after chronic exposure to several HDM lots containing different concentrations of total protein, antigen, and endotoxin (see Table E2, Online Repository). All lots induced AHR in the acute model (see Figure E1, Online Repository). However, after chronic exposure, only half of the lots caused AHR (Figure 1C); moreover, the response magnitude and type were not equivalent. HDM0 and HDM5 increased total R_rs and E_rs. However, using the Constant Phase Model, HDM0 resistance primarily localized to the central airways (R_n), while HDM5 localized to the peripheral airways and tissue (G). HDM3 induced significantly increased AHR in the Single Compartment Model but failed to show AHR in the Constant Phase Model. Furthermore, the airway response to chronic HDM exposure did not appear to correlate with either Der P1 or endotoxin levels, as the three lots that induced AHR contained different ratios of these components, and lots with similar ratios failed to develop AHR. To test if the combination of antigen and endotoxin concentrations are necessary for AHR, we normalized the dose of HDM1 to contain equal Der P1 and endotoxin content to HDM0 (HDM0→HDM1) (see Table E3, Online Repository). Chronic exposure to the normalized dose of HDM1 failed to rescue the AHR phenotype (see Figure E2, Online Repository), suggesting that other components of the extract may be responsible for the development of AHR in this model. Another possible explanation is that the different lots of HDM induced different levels of sensitization. However, to directly address this question we would need to know the levels of HDM-specific IgE prior to the challenge phase and these lots are no longer available.

Figure 1. Physiological response to acute and chronic HDM exposure.

Acute HDM challenge (A) induced significant AHR. Chronic exposure (B) to HDM0 induced significant AHR, while HDM1 did not develop AHR. Different lots of HDM induced variable effects on the development of AHR using two models of the lung (C).

^#P<0.05 vs. HDM1; *P<0.05 vs. PBS controls; n=6–12/group.

We then explored lot-dependent effects on chronic HDM-induced inflammation, allergic antibody production, and lung injury. All lots increased bronchoalveolar lavage fluid (BALF) cellularity compared to controls; however, the cell type varied greatly between groups (see Table E4, Online Repository). Extracts with “high” Der P1 content increased eosinophils, while “high” endotoxin extracts increased neutrophils when compared to “low” Der P1 or endotoxin extracts, respectively. All HDM lots increased serum IgE and BALF albumin. However, HDM5 challenge resulted in the highest serum IgE and total protein in the BALF compared to the other HDM lots (Figure 2A). Alternatively, chronic exposure to different HDM lots altered inflammatory mediator secretion (Figure 2B). For example, HDM5 increased all measured cytokines/chemokines, but interleukin-5 (IL-5) was elevated only in other high DerP1-containing extracts (HDM0, HDM2, and HDM3). Additionally, classic T2 immune response-mediated allergic asthma markers (eosinophilic airway inflammation, serum IgE, total protein, and IL-5) correlated with Der P1 concentration in each HDM dose (see Figure E3, Online Repository). Alternatively, the endotoxin content only associated with the percentage of BALF neutrophils (data not shown). These results are consistent with the development of Th1 and Th2 responses after exposure to endotoxin and allergen challenge, respectively.

Figure 2. Chronic HDM exposure results in the development of allergic asthma.

Markers of allergic asthma and lung injury (A) and pro-inflammatory cytokines/chemokines (B) were measured 48h after the final HDM challenge. Lung tissue inflammation, mucus secretion, and peribronchial collagen deposition (C) were determined from stained lung sections.

*P<0.05 vs. PBS controls; ^#P<0.05 vs. all other groups; n=6–12/group.

Histological evaluation of the lung tissue after chronic HDM exposure demonstrated significantly increased peribronchial inflammation and mucus secretion, regardless of the lot number compared to saline-challenged control animals (Figure 2C). However, most lots did not induce airway fibrosis. Only HDM5 challenge resulted in increased peribronchial collagen associated deposition, consistent with the elevated inflammation and robust AHR seen in these animals.

Commercially available samples of individual D. pteronyssinus HDM extracts typically report the total protein, Der P1 allergen, and endotoxin levels. However, extracts can also contain unreported levels of other Der P family allergens, as well as proteases, chitins, and β-glucans, which can stimulate innate immune system responses (9). While markers of allergic inflammation correlated with Der P1 content and percent neutrophilia with endotoxin content, AHR and collagen deposition did not correlate with either DerP1 or endotoxin content. Additionally, normalizing the extract dose to contain equal concentrations of Der P1 and endotoxin did not reciprocate inflammatory or AHR responses. These data suggest components other than Der P1 or endotoxin within the HDM extract lots may contribute to chronic HDM airway responses, and highlight an important area for future mechanistic studies.

In conclusion, while allergen extracts are becoming a powerful tool for studying allergy, asthma, and airway disease in animal models, our data demonstrates that these extracts are complex mixtures that can result in variable phenotypic responses, and studies should proceed with caution. Due to the variability of components within the extracts, it is important to describe both the lot characteristics as well and the methods used to normalize dosing so that concentrations of specific components can be calculated and compared between studies. Additionally, thorough characterization of additional lot components may be needed for mechanistic studies. Rigorous reporting of these parameters will not only provide transparency to the research, but will also allow for more accurate comparison of results and reproducibility of published work.