Type 2/Th2‐driven inflammation impairs olfactory sensory neurogenesis in mouse chronic rhinosinusitis model

Abstract Background Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is a chronic inflammatory disease often accompanied by impairment of sense of smell. This symptom has been somewhat overlooked, and its relationship to inflammatory cytokines, tissue compression, neuronal loss, and neurogenesis is still unclear. Methods In order to elucidate potential mechanisms leading to CRS in humans, we have established a type 2/T helper type 2 cell (Th2)‐mediated allergic CRS mouse model, based on house dust mite (HDM) and Staphylococcus aureus enterotoxin B (SEB) sensitization. The inflammatory status of the olfactory epithelium (OE) was assessed using histology, biochemistry, and transcriptomics. The sense of smell was evaluated by studying olfactory behavior and recording electro‐olfactograms (EOGs). Results After 22 weeks, a typical type 2/Th2‐mediated inflammatory profile was obtained, as demonstrated by increased interleukin (IL)‐4, IL‐5, and IL‐13 in the OE. The number of mast cells and eosinophils was increased, and infiltration of these cells into the olfactory mucosa was also observed. In parallel, transcriptomic and histology analyses indicated a decreased number of immature olfactory neurons, possibly due to decreased renewal. However, the number of mature sensory neurons was not affected and neither the EOG nor olfactory behavior was impaired. Conclusion Our mouse model of CRS displayed an allergic response to HDM + SEB administration, including the type 2/Th2 inflammatory profile characteristic of human eosinophilic CRSwNP. Although the sense of smell did not appear to be altered in these conditions, the data reveal the influence of chronic inflammation on olfactory neurogenesis, suggesting that factors unique to humans may be involved in CRSwNP‐associated anosmia.


| INTRODUCTION
Chronic rhinosinusitis (CRS) is a very common disease, reportedly affecting approximately 12% of the general population 1 with significant direct medical costs. CRS is defined in adults as a local inflammation of the nasal and paranasal sinus mucosae with symptoms including nasal congestion, loss of smell, rhinorrhea, and facial pain that persists for at least 12 consecutive weeks. 1 There are two main phenotypes of CRS: CRS with nasal polyps (CRSwNP), which typically has an eosinophilic component, and CRS without nasal polyps (CRSsNP), which has a neutrophilic component. 2 Although the causes of CRSwNP are still unclear, suspected pathogenic mechanisms include an innate barrier defect, primary infections that damage the mucosal barrier and promote its permeability, and dysfunction of several components of immunity. 3 Although still a matter of debate, 4 fungal colonization has been proposed to promote dysregulation of the sinonasal microbiome. Association of CRSwNP with local colonization by Staphylococcus aureus has been reported, 5 and the toxins secreted by S. aureus that act as superantigens have been shown to amplify immune responses and shift inflammation toward the type 2/T helper type 2 cell (Th2) profile, including eosinophil infiltration and activation. [6][7][8] While systemic atopy is not strongly associated with CRSwNP, elevations of IgE in nasal secretions and tissue indicate a localized allergic condition. 3 Levels of IgE antibodies to Staphylococcal enterotoxin superantigens in nasal polyp tissue are thought to be related to disease severity. [5][6][7] It is generally well accepted that the immune system plays a major role in CRS development. 2,3,9 Infiltration of innate immune cells and increased levels of pro-inflammatory chemokines and cytokines have been detected in nasal polyps of CRS patients. 3 The injured epithelium releases cytokines interleukin IL-25 and IL-33, and thymic stromal lymphopoietin (TSLP), collectively known as alarmins, which direct Th2-polarized inflammatory response by inducing the production of type 2 cytokines, IL-5, IL-4, and IL-13. 2

G R A P H I C A L A B S T R A C T
Mice intranasally exposed to house dust mite extract + staphylococcus aureus enterotoxin B develop allergic chronic sinusitis (CRS) with a local type 2/Th2 response. Type 2/Th2 cytokine release associates with eosinophil and mast cell infiltration, decreased immature OSN in the olfactory epithelium, and no loss of smell. Although sense of smell did not appear to be altered in this model, the data reveal the influence of chronic inflammation on olfactory neurogenesis Patients with CRS generally suffer from many symptoms, including nasal obstruction, congestion, nasal discharge, facial pressure and pain, headache, and reduction or even loss of smell (hyposmia or anosmia, respectively). 2 Impairment in sense of smell, one of the most troublesome symptoms in patients with CRSwNP, is correlated with disease severity and may be the first sign of disease reoccurrence. 10 This particular symptom has a substantial impact on quality of life and is often overlooked in disease management. The degree of olfactory dysfunction varies across CRS phenotypes. Clinical studies report that olfactory loss is more prevalent in CRSwNP patients than in CRSsNP patients. 11 Although impaired olfaction in CRS is associated with conductive and sensorineural components as well as local inflammation, 12,13 its exact mechanisms are still not fully understood. Nasal airflow reduction, edema-induced compression of olfactory nerves, or hypersecretion of Bowman's glands with altered ion concentrations in the olfactory mucus likely contribute to reducing the sense of smell. 12,14 It has also been suggested that inflammatory mediators could have direct neurotoxic actions on olfactory sensory neurons (OSNs). 12,13,15,16 OSNs with abnormal morphologies and potential functional defects, 17 as well as higher numbers of immature neurons, suggesting an attempt at regeneration, 18 have also been reported in CRS patients.
Because biopsies from CRSwNP patients are somewhat difficult to obtain, several animal models have been developed to study CRS, including allergy models. Ovalbumin (OVA) administration combined with or without low doses of Staphylococcal enterotoxin B (SEB) has been shown to induce eosinophil infiltration, to release type 2 cytokines from the olfactory mucosa, and to form structures that resemble nasal polyps. 19 Recently, house dust mite (HDM), a well-known allergen in humans, was used in combination with SEB in an allergic CRS mouse model to induce nasal inflammation resembling that observed in CRSwNP patients, such as infiltration of eosinophils, mast cells, and polypoid lesions. 20 However, these animal studies did not directly measure the sense of smell or the OSN turnover within the olfactory epithelium (OE).
In our present study, we used HDM and SEB as inducers of allergic CRS in mice to investigate the link between the inflammatory state and the OE structure, as well as the impact of type 2/ Th2 inflammation on olfactory function. Our data demonstrate an elevation of type 2/Th2 markers, concomitant with changes in other transcriptomic and histology markers, and a decrease in immature olfactory neuron number and renewal without affecting the sense of smell in mice.    Table 1). Expression of the chemokine eotaxin 1 was dramatically and significantly increased (10-fold; P < 0.001 compared to control group) ( Table 1). mRNA analysis of additional inflammatory markers in the HDM + SEB-treated OE compared to control OE further revealed an upward trend for the expression of alarmins (eg, IL-25 and TSLP), a significant (P < 0.001) increase in IL-13 receptor subunits (6.8-fold in IL-13Rα 2 and 1.3-fold in IL-13Rα 1 , Table 1), and a significant increase in IL-10 (1.7-fold; P < 0.001).

| MATERIALS AND METHODS
These data were corroborated by protein titration (Figure 2A   F I G U R E 3 Eosinophils and mast cell counts in (A) olfactory mucosa and in (B) respiratory mucosa. Eosinophil infiltration was evaluated using Sirius red staining and quantified by pixel count (number of red pixels/total number of pixels) and expressed as a percentage in an olfactory or respiratory area previously defined. Mast cells were stained based on IHC using mast cell tryptase antibody. Positive cells were counted as a number of cells per squared millimeter. Graphs represent median with interquartile range of eight mice (plots) for each group. Statistical differences between groups were assessed using the Wilcoxon-Mann-Whitney U test, and P values below P < 0.05 were considered statistically significant. in HDM + SEB group compared to control (P < 0.001; Table 1). In contrast, expression of the horizontal basal cell (HBC) markers TRP63 and RGS1 was slightly increased compared to controls (1.4fold and 1.6-fold, respectively).

| HDM + SEB treatment does not induce OSN dysfunction and loss of smell
The heptanal-evoked electro-olfactogram (EOG) characteristically displayed a fast depolarization of approximately 1 mV in amplitude followed by a slow and progressive repolarization lasting about 10 seconds ( Figure 5A). Electro-olfactograms amplitudes observed in each group were not significantly different (P = 0.4536) overall heptanal doses tested ( Figure 5B).

After 22 weeks of allergen treatment, a buried food test was
performed and the median latency to discover the hidden food in HDM + SEB-treated mice group was not significantly different compared to control mice (P = 0.4365) ( Figure 5C).
The performance of the mice changed over the course of the study. The latency to discover hidden food declined over time in both HDM + SEB-treated and control groups ( Figure 5C). A sham trial was performed as a negative control at Week 13, with an odorfree polystyrene pellet; mice were unable to find this odor-free pellet (latency of 900s ± 0 median absolute deviation) ( Figure 5C), suggesting that mice relied on their sense of smell to find buried pellets and that they were not overtrained to burrow and find any object regardless of its odor.

| DISCUSSION
To our knowledge, this study is the first to evaluate the impact of In our present study, we combined and adapted the mouse models developed by Kim et al 19 and Khalmuratova et al. 20 In order to induce a robust type 2/Th2 inflammatory profile, the impact of several factors (eg, mouse strain, type of allergen, anesthesia, sex, age, and stress 21 ) that could modulate allergy development following airway exposure of nonsensitized laboratory rodents to antigen was considered in our experimental procedure. BALB/cByJ female mice were chosen because it has been established that they have a more pronounced type 2/Th2 response in dermatitis or airway allergy inflammation models. 22,23 However, the impact of allergen exposure regimen (dose and duration) on tolerance, a reaction that can occur during a long period of continuous allergen administration, was more difficult to ascertain. To decrease tolerance, we introduced an interruption in the allergen (HDM) administration and monitored IL-10 mRNA levels, which are known as a tolerogenic marker. However, after the immunization pause, IL-10 mRNA levels were still up-regulated, while type 2/Th2 markers remained elevated. These data are in good agreement with those of a long-term study by Kim et al.,24 where mice treated with OVA + SEB presented increased type 2/ Th2 inflammation markers despite a twofold increase in IL-10 cytokine levels, suggesting an incomplete immune tolerance mechanism.
House dust mite is an allergen rarely used to induce allergic CRS in animals, but, as shown by Khalmuratova et al 20 its use may represent an improvement of CRS models, as HDM induces a more robust inflammatory profile than OVA, enhances the infiltration of mast cells known to be involved in the development of CRSwNP, and is more relevant to human disease than OVA. 2,25 In their HDM CRS murine model study, 20 Khalmuratova et al reported an elevation of total serum IgE, as well as increased eosinophil and mast cell numbers in the nasal mucosa. Further describing this model, our transcriptomic analysis showed an increase in inflammatory markers related to type 2/Th2 inflammation in the OE (Table 1), including IL-4, IL-13, and IL-5 mRNA levels. These results were confirmed at the protein level. In contrast, no change in the level of these three cytokines was observed in the serum. These results strongly suggest that our CRS mice model presents a type 2/ Th2 inflammatory profile restricted to the nasal cavity. Focusing on the OE and combining two different stimuli (protease activity with HDM and bacterial protein with SEB), we found a strong increase in IL-33 receptor (ST2) mRNA and a slight increase in IL-25 mRNA without affecting TSLP and IL-33 mRNA levels (Table 1). In our model, the cellular sources of cytokine release have yet to be determined. Recently, in a short-term (3 months) mouse model where CRSwNP was induced by OVA and SEB, a correlation between the up-regulation of IL-25 and TSLP was observed, suggesting that IL-25 could drive TSLP release, leading to type 2/Th2 inflammation. However, this result was not reported in a long-term (6 months) model. 24 These observations pointed to these innate inflammatory markers as participants in the early stages of the disease that drive inflammation toward type 2/Th2 response. It is now clearly established in the literature that innate inflammation markers (eg, alarmin molecules such as IL-25, TSLP, and IL-33) are involved in the generation of CRS. 9,26 In our present study, the structure of the OE was similar when comparing HDM + SEB-treated and control groups, while previous studies have reported differences. 20,24 The thickness of the OE was unchanged, in contrast to the data shown by Khalmuratova et al. 20 This discrepancy could possibly be due to the fact that we measured neurons. 28 Reduced expression of GAP43 has been associated with impaired development and maturation of olfactory neurons in severe neurotoxic or inflammatory conditions. 16,29 Elevations in TNF release are known to induce apoptosis of immature and mature OSNs. 29,30 However, in our present study, we did not observe any change in TNF mRNA ( performance in an olfactory test. Although human and murine olfactory mucosa cytoarchitectures are similar, their relative surfaces and cell subpopulation ratios display some differences. 44 For example, the total number of olfactory neurons is much higher in mice than in humans 45 and could explain why olfactory function in rodents appears to be more resistant to inflammatory challenges.
In conclusion, our study presents a mouse model of allergic CRS describing for the first time a decrease in immature olfactory neurons associated with a type 2/Th2 response within the olfactory area. Our findings strengthen the emerging view that olfactory loss may not only result from physical features, such as mechanical nasal obstruction, but also involve an inflammatory component. In agreement with and reinforcing previously published data on the potential impact of inflammatory cytokines on OSN regeneration, these results also raise the question of a potential toxicity of type 2/Th2 inflammatory cells on olfactory signal transmission, potentially opening new perspectives to explain human olfactory dysfunction in CRSwNP.

ACKNOWLEDG MENTS
The authors would like to thank Isabel Lefevre for a critical reading of the manuscript and Valérie Martin for her statistical assistance.