Diesel exhaust particles increase nasal symptoms and IL-17A in house dust mite-induced allergic mice

Diesel exhaust particles (DEPs), traffic-related air pollutants, are considered environmental factors adversely affecting allergic diseases. However, the immunological basis for the adjuvant effects of DEP in allergic rhinitis (AR) remains unclear. Therefore, this study aimed to investigate the effect of DEP exposure on AR using a mouse model. BALB/c mice sensitized to house dust mite (HDM) were intranasally challenged with HDM in the presence and absence of DEP. Allergic symptom scores, serum total and HDM-specific immunoglobulins (Igs), eosinophil infiltration in the nasal mucosa, cytological profiles in bronchoalveolar lavage fluid (BALF), and cytokine levels in the nasal mucosa and spleen cell culture were analyzed. Mice co-exposed to HDM and DEP showed increased allergic symptom scores compared with mice exposed to HDM alone. Reduced total IgE and HDM-specific IgE and IgG1 levels, decreased eosinophil infiltration in the nasal mucosa, and increased proportion of neutrophils in BALF were found in mice co-exposed to HDM and DEP. Interleukin (IL)-17A level was found to be increased in the nasal mucosa of the co-exposure group compared with that in the HDM-exposed group. The levels of IL-4, IL-13, interferon-γ, IL-25, IL-33, and TSLP expression showed no difference between the groups with and without DEP treatment. Increased expression of IL-17A in the nasal mucosa may contribute to DEP-mediated exacerbation of AR in HDM-sensitized murine AR model.

. Symptom scores. (A) Nasal rubbing, (B) sneezing, and (C) total symptom score of each group. Data are expressed as mean ± standard error mean. *p < 0.05, **p < 0.01. www.nature.com/scientificreports/ The cellular profiles in bronchoalveolar lavage fluid. Figure 4 shows the effect of DEP treatment on the cellular profiles in bronchoalveolar lavage fluid (BALF). The HDM and DEP co-exposure group showed a significant increase in the proportion of neutrophils in the BALF as compared with the PBS, DEP, and HDMexposed groups (p value ≤ 0.001, 0.028, and 0.015, respectively). The proportion of eosinophils in the BALF showed no difference between the groups.
Cytokines in nasal mucosa. The mRNA levels of the inflammatory cytokines (Fig. 5) in the nasal mucosa were determined. Although the IL-4 showed significantly higher levels in the HDM-exposed group and the HDM and DEP co-exposed group as compared with the PBS and DEP-exposed groups, the HDM and DEP co-exposed group showed lower levels than the HDM-exposed group. However, the difference was statistically insignificant (p value = 0.151). IL-13 showed higher levels in the HDM-exposed group compared to the HDM and DEP co-exposed group; however, the difference was not significant (p value = 0.310). The mRNA levels of IFN-γ decreased in the HDM-exposed and the HDM and DEP co-exposed group. The concentration of IL-17A showed significantly higher levels in the DEP-exposed and the HDM and DEP co-exposed groups as compared with the PBS group (all p value = 0.008). In addition, the concentration of IL-17A were higher in the HDM and DEP co-exposed mice group compared to mice exposed only to HDM, which, however, did not reach statisti-  . Differential counts in BALF. Significantly increased proportion of neutrophils in the BALF is seen in the HDM and DEP co-exposed group (group D). *p < 0.05, ***p < 0.001. Cytokines in spleen cell culture. Figure 6 shows the levels of systemic cytokines from spleen cell culture.

Scientific Reports
The levels of IL-4 and IFN-γ showed no difference among the groups. IL-17A level was significantly increased in the DEP-exposed group compared with that in the HDM-exposed group (p value = 0.032). However, there was no difference found between the HDM-exposed group and the HDM and DEP co-exposed group (p value = 0.421). Interestingly, splenocytes from mice co-exposed to HDM and DEP demonstrated a significantly higher level of IL-25 secretion compared with mice exposed only to PBS, DEP, and HDM (all p values = 0.008).

Discussion
This study evaluated the effect of co-exposure to DEP, a major airborne fine particulate pollutant, on the HDMsensitized AR mouse model. DEP co-exposure was associated with increased AR symptoms. Histological analysis showed that eosinophil infiltration was decreased in the nasal tissue and the neutrophil infiltration was increased in the BALF, upon co-exposure to DEP. Furthermore, increased IL-17A levels in nasal mucosa and IL-25 levels in the spleen cell culture were identified with concomitant exposure to DEP and HDM. Recently, epidemiological evidence has extensively linked exposure to DEPs with development, progression, and exacerbation of AR [14][15][16] . The association between air pollution and increased risk of allergic disease might contribute to the dramatic increase in the incidence of allergic airway disease in industrialized countries [17][18][19] . The Cincinnati Childhood Allergy and Air Pollutions Study, a longitudinal prospective study, investigated DEP exposure in children and reported an increased prevalence of wheezing in the exposed children 20 . Furthermore, concomitant DEP and HDM exposure in murine models have been proven to enhance allergic responses [21][22][23] . Consistent with previous studies, this study showed that co-exposure to HDM and DEP exacerbated allergic symptoms. In addition, the daily symptoms observed in the HDM and DEP co-exposed group showed higher scores than the HDM-exposed group on all days, starting from the first day of the nasal challenge (data not shown).
However, the immunological mechanisms by which DEPs contribute to the exacerbation of AR remain poorly understood. A number of biological mechanisms have been proposed, including enhancement of T H 2 inflammation, endotoxin-mediated damage, free radical production, oxidative stress, and the covalent modification of important cellular biomolecules 24 . Previous studies have indicated that co-exposure to an allergen and DEP could enhance T H 2 responses with upregulated IgE production and eosinophilia 21,23 . Brandt et al. reported that repeated co-exposure to both HDM and DEP induced a significantly stronger effector T H 2 response, characterized by increased BALF levels of T H 2 cytokines and eosinophils 15 . Furthermore, excess production of reactive oxygen species (ROS) caused by inhalation of air pollutants has been shown to induce oxidative stress and result in allergic response [25][26][27] . Fukuoka et al. reported that DEP treatment disrupts tight junctions and impairs barrier function in the nasal epithelial cells; thus, individuals exposed to DEP become predisposed to allergen-induced rhinitis 22 .
Recently, the discovery of Th17 cells has added additional layers of complexity to the older T H 1/T H 2 balance paradigm of AR pathogenesis 28 . IL-17A, a key cytokine of T H 17 cells, is a proinflammatory cytokine, and is known to induce tissue neutrophilia [29][30][31] . IL-17A and Th17 cells have been associated with neutrophilic inflammation www.nature.com/scientificreports/ and more severe asthma phenotypes, and have been correlated with the exacerbation of inflammation and the severity of airway hyperresponsiveness 32-34 . Brandt et al. have demonstrated that IL-17A contributes to asthma exacerbation and that co-exposure to HDM and DEP increases IL-17A producing T-cells 35 . They also reported that children with allergic asthma when exposed to high levels of DEP had nearly six times higher levels of serum IL-17A compared with children exposed to low levels of DEP 35 . The present study demonstrated that coexposure to HDM and DEP significantly increased IL-17A level in nasal mucosa compared with mice exposed to HDM alone. In addition, the decreased eosinophil infiltration seen in histological analysis of nasal mucosa, and increased proportion of neutrophils seen in BALF analysis can be explained by increased IL-17A levels in nasal mucosa.
In the present study, systemic cytokines obtained via spleen cell culture showed increased levels of IL-25 (also known as IL-17E) upon DEP co-exposure. Recently, IL-25 was reported to have critical roles in promoting T H 2-mediated inflammation [36][37][38] . IL-25 seems to increase in cultured splenocytes, since local DEP stimulation was added to an existing HDM-induced AR mouse model formed through the intraperitoneal and nasal challenge in our study. However, the increased secretion of IL-25 and the specific role of IL-25 in DEP-treated HDM-sensitized AR mice should be explored by further research.
The present study have several inherent limitations. First, even though the murine model is well-established, it cannot fully reproduce human AR. Second, it is proposed that the amount and type of allergen are important when evaluating the effect of DEP on AR. The relationship is complex and depends on the timing and duration of the exposure, as well as the dose and co-exposures 39 . The present study developed a mouse model with exposure to a clinically relevant allergen, HDM, and DEPs. HDM is one of the major inhalant allergens, and some studies have estimated that 10-20% of the population of any given country is allergic to HDM [40][41][42] . Further detailed studies are required to clarify the effect of DEP on AR using different allergens and various conditions of AR. Third, the immunological data were slightly T H 2 prone in the PBS and DEP groups, since all mice were sensitized to HDM using intraperitoneal injections on days 0 and 7. However, under this study's more identically controlled conditions, it could show more specific effects of DEP. Finally, BALF analysis suggested the possibility of an association between Th17 and increased neutrophil proportion; however, there was no evidence of neutrophils in the nasal tissue.
In conclusion, this study investigated the effect of DEP on HDM-sensitized AR in a mouse model. Particularly interesting finding was the increased expression of IL-17A in the nasal tissues instead of T H 2 pathway. These findings can provide a new insight into the relationship between DEP and HDM-induced AR.

Methods
Mice. Six-week-old female BALB/c mice, weighing 16-19 g, were bred in pathogen-free conditions. All animal experiments in the present study followed the guidelines and ethics of the Institutional Animal Care and Use Committee (IACUC) of the Biomedical Research Institute of Seoul National University Hospital and were approved by the committee (IACUC number: 19-0014-S1A0). The study was carried out in compliance with the ARRIVE guidelines. www.nature.com/scientificreports/ Induction of allergic rhinitis in the murine model. The mice were divided into four groups, each consisting of 10 mice, as follows: Group A, PBS-exposed group; Group B, DEP-exposed group; Group C, HDMexposed group; and Group D, HDM and DEP co-exposed group. Allergen sensitization and challenge for the development of HDM-sensitized AR murine model are summarized in Fig. 7. Briefly, the mice were sensitized with an intraperitoneal injection of 100 μg HDM (Stallergenes Greer, Antony, France; #XPB82D3A25, D. pteronyssinus) complexed with 2 mg of aluminum hydroxide (alum) in 300 μL of PBS on days 0 and 7. The mice were then given 10 μg HDM extract (composed of 3.4 μg of protein, 0.086 μg of Der p 1, and 121.8 EU of endotoxin) in 20 μL of 0.05% Tween 80-PBS intranasally for 7 consecutive days starting day 14 to day 20. The mice in the PBSexposed group were injected with HDM intraperitoneally and were intranasally challenged with 20 μL of 0.05% Tween 80-PBS instead of HDM on the same schedule. After sensitization with HDM, the mice in DEP-exposed group were intranasally administered with 100 μg DEP (National Institute of Standards & Technology, Gaithersburg, MD, USA; standard reference material 2975) suspended in 20 μL of 0.05% Tween 80-PBS on days 11-20 without HDM. The mice in the HDM and DEP co-exposed group were administered with DEP intranasally on days 11-20, and with HDM on days 14-20. HDM was given 2 h after administration of DEP on days 14-20.
Symptom scores. The frequencies of nasal rubbing and sneezing behaviors were recorded by blinded observers during a 15-min period to evaluate early allergic responses on day 20, after the last intranasal challenge in each group. Total symptom score was calculated as the sum of nasal rubbing and sneezing. Mice were then sacrificed 24 h after the last challenge.
Serum levels of total and HDM-specific immunoglobulins. Serum samples were collected from mice at the time of sacrifice. Serum samples were stored at − 80 °C until further use. Serum levels of total IgE and HDM-specific IgE, IgG 1 , and IgG 2a were measured by enzyme-linked immunosorbent assay (ELISA) as described previously 43,44 . The total IgE levels were analyzed by coating the plates overnight with anti-mouse IgE capture monoclonal antibody (mAb) (BD PharMingen, San Jose, CA, USA) at 4 °C. The plate was washed with PBST (PBS containing Figure 7. Experimental protocol. BALB/c mice were sensitized with intraperitoneal injection of HDM and alum on days 0 and 7. The mice in group A were intranasally challenged with PBS for seven consecutive days from day 14 to day 20. The mice exposed to DEP alone (group B) were treated with nasal instillation of DEP on days 11-20 without HDM. The mice in group C were intranasally challenged with HDM on days 14-20. The mice co-exposed to HDM and DEP (group D) were treated intranasally with DEP on days 11-20 and HDM on days 14-20 (2 h after administration of DEP). On day 20, the frequencies of nasal rubbing and sneezing behaviors were recorded for 15 min after the last intranasal challenge. Mice were sacrificed 24 h after the final challenge. www.nature.com/scientificreports/ 0.05% Tween-20) three times and nonspecific antigen-antibody reactions were blocked by incubation with 300 μL of 3% bovine serum albumin (BSA) per well for 1 h at room temperature. Serum samples were added to the 96-well plates along with purified mouse IgE isotype (BD PharMingen) used as a standard, and the plates were incubated for 3 h at 4 °C. The total IgE was detected by adding anti-mouse IgE-horseradish peroxidase (HRP) (#1130-055, Southern Biotechnology, Birmingham, AL, USA) to the plates. The HDM-specific IgE, IgG 1 , and IgG 2a were analyzed by coating the plates with 100 μg/mL HDM at 4 °C overnight. The plates were washed three times with PBST, and nonspecific antigen-antibody reactions were blocked by incubation with 300 μL of 3% BSA per well for 1 h at room temperature. Serum samples were added to the plates and incubated for 2 h at room temperature. The HDM-specific IgE, IgG 1 , and IgG 2a were detected by incubating each plate with biotin rat anti-mouse IgE, IgG 1 , and IgG 2a (BD PharMingen, San Jose, CA, USA). The plates were then washed three times and incubated with Streptavidin-HRP (BD PharMingen) for 30 min at room temperature. The reactions were developed using 3,3′,5,5′-tetramethylbenzidine (TMB) peroxidase substrate (Seracare life science, Milllford, MA, USA) and terminated by adding 1 M HCl. Optical density (OD) was measured in a microplate reader at 450 nm.
Evaluation of histological findings in the nasal mucosa. The nasal histology of the mice was evaluated by fixing the heads of five mice in each group with 10% formaldehyde solution. The nasal tissues were decalcified with ethylenediaminetetraacetic acid (EDTA) solution, embedded in paraffin, sectioned at 4 μm thickness, and stained with Sirius red to visualize eosinophils. The infiltrating eosinophils were counted in five fields of the nasal septal mucosa by a single-blinded observer under a light microscope (400× magnification). Eosinophils were morphologically defined by the presence of eosinophilic granules in the cytoplasm and the presence of a two-lobed nucleus 45 .

Analysis of bronchoalveolar lavage fluid.
The effect of DEP on the HDM-induced recruitment of inflammatory cells was evaluated by measuring the proportion of macrophages, lymphocytes, neutrophils, eosinophils, and basophils in the BALF. The trachea was exposed after the sacrifice on day 21 and was cannulated with a 22-gauge catheter. The lungs were lavaged with 1 mL PBS, and the BALF was collected. The BALF was centrifuged at 1800 rpm at 4 °C for 10 min and the pellets were resuspended in PBS to measure the differential cell counts in the BALF. The differential cell count was determined by centrifuging the BALF at 1000 rpm at room temperature for 5 min using cyto-centrifugation (Cytopro 7620, Wescor, Logan, UT, USA), followed by staining using a Diff-Quik stain (Sysmex, Chuo-ku, Kobe, Japan), and fixation on slides. The relative numbers of different types of leukocytes were determined from the slides using a cell counter. Data were expressed as the ratio of the specific cell number to the total cell number. Measurement of cytokines in the spleen cell culture. Cytokine ELISA was performed as previously described 44,46 . Briefly, spleen single-cell suspensions were plated in 24-well cell culture plates at a final concentration of 5 × 10 6 cells/well using RPMI 1640 containing 10% fetal bovine serum (FBS) supplemented with 100 units/mL penicillin and 100 μg/mL streptomycin (Gibco, Grand Island, NY, USA). The cells were incubated in a CO 2 incubator at 37 °C for 72 h and stimulated with HDM for 72 h. The culture supernatant was collected and stored at − 80 °C until cytokines were measured. Cytokine levels in the culture supernatant were assayed using a DuoSet ELISA kit (R&D Systems) according to the manufacturer's protocol. The concentrations of IL-4, IFN-γ, IL-17A, and IL-25 were determined by interpolation from a standard curve after measuring the OD at 450 nm, and the data were expressed in pg/mL.

Statistical analysis.
The data are presented as means ± standard error of the mean (SEM). The statistical analyses used in this study include the Kruskal-Wallis test and the Mann-Whitney U test with the 2-tailed test for unpaired comparisons. Kruskal-Wallis tests were used to establish significant intergroup variability when comparisons were made between the groups. Mann-Whitney U tests (2-tailed) were then used for betweengroup comparisons. Statistical significance was considered when p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Error bars represent the SEM. Statistical analysis was performed using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA).

Data availability
All the data generated and/or analyzed during the current study are included in this article and are available from the corresponding author on reasonable request.