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Morphometric analysis of inflammation in bronchial biopsies following exposure to inhaled diesel exhaust… Hosseini, Ali; Hirota, Jeremy A.; Hackett, Tillie L.; McNagny, Kelly M.; Wilson, Susan J.; Carlsten, Chris Jan 13, 2016

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RESEARCH Open AccessMorphometric analysis of inflammation inbronchial biopsies following exposure toinhaled diesel exhaust and allergenchallenge in atopic subjectsAli Hosseini1,2,5, Jeremy A. Hirota1,2,5, Tillie L. Hackett2, Kelly M. McNagny3, Susan J. Wilson4 and Chris Carlsten1,2,5*AbstractBackground: Allergen exposure and air pollution are two risk factors for asthma development and airwayinflammation that have been examined extensively in isolation. The impact of combined allergen and dieselexhaust exposure has received considerably less attention. Diesel exhaust (DE) is a major contributor to ambientparticulate matter (PM) air pollution, which can act as an adjuvant to immune responses and augment allergicinflammation. We aimed to clarify whether DE increases allergen-induced inflammation and cellular immuneresponse in the airways of atopic human subjects.Methods: Twelve atopic subjects were exposed to DE 300 μg.m−3 or filtered air for 2 h in a blinded crossover studydesign with a four-week washout period between arms. One hour following either filtered air or DE exposure, subjectswere exposed to allergen or saline (vehicle control) via segmental challenge. Forty-eight hours post-allergen or controlexposure, bronchial biopsies were collected. The study design generated 4 different conditions: filtered air + saline(FAS), DE + saline (DES), filtered air + allergen (FAA) and DE + allergen (DEA). Biopsies sections were immunostainedfor tryptase, eosinophil cationic protein (ECP), neutrophil elastase (NE), CD138, CD4 and interleukin (IL)-4. The percentpositivity of positive cells were quantified in the bronchial submucosa.Results: The percent positivity for tryptase expression and ECP expression remained unchanged in the bronchialsubmucosa in all conditions. CD4 % positive staining in DEA (0.311 ± 0.060) was elevated relative to FAS (0.087 ± 0.018;p = 0.035). IL-4 % positive staining in DEA (0.548 ± 0.143) was elevated relative to FAS (0.127 ± 0.062; p = 0.034).CD138 % positive staining in DEA (0.120 ± 0.031) was elevated relative to FAS (0.017 ± 0.006; p = 0.015), DES(0.044 ± 0.024; p = 0.040), and FAA (0.044 ± 0.008; p = 0.037). CD138 % positive staining in FAA (0.044 ± 0.008)was elevated relative to FAS (0.017 ± 0.006; p = 0.049). NE percent positive staining in DEA (0.224 ± 0.047) waselevated relative to FAS (0.045 ± 0.014; p = 0.031).Conclusions: In vivo allergen and DE co-exposure results in elevated CD4, IL-4, CD138 and NE in the respiratorysubmucosa of atopic subjects, while eosinophils and mast cells are not changed.Trial registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01792232.Keywords: Particulate matter, Segmental allergen challenge, Airway inflammation, GMA immunohistochemistry, IL-4,ECP, Tryptase, CD4, Neutrophil elastase, CD138 (syndecan-1)* Correspondence: carlsten@mail.ubc.ca1Department of Medicine, Division of Respiratory Medicine, Chan-YeungCentre for Occupational and Environmental Respiratory Disease, University ofBritish Columbia, Vancouver, BC V5Z 1M9, Canada2Institute for Heart and Lung Health, University of British Columbia,Vancouver, BC V6Z 1Y6, CanadaFull list of author information is available at the end of the article© 2016 Hosseini et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Hosseini et al. Particle and Fibre Toxicology  (2016) 13:2 DOI 10.1186/s12989-016-0114-zBackgroundAccording to the World Health Organization (WHO),7 million deaths were attributed to air pollution in2012, representing 1 in 8 deaths worldwide and makingair pollution the biggest single environmental health risk[1, 2]. The mortality was more than twice the 2008 esti-mates, confirming that air pollution is increasingly takinga toll on human health [2]. The WHO results are substan-tiated by numerous epidemiological studies that haveestablished a significant association between exposure toambient air particulate matter (PM) and increases in mor-tality and morbidity due to cardiovascular and respiratorydiseases [3–5].Asthma is a major public health problem and it is esti-mated that 300 million people suffer from asthma aroundthe globe, with 250,000 annual deaths and more than 2million annual emergency room visits in the U.S. [6, 7].Asthma is commonly defined as a chronic inflammatorycondition characterized by airway inflammation, reversibleairway obstruction, and increased airway responsive-ness leading to symptoms such as wheezing, coughing,shortness of breath, and chest tightness [8]. Airwayinflammation in asthmatic patients may stem from ahypersensitivity of the respiratory tract to triggers likeallergens and air pollutants resulting in accumulationof chronic inflammatory cells in the airway wall [9].Specific cures for asthma remain elusive and therefore,in order to develop new therapeutic strategies and in-form health policy on air quality, we require a greaterunderstanding of the mechanisms behind how environ-mental exposures can trigger asthma attacks [10].Toxicological studies have shown that ambient airbornePM can induce the production of cytokines and oxidantsthat initiate airway inflammation [11]. PM may have directeffects on the pulmonary system, including induction ofan inflammatory response, exacerbation of existing airwaydisease or impairment of pulmonary defense mechanisms[12]. Epidemiologic reports have indicated that there is ahigher prevalence of asthmatic and allergic symptoms inpeople who live in close proximity to major roads relativeto those in more distant locations [13–15]. Diesel exhaust(DE) is a main contributor to ambient PM air pollution[16]. It has been suggested that exposure to DE can triggerT-helper type 2 (Th2) immune responses which are directlyassociated with the development and exacerbation ofallergic asthma [17]. Consistent with observational studies,animal and human nasal models have demonstrated thatDE can act to augment allergic immune responses [18–21].The chronic airway inflammation in allergic asthma ischaracterized by activation of mast cells, type 2 innatelymphoid cells (ILC2), T cells and infiltration of activatedeosinophils and basophils [22]. Inhalation of allergen,results in Th2 cell activation and secretion of inflamma-tory cytokines such as interleukin (IL)-4, IL-5, IL-9 andIL-13, which are considered to play an important role inthe mucus hyper-secretion, thickening and contractionof airway smooth muscle in atopic asthmatic patients[22–25]. Asthma is a phenotypically heterogeneous dis-order and, over the years, many different clinical sub-types of asthma have been described [26] and classifiedby trigger or symptoms such as allergic, non-allergic,exercise-induced and cold-induced [27]. In our currentstudy, we have focused on classic allergic asthma, andthe study design and interpretation followed accordingly.It has been hypothesized that environmental allergensimpose a greater effect in the presence of DE exhaustbut the exact mechanism behind this synergy is still notclear. Our study investigated the impact of DE exposureon allergen-induced airway inflammation as assessed inthe submucosa of bronchial biopsies. Our hypothesiswas that allergen exposure results in an increase in air-way inflammation in the submucosa that was synergis-tically increased with DE exposure. We used a blindedcrossover study design in atopic human subjects withsegmental allergen challenge following controlled expos-ure to either filtered air or freshly generated DE. Ourresults demonstrate that controlled allergen exposureresulted in no increases in submucosa mast cells oreosinophils, 48 h post-exposure, in either filtered air orDE exposed conditions. In contrast, combination allergenand DE exposure resulted in an increase in submucosaCD4, IL-4, CD138 and NE relative to filtered air andsaline. Our results suggest a role for CD4+ and Th2 im-mune responses in airway inflammation in response tocombination exposures that may not be observed in singleexposure studies in atopic subjects.ResultsSubject characteristicsStudy subject gender, age, height, weight, body mass index(BMI), forced expiratory volume in 1 s (FEV1), methacho-line PC20, and allergen used for segmental allergen chal-lenge are described in Table 1. The study involved samplesfrom 7 female subjects and 5 male subjects. Samples fromall 12 subjects were used for IL-4 immunostaining andmorphometric analysis. For tryptase, eosinophil cationicprotein (ECP), CD4, CD138 and neutrophil elastase (NE)staining, two bronchial biopsies did not contain sufficientsubmucosa area, resulting in only 10 subjects available forcomparison across the four experimental conditions forthese endpoints.Submucosal changes within bronchial biopsies inducedby single exposure or co-exposureTryptase and ECP are unchanged by the combination ofdiesel exhaust and allergen (DEA)Analysis of tryptase-positive staining in bronchial mucosarevealed distinct and strong granular cytoplasmic stainingHosseini et al. Particle and Fibre Toxicology  (2016) 13:2 Page 2 of 14in cells with isolated observations of extracellular staining(Fig. 1b). Quantification of the tryptase positively stainedpixels, demonstrated no differences between any experi-mental conditions (Fig. 1c) although there was a trend toincreased staining in the DEA (diesel exhaust + allergen)samples.Analysis of ECP-positive staining in bronchial mucosarevealed distinct cytoplasmic localization in cells withpervasive observations of extracellular staining suggestingeosinophil degranulation (Fig. 2b). Quantification of theECP-positive pixels, demonstrated no differences betweenexperimental conditions (Fig. 2c).Neutrophil elastase is elevated by DEAAnalysis of NE-positive staining in bronchial mucosarevealed distinct immunohistochemical localization ofelastase in neutrophils with no observations of extra-cellular staining (Fig. 3b). Quantification of the NE-Table 1 Subject characteristicsSubject Gender Age (years) Height (cm) Weight (kg) BMI FEV1 (% of pred) Methacholine PC20 (mg/mL) Positive SPT1 F 20 158 54.6 22 104 13.85 HDM2 F 31 173 70 24 113 >16 HDM3a M 32 161 68 26 123 30.74 Pacific Grasses4 F 34 157 55 22 79 0.23 Pacific Grasses5 M 27 178 70 22 105 >128 HDM6 F 25 173 73 25 117 >128 HDM7a M 27 186 85 25 107 87.64 Pacific Grasses8 F 46 165 65 24 63 >128 HDM9a F 31 146 50 23 103 0.26 Birch10 M 28 176 90 29 100 19.12 Pacific Grasses11a M 23 169 83 29 104 2.41 Pacific Grasses12 F 23 172 96 32 101 54.32 HDMMean (SD) 29 (7) 168 (11) 72 (15) 25 (3) 102 (16)M male, F female, BMI body mass index, FEV1 forced expiratory volume in one second, % of pred percentage of predicted, PC20 provocative concentration causinga 20 % fall in FEV1, SPT skin prick test, HDM house dust mite allergenaPrevious smokerACBFig. 1 Immunohistochemical staining of tryptase-positive mast cells in human bronchial submucosa tissue. a Representative 20X image of positivestaining using mAb AA1 for tryptase with positive staining in red with Mayer’s hematoxylin counterstain in blue from a subject exposed to FAA.b Zoom region (40X) highlighted in panel a black box. c Positive pixel count quantification of submucosa region for tryptase staining. Data areexpressed as mean ± SEM. n = 10 for each experimental conditionHosseini et al. Particle and Fibre Toxicology  (2016) 13:2 Page 3 of 14ACBFig. 2 Immunohistochemical staining of ECP-positive eosinophils in human bronchial submucosa tissue. a Representative 20X image of positivestaining using mAb EG2 for ECP with positive staining in red with Mayer’s hematoxylin counterstain in blue from a subject exposed to FAA.b Zoom region (40X) highlighted in panel a black box. c Positive pixel count quantification of submucosa region for ECP staining. Data areexpressed as mean ± SEM. n =10 for each experimental conditionABCFig. 3 Immunohistochemical staining of elastase-positive neutrophils in human bronchial submucosa tissue. a Representative 20X image of posi-tive staining using mAb NP57 for NE with positive staining in red with Mayer’s hematoxylin counterstain in blue from a subject exposed to FAA.b Zoom region (40X) as highlighted in black box of panel a. c Positive pixel count quantification of submucosa region for NE staining. Data areexpressed as mean ± SEM. n = 10 for each experimental condition. *p < 0.05Hosseini et al. Particle and Fibre Toxicology  (2016) 13:2 Page 4 of 14stained pixels demonstrated a significant increase (p =0.031) in staining after DEA (0.224 ± 0.047) relative toFAS (0.045 ± 0.014). There were no significant differ-ences between FAS vs. DES (0.077 ± 0.024, p > 0.999)or FAA (0.229 ± 0.069, p = 0.175) (Fig. 3c).CD138 positive cells are elevated by FAA and DEAAnalysis of CD138-positive staining in bronchial mu-cosa revealed distinct membrane staining in plasmacells (Fig. 4b). Quantification of the CD138-positive pixelsdemonstrated a significant increase in staining after DEA(0.120 ± 0.031) relative to FAS (0.017 ± 0.006, p = 0.015),DES (0.044 ± 0.024, p = 0.040), and FAA (0.044 ± 0.008,p = 0.037). CD138 positive staining in FAA was elevatedrelative to FAS (0.017 ± 0.006; p = 0.049) (Fig. 4c).CD4 positive cells are significantly elevated by DEAAnalysis of CD4-positive staining in bronchial mucosarevealed distinct membrane staining in T cells (Fig. 5b).Quantification of the CD4-positive pixels demonstrateda significant (p = 0.035) increase in staining after DEA(0.311 ± 0.060) relative to FAS (filtered air + saline [control(vehicle) for allergen]; 0.087 ± 0.018).IL-4 expression is elevated by DEAAnalysis of IL-4-positive staining in bronchial mucosarevealed distinct immunohistochemical localization of IL-4in cells with no observations of extracellular staining(Fig. 6b). Quantification of the IL-4-stained pixels, dem-onstrated a significant increase (p = 0.034) in stainingfor DEA samples (0.548 ± 0.143) relative to FAS sam-ples (0.127 ± 0.062). There were no significant differ-ences between FAS vs. DES (0.353 ± 0.088, p = 0.086)or FAA (0.426 ± 0.130, p = 0.150).Finally, Pearson correlation coefficients matrix wasperformed for each of the endpoints versus each other(Additional file 1: Tables S1 and S2). No endpoints weresignificantly correlated under DEA, suggesting potentialdistinctive biology associated with the endpoints inducedby DEA. NE and CD138 were positively correlated (r =0.76, p < 0.01) after DES. After FAA, tryptase and CD138were positively correlated (r = 0.64, p < 0.04) and ECP andIL-4 were also positively correlated (r = 0.61, p < 0.05).DiscussionTo our knowledge this is the first blinded crossoverhuman study using controlled exposures to a combin-ation of DE and allergens to investigate the lower airwayinflammatory responses in bronchial biopsies in atopicindividuals. Considerable evidence suggests that that theeffects of DE and allergens are synergistic [28]. Weprovide further evidence for synergy by demonstratingthat a combination of DE and allergen (but not DE orallergen alone) results in augmented CD4, IL-4, NE andCD138. Our results suggest that studies examining aller-gen or air pollution in isolation, including those relatedACBFig. 4 Immunohistochemical staining of CD138-positive plasma cells in human bronchial submucosa tissue. a Representative 20X image of posi-tive staining using mAb B-A38 for CD138 with positive staining in red with Mayer’s hematoxylin counterstain in blue from a subject exposed toFAA. b Zoom region (40X) highlighted in black box of panel a. c Positive pixel count quantification of submucosa region for CD138 staining. Dataare expressed as mean ± SEM. n = 9 for each experimental condition. *p < 0.05; **p < 0.01Hosseini et al. Particle and Fibre Toxicology  (2016) 13:2 Page 5 of 14A BCFig. 6 Immunohistochemical staining of IL-4-positive cells in human bronchial submucosa tissue. a Representative 20X image of positive stainingusing mAb 4D9 for IL-4 with positive staining in red with Mayer’s hematoxylin counterstain in blue from a subject exposed to FAA. b Zoom re-gion (40X) highlighted in black box of panel a. c Positive pixel count quantification of submucosa region for IL-4 staining. Data are expressedas mean ± SEM. n = 12 for each experimental condition. *p < 0.05A BCFig. 5 Immunohistochemical staining of CD4-positive T cells in human bronchial submucosa tissue. a Representative 20X image of positive stain-ing using mAb 4B12 for CD4 with positive staining in red with Mayer’s hematoxylin counterstain in blue from a subject exposed to FAA.b Zoom region (40X) highlighted in black box of panel a. c Positive pixel count quantification of submucosa region for CD4 staining. Data areexpressed as mean ± SEM. n = 10 for each experimental condition. *p < 0.05Hosseini et al. Particle and Fibre Toxicology  (2016) 13:2 Page 6 of 14to drug development, may underestimate the real-worldimpact of heterogeneous environmental exposures onairway inflammation in atopic humans [29–31]. Ourresults have implications for health policy aimed at pro-tecting air quality for vulnerable populations [32], recog-nizing that the concentration of DE 300 μg.m−3 used inour study is most relevant in the context of occupationalexposure in Western countries or the daily average levelof PM in some developing countries in the world suchas China and India [33, 34].Mast cells are known to participate in allergic inflam-mation [35]. Previous studies have shown increases thenumber of mast cells in healthy volunteers at 6 h [36]and 18 h [37] after exposure to DE. Tryptase is the mostplentiful granule constituent in mature and activatedmast cells [38], thus we stained for tryptase as it is areliable biomarker of mast cell presence and activation.Basophils are the only other cell type that express tryp-tase, but their frequency is known to be considerablylower than that of in mast cells [39] and tryptase levelsin human basophils are less than 1 % of those in tissuemast cells [40]. Our results demonstrate that 48 h fol-lowing co-exposure to DE and allergen, there was noelevations in tryptase positive cells in the submucosa.Eosinophils are another pro-inflammatory white bloodcell type that differentiates from myeloid progenitors inthe bone marrow; mature eosinophils travel throughblood vessels to mucosal surfaces throughout the body[41]. We examined ECP as a selective marker for eosino-phils, as this protein is largely restricted to these cells;there is evidence that ECP can be detected in neutro-phils [42, 43], but rarely and weakly [44]. Staining ECP(rather than MBP or EPO) as a markers of intensity ofeosinophilic inflammation of the airways in allergic dis-eases [45], allowed us to compare our results with otherDE controlled exposure studies [46, 47].Our data demonstrate that in atopic individuals, com-bination exposure (DE followed by allergen) does notimpact ECP expression in bronchial submucosa. This isconsistent with a previous controlled DE exposure studythat showed no significant increase in the count of tissueeosinophils at 18 h post-exposure [37], although studiesexamining bronchoalveolar lavage [48] and induced spu-tum [47] have shown increases at 6 h post-exposure.We chose a later time point 48 h for sampling tissuebiopsies, thus there is potential that early inflammatorycell recruitment and activation was missed due to reso-lution of the inflammatory event. A previous study utiliz-ing segmental allergen challenge was aimed to elucidatethe time course of inflammatory events in allergic airwaydiseases. They have shown that the level of tryptase inBAL increases immediately within 12 min following thesegmental allergen broncho-provocation with ragweed inallergic rhinitis patients, but this signal was resolved by48 h. In contrast, ECP levels were only increased at thelate time point [49]. This leads us to speculate that in ourtissue biopsies, ECP was released from the tissue eosino-phils and by 48 h was measureable only in BAL [50].Neutrophilic inflammation is associated with progres-sion and the development of chronic respiratory diseases,such as severe asthma [51]. Neutrophils are granulocytesand one of the first responders to the environmentalinsults and migrate to the site. In neutrophils, neutrophilelastase (NE) contributes mainly to digestion of ingestedforeign particles, chemotaxis, infiltration and tissue re-modeling by degrading connective tissue proteins suchas elastin and collagens [52]. Exposure to DEP is as-sociated with accumulation of neutrophils and it hasbeen shown that DEP can activate neutrophils andaugment the expression of NE and other mediators oftissue destruction [53, 54].We demonstrate that NE expression is increased byDEA, suggesting neutrophilic inflammation is inducedby the combined exposure to DE and allergen. Our dataare consistent with our own study of combined exposure[50], in which BAL neutrophils were increased by DEplus allergen, but contrast somewhat with previous hu-man controlled studies that reported increased neutro-phils associated with DE in various compartments atmultiple timepoints through 24 h post-exposure [55],since we showed so similar effect (assessing DES alone)at 48 h.CD138 (syndecan-1) is a cell surface proteoglycan andpredominantly is expressed on mature plasma cells andweakly in epithelial cells. CD138 is highly sensitive andspecific marker for identification of plasma cells and plasmacell differentiation. CD138 modulates cell growth, differen-tiation, adhesion and migration, thus plays important rolesin the regulation of inflammatory responses [56].Aggregation of CD138+ plasma cells were identified inpulmonary fibrosis [57] and in the lung submucosa ofsevere asthmatics with increased inflammatory lympho-cytes infiltrates [58]. Increased frequencies of CD138+IgE+ cells were detected in the lamina propria of thenasal mucosal biopsies from allergic patients [59]. Alsothe number of CD138+ IgE+ cells was positively corre-lated with the IgE serum titres in atopic individuals [60].Exposure to environmental allergens triggers Th2 cellsdifferentiation and production of IL-4 and IL-13 whichproliferate and differentiate B cells into plasma cells andswitch to IgE synthesis [61]. It has been shown thatinhalation allergen challenge significantly increases thenumber of CD138+ IgE-secreting cells in murine lungs[62]. CD138 immunostaining is proven to be an excel-lent indicative of IgE+ cells in the lung tissue [63]. CD138+cells were detectable in bronchial biopsies at 24 h butnot in BAL after segmental allergen challenge in atopicasthmatics [64].Hosseini et al. Particle and Fibre Toxicology  (2016) 13:2 Page 7 of 14We demonstrate that CD138 expression is increasedin DEA vs. FAS, DES and FAA, suggesting plasmacytosisis induced by DE added to allergen. CD138 expressionin FAA was elevated relative to FAS, suggesting theeffect of allergen challenge in submucosal plasmacytosis.Our data are consistent with a previous human nasalmodel that have confirmed that co-administration of DEparticles and allergen stimulate an increase in level ofallergen specific IgE in nasal lavage samples [21] and atrend in our own study of BAL from DE-allergen co-exposure [50].CD4+ T-cells play an essential role in adaptive allergicimmune responses. CD4 is a transmembrane glycopro-tein selectively expressed on the surface of helper T-cellsand plays an important role in the regulation of T-cellsignalling and its functional consequences [65]. Follow-ing activation, naive CD4+ T-cells differentiate into oneof the sub-types of T-helper cells: Th1 [66], Th2 [67],Th9 [68], Th17 [69], or Th22 [70], depending on thenature of antigen and the cytokines present in the sur-rounding milieu [71]. Th2 cells secrete IL-4, IL-5, IL-6,IL-9, IL-10 and IL-13 and induce eosinophil activationand differentiation; Th2 cells are more proficient B-cellhelpers and can stimulate IgG1 and IgE production.Thus, they are well positioned to play substantial role inthe pathogenesis of allergic inflammation. In the presentwork, we explored whether co-exposure to DE and aller-gen induced recruitment of CD4+ cells in human lungtissue. We demonstrate that the number of CD4+ cellssignificantly increased only in DEA vs. FAS (p = 0.035),which suggest an interaction between DE and allergen.In a single exposure model in healthy subjects, it hasbeen previously argued that DE does not alter thenumber of CD3+, CD4+ and CD8+ lymphocytes in thebronchial tissue at 18 h post-exposure [72].IL-4 is known to be an important cytokine in thedevelopment of allergic inflammation; it provides thefirst signal that initiates B-cell class switching to IgEproduction [73]. IL-4 can further enhance IgE-mediatedimmune responses by up-regulating the expression oflow-affinity IgE receptor (FcεRII/CD23) on B-lymphocytesand macrophages and the high-affinity IgE receptor(FcεRI) on mast cells [74, 75]. IL-4 induces the differenti-ation of naive T lymphocytes into Th2 cells which secretemore IL-4, IL-5, and IL-13, maintaining a suitable envir-onment for further Th2 cells differentiation [76, 77]. IL-4can also stimulate the expression of vascular cell adhesionmolecule-1 (VCAM-1) on endothelial cells, which leads toenhanced migration of T-cells, eosinophils, macrophagesand mast cells to inflamed tissue [78]. To for Th2 cells butIL-4 is a signature cytokine of type 2 immunity. IL-4 isknown as a positive feedback cytokine for Th2 cell differ-entiation that stimulates the differentiation of naiveCD4+ cells into IL-4-secreting Th2 cells [79, 80]. It hasbeen shown that human mast cells are one of themajor sources of IL-4 in the skin, nasal and bronchialtissue [81–83]. IL-4 is able to induce the developmentof Th2 cells; thus, this stored and preformed IL-4within mast cell granules has an important influenceduring the initiation and maintenance of the allergicimmunological response. Basophils [84], naive T cells[85] and innate lymphoid cells [86] are also immediatesource of IL-4 upstream of Th2 cells differentiation. IL-4contributes to airway obstruction in asthma via the induc-tion of mucus hypersecretion in mice and human celllines, and increases the release of several pro-inflammatorycytokines such as IL-6, GM-CSF and eotaxin from humanlung fibroblasts [87]. IL-4 is a major factor in the recruit-ment and activation of inflammatory cells that may contrib-ute to inflammation and lung remodeling in chronicasthma [88].We demonstrate that IL-4 expression is increased inDEA vs. FAS (p = 0.034), suggesting a Th2 immune re-sponse is induced by DE and allergen that is not observedwith isolated (single) exposures. Our data are consistentwith in vivo animal, in vitro, and human studies that haveconfirmed DE has strong pro-Th2 effect [89, 90]. Humannasal challenge studies have shown that co-administrationof DE particles and allergen stimulate a Th2 immune re-sponse in nasal wash samples 4 days post-challenge [21,91, 92]. There is one conflicting study that has shown nodifferences in expression of IL-4 in the bronchial sub-mucosa but its authors mention that the bronchial tissuein their study was assessed at a single time point 6 h post-DE exposure [93] while most of the cytokine changes ob-served in previous human nasal studies and animal expos-ure studies were found 24 to 48 h post-exposure [94]. Aprevious study also evaluated the effects of diesel exhaustinhalation in enhancing allergic immunologic responses inlower airways [95]. Consistent with our results, they simi-larly found an increase in the IL-4 level (by 1.7-fold, whichwas close to statistical significance); they also found anon-significant elevation in the number of eosinophils ininduced sputum due to DE exposure. However, thereare some fundamental differences between their modeland our current study a) their allergen challenge was per-formed by inhalation but we challenged our subjects seg-mentally, with saline control simultaneously, which conferssome advantages and limitations; b) the diesel exhaust con-centration that we used was ~300 μg.m−3 PM2.5 whiletheirs was ~100 μg.m−3; c) they analyzed sputum andblood that were acquired 22 h post-exposure while we ana-lyzed endobronchial biopsies that were obtained 48 h post-exposure.A primary strength of our study is that it is a double-blinded cross-over study that fundamentally eliminatestypical confounding covariates, since each subject servesas his/her own control. Our study does have limitations,Hosseini et al. Particle and Fibre Toxicology  (2016) 13:2 Page 8 of 14however. One limitation is generalizability. For example,given the specific gap (one hour) between inhalationexposure and segmental allergen, it is difficult to knowwhether similar findings would occur with simultaneousexposure or some other gap, but we effectively considerthis “co-exposure” given that the particles from DE willremain in the airways for hours after inhalation and thusthen be present when the allergen is inhaled (though,admittedly, the exact dynamics therein are unknownand an important future direction for our work). An-other concern is whether airway changes relevant toour hypotheses can be induced by the bronchoscopyprocedure itself. Investigative bronchoscopy and bron-chial provocation challenge are commonly used tech-niques in airway inflammation research [96]. While FEV1and PEFR dropped due to bronchoscopy with lavage andbiopsies, both returned to baseline within 2 to 24 h[97, 98]. Accordingly, we doubt that significant inflam-mation from these procedures persists through 48 h.ConclusionsIn summary, we have demonstrated for the first timethat acute exposure to DE followed by segmental aller-gen challenge increases the submucosal recruitment ofCD4 cells, CD138-positive plasma cells and expressionof neutrophil elastase and IL-4 in the submucosa of atopichuman subjects. Our study design and results suggest thatexperimental data from complex exposures can capturereal-world exposures that enlighten our understanding ofbiology and maybe inform those concerned with publichealth and policy based on complex exposures.MethodsSubject recruitmentTwelve atopic subjects (19–49 years old) were screened,informed of the protocol, procedures, and potential risk,and agreed to participate in the study (ClinicalTrials.govidentifier: NCT01792232). The UBC Research Ethics Boardapproved the study protocol and informed consent form.Allergic sensitization to house dust mite (HDM), birch andPacific grasses were diagnosed by skin prick test. Birch-sensitive subjects were not studied in the birch season(February-April) and grass-sensitive subjects were not stud-ied in the Pacific grasses season (May-August). Subjectcharacteristics are described in Table 1.Exposure protocolEach subject was exposed for 120 min to filtered air(FA; the control for diesel exhaust) or freshly gener-ated diesel exhaust (DE PM2.5 300 μg.m−3; Table 2) ina double-blinded crossover experiment, with the two dis-tinct visits randomized and counter-balanced to order,with a four-week washout period between each condition(Fig. 7). In this study design, each subject serves as his/herown control.During the two-hour exposure, the subject was askedto exercise on a bicycle ergometer for a total of 30 min(2 × 15 min at ~60 rpm cadence and ~25 W of resist-ance) to increase ventilation-heart rate and mimic modestintermittent activity.Bronchoscopy procedureSegmental allergen challenge (Bronchoscopy #1)One hour following each exposure to DE or FA, segmen-tal allergen challenge was performed through standardfiberoptic bronchoscopy procedure. Bronchoscopy wasused to deliver a diluent-controlled solution of the positiveskin prick allergen extract, at a concentration 10-foldlower than the dose producing a positive wheal ≥3 mm,into a right lower lobe segment. A 5 mL diluent controlwas delivered into a left lower lobe segment.Endobronchial biopsies (Bronchoscopy #2)The second bronchoscopy was done 48 h post-allergenchallenge, endobronchial biopsy specimens (size ≤2 mm)were obtained from the same segments exposed to aller-gen or saline.Following an approximately 1 month washout period,subjects returned and received the second two-hour ex-posure, followed by a bronchoscopy during which aller-gen and saline were administered to opposite lungs anddifferent segments than those during the first exposure.Thus, endobronchial biopsies for each of the 4 differentcrossover conditions was created: 1) FAS: filtered air +saline, 2) DES: DE + saline, 3) FAA: filtered air + allergenand 4) DEA: DE + allergen.Bronchial biopsies processingThe endobronchial biopsies were immediately added to ice-cold acetone containing the protease inhibitors iodoaceta-mide (20 mM) and phenylmethylsulfonylfluoride (PMSF;2 mM, Sigma, Oakville, ON) and fixed at −20 °C overnight(16–24 h). The next day, biopsies were transferred to freshTable 2 Inhaled exposure characteristicsCondition PM2.5 (μg/m3) Particle number (#/cm3) CO (ppm) NO (ppb) NOx (ppb) NO2 (ppb) NO2/PM# (μg/#)FA 8.2 (6.9) 1750.4 (235.1) 2.8 (0.1) 25.3 (5.0) 71.1 (9.8) 45.9 (7.7) 4.9 × 10−9DE 302.0 (30.5) 5.4 × 105 (6.4 × 104) 14.1 (2.0) 8665.5 (1287.1) 9185.3 (1366.1) 519.7 (118.6) 1.8 × 10−9Values are presented as mean (SD)FA filtered air, DE diesel exhaustHosseini et al. Particle and Fibre Toxicology  (2016) 13:2 Page 9 of 14acetone and then to methyl benzoate (Sigma, Oakville,ON) for 15 min each at room temperature, before infiltra-tion with glycol methacrylate (GMA) resin as previouslydescribed at Britten et al. [99].Glycol methacrylate acrylic resin (GMA) embeddingGlycol methacrylate acrylic resin (GMA) is a hydrophilicplastic resin which provides a number of advantagesover frozen and paraffin-embedding techniques [100].The JB-4 Embedding Kit (Polysciences, Warrington, PA)was used for embedding in GMA with some modifica-tion to its original manufacturer’s instructions describedin details at Wilson et al. [100]. For polymerization ofGMA resin, benzoyl peroxide was added to the airtightembedding capsule containing tissue sample and keptfor 48 h at 4 °C to increase polymerization of GMA. Thepolymerized resin block was stored desiccated at −20 °Cfreezer until used for IHC staining.Biopsy quality evaluationGMA blocks containing each biopsy were removed fromembedding capsules and excess resin trimmed to form atrapezium shape around the tissue. Sections were cut at2 μm using an ultra-microtome (Leica EM UC6 at JHRCHistology lab) and floated on distilled water (dH2O) andpicked up onto 10 % poly-l-lysine (PLL)-coated slides(Fisher Scientific, Ottawa, ON). Slides were left on a hotplate to completely dry out, followed by addition of onedrop of toluidine blue stain for 2 min following bydrying and mounting in DPX (Sigma, Oakville, ON) forsubsequent examination under light microscope to checkbiopsy quality. In order to qualify for immunohistochemi-cal analysis the tissue section must have a minimum of0.46 mm2 of submucosal tissue (lamina propria), exclud-ing smooth muscle and glands [101]. If the section wasfound to be of poor standard, further sections were cutand reassessed. If no such level with acceptable histo-logical standard was found in the biopsy, then the biopsywas excluded from further IHC analysis.Controls for ImmunohistochemistryTonsil tissue samples removed from patients (kindly pro-vided by Dr. Andrew Thamboo, Dept. of Otolaryngology(ENT) at St. Paul's Hospital) were used as positive controland staining with isotype-matched controls was used asnegative control (Additional file 2: Figure S1). Endogenousperoxidase was blocked with a 100 μl of 30 % H2O2 solu-tion in 10 mL of sodium azide and endogenous avidin andbiotin were blocked using a commercially available kitfrom Vector Labs (Vector Laboratories, Burlington, On-tario). Colour was developed using a VECTASTAINElite ABC Kit (Vector Laboratories, Burlington,Ontario).Immunohistochemistry (IHC) on endobronchial biopsiesIHC was used to determine the number of CD4+, IL-4+cells, CD138+ plasma cells, elastase-positive (NE+) neutro-phils and activated (EG2+) eosinophils and tryptase-positive(AA1+) mast cells in the lamina propria in endobronchialbiopsies (Table 3).0 h 2 h 3 hDE (300 µg.m-3 of PM 2.5) or FA   inhalationBronchoscopy #2Bronchoscopy #148 hSegmental allergen instillation Endobronchial biopsiesFig. 7 Schematic of exposure protocol. Study subjects were exposed to DE (300 μg.m−3 of PM2.5) or FA (filtered air) for 2 h. One hour post-exposure, a segmental allergen challenge was performed with allergen or saline vehicle in the right upper and middle lobe or left lingular lobe.Forty-eight hours post-exposure endobronchial biopsies were obtained via bronchoscopy. The process was repeated following a washout periodof 1 month with exposure conditions reversed compared to the first visitTable 3 Percent positivity for inflammatory biomarkers’ expression in the lung submucosa. Data are expressed as mean ± standarderror of the mean (SEM)Exposure condition AA1 ECP NE CD138 CD4 IL-4FAS 0.460 ± 0.053 0.308 ± 0.102 0.045 ± 0.014 0.017 ± 0.006 0.045 ± 0.014 0.127 ± 0.062DES 0.681 ± 0.153 0.471 ± 0.175 0.077 ± 0.024 0.044 ± 0.024 0.045 ± 0.014 0.353 ± 0.088FAA 0.646 ± 0.128 0.553 ± 0.109 0.229 ± 0.069 0.044 ± 0.008* 0.045 ± 0.014 0.426 ± 0.130DEA 0.738 ± 0.159 0.487 ± 0.201 0.224 ± 0.047* 0.120 ± 0.031*, **, *** 0.045 ± 0.014* 0.548 ± 0.143*FAS filtered air + saline, DES DE + saline, FAA filtered air + allergen, DEA DE + allergen, AA1 tryptase, ECP eosinophil cationic protein, NE neutrophil elastase*p < 0.05, compared with FAS; **p < 0.05, compared with DES; ***p < 0.05, compared with DEAHosseini et al. Particle and Fibre Toxicology  (2016) 13:2 Page 10 of 14Semi-thin sections were cut at 2 μm from endo-bronchial biopsies and immunostained with monoclo-nal primary antibodies (Table 4). The immunostainingprocedure followed has been described previously [100].Briefly, endogenous peroxidase was blocked using a 100 μlof 30 % H2O2 solution in 10 mL of sodium azide andendogenous biotin were blocked using a commerciallyavailable kit from Vector Labs (Vector Laboratories,Burlington, Ontario). Mouse anti-human monoclonalantibodies were applied at appropriate dilutions (Table 4)and incubated with coverslip 20–22 h at room temperature.After washing with Tris-buffered saline (TBS, 3 × 5 min),the sections were incubated with the biotinylated rabbitanti-mouse secondary antibody (Dako, Burlington, Ontario)for 2 h. ABC (avidin-biotin complex) kit (Vector La-boratories, Burlington, Ontario) was applied after fur-ther washing with TBS (3 × 5 min), and incubated for2 h. The positive staining were then visualized usingAEC (3-amino-9-ethylcarbazole) chromogen kit (Bio-Genex, Fremont, CA) and counterstained with Mayer'sHematoxylin (Dako, Burlington, Ontario).Quantification of ImmunohistochemistryIHC slides were scanned by Aperio ScanScope XT(Aperio Technologies, Vista, CA) at 40X magnifica-tion. Morphometric and immunohistochemical analysiswere performed on the digital images (0.25 μm/pixel)using Aperio® ImageScope™ (version 11.2.0.780). The in-corporated Positive Pixel Count (PPC) algorithm (version9.1) was used to quantify inflammatory cells that stainedpositive (positive pixels) for each antibody of interest. Toquantify positive pixels, a hue value of 0.0 (red) and huewidth of 0.5 was used, and all the three intensity ranges(weak, positive, and strong) of staining were considered aspositive (Fig. 8). The number of positive pixels was dividedby the total number of pixels (positive and negative) in theanalyzed area, and multiplied by 100, to calculate thepercentage of positive pixels.Numberof PositivepixelsTotalnumberof PositivepixelsþNegativepixels 100 %Positivityð ÞFor tryptase, ECP, NE, CD138, CD4 and IL-4, theamount of a positive stain in an image and the area ofthe submucosa were measured. The positive pixel countswere expressed as percent (%) positive pixel/mm2 in thesubmucosa.Statistical analysisOne-way repeated measures ANOVA with Bonferronimultiple comparison post hoc tests and Pearson correl-ation coefficients matrix were performed using Graph-Pad Prism® 6 software (GraphPad Software Inc., La Jolla,CA). Combined Robust regression and Outlier removalmethod (ROUT test) was used and an outlier in CD138dataset was identified and removed. A p-value of <0.05was considered statistically significant. Error bars shownrepresent the standard error of mean (±SEM).Table 4 List of primary monoclonal antibodies are used for IHC stainingAntibody Marker Cell Concentration (μg.mL−1) Catalog no. SourceAA1 Tryptase Mast cells 0.1 ab2378 Abcam Inc., Toronto, ONEG2 (614) ECP Eosinophils 0.07 514-121 Diagnostics, Uppsala, SWIL-4 (4D9) IL-4 Th2, mast cells 20.0 211-44-134AX Amsbio, Cambridge, UKCD4 (4B12) CD4 Helper T cells 8.0 M731001-2 Dako, Burlington, ONCD138 (B-A38) Syndecan-1 Plasma cells 1.0 MCA2459GA AbD Serotec, Raleigh, NCNE (NP57) Elastase 2 Neutrophils 0.1 M075201-2 Dako, Burlington, ONBAFig. 8 Demonstration of image analysis using Aperio® ImageScope™ software. A positive pixel count algorithm was used to quantify positivestaining in the submucosa (blue region) of bronchial biopsies for tryptase, ECP, NE, CD138, CD4, and IL-4. The airway epithelium was not examinedand positive staining in the epithelium was excluded from analysis. a Representative image of tryptase positive staining from a subject exposedto FAA, Black arrows denote positive staining in submucosa area that are selected by the positive pixel count. b Image from a with submucosaregion selected by manual trace followed by positive pixel count recognition of tryptase stain (red colour) within submucosa region (blue colour)Hosseini et al. Particle and Fibre Toxicology  (2016) 13:2 Page 11 of 14Additional filesAdditional file 1: Tables S1 and S2. Pearson correlation coefficientsmatrix for inflammatory biomarkers’ expression in the lung submucosaafter single or co-exposure to diesel exhaust and allergen. (PDF 213 kb)Additional file 2: Figure S1. Immunohistochemical staining of positiveand negative controls. A) Representative 40X image of positive stainingusing mAb AA1 for tryptase in human tonsil tissue; B) Representative 40Ximage of positive staining using mAb EG2 for ECP in human tonsil tissue;C) Representative 40X image of positive staining using mAb NP57 forneutrophil elastase in human tonsil tissue; D) Representative 40X imageof positive staining using mAb B-A38 for CD138 in human lung tissue;E) Representative 40X image of positive staining using mAb 4B12 for CD4in human tonsil tissue; F) Representative 40X image of positive stainingusing mAb 4D9 for IL-4 in human tonsil tissue; G) Representative 40Ximage of isotype control using mAb mouse IgG1 (0.07 μg.mL−1) inhuman lung tissue; H) Representative 40X image of isotype controlstaining using mAb mouse IgG1 (20.0 μg.mL−1) in human lung tissue.(PDF 482 kb)AbbreviationsBAL: bronchoalveolar lavage; BMI: body mass index; DE: diesel exhaust;DEA: diesel exhaust and allergen; DES: diesel exhaust and saline;ECP: eosinophil cationic protein; EPO: eosinophil peroxidase; FA: filteredair; FAA: filtered air and allergen; FAS: filtered air and saline; FEV1: forcedexpiratory volume in 1 s; GMA: glycol methacrylate resin; GM-CSF: granulocyte-macrophage colony-stimulating factor; HDM: house dust mite;IgE: immunoglobulin E; IHC: immunohistochemistry; MBP: major basicprotein; NE: neutrophil elastase; PM: particulate matter; PM2.5: particulatematter less than 2.5 μm in diameter; Th2: T helper cell type-2; VCAM-1: vascularcell adhesion molecule-1; μm: micrometer.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsAH was responsible for monitoring the subjects during voluntary exposures,carried out the immunohistochemistry experiments, performed imageanalysis and data collection, conducted the statistical analysis and datainterpretation, and drafted the manuscript. CC, JH, KM, TH and SW providedintellectual input throughout the study and contributed to revising the draftmanuscript. CC supervised the work, performed the bronchoscopyprocedure and participated in the design and coordination of the study. Allauthors read and approved the final manuscript.AcknowledgementsThis study was funded by the Canadian Institutes of Health Research (CIHR)and AllerGen NCE Inc. AH was supported by CIHR TransplantationScholarship Training Program. The authors would like to thank the ElectronMicroscopy Core Facility, Histology Core and Imaging & Graphic Services ofthe UBC Centre for Heart Lung Innovation at St. Paul's Hospital, Vancouver,Canada.Author details1Department of Medicine, Division of Respiratory Medicine, Chan-YeungCentre for Occupational and Environmental Respiratory Disease, University ofBritish Columbia, Vancouver, BC V5Z 1M9, Canada. 2Institute for Heart andLung Health, University of British Columbia, Vancouver, BC V6Z 1Y6, Canada.3Biomedical Research Centre, University of British Columbia, Vancouver, BCV6T 1Z3, Canada. 4Histochemistry Research Unit, Faculty of Medicine,University of Southampton, Southampton S016 6YD, UK. 5The Lung Center,Vancouver General Hospital (VGH) – Gordon and Leslie Diamond Health CareCentre, 2775 Laurel Street, 7th floor, Vancouver, BC V5Z 1M9, Canada.Received: 17 August 2015 Accepted: 6 January 2016References1. 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