Role of Innate Immune System in Environmental Lung Diseases.

The lung mucosa functions as a principal barrier between the body and inhaled environmental irritants and pathogens. Precise and targeted surveillance mechanisms are required at this lung-environment interface to maintain homeostasis and preserve gas exchange. This is performed by the innate immune system, a germline-encoded system that regulates initial responses to foreign irritants and pathogens. Environmental pollutants, such as particulate matter (PM), ozone (O3), and other products of combustion (NO2, SO3, etc.), both stimulate and disrupt the function of the innate immune system of the lung, leading to the potential for pathologic consequences. PURPOSE OF REVIEW: The purpose of this review is to explore recent discoveries and investigations into the role of the innate immune system in responding to environmental exposures. This focuses on mechanisms by which the normal function of the innate immune system is modified by environmental agents leading to disruptions in respiratory function. RECENT FINDINGS: This is a narrative review of mechanisms of pulmonary innate immunity and the impact of environmental exposures on these responses. Recent findings highlighted in this review are categorized by specific components of innate immunity including epithelial function, macrophages, pattern recognition receptors, and the microbiome. Overall, the review supports broad impacts of environmental exposures to alterations to normal innate immune functions and has important implications for incidence and exacerbations of lung disease. The innate immune system plays a critical role in maintaining pulmonary homeostasis in response to inhaled air pollutants. As many of these agents are unable to be mitigated, understanding their mechanistic impact is critical to develop future interventions to limit their pathologic consequences.

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury.

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, α and ß. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Early life microbiome perturbation alters pulmonary responses to ozone in male mice.

Early life changes in the microbiome contribute to the development of allergic asthma, but little is known about the importance of the microbiome for other forms of asthma. Ozone is a nonatopic asthma trigger that causes airway hyperresponsiveness and neutrophil recruitment to the lungs. The purpose of this study was to test the hypothesis that early life perturbations in the gut microbiome influence subsequent responses to ozone. To that end, we placed weanling mouse pups from The Jackson Laboratories or from Taconic Farms in sex-specific cages either with other mice from the same vendor (same-housed) or with mice from the opposite vendor (cohoused). Mice were maintained with these cagemates until use. The gut microbial community differs in mice from Jackson Labs and Taconic Farms, and cohousing mice transfers fecal microbiota from one mouse to another. Indeed, 16S rRNA sequencing of fecal DNA indicated that differences in the gut microbiomes of Jackson and Taconic same-housed mice were largely abolished when the mice were cohoused. At 10-12 weeks of age, mice were exposed to room air or ozone (2 ppm for 3 hr). Compared to same-housed mice, cohoused male but not female mice had reduced ozone-induced airway hyperresponsiveness and reduced ozone-induced increases in bronchoalveolar lavage neutrophils. Ozone-induced airway hyperresponsiveness was greater in male than in female mice and the sex difference was largely abolished in cohoused mice. The data indicate a role for early life microbial perturbations in pulmonary responses to a nonallergic asthma trigger.

Locally instructed CXCR4hi neutrophils trigger environment-driven allergic asthma through the release of neutrophil extracellular traps.

Low exposure to microbial products, respiratory viral infections and air pollution are major risk factors for allergic asthma, yet the mechanistic links between such conditions and host susceptibility to type 2 allergic disorders remain unclear. Through the use of single-cell RNA sequencing, we characterized lung neutrophils in mice exposed to a pro-allergic low dose of lipopolysaccharide (LPS) or a protective high dose of LPS before exposure to house dust mites. Unlike exposure to a high dose of LPS, exposure to a low dose of LPS instructed recruited neutrophils to upregulate their expression of the chemokine receptor CXCR4 and to release neutrophil extracellular traps. Low-dose LPS-induced neutrophils and neutrophil extracellular traps potentiated the uptake of house dust mites by CD11b+Ly-6C+ dendritic cells and type 2 allergic airway inflammation in response to house dust mites. Neutrophil extracellular traps derived from CXCR4hi neutrophils were also needed to mediate allergic asthma triggered by infection with influenza virus or exposure to ozone. Our study indicates that apparently unrelated environmental risk factors can shape recruited lung neutrophils to promote the initiation of allergic asthma.

Limonene and its ozone-initiated reaction products attenuate allergic lung inflammation in mice.

Inhalation of indoor air pollutants may cause airway irritation and inflammation and is suspected to worsen allergic reactions. Inflammation may be due to mucosal damage, upper (sensory) and lower (pulmonary) airway irritation due to activation of the trigeminal and vagal nerves, respectively, and to neurogenic inflammation. The terpene, d-limonene, is used as a fragrance in numerous consumer products. When limonene reacts with the pulmonary irritant ozone, a complex mixture of gas and particle phase products is formed, which causes sensory irritation. This study investigated whether limonene, ozone or the reaction mixture can exacerbate allergic lung inflammation and whether airway irritation is enhanced in allergic BALB/cJ mice. Naïve and allergic (ovalbumin sensitized) mice were exposed via inhalation for three consecutive days to clean air, ozone, limonene or an ozone-limonene reaction mixture. Sensory and pulmonary irritation was investigated in addition to ovalbumin-specific antibodies, inflammatory cells, total protein and surfactant protein D in bronchoalveolar lavage fluid and hemeoxygenase-1 and cytokines in lung tissue. Overall, airway allergy was not exacerbated by any of the exposures. In contrast, it was found that limonene and the ozone-limonene reaction mixture reduced allergic inflammation possibly due to antioxidant properties. Ozone induced sensory irritation in both naïve and allergic mice. However, allergic but not naïve mice were protected from pulmonary irritation induced by ozone. This study showed that irritation responses might be modulated by airway allergy. However, aggravation of allergic symptoms was observed by neither exposure to ozone nor exposure to ozone-initiated limonene reaction products. In contrast, anti-inflammatory properties of the tested limonene-containing pollutants might attenuate airway allergy.

Regulation of IL-17A expression in mice following subacute ozone exposure.

Exposure to subacute ozone (O3) causes pulmonary neutrophil recruitment. In mice, this recruitment requires IL-17A. Ozone also causes expression of IL-23 and IL-1, which can induce IL-17A. The purpose of this study was to examine the hypothesis that IL-23 and IL-1 contribute to IL-17A expression and subsequent neutrophil recruitment after subacute O3 exposure. Wild-type, IL-23(-/-), and Flt3l(-/-) mice were exposed to air or 0.3 ppm O3 for 72 h. Flt3l(-/-) mice lack conventional dendritic cells (cDC) that can express IL-23 and IL-1. Other wild-type mice were pre-treated with saline or the IL-1R1 antagonist anakinra prior to O3 exposure. After exposure, bronchoalveolar lavage (BAL) was performed and lung tissue harvested. The results indicated that pulmonary Il17a mRNA abundance and IL-17A(+) F4/80(+) cells were significantly reduced in O3-exposed IL-23(-/-) vs in wild-type mice. In contrast, anakinra had no effect on Il23a or Il17a pulmonary mRNA abundance or on BAL concentrations of the neutrophil survival factor G-CSF, but anakinra did reduce BAL neutrophil numbers, likely because anakinra also reduced BAL IL-6. Compared to air, O3 caused a significant increase in DC numbers in wild-type, but not in Flt3(-/-) mice. However, there was no significant difference in Il23a or Il17a mRNA abundance or in BAL neutrophil count in O3-exposed Flt3(-/-) vs in wild-type mice. From these results, it was concluded that IL-23 but not IL-1 contributes to the IL-17A expression induced by subacute O3 exposure. Induction of IL-23 by O3 does not appear to require cDC.

Allergens, air pollutants, and childhood allergic diseases.

BACKGROUND: The synergistic effect of allergens and air pollutants on the risk of allergic diseases is unclear. OBJECTIVE: To evaluate the joint effect of outdoor pollutants and indoor allergens on the risk of allergic diseases. METHODS: We enrolled 2661 kindergarten children from the CEAS cohort. Data on allergic diseases and environmental exposure were collected. Skin prick tests were performed. Individual exposure to air pollution was estimated using a geographic information system with the mean concentration of air pollutants. Multiple logistic regression analysis was performed to estimate the association between air pollutants, allergen exposure and the risk of allergic diseases with adjustments for potential confounders. RESULTS: Overall, 12.6% of the children had asthma, 30.0% had allergic rhinitis (AR), and 14.4% had atopic dermatitis (AD). Mite sensitization significantly increased the risk of AD, AR, and asthma (OR (95%CI) 2.15 (1.53-3.03), 1.94 (1.46-2.58), and 2.31 (1.63-3.29), respectively). Exposure to PM10, PM(2.5), CO, and O(3) was associated with asthma (OR (95% CI) 1.39 (1.03-1.87), 1.45 (1.07-1.97), 1.36 (1.01-1.83), and 0.68 (0.51-0.92), respectively). PM(2.5) may have increased the risk of AR (OR (95% CI) 1.54 (1.03-2.32). Mite sensitization showed a synergistic effect with PM(2.5) on the development of asthma (p < 0.001). Moreover, mite allergens may modify the effect of air pollutants on allergic diseases. CONCLUSION: Dust mites and PM(2.5) play an important role on the risk of asthma and AR. Exposure to PM(2.5) and mite allergens had a synergistic effect on the development of asthma. Avoiding co-exposure to allergens and air pollutants is important.

Interaction with epithelial cells modifies airway macrophage response to ozone.

The initial innate immune response to ozone (O3) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell-Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell-derived signals are important determinants of Mac immunophenotype and response to O3.

Pediatric asthma and ambient pollutant levels in industrializing nations.

Asthma is one of the most common chronic diseases in childhood and its prevalence has been increasing within industrializing nations. The contribution of ambient pollutants to asthma symptomatology has been explored in some countries through epidemiological investigations, molecular analysis and monitoring functional outcomes. The health effects of rising environmental pollution have been of increasing concern in industrializing nations with rising urbanization patterns. This review article provides an overview of the link between pediatric asthma and exposure to rising sources of urban air pollution. It primarily focuses on the asthma-specific effects of sulfur dioxide, nitrogen dioxide, ozone and particulate matter. Worldwide trends of asthma prevalence are also provided which detail the prominent rise in asthma symptoms in many urban areas of Africa, Latin America and Asia. The molecular and functional correlation of ambient pollutants with asthma-specific airway inflammation in the pediatric population are also highlighted. The final aspect of the review considers the correlation of motor vehicle, industrial and cooking energy sources, ascribed as the major emitters among the pollutants in urban settings, with asthma epidemiology in children.

Effects of ozone exposure mediated by BEAS-2B cells on T cells activation: a possible link between environment and asthma.

OBJECTIVE: To explore the possible link between ozone and asthma through analyzing Th1/Th2 differentiation of T cells following incubation with conditioned medium from the BEAS-2B cells exposed to ozone in vitro. METHOD: Bronchial epithelial cell line, BEAS-2B, was cultured using an air-liquid interface culture system in a CO2 incubator and exposed to 0 or 0.16 or 0.25 mg/m3 of ozone for 8 h. The amounts of IL-1ß, IL-6 and RANTES in the cell supernatant were detected. The cell culture supernatants were collected and used as conditioned medium in the next experiment. T cells from children recruited were incubated with conditioned medium for 12 h. Activation rate of CD69 and Th1/Th2/Th17 differentiation were analyzed. RESULTS: BEAS-2B cells exposed to different ozone concentrations showed morphological changes. Cells exposed to 0.16 and 0.25 mg/m3 ozone produced higher amounts of IL-1ß, IL-6 and RANTES than that in the control group. Children with allergic asthma had upregulated expression of genes related with asthma, including CCL2, CCR4, CXCL2, CYSLTR1, IL12RB2, IL13RA2, IL18R1, IL1B, IL8, IL8RB and TNFSF13.CD69 expression in T cells was significantly elevated irrespective of ozone exposure in children with allergic asthma. Following ozone exposure, in asthmatic children group, expression levels of cytokines of Th1 cells were collectively higher than those from Th2 cells. Ozone-exposed conditioned media could slightly increase all the Th1, Th2 and Th17 cytokines in T cells from allergic asthmatic children. CONCLUSIONS: Our results suggested that Th1 cells activation might be predominant over Th2 activation upon ozone exposure in asthmatic children, which might help to clarify the mechanisms of asthma related to environmental factors like ozone.

Inhaled environmental allergens and toxicants as determinants of the asthma phenotype.

The driving environmental factors behind the development of the asthma phenotype remain incompletely studied and understood. Here, we present an overview of inhaled allergic/atopic and mainly nonallergic/nonatopic or toxicant shapers of the asthma phenotype, which are present in both the indoor and outdoor environment around us. The inhaled allergic/atopic factors include fungus, mold, animal dander, cockroach, dust mites, and pollen; these allergic triggers and shapers of the asthma phenotype are considered in the context of their ability to drive the immunologic IgE response and potentially induce interactions between the innate and adaptive immune responses, with special emphasis on the NADPH-dependent reactive oxygen-species-associated mechanism of pollen-associated allergy induction. The inhaled nonallergic/nonatopic, toxicant factors include gaseous and volatile agents, such as sulfur dioxide, ozone, acrolein, and butadiene, as well as particulate agents, such as rubber tire breakdown particles, and diesel exhaust particles. These toxicants are reviewed in terms of their relevant chemical characteristics and hazard potential, ability to induce airway dysfunction, and potential for driving the asthma phenotype. Special emphasis is placed on their interactive nature with other triggers and drivers, with regard to driving the asthma phenotype. Overall, both allergic and nonallergic environmental factors can interact to acutely exacerbate the asthma phenotype; some may also promote its development over prolonged periods of untreated exposure, or possibly indirectly through effects on the genome. Further therapeutic considerations should be given to these environmental factors when determining the best course of personalized medicine for individuals with asthma.

A review on human health perspective of air pollution with respect to allergies and asthma.

The increase in cases of asthma and allergies has become an important health issue throughout the globe. Although these ailments were not common diseases a few short decades ago, they are now affecting a large part of the population in many regions. Exposure to environmental (both outdoor and indoor) pollutants may partially account for the prevalence of such diseases. In this review, we provide a multidisciplinary review based on the most up-to-date survey of literature regarding various types of airborne pollutants and their associations with asthma-allergies. The major pollutants in this respect include both chemical (nitrogen dioxide, ozone, sulfur dioxide, particulate matter, and volatile organic compounds) and biophysical parameters (dust mites, pet allergens, and mold). The analysis was extended further to describe the development of these afflictions in the human body and the subsequent impact on health. This publication is organized to offer an overview on the current state of research regarding the significance of air pollution and its linkage with allergy and asthma.

Efecto modulador de la ozonoterapia sobre la actividad del sistema inmune / Modulator effect of ozone therapy over immune system activity

El empleo de la medicina biológica ha permitido aplicar terapias sistémicas beneficiosas donde el cuerpo es tratado como un todo. La ozonoterapia es una técnica interesante en el campo de aplicación de la medicina biológica; es un proceder terapéutico, seguro, natural y factible, con resultados alentadores en el tratamiento de enfermedades inmunológicas, por el impacto social y económico que se genera tras su aplicación. Existen evidencias experimentales que aseveran esta afirmación. En este artículo se describen las propiedades inmunomoduladoras de la ozonoterapia, al caracterizar los efectos biológicos del ozono sobre las células del sistema inmune, mediadores solubles y otros tipos celulares

The employment of biological medicine has allowed the application of beneficial and systemic therapies where the body is treated as a whole. The ozone therapy is a modality of the biological medicine, it is a natural therapeutical procedure that has represented an extremely safe medical therapy, free from side effects, an effective and feasible method in the field of treatment of immunological diseases, with encouraging results for the social and economic impact that is generated after its application. There are experimental evidences that assert this statement. We hereby describe the immunomodulatory property of ozone therapy, the biological effects of the ozone on the cells of the immune system cells, other cellular types and different immune mediators

Efecto modulador de la ozonoterapia sobre la actividad del sistema inmune / Modulator effect of ozone therapy over immune system activity

El empleo de la medicina biológica ha permitido aplicar terapias sistémicas beneficiosas donde el cuerpo es tratado como un todo. La ozonoterapia es una técnica interesante en el campo de aplicación de la medicina biológica; es un proceder terapéutico, seguro, natural y factible, con resultados alentadores en el tratamiento de enfermedades inmunológicas, por el impacto social y económico que se genera tras su aplicación. Existen evidencias experimentales que aseveran esta afirmación. En este artículo se describen las propiedades inmunomoduladoras de la ozonoterapia, al caracterizar los efectos biológicos del ozono sobre las células del sistema inmune, mediadores solubles y otros tipos celulares(AU)

The employment of biological medicine has allowed the application of beneficial and systemic therapies where the body is treated as a whole. The ozone therapy is a modality of the biological medicine, it is a natural therapeutical procedure that has represented an extremely safe medical therapy, free from side effects, an effective and feasible method in the field of treatment of immunological diseases, with encouraging results for the social and economic impact that is generated after its application. There are experimental evidences that assert this statement. We hereby describe the immunomodulatory property of ozone therapy, the biological effects of the ozone on the cells of the immune system cells, other cellular types and different immune mediators(AU)

[Environmental pollution and allergy: immunological mechanisms]. / Pollution environnementale et allergie : mécanismes immunologiques.

Airborne pollutants, both particulate and gaseous, represent a major environmental factor promoting allergic sensitization and disease expression. These adverse effects of particulate matter are highly dependent upon the nature and size of the particles, their content of chemicals and metals, and the subject's genetic makeup. Diesel exhaust and gases, in particular ozone, have been shown to exacerbate cellular inflammation and to act as mucosal adjuvants to skew the immune response to inhaled antigens toward a Th2-like phenotype. Growing evidence suggests that mechanisms of pollutant-induced amplification of the allergic reaction depend on oxidative stress that is under the control of susceptibility genes, as well as epigenetic mechanisms.

Genes of innate immunity and the biological response to inhaled ozone.

Ambient ozone has a significant impact on human health. We have made considerable progress in understanding the fundamental mechanisms that regulate the biological response to ozone. It is increasingly clear that genes of innate immunity play a central role in both infectious and noninfectious lung disease. The biological response to ambient ozone provides a clinically relevant environmental exposure that allows us to better understand the role of innate immunity in noninfectious airways disease. In this brief review, we focus on (1) specific cell types in the lung modified by ozone, (2) ozone and oxidative stress, (3) the relationship between genes of innate immunity and ozone, (4) the role of extracellular matrix in reactive airways disease, and (5) the effect of ozone on the adaptive immune system. We summarize recent advances in understanding the mechanisms that ozone contributes to environmental airways disease.

Epithelial cells, the "switchboard" of respiratory immune defense responses: effects of air pollutants.

"Epimmunome", a term introduced recently by Swamy and colleagues, describes all molecules and pathways used by epithelial cells (ECs) to instruct immune cells. Today, we know that ECs are among the first sites within the human body to be exposed to pathogens (such as influenza viruses) and that the release of chemokine and cytokines by ECs is influenced by inhaled agents. The role of the ECs as a switchboard to initiate and regulate immune responses is altered through air pollutant exposure, such as ozone, tobacco smoke and diesel exhaust emissions. The details of the interplay between ECs and immune cells are not yet fully understood and need to be investigated further. Co-culture models, cell specific genetically-modified mice and the analysis of human biopsies provide great tools to gain knowledge about potential mechanisms. Increasing our understanding about the role of ECs in respiratory immunity may yield novel therapeutic targets to modulate downstream diseases.

Allergen and ozone exacerbate serotonin-induced increases in airway smooth muscle contraction in a model of childhood asthma.

BACKGROUND: Serotonin (5-HT) modulates cholinergic neurotransmission and exacerbates airway smooth muscle (ASM) contraction in normal animal and nonasthmatic human tissue. Exposure to house dust mite allergen (HDMA) and ozone (O(3)) leads to airway hyperreactivity and 5-HT-positive cells in the airway epithelium of infant rhesus monkeys. Research shows that concomitant exposure in allergic animals has an additive effect on airway hyperreactivity. OBJECTIVES: In this study, the hypothesis is that the exposure of allergic infant rhesus monkeys to HDMA, O(3) and in combination, acting through 5-HT receptors, enhances 5-HT modulation of postganglionic cholinergic ASM contraction. METHODS: Twenty-four HDMA-sensitized infant monkeys were split into 4 groups at the age of 1 month, and were exposed to filtered air (FA), HDMA, O(3) or in combination (HDMA+O(3)). At the age of 6 months, airway rings were harvested and postganglionic, and parasympathetic-mediated ASM contraction was evaluated using electrical-field stimulation (EFS). RESULTS: 5-HT exacerbated the EFS response within all exposure groups, but had no effect in the FA group. 5-HT(2), 5-HT(3) and 5-HT(4) receptor agonists exacerbated the response. 5-HT concentration-response curves performed after incubation with specific receptor antagonists confirmed the involvement of 5-HT(2), 5-HT(3) and 5-HT(4) receptors. Conversely, a 5-HT(1) receptor agonist attenuated the tension across all groups during EFS, and in ASM contracted via exogenous acetylcholine. CONCLUSIONS: HDMA, O(3) and HDMA+O(3) exposure in a model of childhood allergic asthma enhances 5-HT exacerbation of EFS-induced ASM contraction through 5-HT(2), 5-HT(3) and 5-HT(4) receptors. A nonneurogenic inhibitory pathway exists, unaffected by exposure, mediated by 5-HT(1) receptors located on ASM.

Increased CCL24/eotaxin-2 with postnatal ozone exposure in allergen-sensitized infant monkeys is not associated with recruitment of eosinophils to airway mucosa.

Epidemiology supports a causal link between air pollutant exposure and childhood asthma, but the mechanisms are unknown. We have previously reported that ozone exposure can alter the anatomic distribution of CD25+ lymphocytes in airways of allergen-sensitized infant rhesus monkeys. Here, we hypothesized that ozone may also affect eosinophil trafficking to allergen-sensitized infant airways. To test this hypothesis, we measured blood, lavage, and airway mucosa eosinophils in 3-month old monkeys following cyclical ozone and house dust mite (HDM) aerosol exposures. We also determined if eotaxin family members (CCL11, CCL24, CCL26) are associated with eosinophil location in response to exposures. In lavage, eosinophil numbers increased in animals exposed to ozone and/or HDM. Ozone+HDM animals showed significantly increased CCL24 and CCL26 protein in lavage, but the concentration of CCL11, CCL24, and CCL26 was independent of eosinophil number for all exposure groups. In airway mucosa, eosinophils increased with exposure to HDM alone; comparatively, ozone and ozone+HDM resulted in reduced eosinophils. CCL26 mRNA and immunofluorescence staining increased in airway mucosa of HDM alone animals and correlated with eosinophil volume. In ozone+HDM animal groups, CCL24 mRNA and immunofluorescence increased along with CCR3 mRNA, but did not correlate with airway mucosa eosinophils. Cumulatively, our data indicate that ozone exposure results in a profile of airway eosinophil migration that is distinct from HDM mediated pathways. CCL24 was found to be induced only by combined ozone and HDM exposure, however expression was not associated with the presence of eosinophils within the airway mucosa.