Angela M. Gleason

Toll-Like Receptor 2 Regulates Organic Dust–Induced Airway Inflammation

Organic dust exposure in agricultural environments results in significant airway inflammatory diseases. Gram-positive cell wall components are present in high concentrations in animal farming dusts, but their role in mediating dust-induced airway inflammation is not clear. This study investigated the role of Toll-like receptor (TLR) 2, a pattern recognition receptor for gram-positive cell wall products, in regulating swine facility organic dust extract (DE)-induced airway inflammation in mice. Isolated lung macrophages from TLR2 knockout mice demonstrated reduced TNF-α, IL-6, keratinocyte chemoattractant/CXCL1, but not macrophage inflammatory protein-2/CXCL2 expression, after DE stimulation ex vivo. Next, using an established mouse model of intranasal inhalation challenge, we analyzed bronchoalveolar lavage fluid and lung tissue in TLR2-deficient and wild-type (WT) mice after single and repetitive DE challenge. Neutrophil influx and select cytokines/chemokines were significantly lower in TLR2-deficient mice at 5 and 24 hours after single DE challenge. After daily exposure to DE for 2 weeks, there were significant reductions in total cellularity, neutrophil influx, and TNF-α, IL-6, CXCL1, but not CXCL2 expression, in TLR2-deficient mice as compared with WT animals. Lung pathology revealed that bronchiolar inflammation, but not alveolar inflammation, was reduced in TLR2-deficient mice after repetitive exposure. Airway hyperresponsiveness to methacholine after dust exposure was similar in both groups. Finally, airway inflammatory responses in WT mice after challenge with a TLR2 agonist, peptidoglycan, resembled DE-induced responses. Collectively, these results demonstrate that the TLR2 pathway is important in regulating swine facility organic dust-induced airway inflammation, which suggests the importance of TLR2 agonists in mediating large animal farming-induced airway inflammatory responses.

αβ T Cells and a Mixed Th1/Th17 Response are Important in Organic Dust-Induced Airway Disease

BACKGROUND Organic dust exposure in agricultural environments induces an inflammatory response that attenuates over time, yet repetitive dust exposures result in chronic lung diseases. Animal models resembling this chronic lung inflammatory response have been developed, yet the underlying cellular mechanisms are not well defined. OBJECTIVE Because mice repetitively exposed to organic dust extracts (DE) display increased CD3+ T cell lung infiltrates, we sought to determine the phenotype and importance of these cells. METHODS Mice received swine confinement DE repetitively for 3 weeks by established intranasal inhalation protocol. Studies were conducted with peptidoglycan (PGN) because it is a major DE component in large animal farming environments and has shared similar biologic effects with DE. Enumeration of T cells and intracellular cytokine profiles were conducted by flow cytometry techniques. Whole lung homogenate cytokines were analyzed by multiplex immunoassay. T cell receptor (TCR) αβ knockouts were used to determine the functional importance of αβ-expressing T cells. RESULTS DE increased lung-associated CD3+CD4+ T cells and interleukin (IL)-17 (but not IL-4, interferon [IFN]-γ, IL-10) producing CD4+ T cells. PGN treatment resulted in increased IL-17 and IFN-γ producing CD4+ T cells and IFN-γ producing CD8+ T cells. Both DE and PGN augmented expression of cytokines associated with Th1 and Th17 polarization in lung homogenates. DE-induced lung mononuclear aggregates and bronchiolar compartment inflammation were significantly reduced in TCR knockout animals; however, neutrophil influx and alveolar compartment inflammation were not affected. CONCLUSION Studies demonstrated that DE and PGN exposure promote a Th1/Th17 lung microenvironment and that αβ-expressing T cells are important in mediating DE-induced lung pathologic conditions.

CD11c+/CD11b+ Cells Are Critical for Organic Dust–Elicited Murine Lung Inflammation

Organic dust exposure in the agricultural industry results in significant lung disease. Macrophage infiltrates are increased in the lungs after organic dust exposures, yet the phenotype and functional importance of these cells remain unclear. Using an established intranasal inhalation murine model of dust-induced lung inflammation, animals were treated once or daily for 3 weeks with swine confinement organic dust extract (DE). Repetitive DE treatment for 3 weeks resulted in significant increases in CD11c(+)/CD11b(+) macrophages in whole lung-associated tissue. These cells displayed increased costimulatory molecule (CD80 and CD86) expression, enhanced phagocytic ability, and an increased production of IL-6, CXCL1, and CXCL2. Similar findings were observed with the CD11c(+)/CD11b(+) macrophage infiltrate after repetitive exposure to peptidoglycan, a major DE component. To determine the functional importance of macrophages in mediating DE-induced airway inflammation, lung macrophages were selectively depleted using a well-established intranasal clodronate liposome depletion/suicide strategy. First, macrophage depletion by clodronate liposomes resulted in significant reductions in airway neutrophil influx and TNF-α and IL-6 production after a single exposure to DE. In contrast, after repetitive 3-week exposure to DE, airway lavage fluid and lung tissue neutrophils were significantly increased in clodronate liposome-treated mice compared with control mice. A histological examination of lung tissue demonstrated striking increases in alveolar and bronchiolar inflammation, as well as in the size and distribution of cellular aggregates in clodronate-liposome versus saline-liposome groups repetitively exposed to DE. These studies demonstrate that DE elicits activated CD11c(+)/CD11b(+) macrophages in the lung, which play a critical role in regulating the outcome of DE-induced airway inflammation.

Myeloid Differentiation Factor 88–Dependent Signaling Is Critical for Acute Organic Dust–Induced Airway Inflammation in Mice

Organic dust exposure within agricultural environments results in airway diseases. Toll-like receptor 2 (TLR2) and TLR4 only partly account for the innate response to these complex dust exposures. To determine the central pathway in mediating complex organic dust-induced airway inflammation, this study targeted the common adaptor protein, myeloid differentiation factor 88 (MyD88), and investigated the relative contributions of receptors upstream from this adaptor. Wild-type, MyD88, TLR9, TLR4, IL-1 receptor I (RI), and IL-18R knockout (KO) mice were challenged intranasally with organic dust extract (ODE) or saline, according to an established protocol. Airway hyperresponsiveness (AHR) was assessed by invasive pulmonary measurements. Bronchoalveolar lavage fluid was collected to quantitate leukocyte influx and cytokine/chemokine (TNF-α, IL-6, chemokine [C-X-C motif] ligands [CXCL1 and CXCL2]) concentrations. Lung tissue was collected for histopathology. Lung cell apoptosis was determined by a terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and lymphocyte influx and intercellular adhesion molecule-1 (ICAM-1) expression were assessed by immunohistochemistry. ODE-induced AHR was significantly attenuated in MyD88 KO mice, and neutrophil influx and cytokine/chemokine production were nearly absent in MyD88 KO animals after ODE challenges. Despite a near-absent airspace inflammatory response, lung parenchymal inflammation was increased in MyD88 KO mice after repeated ODE exposures. ODE-induced epithelial-cell ICAM-1 expression was diminished in MyD88 KO mice. No difference was evident in the small degree of ODE-induced lung-cell apoptosis. Mice deficient in TLR9, TLR4, and IL-18R, but not IL-1IR, demonstrated partial protection against ODE-induced neutrophil influx and cytokine/chemokine production. Collectively, the acute organic dust-induced airway inflammatory response is highly dependent on MyD88 signaling, and is dictated, in part, by important contributions from upstream TLRs and IL-18R.

Organic dust augments nucleotide-binding oligomerization domain expression via an NF-κB pathway to negatively regulate inflammatory responses

Nucleotide-binding oligomerization domain 2 (NOD2) is involved in innate immune responses to peptidoglycan degradation products. Peptidoglycans are important mediators of organic dust-induced airway diseases in exposed agriculture workers; however, the role of NOD2 in response to complex organic dust is unknown. Monocytes/macrophages were exposed to swine facility organic dust extract (ODE), whereupon NOD2 expression was evaluated by real-time PCR and Western blot. ODE induced significant NOD2 mRNA and protein expression at 24 and 48 h, respectively, which was mediated via a NF-κB signaling pathway as opposed to a TNF-α autocrine/paracrine mechanism. Specifically, NF-κB translocation increased rapidly following ODE stimulation as demonstrated by EMSA, and inhibition of the NF-κB pathway significantly reduced ODE-induced NOD2 expression. However, there was no significant reduction in ODE-induced NOD2 gene expression when TNF-α was inhibited or absent. Next, it was determined whether NOD2 regulated ODE-induced inflammatory cytokine production. Knockdown of NOD2 expression by small interfering RNA resulted in increased CXCL8 and IL-6, but not TNF-α production in response to ODE. Similarly, primary lung macrophages from NOD2 knockout mice demonstrated increased IL-6, CXCL1, and CXCL1, but not TNF-α, expression. Lastly, a higher degree of airway inflammation occurred in the absence of NOD2 following acute (single) and repetitive (3 wk) ODE exposure in an established in vivo murine model. In summary, ODE-induced NOD2 expression is directly dependent on NF-κB signaling, and NOD2 is a negative regulator of complex, organic dust-induced inflammatory cytokine/chemokine production in mononuclear phagocytes.

Pattern recognition scavenger receptor A/CD204 regulates airway inflammatory homeostasis following organic dust extract exposures

Abstract Exposure to agriculture organic dusts, comprised of a diversity of pathogen-associated molecular patterns, results in chronic airway diseases. The multi-functional class A macrophage scavenger receptor (SRA)/CD204 has emerged as an important class of pattern recognition receptors with broad ligand binding ability. The objective was to determine the role of SRA in mediating repetitive and post-inflammatory organic dust extract (ODE)-induced airway inflammation. Wild-type (WT) and SRA knockout (KO) mice were intra-nasally treated with ODE or saline daily for 3 weeks and immediately euthanized or allowed to recover for 1 week. Results show that lung histopathologic changes were increased in SRA KO mice as compared to WT following repetitive ODE exposures marked predominately by increased size and distribution of lymphoid aggregates. After a 1-week recovery from daily ODE treatments, there was significant resolution of lung injury in WT mice, but not SRA KO animals. The increased lung histopathology induced by ODE treatment was associated with decreased accumulation of neutrophils, but greater accumulation of CD4+ T-cells. The lung cytokine milieu induced by ODE was consistent with a TH1/TH17 polarization in both WT and SRA KO mice. Overall, the data demonstrate that SRA/CD204 plays an important role in the normative inflammatory lung response to ODE, as evidenced by the enhanced dust-mediated injury viewed in the absence of this receptor.

Organic Dust, Lipopolysaccharide, and Peptidoglycan Inhalant Exposures Result in Bone Loss/Disease

Skeletal health consequences associated with chronic inflammatory respiratory disease, and particularly chronic obstructive pulmonary disease (COPD), contribute to overall disease morbidity. Agricultural environmental exposures induce significant airway diseases, including COPD. However, animal models to understand inhalant exposure-induced lung injury and bone disease have not been described. Using micro-computed tomography (micro-CT) imaging technology and histology, bone quantity and quality measurements were investigated in mice after repetitive intranasal inhalation exposures to complex organic dust extracts (ODEs) from swine confinement facilities. Comparison experiments with LPS and peptidoglycan (PGN) alone were also performed. After 3 weeks of repetitive ODE inhalation exposure, significant loss of bone mineral density and trabecular bone volume fraction was evident, with altered morphological microarchitecture changes in the trabecular bone, compared with saline-treated control animals. Torsional resistance was also significantly reduced. Compared with saline treatment, ODE-treated mice demonstrated decreased collagen and proteoglycan content in their articular cartilage, according to histopathology. Significant bone deterioration was also evident after repetitive intranasal inhalant treatment with LPS and PGN. These findings were not secondary to animal distress, and not entirely dependent on the degree of induced lung parenchymal inflammation. Repetitive LPS treatment demonstrated the most pronounced changes in bone parameters, and PGN treatment resulted in the greatest lung parenchymal inflammatory changes. Collectively, repetitive inhalation exposures to noninfectious inflammatory agents such as complex organic dust, LPS, and PGN resulted in bone loss. This animal model may contribute to efforts toward understanding the mechanisms and evaluating the therapeutics associated with adverse skeletal health consequences after subchronic airway injury.

Chronic obstructive pulmonary disease patients have greater systemic responsiveness to ex vivo stimulation with swine dust extract and its components versus healthy volunteers

Chronic obstructive pulmonary disease (COPD) is characterized by an airway and systemic inflammatory response. Bioaerosols/organic dusts are important agricultural pollutants that may lead to COPD. These environments are complex, containing a rich source of various microbial components. The objective of this study was to determine whether individuals with COPD have enhanced systemic responsiveness to settled swine facility organic dust extract (ODE) or its main pathogenic components (peptidoglycan [PGN], lipopolysaccharide [LPS]) versus healthy volunteers. A modified whole blood assay (WBA) that included occupational levels of ODE and concentrations of LPS and PGN found in ODE was used to determine systemic responsiveness (mediator release), and sputum inflammatory markers were measured to explore for systemic and airway associations. Sputum samples were evaluated for cell counts, and tumor necrosis factor (TNF)-α, interleukin (IL)-8/CXCL8, IL-6, and IL-10. Ex vivo whole blood stimulation with ODE, LPS, and PGN each resulted in significant mediator release in all subjects, with the highest occurring with ODE; PGN resulted in significantly enhanced TNF-α and IL-8 as compared to LPS. COPD subjects demonstrated greater systemic responsiveness using the modified WBA versus healthy controls. Within COPD subjects, blood baseline TNF-α, IL-8, and IL-10 and ODE-, PGN-, and LPS-stimulated IL-8 levels significantly correlated with lung function. In conclusion, dust-induced mediator release was robust, and PGN, in part, resembled dust-induced mediator release. Subjects with COPD demonstrated increased mediator release following ex vivo whole blood stimulation with bioaerosol components, suggesting that circulating blood cells in COPD subjects may be primed to respond greater to microbial/inflammatory insult.

Vitamin D treatment modulates organic dust-induced cellular and airway inflammatory consequences.

Exposure to organic dusts elicits airway inflammatory diseases. Vitamin D recently has been associated with various airway inflammatory diseases, but its role in agricultural organic dust exposures is unknown. This study investigated whether vitamin D reduces organic dust–induced inflammatory outcomes in cell culture and animal models. Organic dust extracts obtained from swine confinement facilities induced neutrophil chemokine production (human IL‐8, murine CXCL1/CXCL2). Neutrophil chemokine induction was reduced in human blood monocytes, human bronchial epithelial cells, and murine lung slices pretreated with 1,25‐(OH)2D3. Intranasal inhalation of organic dust extract induced neutrophil influx, and CXCL1/CXCL2 release was also decreased in mice fed a relatively high vitamin D diet as compared to mice fed a low vitamin D diet. These findings were associated with reduced tracheal epithelial cell PKCα and PKCε activity and whole lung TLR2 and TLR4 gene expression. Collectively, vitamin D plays a role in modulating organic dust–induced airway inflammatory outcomes.

Protein kinase C epsilon is important in modulating organic-dust-induced airway inflammation.

ABSTRACT Organic dust samples from swine confinement facilities elicit pro-inflammatory cytokine/chemokine release from bronchial epithelial cells and monocytes, dependent, in part, upon dust-induced activation of the protein kinase C (PKC) isoform, PKCε. PKCε is also rapidly activated in murine tracheal epithelial cells following in vivo organic dust challenges, yet the functional role of PKCε in modulating dust-induced airway inflammatory outcomes is not defined. Utilizing an established intranasal inhalation animal model, experiments investigated the biologic and physiologic responses following organic dust extract (ODE) treatments in wild-type (WT) and PKCε knock-out (KO) mice. We found that neutrophil influx increased more than twofold in PKCε KO mice following both a one-time challenge and 3 weeks of daily challenges with ODE as compared with WT mice. Lung pathology revealed increased bronchiolar and alveolar inflammation, lymphoid aggregates, and T cell influx in ODE-treated PKCε KO mice. Airway hyperresponsiveness to methacholine increased in PKCε KO + ODE to a greater magnitude than WT + ODE animals. There were no significant differences in cytokine/chemokine release elicited by ODE treatment between groups. However, ODE-induced nitric oxide (NO) production differed in that ODE exposure increased nitrate levels in WT mice but not in PKCε KO mice. Moreover, ODE failed to upregulate NO from ex vivo stimulated PKCε KO lung macrophages. Collectively, these studies demonstrate that PKCε-deficient mice were hypersensitive to organic dust exposure and suggest that PKCε is important in the normative lung inflammatory response to ODE. Dampening of ODE-induced NO may contribute to these enhanced inflammatory findings.