Lung-delivered IL-10 therapy elicits beneficial effects via immune modulation in organic dust exposure-induced lung inflammation.

Efficacious therapeutic options capable of resolving inflammatory lung disease associated with environmental and occupational exposures are lacking. This study sought to determine the preclinical therapeutic potential of lung-delivered recombinant interleukin (IL)-10 therapy following acute organic dust exposure in mice. Here, C57BL/6J mice were intratracheally instilled with swine confinement organic dust extract (ODE) (12.5%, 25%, 50% concentrations) with IL-10 (1 µg) treatment or vehicle control intratracheally-administered three times: 5 hr post-exposure and then daily for 2 days. The results showed that IL-10 treatment reduced ODE (25%)-induced weight loss by 66% and 46% at Day 1 and Day 2 post-exposure, respectively. IL-10 treatment reduced ODE (25%, 50%)-induced lung levels of TNFα (-76%, -83% [reduction], respectively), neutrophil chemoattractant CXCL1 (-51%, -60%), and lavage fluid IL-6 (-84%, -89%). IL-10 treatment reduced ODE (25%, 50%)-induced lung neutrophils (-49%, -70%) and recruited CD11cintCD11b+ monocyte-macrophages (-49%, -70%). IL-10 therapy reduced ODE-associated expression of antigen presentation (MHC Class II, CD80, CD86) and inflammatory (Ly6C) markers and increased anti-inflammatory CD206 expression on CD11cintCD11b+ cells. ODE (12.5%, 25%)-induced lung pathology was also reduced with IL-10 therapy. In conclusion, the studies here showed that short-term, lung-delivered IL-10 treatment induced a beneficial response in reducing inflammatory consequences (that were also associated with striking reduction in recruited monocyte-macrophages) following acute complex organic dust exposure.

Targeting transitioning lung monocytes/macrophages as treatment strategies in lung disease related to environmental exposures.

BACKGROUND: Environmental/occupational exposures cause significant lung diseases. Agricultural organic dust extracts (ODE) and bacterial component lipopolysaccharide (LPS) induce recruited, transitioning murine lung monocytes/macrophages, yet their cellular role remains unclear. METHODS: CCR2 RFP+ mice were intratracheally instilled with high concentration ODE (25%), LPS (10 µg), or gram-positive peptidoglycan (PGN, 100 µg) for monocyte/macrophage cell-trafficking studies. CCR2 knockout (KO) mice and administration of intravenous clodronate liposomes strategies were employed to reduce circulating monocytes available for lung recruitment following LPS exposure. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected. Pro-inflammatory and/or pro-fibrotic cytokines, chemokines, and lung extracellular matrix mediators were quantitated by ELISA. Infiltrating lung cells including monocyte/macrophage subpopulations, neutrophils, and lymphocytes were characterized by flow cytometry. Lung histopathology, collagen content, vimentin, and post-translational protein citrullination and malondialdehyde acetaldehyde (MAA) modification were quantitated. Parametric statistical tests (one-way ANOVA, Tukey'smultiple comparison) and nonparametric statistical (Kruskal-Wallis, Dunn's multiple comparison) tests were used following Shapiro-Wilk testing for normality. RESULTS: Intratracheal instillation of ODE, LPS, or PGN robustly induced the recruitment of inflammatory CCR2+ CD11cintCD11bhi monocytes/macrophages and both CCR2+ and CCR2- CD11c-CD11bhi monocytes at 48 h. There were also increases in CCR2+ CD4+ and CD8+ T cells and NK cells. Despite reductions in LPS-induced lung infiltrating CD11cintCD11bhi cells (54% reduction), CCR2 knockout (KO) mice were not protected against LPS-induced inflammatory and pro-fibrotic consequences. Instead, compensatory increases in lung neutrophils and CCL2 and CCL7 release occurred. In contrast, the depletion of circulating monocytes through the administration of intravenous clodronate (vs. vehicle) liposomes 24 h prior to LPS exposure reduced LPS-induced infiltrating CD11cintCD11bhi monocyte-macrophage subpopulation by 59% without compensatory changes in other cell populations. Clodronate liposome pre-treatment significantly reduced LPS-induced IL-6 (66% reduction), matrix metalloproteinases (MMP)-3 (36%), MMP-8 (57%), tissue inhibitor of metalloproteinases (61%), fibronectin (38%), collagen content (22%), and vimentin (40%). LPS-induced lung protein citrullination and MAA modification, post-translational modifications implicated in lung disease, were reduced (39% and 48%) with clodronate vs. vehicle liposome. CONCLUSION: Highly concentrated environmental/occupational exposures induced the recruitment of CCR2+ and CCR2- transitioning monocyte-macrophage and monocyte subpopulations and targeting peripheral monocytes may reduce the adverse lung consequences resulting from exposures to LPS-enriched inhalants.

Combined repetitive inhalant endotoxin and collagen-induced arthritis drive inflammatory lung disease and arthritis severity in a testosterone-dependent manner.

Respiratory-related diseases are a leading cause of death in rheumatoid arthritis (RA) and are disproportionately higher in men, which may be attributable to environmental risk factors. Animal studies have demonstrated potentiated autoimmunity, arthritis, and profibrotic/inflammatory lung disease with a combination of airborne exposures and collagen-induced arthritis (CIA). This study aimed to determine whether hormone-dependent differences explained these observations. Arthritis-prone male intact and castrated DBA/1J mice received intranasal inhalation of lipopolysaccharide (LPS) daily for 5 wk and CIA induction. Arthritis scores and serum pentraxin-2 levels were increased in castrated versus intact mice. In contrast, airway cell influx, lung tissue infiltrates, and lung levels of proinflammatory and profibrotic markers (C5a, IL-33, and matrix metalloproteinases) were reduced in castrated versus intact mice. CIA + LPS-induced lung histopathology changes and the expression of lung autoantigens including malondialdehyde acetaldehyde (MAA)- and citrulline (CIT)-modified proteins and vimentin were reduced in castrated animals. There were no differences in serum anti-MAA or anti-CIT protein antibody (ACPA) levels or serum pentraxin levels between groups. Testosterone replacement led to a reversal of several lung inflammatory/profibrotic endpoints noted earlier in castrated male CIA + LPS-treated mice with testosterone supplementation promoting neutrophil influx, MAA expression, and TNF-α, IL-6, and MMP-9. These findings imply that testosterone contributes to lung and arthritis inflammatory responses following CIA + LPS coexposure, but not to systemic autoantibody responses. The CIA + LPS model provides a paradigm for investigations focused on the mechanistic underpinnings for epidemiologic and phenotypic sex differences in RA-related lung disease.NEW & NOTEWORTHY Our study shows that testosterone acts as a key immunomodulatory hormone contributing to critical features of rheumatoid arthritis (RA)-associated lung disease in the setting of airborne endotoxin (lipopolysaccharide; LPS) exposures and concomitant arthritis induction in mice. The exaggerated airway inflammation observed following combined exposures in male mice was accompanied by increases in profibrotic mediators, netosis, and increased expression of lung autoantigens, all relevant to the pathogenesis of lung disease in arthritis.

Expansion of distinct peripheral blood myeloid cell subpopulations in patients with rheumatoid arthritis-associated interstitial lung disease.

OBJECTIVES: Interstitial lung disease (ILD) is associated with significant mortality in rheumatoid arthritis (RA) patients with key cellular players remaining largely unknown. This study aimed to characterize inflammatory and myeloid derived suppressor cell (MDSC) subpopulations in RA-ILD as compared to RA, idiopathic pulmonary fibrosis (IPF) without autoimmunity, and controls. METHODS: Peripheral blood was collected from patients with RA, RA-ILD, IPF, and controls (N = 60, 15/cohort). Myeloid cell subpopulations were identified phenotypically by flow cytometry using the following markers:CD45,CD3,CD19,CD56,CD11b,HLA-DR,CD14,CD16,CD15,CD125,CD33. Functionality of subsets were identified with intracellular arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS) expression. RESULTS: There was increased intermediate (CD14++CD16+) and nonclassical (CD14+/-CD16++) and decreased classical (CD14++CD16-) monocytes in RA, RA-ILD, and IPF vs. control. Intermediate monocytes were higher and classical monocytes were lower in RA-ILD vs. RA but not IPF. Monocytic (m)MDSCs were higher in RA-ILD vs. control and RA but not IPF. Granulocytic (g)MDSCs did not significantly differ. In contrast, neutrophils were increased in IPF and RA-ILD patients with elevated expression of Arg-1 sharing similar dimensional clustering pattern. Eosinophils were increased in RA-ILD vs. controls, RA and IPF. Across cohorts, iNOS was decreased in intermediate/nonclassical monocytes but increased in mMDSCs vs. classical monocytes. In RA-ILD, iNOS positive mMDSCs were increased versus classic monocytes. CONCLUSIONS: Myeloid cell subpopulations are significantly modulated in RA-ILD patients with expansion of CD16+ monocytes, mMDSCs, and neutrophils, a phenotypic profile more aligned with IPF than other RA patients. Eosinophil expansion was unique to RA-ILD, potentially facilitating disease pathogenesis and providing a future therapeutic target.

Post-endotoxin exposure-induced lung inflammation and resolution consequences beneficially impacted by lung-delivered IL-10 therapy.

Although lung diseases typically result from long-term exposures, even a robust, one-time exposure can result in long-lasting consequences. Endotoxin is a ubiquitous environmental/occupational inflammatory agent often used to model airway inflammation. Using a murine model, the return to lung homeostasis following high dose inhalant lipopolysaccharide (LPS, 10-100 µg) exposure were delineated over 2 weeks. LPS-induced rapid weight loss, release of proinflammatory mediators, and inflammatory cell influx with prolonged persistence of activated macrophages CD11c+CD11b+ and recruited/transitioning CD11cintCD11b+ monocyte-macrophages out to 2 weeks. Next, lung-delivered recombinant (r) interleukin (IL)-10 was intratracheally administered for 3 doses initiated 5 h following LPS (10 µg) exposure for 2 days. IL-10 therapy reduced LPS-induced weight loss and increased blood glucose levels. Whereas there was no difference in LPS-induced bronchoalveolar lavage airway fluid cellular influx, total lung cell infiltrates were reduced (37%) with rIL-10 treatment. Post-LPS exposure treatment with rIL-10 strikingly reduced lavage fluid and lung homogenate levels of tumor necrosis factor-α (88% and 93% reduction, respectively), IL-6 (98% and 94% reduction), CXCL1 (66% and 75% reduction), and CXCL2 (47% and 67% reduction). LPS-induced recruited monocyte-macrophages (CD11cintCD11b+) were reduced (68%) with rIL-10. Correspondingly, LPS-induced lung tissue CCR2+ inflammatory monocyte-macrophage were reduced with rIL-10. There were also reductions in LPS-induced lung neutrophils, lymphocyte subpopulations, collagen content, and vimentin expression. These findings support the importance of studying resolution processes for the development of treatment after unintended environmental/occupational biohazard exposures. Short-term, lung-delivered rIL-10 favorably hastened inflammatory recovery processes following acute, high dose inhalant LPS exposure.

Relationships of serum CC16 levels with smoking status and lung function in COPD.

BACKGROUND: The club cell secretory protein (CC16) has anti-inflammatory and antioxidant effects, and low CC16 serum levels have been associated with both risk and progression of COPD, yet the interaction between smoking and CC16 on lung function outcomes remains unknown. METHODS: Utilizing cross-sectional data on United States veterans, CC16 serum concentrations were measured by ELISA and log transformed for analyses. Spirometry was conducted and COPD status was defined by post-bronchodilator FEV1/FVC ratio < 0.7. Smoking measures were self-reported on questionnaire. Multivariable logistic and linear regression were employed to examine associations between CC16 levels and COPD, and lung function with adjustment for covariates. Unadjusted Pearson correlations described relationships between CC16 level and lung function measures, pack-years smoked, and years since smoking cessation. RESULTS: The study population (N = 351) was mostly male, white, with an average age over 60 years. An interaction between CC16 and smoking status on FEV1/FVC ratio was demonstrated among subjects with COPD (N = 245, p = 0.01). There was a positive correlation among former smokers and negative correlation among current or never smokers with COPD. Among former smokers with COPD, CC16 levels were also positively correlated with years since smoking cessation, and inversely related with pack-years smoked. Increasing CC16 levels were associated with lower odds of COPD (ORadj = 0.36, 95% CI 0.22-0.57, Padj < 0.0001). CONCLUSIONS: Smoking status is an important effect modifier of CC16 relationships with lung function. Increasing serum CC16 corresponded to increases in FEV1/FVC ratio in former smokers with COPD versus opposite relationships in current or never smokers. Additional longitudinal studies may be warranted to assess relationship of CC16 with smoking cessation on lung function among subjects with COPD.

Increased susceptibility to organic dust exposure-induced inflammatory lung disease with enhanced rheumatoid arthritis-associated autoantigen expression in HLA-DR4 transgenic mice.

Immunogenetic as well as environmental and occupational exposures have been linked to the development of rheumatoid arthritis (RA), RA-associated lung disease, and other primary lung disorders. Importantly, various inhalants can trigger post-translational protein modifications, resulting in lung autoantigen expression capable of stimulating pro-inflammatory and/or pro-fibrotic immune responses. To further elucidate gene-environment interactions contributing to pathologic lung inflammation, we exploited an established model of organic dust extract (ODE) exposure with and without collagen-induced arthritis (CIA) in C57BL/6 wild type (WT) versus HLA-DR4 transgenic mice. ODE-induced airway infiltration driven by neutrophils was significantly increased in DR4 versus WT mice, with corresponding increases in bronchoalveolar lavage fluid (BALF) levels of TNF-⍺, IL-6, and IL-33. Lung histopathology demonstrated increased number of ectopic lymphoid aggregates comprised of T and B cells following ODE exposure in DR4 mice. ODE also induced citrullination, malondialdehyde acetaldehyde (MAA) modification, and vimentin expression that co-localized with MAA and was enhanced in DR4 mice. Serum and BALF anti-MAA antibodies were strikingly increased in ODE-treated DR4 mice. Coupling ODE exposure with Type II collagen immunization (CIA) resulted in similarly augmented pro-inflammatory lung profiles in DR4 mice (relative to WT mice) that was accompanied by a profound increase in infiltrating lung CD4+ and CD8+ T cells as well as CD19+CD11b+ autoimmune B cells. Neither modeling strategy induced significant arthritis. These findings support a model in which environmental insults trigger enhanced post-translational protein modification and lung inflammation sharing immunopathological features with RA-associated lung disease in the selected immunogenetic background of HLA-DR4 mice.

The impact of airborne endotoxin exposure on rheumatoid arthritis-related joint damage, autoantigen expression, autoimmunity, and lung disease.

Airborne biohazards are risk factors in the development and severity of rheumatoid arthritis (RA) and RA-associated lung disease, yet the mechanisms explaining this relationship remain unclear. Lipopolysaccharide (LPS, endotoxin) is a ubiquitous inflammatory agent in numerous environmental and occupational air pollutant settings recognized to induce airway inflammation. Combining repetitive LPS inhalation exposures with the collagen induced arthritis (CIA) model, DBA1/J mice were assigned to either: sham (saline injection/saline inhalation), CIA (CIA/saline), LPS (saline/LPS 100 ng inhalation), or CIA + LPS for 5 weeks. Serum anti-citrullinated (CIT) protein antibody (ACPA) and anti-malondialdehyde-acetaldehyde (MAA) antibodies were strikingly potentiated with co-exposure (CIA + LPS). CIT- and MAA-modified lung proteins were increased with co-exposure and co-localized across treatment groups. Inhaled LPS exacerbated arthritis with CIA + LPS > LPS > CIA versus sham. Periarticular bone loss was demonstrated in CIA and CIA + LPS but not in LPS alone. LPS induced airway inflammation and neutrophil infiltrates were reduced with co-exposure (CIA + LPS). Potentially signaling transition to pro-fibrotic processes, there were increased infiltrates of activated CD11c+CD11b+ macrophages and transitioning CD11c+CD11bint monocyte-macrophage populations with CIA + LPS. Moreover, several lung remodeling proteins including fibronectin and matrix metalloproteinases as well as complement C5a were potentiated with CIA + LPS compared to other treatment groups. IL-33 concentrations in lung homogenates were enhanced with CIA + LPS with IL-33 lung staining driven by LPS. IL-33 expression was also significantly increased in lung tissues from patients with RA-associated lung disease (N = 8) versus controls (N = 7). These findings suggest that patients with RA may be more susceptible to developing interstitial lung disease following airborne biohazard exposures enriched in LPS.

High-throughput analysis of lung immune cells in a combined murine model of agriculture dust-triggered airway inflammation with rheumatoid arthritis.

Rheumatoid arthritis (RA)-associated lung disease is a leading cause of mortality in RA, yet the mechanisms linking lung disease and RA remain unknown. Using an established murine model of RA-associated lung disease combining collagen-induced arthritis (CIA) with organic dust extract (ODE)-induced airway inflammation, differences among lung immune cell populations were analyzed by single cell RNA-sequencing. Additionally, four lung myeloid-derived immune cell populations including macrophages, monocytes/macrophages, monocytes, and neutrophils were isolated by fluorescence cell sorting and gene expression was determined by NanoString analysis. Unsupervised clustering revealed 14 discrete clusters among Sham, CIA, ODE, and CIA+ODE treatment groups: 3 neutrophils (inflammatory, resident/transitional, autoreactive/suppressor), 5 macrophages (airspace, differentiating/recruited, recruited, resident/interstitial, and proliferative airspace), 2 T-cells (differentiating and effector), and a single cluster each of inflammatory monocytes, dendritic cells, B-cells and natural killer cells. Inflammatory monocytes, autoreactive/suppressor neutrophils, and recruited/differentiating macrophages were predominant with arthritis induction (CIA and CIA+ODE). By specific lung cell isolation, several interferon-related and autoimmune genes were disproportionately expressed among CIA and CIA+ODE (e.g. Oasl1, Oas2, Ifit3, Gbp2, Ifi44, and Zbp1), corresponding to RA and RA-associated lung disease. Monocytic myeloid-derived suppressor cells were reduced, while complement genes (e.g. C1s1 and Cfb) were uniquely increased in CIA+ODE mice across cell populations. Recruited and inflammatory macrophages/monocytes and neutrophils expressing interferon-, autoimmune-, and complement-related genes might contribute towards pro-fibrotic inflammatory lung responses following airborne biohazard exposures in setting of autoimmune arthritis and could be predictive and/or targeted to reduce disease burden.

MyD88 regulates a prolonged adaptation response to environmental dust exposure-induced lung disease.

BACKGROUND: Environmental organic dust exposures enriched in Toll-like receptor (TLR) agonists can reduce allergic asthma development but are associated with occupational asthma and chronic bronchitis. The TLR adaptor protein myeloid differentiation factor88 (MyD88) is fundamental in regulating acute inflammatory responses to organic dust extract (ODE), yet its role in repetitive exposures is unknown and could inform future strategies. METHODS: Wild-type (WT) and MyD88 knockout (KO) mice were exposed intranasally to ODE or saline daily for 3 weeks (repetitive exposure). Repetitively exposed animals were also subsequently rested with no treatments for 4 weeks followed by single rechallenge with saline/ODE. RESULTS: Repetitive ODE exposure induced neutrophil influx and release of pro-inflammatory cytokines and chemokines were profoundly reduced in MyD88 KO mice. In comparison, ODE-induced cellular aggregates, B cells, mast cell infiltrates and serum IgE levels remained elevated in KO mice and mucous cell metaplasia was increased. Expression of ODE-induced tight junction protein(s) was also MyD88-dependent. Following recovery and then rechallenge with ODE, inflammatory mediators, but not neutrophil influx, was reduced in WT mice pretreated with ODE coincident with increased expression of IL-33 and IL-10, suggesting an adaptation response. Repetitively exposed MyD88 KO mice lacked inflammatory responsiveness upon ODE rechallenge. CONCLUSIONS: MyD88 is essential in mediating the classic airway inflammatory response to repetitive ODE, but targeting MyD88 does not reduce mucous cell metaplasia, lymphocyte influx, or IgE responsiveness. TLR-enriched dust exposures induce a prolonged adaptation response that is largely MyD88-independent. These findings demonstrate the complex role of MyD88-dependent signaling during acute vs. chronic organic dust exposures.

Amphiregulin modulates murine lung recovery and fibroblast function following exposure to agriculture organic dust.

Exposure to agricultural bioaerosols can lead to chronic inflammatory lung diseases. Amphiregulin (AREG) can promote the lung repair process but can also lead to fibrotic remodeling. The objective of this study was to determine the role of AREG in altering recovery from environmental dust exposure in a murine in vivo model and in vitro using cultured human and murine lung fibroblasts. C57BL/6 mice were intranasally exposed to swine confinement facility dust extract (DE) or saline daily for 1 wk or allowed to recover for 3-7 days while being treated with an AREG-neutralizing antibody or recombinant AREG. Treatment with the anti-AREG antibody prevented resolution of DE exposure-induced airway influx of total cells, neutrophils, and macrophages and increased levels of TNF-α, IL-6, and CXCL1. Neutrophils and activated macrophages (CD11c+CD11bhi) persisted after recovery in lung tissues of anti-AREG-treated mice. In murine and human lung fibroblasts, DE induced the release of AREG and inflammatory cytokines. Fibroblast recellularization of primary human lung mesenchymal matrix scaffolds and wound closure was inhibited by DE and enhanced with recombinant AREG alone. AREG treatment rescued the DE-induced inhibitory fibroblast effects. AREG intranasal treatment for 3 days during recovery phase reduced repetitive DE-induced airway inflammatory cell influx and cytokine release. Collectively, these studies demonstrate that inhibition of AREG reduced, whereas AREG supplementation promoted, the airway inflammatory recovery response following environmental bioaerosol exposure, and AREG enhanced fibroblast function, suggesting that AREG could be targeted in agricultural workers repetitively exposed to organic dust environments to potentially prevent and/or reduce disease.

Combined Collagen-Induced Arthritis and Organic Dust-Induced Airway Inflammation to Model Inflammatory Lung Disease in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is characterized by extra-articular involvement including lung disease, yet the mechanisms linking the two conditions are poorly understood. The collagen-induced arthritis (CIA) model was combined with the organic dust extract (ODE) airway inflammatory model to assess bone/joint-lung inflammatory outcomes. DBA/1J mice were intranasally treated with saline or ODE daily for 5 weeks. CIA was induced on days 1 and 21. Treatment groups included sham (saline injection/saline inhalation), CIA (CIA/saline), ODE (saline/ODE), and CIA + ODE (CIA/ODE). Arthritis inflammatory scores, bones, bronchoalveolar lavage fluid, lung tissues, and serum were assessed. In DBA/1J male mice, arthritis was increased in CIA + ODE > CIA > ODE versus sham. Micro-computed tomography (µCT) demonstrated that loss of BMD and volume and deterioration of bone microarchitecture was greatest in CIA + ODE. However, ODE-induced airway neutrophil influx and inflammatory cytokine/chemokine levels in lavage fluids were increased in ODE > CIA + ODE versus sham. Activated lung CD11c+ CD11b+ macrophages were increased in ODE > CIA + ODE > CIA pattern, whereas lung hyaluronan, fibronectin, and amphiregulin levels were greatest in CIA + ODE. Serum autoantibody and inflammatory marker concentrations varied among experimental groups. Compared with male mice, female mice showed less articular and pulmonary disease. The interaction of inhalation-induced airway inflammation and arthritis induction resulted in compartmentalized responses with the greatest degree of arthritis and bone loss in male mice with combined exposures. Data also support suppression of the lung inflammatory response, but increases in extracellular matrix protein deposition/interstitial disease in the setting of arthritis. This coexposure model could be exploited to better understand and treat RA-lung disease. © 2019 American Society for Bone and Mineral Research.

Ovalbumin-sensitized mice have altered airway inflammation to agriculture organic dust.

Agriculture exposures are associated with reducing the risk of allergy and asthma in early life; yet, repeated exposures later in life are associated with chronic bronchitis and obstructive pulmonary diseases. The objective of this study was to investigate the airway inflammatory response to organic dust extract (ODE) in mice with established ovalbumin (OVA)-induced experimental asthma. C57BL/6 mice were either OVA sensitized/aerosol-exposed or saline (Sal) sensitized/aerosol-challenged. Both groups were then subsequently challenged once with intranasal saline or swine confinement ODE to obtain 4 treatment groups of Sal-Sal, Sal-ODE, OVA-Sal, and OVA-ODE. Airway hyper-responsiveness (AHR) to methacholine, bronchiolar lavage fluid, lung tissues, and serum were collected. Intranasal inhalation of ODE in OVA-treated (asthmatic) mice (OVA-ODE) increased AHR and total cellular influx marked by elevated neutrophil and eosinophil counts. Flow cytometry analysis further demonstrated that populations of CD11chi dendritic cells (DC), CD3+ T cells, CD19+ B cells, and NKp46+ group 3 innate lymphoid cells (ILC3) were increased in lavage fluid of OVA-ODE mice as compared to ODE or OVA alone. Alveolar macrophages, DC, and T cells were significantly increased with co-exposure to OVA-ODE as compared to OVA alone. Lung ILC2 and ILC3 were only increased in OVA-Sal mice. Cytokine/chemokine levels varied with exposure to OVA-ODE reflecting an additive mixture of the pro- and allergic-inflammatory profiles. Collectively, ODE increased airway inflammatory cells and chemotactic mediator release in allergic (OVA) sensitized mice to suggest that persons with allergy/asthma be identified and warned prior to the occupational exposure of potentially worsening airway disease.

MyD88 controls airway epithelial Muc5ac expression during TLR activation conditions from agricultural organic dust exposure.

Inflammation from airborne microbes can overwhelm compensatory mucociliary clearance mechanisms, leading to mucous cell metaplasia. Toll-like receptor (TLR) activation via myeloid differentiation factor 88 (MyD88) signaling is central to pathogen responses. We have previously shown that agricultural organic dust extract (ODE), with abundant microbial component diversity, activates TLR-induced airway inflammation. With the use of an established model, C57BL/6J wild-type (WT) and global MyD88 knockout (KO) mice were treated with intranasal inhalation of ODE or saline, daily for 1 wk. ODE primarily increased mucin (Muc)5ac levels relative to Muc5b. Compared with ODE-challenged WT mice, ODE-challenged, MyD88-deficient mice demonstrated significantly increased Muc5ac immunostaining, protein levels by immunoblot, and expression by quantitative PCR. The enhanced Muc5ac levels in MyD88-deficient mice were not explained by differences in the differentiation program of airway secretory cells in naïve mice. Increased Muc5ac levels in MyD88-deficient mice were also not explained by augmented inflammation, IL-17A, or neutrophil elastase levels. Furthermore, the enhanced airway mucins in the MyD88-deficient mice were not due to defective secretion, as the mucin secretory capacity of MyD88-KO mice remained intact. Finally, ODE-induced Muc5ac levels were enhanced in MyD88-deficient airway epithelial cells in vitro. In conclusion, MyD88 deficiency enhances airway mucous cell metaplasia under environments with high TLR activation.

Sex differences impact the lung-bone inflammatory response to repetitive inhalant lipopolysaccharide exposures in mice.

Skeletal health consequences associated with inflammatory diseases of the airways significantly contribute to morbidity. Sex differences have been described independently for lung and bone diseases. Repetitive inhalant exposure to lipopolysaccharide (LPS) induces bone loss and deterioration in male mice, but comparison effects in females are unknown. Using an intranasal inhalation exposure model, 8-week-old C57BL/6 male and female mice were treated daily with LPS (100 ng) or saline for 3 weeks. Bronchoalveolar lavage fluids, lung tissues, tibias, bone marrow cells, and blood were collected. LPS-induced airway neutrophil influx, interleukin (IL)-6 and neutrophil chemoattractant levels, and bronchiolar inflammation were exaggerated in male animals as compared to female mice. Trabecular bone micro-CT imaging and analysis of the proximal tibia were conducted. Inhalant LPS exposures lead to deterioration of bone quality only in male mice (not females) marked by decreased bone mineral density, bone volume/tissue volume ratio, trabecular thickness and number, and increased bone surface-to-bone volume ratio. Serum pentraxin-2 levels were modulated by sex differences and LPS exposure. In proof-of-concept studies, ovarectomized female mice demonstrated LPS-induced bone deterioration, and estradiol supplementation of ovarectomized female mice and control male mice protected against LPS-induced bone deterioration findings. Collectively, sex-specific differences exist in LPS-induced airway inflammatory consequences with significant differences found in bone quantity and quality parameters. Male mice demonstrated susceptibility to bone loss and female animals were protected, which was modulated by estrogen. Therefore, sex differences influence the biologic response in the lung-bone inflammatory axis in response to inhalant LPS exposures.

A role for B cells in organic dust induced lung inflammation.

BACKGROUND: Agriculture organic dust exposures induce lung disease with lymphoid aggregates comprised of both T and B cells. The precise role of B cells in mediating lung inflammation is unknown, yet might be relevant given the emerging role of B cells in obstructive pulmonary disease and associated autoimmunity. METHODS: Using an established animal model, C57BL/6 wild-type (WT) and B-cell receptor (BCR) knock-out (KO) mice were repetitively treated with intranasal inhalation of swine confinement organic dust extract (ODE) daily for 3 weeks and lavage fluid, lung tissues, and serum were collected. RESULTS: ODE-induced neutrophil influx in lavage fluid was not reduced in BCR KO animals, but there was reduction in TNF-α, IL-6, CXCL1, and CXCL2 release. ODE-induced lymphoid aggregates failed to develop in BCR KO mice. There was a decrease in ODE-induced lung tissue CD11c+CD11b+ exudative macrophages and compensatory increase in CD8+ T cells in lavage fluid of BCR KO animals. Compared to saline, there was an expansion of conventional B2-, innate B1 (CD19+CD11b+CD5+/-)-, and memory (CD19+CD273+/-CD73+/-) B cells following ODE exposure in WT mice. Autoreactive responses including serum IgG anti-citrullinated protein antibody (ACPA) and anti-malondialdehyde-acetaldehyde (MAA) autoantibodies were increased in ODE treated WT mice as compared to saline control. B cells and serum immunoglobulins were not detected in BCR KO animals. CONCLUSIONS: Lung tissue staining for citrullinated and MAA modified proteins were increased in ODE-treated WT animals, but not BCR KO mice. These studies show that agriculture organic dust induced lung inflammation is dependent upon B cells, and dust exposure induces an autoreactive response.

Reduced microRNA-503 expression augments lung fibroblast VEGF production in chronic obstructive pulmonary disease.

Alterations in microRNA (miRNA) expression may contribute to COPD pathogenesis. In COPD, lung fibroblast repair functions are altered in multiple ways, including extracellular mediator release. Our prior study revealed miR-503 expression is decreased in COPD lung fibroblasts, although the exact role played by miR-503 is undetermined. The current study examined a role of miR-503 in cytokine, growth factor and fibronectin production by lung fibroblasts from patients with and without COPD. Primary adult lung fibroblasts were isolated from patients with or without COPD. MiR-503 expression and interleukin (IL)-6, -8, PGE2, HGF, KGF, VEGF and fibronectin release were examined with or without inflammatory cytokines, IL-1ß and tumor necrosis factor (TNF)-α. MiR-503 expression was decreased in COPD lung fibroblasts. The expression of miR-503 was positively correlated with %FVC, %FEV1, and %DLco as well as IL-6, -8, PGE2, HGF, KGF, and VEGF in the absence or presence of IL-1ß/TNF-α. In addition, IL-8 and VEGF release from COPD lung fibroblasts were increased compared to those from control. Exogenous miR-503 inhibited VEGF release from primary adult and fetal lung fibroblasts but not IL-8 release. As expected, COPD fibroblasts proliferated more slowly than control fibroblasts. MiR-503 did not affect proliferation of either control or COPD lung fibroblasts. MiR-503 inhibition of VEGF protein production and mRNA was mediated by direct binding to the 3' untranslated region of VEGF mRNA. Endogenous miR-503 was differently regulated by exogenous stimulants associated with COPD pathogenesis, including IL-1ß/TNF-α, TGF-ß1 and PGE2. Endogenous miR-503 inhibition augmented VEGF release by IL-1ß/TNF-α and TGF-ß1 but not by PGE2, demonstrating selectivity of miR-503 regulation of VEGF. In conclusions, reduced miR-503 augments VEGF release from lung fibroblasts from patients with COPD. Since VEGF contributes to disturbed vasculature in COPD, altered miR-503 production might play a role in modulating fibroblast-mediated vascular homeostasis in COPD.

Post-injury and resolution response to repetitive inhalation exposure to agricultural organic dust in mice.

Inhalation of organic dusts in agricultural environments causes airway inflammatory diseases. Despite advances in understanding the airway response to dust-induced inflammation, less is known about the transition from lung injury to repair and recovery. The objective of this study was to define the post-inflammation homeostasis events following organic dust-induced lung injury. Using an established protocol, mice were intranasally treated with swine confinement facility organic dust extract (ODE) daily for 3 weeks (repetitive exposure) or treated daily with ODE for 3 weeks followed by no treatment for 1-4 weeks (recovery period) whereupon lavage fluid, lung tissue, and sera were processed. During recovery period, a significant decrease was observed in ODE-induced neutrophil levels after 1 week, lymphocytes at 2 weeks, and macrophages at 4 weeks in the lavage fluid. ODE-induced lung cellular aggregates and bronchiolar compartment inflammation were diminished, but persisted for 4 weeks post-injury. Alveolar inflammation resolved at 3 weeks. ODE-induced lung neutrophils were cleared by 3 weeks, B-cells by 2 weeks, and CD3+CD4+ and CD3+CD8+ T cells by 4 week recovery period. Collectively, these results identify important processes during recovery period following agricultural dust-induced inflammation, and present possible strategies for improving lung repair and resolution.