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.

Phenocopy behavioral variant frontotemporal dementia: A case study.

Objective: Behavioral variant frontotemporal dementia (bvFTD) is a neurodegenerative condition characterized by progressive changes in behavior, cognition, and day-to-day functioning. Progression of the disease usually leads to death 3-5 years after diagnosis. However, there are reports of individuals who are initially diagnosed with bvFTD but fail to progress. These individuals are thought to have what is becoming known as phenocopy bvFTD (phFTD). Methods: This manuscript reviews a single case study of a 68-year-old male Veteran who was diagnosed with bvFTD in 2010, which has not progressed over time. Results: Review of serial neuropsychological evaluations was broadly normal with mild evidence of executive dysfunction with minimal reliable change in his performances from 2015, 2017, and 2022 evaluations. He also has not developed neuroimaging evidence of FTD. Conclusions: This case illustrates the importance of monitoring individuals over time and incorporating neuroimaging data into the diagnosis. We believe this Veteran's presentation is most consistent with what has been described as phFTD.

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.

Validation of clinical acceptability of deep-learning-based automated segmentation of organs-at-risk for head-and-neck radiotherapy treatment planning.

Introduction: Organ-at-risk segmentation for head and neck cancer radiation therapy is a complex and time-consuming process (requiring up to 42 individual structure, and may delay start of treatment or even limit access to function-preserving care. Feasibility of using a deep learning (DL) based autosegmentation model to reduce contouring time without compromising contour accuracy is assessed through a blinded randomized trial of radiation oncologists (ROs) using retrospective, de-identified patient data. Methods: Two head and neck expert ROs used dedicated time to create gold standard (GS) contours on computed tomography (CT) images. 445 CTs were used to train a custom 3D U-Net DL model covering 42 organs-at-risk, with an additional 20 CTs were held out for the randomized trial. For each held-out patient dataset, one of the eight participant ROs was randomly allocated to review and revise the contours produced by the DL model, while another reviewed contours produced by a medical dosimetry assistant (MDA), both blinded to their origin. Time required for MDAs and ROs to contour was recorded, and the unrevised DL contours, as well as the RO-revised contours by the MDAs and DL model were compared to the GS for that patient. Results: Mean time for initial MDA contouring was 2.3 hours (range 1.6-3.8 hours) and RO-revision took 1.1 hours (range, 0.4-4.4 hours), compared to 0.7 hours (range 0.1-2.0 hours) for the RO-revisions to DL contours. Total time reduced by 76% (95%-Confidence Interval: 65%-88%) and RO-revision time reduced by 35% (95%-CI,-39%-91%). All geometric and dosimetric metrics computed, agreement with GS was equivalent or significantly greater (p<0.05) for RO-revised DL contours compared to the RO-revised MDA contours, including volumetric Dice similarity coefficient (VDSC), surface DSC, added path length, and the 95%-Hausdorff distance. 32 OARs (76%) had mean VDSC greater than 0.8 for the RO-revised DL contours, compared to 20 (48%) for RO-revised MDA contours, and 34 (81%) for the unrevised DL OARs. Conclusion: DL autosegmentation demonstrated significant time-savings for organ-at-risk contouring while improving agreement with the institutional GS, indicating comparable accuracy of DL model. Integration into the clinical practice with a prospective evaluation is currently underway.

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.

Cross-Site Scheduling of Endoscopic Procedures Improves Efficiency While Maintaining Patient Safety.

BACKGROUND AND AIM: Accuracy in scheduling complex procedures is improved through technology to aid nonmedically trained allied health professionals. We used a new computer technology to assess whether a single coordinator could schedule endoscopic procedures across sites of a multisite academic medical institution, thus improving efficiency within the clinic overall. METHODS: A multidisciplinary team designed a cross-site scheduling model. The first phase involved accurately identifying those procedures that were appropriate for nontrained coordinators to schedule. A pilot study with gastroenterology staff was implemented and evaluated and then rolled out to non-gastroenterology staff. RESULTS: A significant decrease in call volumes occurred which in turn led to a decrease from >100 to 38 seconds in average speed to answer (ASA). A total of 115 hours of manpower was saved with the efficiency of being able to schedule without the need for a second coordinator. CONCLUSIONS: Efficiencies in call volume and ASA led to substantial time and money savings. Because of the continued involvement of multiple work groups, changes were seen as favorable rather than burdensome. Such technology could be used across other disciplines where routine procedures or tests require specific scheduling knowledge.

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

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.

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.

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)(2) D(3) . 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.

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.

αß 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) αß knockouts were used to determine the functional importance of αß-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 αß-expressing T cells are important in mediating DE-induced lung pathologic conditions.

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

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.

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.

Stress reactivity in young marmosets (Callithrix geoffroyi): ontogeny, stability, and lack of concordance among co-twins.

Variation in response styles in the hypothalamic-pituitary-adrenal (HPA) axis are known to be predictors of short- and long-term health outcomes. The nature of HPA responses to stressors changes with developmental stage, and some components of the stress response exhibit long-term individual consistency (i.e., are trait-like) while others are transient or variable (i.e., state-like). Here we evaluated the response of marmoset monkeys (Callithrix geoffroyi) to a standardized social stressor (social separation and exposure to a novel environment) at three different stages of development: juvenile, subadult, and young adult. We monitored levels of urinary cortisol (CORT), and derived multiple measures of HPA activity: Baseline CORT, CORT reactivity, CORT Area Under the Curve (AUC), and CORT regulation. Juvenile marmosets exhibited the most dramatic stress response, had higher AUCs, and tended to show poorer regulation. While baseline CORT and CORT regulation were not consistent within an individual across age, CORT reactivity and measures of AUC were highly correlated across time; i.e., individuals with high stress reactivity and AUC as juveniles also had high measures as subadults and adults, and vice-versa. Marmoset co-twins did not exhibit similar patterns of stress reactivity. These data suggest that regardless of the source of variation in stress response styles in marmosets, individually-distinctive patterns are established by six months of age, and persist for at least a year throughout different phases of marmoset life history.

Organic dust augments nucleotide-binding oligomerization domain expression via an NF-{kappa}B 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.

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.