Shifted T-cell polarisation after agricultural dust exposure in mice and men

RATIONALE: A low prevalence of asthma and atopy has been shown in farmers and agricultural workers. However, in these workers, a higher prevalence of respiratory symptoms has been reported, in which T helper 1 (Th1) and/or Th17 responses may play a role. AIM: We investigated the effect of exposure to dust extracts (DEs) from different farms on airway inflammation and T-cell polarisation in a mouse model and assessed T-cell polarisation in agricultural workers from the same farms. METHODS: DEs were prepared from settled dust collected at cattle and pig farms and bulb and onion industries. Mice were exposed to phosphate-buffered saline (PBS), DEs, house dust mite (HDM) or HDM+DE via nasal instillation, four times per week during 5 weeks. Hyperresponsiveness, airway inflammation, IgE levels and T-cell polarisation were assessed. Th-cell and T cytotoxic (Tc)-cell subsets were investigated in peripheral blood samples from 33 agricultural workers and 9 non-exposed controls. RESULTS: DEs induced interleukin(IL)-17, IL-1β and IL-6 in mouse lung homogenates. DE-exposed mice had more mixed inflammatory infiltrates in the lungs, and more neutrophils compared with PBS-exposed mice. DEs protected against the HDM-induced Th2 response and methacholine hyperresponsiveness. Interestingly, occupationally exposed humans had higher frequencies of Th cells spontaneously expressing IL-17 and interferon γ compared with controls. CONCLUSION: Chronic exposure to different types of farm dust induces a Th/Tc-17 inflammatory response in mice and agricultural workers. This may contribute to the low prevalence of Th2-related diseases but may constitute a risk for other chronic respiratory diseases

Fibrocytes are increased in lung and peripheral blood of patients with idiopathic pulmonary fibrosis

Background: Fibrocytes are implicated in Idiopathic Pulmonary Fibrosis (IPF) pathogenesis and increased proportions in the circulation are associated with poor prognosis. Upon tissue injury, fibrocytes migrate to the affected organ. In IPF patients, circulating fibrocytes are increased especially during exacerbations, however fibrocytes in the lungs have not been examined. Therefore, we sought to evaluate if fibrocytes can be detected in IPF lungs and we compare percentages and phenotypic characteristics of lung fibrocytes with circulating fibrocytes in IPF. Methods: First we optimized flow cytometric detection circulating fibrocytes using a unique combination of intra- and extra-cellular markers to establish a solid gating strategy. Next we analyzed lung fibrocytes in single cell suspensions of explanted IPF and control lungs and compared characteristics and numbers with circulating fibrocytes of IPF. Results: Using a gating strategy for both circulating and lung fibrocytes, which excludes potentially contaminating cell populations (e.g. neutrophils and different leukocyte subsets), we show that patients with IPF have increased proportions of fibrocytes, not only in the circulation, but also in explanted end-stage IPF lungs. These lung fibrocytes have increased surface expression of HLA-DR, increased intracellular collagen-1 expression, and also altered forward and side scatter characteristics compared with their circulating counterparts. Conclusions: These findings demonstrate that lung fibrocytes in IPF patients can be quantified and characterized by flow cytometry. Lung fibrocytes have different characteristics than circulating fibrocytes and represent an intermediate cell population between circulating fibrocytes and lung fibroblast. Therefore, more insight in their phenotype might lead to specific therapeutic targeting in fibrotic lung diseases

Enhanced Bruton's tyrosine kinase in B-cells and autoreactive IgA in patients with idiopathic pulmonary fibrosis

RATIONALE: Idiopathic Pulmonary Fibrosis (IPF) is thought to be triggered by repeated alveolar epithelial cell injury. Current evidence suggests that aberrant immune activation may contribute. However, the role of B-cell activation remains unclear. We determined the phenotype and activation status of B-cell subsets and evaluated the contribution of activated B-cells to the development of lung fibrosis both in humans and in mice. METHODS: B-cells in blood, mediastinal lymph node, and lung single-cell suspensions of IPF patients and healthy controls (HC) were characterized using 14-color flow cytometry. Mice were exposed to bleomycin to provoke pulmonary fibrosis. RESULTS: More IgA+ memory B-cells and plasmablasts were found in blood (n = 27) and lungs (n = 11) of IPF patients compared to HC (n = 21) and control lungs (n = 9). IPF patients had higher levels of autoreactive IgA in plasma, which correlated with an enhanced decline of forced vital capacity (p = 0.002, r = - 0.50). Bruton's tyrosine kinase expression was higher in circulating IPF B-cells compared to HC, indicating enhanced B-cell activation. Bleomycin-exposed mice had increased pulmonary IgA+ germinal center and plasma cell proportions compared to control mice. The degree of lung fibrosis correlated with pulmonary germinal center B-cell proportions (p = 0.010, r = 0.88). CONCLUSION: Our study demonstrates that IPF patients have more circulating activated B-cells and autoreactive IgA, which correlate with disease progression. These B-cell alterations were also observed in the widely used mouse model of experimental pulmonary fibrosis. Autoreactive IgA could be useful as a biomarker for disease progression in IPF

Characterization of macrophage phenotypes in three murine models of house-dust-mite-induced asthma

In asthma, an important role for innate immunity is increasingly being recognized. Key innate immune cells in the lungs are macrophages. Depending on the signals they receive, macrophages can at least have an M1, M2, or M2-like phenotype. It is unknown how these macrophage phenotypes behave with regard to (the severity of) asthma. We have quantified the phenotypes in three models of house dust mite (HDM-) induced asthma (14, 21, and 24 days). M1, M2, and M2-like phenotypes were identified by interferon regulatory factor 5 (IRF5), YM1, and IL-10, respectively. We found higher percentages of eosinophils in HDM-exposed mice compared to control but no differences between HDMmodels. T cell numbers were higher after HDM exposure and were the highest in the 24-day HDM protocol. Higher numbers of M2 macrophages after HDM correlated with higher eosinophil numbers. In mice with less severe asthma, M1 macrophage numbers were higher and correlated negatively with M2 macrophages numbers. Lower numbers of M2-like macrophages were found after HDM exposure and these correlated negatively with M2 macrophages. The balance between macrophage phenotypes changes as the severity of allergic airway inflammation increases. Influencing this imbalanced relationship could be a novel approach to treat asthma

Effect of chronic bile duct obstruction and LPS upon targeting of naproxen to the liver using naproxen-albumin conjugate

Naproxen covalently linked to human serum albumin (NAP-HSA) is efficiently targeted to endothelial and Kupffer cells of the liver and may offer a new therapeutic approach in the treatment of liver disease associated with inflammatory processes. In the present investigation we explored the pharmacokinetic behaviour of targeted and non-targeted naproxen as well as the pharmacokinetic properties of the active metabolite, Naproxen-lysine (Nap-lysine), in rats rendered fibrotic by bile duct ligation (BDL) for 4 weeks. Furthermore, we studied the effect of endotoxemia, experimentally induced by intravenous injection of 800 mu g/kg lipopolysaccaride (LPS) upon the pharmacokinetics of these agents in order to investigate the feasibility of targeting naproxen to nonparenchymal cells in the inflamed and fibrotic liver. Our studies demonstrate that liver disease altered the pharmacokinetic behaviour of the different naproxen compounds. Thus, initial plasma concentrations of NAP-HSA and naproxen were markedly lower in BDL rats accompanied by an increase of the volume of distribution during the terminal elimination phase(Vd(beta) BDL vs control 114 +/- 63 vs 50 +/- 7 and 202 +/- 24 vs 115 +/- 11 ml/kg for naproxen and NAP-HSA, respectively). After injection of LPS, no significant change in the pharmacokinetics of NAP-HSA was found whereas the naproxen treated control animals showed an increase in the terminal volume of distribution (176 +/- 34 vs 115 +/- 11 ml/kg) as well as an elevation of the plasma half-life (171 +/- 27 vs 116 +/- 14 min). The feasibility of targeting naproxen to the chronically diseased liver could be clearly demonstrated: 15 min after administration of the conjugate 46% and 55% of the administered dose was found in the liver of CTR and BDL rats, whereas after injection of free naproxen only 5% and 12% of the dose was detected in liver tissue, respectively.We conclude that targeting albumin-linked naproxen to non-parenchymal cells in the liver is still feasible under the pathological conditions induced in the present study. Liver fibrosis induced significant alterations in the pharmacokinetic behaviour of the studied compounds.</p

Effect of chronic bile duct obstruction and LPS upon targeting of naproxen to the liver using naproxen-albumin conjugate

Naproxen covalently linked to human serum albumin (NAP-HSA) is efficiently targeted to endothelial and Kupffer cells of the liver and may offer a new therapeutic approach in the treatment of liver disease associated with inflammatory processes. In the present investigation we explored the pharmacokinetic behaviour of targeted and non-targeted naproxen as well as the pharmacokinetic properties of the active metabolite, Naproxen-lysine (Nap-lysine), in rats rendered fibrotic by bile duct ligation (BDL) for 4 weeks. Furthermore, we studied the effect of endotoxemia, experimentally induced by intravenous injection of 800 mu g/kg lipopolysaccaride (LPS) upon the pharmacokinetics of these agents in order to investigate the feasibility of targeting naproxen to nonparenchymal cells in the inflamed and fibrotic liver. Our studies demonstrate that liver disease altered the pharmacokinetic behaviour of the different naproxen compounds. Thus, initial plasma concentrations of NAP-HSA and naproxen were markedly lower in BDL rats accompanied by an increase of the volume of distribution during the terminal elimination phase(Vd(beta) BDL vs control 114 +/- 63 vs 50 +/- 7 and 202 +/- 24 vs 115 +/- 11 ml/kg for naproxen and NAP-HSA, respectively). After injection of LPS, no significant change in the pharmacokinetics of NAP-HSA was found whereas the naproxen treated control animals showed an increase in the terminal volume of distribution (176 +/- 34 vs 115 +/- 11 ml/kg) as well as an elevation of the plasma half-life (171 +/- 27 vs 116 +/- 14 min). The feasibility of targeting naproxen to the chronically diseased liver could be clearly demonstrated: 15 min after administration of the conjugate 46% and 55% of the administered dose was found in the liver of CTR and BDL rats, whereas after injection of free naproxen only 5% and 12% of the dose was detected in liver tissue, respectively. We conclude that targeting albumin-linked naproxen to non-parenchymal cells in the liver is still feasible under the pathological conditions induced in the present study. Liver fibrosis induced significant alterations in the pharmacokinetic behaviour of the studied compounds