TNFR1/phox interaction and TNFR1 mitochondrial translocation Thwart silica-induced pulmonary fibrosis.

Macrophages play a fundamental role in innate immunity and the pathogenesis of silicosis. Phagocytosis of silica particles is associated with the generation of reactive oxygen species (ROS), secretion of cytokines, such as TNF, and cell death that contribute to silica-induced lung disease. In macrophages, ROS production is executed primarily by activation of the NADPH oxidase (Phox) and by generation of mitochondrial ROS (mtROS); however, the relative contribution is unclear, and the effects on macrophage function and fate are unknown. In this study, we used primary human and mouse macrophages (C57BL/6, BALB/c, and p47(phox-/-)) and macrophage cell lines (RAW 264.7 and IC21) to investigate the contribution of Phox and mtROS to silica-induced lung injury. We demonstrate that reduced p47(phox) expression in IC21 macrophages is linked to enhanced mtROS generation, cardiolipin oxidation, and accumulation of cardiolipin hydrolysis products, culminating in cell death. mtROS production is also observed in p47(phox-/-) macrophages, and p47(phox-/-) mice exhibit increased inflammation and fibrosis in the lung following silica exposure. Silica induces interaction between TNFR1 and Phox in RAW 264.7 macrophages. Moreover, TNFR1 expression in mitochondria decreased mtROS production and increased RAW 264.7 macrophage survival to silica. These results identify TNFR1/Phox interaction as a key event in the pathogenesis of silicosis that prevents mtROS formation and reduces macrophage apoptosis.

LPS-treated macrophage cytokines repress surfactant protein-B in lung epithelial cells.

In the mouse lung, Escherichia coli LPS can decrease surfactant protein-B (SFTPB) mRNA and protein concentrations. LPS also regulates the expression, synthesis, and concentrations of a variety of gene and metabolic products that inhibit SFTPB gene expression. The purpose of the present study was to determine whether LPS acts directly or indirectly on pulmonary epithelial cells to trigger signaling pathways that inhibit SFTPB expression, and whether the transcription factor CCAAT/enhancer binding protein (C/EBP)-ß (CEBPB) is a downstream inhibitory effector. To investigate the mechanism of SFTPB repression, the human pulmonary epithelial cell lines NCI-H441 (H441) and NCI-H820 (H820) and the mouse macrophage-like cell line RAW264.7 were treated with LPS. Whereas LPS did not decrease SFTPB transcripts in H441 or H820 cells, the conditioned medium of LPS-treated RAW264.7 cells decreased SFTPB transcripts in H441 and H820 cells, and inhibited SFTPB promoter activity in H441 cells. In the presence of neutralizing anti-tumor necrosis factor (TNF) antibodies, the conditioned medium of LPS-treated RAW264.7 cells did not inhibit SFTPB promoter activity. In H441 cells treated with recombinant TNF protein, SFTPB transcripts decreased, whereas CEBPB transcripts increased and the transient coexpression of CEBPB decreased SFTPB promoter activity. Further, CEBPB short, interfering RNA increased basal SFTPB transcripts and countered the decrease of SFTPB transcripts by TNF. Together, these findings suggest that macrophages participate in the repression of SFTPB expression by LPS, and that macrophage-released cytokines (including TNF) regulate the transcription factor CEBPB, which can function as a downstream transcriptional repressor of SFTPB gene expression in pulmonary epithelial cells.

A multi-cyclone sampling array for the collection of size-segregated occupational aerosols.

In this study a serial multi-cyclone sampling array capable of simultaneously sampling particles of multiple size fractions, from an occupational environment, for use in in vivo and in vitro toxicity studies and physical/chemical characterization, was developed and tested. This method is an improvement over current methods used to size-segregate occupational aerosols for characterization, due to its simplicity and its ability to collect sufficient masses of nano- and ultrafine sized particles for analysis. This method was evaluated in a chamber providing a uniform atmosphere of dust concentrations using crystalline silica particles. The multi-cyclone sampling array was used to segregate crystalline silica particles into four size fractions, from a chamber concentration of 10 mg/m(3). The size distributions of the particles collected at each stage were confirmed, in the air, before and after each cyclone stage. Once collected, the particle size distribution of each size fraction was measured using light scattering techniques to further confirm the size distributions. As a final confirmation, scanning electron microscopy was used to collect images of each size fraction. The results presented here, using multiple measurement techniques, show that this multi-cyclone system was able to successfully collect distinct size-segregated particles at sufficient masses to perform toxicological evaluations and physical/chemical characterization.

Occupational Histoplasmosis: Epidemiology and Prevention Measures.

In areas where Histoplasma is endemic in the environment, occupations involving activities exposing workers to soil that contains bird or bat droppings may pose a risk for histoplasmosis. Occupational exposures are frequently implicated in histoplasmosis outbreaks. In this paper, we review the literature on occupationally acquired histoplasmosis. We describe the epidemiology, occupational risk factors, and prevention measures according to the hierarchy of controls.

Differential activation of RAW 264.7 macrophages by size-segregated crystalline silica.

BACKGROUND: Occupational exposure to crystalline silica is a well-established occupational hazard. Once in the lung, crystalline silica particles can result in the activation of alveolar macrophages (AM), potentially leading to silicosis, a fibrotic lung disease. Because the activation of alveolar macrophages is the beginning step in a complicated inflammatory cascade, it is necessary to define the particle characteristics resulting in this activation. The aim of this research was to determine the effect of the size of crystalline silica particles on the activation of macrophages. METHODS: RAW 264.7 macrophages were exposed to four different sizes of crystalline silica and their activation was measured using electron microscopy, reactive oxygen species (ROS) generation by mitochondria, and cytokine expression. RESULTS: These data identified differences in particle uptake and formation of subcellular organelles based on particle size. In addition, these data show that the smallest particles, with a geometric mean of 0.3 µm, significantly increase the generation of mitochondrial ROS and the expression of cytokines when compared to larger crystalline silica particles, with a geometric mean of 4.1 µm. CONCLUSION: In summary, this study presents novel data showing that crystalline silica particles with a geometric mean of 0.3 µm enhance the activation of AM when compared to larger silica particles usually represented in in vitro and in vivo research.

Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs.

Mesenchymal stem cells (MSCs) and macrophages are fundamental components of the stem cell niche and function coordinately to regulate haematopoietic stem cell self-renewal and mobilization. Recent studies indicate that mitophagy and healthy mitochondrial function are critical to the survival of stem cells, but how these processes are regulated in MSCs is unknown. Here we show that MSCs manage intracellular oxidative stress by targeting depolarized mitochondria to the plasma membrane via arrestin domain-containing protein 1-mediated microvesicles. The vesicles are then engulfed and re-utilized via a process involving fusion by macrophages, resulting in enhanced bioenergetics. Furthermore, we show that MSCs simultaneously shed micro RNA-containing exosomes that inhibit macrophage activation by suppressing Toll-like receptor signalling, thereby de-sensitizing macrophages to the ingested mitochondria. Collectively, these studies mechanistically link mitophagy and MSC survival with macrophage function, thereby providing a physiologically relevant context for the innate immunomodulatory activity of MSCs.

Systemic inhibition of NF-kappaB activation protects from silicosis.

BACKGROUND: Silicosis is a complex lung disease for which no successful treatment is available and therefore lung transplantation is a potential alternative. Tumor necrosis factor alpha (TNFalpha) plays a central role in the pathogenesis of silicosis. TNFalpha signaling is mediated by the transcription factor, Nuclear Factor (NF)-kappaB, which regulates genes controlling several physiological processes including the innate immune responses, cell death, and inflammation. Therefore, inhibition of NF-kappaB activation represents a potential therapeutic strategy for silicosis. METHODS/FINDINGS: In the present work we evaluated the lung transplant database (May 1986-July 2007) at the University of Pittsburgh to study the efficacy of lung transplantation in patients with silicosis (n = 11). We contrasted the overall survival and rate of graft rejection in these patients to that of patients with idiopathic pulmonary fibrosis (IPF, n = 79) that was selected as a control group because survival benefit of lung transplantation has been identified for these patients. At the time of lung transplantation, we found the lungs of silica-exposed subjects to contain multiple foci of inflammatory cells and silicotic nodules with proximal TNFalpha expressing macrophage and NF-kappaB activation in epithelial cells. Patients with silicosis had poor survival (median survival 2.4 yr; confidence interval (CI): 0.16-7.88 yr) compared to IPF patients (5.3 yr; CI: 2.8-15 yr; p = 0.07), and experienced early rejection of their lung grafts (0.9 yr; CI: 0.22-0.9 yr) following lung transplantation (2.4 yr; CI:1.5-3.6 yr; p<0.05). Using a mouse experimental model in which the endotracheal instillation of silica reproduces the silica-induced lung injury observed in humans we found that systemic inhibition of NF-kappaB activation with a pharmacologic inhibitor (BAY 11-7085) of IkappaB alpha phosphorylation decreased silica-induced inflammation and collagen deposition. In contrast, transgenic mice expressing a dominant negative IkappaB alpha mutant protein under the control of epithelial cell specific promoters demonstrate enhanced apoptosis and collagen deposition in their lungs in response to silica. CONCLUSIONS: Although limited by its size, our data support that patients with silicosis appear to have poor outcome following lung transplantation. Experimental data indicate that while the systemic inhibition of NF-kappaB protects from silica-induced lung injury, epithelial cell specific NF-kappaB inhibition appears to aggravate the outcome of experimental silicosis.

Molecular and functional properties of lung SP cells.

Previous analysis of lung injury and repair has provided evidence for region-specific stem cells that maintain proximal and distal epithelial compartments. However, redundant expression of lineage markers by cells at several levels of the stem cell hierarchy has complicated phenotypic and functional characterization of clonogenic airway cells. Based on the demonstration that rapid efflux of the DNA dye Hoechst 33342 can be used to prospectively purify long-term repopulating hematopoietic stem cells, we hypothesized that lung cells with similar biochemical properties would be enriched for clonogenic progenitors. We demonstrate that Hoechst-dim side population (SP) cells isolated from proximal and distal compartments of the mouse lung were relatively small and agranular, exhibited low red and green autofluorescence, and that the SP fraction was highly enriched in clonogenic cells. Quantitative RT-PCR indicated that vimentin mRNA was enriched and that epithelial markers were depleted in these preparations of SP cells. Bleomycin exposure was associated with decreased clonogenicity among alveolar SP and suggested that SP cell function was compromised under profibrotic conditions. We conclude that the SP phenotype is common to clonogenic cells at multiple airway locations and suggest that Hoechst efflux is a property of cells expressing a wound-repair phenotype.