Shigeaki Ito

Ciliatoxicity in human primary bronchiolar epithelial cells after repeated exposure at the air-liquid interface with native mainstream smoke of K3R4F cigarettes with and without charcoal filter

Mucociliary clearance is the primary physical mechanism to protect the human airways against harmful effects of inhaled particles. Environmental factors play a significant role in the impairment of this defense mechanism, whereas cigarette smoke is discussed to be one of the clinically most important causes. Impaired mucociliary clearance in smokers has been connected to changes in ciliated cells such as decreased numbers, altered structure and beat frequency. Clinical studies have shown that cilia length is reduced in healthy smokers and that long-term exposure to cigarette smoke leads to reduced numbers of ciliated cells in mice. We present an in vitro model of primary normal human bronchiolar epithelial (NHBE) cells with in vivo like morphology to study the influence of cigarette mainstream smoke on ciliated cells. We exposed mucociliary differentiated cultures repeatedly to non-toxic concentrations of mainstream cigarette smoke (4 cigarettes, 5 days/week, 8 repetitions in total) at the air-liquid interface. Charcoal filter tipped cigarettes were compared to those being equipped with standard cellulose acetate filters. Histopathological analyses of the exposed cultures showed a reduction of cilia bearing cells, shortening of existing cilia and finally disappearance of all cilia in cigarette smoke exposed cells. In cultures exposed to charcoal filtered cigarette smoke, little changes in cilia length were seen after four exposure repetitions, but those effects were reversed after a two day recovery period. Those differences indicate that volatile organic compounds, being removed by the charcoal filter tip, affect primary bronchiolar epithelial cells concerning their cilia formation and function comparable with the in vivo situation. In conclusion, our in vitro model presents a valuable tool to study air-borne ciliatoxic compounds.

Structure of Novel Enzyme in Mannan Biodegradation Process 4-O-β-D-Mannosyl-D- Glucose Phosphorylase MGP

The crystal structure of a novel component of the mannan biodegradation system, 4-O-β-D-mannosyl-D-glucose phosphorylase (MGP), was determined to a 1.68-Å resolution. The structure of the enzyme revealed a unique homohexameric structure, which was formed by using two helices attached to the N-terminus and C-terminus as a tab for sticking between subunits. The structures of MGP complexes with genuine substrates, 4-O-β-D-mannosyl-D-glucose and phosphate, and the product D-mannose-1-phosphate were also determined. The complex structures revealed that the invariant residue Asp131, which is supposed to be the general acid/base, did not exist close to the glycosidic Glc-O4 atom, which should be protonated in the catalytic reaction. Also, no solvent molecule that might mediate a proton transfer from Asp131 was observed in the substrate complex structure, suggesting that the catalytic mechanism of MGP is different from those of known disaccharide phosphorylases.

The mannobiose-forming exo-mannanase involved in a new mannan catabolic pathway in Bacteroides fragilis

We have proposed a new mannan catabolic pathway in Bacteroides fragilis NCTC 9343 that involves a putative mannanase ManA in glycoside hydrolase family 26 (BF0771), a mannobiose and/or sugar transporter (BF0773), mannobiose 2-epimerase (BF0774), and mannosylglucose phosphorylase (BF0772). If this hypothesis is correct, ManA has to generate mannobiose from mannans as the major end product. In this study, the BF0771 gene from the B. fragilis genome was cloned and expressed in Escherichia coli cells. The expressed protein was found to produce mannobiose exclusively from mannans and initially from manno-oligosaccharides. Production of 4-O-β-d-glucopyranosyl-d-mannose or 4-O-β-d-mannopyranosyl-d-glucose from mannans was not detectable. The results indicate that this enzyme is a novel mannobiose-forming exo-mannanase, consistent with the new microbial mannan catabolic pathway we proposed.

Repeated whole cigarette smoke exposure alters cell differentiation and augments secretion of inflammatory mediators in air-liquid interface three-dimensional co-culture model of human bronchial tissue.

In vitro models of human bronchial epithelium are useful for toxicological testing because of their resemblance to in vivo tissue. We constructed a model of human bronchial tissue which has a fibroblast layer embedded in a collagen matrix directly below a fully-differentiated epithelial cell layer. The model was applied to whole cigarette smoke (CS) exposure repeatedly from an air-liquid interface culture while bronchial epithelial cells were differentiating. The effects of CS exposure on differentiation were determined by histological and gene expression analyses on culture day 21. We found a decrease in ciliated cells and perturbation of goblet cell differentiation. We also analyzed the effects of CS exposure on the inflammatory response, and observed a significant increase in secretion of IL-8, GRO-α, IL-1β, and GM-CSF. Interestingly, secretion of these mediators was augmented with repetition of whole CS exposure. Our data demonstrate the usefulness of our bronchial tissue model for in vitro testing and the importance of exposure repetition in perturbing the differentiation and inflammation processes.

Application of a direct aerosol exposure system for the assessment of biological effects of cigarette smoke and novel tobacco product vapor on human bronchial epithelial cultures

ABSTRACT Recent advancements in in vitro exposure systems and cell culture technology enable direct exposure to cigarette smoke (CS) of human organotypic bronchial epithelial cultures. MucilAir organotypic bronchial epithelial cultures were exposed, using a Vitrocell exposure system, to mainstream aerosols from the 3R4F cigarette or from a recently developed novel tobacco vapor product (NTV). The exposure aerosol dose was controlled by dilution flow and the number of products smoked; there were five exposure conditions for 3R4F smoke and three for NTV vapor. The amount of nicotine delivered to the tissues under each condition was analyzed and that of the total particulate matter (TPM) was estimated using nicotine data. The nicotine dose was similar for the two products at the highest dose, but the estimated TPM levels from the NTV were 3.7 times the levels from the 3R4F. Following 3R4F smoke exposure, a dose dependent increase was observed in cytotoxicity, cytokine secretion, and differential gene expression. However, no changes were detected in these endpoints following NTV vapor exposure, suggesting the biological effects of NTV vapor are lower than those of conventional combustible CS. Our study design, which includes collection of biological data and dosimetry data, is applicable to assessing novel tobacco products. HIGHLIGHTSBiological effects of aerosols from the 3R4F reference cigarette and a novel tobacco vapor product (NTV) were analyzed.Human organotypic bronchial epithelial cultures were exposed to the aerosols using a direct aerosol exposure system.The 3R4F smoke increased cytotoxicity, cytokine secretion, and differential gene expression, depending on the exposure dose.No changes were detected in any of the analyzed endpoints following NTV vapor exposure. Abbreviations: AK: adenylate kinase; CS: cigarette smoke; DEGs: differentially expressed genes; FDR: false discovery rate; HCI: Health Canada Intense; ISO: International Organization for Standardization; NTV: novel tobacco vapor product; TPM: total particulate matter.

Metaplastic phenotype in human primary bronchiolar epithelial cells after repeated exposure to native mainstream smoke at the air-liquid interface

3D constructs composed of primary normal differentiated human bronchiolar epithelial (NHBE) cells as mono- or co-culture in combination with normal human lung fibroblasts were exposed repeatedly at the air-liquid interface with non-lethal concentrations of mainstream cigarette smoke (4 cigarettes a day, 5days/week, 13 times repetition in total) to build up a permanent burden on the cells. Samples were taken after 4, 8 and 13 times of repeated smoke exposure and the cultures were analyzed by histopathological methods In comparison with the clean air exposure (process control) and incubator control cells the cigarette smoke exposed cultures showed a reduction of cilia bearing as well as mucus producing cells. In both mono- as well as co-cultures, hyperplasia was induced showing different histological cell types (undifferentiated secretory and squamous cell types). At the end of the exposure phase, we observed the development of non-hyperplastic areas strongly positive to CK13 antibody, commonly seen in squamous cells as a marker for non-cornified squamous epithelium, thus suggesting a transition of the normal bronchial epithelial cells towards metaplastic cells. The control cultures (clean air exposed and incubator cells) showed no comparable phenotypic changes. In conclusion, our in vitro model presents a valuable tool to study the induction of metaplastic alterations after exposure to airborne material.

Multi-omics analysis: Repeated exposure of a 3D bronchial tissue culture to whole-cigarette smoke

Cigarette smoke (CS) is a major risk factor in the development of chronic inflammatory lung diseases such as chronic obstructive pulmonary disease. A comprehensive investigation of the biological impacts of chronic CS exposure on lung tissue is therefore important for understanding the pathogenesis of lung disease. We used three-dimensional (3D) organotypic human bronchial tissue cultures and metabolomics, transcriptomics, and proteomics to investigate changes in biological processes affected by repeated whole-CS exposure. We found that CS perturbed central carbon metabolism in relation with oxidative stress responses. Epidermal growth factor receptor, which is involved in the early-stage pathogenesis of airway diseases, was identified as a key regulator of the perturbed processes. Proteomic analysis of proteins in the apical surface liquid of the 3D bronchial tissue cultures indicated that repeated whole-CS exposure induced alterations in the secretion of several known biomarkers of airway diseases, including mucins and matrix metalloproteinases. These findings are consistent with observations from lung disease patients. Overall, our results suggest that 3D bronchial tissue cultures can provide valuable information on tissue-specific alterations in biological processes induced by chronic exposure to CS.

Effects of repeated cigarette smoke extract exposure over one month on human bronchial epithelial organotypic culture

Cigarette smoke is a known risk factor for inflammatory diseases in the respiratory tract, and inflammatory exacerbation is considered pivotal to the pathogenesis of these diseases. Here, we performed two repeated exposure studies in which we exposed human bronchial epithelial tissues in an organotypic culture model to cigarette smoke extract (CSE); the first study was conducted over a four-day period to determine the suitable dose range for the extended exposure period, and the second was a one-month exposure study to elucidate the exposure-by-exposure effects in bronchial tissues. We focused on matrix metalloproteinase (MMP)-9 and -1/3 and the inflammatory cytokines interleukin (IL)-8 and growth factor related oncogene to evaluate the transition into an inflammatory state. Even at CSE doses with no or low toxicity for a single exposure, the repetition of exposure induced cumulative effects on both the inflammatory responses, specifically the IL-8 and MMPs levels, and tissue morphology. Interestingly, untreated controls initially had relatively high baseline levels of these secreted proteins; these levels gradually declined, after which they showed periodic level changes, suggesting an acclimation period may be needed for this system. These results demonstrate the usability of this system for the elucidation of sub-chronic effects in vitro.

Oxidative stress responses in human bronchial epithelial cells exposed to cigarette smoke and vapor from tobacco- and nicotine-containing products.

ABSTRACT The use of novel tobacco‐ and nicotine‐containing vapor products that do not combust tobacco leaves is on the rise worldwide. The emissions of these products typically contain lower numbers and levels of potentially harmful chemicals compared with conventional cigarette smoke. These vapor products may therefore elicit fewer adverse biological effects. We compared the effects of emissions from different types of such products, i.e., our proprietary novel tobacco vapor product (NTV), a commercially available heat‐not‐burn tobacco product (HnB), and e‐cigarette (E‐CIG), and a combustible cigarette in a human bronchial epithelial cell line. The aqueous extract (AqE) of the test product was prepared by bubbling the produced aerosol into medium. Cells were exposed to the AqEs of test products, and then glutathione oxidation, Nrf2 activation, and secretion of IL‐8 and GM‐CSF were examined. We found that all endpoints were similarly perturbed by exposure to each AqE, but the effective dose ranges were different between cigarette smoke and the tobacco‐ and nicotine‐containing vapors. These results demonstrate that the employed assays detect differences between product exposures, and thus may be useful to understand the relative potential biological effects of tobacco‐ and nicotine‐containing products. HighlightsBEAS‐2B was exposed to cigarette and three different novel tobacco aerosols.Biological effects on oxidative stress and inflammatory responses were examined.Effective dose range of novel tobacco aerosols was higher than cigarette smoke.