Formaldehyde exposure induces differentiation of regulatory T cells via the NFAT-mediated T cell receptor signalling pathway in Yucatan minipigs.

The use of minipigs (Sus scrofa) as a platform for toxicological and pharmacological research is well established. In the present study, we investigated the effect of formaldehyde (FA) exposure on helper T cell-mediated splenic immune responses in Yucatan minipigs. The minipigs were exposed to different inhaled concentrations of FA (0, 2.16, 4.62, or 10.48 mg/m3) for a period of 2 weeks. Immune responses elicited by exposure to FA were determined by assessing physiological parameters, mRNA expression, and cytokine production. Additionally, the distribution of helper T cells and regulatory T (Treg) cells and expression of NFAT families, which are well-known T cell receptor signalling proteins associated with regulatory T cell development, were evaluated. Exposure to FA suppressed the expression of genes associated with Th1 and Th2 cells in minipigs in a concentration-dependent manner. The subsequent production of cytokines also declined post-FA exposure. Furthermore, exposure to FA induced the differentiation of CD4+ Foxp3+ Treg cells with divergent expression levels of NFAT1 and NFAT2. These results indicated that exposure to FA increased the Treg cell population via the NFAT-mediated T cell receptor signalling pathway, leading to suppression of effector T cell activity with a decline in T cell-related cytokine production.

Rhinovirus infection in murine chronic allergic rhinosinusitis model.

BACKGROUND: Patients with chronic rhinosinusitis (CRS) commonly experience aggravation of their symptoms after viral upper respiratory infection (URI). Rhinovirus (RV) is the most common URI-causing virus. However, there is a lack of a mouse model of RV infection and in vivo studies investigating the effect of RV infection on CRS. METHODS: A mouse model of chronic allergic rhinosinusitis (CARS) was established by sensitizing to ovalbumin (OVA) through intraperitoneal injection followed by nasal challenges with OVA for 5 weeks. Both control and CARS mice were euthanized at 48 hours after infection with minor group RV serotype 1B (RV1B). Sinonasal complex samples were evaluated histologically; and interleukin (IL)-6, macrophage inflammatory protein (MIP)-2, IL-13, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were measured in the nasal lavage fluid. The RV1B-infected areas in control and CARS mice were identified using immunofluorescence. RESULTS: In the infected control mice group, RV1B increased secretory hyperplasia in the sinonasal mucosa and the production of proinflammatory cytokines including INF-γ, MIP-2, and IL-13. Immunohistochemical analysis of nasal mucosa from RV1B-infected mice presented abundant RV1B staining, which was distributed between the epithelium and the lamina propria. In the CARS group, the RV1B-infected area per unit was significantly higher than that in control mice. However, RV1B infection neither increased the proinflammatory cytokine secretion nor worsened the histology significantly. CONCLUSION: We successfully established a mouse model of upper airway RV infection by nasal inoculation with RV1B. Although there was histologically-proven increased RV infection in the CARS model, the infection did not intensify sinonasal inflammation.

Establishment of a mouse model for pulmonary inflammation and fibrosis by intratracheal instillation of polyhexamethyleneguanidine phosphate.

Although several animal models have been developed to study human pulmonary fibrosis, lack of a perfect model has raised the need for various animal models of pulmonary fibrosis. In this study, we evaluated the pulmonary effect of polyhexamethyleneguanidine phosphate instillation into the lungs of mice to determine the potential of these mice as a murine model of pulmonary fibrosis. Intratracheal instillation of polyhexamethyleneguanidine phosphate induced severe lung inflammation manifested by the infiltration of mononuclear cells and neutrophils and increased production of IL-6, TNF-α, CCL2 and CXCL1. The lung inflammation gradually increased until 28 days after polyhexamethyleneguanidine phosphate exposure, and increases of collagen deposition and TGF-ß production, which are indicators of pulmonary fibrosis, were seen. Our study showed that intratracheal instillation of polyhexamethyleneguanidine phosphate induces pulmonary inflammation and fibrosis in mice.

Oral intake of Lactobacillus rhamnosus M21 enhances the survival rate of mice lethally infected with influenza virus.

BACKGROUND: Influenza viruses cause acute respiratory disease. Because of the high genetic variability of viruses, effective vaccines and antiviral agents are limited. Considering the fact that the site of influenza virus entry is the mucosa of the upper respiratory tract, probiotics that can enhance mucosal immunity as well as systemic immunity could be an important source of treatment against influenza infection. METHODS: Mice were fed with Lactobacillus rhamnosus M21 or skim milk and were challenged with influenza virus. The resulting survival rate, lung inflammation, and changes in the cytokine and secretory immunoglobulin A (sIgA) levels were examined. RESULTS: Because of infection (influenza virus), all the mice in the control group and 60% of the mice in the L. rhamnosus M21 group died; however, the remaining 40% of the mice fed with L. rhamnosus M21 survived the infection. Pneumonia was severe in the control group but moderate in the group treated with L. rhamnosus M21. Although there were no significant changes in the proinflammatory cytokines in the lung lysates of mice collected from both groups, levels of interferon-γ and interleukin-2, which are representative cytokines of type I helper T cells, were significantly increased in the L. rhamnosus M21-treated group. An increase in sIgA as well as the diminution of inflammatory cells in bronchoalveolar lavage fluid was also observed in the L. rhamnosus M21-treated group. CONCLUSION: These results demonstrate that orally administered L. rhamnosus M21 activates humoral as well as cellular immune responses, conferring increased resistance to the host against influenza virus infection.

Polyhexamethylene guanidine phosphate aerosol particles induce pulmonary inflammatory and fibrotic responses.

Polyhexamethylene guanidine (PHMG) phosphate was used as a disinfectant for the prevention of microorganism growth in humidifiers, without recognizing that a change of exposure route might cause significant health effects. Epidemiological studies reported that the use of humidifier disinfectant containing PHMG-phosphate can provoke pulmonary fibrosis. However, the pulmonary toxicity of PHMG-phosphate aerosol particles is unknown yet. This study aimed to elucidate the toxicological relationship between PHMG-phosphate aerosol particles and pulmonary fibrosis. An in vivo nose-only exposure system and an in vitro air-liquid interface (ALI) co-culture model were applied to confirm whether PHMG-phosphate induces inflammatory and fibrotic responses in the respiratory tract. Seven-week-old male Sprague-Dawley rats were exposed to PHMG-phosphate aerosol particles for 3 weeks and recovered for 3 weeks in a nose-only exposure chamber. In addition, three human lung cells (Calu-3, differentiated THP-1 and HMC-1 cells) were cultured at ALI condition for 12 days and were treated with PHMG-phosphate at set concentrations and times. The reactive oxygen species (ROS) generation, airway barrier injuries and inflammatory and fibrotic responses were evaluated in vivo and in vitro. The rats exposed to PHMG-phosphate aerosol particles in nanometer size showed pulmonary inflammation and fibrosis including inflammatory cytokines and fibronectin mRNA increase, as well as histopathological changes. In addition, PHMG-phosphate triggered the ROS generation, airway barrier injuries and inflammatory responses in a bronchial ALI co-culture model. Those results demonstrated that PHMG-phosphate aerosol particles cause pulmonary inflammatory and fibrotic responses. All features of fibrogenesis by PHMG-phosphate aerosol particles closely resembled the pathology of fibrosis that was reported in epidemiological studies. Finally, we expected that PHMG-phosphate infiltrated into the lungs in the form of aerosol particles would induce an airway barrier injury via ROS, release fibrotic inflammatory cytokines, and trigger a wound-healing response, leading to pulmonary fibrosis. A simultaneous state of tissue destruction and inflammation caused by PHMG-phosphate had whipped up a "perfect storm" in the respiratory tract.

Polyhexamethyleneguanidine phosphate induces severe lung inflammation, fibrosis, and thymic atrophy.

Polyhexamethyleneguanidine phosphate (PHMG-P) has been widely used as a disinfectant because of its strong bactericidal activity and low toxicity. However, in 2011, the Korea Centers for Disease Control and Prevention and the Ministry of Health and Welfare reported that a suspicious outbreak of pulmonary disease might have originated from humidifier disinfectants. The purpose of this study was to assess the toxicity of PHMG-P following direct exposure to the lung. PHMG-P (0.3, 0.9, or 1.5 mg/kg) was instilled into the lungs of mice. The levels of proinflammatory markers and fibrotic markers were quantified in lung tissues and flow cytometry was used to evaluate T cell distribution in the thymus. Administration of PHMG-P induced proinflammatory cytokines elevation and infiltration of immune cells into the lungs. Histopathological analysis revealed a dose-dependent exacerbation of both inflammation and pulmonary fibrosis on day 14. PHMG-P also decreased the total cell number and the CD4(+)/CD8(+) cell ratio in the thymus, with the histopathological examination indicating severe reduction of cortex and medulla. The mRNA levels of biomarkers associated with T cell development also decreased markedly. These findings suggest that exposure of lung tissue to PHMG-P leads to pulmonary inflammation and fibrosis as well as thymic atrophy.

Standardization of bronchoalveolar lavage method based on suction frequency number and lavage fraction number using rats.

Bronchoalveolar lavage (BAL) is a useful tool in researches and in clinical medicine of lung diseases because the BAL fluid contains biochemical and cytological indicators of the cellular response to infection, drugs, or toxicants. However, the variability among laboratories regarding the technique and the processing of the BAL material limits clinical research. The aim of this study was to determine the suction frequency and lavage fraction number necessary to reduce the variability in lavage using male Sprague-Dawley rats. We compared the total cell number and protein level of each lavage fraction and concluded that more cells and protein can be obtained by repetitive lavage with a suction frequency of 2 or 3 than by lavage with a single suction. On the basis of total cell recovery, approximately 70% of cells were obtained from fractions 1~3. The first lavage fraction should be used for evaluation of protein concentration because fractions 2~5 of lavage fluid were diluted in manifolds. These observations were confirmed in bleomycin-induced inflamed lungs of rats. We further compared the BAL data from the whole lobes with data from the right lobes and concluded that BAL data of the right lobes represented data of the whole lobes. However, this conclusion can only be applied to general lung diseases. At the end, this study provides an insight into the technical or analytical problems of lavage study in vivo.

LC50 Determination of tert-Butyl Acetate using a Nose Only Inhalation Exposure in Rats.

tert-Butyl acetate (TBAc) is an organic solvent, which is commonly used in architectural coatings and industrial solvents. It has recently been exempted from the definition of a volatile organic compound (VOC) by the Air Resources Board (ARB) . Since the use of TBAc as a substitute for other VOCs has increased, thus its potential risk in humans has also increased. However, its inhalation toxicity data in the literature are very limited. Hence, inhalation exposure to TBAc was carried out to investigate its toxic effects in this study. Adult male rats were exposed to TBAc for 4 h for 1 day by using a nose-only inhalation exposure chamber (low dose, 2370 mg/m(3) (500 ppm) ; high dose, 9482 mg/m(3) (2000 ppm) ) . Shamtreated control rats were exposed to clean air in the inhalation chamber for the same period. The animals were killed at 2, 7, and 15 days after exposure. At each time point, body weight measurement, bronchoalveolar lavage fluid (BALF) analysis, histopathological examination, and biochemical assay were performed. No treatment-related abnormal effects were observed in any group according to time course. Based on those findings, the median lethal concentration (LC50) of TBAc was over 9482 mg/m(3) in this study. According to the MSDS, the 4 h LC50 for TBAc for rats is over 2230 mg/m(3). We suggested that this value is changed and these findings may be applied in the risk assessment of TBAc which could be beneficial in a sub-acute study.