Asian sand dust enhances murine lung inflammation caused by Klebsiella pneumoniae

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Abstract

Inhaling concomitants from Asian sand dust (ASD) may result in exacerbation of pneumonia by the pathogen. The exacerbating effect of ASD on pneumonia induced by Klebsiella pneumoniae (KP) was investigated in ICR mice. The organic substances adsorbed onto ASD collected from the atmosphere of Iki-island in Japan were excluded by heat treatment at 360 °C for 30 min. ICR mice were instilled intratracheally with ASD at doses of 0.05 mg or 0.2 mg/mouse four times at 2-week intervals (total dose of 0.2 mg or 0.8 mg/mouse) and were administrated with ASD in the presence or absence of KP at the last intratracheal instillation. Pathologically, ASD caused exacerbation of pneumonia by KP as shown by increased inflammatory cells within the bronchiolar and the alveolar compartments. ASD enhanced the neutrophil number dose dependently as well as the expression of cytokines (IL-1β, IL-6, IL-12, IFN-γ, TNF-α) and chemokines (KC, MCP-1, MIP-1α) related to KP in BALF. In an in vitro study using RAW264.7 cells, combined treatment of ASD and KP increased gene expression of IL-1β, IL-6, IFN-β, KC, MCP-1, and MIP-1α. The same treatment tended to increase the protein level of IL-1β, TNF-α and MCP-1 in a culture medium compared to each treatment alone. The combined treatment tended to increase the gene expression of Toll-like receptor 2 (TLR2), and NALP3, ASC and caspase-1 compared with KP alone. These results suggest that the exacerbation of pneumonia by ASD + KP was due to the enhanced production of pro-inflammatory mediators via activation of TLR2 and NALP3 inflammasome pathways in alveolar macrophages.

Introduction

It is well known that sand storms arise in almost all areas in the world, especially Arizona, Brisbane, the Sahara and the Asian region. Asian sand dust (ASD) storms arise from the Gobi Desert, the Taklimakan Desert, and loess areas of interior China during the spring season. ASD aerosol spreads through downwind areas, such as East China, the Korean Peninsula, and Japan, as well as across the Pacific Ocean to the United States (Duce et al., 1980, Husar et al., 2001, Kim et al., 2001). ASD reportedly is transported one full circuit around the globe (Uno et al., 2009). ASD contains various chemical species, such as sulfate (SO42 ) and nitrate (NO3) derived from air pollutants (SO2, NO2) as well as microbial agents, including bacteria, fungi, fungal spores, and viruses (Chen et al., 2010, Kobayashi et al., 2010, Maki et al., 2010). Recently the microbial communities transported with ASD have attracted much attention as bioaerosols (biological particles) affecting ecosystem and human health in downwind areas.

The airborne sand dust and the microorganisms mentioned above may cause adverse effects on respiratory health and increase mortality and morbidity. Epidemiologic studies have demonstrated that dust storm events caused an increase of hospitalization for pneumonia in China (Meng and Lu, 2007) and an increase in daily mortality in Seoul, Korea (Kwon et al., 2002). In Taiwan, there have been reports that ASD events coincided with an increase of mortality, emergency treatment for cardiovascular disease and hospitalization for pneumonia (Bell et al., 2008, Chan et al., 2008, Chen et al., 2004). In Japan, the deterioration of Japanese cedar pollinosis and seasonal allergic rhinitis (Sato, 2009), and exacerbation of adult asthma (Watanabe et al., 2011) and child asthma (Kanatani et al., 2010) occurred during a dust storm event also have been reported. Therefore, experimental studies to confirm the epidemiological results, suggesting that ASD causes an increase in the incidence of pneumonia induced by microbial agents during a dust storm event, are in order.

Inhalation of highly pathogenic bacteria or influenza viruses, which might be transported by ASD, presents a significant threat to human health. Our previous studies have shown that intratracheal instillation of ASD caused bronchitis and alveolitis, and clearly increased neutrophils along with its relevant chemokines and Th1 relevant cytokines in bronchoalveolar lavage fluid (BALF), whereas ASD heated at 360 °C to exclude toxic materials (microbiological materials, sulfate, nitrate, etc.) caused considerably fewer effects (He et al., 2010a). We suggest that the microbial components attached to ASD play an important role in the lung inflammation caused by ASD. However, whether the Asian sand particle itself causes the deterioration into pneumonia induced by pathogenic bacteria is unknown. Klebsiella pneumoniae (KP) is a gram-negative bacterium with a world-wide distribution and the most common cause of human pneumonia infections (Podschun and Ullmann, 1998). In the present study, the exacerbating effect of ASD on pneumonia induced by KP was investigated in ICR mice. The investigation included examination of pathologic change in the mice lungs, cytological alteration in bronchoalveolar lavage fluids (BALF), and changes in inflammatory cytokines and chemokines in BALF.

Alveolar macrophages are key cells for protective pulmonary defense against inflammation (Broug-Holub et al., 1997). Toll-like receptors (TLRs) that belong to the pattern recognition receptors (PRRs) family in antigen presenting cells like macrophages or dendritic cells have been the molecules involved in the recognition of pathogen-associated molecular patterns (PAMP) that lead to the inflammatory process (Mariathasan and Monack, 2007). NALP3 (NACHT domain, leucine-rich repeat, and pyrin domain-containing protein 3), which belongs to the Nod-like receptor (NLR) family in antigen presenting cells is an essential component of inflammasomes triggered by microbial ligands like peptidoglycan, danger-associated molecular patterns, and crystals, to form NALP3 inflammasome (Pétrilli et al., 2007). The NALP3 inflammasome activates caspase-1 (Agostini et al., 2004) for processing and secretion of the cytokines interleukin (IL)-1β (Fritz et al., 2006, Mayor et al., 2007).

In the first in vitro experiment, the gene expression of TLR2, TLR4 and pro-inflammatory cytokines secreted into the culture medium was measured in RAW264.7 cells infected with KP plus anti-TLR2 and anti-TLR4 antibodies to investigate the role of TLRs in signaling a pathway for pro-inflammatory cytokines production. In the second in vitro experiment, the gene expression of TLR2, TLR4, NALP3, ASC, caspase-1 and IL-1β in RAW264.7 cells was measured in the presence of ASD and/or KP to investigate the role of TLRs and NALP3 inflammasome in the deterioration of pneumonia by ASD. The cytokine levels secreted into the culture medium by RAW264.7 cells were also measured.

Section snippets

Animals

A total of 96 male ICR mice (5 weeks old) were purchased from Charles River Japan, Inc. (Kanagawa, Japan). After 1 week, the sick mice, mice with abnormal body weight, and mice stressed from different environmental breeding were screened out. The remaining mice were subsequently fed a commercial diet CE-2 (CLEA Japan, Inc., Tokyo, Japan) and given water ad libitum. Mice were housed in plastic cages lined with soft wood chips. The cages were placed in a conventional room, which was air conditioned

Contents of chemical elements in ASD

As previously reported (He et al., 2010a), the chemical elements in the ASD sample used in this study were 61.8% SiO2, 13.6% Al2O3, 5.7% Fe2O3, 5.4% CaO, 3.3% MgO, 0.01% TiO2, and 2.6% K2O. The size distribution peak of ASD was observed at 4.7 μm.

Enhancement of cell numbers in BALF by ASD

To estimate the effects of ASD on the lung inflammation caused by KP, the cellular profile of BALF was examined (Fig. 1). ASD0.2 and ASD0.8 caused little increase in the number of neutrophils in BALF. ASD0.8 alone (p < 0.001) and ASD0.2 + KP (p < 0.01)

Discussion

ASD has been shown to adsorb many kinds of microorganisms (Chen et al., 2010, Kobayashi et al., 2010, Maki et al., 2010) some of which are highly pathogenic. The exacerbation of pneumonia incidence by Asian sand particles in murine lungs was investigated using KP.

The present study has demonstrated that administration of ASD exacerbated pneumonia incidence in KP infected mice, which is evidenced by cellular profiles of BALF and pathological examinations. As to overall trends, these changes were

Conflict of interest statements

The authors do not have any conflict to interest to disclose.

Acknowledgments

We appreciate the vital contribution of students at Oita University of Nursing and Health Sciences in this research. This study was supported in part by a grant (No. 21651010) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by Ministry of the Environment in Japan. This work was partly supported by the Global Environment Research Fund (B-0901) of the Ministry of the Environment, Japan.

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  • Cited by (0)

    1

    Present address: Oita University of Nursing and Health Sciences, 2944-9 Megusuno Oita City, Oita Prefecture, 870-1201, Japan.

    2

    Professor Yamamoto has passed away for gastric cancer in December, 2010.

    3

    Present address: Department of Public Health and Molecular Toxicology, School of Pharmacy, Kitasato University, 108-8641, Tokyo, Japan.

    4

    Present address: Environmental Health Division, Department of Environmental Engineering, Graduate School of Engineering, Kyoto University, Kyoto daigaku-Katsura, Nishikyo-ku, Kyoto 615-8530, Japan.

    5

    Present address: National Institute for Minamata Disease, Department of Basic Medical Sciences, 4058-18 Hama, Minamata City, Kumamoto, 867-0008, Japan.

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