Original ArticleTNF-α inhibits the growth of Legionella pneumophila in airway epithelial cells by inducing apoptosis
Introduction
Legionella pneumophila (Lp) infection is characterized by life-threatening pneumonia that is caused by the inhalation of contaminated aerosols. Despite the availability of appropriate antibiotics, Lp infection can become severe in a short period and often requires intensive care. Therefore, it is important to determine mechanisms underlying the pathogenesis of Lp infection to develop better strategies for its prevention and treatment.
Although Lp mainly infects and replicates in alveolar macrophages, it also infects and replicates in non-phagocytic cells such as lung epithelial cells [1], [2], [3]. Because airway epithelial cells and alveolar macrophages come in contact with inhaled pathogens, they may be involved in the pathogenesis of Lp infection. Previously, we reported that lung epithelial cells produced mucins in response to Lp infection and that this response could exert protective effects against Lp infection [3]. However, information on the relationship between Lp and lung epithelial cells is scarce and the role of these cells in Lp infection is unclear.
Cell death is suggested to be involved in the pathogenesis of Lp infection. Lp-infected macrophages undergo cell death through apoptosis [4], [5], pyroptosis [6], and programmed necrosis [7]. Proinflammatory mediators, including IL-1β and high-mobility group box 1, released by dying cells induce inflammation. Therefore, Lp-induced cell death is an important mechanism underlying severe inflammation in patients with Lp infection. However, information on Lp-induced cell death of lung epithelial cells is unavailable.
TNF-α, a proinflammatory cytokine, is rapidly released by various immune cells upon pathogen recognition. TNF-α exerts protective effects against invasive pathogens, especially intracellular pathogens, and inhibits the growth of Lp in macrophages [8]. However, TNF-α is also involved in chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease and is a therapeutic target for treating these diseases because it is a major cause of inflammation. Therefore, TNF-α inhibitors that provide significant benefits such as reduction of disease activity and improvement of symptoms are used for treating patients with the above diseases. However, patients treated with TNF-α inhibitors are at an increased risk of bacterial, fungal, and viral infections [9], [10] because important initial responses to invasive pathogens may be suppressed in these patients. Lp is a pathogen of those risk-elevated infection and the U.S. Food and Drug Administration added Lp into the emerging pathogen list in the patients under the anti-TNF-α therapy [11]. Fatal cases of Lp infection have been observed in patients undergoing anti-TNF-α therapy [12]. Moreover, incidence of Lp-induced pneumonia is high in patients undergoing anti-TNF-α therapy [13].
TNF-α inhibits intracellular bacterial proliferation in and cell death of Lp-infected macrophages. However, the role of TNF-α in lung epithelial cells is unknown. Therefore, we examined the effect of TNF-α on bacterial growth in and cell death of lung epithelial cells in the present study.
Section snippets
Bacterial strain
Lp NUL1 strain (serogroup 1), which was clinically isolated from the sputum of a patient with L. pneumophila pneumonia, was used in this study [3], [7]. The bacteria were stored at −80 °C in a Microbank system (Pro-Lab Diagnostics, Ontario, Canada) until further use. The bacteria were cultured on Wadowsky–Yee–Okuda agar plates (Eiken Kagaku, Tokyo, Japan) for 3 days. Next, a single colony was subcultured in 3 mL buffered yeast extract (BYE) broth for 12 h at 37 °C under constant shaking. The
TNF-α inhibits the intracellular growth of Lp in airway epithelial cells
Bacterial count in Lp-infected NCI-H292 cells was measured (Fig. 1A). Bacterial count was 2.93 ± 0.04 log10 cfu/mL at 0 h and increased to 6.31 ± 0.02 log10 cfu/mL at 48 h after incubation with Lp in a time-dependent manner (p < 0.01 compared with bacterial count at 0 h). We also confirmed that Lp did not replicate in the culture medium alone (data not shown).
Effects of TNF-α on Lp growth were examined at 24 and 48 h. At 24 h, no significant differences were observed in bacterial count in cells
Discussion
Lp causes community-acquired pneumonia. However, it is also recognized as a re-emerging pathogen in patients undergoing anti-TNF-α therapy. Respiratory tract epithelial cells function as important barriers against airborne infections and are a target of Lp infection. In the present study, we examined the effects of TNF-α on Lp-infected airway epithelial cells.
The relationship between Lp and alveolar macrophages has been mainly discussed in the pathogenesis of Lp infection from the early stage
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgements
This research was partly supported by a Grant-in-Aid for Scientific Research (No. 15K09572 to K.Y.) and Grant-in-Aid for Young Scientists (B) (Nos. 23791137 and 30580360 to Y.M.) from the Japan Society for the Promotion of Science.
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