Mol. Cells 2015; 38(7): 610-615
Published online June 18, 2015
https://doi.org/10.14348/molcells.2015.2328
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: songmic@sch.ac.kr
Alveolar epithelial cells have been functionally implicated in
Keywords anti-tuberculosis, inducible nitric oxide synthase (iNOS),
Little is known about the role of alveolar epithelial cells in the pathogenesis of TB. One of the likely mediators of anti-mycobacterial activity is nitric oxide (NO), which is produced by oxidation of L-arginine by the enzyme nitric oxide synthase (NOS) (Stuer et al., 1989). In macrophages, NO, with other toxic superoxide radicals within acidic phagosomes, is vital in limiting mycobacteria (Chan et al., 1992). However, excessive generation of NO may lead to cytotoxic effects and DNA damage, which may lead to cell death through activation of p53 and poly (ADP-ribose) polymerase (Eizirik et al., 1996; Ignarro, 2000). The role of NO in alveolar epithelial cells during mycobacteria infection is not well elucidated.
Ursolic acid (UA; 3-beta-3-hydroxy-urs-12-ene-28-oic-acid) is a pentacyclic triterpenoid carboxylic acid with several biological and pharmacological effects, including anti-inflammatory, anti-oxidant, anti-proliferative, anti-cancer, anti-mutagenic, anti-atherosclerotic, anti-hypertensive, anti-leukemic, and antiviral activities in a number of experimental systems (Ikeda et al., 2008; Tsai and Yin, 2008). Additionally, recent studies demonstrated the anti-TB effects of UA through immunomodulation and activation of intracellular mycobactericidal activity (Jim?nez-Arellanes et al., 2013; Podder et al., 2015). UA is found in a number of foods, including apples, basil, bilberries, cranberries, elder flower, peppermint, rosemary, lavender, oregano, thyme, hawthorn, and prunes (Liu, 1995). Natural compounds enriched in terpenoids have a strong potential to act as inhibitors of the activation of the immune-regulatory transcription factor, nuclear factor-kappa B (NF-κB) (De las Heras et al., 2003).
The present study involved alveolar epithelial A549 cells. This cell line has been used to study various intracellular pathogens such as
Type II human alveolar A549 cells were purchased from American Type Culture Collection (USA). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% (v/v) antibiotic/antimycotic cocktail (100°U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B; Invitrogen, USA) in a humidified atmosphere of 5% CO2 at 37°C. Cells were seeded (5 × 105 cells/ml) in six-well tissue culture plates overnight until they reached a confluence of 75?85%. Immediately before infection or treatment, cells were replaced with serum-free DMEM media. UA and the competitive nitric oxide synthase inhibitor,
A549 cells (1 × 105 cells/ml) were grown in a six-well tissue culture plate overnight. The culture medium was removed and replenished by warm DMEM without FBS. Cells were infected with
The cell viability assay is based on the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan crystal by mitochondrial dehydrogenase enzyme. A549 cells were grown in a 96-well tissue culture plate overnight to a confluence of about 80%. The cells were infected and/or treated as required. Following incubation, 20 μl of 5 mg/ml MTT were added to 200 μl of cell suspension and incubated at 37°C for 4 h. The medium was aspirated and the purple formazan crystal was dissolved by adding 100 μl of dimethyl sulfoxide. After 30 min of incubation at 37°C in the dark, the absorbance was measured at 570 nm using a Victor™ X3 Multilabel reader (Perkin Elmer, USA).
The concentration of nitrite produced as a means to measure NO was measured using Griess reagent system (Promega, USA). In brief, supernatants of infected and/or treated cells for specified time points were collected and centrifuged at 400 ×
A549 cells were seeded, infected with
Cytosolic and nuclear proteins were extracted according to the company protocol (BioVision, USA). Briefly, cells were harvested by centrifugation after the desired infection and/or treatment for desired time points. Cytosol extraction buffer A (CEB-A) was added (200 μl) to the cell pellet, vortexed vigorously, and incubated on ice for 10 min. Cytosol extraction buffer B (CEB-B) was added (11 μl), vortexed vigorously, centrifuged at 16,000×
Cells were harvested following the desired infection and/or treatment, and proteins were collected by using RIPA lysis buffer containing protease inhibitor cocktail (Santa Cruz Bio-technology, USA). The protein concentration was quantified using a BCA protein assay kit (Pierce, USA). Proteins were separated using a 4?20% sodium dodecyl sulfate polyacrylamide electrophoresis gradient gel (Mini-PROTEAN? TGX™ Precast Gel; Bio-Rad, USA) at 100 V for 1.30 h. The separated proteins were transferred to a Trans-Blot SD Semi-Dry Cell polyvinylidene fluoride membrane (Bio-Rad) at 15 V for 1 h. The membranes were blocked by incubation with 5% dried skim milk in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST) for 1 h at room temperature. Membranes were incubated overnight at 4°C with primary antibody against iNOS, nuclear factor-kappa B (NF-κB), P65, Lamin B, α-tubulin (all from Santa Cruz Biotechnology), or β-actin (Abcam, USA). A second incubation was carried out with horseradish peroxidase-conjugated secondary anti-rabbit IgG and anti-mouse IgG (Santa Cruz Biotechnology) for 1.5 h at room temperature. The bound antibodies were visualized using enhanced chemiluminescence western blotting detection reagents (Bio-Rad) and images were acquired using a ChemiDoc™ XRS+ System with Image Lab™ software (Bio-Rad).
Cells were harvested following infection and/or treatment. Total mRNA was collected, quantified, and checked for purity, cDNA was prepared, and qRT-PCR was performed as described previously (Kim et al., 2013). The primers used for human tumor necrosis factor-α (TNF-α) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were 5′-TCTTCTCGAACCCCGAGTGA-3′, 5′-CCTCTGATGGCACCACCAG-3′ and 5′-TCCCATCACCATCTTCCA-3′, 5′-CATCACGCCACAGTTTCC-3′, respectively. The primer for human interleukin-6 (IL-6) was purchased from Bioneer, catalog no. N-1063 (Korea). The assay results were normalized to the endogenous control gene GAPDH.
At least three individual experiments were conducted. Differences between groups were analyzed using one-way analysis of variance followed by the Student’s
To detect successful infection of
NO production was induced in A549 cells upon exposure to
Next, we investigated cell viability following
To correlate NO generation with intracellular mycobactericidal activity in infected A549 cells, MGIT TTD (Fig. 2C), and CFU counts (Fig. 2D) were performed for 0?72 h. The number of bacteria increased, as evident by decreased TTD (days) but increased CFU/ml with time. Therefore, the level of NO generated following
Up-regulation of iNOS was observed in
To investigate the involvement of the NF-κB pathway, cytosolic and nuclear extracts were collected after A549 cells were infected and/or treated with UA. Western blots showed that the nuclear NF-κB levels started to increase at 48 h, which was very significantly evident at 72 h. Conversely, the cytosolic levels were reduced at 72 h in
The innate immune system is the first line of defense against pathogens before the adaptive immune system takes part. Once inhaled, mycobacteria enter the lung and infect macrophages (Ellner, 1997; Fenton and Vermeulen, 1996; Fulton et al., 1998; Rich et al., 1997). Thus far, studies have mainly focused on the pathogenesis of mycobacteria in alveolar macrophages. However, it is likely that mycobacteria also invade alveolar epithelial cells during TB infection. Mycobacteria can successfully invade and replicate within type II alveolar epithelial cells (Bermudez and Goodman, 1996; Garcia-Perez et al., 2003). Therefore, epithelial cells are not innocent bystanders; rather, they have significant roles in innate immunity and inflammatory responses (Gribar et al., 2008). Our data demonstrate that mycobacteria successfully infect and replicate in alveolar epithelial A549 cells. Along with anti-carcinogenic, anti-inflammatory, antioxidant, and pro-apoptotic properties, UA also has anti-TB potential (Jim?nez-Arellanes et al., 2013; Podder et al., 2015). Our goal was to detect the role of UA in mycobacteria-infected alveolar epithelial A549 cells in the context of NO generation and cell viability. NO plays an important role in inflammation, where it is produced by iNOS, which is responsive to interferon-gamma (IFN-γ). IFN-γ stimulation of A549 cells increases NO production (Guzik et al., 2003; Xie and Nathan, 1994). Therefore, NO that is produced due to mycobacterial infection in A549 cells may be attributed to the
The anti-carcinogenic, anti-inflammatory, and pro-apoptotic activities of UA are due to its ability to inhibit the immunoregulatory transcription factor, NF-κB, in response to a variety of carcinogens and inflammatory agents (Shishodia et al., 2003). After taking into account that UA is a triterpenoid carboxylic acid, it is not surprising that this compound possesses potent anti-inflammatory and cytotoxic activities and is a potent inhibitor of NF-κB activation (De las Heras et al., 2003; Yang et al., 2002; 2003). UA partially inhibited the activation of NF-κB and its downstream pro-inflammatory cytokines, TNF-α and IL-6, in mycobacteria-infected alveolar epithelial cells. The observations indicate the anti-inflammatory potential of UA and support the previous description of the activation of NF-κB 48 h after
Our results strongly suggest that alveolar epithelial cells act as the first line of defense against mycobacteria by inducing NO generation. The levels of NO generated directly following infection of A549 cells with mycobacteria are insufficient to kill intracellular mycobacteria. However the bacterial count increased with time. Interestingly, the increased production of NO due to mycobacterial infection showed an increasing cytotoxicity in A549 cells. This cytotoxic effect was partially reversed by treating cells with UA following mycobacterial infection, which is attributed to the reduction of NO production in infected A549 cells. The induced activation of the immunoregulatory transcription factor, NF-κB following infection was also significantly quenched by UA. Taken together, our data reveal the critical role of NO in mycobacterial infection and the protective role of UA in mycobacteria-infected A549 cells.
Mol. Cells 2015; 38(7): 610-615
Published online July 31, 2015 https://doi.org/10.14348/molcells.2015.2328
Copyright © The Korean Society for Molecular and Cellular Biology.
Tamanna Zerin1, Minjung Lee1, Woong Sik Jang2, Kung-Woo Nam3, and Ho-yeon Song1,*
1Department of Microbiology, School of Medicine, Soonchunhyang University, Cheonan 330-090, Korea, 2Regional Innovation Center, Soonchunhyang University, Asan 336-745, Korea, 3Department of Life Science and Biotechnology, College of Natural Science, Soonchunhyang University, Asan 336-745, Korea
Correspondence to:*Correspondence: songmic@sch.ac.kr
Alveolar epithelial cells have been functionally implicated in
Keywords: anti-tuberculosis, inducible nitric oxide synthase (iNOS),
Little is known about the role of alveolar epithelial cells in the pathogenesis of TB. One of the likely mediators of anti-mycobacterial activity is nitric oxide (NO), which is produced by oxidation of L-arginine by the enzyme nitric oxide synthase (NOS) (Stuer et al., 1989). In macrophages, NO, with other toxic superoxide radicals within acidic phagosomes, is vital in limiting mycobacteria (Chan et al., 1992). However, excessive generation of NO may lead to cytotoxic effects and DNA damage, which may lead to cell death through activation of p53 and poly (ADP-ribose) polymerase (Eizirik et al., 1996; Ignarro, 2000). The role of NO in alveolar epithelial cells during mycobacteria infection is not well elucidated.
Ursolic acid (UA; 3-beta-3-hydroxy-urs-12-ene-28-oic-acid) is a pentacyclic triterpenoid carboxylic acid with several biological and pharmacological effects, including anti-inflammatory, anti-oxidant, anti-proliferative, anti-cancer, anti-mutagenic, anti-atherosclerotic, anti-hypertensive, anti-leukemic, and antiviral activities in a number of experimental systems (Ikeda et al., 2008; Tsai and Yin, 2008). Additionally, recent studies demonstrated the anti-TB effects of UA through immunomodulation and activation of intracellular mycobactericidal activity (Jim?nez-Arellanes et al., 2013; Podder et al., 2015). UA is found in a number of foods, including apples, basil, bilberries, cranberries, elder flower, peppermint, rosemary, lavender, oregano, thyme, hawthorn, and prunes (Liu, 1995). Natural compounds enriched in terpenoids have a strong potential to act as inhibitors of the activation of the immune-regulatory transcription factor, nuclear factor-kappa B (NF-κB) (De las Heras et al., 2003).
The present study involved alveolar epithelial A549 cells. This cell line has been used to study various intracellular pathogens such as
Type II human alveolar A549 cells were purchased from American Type Culture Collection (USA). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% (v/v) antibiotic/antimycotic cocktail (100°U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B; Invitrogen, USA) in a humidified atmosphere of 5% CO2 at 37°C. Cells were seeded (5 × 105 cells/ml) in six-well tissue culture plates overnight until they reached a confluence of 75?85%. Immediately before infection or treatment, cells were replaced with serum-free DMEM media. UA and the competitive nitric oxide synthase inhibitor,
A549 cells (1 × 105 cells/ml) were grown in a six-well tissue culture plate overnight. The culture medium was removed and replenished by warm DMEM without FBS. Cells were infected with
The cell viability assay is based on the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan crystal by mitochondrial dehydrogenase enzyme. A549 cells were grown in a 96-well tissue culture plate overnight to a confluence of about 80%. The cells were infected and/or treated as required. Following incubation, 20 μl of 5 mg/ml MTT were added to 200 μl of cell suspension and incubated at 37°C for 4 h. The medium was aspirated and the purple formazan crystal was dissolved by adding 100 μl of dimethyl sulfoxide. After 30 min of incubation at 37°C in the dark, the absorbance was measured at 570 nm using a Victor™ X3 Multilabel reader (Perkin Elmer, USA).
The concentration of nitrite produced as a means to measure NO was measured using Griess reagent system (Promega, USA). In brief, supernatants of infected and/or treated cells for specified time points were collected and centrifuged at 400 ×
A549 cells were seeded, infected with
Cytosolic and nuclear proteins were extracted according to the company protocol (BioVision, USA). Briefly, cells were harvested by centrifugation after the desired infection and/or treatment for desired time points. Cytosol extraction buffer A (CEB-A) was added (200 μl) to the cell pellet, vortexed vigorously, and incubated on ice for 10 min. Cytosol extraction buffer B (CEB-B) was added (11 μl), vortexed vigorously, centrifuged at 16,000×
Cells were harvested following the desired infection and/or treatment, and proteins were collected by using RIPA lysis buffer containing protease inhibitor cocktail (Santa Cruz Bio-technology, USA). The protein concentration was quantified using a BCA protein assay kit (Pierce, USA). Proteins were separated using a 4?20% sodium dodecyl sulfate polyacrylamide electrophoresis gradient gel (Mini-PROTEAN? TGX™ Precast Gel; Bio-Rad, USA) at 100 V for 1.30 h. The separated proteins were transferred to a Trans-Blot SD Semi-Dry Cell polyvinylidene fluoride membrane (Bio-Rad) at 15 V for 1 h. The membranes were blocked by incubation with 5% dried skim milk in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST) for 1 h at room temperature. Membranes were incubated overnight at 4°C with primary antibody against iNOS, nuclear factor-kappa B (NF-κB), P65, Lamin B, α-tubulin (all from Santa Cruz Biotechnology), or β-actin (Abcam, USA). A second incubation was carried out with horseradish peroxidase-conjugated secondary anti-rabbit IgG and anti-mouse IgG (Santa Cruz Biotechnology) for 1.5 h at room temperature. The bound antibodies were visualized using enhanced chemiluminescence western blotting detection reagents (Bio-Rad) and images were acquired using a ChemiDoc™ XRS+ System with Image Lab™ software (Bio-Rad).
Cells were harvested following infection and/or treatment. Total mRNA was collected, quantified, and checked for purity, cDNA was prepared, and qRT-PCR was performed as described previously (Kim et al., 2013). The primers used for human tumor necrosis factor-α (TNF-α) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were 5′-TCTTCTCGAACCCCGAGTGA-3′, 5′-CCTCTGATGGCACCACCAG-3′ and 5′-TCCCATCACCATCTTCCA-3′, 5′-CATCACGCCACAGTTTCC-3′, respectively. The primer for human interleukin-6 (IL-6) was purchased from Bioneer, catalog no. N-1063 (Korea). The assay results were normalized to the endogenous control gene GAPDH.
At least three individual experiments were conducted. Differences between groups were analyzed using one-way analysis of variance followed by the Student’s
To detect successful infection of
NO production was induced in A549 cells upon exposure to
Next, we investigated cell viability following
To correlate NO generation with intracellular mycobactericidal activity in infected A549 cells, MGIT TTD (Fig. 2C), and CFU counts (Fig. 2D) were performed for 0?72 h. The number of bacteria increased, as evident by decreased TTD (days) but increased CFU/ml with time. Therefore, the level of NO generated following
Up-regulation of iNOS was observed in
To investigate the involvement of the NF-κB pathway, cytosolic and nuclear extracts were collected after A549 cells were infected and/or treated with UA. Western blots showed that the nuclear NF-κB levels started to increase at 48 h, which was very significantly evident at 72 h. Conversely, the cytosolic levels were reduced at 72 h in
The innate immune system is the first line of defense against pathogens before the adaptive immune system takes part. Once inhaled, mycobacteria enter the lung and infect macrophages (Ellner, 1997; Fenton and Vermeulen, 1996; Fulton et al., 1998; Rich et al., 1997). Thus far, studies have mainly focused on the pathogenesis of mycobacteria in alveolar macrophages. However, it is likely that mycobacteria also invade alveolar epithelial cells during TB infection. Mycobacteria can successfully invade and replicate within type II alveolar epithelial cells (Bermudez and Goodman, 1996; Garcia-Perez et al., 2003). Therefore, epithelial cells are not innocent bystanders; rather, they have significant roles in innate immunity and inflammatory responses (Gribar et al., 2008). Our data demonstrate that mycobacteria successfully infect and replicate in alveolar epithelial A549 cells. Along with anti-carcinogenic, anti-inflammatory, antioxidant, and pro-apoptotic properties, UA also has anti-TB potential (Jim?nez-Arellanes et al., 2013; Podder et al., 2015). Our goal was to detect the role of UA in mycobacteria-infected alveolar epithelial A549 cells in the context of NO generation and cell viability. NO plays an important role in inflammation, where it is produced by iNOS, which is responsive to interferon-gamma (IFN-γ). IFN-γ stimulation of A549 cells increases NO production (Guzik et al., 2003; Xie and Nathan, 1994). Therefore, NO that is produced due to mycobacterial infection in A549 cells may be attributed to the
The anti-carcinogenic, anti-inflammatory, and pro-apoptotic activities of UA are due to its ability to inhibit the immunoregulatory transcription factor, NF-κB, in response to a variety of carcinogens and inflammatory agents (Shishodia et al., 2003). After taking into account that UA is a triterpenoid carboxylic acid, it is not surprising that this compound possesses potent anti-inflammatory and cytotoxic activities and is a potent inhibitor of NF-κB activation (De las Heras et al., 2003; Yang et al., 2002; 2003). UA partially inhibited the activation of NF-κB and its downstream pro-inflammatory cytokines, TNF-α and IL-6, in mycobacteria-infected alveolar epithelial cells. The observations indicate the anti-inflammatory potential of UA and support the previous description of the activation of NF-κB 48 h after
Our results strongly suggest that alveolar epithelial cells act as the first line of defense against mycobacteria by inducing NO generation. The levels of NO generated directly following infection of A549 cells with mycobacteria are insufficient to kill intracellular mycobacteria. However the bacterial count increased with time. Interestingly, the increased production of NO due to mycobacterial infection showed an increasing cytotoxicity in A549 cells. This cytotoxic effect was partially reversed by treating cells with UA following mycobacterial infection, which is attributed to the reduction of NO production in infected A549 cells. The induced activation of the immunoregulatory transcription factor, NF-κB following infection was also significantly quenched by UA. Taken together, our data reveal the critical role of NO in mycobacterial infection and the protective role of UA in mycobacteria-infected A549 cells.