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International Journal of Mycobacteriology

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Available at www.sciencedirect.com

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Differential signaling of inducible nitric oxide synthase induction in Mycobacterium tuberculosis infected alveolar epithelial cell line A549 in response to cytokines IFN-c, TNF-a and IL-1b Sugata Roy 1, Sadhna Sharma 2, Monika Sharma 2, Mridula Bose

*

Dept. of Microbiology, V.P. Chest Institute, University of Delhi, Delhi 110007, India

A R T I C L E I N F O

A B S T R A C T

Article history:

Background: In earlier studies, it was shown that ex vivo Mycobacterium tuberculosis-infected

Received 23 December 2013

type II alveolar epithelial cells generate de novo nitric oxide (NO), but the mycobactericidal

Received in revised form

quantity of NO was released only by stimulation of these cells with proinflammatory cyto-

13 January 2014

kines, i.e. IFN-c, TNF-a and IL-1b. In the present communication, it was demonstrated that

Accepted 16 January 2014

M. tuberculosis-infected/mycobacterial antigens stimulated cells utilize both, JAK-STAT and

Available online 21 February 2014

NF-jB pathways for the induction of inducible Nitric Oxide Synthase (iNOS) mRNA and NO production.

Keywords:

Methods: Alveolar epithelial cell line A549 were either infected with M. tuberculosis or stim-

Alveolar epithelial cell line A549

ulated with M. tuberculosis components. Confocal microscopy, NO estimation and EMSA

iNOS gene

were performed on the infected/stimulated A549 cells.

STAT-1

Results: Nuclear extracts prepared from M. tuberculosis infected A549 cells alone or stimu-

NF-jB

lated with IFN-c or a combination of three cytokines (IFN-c, TNF-a and IL-1b) formed DNA protein complexes with probes from both 5.2 kb region (specific for binding of STAT-1 protein) and 5.8 kb region (specific for binding of both STAT-1 and NF-jB) of the iNOS promoter. However, TNF-a or IL-1b stimulated M. tuberculosis-infected A549 cells showed no protein DNA complexes with construct from 5.2 kb region. Conclusions: This differential response indicated that TNF-a/IL-1b does not allow STAT-1 production or its translocation to nucleus in M. tuberculosis-infected A549 cells in the absence of IFN-c. This differential signaling of iNOS induction in M. tuberculosis-infected alveolar epithelial cells by cytokines may be responsible for controlled production of NO intracellularly. 2014 Published by Elsevier Ltd. on behalf of Asian-African Society for Mycobacteriology.

* Corresponding author. Tel.: +91 11 27667667; fax: +91 11 27667420. E-mail addresses: [email protected] (S. Roy), [email protected] (S. Sharma), [email protected] (M. Sharma), [email protected] (M. Bose). 1 Present address: Omics Molecular Interaction Research Unit, LSA Technology Development Group, RIKEN Yokohama Institute, 1-7-22 Suehiro-Cho, Tsurumi-Ku, Yokohama 230-0045, Japan. Tel.: +81 45 503 9222x3486. 2 Present address: Dept. of Zoology, Miranda House, University of Delhi, Delhi 110007, India. Tel.: +91 11 27667367. http://dx.doi.org/10.1016/j.ijmyco.2014.01.008 2212-5531/ 2014 Published by Elsevier Ltd. on behalf of Asian-African Society for Mycobacteriology.

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Introduction

Material and methods

The pulmonary epithelial cells which cover a surface area of 70 m2 act as the innate immune defense to inhaled pathogens [34]. The immune response in the lungs is complex and there is growing evidence that nitric oxide (NO) plays an important role [1,20,27]. Inducible nitric oxide synthase (iNOS) is the enzyme that leads to secretion of nitric oxide. In a mouse model of tuberculosis (TB) numerous experiments have demonstrated a critical mycobactericidal role played by NO which was further strengthened by a study in a genetically disrupted iNOS mouse strain [7,21]. In contrast, the role of NO in human TB is still a subject of active research. The recent knowledge that alveolar epithelial cell line A549 is a robust NO producer initiated many studies to investigate the Mycobacterium tuberculosis–A549 cell interaction. It has been demonstrated that human type II alveolar epithelial cell line A549 could express inducible NO synthase in response to a combination of three proinflammatory cytokines IL-1b, TNF-a and IFN-c. iNOS induction leads to continuous NO production until the time the enzyme is degraded [8,16,18,21]. These cytokines or other inducers enhance or inhibit iNOS expression by activating or blocking ‘cell type’ and ‘species specific’ signal transduction pathways [18,29]. The iNOS promoters of all the species contain a TATA box which is about 30 bp from the start site of transcription. All mammalian iNOS promoters possess binding sites for the transcription factor NF-jB near the TATA box. The mammalian promoters also display binding sites at position 900 for transcription factors induced by IFN-c [6,36,38]. A 50-fold higher induction was seen when 8.3 kb fragment of iNOS promoter is used where the transcription sites seem to be located upstream of 3.8 kb of 5 0 region of human iNOS gene [6,12,22]. All mammalian iNOS promoters show homologies with GAS, the binding site for IFN-c regulated transcription factor STAT-1a [18] and involve IFN-cJAK2-STAT-1a pathway in iNOS induction [8,15,18,19,23]. Many compounds were shown to inhibit iNOS expression by blocking STAT-1a activation [3,17] whereas extracellular regulated kinases ERK1/ERK2 were shown to contribute to iNOS induction by activation of STAT-1a in IFN-c stimulated cells [3]. The human iNOS promoter was found to have a bi-functional NF-jB/STAT-1a motif at 5.8 kb and a STAT-1a-specific responsive element at 5.2 kb by site directed mutagenesis [8]. STAT1a either by directly binding to the iNOS promoter or indirectly inducing IRF-1 activity led to iNOS induction [18,16]. The transcription factor NF-jB also seems to be a central target for induction or inhibition of iNOS expression in different cell types. LPS and cytokines like IL-1b or TNF-a induce iNOS expression [11,13], whereas glucocorticoids and transforming growth factor inhibit iNOS expression by modulating NF-jB [14,24,37]. Multiple NF-jB binding sites between positions 5.2 and 6.5 kb of human iNOS promoter has been shown [33]. A lot of studies show that NO produced by human macrophages and also by epithelial cells has antimycobacterial effects, including the work in the laboratory used in this study [4,5,25,28,30,33]. In the present work, it was attempted to find out whether NO produced by M. tuberculosis-infected alveolar epithelial cells follow the same regulatory mechanism as followed by cytokine-stimulated alveolar epithelial cells.

Cell line Human pulmonary alveolar epithelial cell line A549 was cultured as described in the previous publications [28,30].

Mycobacteria M. tuberculosis H37Rv (American-type culture collection) was maintained as described in the previous publications [28,30].

M. tuberculosis H37Rv infected A549 cell lines alone or with cytokine treatment A549 cells grown in six well tissue culture wells were infected with mycobacteria and stimulated with cytokines as described in the previous publications [28,30]. To demonstrate invasion of A549 cells by M. tuberculosis, the infected monolayers were treated with 0.1% triton-X to release the internalized mycobacteria as described in an earlier publication [28]. The infected monolayers were also processed for Laser Scanning Confocal Microscopy. For some experiments, alveolar epithelial cells were infected with M. tuberculosis H37Rv followed by stimulation with three prime cytokines IFN-c, TNF-a and IL-1b as described earlier [28]. NO assays and nuclear protein preparations were done at 48 h, as this time period was midpoint of the experimental duration and a sufficient amount of NO was elaborated in all experimental conditions at this time point. The cell growth and viability of infected and cytokine-treated A549 cell line was done using XTT assays as described in the previous publications [28,30].

Gamma-irradiated H37Rv or H37Rv components treated A549 cells Gamma-irradiated H37Rv or H37Rv components-treated A549 cells were used for experiments as described earlier [28,30].

Confocal microscopy To confirm the intracellular presence of M. tuberculosis in A549 cells, the infected monolayers were processed for confocal microscopy as described in the previous publication [30].

Nitric oxide assay Nitric oxide was measured using Griess reagent using an ELISA Reader at 540 nm as described in earlier publications [28,30].

Preparation of nuclear protein fraction To demonstrate the expression of transcription factor STAT1a and NF-jB, nuclear proteins were extracted from uninfected and infected A549 cells using protocol described in earlier studies [8,22,30].


Electrophoretic mobility shift assay Gel shift assay was performed using synthesized complementary probes as reported in earlier studies [8,30].

Results Invasion by and replication of M. tuberculosis in the alveolar epithelial cells To demonstrate that M. tuberculosis was internalized by alveolar epithelial cells, laser scanning confocal microscopy (LSCM) using Auramine-rhodamine staining was performed. 39% of the cells demonstrated internalized mycobacteria after 6 h of infection and 1–3 bacteria per infected cell were found by LSCM (Fig. A.1). To enumerate the internalized mycobacteria, the infected monolayers were also lysed to release the intracellular mycobacteria. CFU was determined at different time points by inoculating the lysate on LJ slants. The CFU at 6 h was found to be 10.85 + 0.26 and at 100 h it became 13.14 ± 0.42 indicating that mycobacteria not only invade, but can replicate inside the alveolar epithelial cells (Table B.1).

Infection of alveolar epithelial cells with M. tuberculosis leads to signal transduction of iNOS gene through STAT-1 and NFjB Since A549 cell line is an alveolar epithelial cell line and proinflammatory cytokines IFN-c, TNF-a and IL-1b are present in the internal milieu of the infected lung, the A549 cells were stimulated with these cytokines individually or in combination. To determine the presence of transcription factors induced by cytokines, nuclear extracts derived from cytokine-stimulated A549 cells were subjected to EMSA using 22 bp oligonucleotide STAT-1 probes corresponding to the cis-acting DNA elements at 5.2 kb in the hiNOS promoter. Nuclear extracts from untreated cells served as a control. It was observed that nuclear extracts from cells stimulated with IFN-c alone or IFN-c as a part of a cytokine mixture produced DNA protein complex with the wild type oligonucleotide at 5.2 kb. When highly selective mutant DNA sequence of 5.2 kb region of hiNOS promoter was used, the DNA–protein binding was abolished (Fig. A.2). The data suggested

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expression of STAT-1 protein. To study the expression of NFjB in transcription of iNOS gene, 18 bp oligonucleotides, corresponding to the 5.8 kb region of the hiNOS promoter was used. It has been reported that 5.8 kb is a bifunctional region for both STAT-1 and NF-jB and is important for the induction of the iNOS gene. Nuclear extracts from A549 cell line stimulated with a combination of cytokines produced DNA protein complex with the wild type oligonucleotide from the 5.8 kb region. When 5.8 kb NF-jB mutant was used, protein DNA complexes were obtained in IFN-c/cytokine stimulated A549 cells, but not in the TNF-a or IL-1b-stimulated A549 cells as IFN-c act through stimulation of STAT-1 and TNF-a and IL1b bring their effect through NF-jB production. When 5.8 kb STAT1 mutant was used, protein DNA complexes were obtained in TNF-a/IL-1b-stimulated A549 cells (Fig. A.2). The data suggested binding of both STAT-1 and NF-jB protein for iNOS gene expression in cytokine-stimulated A549 cells. The appearance of STAT-1 and NF-jB correlated with the production of nitric oxide by the cytokine stimulated A549 cells. To investigate whether M. tuberculosis H37Rv affect STAT1 and NF-jB DNA binding activity in reference to the induction of iNOS gene in human lung epithelial cells line A549, the induction was analyzed of STAT1 and NF-jB in the infected A549 cells. Gel shift assays using 5.2 and 5.8 kb oligonucleotide demonstrated a good amount of protein DNA complexes which show the expression of both STAT1 and NF-jB in concurrence with NO production by these cells (Fig. A.3, Table B.2). The level of NO produced by M. tuberculosis-infected pulmonary epithelial cells (29.53 ± 5.7 mmol/106 cells) was higher than that produced by TNF-a (19.46 ± 2.9 mmol/106 cells) or IL-1b (21.08 ± 5.9 mmol/106 cells) stimulated uninfected A549 cells indicating simultaneous expression of both the transcription factors leads to enhanced production of NO (Table B.1).

TNF-a regulate the release of NO in M. tuberculosis-infected alveolar epithelial cells To examine the effect of three prime cytokine IFN-c, TNF-a and IL-1b for the induction of STAT1 and NF-jB in the infected A549 cells, EMSA assays were performed on cytokine-stimulated M. tuberculosis-infected A549 cells. When M. tuberculosis-infected cells were stimulated with IFN-c alone or

Auramine rhodamine stained yellowish orange mycobacteria were seen (100X). Fig. A.1 – Laser Scanning Confocal Microscopy micrographs showing M. tuberculosis-infected A549 cells. Yellowish orange mycobacteria were seen that were stained with Auramine-Rhodamine (100X).

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Table B.1 – Relationship between Nitric Oxide production and mycobacterial growth inside A549 cells infected with M. tuberculosis.

Nitric oxide (lmol/L) CFU/ml/well (·105) % Increase in CFU

24 h

48 h

72 h

20.02 ± 5.5 10.26 ± 0.26 3.07%

29.53 ± 5.7 10.62 ± 0.48 7.17%

49.74 ± 7.2 13.10 ± 0.28 24.09%

Percent reduction was calculated from CFU/ml at 6 h after amikacin treatment and washing in each set of experiments (three independent experiments). CFU/ml at 6 h was 10.53 ± 0.71 which was the basal value.

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Fig. A.2 – Mobility shift assay with 5.2 kb oligo in IFNc/ cytokine mixture treated infected A549 cells. Lane 1 free probe; Lane 2 A549 cell only; Lane 3 A549 cells infected with M. tb; Lane 4 A549 cells stimulated with IFN c; Lane 5 A549 cells stimulated with TNFa; Lane 6 A549 cells stimulated with IL-1b; lane 7 A549 cells stimulated with TNFa + IFN c; Lane 8 A549 cells stimulated with cytokine mixture.

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cytokine mixture, the protein DNA complexes were observed using both 5.2 and 5.8 kb oligonucleotide probes. The results were confirmed using specific mutant probes indicating expression of both STAT-1 and NF-jB in these cells. Interestingly, when infected A549 cells were stimulated with TNF-a or IL-1b, no protein DNA complexes were obtained using 5.2 kb probes, but protein DNA complexes were obtained when 5.8 kb wild type, STAT1 mutant and 5.8 kb NF-jB mutant oligonucleotide probes were used (Figs. A.3,A.4,A.5 Table B.2). These results indicate that either STAT-1 is not expressed, or it is not translocated to the nucleus or some conformational change may inhibit its binding to 5.2 kb probe.

Stimulation of alveolar epithelial cells with mycobacterial components also result in signal transduction of iNOS gene through STAT-1 and NF-jB It is documented in the previous communication that mycobacterial components are potent inducers of NO. Here it was observed that nuclear extracts from cells stimulated with mycobacterial components produced DNA protein complex with both the wild type oligonucleotides at 5.2 and 5.8 kb indicating that some constitutive component of M. tuberculosis is responsible for both STAT 1 and NF-jB expression in these cells. Surprisingly, here also cells stimulated with the cell wall component of M. tuberculosis, particularly LAM, did not show any binding with the 5.8 probe (Figs. A.6,A.7). This again indicated differential regulation of iNOS activity by regulating the binding of STAT-1 and NF-jB with the iNOS promoter.

Discussion

Fig. A.3 – Protein DNA complex with 5.8 kb oligo in cytokine stimulated infected A549 cells. Lane 1 free probe; Lane 2 A549 cell only; Lane 3 A549 cells infected with M. tb; Lane 4 A549 cells stimulated with IFN c; Lane 5 A549 cells stimulated with TNFa; Lane 6 A549 cells stimulated with IL1b; Lane 7 A549 cells stimulated with TNFa + IFN c; Lane 8 A549 cells stimulated with cytokine mixture.

The inducible nitric oxide synthase (iNOS) is found to be regulated at all levels, be it transcriptional, post-transcriptional or translational, and this regulation is cell and species-specific. A cell has many regulatory mechanisms for iNOS expression, but once expressed, the enzyme activity is not regulated [18]. It was demonstrated in this study that M. tuberculosis-infected alveolar epithelial cells inducibly transcribe iNOS and secrete NO through a mechanism involving cytoplasmic transcription factors STAT-1 and NF-jB. Thus, alveolar epithelial cells actively participate in the signaling mechanism that enhance the inflammatory response against tubercle bacilli and play an important role in innate immunity. The alveolar macrophages are the primary cells to encounter M. tuberculosis, and roughly thousands of alveolar epithelial cells share the same alveolar space with about 50


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Table B.2 – Mobility Shift Assay and nitric oxide (NO) production under different experimental conditions. Entity

A549 cells

Infected cells

Infected Infected Infected Infected Infected cells + IFN-c cells + TNF-a cells + IL-1b cells + IFN-c + cells + cytokine TNF-a mixture

No production 5.2 kb probe 5.2 kb STAT-1 mutant probe 5.8 kb probe 5.8 kb STAT-1 mutant Probe 5.8 kb NF-jB mutant probe 5.8 kb double mutant probe

13.30 ± 3.4 ve ve ve ve ve ve

29.53 ± 5.7 +ve ve +ve +ve +ve ve

61.27 ± 4.9 +ve ve +ve +ve +ve ve

31.56 ± 3.6 ve ve +ve +ve +ve ve

38.03 ± 6.0 ve ve +ve +ve +ve ve

55.46 ± 6.2 +ve ve +ve +ve +ve ve

56.75±7.7 +ve ve +ve +ve +ve ve

Nuclear proteins were extracted from infected A549 cells stimulated with various cytokines at 48 h (three independent experiments). Oligonucleotide probes were prepared from regions specific for wild type 5.2 and 5.8 kb region along with probes containing mutant sequences. +ve sign indicates protein DNA complex was formed in that experiment. ve sign indicates no protein DNA complex. Cytokine mixture is a mix of IFN-c, TNF-a and IL-1b.

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Fig. A.4 – Protein DNA complex with 5.8 kb NF-jB mutant in cytokine stimulated infected A549 cells. Lane 1 free probe; Lane 2 A549 cell only; Lane 3 A549 cells infected with M. tb; Lane 4 A549 cells stimulated with IFN c; Lane 5 A549 cells stimulated with TNFa; Lane 6 A549 cells stimulated with IL1b; Lane 7 A549 cells stimulated with TNFa + IFN c; Lane 8 A549 cells stimulated with cytokine mixture.

Fig. A.5 – Protein DNA complex with 5.8 kb STAT1 mutant in cytokine stimulated infected A549 cells. Lane 1 free probe; Lane 2 A549 cell only; Lane 3 A549 cells infected with M. tb; Lane 4 A549 cells stimulated with IFN c; Lane 5 A549 cells stimulated with TNFa; Lane 6 A549 cells stimulated with IL1b; Lane 7 A549 cells stimulated with TNFa + IFN c; Lane 8 A549 cells stimulated with cytokine mixture.

mononuclear phagocytes [34]. Therefore, it is plausible to assume that a few M. tuberculosis bacilli may be able to interact with alveolar epithelial cells during the infectious process. These results demonstrated invasion of A549 cells by M. tuberculosis as well as their replication inside the alveolar epithelial cells as indicated by confocal microscopy and CFU assay. Similar to these results, other groups [2,35] have also observed invasion of A549 cells with M. tuberculosis after 4 h of infection. It has been reported recently that live mycobacteria enter alveolar epithelial cells by macropinocytosis [9] by inducing changes in the distribution of actin filaments. These results confirm this as heat-killed M. tuberculosis fail to internalize, which could be due to its failure to induce actin mobilization. Although many bacterial pathogens adhere and enter epithelial cells to facilitate migration to sub-epithelial spaces, it was observed that M. tuberculosis is even capable of replicating inside as the number of intracellular

mycobacteria increased from 10.85 ± 0.26 to 13.14 ± 0.42 after four days of infection. The molecular regulation of the human iNOS gene contrasts markedly with that of the murine and rat responsiveness [10,26]. These cytokines activate their respective signal transduction pathways, and through functional NF-jB and STAT1a elements located upstream of 4.7 kb, initiate the transcriptional machinery required for human iNOS expression. It has been demonstrated that 5.2 kb region in hiNOS promoter is the STAT1 binding site and is important for induction of NO synthase gene in human alveolar epithelial cells. Additionally, critical NF-jB elements have been localized far upstream in the human inducible NO synthase gene promoter from 5.2 to 6.1 kb and at 8.3 kb. A recent report demonstrated that the critical NF-jB elements, required for the transcriptional induction of hiNOS, are localized at the 5.8 kb hiNOS promoter. This 5.8 kb region was found to be bifunctional NF-jB/STAT1 motif [8].

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Fig. A.6 – Mycobacterial Antigenic components of H37Rv induced STAT1 induction in A549 cells using 5.2 probe. Lane 1 free probe; Lane 2 A549 cell only; Lane 3 A549 cells stimulated with WCL; Lane 4 A549 cells stimulated with MEM; Lane 5 A549 cells stimulated with CW; Lane 6 A549 cells stimulated with CYT; Lane 7 A549 cells stimulated with LAM.

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Fig. A.7 – Protein DNA complex with 5.8 kb oligo probe in A549 cells stimulated with Mycobacterial Antigenic components of H37Rv. Lane 1 free probe; Lane 2 A549 cell only; Lane 3 A549 cells stimulated with WCL; Lane 4 A549 cells stimulated with MEM; Lane 5 A549 cells stimulated with CW; Lane 6 A549 cells stimulated with CYT; Lane 7 A549 cells stimulated with LAM.

These results demonstrated the expression of STAT1 transcription factor using the 5.2 kb oligonucleotide probe in M. tuberculosis-infected A549 cells. The results were confirmed using mutant oligonucleotide of 5.2 kb. Interestingly, when the M. tuberculosis-infected cells were stimulated with TNF-a or IL-1b, no protein DNA complex with oligo-probe of 5.2 kb was observed, but the complex was observed when either IFN-c alone or cytokine mixture was used. The levels of NO production were comparable in M. tuberculosis-infected, or TNF-a or IL-1b stimulated M. tuberculosis-infected A549 cells, whereas these levels were much higher in the IFN-c stimulated M. tuberculosis-infected A549 cells. It has been

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observed in this lab that M. tuberculosis-infected A549 cells are capable of secreting cytokines like IFN-c and TNF-a; this endogenous production may be sufficient to induce the iNOS enzyme and subsequent NO release. As M. tuberculosis-infected macrophages along with T cells are the main source of these cytokines, alveolar epithelial cells are further stimulated to produce more NO and thus restrict the growth of M. tuberculosis [28]. The inhibition of binding of STAT-1 to 5.2 kb probe could result from possibilities of direct capture of STAT-1 by protein–protein interactions, or by inhibition of nuclear translocation of STAT-1 probably by inhibiting ERK protein kinases or by enhancing the expression of specific inhibitors of STAT-1 [18]. The NF-jB sequence motif is recognized by members of the Rel homology family, including NF-jB 1,NF-jB 2, Rel A, cRel and Rel B. Various forms of the jB sequence motif have been shown to exhibit a differential affinity for and functional response to different dimeric combination of Rel family proteins. Furthermore, members of the Rel family have been shown to physically and functionally interact with members of the other transcription family [31,32]. As involvement of NF-jB is implicated in iNOS induction following TNF-a stimulation, NF-jB or other homologous transcription factors may be responsible for squelching of STAT-1a in the infected cells following TNF-a stimulation. Interestingly, protein DNA complexes were obtained using 5.8 kb oligonucleotide probe in M. tuberculosis-infected A549 cells which established involvement of both STAT1 and NF-jB in these cells. This was later confirmed by using 5.8 kb NF-jB mutant and 5.8 kb STAT1 mutant. The finding that DNA–protein complexes were observed even when mutant 5.8 kb NF-jB probe was used indicates that active STAT-1 was present. It may indicate that M. tuberculosis-infected A549 cells in the presence of TNF-a may show some conformational changes in STAT-1 so that it can bind to wild 5.8 kb and mutant 5.8 kb NF-jB probes but not to the wild type 5.2 jB probes. Although these possibilities have been observed in response to infection by different microorganisms leading to inhibition of IFN-c responsive genes, additional experiments will be necessary to determine which of the methods may account for the inhibition of binding of STAT-1 in TNF-a stimulated M. tuberculosis-infected cells. It was observed bioinformatically using DNA-Star software and CDC-Blast program that some of the M. tuberculosis proteins show about 32% homology to the DNA binding domain of STAT-1 protein; these M. tuberculosis proteins may interfere with STAT-1 binding with DNA probes, but needs more experimental data for support. This data suggested that production of NO in M. tuberculosis-infected A549 cells utilizes both STAT-1-Janus kinase (JAK) and NF-jB pathways. Involvement of STAT-JAK kinase and NF-jB pathways for the induction of NO synthase gene in infected A549 cells shows that these cells behave as immune cells and may supplement macrophages in countering tubercle bacilli. Another interesting observation was that c-irradiated M. tuberculosis and mycobacterial components, namely whole cell lysate, cell wall fraction, cytosol fraction of H37Rv-stimulated A549 cells, expressed both STAT-1 and NF-jB, whereas cell wall components, particularly LAM, could only express STAT-1.


Conclusion These findings indicate that alveolar epithelial cells have the full machinery to respond to mycobacterial challenges whether in the form of whole particulate M. tuberculosis or its antigens present in the lung milieu following infection. The differential signaling of NO production may be generated by diversity in the control of gene expression during M. tuberculosis infection.

Conflict of interest There is no conflict of interest to declare.

Acknowledgements The authors acknowledge the financial support provided by the Indian Council of Medical Research, India.

R E F E R E N C E S

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