Potential of rifamides to inhibit TNF-induced NF-{kappa}B activation

Ali A. Pahlevana,b, David J. M. Wrighta,*, Laura Bradleyb, Clive Smithb and Brian M. J. Foxwellb

a Infectious Diseases and Microbiology, Charing Cross Campus, Imperial College; b Cytokine Biology and Signal Transduction Laboratory, Kennedy Institute of Rheumatology, London, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Rifamides are important components in the treatment of tuberculosis. However, it is well documented that these drugs can have immunosuppressive activity, a property of these drugs that is particularly relevant to AIDS patients. In this study, we have shown that a number of rifamide analogues have the potential to block tumour necrosis factor (TNF)- or phorbol myristate acetate-induced NF-{kappa}B activation. As TNF is important in the host defence against tuberculosis, suppression of this activity may provide a potential mechanism of rifamide immunosuppressive activity.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Rifampicin is an antibiotic that constitutes an essential part of pulmonary tuberculosis therapy.1 The immunosuppressive role of rifamides has been well described in man.2 Clinically, an increase in tuberculosis recurrence occurs among HIV-1 patients after treating concomitant tuberculosis; it is possible that this increase is related to the use of rifampicin.3 The mechanism of this immunosuppressive activity is unknown, although in vitro studies by Calleja et al.2 indicate that rifampicin may have steroid-like activities.

The Mycobacterium ulcerans toxin has been shown to be immunosuppressive and block NF-{kappa}B activation by tumour necrosis factor (TNF).4 As the structure of M. ulcerans toxin, a polyketide,5 is related to that of rifamides we decided to investigate whether these drugs inhibited NF-{kappa}B activation, particularly in response to TNF, a cytokine important to the host defence against bacterial infection.6 We now show that rifamides can inhibit TNF-induced NF-{kappa}B activation, a result that may help explain the immunosuppressive properties of these drugs.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Bacterial strains and reagents

The M. ulcerans pathogenic strain (7634) from an original clinical isolate was provided by Dr J. L. Stanford (Middlesex Hospital, London, UK). Mycobacteria were grown in Dubos broth base medium (Difco, Oxford, UK) as previously described.4 Rifamides were obtained from Sigma (Poole, UK), or were given by Marion-Roussel, Romainville, France (rifapentine) and Pharmacia, Milton Keynes, UK (rifabutin).

Preparation of M. ulcerans culture filtrate (CF) and ethyl acetate extracted filtrate (EAEF)

Culture filtrates (CFs) were prepared as described by Pahlevan et al.4 CF was then extracted twice with ethyl acetate. The organic phase was collected and concentrated by evaporation, and hence termed ethyl acetate extracted filtrate (EAEF). EAEF had the same activities as our previously partially purified M. ulcerans toxin, aHDL,4 or a chemically defined extracted factor from M. ulcerans CF termed MUPT (M. ulcerans polyketide toxin) (results not shown).4,7

Cell culture and assay for ß-galactosidase

The human T cell line Jurkat subclone 3KB5.2 (NF-{kappa}B) containing the NF-{kappa}B ß-galactosidase reporter gene (given by Prof. Herzenberg, Stanford, CA, USA) was maintained as described previously.4 The cells were washed twice and resuspended at 2 x 106 cells/mL in complete RPMI, without phenol red. One hundred microlitres per well of cell suspension were added to a 96-well plate and treated in the presence or absence of compounds with stimuli: TNF (20 ng/mL) or phorbol myristate acetate (PMA) (50 ng/mL) in triplicate overnight. The cells were lysed and ß-galactosidase activity was measured as described previously.4

Electrophoretic mobility shift assay (EMSA)

Following activation of cells with TNF or PMA for 30 min, cells were lysed and nuclear proteins were extracted and assayed for NF-{kappa}B DNA binding activity using an oligonucleotide encoding the NF-{kappa}B binding sequence as described previously.4


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
A Jurkat T cell line incorporating an NF-{kappa}B-driven ß-galactosidase reporter gene construct was used to investigate the effect of rifamides on TNF- and PMA-induced NF-{kappa}B activation. A variety of rifamide analogues, rifamycin B, rifapentine, rifamycin SV, rifabutin and rifampicin were capable of inhibiting both TNF- and PMA-induced NF-{kappa}B activation (Figure 1aGo–e). This was different from that obtained with M. ulcerans toxin, which, although inhibiting TNF-induced NF-{kappa}B activity (Figure 1fGo), synergized with PMA (Figure 1fGo, and Pahlevan et al.4). Studies of DNA binding activity by EMSA showed that the rifamides rifapentine and rifampicin did not inhibit TNF- or PMA-induced NF-{kappa}B binding activity (Figure 2Go). Similar preliminary data were obtained for the other rifamide analogues (data not shown). The data would indicate that the rifamides are blocking NF-{kappa}B activation after DNA binding, possibly during the NF-{kappa}B gene transactivation process. These results are also different from M. ulcerans toxin, which does inhibit TNF-induced NF-{kappa}B DNA binding activity.4 The specific target for the inhibitory effect on NF-{kappa}B is still to be elucidated.



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Figure 1. Jurkat cells were stimulated with TNF 20 ng/mL ({square}) or PMA 50 ng/mL (), as described in Materials and methods, in the absence or presence of the given concentrations of rifamides. After overnight culture, the cells were harvested and ß-galactosidase activity measured. The data represent the mean of triplicate points ± s.d. The study is representative of three separate experiments.

 


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Figure 2. Jurkat cells were treated with TNF (20 or 50 ng/mL), PMA (100 ng/mL) in the presence of rifapentine (Rp) or rifampicin (Rc). After 30 min incubation, cells were harvested and extracts prepared for NF-{kappa}B DNA binding activity by EMSA analysis. This study is representative of three separate experiments. NS-band, non-specific band.

 
The ability of rifamides to block NF-{kappa}B activation is potentially in agreement with Calleja et al.,2 who found that rifampicin inhibited the activation of an IL-2 promoter– reporter gene construct activated by PMA/ionomycin in Jurkat cells. Although the targets of rifampicin were not defined, there are NF-{kappa}B binding sites in the IL-2 promoter. However, Jaffuel et al.8 showed no effect of rifampicin on NF-{kappa}B activation using a reporter gene system in the A549 cell line. There could be many reasons for this discrepancy with our data. In the Jaffuel et al.8 study, no physiological stimulus was used; rather, activation was induced by co-transfection of a plasmid expressing the p65 NF-{kappa}B subunit. If rifamides inhibit the pathways regulating the activation of the p65 subunit, this experiment may fail to show an effect. Also, only one concentration of rifamide was used (10-6 M), which is equivalent to the lowest concentration used in our experiments that was ineffective (Figure 1Go). Lastly, there may be differences between cell lines of different origin.

Intracellular concentrations of rifamides other than rifampicin may be much higher than serum levels,9,10 therefore, the highest concentration tested in our experiments may prove clinically significant and more likely to be immunosuppressive. This effect may impinge on the management of tuberculosis and is, perhaps, especially significant in patients with concurrent AIDS, where additional immunosuppression may cause reoccurrence (see preliminary findings reviewed by Corbett et al.1). There have been relapses reported when rifapentine was used to treat tuberculosis in HIV patients.10 It is possible that this rifamide has an immunosuppressive effect, which by encouraging recurrences promotes the development of rifamide monoresistance in these patients. Although the rifamides share with the M. ulcerans toxin the ability to inhibit NF-{kappa}B activity, the data would indicate that the mechanisms by which they act are distinct, possibly linked to the different side chain structures. Such structural differences may also explain our observation that rifamides, unlike M. ulcerans toxin,4 were unable to block LPS-induced TNF production from human peripheral blood monocytes (data not shown). The result would discount another potential mechanism of rifamide immunosuppression.

In summary, these data show that rifamides have the potential to block NF-{kappa}B activation by TNF, which could provide a mechanism for the immunosuppressive properties of these drugs.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Supported by The Wellcome Trust and The Arthritis Research Campaign.


    Notes
 
* Correspondence address. Cell and Molecular Biology Section, Division of Biomedical Sciences, Alexander Fleming Building, Imperial College of Science, Technology and Medicine, Exhibition Road, London SW7 2AZ, UK. Tel: +44-20-7594-3039; Fax: +44-20-8846-7261; E-mail: d.j.wright{at}ic.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Corbett, E. L., Mallory, K. F., Grant, A. D., Churchyard, G. J. & De Cock, K. M. (2001). HIV-1 infection and risk of tuberculosis after rifampicin treatment. Lancet 357, 957–8.

2 . Calleja, C., Pascussi, J. M., Mani, J. C., Maurel, P. & Vilarem, M. J. (1998). The antibiotic rifampicin is a nonsteroidal ligand and activator of the human glucocorticoid receptor. Nature Medicine 4, 92–6.[ISI][Medline]

3 . Fitzgerald, D. W., Desvarieux, M., Severe, P., Joseph, P., Johnson, W. D., Jr & Pape, J. W. (2000). Effect of post-treatment isoniazid on prevention of recurrent tuberculosis in HIV-1-infected individuals: a randomised trial. Lancet 356, 1470–4.[ISI][Medline]

4 . Pahlevan, A. A., Wright, D. J., Andrews, C., George, K. M., Small, P. L. & Foxwell, B. M. (1999). The inhibitory action of Mycobacterium ulcerans soluble factor on monocyte/T cell cytokine production and NF-{kappa}B function. Journal of Immunology 163, 3928–35.[Abstract/Free Full Text]

5 . George, K. M., Chatterjee, D., Gunawardana, G., Welty, D., Hayman, J., Lee, R. et al. (1999). Mycolactone: a polyketide toxin from Mycobacterium ulcerans required for virulence. Science 283, 854–7.[Abstract/Free Full Text]

6 . Nunez Martinez, O., Ripoll Noiseux, C., Carneros Martin, J. A., Gonzalez Lara, V. & Gregorio Maranon, H. G. (2001). Reactivation tuberculosis in a patient with anti-TNF-{alpha} treatment. American Journal of Gastroenterology 96, 1665–6.[ISI][Medline]

7 . George, K. M., Barker, L. P., Welty, D. M. & Small, P. L. (1998). Partial purification and characterization of biological effects of a lipid toxin produced by Mycobacterium ulcerans. Infection and Immunity 66, 587–93.[Abstract/Free Full Text]

8 . Jaffuel, D., Demoly, P., Gougat, C., Balaguer, P., Mautino, G., Godard, P. et al. (2000). Transcriptional potencies of inhaled glucocorticoids. American Journal of Respiratory and Critical Care Medicine 162, 57–63.[Abstract/Free Full Text]

9 . Parenti, F. & Lancini, G. (1997). Rifamycins. In Antibiotics and Chemotherapy; Anti-infective Agents and their Use in Therapy, (O'Grady, F., Lambert, H. P., Finch, T. G. & Greenwood, D., Eds), pp. 453–9. Churchill Livingstone, New York.

10 . Vernon, A., Burman, W., Benator, D., Khan, A. & Bozeman, L. (1999). Acquired rifamycin monoresistance in patients with HIV-related tuberculosis treated with once-weekly rifapentine and isoniazid. Tuberculosis Trials Consortium. Lancet 353, 1843–7.[ISI][Medline]

Received 18 October 2001; returned 27 November 2001; revised 10 December 2001; accepted 10 December 2001





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