Role of Human Neutrophil Peptide 1 as a Possible Adjunct to Antituberculosis Chemotherapy

Transcription

1 MAJOR ARTICLE Role of Human Neutrophil Peptide 1 as a Possible Adjunct to Antituberculosis Chemotherapy Anjana Kalita, Indu Verma, and G. K. Khuller Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India We report the role of human neutrophil peptide (HNP) 1 as an adjunct to antituberculosis (anti-tb) drugs. The combination of HNP-1, isoniazid, and rifampicin was evaluated against Mycobacterium tuberculosis H 37 Rv in vitro, ex vivo, and in vivo, and synergism was observed on the basis of reductions in minimum inhibitory concentrations (MICs) of these agents. In vitro results revealed 11-log unit reductions even when HNP-1 and anti-tb drugs were used at 1/16 MICs. This combination was also found to be bactericidal against intracellular mycobacteria even at 1/8 MICs of HNP-1 and drugs. HNP-1 used in conjunction with anti-tb drugs resulted in significant clearance of bacterial load from lungs, liver, and spleen of infected, compared with control animals. The effective therapeutic dosage of drugs could be reduced to half by supplementing HNP-1 in the therapeutic schedule. These results clearly suggest that HNP-1 can be used as adjunct chemotherapy with conventional drugs against TB. Although the incidence of tuberculosis in developed countries has declined during past decades, the emergence of HIV and multidrug-resistant (MDR) strains of Mycobacterium tuberculosis has reversed the trend. To counteract the emerging threat of MDR M. tuberculosis, it is necessary not only to enhance our knowledge about the mechanisms of resistance but also to find new approaches to treat tuberculosis (TB). In this context, antimicrobial peptides (AMPs) hold a promising future for the treatment of TB. Among naturally occurring AMPs, defensins form a unique family of cysteine-rich cationic polypeptides with 3 4 disulfide bridges [1]. Defensins present in the azurophilic granules of human neutrophils are called human neutrophil peptides (HNPs) and have been identified as HNP1 4 [2]. HNP-1 has been reported to exhibit a broad spectrum of in vitro killing activity against various targets, including bacteria, fungi, enveloped viruses, parasites Received 28 October 2003; accepted 13 April 2004; electronically published 15 September Financial support: Indian Council of Medical Research, New Delhi; Postgraduate Institute of Medical Education and Research, Chandigarh. Reprints or correspondence: Prof. G. K. Khuller, Dept. of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh , India (gkkhuller@yahoo.co.in). The Journal of Infectious Diseases 2004; 190: by the Infectious Diseases Society of America. All rights reserved /2004/ $15.00 [3], and mycobacteria [4]. The only defining characteristics of their antimicrobial activity are their action on target membranes. The resistance to antimycobacterial drugs is mainly considered to be due to a peculiar mycobacterial cell envelope, which acts as a barrier to the entry of drugs. The unusually lipid-rich cell-wall structure of mycobacteria makes the cell surface hydrophobic, which causes the aggregation of cells and reduces its permeability to a variety of molecules, including anti-tb drugs [5]. It has been reported elsewhere [6] that HNP- 1 has powerful bactericidal activity against M. tuberculosis that is mediated by the increased permeability of the mycobacterial cell envelope. Hence, HNP-1 is perceived to be one of the promising solutions to the growing problem of resistance to conventional antibiotics in mycobacteria, particularly by facilitating the entry of drugs into the mycobacterial cell. It is of interest that various studies have shown that the emergence of resistance against AMPs is less probable than that observed for conventional antibiotics, which provides the impetus to develop AMPs as useful therapeutic agents for infectious diseases [7]. In addition, AMPs have been shown to have synergistic activity in combination with various antibiotics against a variety of infectious pathogens [8, 9]. However, until now, no information has been available on the combined activity of HNP-1 along with conventional anti-tb drugs JID 2004:190 (15 October) Kalita et al.

2 MATERIALS AND METHODS Source and maintenance of mycobacterial culture. M. tuberculosis H 37 Rv (originally obtained from National Collection of Type Cultures, London) was maintained on Lowenstein Jensen s medium and subsequently grown in Youman s modified medium that contained 0.05% Tween-80 [10] on an orbital shaker at 100 rpm at 37 C. For the enumeration of viable counts of M. tuberculosis, 2.5-g agar powder was added to the medium and supplemented with 1% (wt/vol) filter-sterilized bovine serum albumin. Animals. BALB/c mice of either sex (4 5 weeks old) weighing g were obtained from the central animal house of the institute and were maintained in the departmental animal house. Mice were fed a standard pellet diet and water ad libitum. HNP-1 and anti-tb drugs. HNP-1 was obtained from Peptide Institute. It was dissolved in 0.01% acetic acid as a stock of 100 mg/ml stored at 20 C and used within 1 week. Rifampicin and isoniazid were procured from Sigma Chemical. Cell line. Murine macrophage cell line J774. A1 was obtained from National Centre for Cell Sciences (Pune) and maintained in RPMI 1640 medium supplemented with 5% 10% fetal calf serum, 2 mmol/l l-glutamine, and antibiotics (100 U/mL penicillin and 1 mg/ml gentamycin) at 37 C in5%co 2. During the infection of monolayers, RPMI 1640 medium was used without antibiotics. In vitro activity of HNP-1 and anti-tb drugs (isoniazid and rifampicin) against M. tuberculosis H 37 Rv. The antimycobacterial activity of HNP-1, isoniazid, and rifampicin, individually and in combination, was monitored by use of a broth-dilution method. In brief, cells were grown individually in the presence of different concentrations of HNP-1 ( mg/ml) and anti- TB drugs ( mg/ml) in specially designed flat-bottom tubes that contained 5 ml of Youman s medium, followed by the addition of an inoculum of cells of M. tuberculosis H 37 Rv. Growth was monitored during the midlog phase by measuring the optical density at 620 nm. In addition, cell cultures from the midlog phase were diluted appropriately in PBS and plated on Youman s medium supplemented with bovine serum albumin (BSA). After 4 5 weeks, the number of colony-forming units was enumerated, and the data obtained were used for the calculation of the MICs of HNP-1, isoniazid, and rifampicin. To determine the synergistic effect of HNP-1 and anti-tb drugs, the above-mentioned experiment was performed by use of the combination of HNP-1 and drugs at their MICs, as well as serial dilutions of the MICs (1/2, 1/4, 1/8, and 1/16 MIC), as described by Hiefets et al. [11]. Intracellular killing of M. tuberculosis by HNP-1 and anti- TB drugs. The effect of HNP-1, isoniazid, and rifampicin was evaluated against intracellular M. tuberculosis H 37 Rv by use of the murine macrophage cell line J774.A1 [12]. Macrophage cells were cultured at a concentration of cells/ml/well and infected with M. tuberculosis H 37 Rv in the ratio of 1:10 for 2 h in a 24- well culture plate. The infected monolayer was washed 3 times with RPMI 1640 medium, to remove nonphagocytosed mycobacteria. The infected monolayers were then treated with HNP- 1 and anti-tb drugs at different concentrations (10 40 mg/ml HNP-1 and 5 25 mg/ml each anti-tb drug) for 3 days. Acetic acid (0.01%) was added to the control wells. At the end of the treatment period, infected monolayer cells were lysed with 500 ml of chilled 0.25% SDS. Lysates were approximately diluted and plated in solidified Youman s medium supplemented with 1% BSA. After incubation for 4 6 weeks, the number of colonyforming units was counted in control and test plates, to determine the MICs of HNP-1, rifampicin, and isoniazid. To monitor the synergistic effect of HNP-1 and anti-tb drugs, similar studies were performed by use of the combination of HNP-1 with anti-tb drugs at their MICs as well as at serial dilutions of the MICs. All of these combinations of HNP-1 and anti-tb drugs were considered to be synergistic, which resulted in 11 log unit reductions in colony-forming units, compared with control, untreated cells. Therapeutic potential of HNP-1 and anti-tb drugs against experimental TB. Mice were infected intravenously with M. tuberculosis H 37 Rv ( cell/mouse); after 15 days, infection was confirmed by Ziehl-Neelsen staining of lung homogenates. The infected mice were then either treated with HNP-1 and anti-tb drugs, individually or in combination. HNP-1 was administered subcutaneously (sc) at a dose of 5 mg/mouse, and isoniazid and rifampicin were administered sc at a concentration of 25 mg/kg body weight for each drug. HNP-1 was administered once weekly, whereas anti-tb drugs were administered daily for 4 6 weeks. After complete regimens, mice from each group were killed, and different organs were removed aseptically. Organ homogenates were prepared and plated on Youman s agar medium for the enumeration of colony-forming units [13]. In another set of experiments, the combination of HNP-1, isoniazid, and rifampicin was evaluated for its therapeutic potential in mice with TB by use of the procedure described above. However, in combination, in addition to the above-mentioned doses of HNP-1 and anti-tb drugs, lower concentrations were also tested HNP-1 at a dose of 2.5 mg/mouse/week isoniazid and rifampicin, which was equivalent to a dose of 12.5 mg/kg body weight/day for each drug. The therapeutic efficacy of combination regimen was also tested by plating the organ homogenates of infected mice, followed by the enumeration of colony-forming units. Statistical analysis. Statistical analysis was done by 1-way analysis of variance and by unpaired Student s t test. RESULTS In vitro efficacy of a combination of HNP-1 and anti-tb drugs against M. tuberculosis H 37 Rv. The MICs of HNP-1, isoni- HNP-1 as an Adjunct to TB Chemotherapy JID 2004:190 (15 October) 1477

3 Table 1. Effect of human neutrophil peptide (HNP) 1 and anti-tuberculosis drugs expressed as the fractional inhibitory concentration (FIC) in Mycobacterium tuberculosis H 37 Rv. Antimicrobial agents FIC index MIC, mg/ml INH/RIF /0.05 HNP/INH / HNP/RIF / HNP/INH/RIF /0.187/ NOTE. INH, isoniazid; RIF, rifampicin. azid, and rifampicin against M. tuberculosis H 37 Rv were found to be 2.5, 0.3, and 0.2 mg/ml, respectively. The combination of HNP-1 with either of isoniazid or rifampicin was found to be highly effective, as evidenced by reduced MICs (0.31, , and mg/ml) of HNP-1, isoniazid, and rifampicin, respectively, thereby exhibiting a fractional inhibitory concentration (FIC) index of 0.25 (table 1). In the case of the combination of all these antimicrobial agents (HNP-1, isoniazid, and rifampicin), the FIC index was further reduced to 0.187, which suggests that anti-tb drugs act more effectively even at their reduced concentrations in the presence of HNP-1. Activity of HNP-1 and anti-tb drugs against intracellular M. tuberculosis H 37 Rv. Because M. tuberculosis is an intracellular pathogen, the antimycobacterial activity of HNP-1 and anti-tb drugs was evaluated individually, as well as in combination, against M. tuberculosis H 37 Rv grown in the murine macrophage cell line J774 A.1. Isoniazid, rifampicin, and HNP- 1, when used individually, resulted in significant ( P!.001) reductions in colony-forming units, compared with control cells. The effect of anti-tb drugs was monitored in combination with HNP-1 at their MICs and at 2-fold dilutions of MICs (table 2). A significant decrease in the viability ( P!.001) of M. tuberculosis H 37 Rv ( log cfu) was observed in the presence of the combination of anti-tb drugs and HNP-1, compared with cells treated with anti-tb drugs only (isoniazid + rifampicin, log cfu), even at 1/4 MICs. When the concentration of HNP-1 with isoniazid and rifampicin was reduced to even 1/8 MICs, what was probably a synergistic effect was observed, although it was only marginally significant ( P!.005), compared with the combination of drugs alone at the same concentration (i.e., 1/8 MICs). Therapeutic potential of HNP-1, isoniazid, and rifampicin against experimental TB. The therapeutic efficacy of HNP- 1(5mg/mouse/week) and anti-tb drugs alone (25 mg/kg body weight/day) was investigated in terms of a reduction in the number of bacilli in different target organs of mice with TB after 4 weeks of chemotherapy. There was a 0.62-, 1.11-, and 1.18-log unit reduction in lungs, liver, and spleen of infected mice, compared with untreated control mice with anti-tb drugs, and a 1- log unit reduction was seen with HNP-1. However, the reduction of the dose to half (12.5 mg/kg body weight/day) resulted in a less-significant clearance of bacterial load from liver and spleen. Both anti-tb drugs and HNP-1 were also evaluated individually for their efficacy on a 6-week therapeutic schedule, and results comparable to those seen on a 4-week regimen were observed. Hence, therapeutic studies at high and low doses of anti-tb drugs alone or with HNP1-1 were evaluated after 4 weeks of therapy (figure 1). It is of interest that the use of HNP-1 at 5 mg/mouse/week along with low doses of drugs resulted in a significant reduction in bacterial load, compared with mice treated with drugs alone either at high or low doses. Furthermore, HNP-1 at a dose of 2.5 mg/mouse/week in combination with low dosage of anti-tb drugs (i.e., 12.5 mg/kg body weight/day) resulted in significantly better clearance of bacilli than that seen in mice treated with the same dose of drugs alone. However, this combination was found to be comparable to that of the treatment with a high dose of drugs alone (i.e., 25 mg/kg body weight/day) (figure 1). These findings clearly demonstrate the synergistic efficacy of anti-tb drugs with HNP-1 and suggest the use of HNP-1 in therapeutic regimen for TB. DISCUSSION In recent years, several naturally occurring cationic AMPs have gained the attention of researchers as novel therapeutic agents for infectious diseases, including TB. The resistance to antimycobacterial drugs is considered to be due to the peculiar mycobacterial cell envelope. Earlier, Sharma et al. [6] showed that a cationic peptide from human neutrophils (i.e., HNP-1) has strong bactericidal activity against M. tuberculosis, which is probably being mediated by an increased permeability of the my- Table 2. Intracellular killing of Mycobacterium tuberculosis H 37 Rv in the presence of anti-tuberculosis drugs (isoniazid [INH] and rifampicin [RIF]), alone and in combination with human neutrophil peptide (HNP) 1 within the macrophage cell line J774.A1. Group INH + RIF + HNP-1, log cfu INH + RIF, log cfu Control Full MIC a,b a 1/2 MIC a,b a 1/4 MIC a,b a 1/8 MIC a,c a NOTE. Values are the mean SD of 2 independent ex- periments. cfu, colony-forming units. a P!.001, vs. controls. b P!.05, INH + RIF + HNP-1 group vs. INH + RIF groups (unpaired Student s t test). c P!.001, INH + RIF + HNP-1 group vs. INH + RIF (unpaired Student s t test) JID 2004:190 (15 October) Kalita et al.

4 Figure 1. Log reduction in colony-forming units (cfu) in mice with tuberculosis (TB) treated with different combinations of anti-tb drugs and human neutrophil peptide 1. All values are mean SD of 6 8 mice. Control group, untreated mice with TB. Treatment was administered for 4 weeks. cobacterial cell envelope. Thus, HNP-1 can be speculated to be one of the promising solutions to the growing problem of resistance to conventional antibiotics against mycobacteria. The results of in vitro studies demonstrated that the combination of HNP-1 with anti-tb drugs (i.e., isoniazid and rifampicin) resulted in a significant reduction ( P!.001 ) in bacterial load at all the dilutions (i.e., up to 1/16 MIC; FIC index, 0.187), thereby suggesting synergism among these 3 antimicrobial agents. Giacometti et al. [9] also reported the potentiation of the action of macrolide (clarithromycin) by cationic peptides and concluded that this is a complex mechanism that probably involves a peptide induced entrance of large lipophilic and amphiphilic molecules into the cells. Ulvatne et al. [14] also observed the synergism between antimicrobial peptides and antibiotics against Escherichia coli and suggested that it could be due to increased uptake and increased accessibility to the target. In the present study, the synergy between HNP-1 and anti-tb drugs against M. tuberculosis H 37 Rv could be a result of increased permeability of both the mycobacterial cell wall and the cell membrane by HNP-1, as well as increased access to intracellular targets for anti-tb drugs. Because M. tuberculosis is an intracellular pathogen, antimycobacterial activities of HNP-1 and drugs were evaluated in a macrophage model by use of the J774A.1 cell line, which may be a more accurate reflection of natural conditions. The microbicidal activity of anti-tb drugs was found to be potentiated even at their reduced concentrations when they were combined with HNP-1. A number of reports have described the synergistic interaction of anti-tb drugs against intracellular mycobacteria [15, 16]. However, no report is available on the combined effect of cationic peptides and anti-tb drugs. Earlier, Sharma et al. [12] reported that the intracellular killing of M. tuberculosis by HNP-1 could be due to its direct interaction with the bacilli. Thus, the synergy observed between HNP-1 and anti-tb drugs against intracellular mycobacteria is probably due to the direct killing of bacilli by HNP-1 as well as to the increased penetration of drugs into the mycobacterial cells present in the macrophages. These findings make HNP-1 a potential candidate to develop an alternative/adjunct therapeutic strategy against TB. Although there are various reports that have suggested in vivo activity of antimicrobial peptides against a number of infectious diseases [17, 18], the present study is the first to report the combined effect of HNP-1 and anti-tb drugs against experimental TB. The results of combination studies indicated that HNP-1 used along with high and low dosages of isoniazid and rifampicin resulted in a remarkably significant reduction in bacterial load in the organs of infected mice, compared with control mice treated with anti-tb drugs alone (figure 1). These results are supported by the observations of Darveau et al. [19], who also reported the combined activity of antimicrobial peptides with b-lactam antibiotics. The most interesting finding of our study was that the addition of HNP-1 can not only potentiate the effectiveness of anti-tb drugs but also reduce the therapeutic dosage of drugs to half (from 25 to 12.5 mg/kg body weight/ day) while maintaining therapeutic efficacy, compared with drugs alone, against experimental TB (figure 1). This observation seems to be of significance, considering that it may avoid the classic antibiotic resistance seen in M. tuberculosis or it may not allow the selection of resistant strains particularly by improving patient compliance. HNP-1 as an Adjunct to TB Chemotherapy JID 2004:190 (15 October) 1479

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