Effects of Nitric Oxide Synthase Inhibitors on Murine Infection with Mycobacterium tuberculosis

Transcription

1 INFECTION AND IMMUNITY, Feb. 1995, p Vol. 63, No /95/$ Copyright 1995, American Society for Microbiology Effects of Nitric Oxide Synthase Inhibitors on Murine Infection with Mycobacterium tuberculosis J. CHAN, 1 * K. TANAKA, 2 D. CARROLL, 2 J. FLYNN, 4 AND B. R. BLOOM 3 Departments of Medicine 1 and Pathology, 2 and the Howard Hughes Medical Institute, 3 Albert Einstein College of Medicine, Bronx, New York, and Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 4 Received 1 September 1994/Returned for modification 26 September 1994/Accepted 21 October 1994 We have recently demonstrated that the macrophage L-arginine-dependent cytotoxic pathway effectively kills the virulent Erdman strain of Mycobacterium tuberculosis in vitro via the generation of toxic reactive nitrogen intermediates by the enzyme nitric oxide synthase. This report demonstrates that two distinct inhibitors of nitric oxide synthase (aminoguanidine and N G -monomethyl-l-arginine) render similar deleterious effects on tuberculous infection in mice, as assessed by mortality, bacterial burden, and pathological tissue damage, thus confirming the importance of reactive nitrogen intermediates in resistance against M. tuberculosis. The L-arginine-dependent cytotoxic pathway, activated by cytokines and bacterial products (17, 21, 22), mediates the potent antimicrobial function of murine macrophages. Induction of this cytostatic and cytocidal pathway (17, 21, 22) results in the generation of nitric oxide (NO) and related reactive nitrogen intermediates (RNI). This reaction is carried out by the inducible form of NO synthase (inos) of macrophages by using L-arginine as the substrate. Gamma interferon (IFN- ) and tumor necrosis factor alpha have been identified as the key cytokines responsible for induction of this antimicrobial mechanism (8). Phylogenetically diverse pathogens from the bacterial, fungal, protozoan, and helminthic families have been shown to be susceptible to the cytotoxic and cytocidal effects of RNI (17, 21, 22). We have previously demonstrated that murine macrophages activated by IFN- and tumor necrosis factor alpha or Escherichia coli lipopolysaccharide to generate RNI kill Mycobacterium tuberculosis effectively (4). The antimycobacterial activity of these RNI-producing macrophages correlated with the amount of nitrogen oxides generated and was inhibited by NOS inhibitors. The significance of the results of these in vitro experiments has recently been extended to host defense against the tubercle bacillus in vivo (5, 10). Mice with targeted disruption of the IFN- gene (gko) developed fulminant and rapidly fatal M. tuberculosis infections with markedly increased bacillary loads (5, 10). Significantly, these animals are deficient in the expression of inos and in the production of toxic nitrogen oxides (10). In addition, gko are ineffective at controlling infection with the attenuated Mycobacterium bovis BCG vaccine strain (7). A similar exacerbated clinical progression of infection with BCG has been reported for transgenic mice with targeted disruption of the gene encoding the receptor of IFN-, the key enzyme for the induction of macrophage inos (16). In another murine model, transgenic mice with disruption of the IFN regulatory factor 1 gene have also been shown to be more susceptible to BCG infection (15). Therefore, dysfunction of cytokine production or of any regulatory element along the pathway of NOS induction could, in theory, compromise the L-arginine-dependent cytotoxic mechanism, thus increasing the susceptibility to various infectious agents. Although the disease exacerbation of mycobacterial infections observed in these transgenic mouse models * Corresponding author. strongly implicates the RNI pathway in host defense against mycobacteria, cautious interpretation of these data is warranted because of the pleiotropic biological effects of IFN- and IFN regulatory factor 1. We therefore choose an alternative strategy using NOS inhibitors as pharmacological probes to evaluate further the in vivo significance of RNI in host defense against M. tuberculosis. Aminoguanidine (AG) (Sigma) and N G -monomethyl-l-arginine (NMMA) (Chem Biochem, Salt Lake City, Utah) were the NOS inhibitors used in this study. Although information concerning the pharmacodynamics and metabolism of AG (1) and NMMA (19, 23 25) is still incomplete, both compounds have been used extensively in vitro and in vivo to inhibit RNI production (2, 3, 6, 9, 12 14, 18, 20, 21, 26). These two NOS inhibitors differ not only in their chemical structures but also in their effects on the various isoforms of NOS. Thus, AG has been shown to be relatively selective at inhibiting inos: the relative inhibitory effect of AG on inos compared with that on the constitutively expressed isoform is 40:1 (2, 6), while such selectivity has not been shown for NMMA. Remarkably, despite such differences, both AG and NMMA have been independently shown to exacerbate murine listeriosis (2, 3) and malaria (26). Finally, NMMA has also been employed to demonstrate the importance of RNI in resistance to Francisella tularensis (13) and Leishmania major in vivo (9, 18). It is curious, however, that NMMA, when used in another murine listeriosis model in which treatment was administered once at the time of infection, appeared to decrease tissue listerial burden on days 3 and 7 postinfection (14). Although the mechanism underlying the opposing effects of NMMA on listeriosis in two different murine models is presently unclear, these observations imply that RNI have a complex role in infectious diseases. Preliminary experiments were performed to characterize the optimum dose and method of delivery of AG in our murine experimental tuberculosis model by using relatively resistant C57BL/6 mice (Jackson Laboratory). The routes of administration and the doses of AG evaluated included the following: (i) 0.1 mg (in 100 l of phosphate-buffered saline) intraperitoneally (i.p.) twice daily per mouse, (ii) 1% (wt/vol) solution in water given to mice ad lib, and (iii) a daily dose of 0.2 mg delivered as a continuous infusion via surgically implanted osmotic pumps (Alza Corp., Palo Alto, Calif.). Exacerbated tuberculous infection, as measured by mortality and bacillary 736

2 VOL. 63, 1995 NOTES 737 FIG. 1. Treatment with the NOS inhibitors AG (A) and NMMA (B) increased mortality among C57BL/6 mice infected with the virulent Erdman strain of M. tuberculosis. There were eight mice per group for panel A and six mice per group for panel B. Symbols: in panel A, solid square, 0% AG; solid circle, 1% AG; open square, 2.5% AG; in panel B, solid square, no NMMA treatment; open square, 50 mm NMMA in drinking water. FIG. 2. AG treatment increased the bacillary burden in tissues of C57BL/6 mice infected with 10 6 CFU of the virulent Erdman strain of M. tuberculosis. The mice were treated with the following percents AG: 0 (square), 1 (circle), and 2.5 (rhombus). Three animals were studied per group per time point. The numbers of CFU shown represent that from the left upper lobe of the lung (top), the right lobe of the liver (middle), and the caudate quarter of the spleen (bottom). At 12 h after infection, three mice given no AG were studied to quantitate the baseline bacillary load in tissues. Bars represent standard deviations. burden, was observed in mice that received 0.1 mg of AG i.p. twice a day and those that were given the 1% solution of the NOS inhibitor ad lib. Since the osmotic pump can remain in mice for no more than 2 weeks and animals can tolerate implantation of the pump only twice, drug delivery via this device was not feasible for extended studies. Consequently, we chose the oral route of administration of AG, because this mode of drug delivery is relatively noninvasive and much less laborintensive than twice daily i.p. injections. The route of delivery and the dose of NMMA (50 mm in drinking water given to mice ad lib) used in this study have been shown previously (3) to be effective in abrogating NO production by mice infected with Listeria monocytogenes, as assessed by measuring urinary nitrate content. Six- to eight-week-old C57BL/6 female mice were infected intravenously with 10 6 CFU of virulent M. tuberculosis, Erdman strain, that had been recently passaged in animals as previously described (10). Administration of 0, 1, or 2.5% (wt/vol) AG in sterile drinking water as described above was initiated 7 days prior to infection and was continued throughout the study. While there were no deaths among mice receiving normal drinking water after infection with 10 6 CFU of M. tuberculosis, 100% of the animals given 1 or 2.5% AG died by day 91 or 34 postinfection, respectively (Fig. 1A). The mean survival time for mice treated with 1% AG was days, while for animals receiving the higher dose of the NOS inhibitor, it was days. Uninfected mice receiving equivalent amounts of AG survived for up to 120 days without apparent distress. Thus, the mice receiving 1% AG survived longer than those given 2.5% AG, demonstrating a dose-dependent effect of the NOS inhibitor on disease progression. These results strongly suggest that the L-arginine-dependent cytotoxic mechanism plays a significant role in host defense against the tubercle bacillus in vivo. Parallel experiments designed to assess the bacillary burdens in the various groups of mice (by quantitating the number of CFU on 7H10 agar plates [10]) revealed that the tissues of

3 738 NOTES INFECT. IMMUN. FIG. 3. Histopathological studies of lung tissues of M. tuberculosis-infected C57BL/6 mice given 0% (A and C) or 2.5% (B and D) AG. Kinyoun s acid-fast stain (A and B) revealed markedly increased bacillary loads in the lungs of AG-treated mice compared with those of controls. Hematoxylin and eosin staining of these sections (C and D) revealed necrosis in tissues of animals treated with 2.5% AG. Micrographs shown are representative of three mice studied on day 27 postinfection. Magnifications, 400 (A and B) and 100 (C and D). animals receiving 1 or 2.5% AG contained a markedly increased number of CFU of M. tuberculosis over that of controls given plain drinking water (Fig. 2). The most striking difference in tissue content of tubercle bacilli was observed between animals receiving 2.5 or 0% AG: at day 27 postinfection, the lung tissues of infected animals given 2.5% AG contained over 100-fold-more viable bacilli than those of controls receiving no NOS inhibitor (Fig. 2). The lack of difference between tissue bacillary burden of mice treated with AG and that of animals given plain drinking water on day 12 postinfection could be due to the existence of other antimicrobial mechanisms that play a role in host defense against M. tuberculosis. Such antimycobacterial activities, however, are not sufficient in controlling disseminated tuberculous infection in the absence of a functional nitric oxide-generating pathway. Quantitation of viable CFU in tissues also revealed a dose-dependent effect of AG on bacillary burden (Fig. 2). This dose effect is, however, apparent only in the lungs, where the bacillary load of mice given 2.5% AG was over 10-fold higher than that of animals receiving the lower dose of the NOS inhibitor. The reason for the apparent lack of a dose-dependent effect of AG on viable CFU in the spleen and liver is currently unknown. In sum, mortality of AG-treated mice correlates well with tissue bacillary burden; these criteria of disease progression are, in turn, dependent on the dosage of the NOS inhibitor administered. Finally, histological examination of tissues from the various experimental groups of mice revealed comparable morphological appearances of the granulomatous lesions. Marked necrosis, however, was seen in the lung tissues of AG-treated animals at later times postinfection (Fig. 3). The relative abundance of bacilli in tissues from the various groups of animals, as assessed by Kinyoun s acid-fast staining, agreed with results obtained by CFU quantitation (Fig. 3). In order to examine the relationship between disease outcome and the activity of the NO-generating pathway in tuberculous infection, M. tuberculosis-infected, AG-treated mice were bled at various time intervals postinfection, and serum nitrite and nitrate contents (production of these nitrogen oxides has been shown to correlate with in vivo generation of RNI [3, 12]) were quantitated as previously described (10). Significant levels of these nitrogen oxides were detectable even in the experimental group that received the highest dose of AG (data not shown). We reasoned that these results were likely attributable to the relative selective effect of AG on inos, leaving the constitutive forms of the central nervous system and the endothelium uninhibited during tuberculous infection. Consequently, we examined the effect of NMMA on tuberculous infection in mice, since this inhibitor of nitric oxide synthase lacks the AG-like selective effect on inos and has been shown to exacerbate murine listerioses and to abrogate virtually all NOS activity during infection with L. monocytogenes (3). In these experiments, all mouse diets (Zeigler Bros., Inc., Gardners, Pa.) used were free of nitrite, nitrate, and L-arginine (3, 12). The lack of dietary L-arginine has been demonstrated not to affect RNI generation in mycobacterial (12) and listerial infections (3), probably because of the presence of an endogenous pathway for the production of this amino acid (12). Further, progression of infections in these infectious disease models was not affected by this diet (3, 12). Results in Fig. 1B demonstrate that daily administration of NMMA (50 mm in

4 VOL. 63, 1995 NOTES 739 TABLE 1. Treatment of M. tuberculosis-infected C57BL/6 mice with NMMA-abrogated production of RNI Day postinfection Nitrite and nitrate ( M) (mean SEM) a With NMMA Without NMMA a Data shown represent serum nitrite and nitrate contents, quantitated as previously described (8), derived from three or four animals. drinking water given ad lib beginning 3 days prior to infection) markedly increased the mortality of C57BL/6 mice infected with M. tuberculosis. All animals given NMMA died by day 38 postinfection (mean survival time, days), while all infected mice fed normal drinking water survived. The NOS inhibitor did not lead to any apparent distress in uninfected animals during the experiment. Quantitation of serum nitrite and nitrate contents indicated that infected mice fed NMMA had markedly diminished NO production from that of untreated animals, demonstrating a correlation between NO production and effective host defense against M. tuberculosis (Table 1). More detailed analyses of tissue histopathology and CFU quantitation were not feasible because of the high cost of NMMA. Nevertheless, the mortality of M. tuberculosis-infected mice treated with NMMA in this study correlates strikingly with the down-regulation of the L-arginine cytotoxic mechanism during tuberculous infection. Taken together, these studies strongly establish the importance in vivo of RNI in host defense against M. tuberculosis. In other infectious disease models, the NOS inhibitors AG and NMMA have been shown to exacerbate listeriosis (2, 3), leishmaniasis (9, 18), malaria (26), and tularemia (13). The present data independently reinforce the findings of previous studies (9) of transgenic experimental tuberculosis murine models that in vivo expression of inos correlated with protection against mycobacterial infection or disease. It could be argued that unknown nonspecific effects of metabolic inhibitors could confound interpretation of the effects of such antagonists on biochemical pathways such as the L-arginine-dependent cytotoxic mechanism. Considering the known polymorphic biological functions of cytokines, however, the range of nonspecific effects operative in transgenic mouse models using animals with disruption of the genes encoding IFN- or its related regulatory elements is not likely to be less than those of the inos inhibitors employed in this study. Finally, the use of two distinct NOS inhibitors in this study to demonstrate the same deleterious effects of these compounds on murine tuberculous infection argues against the possibility that the observed results are caused merely by nonspecific effects. Perhaps the most definitive proof of the role that the macrophage L-arginine-dependent antimicrobial mechanism plays in infectious diseases awaits the development of transgenic animals that lack the functional inos isoform or the discovery of naturally occurring RNI-deficient phenotypes analogous to that of the chronic granulomatous diseases in humans for reactive oxygen radicals (11). In summary, the present data establish that treatment of M. tuberculosis-infected mice with two distinct inhibitors of NOS profoundly increases mortality, bacterial burden, and pathological tissue damage, thus confirming the importance of RNI in resistance to tuberculous infection. It remains to be established whether this cytocidal mechanism, which has yet to be demonstrated in human monocyte-derived macrophages, is equally important in human resistance to tuberculosis. 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