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Virulent but not Avirulent Mycobacterium tuberculosis Can Evade the Growth Inhibitory Action of a T Helper 1–dependent, Nitric Oxide Synthase 2–independent Defense in Mice

Virulent but not Avirulent Mycobacterium tuberculosis Can Evade the Growth Inhibitory Action of a... Control of infection with virulent Mycobacterium tuberculosis (Mtb) in mice is dependent on the generation of T helper (Th)1-mediated immunity that serves, via secretion of interferon (IFN)- and other cytokines, to upregulate the antimycobacterial function of macrophages of which the synthesis of inducible nitric oxide synthase (NOS)2 is an essential event. As a means to under- standing the basis of Mtb virulence, the ability of gene-deleted mice incapable of making / Phox NOS2 (NOS2 ), gp91 subunit of the respiratory burst NADPH-oxidase complex / / (Phox ), or either enzyme (NOS2/Phox ), to control airborne infection with the aviru- lent R1Rv and H37Ra strains of Mtb was compared with their ability control infection with / / / the virulent H37Rv strain. NOS2 , Phox , and NOS2/Phox mice showed no defi- ciency in ability to control infection with either strain of avirulent Mtb. By contrast, NOS2 mice, but not Phox mice, were incapable of controlling H37Rv infection and died early from neutrophil-dominated lung pathology. Control of infection with avirulent, as well as vir- ulent Mtb, depended on the synthesis of IFN-, and was associated with a substantial increase in the synthesis in the lungs of mRNA for IFN- and NOS2, and with production of NOS2 by macrophages at sites of infection. The results indicate that virulent, but not avirulent, Mtb can overcome the growth inhibitory action of a Th1–dependent, NOS2-independent mecha- nism of defense. Key words: M. tuberculosis • virulence • NOS2 synthase • NADPH-oxidase • Th1 immunity Introduction The onset of expression of acquired, T cell–mediated immu- cells of the inducible isoform of nitric oxide synthase nity to airborne infection with virulent strains of Mycobacte- (NOS)2 that catalyzes high output generation of nitric ox- rium tuberculosis (Mtb)* in genetically resistant mice is evi- ide (NO) from l-arginine. Mice functionally deleted of the denced by inhibition of Mtb growth in the lungs starting at gene for NOS2 fail to control Mtb growth and die much 20 d of infection, after which infection is held at an approx- earlier than wild-type (WT) mice of progressive, fulminat- imately stationary level for a protracted period of time (dis- ing granulocyte-dominated lung pathology (6, 7). Mice cussed in reference 1). It is generally agreed that anti-Mtb treated with inhibitors of NOS2 are likewise incapable of immunity is Th1 mediated predominantly by CD4 and controlling infection with virulent strains of Mtb (6, 8). CD8 T cells via the secretion of IFN- and other Th1 cy- Published studies of antituberculosis immunity in mice tokines (2–5) that function to upregulate the antimicrobial deal almost exclusively with immunity to infection with function of macrophages in which Mtb bacilli reside at sites virulent strains of Mtb that are considered virulent because of infection. The ability of activated macrophages to inhibit of their ability to persist in stationary number and cause growth of virulent Mtb depends on the synthesis by these progressive lung disease, in spite of the acquisition by the host of Th1-mediated immunity. The role of Th1-medi- Address correspondence to Robert J. North, The Trudeau Institute, 100 ated immunity in the control of infection with avirulent Algonquin Ave., PO Box 59, Saranac Lake, NY 12983. Phone: 518-891- strains of Mtb, on the other hand, has received little atten- 3080; Fax: 518-891-5126; E-mail: [email protected] tion. It was considered important for a better understanding *Abbreviations used in this paper: Mtb, Mycobacterium tuberculosis; NO, ni- Phox of virulence, therefore, to know whether the requirements tric oxide; NOS, nitric oxide synthase; phox, gp91 subunit of the re- for control of infection with avirulent strains of Mtb are the spiratory burst NADPH-oxidase complex; WT, wild-type. 991 J. Exp. Med.  The Rockefeller University Press • 0022-1007/2002/10/991/8 $5.00 Volume 196, Number 7, October 7, 2002 991–998 http://www.jem.org/cgi/doi/10.1084/jem.20021186 same as those for the control of infection with virulent was extracted from lungs homogenized in Trizol (Life Sciences), according to the manufacturer’s instructions. The RNA pellet strains. To this end the study presented here used targeted was dissolved in DEPC-treated distilled water. To remove con- gene-deleted mice to determine whether control of infec- taminated genomic DNA, RNA samples were treated with tion with avirulent Mtb, like control of infection with vir- RNase-free DNase I (Ambion) for 1 h at 37C. Aliquots of ulent Mtb, depends on the generation of Th1 immunity RNA samples were then passed through RNeasy minicolumns and an ability of host macrophages to generate NO and re- (QIAGEN), treated with DNA-free kit (Ambion), and stored at active oxygen. The results show that control of infection –70C. RiboGreen™ Quantitation kit (Molecular Probes) was with either of two avirulent strains of Mtb depended on used to quantify RNA for real-time RT-PCR analysis. The assay the generation of Th1 immunity, but did not depend on was repeated three times using different dilutions of the samples. the synthesis of NOS2 by macrophages. They also show Primers and probes were designed with Primer Express Soft- that the ability to generate reactive oxygen and its metabo- ware (PE Biosystems), and purchased from Integrated DNA Technologies. Probes contained a fluorescent dye (6-carboxy- lites was not required to control infection with virulent or fluorescein; FAM) and a quencher (Black Hole Quencher 1; avirulent Mtb. Thus virulence is a property that enables BHQ1). The melting temperature of the hybridized probe Mtb to overcome the growth inhibitory action of a Th1- (70C) was always 10C higher than the PCR primers (57– dependent, NOS2-independent mechanism capable of in- 60C). hibiting the growth of avirulent Mtb. To make RNA standards, each amplicon of IFN- and NOS2 was generated by PCR from WT mouse lung mRNA using the same primers as those used for real-time RT-PCR. The ampli- Materials and Methods cons were cloned behind the T7 RNA polymerase promoter in Mice. WT mice and NOS2 mice, and mice deleted of the the pGEM-T Easy Vector System (Promega). The sequence of Phox Phox gene for the gp91 subunit (9) of the respiratory burst gp91 each cloned amplicon was determined by thermocycler sequenc- subunit of the respiratory burst NADPH-oxidase complex ing. After linearization of plasmid DNA, amplicons were tran- / / (Phox mice), as well as double mutant mice (NOS2/Phox scribed using T7 RNA polymerase (Promega). Template DNA mice), all on a C57BL/6 background were purchased from the was removed by digestion with DNase I, and RNA was purified Trudeau Institute Animal Breeding Facility. The mutant mice by using RNeasy minicolumns and quantified with the Ri- were from breeding stock supplied by C. Nathan (Cornell Uni- boGreen assay. It was determined that 1 g of an average 1,000 / 12 versity Medical College, New York, NY). Phox mice were bp mRNA contained 1.8  10 molecules. To obtain a stan- originally supplied by M. Dinauer (Indiana University Medical dard curve, serial dilutions of each transcript was performed in 9 1 Center, Indianapolis, IN). Mice deleted of the gene for the triplicate to give dilutions ranging from 10 to 10 molecules. / / TCR- chain (TCR-/ mice) and IFN- (IFN- mice) The dilutions were then subjected to real-time RT-PCR analy- on a C57BL/6 background were purchased from The Jackson sis as described below. Laboratory. All mice were used when they were 10 wk old. For real-time RT-PCR, 2 g of RNA was transcribed by us- Bacteria and Infection. The H37Rv (TMC# 102), H37Ra ing a random hexamer and a TaqMan Gold RT-PCR kit (PE (TMC# 201), and R1Rv (TMC# 205) strains of Mtb were orig- Biosystems), according to the manufacturer’s instructions. Real- inally obtained from the Trudeau Mycobacterial Culture Collec- time PCR to enumerate IFN- and NOS2 amplicons was per- tion (TMC), Trudeau Institute. The avirulent H37Ra and viru- formed in the ABI-prism 7700 Sequence Detector. Reaction lent H37Rv strains were derived from the virulent H37strain as conditions were programmed on a dedicated Power Macintosh smooth and rough colony in 1934, respectively (10). The H37 7200 computer. PCR amplification was performed in a total of parental strain was isolated from a human with pulmonary tuber- 25 l containing 10 l of cDNA sample, 2.5 l of 10 Taqman culosis in 1905 (11). The R1Rv strain was derived in 1946 from Buffer A, 5.5 mM MgCl 200 M each of dATP, dCTP, and 2, the virulent R1 strain that was isolated from a human with lung dGTP, 400 M dUTP, 0.25 M of each primer, 0.625 U of disease in 1891 (12). All strains are currently available from the AmpliTaq Gold, and 0.25 U of AmpErase Uracil N-glycosylase American Type Culture Collection. They were grown in suspen- (Perkin Elmer/Applied Biosystems). The reaction also contained sion culture in Proskauer and Beck medium containing 0.01% 0.2 M of detection probe. Amplification was performed in trip- Tween 80. The cultures were harvested after reaching 10 CFU licate wells under the following conditions: 2 min at 50C and 10 per ml, dispersed by ultrasound, and stored in vials in 1 ml lots at min at 94C followed by a total of 40 two-temperature cycles –70C until needed. To infect mice via the respiratory route, a (15 s at 94C and 1 min at 60C). The copy number in each sam- vial was thawed, diluted appropriately in PBS containing 0.01% ple was calculated according to the formula N (Ct-b)/m, where Tween 80, dispersed further by two 5 s bursts of ultrasound, and N is copy number, Ct is the threshold cycle, b is the y-intercept, 10 ml of the diluted culture used to load the nebulizer reservoir of and m is the slope of the standard curve line. an aerosol infection apparatus (Tri Instruments). Exposure to the Histology and Immunocytochemistry. Lungs were fixed in 10% aerosol for 30 min resulted in each mouse being infected with neutral buffered formaldehyde for 18 h. They were then washed 10 CFU. This was determined by enumerating CFU in the for several hours in distilled water, dehydrated in 70 and then lungs 1 d after aerosol exposure. Changes in the numbers of CFU 100% ethanol, and embedded in paraffin according to standard in lungs, livers, and spleens against time of infection was deter- procedures. Paraffin sections were cut at a thickness of 5 or 10 mined by plating 10-fold serial dilutions of whole organ homoge- m with a rotary microtome, stained for acid-fast bacteria with a nates on enriched Middlebrook 7H11 agar, and counting colonies modified basic fuchsin stain (14), and counter stained with meth- after 3 wk incubation at 37C as described previously (13). ylene blue. Immunocytochemical staining for NOS2 involved Real-Time RT-PCR Quantitation of mRNA Synthesis for IFN- reacting 10 m-thick section with 0.1 g/ml of affinity-purified and NOS2. Lungs were harvested at the times indicated in the monospecific rabbit Ig anti–mouse NOS2 (Transduction Labora- Results section, and snap frozen in liquid nitrogen. Total RNA tories) as the primary reagent. After washing, the sections were 992 NOS2 and M. tuberculosis Virulence reacted with biotinylated goat Ig anti–rabbit Ig as the second re- Phox mice also were not deficient in an ability to con- agent. The sections were then reacted with avidin-coupled trol infection with R1Rv during the first 60 d of infection. horseradish peroxidase and with diaminobenzidine as substrate to On the contrary, R1Rv infection in Phox mice was produce a brown reaction product in cells containing NOS2. controlled at about a 1 log lower level than in WT mice, The secondary and tertiary reagents were supplied in kit form indicating that these mice were more resistant than WT (Vectastain ABC kit; Vector Laboratories) and used according to mice, presumably because they developed compensatory the supplier’s instructions. Photomicrographs were taken with a mechanisms of defense. NOS2/Phox mice showed the Nikon Microphot-Fx microscope. same resistance as Phox mice up to day 40. A shortage of NOS2/Phox mice prevented infection in these mice from being followed beyond this time. Results Fig. 3 shows the results of a similar study that tested the / / Susceptibility of NOS2 and Phox Mice to Infection / / resistance of WT, NOS2 , and NOS2/Phox mice to with Virulent Versus Avirulent Mtb. To investigate whether infection with the avirulent H37Ra strain of Mtb. It can be NOS2 and NADPH-oxidase are required for resistance to seen that control of lung infection with H37Ra in NOS2 infection with virulent versus avirulent Mtb strains, WT, mice was the same as in WT mice over 100 d of infec- / / / NOS2 , Phox , and NOS2/Phox mice were in- tion. H37Ra infection progressed for 20–30 d, after fected via the respiratory route with 10 CFU of H37Rv, which it underwent progressive resolution until the experi- R1Rv, or H37Ra, and bacterial growth monitored in ment was terminated. As was the case with R1Rv infec- lungs, livers, and spleens over time. As shown in Fig. 1, tion, NOS2/Phox mice were somewhat more resistant WT mice were capable of controlling growth of H37Rv in to H37Ra infection than WT mice. Growth of H37Ra in their lungs and other organs starting on day 20 of infection, the liver and spleen is not shown, because these organs did and of maintaining infection at an approximately stationary not become infected. level from day 20 until day 60 when the experiment was Control of Infection with Avirulent Mtb Depends on T Cell– terminated. In NOS2 mice, in contrast, H37Rv grew mediated Immunity. The foregoing results clearly show progressively beyond day 20, resulting in death before day that, whereas NOS2 synthesis is needed for control of in- 50. In Phox mice, on the other hand, the course of fection with H37Rv, it is not needed for control of infec- H37Rv infection was essentially the same as in WT mice, tion with R1Rv or H37Ra over a 60-d period. Because whereas in NOS2/Phox mice infection was essentially synthesis of NOS2 by macrophages is induced by Th1 cy- the same as in NOS2 mice. tokines (4), failure to show a role for NOS2 in control of In mice infected with R1Rv (Fig. 2) the results were dif- early infection with R1Rv or H37Ra could mean that re- ferent, in that growth of this organism was essentially the sistance to infection with these avirulent strains is not de- same in the lungs of NOS2 and WT mice over the 60 d pendent on Th1-mediated immunity. This possibility was of the experiment. In both types of mice, R1Rv infection investigated by determining whether infection with R1Rv progressed for 20 d before declining by 1 log by day 60. or H37Ra is exacerbated in gene-deleted mice incapable Figure 2. Growth of R1Rv in lungs, livers, and spleens of WT, / / Phox , NOS2 , and NOS2/ Phox mice infected aerogenically with 10 CFU. Growth of R1Rv was identical in the lungs of WT and NOS2 mice, in that it was con- trolled at day 20, after which infec- Figure 1. Growth of H37Rv in tion slowly resolved. Growth of the lungs, livers, and spleens of WT, R1Rv in Phox and NOS2/ / / / Phox , NOS2 , and NOS2/ Phox mice was controlled at / 2 Phox mice infected with 10 about a 1 log lower level than in CFU aerogenically. Whereas in WT WT mice after which infection and Phox mice growth of slowly resolved. Dissemination of H37Rv was controlled from day 20 R1Rv to the liver and spleen was of infection on, growth of the slower in Phox and NOS2/ pathogen was progressive in Phox mice, although by day 60 / / NOS2 and NOS2/Phox all groups showed similar CFU mice. Means of five mice per time numbers in these organs. Means of point SD. five mice per time point SD. 993 Jung et al. Figure 3. Growth of H37Ra in WT and mutant mice. Results of two experiments (a and b) showing that growth H37Ra in WT and NOS2 mice (a) was identical, in that in both cases H37Ra grew for Figure 5. Number of H37Ra in lungs, 20–30 d in the lung, after which in- fection slowly resolved. H37Ra also livers, and spleens of WT, TCR- , grew similarly in NOS2/Phox and IFN- mice at day 50 of an aero- genic infection initiated with 5  10 CFU. mice, except that infection was con- Infection in the lungs was significantly trolled at a lower level in the latter mice. Means of five mice per time higher in mutant mice. H37Ra did not point SD. disseminate to the liver and spleen in WT mice, but did so in mutant mice incapable of expressing immunity. Means of five mice SD. of making  T cells or IFN-. As can be seen in Figs. 4 and 5. / / TCR-/ and IFN- mice allowed significantly fection, is associated with the expression of Th1-mediated more growth of R1Rv and H37Ra in their lungs and other immunity is to measure IFN- and NOS2 gene expression. organs than WT mice over a 50-d period of infection. It Therefore, real-time RT-PCR was used measure changes will also be noted in the case of H37Ra (Fig. 5) that infec- in the copy number of mRNA for IFN- and NOS2 in tion did not disseminate from the lungs to the liver and the lungs of mice infected with either organism against spleen in WT mice, but did so in mice devoid of  T time of infection. The results show (Fig. 6) that between cells. Thus resistance to R1Rv and H37Ra infection, like days 11 and 20 of H37Rv infection the copy number of resistance to H37Rv infection, almost certainly is depen- IFN- and NOS2 increased 500-fold. Moreover, in- dent on Th1-mediated immunity. creased mRNA synthesis for these two proteins was sus- Increased Synthesis of mRNA for IFN- and NOS2 in Re- tained until day 50 of infection when the experiment was sponse to H37Rv and R1Rv Infection. Because Th1-medi- terminated. IFN- and NOS2 mRNA synthesis also in- ated immunity against Mtb infection is dependent on IFN- creased in the lungs of R1Rv-infected mice between days synthesis by T cells, and NOS2 synthesis by macrophages 11 and 20 of infection, but to a lesser extent than in the (4), an additional way to determine whether acquired resis- case of H37Rv-infected mice. Thus control of H37Rv at a tance to infection with R1Rv, like control of H37Rv in- stationary level and the slow resolution of R1Rv infection were associated with a substantial increase in mRNA syn- thesis of a key cytokine of Th1 cells and a key enzyme of activated macrophages. Figure 6. Results of a real-time RT-PCR study of IFN- and NOS2 gene expression in the lungs of WT mice infected aerogenically with 10 CFU of H37Rv or R1Rv. Copy numbers of mRNA for IFN- and NOS2 per total lung RNA in- crease significantly between days 11 and 20 of infection with either or- ganism, and were sustained from day Figure 4. Number of R1Rv in the lungs, 20 to day 50 when the study was livers, and spleens of WT, TCR- , terminated. However, increased and IFN- mice at day 50 of an aero- IFN- and NOS2 mRNA synthesis genic initiated with 10 CFU. Infection was was significantly lower in the case of exacerbated substantially in all organs of the lungs infected with R1Rv. Means of mutant mice. Means of five mice SD. three mice per time point SD. 994 NOS2 and M. tuberculosis Virulence Discussion Immunocytochemistry of NOS2 Synthesis by Macrophages in Lungs Infected with Virulent Versus Avirulent Mtb. Lung The virulent H37Rv strain of Mtb used in this study is a histopatholgy induced by H37Rv in WT and NOS2 widely investigated laboratory strain that was derived as a mice on day 40 of infection is shown in Fig. 7. Day 40 was smooth colony in 1934 from the parent H37 strain isolated chosen to examine H37Rv-induced pathology because from a patient with active tuberculosis in 1905 (11). It is most NOS2 mice died before day 50. It can be seen apparent from examining past and current literature that (Fig. 7 a) that each site of lung infection in WT mice was H37Rv has not lost virulence for mice since its isolation, evidenced by accumulations of macrophages in close prox- and there is no reason to believe at present that it is less vir- imity to one or more dense aggregates of lymphoid cells. ulent for mice than most recent clinical Mtb isolates. It is Many of the macrophages in these lesions were infected shown here, in agreement with previous studies (1, 15–18) with acid-fast bacilli (Fig. 7 b) and most stained positively that H37Rv, like other virulent Mtb strains grows 4.5 for NOS2 by immunocytochemistry. In NOS2 mice logs in the lungs of mice over 20 d of infection, after which on day 40, in contrast, sites of H37Rv infection were seen its growth is controlled and held at a stationary level by as large focal areas of neutrophil-dominated inflammation specific immunity. The avirulent H37Ra strain was derived (Fig. 7, c and d) in which most alveoli were densely packed from a parental H37 strain as a rough colony at the time of with degenerating neutrophils. Tissue necrosis was obvi- derivation of the H37Rv strain (10). It was classified as ously taking place at these sites, and no cells stained posi- avirulent on the basis of its inability to grow progressively tively for NOS2. and cause disease in laboratory animals. Infection with this Sites of R1Rv lung infection in WT mice (Fig. 8 a) at strain is progressive for 21 d, after which host immunity day 50 were also seen as accumulations of macrophages in causes infection to progressively resolve, as shown here for close proximity to lymphoid cell aggregates. Macrophage mice, and elsewhere for guinea pigs (19). A description of accumulations were somewhat less extensive than in differences between the IS6110 DNA fingerprint patterns H37Rv-induced lesions, and most macrophages stained of H37Rv and H37Ra has been published recently (20). positively for NOS2. However, few macrophages showed The R1Rv strain is an attenuated derivative of a parental the presence of acid-fast bacilli, in keeping with the rela- R1strain (12) that was isolated from a patient with lung dis- tively smaller number of R1Rv bacilli in the lungs of WT ease in 1891. R1Rv is known from a previous study (21) to mice at day 50 of infection. In NOS2 mice, R1Rv- grow progressively in the lungs of mice for 3 wk, before induced lung lesions at day 50 (Fig. 8 b) were larger and its growth is controlled and held at stationary level for a had a different appearance to those in WT mice. Each con- protracted period of time. However, R1Rv, like H37Ra sisted of an extensive accumulation of macrophages sur- grows, at a much slower rate in the lungs of mice than rounded by a partial or complete mantle of darkly stained H37Rv during the first 20 d of infection (21). It is shown lymphoid cells. No cells in these lesions (Fig. 8 c) stained here, in this connection that R1Rv grew 3 logs, and positively for NOS2, and neutrophils were almost com- H37Ra 2 logs in the lungs of mice before infection be- pletely absent. Acid-fast R1Rv bacilli were present in mac- gan to resolve. According to a previous study (21), infec- rophages in the centers of the lesions, but were no more tion with R1Rv eventually stabilizes at a stationary level numerous than in lesions of WT mice at day 50. and causes neither progressive lung pathology nor death. Figure 7. Micrographs of section of lung from a WT / 2 and an NOS2 mouse infected 40 d earlier with 10 CFU of H37Rv aerogenically. Lesions in WT lung at low power (a) consisted of aggregates of macrophages stained for NOS2 (brown) in close proximity to aggre- gates to lymphoid cells (dark blue). At higher power (b) NOS2-stained macrophages were seen to contain acid-fast bacilli (red). In contrast, lesions in NOS2 lungs at low power (c) were seen to be composed of large numbers of neutrophils that at higher power (d) were seen to contain acid-fast bacilli and to be in the process of degeneration. No cells in NOS2 lungs stained for NOS2. Original magnifications: 80 for a and c; 470 for b and d. 995 Jung et al. mice, like that induced by H37Rv, was characterized by accumulations of macrophages in close association with aggregates of lymphoid cells, R1Rv-induced lesion in NOS2 mice were seen as a central core of macrophages surrounded by a thick mantle of lymphoid cells. Another key finding presented is that although NOS2 was not needed for resistance to R1Rv infection, NOS2 was nev- ertheless synthesized by R1Rv-infected macrophages in le- sions in WT mice, as demonstrated by immunocytochem- istry. This agrees with the additional finding that control of infection with R1Rv, like control of H37Rv infection, was preceded by a large increase in the synthesis of mRNA for IFN- and NOS2 in the lungs. The increase was smaller in the case of R1Rv infection, which is in keeping with a log lower level of R1Rv infection. It was not sur- prising to find that maintenance of stationary H37Rv infec- tion and slow resolution of R1Rv infection were associated with a sustained high level of synthesis of IFN- and NOS2 mRNA. Further reason for concluding that immu- nity to R1Rv and H37Ra infection is Th1-mediated is the demonstration that infection with either organism was not controlled by gene-deleted mice incapable of making  T cells or IFN-. It goes without saying that the presence of IFN-–producing T cells at sites of R1Rv infection would be expected to cause activation of Mtb-infected macro- phages at these sites, as evidence by the acquisition by mac- rophages of NOS2, as shown here. The findings with H37Rv serve to confirm results pub- lished by others (6, 7) showing that NOS2 mice are in- capable of controlling Mtb growth in their lungs and other Figure 8. Micrographs of lung lesions in WT and NOS2 mice in- 2 organs after day 20 of infection and quickly succumb to in- fected with 10 CFU of R1Rv aerogenically. At lower power, (a) lesions in WT lungs were composed of aggregates of lymphoid cells (blue) in fection-induced pathology. However, they are in disagree- close association with aggregates of macrophages containing NOS2 ment with those in a recent publications (22) showing that (brown). In the lungs of NOS2 mice at low power (b) lesions were NOS2 mice are not substantially deficient in a capacity seen to consist typically of a central core of macrophages surrounded by a to control early airborne infection with the virulent Erd- mantle of lymphoid cells (blue). At higher power (c) none of the mac- rophages in the central core stained for NOS2, and some of them were man strain of Mtb. This contradictory evidence with Mtb infected with acid-fast bacilli (red). Original magnifications: 92 for a Erdman needs to be considered, however, in light of other and b: 740 for c. publications (6, 7) showing that NOS2 is essential for im- mune control of infection with Mtb Erdman, and that mice incapable of making NOS2 quickly succumb to a very This is contrast to H37Rv stationary infection that causes small aerosol challenge with this strain (7), as do mice in- both progressive pathology and death. This previous find- fected by aerosol with H37Rv (1). The reason why growth ing with R1Rv led to the suggestion (21) that the ability to of Erdman was controlled by NOS2 mice in one labo- persist in infected tissues in the face of acquired host immu- ratory, but not in others is not known. However, in view nity is by itself not a measure of Mtb pathogenicity or viru- of the findings presented here with avirulent strains of Mtb, lence, although persistence is necessary in order for viru- a possibility that needs to be considered is that an Erdman lence to be expressed. strain whose growth is fairly well controlled in the absence A key finding presented here is that, whereas growth of of NOS2 is likely to be less virulent than an Erdman strain H37Rv after day 20 was progressive and quickly lethal for whose growth is progressive. Indeed, it is apparent from NOS2 mice, growth of R1Rv slowly resolved in these published growth curves (22, 23) that the Erdman strain mice and did so to the same extent as in WT mice. R1Rv- that grows less in NOS2 mice multiplies at a slower rate infected WT and NOS2 mice showed no signs of mor- in WT mice than the Erdman strains used by others (18), bidity over 150-d period of observation (unpublished data). and that it gives rise to a lower level of stationary infection Moreover, whereas H37Rv induced a fulminating, neutro- after the expression of Th1 immunity. In fact, an examina- phil-dominated pathology in the lungs of NOS2 mice, tion of published growth curves of the Erdman strain in R1Rv-induced lung pathology that was essentially devoid question shows that it grows at a similar rate and gives rise of neutrophils, although different from that induced in WT to about the same level of infection as the avirulent R1Rv mice. Thus, whereas R1Rv-induced pathology in WT strain used in this study. 996 NOS2 and M. tuberculosis Virulence An additional finding revealed by this study is that the intracellular bacteria. Curr. Opin. Immunol. 13:417–428. 6. McMicking, J.D., R.J. North, R. LaCourse, J.S. Mudget, generation of reactive oxygen by NADPH-oxidase is not S.K. Shah, and C.F. Nathan. 1997. Identification of nitric needed for control of infection with either virulent or avir- oxide synthase as a protective locus against tuberculosis. Proc. ulent Mtb. On the contrary, Phox mice were identical Natl. Acad. Sci. USA. 94:5243–5248. to WT mice in their ability to stabilize H37Rv infection, 7. Scanga, C.A., V.P. Mohan, K. Tanaka, D. Alland, J.L. Flynn, and were more resistant than WT mice to infection with and J. Chan. 2001. The inducible nitric oxide synthase locus R1Rv and H37Ra. Moreover, NOS2/Phox double confers protection against aerogenic challenge of both clinical mutant mice were no more susceptible than NOS2 and laboratory strains of Mycobacterium tuberculosis. Infect. Im- mice to infection with R1Rv or H37Rv. The demonstra- mun. 69:7711–7779. tion that Phox mice were not more susceptible than 8. Flynn, J.L., C.A. Scanga, K.E. Tanaka, and J. Chan. 1998. WT mice to H37Rv infection is in overall agreement with Effects of aminoguanidine on latent murine tuberculosis. J. Immunol. 160:1796–1803. results published by another laboratory (24) showing only a 9. Pollock, J.D., D.A. Williams, M.A. Gifford, L.L. Li, X. Du, transient, small increase in the growth of Mtb in the lungs, / J. Fisherman, S.H. Orkin, C.M. Doerchuck, and M.C. but not in other organs, of Phox mice over WT mice. Dinauer. 1995. Mouse model of X-linked chronic granulo- The results are in disagreement, on the other hand, with a matous disease, an inherited defect in phagocyte superoxide publication (25) showing that Phox mice are signifi- production. Nat. Genet. 9:202–209. cantly more susceptible to lung infection with virulent Mtb 10. Steenken, W., W.H. Oatway, and S.A. Petroff. 1934. Bio- than WT mice, as assessed by Mtb growth in the lungs. logical studies of the tubercle bacillus. III. Dissociation and The reason for this discrepancy is not known and is difficult pathogenicity of the R and S variants of the human tubercle to explain. bacillus (H37). J. Exp. Med. 60:515–543. The NOS2-independent mechanism of defense that is 11. Steenken, W., and L.U. Gardner. 1946. History of H37 strain of tubercle bacillus. Am. Rev. Tuberc. 54:62–66. capable of stabilizing infection with R1Rv and H37Ra was 12. Steenken, W., and L.U. Gardner. 1946. R1 strain of tubercle not identified by this study. It is likely to be the same bacillus, its dissociation and virulence of variants in normal mechanism that enables NOS2-deficient mice to inhibit and silicotic guinea pigs. Am. Rev. Tuberc. 54:51–60. the growth of Mycobacterium avium in major organs (26), and 13. Dunn, P.L., and R.J. North. 1995. Virulence ranking of enables NOS2-deficient macrophages to inhibit growth of some Mycobacterium tuberculosis and Mycobacterium bovis strains this organism in vitro (26). It may also be the same mecha- according to their ability to multiply in the lungs, induce nism that enables human macrophages treated with inhibi- lung pathology and cause mortality. Infect. Immunity. 63: tors of NOS2 to control the growth of H37Ra in vitro 3428–3437. (27). It has been shown by in vitro studies (28) that mouse 14. Ellis, R.C., and L.A. Zabrowarny. 1993. A safer staining macrophages are capable of expressing both NOS2-depen- method for acid fast bacilli. J. Clin. Pathol. 46:559–560. dent and NOS2-independent mechanisms of antimycobac- 15. Schell, R.F., W.F. Ealey, G.E. Harding, and D.W. Smith. 1974. The influence of vaccination on the course of experi- terial defense. mental airborne tuberculosis in mice. J. Reticuloendoth. Soc. In conclusion, by showing that NOS2 mice can con- 16:131–138. trol infection with avirulent, but not virulent Mtb, this 16. North, RJ., L. Ryan, R. LaCourse, T. Mogues and M.E. study suggests that virulence is a property that enables Mtb Goodrich. 1999. Growth of mycobacteria in mice as an un- to multiply in the face of the growth inhibitory action of a reliable indicator of virulence. Infect. Immun. 67:5483–5485. NOS2-independent, Th1-dependent anti-Mtb defense. 17. Manca, C., L. Tsenova, C.E. Barry, A. Bergtold, S. Freeman, P.A. Haslett, J.M. Musser, V.H. Freedman, and G. Kaplan. This work was supported by National Institutes of Health grants 1999. Mycobacterium tuberculosis CDC1551 induces a AI-37844 and HL-64565. more vigorous host response in vivo and in vitro, but is not Submitted: 12 July 2002 more virulent than other clinical isolates. J. Immunol. 162: Revised: 13 August 2002 6740–6746. Accepted: 21 August 2002 18. Kelley, C.L., and F.M. Collins. 1999. Growth of a highly virulent strain of Mycobacterium tuberculosis in mice of differing susceptibility to tuberculosis. Tuberc. Lung Dis. 79:367–370. 19. Alsaadi, A.-I., and D.W. Smith. 1973. The fate of virulent References and attenuated mycobacteria in guinea pigs infected by the 1. Mogues, T., M.E. Goodrich, L. Ryan, R. LaCourse, and respiratory route. Am. Rev. Resp. Dis. 107:1041–1046. R.J. North. 2001. The relative importance of T cell subsets 20. Bifani, P., S. Moghazeh, B. Shopsin, J. Driscoll, A. Raviko- in immunity and immunopathology of airborne Mycobacte- vitch, and B.N. Kreiswirth. 2000. Molecular characterization rium tuberculosis infection in mice. J. Exp. Med. 193:271–380. of Mycobacterium tuberculosis H37Rv/Ra variants. Distinguish- 2. Boom, W.H. 1996. The role of T cell subsets in Mycobacte- ing the mycobacterial laboratory strain. J. Clin. Microbiol. 38: rium tuberculosis infection. Infect. Agents Dis. 5:73–81. 3200–3204. 3. Cooper, A.M., B.M. Saunders, C.D. D’Souza, A.A. Frank, 21. Dunn, P.L., and R.J. North. 1996. Persistent infection with and I.M. Orme. 1997. Bull. Inst. Pasteur. 95:85–95. virulent but not avirulent Mycobacterium tuberculosis in the 4. Flynn, J.L., and J. Chan. 2001. Immunology of tuberculosis. lungs of mice causes progressive pathology. Med. Microbiol. Annu. Rev. Immunol. 19:93–129. 45:103–109. 5. Raupach, B., and S.H. Kaufmann. 2001. Immune response to 22. Cooper, A.M., J.E. Pearl, J.V. Brooks, S. Ehlers, and I.M. 997 Jung et al. Orme. 2000. Expression of the nitric oxide synthase 2 gene is rium tuberculosis using transgenic mice. Tuberc. Lung Dis. 78: not essential for early control of Mycobacterium tuberculosis 237–246. in the murine lung. Infect. Immunity. 68:6879–6882. 26. Gomes, M.S., M. Florido, T.F. Pais, and R. Appelberg. 23. Valway, S.E., M.P.C. Sanchez, T.F. Shinnick, I. Orme, T. 1999. Improved clearance of Mycobacterium avium upon Agerton, D. Hoy, J.S. Jones, H. Westmoreland, and I.M. disruption of the inducible nitric oxide synthase gene. J. Im- Onorato. 1998. An outbreak involving extensive transmis- munol. 162:6734–6739. sion of a virulent strain of Mycobacterium tuberculosis. N. Engl. 27. Aston, C., W.N. Rom, A.T. Talbot, and J. Reibman. 1998. J. Med. 338:633–639. Early inhibition of mycobacterial growth by human alveolar 24. Cooper, A.M., B.H. Segal, A.A. Frank, S.M. Holland, and macrophages is not due to nitric oxide. Am. J. Respr. Crit. I.A. Orme. 2000. Transient loss of resistance to pulmonary Care Med. 157:1943–1950. phox/ tuberculosis in p47 mice. Infect. Immun. 68:1231– 28. Bekker, L.-G., S. Freeman, P.J. Murray, B. Ryffel, and G. 1234. Kaplan. 2001. TNF- controls intracellular mycobacterial 25. Adams, L.B., M.C. Dinauer, D.E. Morgenstern, and J.L. growth by both inducible nitric oxide synthase-dependent Krahenbuhl. 1997. Comparison of the roles of oxygen and and inducible nitric oxide synthase-independent pathways. J. nitrogen intermediates in the host response to mycobacte- Immunol. 166:6728–6734. 998 NOS2 and M. tuberculosis Virulence http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

Virulent but not Avirulent Mycobacterium tuberculosis Can Evade the Growth Inhibitory Action of a T Helper 1–dependent, Nitric Oxide Synthase 2–independent Defense in Mice

Jung, Yu-Jin; LaCourse, Ronald; Ryan, Lynn; North, Robert J.
The Journal of Experimental Medicine , Volume 196 (7) – Oct 7, 2002
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Copyright © 2002, The Rockefeller University Press
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0022-1007
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10.1084/jem.20021186
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Abstract

Control of infection with virulent Mycobacterium tuberculosis (Mtb) in mice is dependent on the generation of T helper (Th)1-mediated immunity that serves, via secretion of interferon (IFN)- and other cytokines, to upregulate the antimycobacterial function of macrophages of which the synthesis of inducible nitric oxide synthase (NOS)2 is an essential event. As a means to under- standing the basis of Mtb virulence, the ability of gene-deleted mice incapable of making / Phox NOS2 (NOS2 ), gp91 subunit of the respiratory burst NADPH-oxidase complex / / (Phox ), or either enzyme (NOS2/Phox ), to control airborne infection with the aviru- lent R1Rv and H37Ra strains of Mtb was compared with their ability control infection with / / / the virulent H37Rv strain. NOS2 , Phox , and NOS2/Phox mice showed no defi- ciency in ability to control infection with either strain of avirulent Mtb. By contrast, NOS2 mice, but not Phox mice, were incapable of controlling H37Rv infection and died early from neutrophil-dominated lung pathology. Control of infection with avirulent, as well as vir- ulent Mtb, depended on the synthesis of IFN-, and was associated with a substantial increase in the synthesis in the lungs of mRNA for IFN- and NOS2, and with production of NOS2 by macrophages at sites of infection. The results indicate that virulent, but not avirulent, Mtb can overcome the growth inhibitory action of a Th1–dependent, NOS2-independent mecha- nism of defense. Key words: M. tuberculosis • virulence • NOS2 synthase • NADPH-oxidase • Th1 immunity Introduction The onset of expression of acquired, T cell–mediated immu- cells of the inducible isoform of nitric oxide synthase nity to airborne infection with virulent strains of Mycobacte- (NOS)2 that catalyzes high output generation of nitric ox- rium tuberculosis (Mtb)* in genetically resistant mice is evi- ide (NO) from l-arginine. Mice functionally deleted of the denced by inhibition of Mtb growth in the lungs starting at gene for NOS2 fail to control Mtb growth and die much 20 d of infection, after which infection is held at an approx- earlier than wild-type (WT) mice of progressive, fulminat- imately stationary level for a protracted period of time (dis- ing granulocyte-dominated lung pathology (6, 7). Mice cussed in reference 1). It is generally agreed that anti-Mtb treated with inhibitors of NOS2 are likewise incapable of immunity is Th1 mediated predominantly by CD4 and controlling infection with virulent strains of Mtb (6, 8). CD8 T cells via the secretion of IFN- and other Th1 cy- Published studies of antituberculosis immunity in mice tokines (2–5) that function to upregulate the antimicrobial deal almost exclusively with immunity to infection with function of macrophages in which Mtb bacilli reside at sites virulent strains of Mtb that are considered virulent because of infection. The ability of activated macrophages to inhibit of their ability to persist in stationary number and cause growth of virulent Mtb depends on the synthesis by these progressive lung disease, in spite of the acquisition by the host of Th1-mediated immunity. The role of Th1-medi- Address correspondence to Robert J. North, The Trudeau Institute, 100 ated immunity in the control of infection with avirulent Algonquin Ave., PO Box 59, Saranac Lake, NY 12983. Phone: 518-891- strains of Mtb, on the other hand, has received little atten- 3080; Fax: 518-891-5126; E-mail: [email protected] tion. It was considered important for a better understanding *Abbreviations used in this paper: Mtb, Mycobacterium tuberculosis; NO, ni- Phox of virulence, therefore, to know whether the requirements tric oxide; NOS, nitric oxide synthase; phox, gp91 subunit of the re- for control of infection with avirulent strains of Mtb are the spiratory burst NADPH-oxidase complex; WT, wild-type. 991 J. Exp. Med.  The Rockefeller University Press • 0022-1007/2002/10/991/8 $5.00 Volume 196, Number 7, October 7, 2002 991–998 http://www.jem.org/cgi/doi/10.1084/jem.20021186 same as those for the control of infection with virulent was extracted from lungs homogenized in Trizol (Life Sciences), according to the manufacturer’s instructions. The RNA pellet strains. To this end the study presented here used targeted was dissolved in DEPC-treated distilled water. To remove con- gene-deleted mice to determine whether control of infec- taminated genomic DNA, RNA samples were treated with tion with avirulent Mtb, like control of infection with vir- RNase-free DNase I (Ambion) for 1 h at 37C. Aliquots of ulent Mtb, depends on the generation of Th1 immunity RNA samples were then passed through RNeasy minicolumns and an ability of host macrophages to generate NO and re- (QIAGEN), treated with DNA-free kit (Ambion), and stored at active oxygen. The results show that control of infection –70C. RiboGreen™ Quantitation kit (Molecular Probes) was with either of two avirulent strains of Mtb depended on used to quantify RNA for real-time RT-PCR analysis. The assay the generation of Th1 immunity, but did not depend on was repeated three times using different dilutions of the samples. the synthesis of NOS2 by macrophages. They also show Primers and probes were designed with Primer Express Soft- that the ability to generate reactive oxygen and its metabo- ware (PE Biosystems), and purchased from Integrated DNA Technologies. Probes contained a fluorescent dye (6-carboxy- lites was not required to control infection with virulent or fluorescein; FAM) and a quencher (Black Hole Quencher 1; avirulent Mtb. Thus virulence is a property that enables BHQ1). The melting temperature of the hybridized probe Mtb to overcome the growth inhibitory action of a Th1- (70C) was always 10C higher than the PCR primers (57– dependent, NOS2-independent mechanism capable of in- 60C). hibiting the growth of avirulent Mtb. To make RNA standards, each amplicon of IFN- and NOS2 was generated by PCR from WT mouse lung mRNA using the same primers as those used for real-time RT-PCR. The ampli- Materials and Methods cons were cloned behind the T7 RNA polymerase promoter in Mice. WT mice and NOS2 mice, and mice deleted of the the pGEM-T Easy Vector System (Promega). The sequence of Phox Phox gene for the gp91 subunit (9) of the respiratory burst gp91 each cloned amplicon was determined by thermocycler sequenc- subunit of the respiratory burst NADPH-oxidase complex ing. After linearization of plasmid DNA, amplicons were tran- / / (Phox mice), as well as double mutant mice (NOS2/Phox scribed using T7 RNA polymerase (Promega). Template DNA mice), all on a C57BL/6 background were purchased from the was removed by digestion with DNase I, and RNA was purified Trudeau Institute Animal Breeding Facility. The mutant mice by using RNeasy minicolumns and quantified with the Ri- were from breeding stock supplied by C. Nathan (Cornell Uni- boGreen assay. It was determined that 1 g of an average 1,000 / 12 versity Medical College, New York, NY). Phox mice were bp mRNA contained 1.8  10 molecules. To obtain a stan- originally supplied by M. Dinauer (Indiana University Medical dard curve, serial dilutions of each transcript was performed in 9 1 Center, Indianapolis, IN). Mice deleted of the gene for the triplicate to give dilutions ranging from 10 to 10 molecules. / / TCR- chain (TCR-/ mice) and IFN- (IFN- mice) The dilutions were then subjected to real-time RT-PCR analy- on a C57BL/6 background were purchased from The Jackson sis as described below. Laboratory. All mice were used when they were 10 wk old. For real-time RT-PCR, 2 g of RNA was transcribed by us- Bacteria and Infection. The H37Rv (TMC# 102), H37Ra ing a random hexamer and a TaqMan Gold RT-PCR kit (PE (TMC# 201), and R1Rv (TMC# 205) strains of Mtb were orig- Biosystems), according to the manufacturer’s instructions. Real- inally obtained from the Trudeau Mycobacterial Culture Collec- time PCR to enumerate IFN- and NOS2 amplicons was per- tion (TMC), Trudeau Institute. The avirulent H37Ra and viru- formed in the ABI-prism 7700 Sequence Detector. Reaction lent H37Rv strains were derived from the virulent H37strain as conditions were programmed on a dedicated Power Macintosh smooth and rough colony in 1934, respectively (10). The H37 7200 computer. PCR amplification was performed in a total of parental strain was isolated from a human with pulmonary tuber- 25 l containing 10 l of cDNA sample, 2.5 l of 10 Taqman culosis in 1905 (11). The R1Rv strain was derived in 1946 from Buffer A, 5.5 mM MgCl 200 M each of dATP, dCTP, and 2, the virulent R1 strain that was isolated from a human with lung dGTP, 400 M dUTP, 0.25 M of each primer, 0.625 U of disease in 1891 (12). All strains are currently available from the AmpliTaq Gold, and 0.25 U of AmpErase Uracil N-glycosylase American Type Culture Collection. They were grown in suspen- (Perkin Elmer/Applied Biosystems). The reaction also contained sion culture in Proskauer and Beck medium containing 0.01% 0.2 M of detection probe. Amplification was performed in trip- Tween 80. The cultures were harvested after reaching 10 CFU licate wells under the following conditions: 2 min at 50C and 10 per ml, dispersed by ultrasound, and stored in vials in 1 ml lots at min at 94C followed by a total of 40 two-temperature cycles –70C until needed. To infect mice via the respiratory route, a (15 s at 94C and 1 min at 60C). The copy number in each sam- vial was thawed, diluted appropriately in PBS containing 0.01% ple was calculated according to the formula N (Ct-b)/m, where Tween 80, dispersed further by two 5 s bursts of ultrasound, and N is copy number, Ct is the threshold cycle, b is the y-intercept, 10 ml of the diluted culture used to load the nebulizer reservoir of and m is the slope of the standard curve line. an aerosol infection apparatus (Tri Instruments). Exposure to the Histology and Immunocytochemistry. Lungs were fixed in 10% aerosol for 30 min resulted in each mouse being infected with neutral buffered formaldehyde for 18 h. They were then washed 10 CFU. This was determined by enumerating CFU in the for several hours in distilled water, dehydrated in 70 and then lungs 1 d after aerosol exposure. Changes in the numbers of CFU 100% ethanol, and embedded in paraffin according to standard in lungs, livers, and spleens against time of infection was deter- procedures. Paraffin sections were cut at a thickness of 5 or 10 mined by plating 10-fold serial dilutions of whole organ homoge- m with a rotary microtome, stained for acid-fast bacteria with a nates on enriched Middlebrook 7H11 agar, and counting colonies modified basic fuchsin stain (14), and counter stained with meth- after 3 wk incubation at 37C as described previously (13). ylene blue. Immunocytochemical staining for NOS2 involved Real-Time RT-PCR Quantitation of mRNA Synthesis for IFN- reacting 10 m-thick section with 0.1 g/ml of affinity-purified and NOS2. Lungs were harvested at the times indicated in the monospecific rabbit Ig anti–mouse NOS2 (Transduction Labora- Results section, and snap frozen in liquid nitrogen. Total RNA tories) as the primary reagent. After washing, the sections were 992 NOS2 and M. tuberculosis Virulence reacted with biotinylated goat Ig anti–rabbit Ig as the second re- Phox mice also were not deficient in an ability to con- agent. The sections were then reacted with avidin-coupled trol infection with R1Rv during the first 60 d of infection. horseradish peroxidase and with diaminobenzidine as substrate to On the contrary, R1Rv infection in Phox mice was produce a brown reaction product in cells containing NOS2. controlled at about a 1 log lower level than in WT mice, The secondary and tertiary reagents were supplied in kit form indicating that these mice were more resistant than WT (Vectastain ABC kit; Vector Laboratories) and used according to mice, presumably because they developed compensatory the supplier’s instructions. Photomicrographs were taken with a mechanisms of defense. NOS2/Phox mice showed the Nikon Microphot-Fx microscope. same resistance as Phox mice up to day 40. A shortage of NOS2/Phox mice prevented infection in these mice from being followed beyond this time. Results Fig. 3 shows the results of a similar study that tested the / / Susceptibility of NOS2 and Phox Mice to Infection / / resistance of WT, NOS2 , and NOS2/Phox mice to with Virulent Versus Avirulent Mtb. To investigate whether infection with the avirulent H37Ra strain of Mtb. It can be NOS2 and NADPH-oxidase are required for resistance to seen that control of lung infection with H37Ra in NOS2 infection with virulent versus avirulent Mtb strains, WT, mice was the same as in WT mice over 100 d of infec- / / / NOS2 , Phox , and NOS2/Phox mice were in- tion. H37Ra infection progressed for 20–30 d, after fected via the respiratory route with 10 CFU of H37Rv, which it underwent progressive resolution until the experi- R1Rv, or H37Ra, and bacterial growth monitored in ment was terminated. As was the case with R1Rv infec- lungs, livers, and spleens over time. As shown in Fig. 1, tion, NOS2/Phox mice were somewhat more resistant WT mice were capable of controlling growth of H37Rv in to H37Ra infection than WT mice. Growth of H37Ra in their lungs and other organs starting on day 20 of infection, the liver and spleen is not shown, because these organs did and of maintaining infection at an approximately stationary not become infected. level from day 20 until day 60 when the experiment was Control of Infection with Avirulent Mtb Depends on T Cell– terminated. In NOS2 mice, in contrast, H37Rv grew mediated Immunity. The foregoing results clearly show progressively beyond day 20, resulting in death before day that, whereas NOS2 synthesis is needed for control of in- 50. In Phox mice, on the other hand, the course of fection with H37Rv, it is not needed for control of infec- H37Rv infection was essentially the same as in WT mice, tion with R1Rv or H37Ra over a 60-d period. Because whereas in NOS2/Phox mice infection was essentially synthesis of NOS2 by macrophages is induced by Th1 cy- the same as in NOS2 mice. tokines (4), failure to show a role for NOS2 in control of In mice infected with R1Rv (Fig. 2) the results were dif- early infection with R1Rv or H37Ra could mean that re- ferent, in that growth of this organism was essentially the sistance to infection with these avirulent strains is not de- same in the lungs of NOS2 and WT mice over the 60 d pendent on Th1-mediated immunity. This possibility was of the experiment. In both types of mice, R1Rv infection investigated by determining whether infection with R1Rv progressed for 20 d before declining by 1 log by day 60. or H37Ra is exacerbated in gene-deleted mice incapable Figure 2. Growth of R1Rv in lungs, livers, and spleens of WT, / / Phox , NOS2 , and NOS2/ Phox mice infected aerogenically with 10 CFU. Growth of R1Rv was identical in the lungs of WT and NOS2 mice, in that it was con- trolled at day 20, after which infec- Figure 1. Growth of H37Rv in tion slowly resolved. Growth of the lungs, livers, and spleens of WT, R1Rv in Phox and NOS2/ / / / Phox , NOS2 , and NOS2/ Phox mice was controlled at / 2 Phox mice infected with 10 about a 1 log lower level than in CFU aerogenically. Whereas in WT WT mice after which infection and Phox mice growth of slowly resolved. Dissemination of H37Rv was controlled from day 20 R1Rv to the liver and spleen was of infection on, growth of the slower in Phox and NOS2/ pathogen was progressive in Phox mice, although by day 60 / / NOS2 and NOS2/Phox all groups showed similar CFU mice. Means of five mice per time numbers in these organs. Means of point SD. five mice per time point SD. 993 Jung et al. Figure 3. Growth of H37Ra in WT and mutant mice. Results of two experiments (a and b) showing that growth H37Ra in WT and NOS2 mice (a) was identical, in that in both cases H37Ra grew for Figure 5. Number of H37Ra in lungs, 20–30 d in the lung, after which in- fection slowly resolved. H37Ra also livers, and spleens of WT, TCR- , grew similarly in NOS2/Phox and IFN- mice at day 50 of an aero- genic infection initiated with 5  10 CFU. mice, except that infection was con- Infection in the lungs was significantly trolled at a lower level in the latter mice. Means of five mice per time higher in mutant mice. H37Ra did not point SD. disseminate to the liver and spleen in WT mice, but did so in mutant mice incapable of expressing immunity. Means of five mice SD. of making  T cells or IFN-. As can be seen in Figs. 4 and 5. / / TCR-/ and IFN- mice allowed significantly fection, is associated with the expression of Th1-mediated more growth of R1Rv and H37Ra in their lungs and other immunity is to measure IFN- and NOS2 gene expression. organs than WT mice over a 50-d period of infection. It Therefore, real-time RT-PCR was used measure changes will also be noted in the case of H37Ra (Fig. 5) that infec- in the copy number of mRNA for IFN- and NOS2 in tion did not disseminate from the lungs to the liver and the lungs of mice infected with either organism against spleen in WT mice, but did so in mice devoid of  T time of infection. The results show (Fig. 6) that between cells. Thus resistance to R1Rv and H37Ra infection, like days 11 and 20 of H37Rv infection the copy number of resistance to H37Rv infection, almost certainly is depen- IFN- and NOS2 increased 500-fold. Moreover, in- dent on Th1-mediated immunity. creased mRNA synthesis for these two proteins was sus- Increased Synthesis of mRNA for IFN- and NOS2 in Re- tained until day 50 of infection when the experiment was sponse to H37Rv and R1Rv Infection. Because Th1-medi- terminated. IFN- and NOS2 mRNA synthesis also in- ated immunity against Mtb infection is dependent on IFN- creased in the lungs of R1Rv-infected mice between days synthesis by T cells, and NOS2 synthesis by macrophages 11 and 20 of infection, but to a lesser extent than in the (4), an additional way to determine whether acquired resis- case of H37Rv-infected mice. Thus control of H37Rv at a tance to infection with R1Rv, like control of H37Rv in- stationary level and the slow resolution of R1Rv infection were associated with a substantial increase in mRNA syn- thesis of a key cytokine of Th1 cells and a key enzyme of activated macrophages. Figure 6. Results of a real-time RT-PCR study of IFN- and NOS2 gene expression in the lungs of WT mice infected aerogenically with 10 CFU of H37Rv or R1Rv. Copy numbers of mRNA for IFN- and NOS2 per total lung RNA in- crease significantly between days 11 and 20 of infection with either or- ganism, and were sustained from day Figure 4. Number of R1Rv in the lungs, 20 to day 50 when the study was livers, and spleens of WT, TCR- , terminated. However, increased and IFN- mice at day 50 of an aero- IFN- and NOS2 mRNA synthesis genic initiated with 10 CFU. Infection was was significantly lower in the case of exacerbated substantially in all organs of the lungs infected with R1Rv. Means of mutant mice. Means of five mice SD. three mice per time point SD. 994 NOS2 and M. tuberculosis Virulence Discussion Immunocytochemistry of NOS2 Synthesis by Macrophages in Lungs Infected with Virulent Versus Avirulent Mtb. Lung The virulent H37Rv strain of Mtb used in this study is a histopatholgy induced by H37Rv in WT and NOS2 widely investigated laboratory strain that was derived as a mice on day 40 of infection is shown in Fig. 7. Day 40 was smooth colony in 1934 from the parent H37 strain isolated chosen to examine H37Rv-induced pathology because from a patient with active tuberculosis in 1905 (11). It is most NOS2 mice died before day 50. It can be seen apparent from examining past and current literature that (Fig. 7 a) that each site of lung infection in WT mice was H37Rv has not lost virulence for mice since its isolation, evidenced by accumulations of macrophages in close prox- and there is no reason to believe at present that it is less vir- imity to one or more dense aggregates of lymphoid cells. ulent for mice than most recent clinical Mtb isolates. It is Many of the macrophages in these lesions were infected shown here, in agreement with previous studies (1, 15–18) with acid-fast bacilli (Fig. 7 b) and most stained positively that H37Rv, like other virulent Mtb strains grows 4.5 for NOS2 by immunocytochemistry. In NOS2 mice logs in the lungs of mice over 20 d of infection, after which on day 40, in contrast, sites of H37Rv infection were seen its growth is controlled and held at a stationary level by as large focal areas of neutrophil-dominated inflammation specific immunity. The avirulent H37Ra strain was derived (Fig. 7, c and d) in which most alveoli were densely packed from a parental H37 strain as a rough colony at the time of with degenerating neutrophils. Tissue necrosis was obvi- derivation of the H37Rv strain (10). It was classified as ously taking place at these sites, and no cells stained posi- avirulent on the basis of its inability to grow progressively tively for NOS2. and cause disease in laboratory animals. Infection with this Sites of R1Rv lung infection in WT mice (Fig. 8 a) at strain is progressive for 21 d, after which host immunity day 50 were also seen as accumulations of macrophages in causes infection to progressively resolve, as shown here for close proximity to lymphoid cell aggregates. Macrophage mice, and elsewhere for guinea pigs (19). A description of accumulations were somewhat less extensive than in differences between the IS6110 DNA fingerprint patterns H37Rv-induced lesions, and most macrophages stained of H37Rv and H37Ra has been published recently (20). positively for NOS2. However, few macrophages showed The R1Rv strain is an attenuated derivative of a parental the presence of acid-fast bacilli, in keeping with the rela- R1strain (12) that was isolated from a patient with lung dis- tively smaller number of R1Rv bacilli in the lungs of WT ease in 1891. R1Rv is known from a previous study (21) to mice at day 50 of infection. In NOS2 mice, R1Rv- grow progressively in the lungs of mice for 3 wk, before induced lung lesions at day 50 (Fig. 8 b) were larger and its growth is controlled and held at stationary level for a had a different appearance to those in WT mice. Each con- protracted period of time. However, R1Rv, like H37Ra sisted of an extensive accumulation of macrophages sur- grows, at a much slower rate in the lungs of mice than rounded by a partial or complete mantle of darkly stained H37Rv during the first 20 d of infection (21). It is shown lymphoid cells. No cells in these lesions (Fig. 8 c) stained here, in this connection that R1Rv grew 3 logs, and positively for NOS2, and neutrophils were almost com- H37Ra 2 logs in the lungs of mice before infection be- pletely absent. Acid-fast R1Rv bacilli were present in mac- gan to resolve. According to a previous study (21), infec- rophages in the centers of the lesions, but were no more tion with R1Rv eventually stabilizes at a stationary level numerous than in lesions of WT mice at day 50. and causes neither progressive lung pathology nor death. Figure 7. Micrographs of section of lung from a WT / 2 and an NOS2 mouse infected 40 d earlier with 10 CFU of H37Rv aerogenically. Lesions in WT lung at low power (a) consisted of aggregates of macrophages stained for NOS2 (brown) in close proximity to aggre- gates to lymphoid cells (dark blue). At higher power (b) NOS2-stained macrophages were seen to contain acid-fast bacilli (red). In contrast, lesions in NOS2 lungs at low power (c) were seen to be composed of large numbers of neutrophils that at higher power (d) were seen to contain acid-fast bacilli and to be in the process of degeneration. No cells in NOS2 lungs stained for NOS2. Original magnifications: 80 for a and c; 470 for b and d. 995 Jung et al. mice, like that induced by H37Rv, was characterized by accumulations of macrophages in close association with aggregates of lymphoid cells, R1Rv-induced lesion in NOS2 mice were seen as a central core of macrophages surrounded by a thick mantle of lymphoid cells. Another key finding presented is that although NOS2 was not needed for resistance to R1Rv infection, NOS2 was nev- ertheless synthesized by R1Rv-infected macrophages in le- sions in WT mice, as demonstrated by immunocytochem- istry. This agrees with the additional finding that control of infection with R1Rv, like control of H37Rv infection, was preceded by a large increase in the synthesis of mRNA for IFN- and NOS2 in the lungs. The increase was smaller in the case of R1Rv infection, which is in keeping with a log lower level of R1Rv infection. It was not sur- prising to find that maintenance of stationary H37Rv infec- tion and slow resolution of R1Rv infection were associated with a sustained high level of synthesis of IFN- and NOS2 mRNA. Further reason for concluding that immu- nity to R1Rv and H37Ra infection is Th1-mediated is the demonstration that infection with either organism was not controlled by gene-deleted mice incapable of making  T cells or IFN-. It goes without saying that the presence of IFN-–producing T cells at sites of R1Rv infection would be expected to cause activation of Mtb-infected macro- phages at these sites, as evidence by the acquisition by mac- rophages of NOS2, as shown here. The findings with H37Rv serve to confirm results pub- lished by others (6, 7) showing that NOS2 mice are in- capable of controlling Mtb growth in their lungs and other Figure 8. Micrographs of lung lesions in WT and NOS2 mice in- 2 organs after day 20 of infection and quickly succumb to in- fected with 10 CFU of R1Rv aerogenically. At lower power, (a) lesions in WT lungs were composed of aggregates of lymphoid cells (blue) in fection-induced pathology. However, they are in disagree- close association with aggregates of macrophages containing NOS2 ment with those in a recent publications (22) showing that (brown). In the lungs of NOS2 mice at low power (b) lesions were NOS2 mice are not substantially deficient in a capacity seen to consist typically of a central core of macrophages surrounded by a to control early airborne infection with the virulent Erd- mantle of lymphoid cells (blue). At higher power (c) none of the mac- rophages in the central core stained for NOS2, and some of them were man strain of Mtb. This contradictory evidence with Mtb infected with acid-fast bacilli (red). Original magnifications: 92 for a Erdman needs to be considered, however, in light of other and b: 740 for c. publications (6, 7) showing that NOS2 is essential for im- mune control of infection with Mtb Erdman, and that mice incapable of making NOS2 quickly succumb to a very This is contrast to H37Rv stationary infection that causes small aerosol challenge with this strain (7), as do mice in- both progressive pathology and death. This previous find- fected by aerosol with H37Rv (1). The reason why growth ing with R1Rv led to the suggestion (21) that the ability to of Erdman was controlled by NOS2 mice in one labo- persist in infected tissues in the face of acquired host immu- ratory, but not in others is not known. However, in view nity is by itself not a measure of Mtb pathogenicity or viru- of the findings presented here with avirulent strains of Mtb, lence, although persistence is necessary in order for viru- a possibility that needs to be considered is that an Erdman lence to be expressed. strain whose growth is fairly well controlled in the absence A key finding presented here is that, whereas growth of of NOS2 is likely to be less virulent than an Erdman strain H37Rv after day 20 was progressive and quickly lethal for whose growth is progressive. Indeed, it is apparent from NOS2 mice, growth of R1Rv slowly resolved in these published growth curves (22, 23) that the Erdman strain mice and did so to the same extent as in WT mice. R1Rv- that grows less in NOS2 mice multiplies at a slower rate infected WT and NOS2 mice showed no signs of mor- in WT mice than the Erdman strains used by others (18), bidity over 150-d period of observation (unpublished data). and that it gives rise to a lower level of stationary infection Moreover, whereas H37Rv induced a fulminating, neutro- after the expression of Th1 immunity. In fact, an examina- phil-dominated pathology in the lungs of NOS2 mice, tion of published growth curves of the Erdman strain in R1Rv-induced lung pathology that was essentially devoid question shows that it grows at a similar rate and gives rise of neutrophils, although different from that induced in WT to about the same level of infection as the avirulent R1Rv mice. Thus, whereas R1Rv-induced pathology in WT strain used in this study. 996 NOS2 and M. tuberculosis Virulence An additional finding revealed by this study is that the intracellular bacteria. Curr. Opin. Immunol. 13:417–428. 6. McMicking, J.D., R.J. North, R. LaCourse, J.S. Mudget, generation of reactive oxygen by NADPH-oxidase is not S.K. Shah, and C.F. Nathan. 1997. Identification of nitric needed for control of infection with either virulent or avir- oxide synthase as a protective locus against tuberculosis. Proc. ulent Mtb. On the contrary, Phox mice were identical Natl. Acad. Sci. USA. 94:5243–5248. to WT mice in their ability to stabilize H37Rv infection, 7. Scanga, C.A., V.P. Mohan, K. Tanaka, D. Alland, J.L. Flynn, and were more resistant than WT mice to infection with and J. Chan. 2001. The inducible nitric oxide synthase locus R1Rv and H37Ra. Moreover, NOS2/Phox double confers protection against aerogenic challenge of both clinical mutant mice were no more susceptible than NOS2 and laboratory strains of Mycobacterium tuberculosis. Infect. Im- mice to infection with R1Rv or H37Rv. The demonstra- mun. 69:7711–7779. tion that Phox mice were not more susceptible than 8. Flynn, J.L., C.A. Scanga, K.E. Tanaka, and J. Chan. 1998. WT mice to H37Rv infection is in overall agreement with Effects of aminoguanidine on latent murine tuberculosis. J. Immunol. 160:1796–1803. results published by another laboratory (24) showing only a 9. Pollock, J.D., D.A. Williams, M.A. Gifford, L.L. Li, X. Du, transient, small increase in the growth of Mtb in the lungs, / J. Fisherman, S.H. Orkin, C.M. Doerchuck, and M.C. but not in other organs, of Phox mice over WT mice. Dinauer. 1995. Mouse model of X-linked chronic granulo- The results are in disagreement, on the other hand, with a matous disease, an inherited defect in phagocyte superoxide publication (25) showing that Phox mice are signifi- production. Nat. Genet. 9:202–209. cantly more susceptible to lung infection with virulent Mtb 10. Steenken, W., W.H. Oatway, and S.A. Petroff. 1934. Bio- than WT mice, as assessed by Mtb growth in the lungs. logical studies of the tubercle bacillus. III. Dissociation and The reason for this discrepancy is not known and is difficult pathogenicity of the R and S variants of the human tubercle to explain. bacillus (H37). J. Exp. Med. 60:515–543. The NOS2-independent mechanism of defense that is 11. Steenken, W., and L.U. Gardner. 1946. History of H37 strain of tubercle bacillus. Am. Rev. Tuberc. 54:62–66. capable of stabilizing infection with R1Rv and H37Ra was 12. Steenken, W., and L.U. Gardner. 1946. R1 strain of tubercle not identified by this study. It is likely to be the same bacillus, its dissociation and virulence of variants in normal mechanism that enables NOS2-deficient mice to inhibit and silicotic guinea pigs. Am. Rev. Tuberc. 54:51–60. the growth of Mycobacterium avium in major organs (26), and 13. Dunn, P.L., and R.J. North. 1995. Virulence ranking of enables NOS2-deficient macrophages to inhibit growth of some Mycobacterium tuberculosis and Mycobacterium bovis strains this organism in vitro (26). It may also be the same mecha- according to their ability to multiply in the lungs, induce nism that enables human macrophages treated with inhibi- lung pathology and cause mortality. Infect. Immunity. 63: tors of NOS2 to control the growth of H37Ra in vitro 3428–3437. (27). It has been shown by in vitro studies (28) that mouse 14. Ellis, R.C., and L.A. Zabrowarny. 1993. A safer staining macrophages are capable of expressing both NOS2-depen- method for acid fast bacilli. J. Clin. Pathol. 46:559–560. dent and NOS2-independent mechanisms of antimycobac- 15. Schell, R.F., W.F. Ealey, G.E. Harding, and D.W. Smith. 1974. The influence of vaccination on the course of experi- terial defense. mental airborne tuberculosis in mice. J. Reticuloendoth. Soc. In conclusion, by showing that NOS2 mice can con- 16:131–138. trol infection with avirulent, but not virulent Mtb, this 16. North, RJ., L. Ryan, R. LaCourse, T. Mogues and M.E. study suggests that virulence is a property that enables Mtb Goodrich. 1999. Growth of mycobacteria in mice as an un- to multiply in the face of the growth inhibitory action of a reliable indicator of virulence. Infect. Immun. 67:5483–5485. NOS2-independent, Th1-dependent anti-Mtb defense. 17. Manca, C., L. Tsenova, C.E. Barry, A. Bergtold, S. Freeman, P.A. Haslett, J.M. Musser, V.H. Freedman, and G. Kaplan. This work was supported by National Institutes of Health grants 1999. Mycobacterium tuberculosis CDC1551 induces a AI-37844 and HL-64565. more vigorous host response in vivo and in vitro, but is not Submitted: 12 July 2002 more virulent than other clinical isolates. J. Immunol. 162: Revised: 13 August 2002 6740–6746. Accepted: 21 August 2002 18. Kelley, C.L., and F.M. Collins. 1999. Growth of a highly virulent strain of Mycobacterium tuberculosis in mice of differing susceptibility to tuberculosis. Tuberc. Lung Dis. 79:367–370. 19. Alsaadi, A.-I., and D.W. 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J. nitrogen intermediates in the host response to mycobacte- Immunol. 166:6728–6734. 998 NOS2 and M. tuberculosis Virulence

Journal

The Journal of Experimental Medicine – Pubmed Central

Published: Oct 7, 2002

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