Introduction

Mycobacterium tuberculosis is principally a pulmonary pathogen causing two million deaths worldwide annually¹. In addition to the increasing incidence of TB, there has been a global emergence of drug resistant strains, posing a threat to existing therapeutic possibilities². The case fatality rate for multidrug-resistant tuberculosis (MDR-TB) is 40-60%, which equals the case fatality rate for untreated TB³. Hence, newer strategies to combat it are required.

Tuberculosis is acquired through the inhalation of droplets containing live M. tuberculosis and the lung is the major site of disease activity. The mechanism responsible for early control of bacillary growth relies on phagocytic cell activation, which is in part modulated by the in vivo cytokine environment. IFN--activated murine macrophages control M. tuberculosis via an oxidative, arginine-dependent mechanism that involves the generation of reactive nitrogen intermediates. The inhibition of macrophage nitrite production by arginine analogues correlates with the loss of antimicrobial activity. MacMicking et al. have conclusively identified inducible nitric oxide synthase (iNOS or NOS2) as a protective locus against tuberculosis⁴. Inducible nitric oxide synthase is a haemoprotein that catalyses the oxidation of L-arginine to nitric oxide and citrulline. The nitric oxide (NO) produced is a short lived, readily diffusible molecule of great biological importance and forms nitrite as a stable product^{5, 6}. Inducible nitric oxide synthase is capable of producing a large amount of NO when induced by mediators such as IFN-, TNF-, IL-1, IL-2, IL-6, lipopolysaccharide (LPS) and a number of micro-organisms⁷. Moreover, Nathan and Shiloh⁶, made a preliminary observation that drugs that appear to cure M. tuberculosis infection in immunocompetent mice failed to do so in iNOS deficient mice, suggesting that tuberculocidal drugs may be effective in vivo only with the help from iNOS derived NO. Although there are conflicting reports as to whether human monocytes/macrophages are able to kill M. tuberculosis in an iNOS dependent manner^{8, 9, 10, 11, 12, 13}, in an earlier communication from our laboratory we have reported that proinflammatory cytokine stimulation of monocytes/ex vivo matured macrophages from active tuberculosis patients leads to nitric oxide (NO) production that brings down CFU of M. tuberculosis in these cells¹⁴. To investigate the immunodepression in MDR-TB cases, we assayed nitric oxide release and release of other monocyte-derived cytokines like TNF- and IL-12 from the peripheral blood monocytes of MDR-TB cases as an in vitro correlate of immune competence. Healthy controls and fresh active pulmonary tuberculosis cases were also included for comparison. We find that peripheral blood monocytes from MDR-TB patients release a very low level of NO as well as TNF- and IL-12 in response to M. tuberculosis infection or M. tuberculosis components even after stimulation with IFN-.

Materials and Methods

Subjects

Fourteen confirmed newly diagnosed pulmonary TB patients and 11 MDR-TB patients were taken up, from RB TB Hospital, Kingsway Camp, Delhi for the study. The study was approved by the Ethical Committee of the Institute. The newly diagnosed TB patients (mean age 42.7 3.2 years, four females and 10 males) participated in this study within 1 month of beginning the antituberculous drug treatment. Their diagnosis were bacteriologically confirmed as sputum positive active TB and these patients exhibited minimal to moderate TB in their chest X-rays. They were given the first line drugs, isoniazid, rifampicin, pyrazinamide and ethambutol as per the Revised National Tuberculosis Control Programme (RNTCP), as recommended by the World Health Organization (WHO).

Eleven patients (mean age 33.6 3.7 years, five females and six males) were clinically and bacteriologically categorized as suffering from active sputum positive MDR-TB with an average duration of illness of 3.0 0.2 years. All of the patients had parenchymal TB, but none had miliary or pleural TB. None of the patients had a previous history of diabetes mellitus or steroid therapy and all were HIV negative. These patients had moderate to advanced radiographic abnormalities upon chest X-ray examination. All of them were infected by tubercle bacilli that were resistant to rifampicin and isoniazid. Fluoroquinolones (ofloxacin, ciprofloxacin), aminoglycosides (kanamycin), p-aminosalicylate (PAS), ethionamide and prothionamide, the second line drugs, were used to treat these patients. A complete history was taken and a physical examination was performed on each patient by one of the investigators.

Eleven age- and sex-matched normal control subjects (mean age 36.3 4.3 years, seven females and four males) were also included in the study. They had no previous history of clinical TB or any other disease. All the controls had received Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccination at birth. All were tuberculin positive.

Isolation of PBMC and monocytes

Venous blood from tuberculosis patients were drawn into heparinized syringes, diluted 1:2 in RPMI 1640, layered on to ficoll-hypaque and centrifuged for 30 min at 400g. PBMC were isolated from the interface and washed in RPMI 1640. Approximately 2 10⁶ PBMC were placed in 6-well culture plates and incubated overnight at 37°C. The next day nonadherent cells were removed by washing in RPMI 1640. Adherent cells were 95% monocytes (PB Mn) as determined by esterase staining. These cells were then infected with M. tuberculosis or stimulated with mycobacterial antigens.

Mycobacterial culture

Mycobacterium tuberculosis H37Rv was maintained in Lowenstein-Jensen medium slants. They were then grown in Middlebrook 7H9 broth (Difco, BD Diagnostic Systems, Sparks, MN, USA) at 37°C for 2 weeks. The mid-log phase cultures were pelleted, resuspended in RPMI medium and passed through a 8 filter to form single cell suspension and a density of 2 10⁵ bacteria/mL (at a multiplicity of infection, MOI, 1:10) was used for infection studies.

Mycobacterial components

Mycobacterium tuberculosis H37Rv components, for example whole cell lysate (WCL), lipoarabinomannan (LAM) and culture filtrate protein (CFP) were kindly provided by Dr JT Belisle, Colorado State University, Colorado, USA.

Monocyte infection or stimulation

The adherent monocytes, isolated as in the preceding, were recharged with complete RPMI medium supplemented with 10% FCS and 10 g/mL of gentamicin and were infected overnight. They were washed repeatedly the next morning to remove the extracellular bacteria and incubated further for another 48 h. The overnight infection of monocytes with M. tuberculosis H37Rv did not affect the viability of the monocytes as was seen by XTT assay¹⁵ (data not shown).

The number of ingested mycobacteria was calculated by lysing the infected cells from one of the wells in 1% Triton X. Serial dilutions of the lysate were inoculated in LJ medium slants in duplicates. The slants were incubated at 37°C for three weeks. At the end of three weeks, slants were taken out and colony forming units were calculated from the number of mycobacterial colonies on the LJ slants, taking into consideration the dilution factors. Ten to 15 mycobacteria were found per cell and 80% of monocytes were infected (data not shown).

Or in another set of experiments, the adherent monocytes were exposed to mycobacterial components at the following concentrations: WCL (10 g/mL), LAM (5 g/mL), CFP (5 g/mL), and then cultured for 48 h after stimulation. These concentrations were found to be optimal (data not shown).

The concentrations of nitric oxide and cytokines in the culture supernatant were measured at 48 h; in both mycobacteria-infected monocytes as well as mycobacterial component-stimulated monocytes the production of these cytokines was maximal at this time point.

Measurement of NO concentration

Concentration of nitrite produced by monocytes as a measure of the production of NO was determined at 540 nm using Griess reagent in a spectrophotometer. Briefly, 100 L supernatant was removed from culture wells, centrifuged at 400g for 10 min to make it cell free and incubated with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochloride, 2.5% H₃ PO₄) at room temperature for 10 min¹⁶. Concentration of nitrite was determined by using sodium nitrite as standard.

Cytokines assay

The cytokines TNF- and IL-12p40 were assayed in cell culture supernatants at 48 h by a solid phase sandwich ELISA using matched antibody pairs according to the manufacturer's instructions (Diaclone Research, Besançon, France). Briefly, the culture supernatants were added to the cytokine specific monoclonal antibody coated wells along with biotinylated cytokine specific monoclonal antibody. After incubation and washing, the enzyme (strepavidin peroxidase) was added. After incubation and washing to remove unbound enzyme, a substrate solution was added to induce a coloured reaction product. The intensity of this coloured product was directly proportional to the concentration of cytokine present in the samples. The minimum detectable dose of IL-12p40 was less than 20 pg/mL and that of TNF- was less than 10 pg/mL.

Statistical analysis

The results are presented as mean SEM. ANOVA (one-way analysis of variance) was used to compare the NO and cytokine protein estimations when three groups were used for comparisons and Student's t-test when two groups are used for comparisons using Graph Pad Prism software (Graph Pad Software, San Diego, CA, USA). Pearson's coefficient of correlation was calculated between various parameters.

Results

As blood monocytes are precursors of alveolar macrophages, we have tried to understand the regulatory effect of M. tuberculosis infection on the nitric oxide and cytokine production in these cells. As an interaction of T lymphocytes and macrophages is essential for protective immune response, the effect of IFN-, a product of T cells, on NO production from these cells was also studied.

Induction of nitric oxide in monocytes infected with M. tuberculosis and stimulated with IFN- in patients and controls

Production of nitric oxide in response to M. tuberculosis infection was determined using Griess Reagent in the supernatant of cultured peripheral blood monocytes. The kinetics of secretion showed an optimal production at 48 h after infection, therefore this time period was considered for all further observations. The mean spontaneous NO production from normal control subjects was 18.55 4.52 mol/L (Figure 1). All but one had NO levels within 27.6 mol/L (mean + 2 SEM). Any value above this was taken as raised. It was observed that infected cells from fresh, active TB patients showed significantly raised production of nitric oxide (74.36 5.54 mol/L; p < 0.001) as compared to that of infected cells from healthy donors (31.83 4.52 mol/L). When the infected cells were stimulated with exogenous IFN-, production of nitric oxide was further enhanced in cells from active TB patients (80.11 5.23 mol/L; p < 0.0001) as compared to similarly treated cells from healthy controls (34.32 6.61 mol/L). All the active TB patient's monocytes showed significantly raised level of NO production following M. tuberculosis infection (P < 0.001) or IFN- stimulation (P = 0.0001) when compared in a group with normal controls and MDR-TB patients using ANOVA (Figure 1). Addition of N^G mono methyl arginine (N^G MMA), an analogue of arginine, to the culture supernatant greatly inhibited the production of nitric oxide in these cells (data not shown).

Figure 1.

The production of nitric oxide (NO), TNF- and IL-12 by cultured peripheral blood monocytes in healthy subjects (n = 11) and fresh active TB (n = 14) and multidrug-resistant tuberculosis (MDR-TB) patients (n = 11) before and after ex vivo infection with Mycobacterium tuberculosis and following IFN- stimulation. Mean values are indicated. (***) p < 0.001; (##) p = 0.0001; () p < 0.0005; () p < 0.005; () p < 0.0005 by a one-way ANOVA test. , normal; , active TB; , MDR-TB.

Full figure and legend (27K)

Both infected as well as IFN- stimulated infected cells from active TB patients showed significant (P < 0.001) production of nitric oxide as compared to that of uninfected cells (33.65 5.46 mol/L) from the same patients. However, the monocytes from MDR-TB patients demonstrated significantly lower levels of NO release (P < 0.001) when compared to the same from the fresh, active cases. Even their spontaneous NO production (21.7 6.79 mol/L) or NO production from M. tuberculosis infected (25.56 8.36 mol/L), or from IFN- stimulated monocytes (20.51 8.20 mol/L) was less when compared to normal healthy control monocytes under similar conditions (Figure 1).

Relationship between the generation of NO, TNF- and IL-12 by cultured peripheral blood monocytes

As both TNF- and IL-12 from monocyte/macrophage are considered essential for the protective immune response, we assayed the production of TNF- and IL-12 by the monocytes in our experimental system. IL-12 is responsible for production of IFN- by T cells and NK cells and thus bridges the innate and adaptive immune response. The mean production of TNF- and IL-12 from uninfected cells of healthy controls was 87.18 35.82 pg/mL and 289.1 92.21 pg/mL, respectively (Figure 1). The monocytes from active TB patients when infected with M. tuberculosis secreted significant amount of TNF- (511.3 69.23 pg/mL) in comparison to infected cells from healthy donors (117.7 69.9 pg/mL) and that from MDR-TB patients (157.9 74.23 pg/mL; p < 0.001) and secreted even higher levels after treatment of these cells with IFN- (P < 0.005). Although the production of IL-12 from infected cells of active TB patients (669.6 145.9 pg/mL) was higher as compared to that from infected cells of normal controls (309.5 11.06 pg/mL), it was not significant statistically. In addition, a definite and significant correlation (P < 0.005) has been observed between the levels of nitrite generated and TNF- production by monocytes of all the individuals. This correlation was valid for every set of experiments with peripheral blood monocytes, whether uninfected, infected or IFN- stimulated and infected (Figure 2). No significant correlation was observed between NO production and IL-12 production in any of the group although a positive trend was observed.

Figure 2.

The correlation between NO and TNF- production from (a) uninfected cultured monocytes (^r_p = 0.44; p < 0.005); (b) M. tuberculosis infected monocytes (^r_p = 0.52; p < 0.001); and (c) IFN- stimulated infected monocytes (^r_p = 0.46; p < 0.005). The number of patients and the significance are indicated. Statistical analysis was performed using Pearson's correlation. , normal controls (n =11); , active TB (n = 14); , MDR-TB (n = 11).

Full figure and legend (15K)

Subcellular fractions are sufficient to induce NO, TNF- and IL-12 production

To determine whether NO induction by M. tuberculosis was due to a bacterial component induced after infection of monocytes or could be due to a constitutively expressed bacterial component, we substituted killed (-irradiated) M. tuberculosis for live bacteria. We observed that killed mycobacteria could also induce NO production (data not shown).

The finding that killed M. tuberculosis was capable of causing NO induction indicated a possibility that one or more preformed components of the bacteria may be sufficient for initiating a pathway that results in NO inductive response. In a subsequent set of experiments we found that WCL, CFP as well as LAM were able to induce NO indicating thereby that essential bacterial components were able to exert its effect even when they were not presented in the form of whole particulate bacteria.

Here again, the cells from active tuberculosis patients showed significant and maximum production of NO following stimulation with any of the three antigens used (WCL = 78.24 6.17 mol/L; CFP = 66.54 6.02 mol/L; LAM = 76.21 mol/L) in comparison to unstimulated cells from these patients (33.65 5.45 mol/L; p = < 0.001) (Figure 3). Although cells from healthy controls could produce a good amount of NO after stimulation with mycobacterial components (WCL = 47.80 11.73 mol/L; CFP = 47.70 11.43 mol/L; LAM = 42.00 10.19 mol/L), the response of monocytes from MDR-TB patients in similar conditions was minimal (WCL = 25.86 4.41 mol/L; CFP = 18.91 3.94 mol/L; LAM = 27.86 6.17 mol/L) and was comparable to spontaneous release of NO from unstimulated cells of healthy normal controls (18.55 4.52 mol/L). The mycobacterial components were able to induce good amounts of cytokine TNF- and IL-12 in the cultured monocytes of the patients and controls and showed the same trend as was seen with NO production (Figure 3).

Figure 3.

The production of NO, TNF- and IL-12 after ex vivo stimulation of peripheral blood monocytes with M. tuberculosis H37Rv components. Whole cell lysate (WCL); culture filtrate protein (CFP); lipoarabinomannan (LAM). The production of NO and TNF- is significantly higher in active TB patients when compared either with healthy controls or MDR-TB patients using one-way analysis of variance (P < 0.005). , healthy; , active TB; , MDR-TB.

Full figure and legend (34K)

Discussion

To what extent the pathogenesis of tuberculosis depends on the host factors is an important issue in tuberculosis research. The current study was designed to determine the ability of peripheral blood monocytes from primary active and MDR-TB patients to produce NO and to find whether induction of NO was in coordination with the production of proinflammatory cytokines TNF- and IL-12. NO plays an important role in resistance to M. tuberculosis infection as evidenced both in experimental and human tuberculosis^{17, 18, 19, 20, 21}. Pretreatment with NOS2 inhibitors profoundly increases mortality, bacterial burden and pathological tissue damage in mice infected with M. tuberculosis²². Cytokines have multiple actions that play a role in host response to M. tuberculosis, including local granuloma formation, inflammation and delayed hypersensitivity. TNF- acts as chemotactic factor to attract inflammatory cells, such as T-lymphocytes, into the lower respiratory tract and, acting synergistically with IFN-, contributes to the activation of macrophages and mycobacterial growth inhibition^{23, 24}. In our study we have found an upregulation of NO, TNF- and IL-12 from peripheral blood monocytes of the active tuberculosis patients following M. tuberculosis infection or the stimulation of these cells with mycobacterial components signifying immune competence of these cells. The amount of NO production from cultured peripheral blood monocytes correlated highly with the levels of TNF- released into the culture medium which suggests that the release of the two may be cross-regulatory. Although the production of NO and IL-12 by cultured monocytes did not correlate significantly, a definite positive trend was observed. Our results also corroborate with some earlier studies that showed that NO modulates release of TNF- and vice-versa in the peripheral blood monocytes of patients with active TB²⁵. This response is, however, not always protective. Sustained release of NO and TNF- following M. tuberculosis infection of macrophages could lead to self-tissue destruction, ultimately culminating in progressive disease. High level NO and TNF- production is also shown to be responsible for apoptosis of macrophages infected with M. tuberculosis and thus can reduce the protective capacity of the host. It is the kinetics and the balance of these cytokines secreted by mononuclear phagocytes, before T-cell activation, that is critical in regulating subsequent immune responses and control of mycobacterial growth in patients with the active disease²⁶. Our results reveal that the peripheral blood monocytes from MDR-TB patients did not produce an optimum amount of NO and other cytokines, either spontaneously or following M. tuberculosis infection/mycobacterial component stimulation. These mononuclear cells from MDR-TB patients could have become anergic due to the repetitive chronic antigenic stimulation in vivo. Additionally, it may be possible that these patients' monocytes released low levels of IL-12 that may have resulted in diminished IFN- production leading to suboptimal or significantly impaired monocyte/macrophage activation in the MDR-TB cases. However, when the monocytes from these patients were exposed to IFN- in the presence of M. tuberculosis, the release of nitric oxide did not go up beyond basal levels. Therefore, MDR-TB patients' desensitization may not be the result of a defect in the production of IFN-, but may involve an IFN- receptor defect or the subsequent steps of the signal transduction pathway of IFN-, that ultimately induce NO release. This defect in the immune responsiveness of these patients could also be responsible for the ineffectiveness of chemotherapy in these patients. According to the results of our study, these cells are possibly incapable of producing a sufficient amount of NO as well as TNF-, a highly potent proinflammatory cytokine, and therefore the mycobactericidal capacity is significantly depressed, which in turn may cause unsuccessful chemotherapy leading to MDR-TB, at least in some patients. However, it may also be possible that inadequate/irregular treatment in a patient causes the tubercle bacilli to become drug resistant thereby resulting in chronic bacillary and excess antigenic load. This could be responsible for development of immune tolerance and reduced ability to respond to further stimulation to release bactericidal molecules such as nitric oxide or proinflammatory cytokines such as TNF-. Our hypothesis may be tenable as chemotherapy was generally found to be successful in immunocompetent rather than in immunodeficient patients⁶. Moreover, it was also reported that chemotherapy that appears to cure M. tuberculosis infection in immunocompetent mice fails to do so in NOS2-deficient mice^{4, 6}. Effective delivery of reactive nitrogen intermediates in such MDR-TB patients could provide a new immunotherapeutic strategy to contain drug resistant mycobacterial infection. In addition, our observations do seem to raise the interesting possibility that strong TNF- and NO production from peripheral blood monocytes of the tuberculosis patients may be used as an indicator to predict who will respond well to therapy, regardless of their drug sensitivity. However, this will require a prospective study on a larger population to be clinically useful.

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Acknowledgements

We are thankful to Department of Science and Technology, Government of India and Indian Council of Medical Research, India for providing the financial support.