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The 6-Kilodalton Early Secreted Antigenic Target-Responsive, Asymptomatic Contacts of Tuberculosis Patients Express Elevated Levels of Inter
     Armauer Hansen Research Institute, Addis Ababa, Ethiopia

    Department of Tuberculosis Immunology, Statens Serum Institute, Copenhagen, Denmark

    Centre for Infectious Diseases and International Health, Windeyer Institute of Medical Sciences, Royal Free and University College Medical School, London, United Kingdom

    ABSTRACT

    It is well known that the majority of healthy individuals exposed to Mycobacterium tuberculosis do not become clinically ill. We have previously shown that in recently exposed healthy contacts of tuberculosis (TB) patients, a strong immune response to the M. tuberculosis 6-kDa early secreted antigenic target (ESAT-6) virulence factor correlated with a higher risk of subsequent disease, although the mechanism was unclear at that time. Inspired by recent reports that elevated expression of interleukin-4 (IL-4) in health care workers exposed to M. tuberculosis also correlated with a higher risk of their subsequently developing disease, we examined expression of IL-4, its competitive antagonist IL-42, and gamma interferon (IFN-) in healthy household contacts of TB patients from Ethiopia. We then compared cytokine expression to their recognition of ESAT-6 (which is largely restricted to members of the tuberculosis complex and which serves as a reliable marker of infection) or to Ag85A (an antigen that is conserved among the mycobacteria and serves as a nonspecific control). Our study shows that in these recently exposed individuals, there is a correlation between a strong response to ESAT-6 and elevated expression of IL-4. Further, elevated expression of IL-4 is associated with lower expression of its antagonistic splice variant IL-42 and with the Th1 cytokine IFN-, suggesting that in these at-risk individuals, immunity is skewed away from a protective Th1 response, even before the development of clinical symptoms.

    INTRODUCTION

    With new vaccines for tuberculosis (TB) currently entering clinical trials (6), assessing correlates of immunity has become a crucial goal. It has been known for some time that a Th1 response is required to contain infection; the susceptibility of mice or humans with defects in gamma interferon (IFN-) or interleukin-12 (IL-12) recognition or production is profound (2, 3, 14). The necessary role of CD4 cells is also made obvious by the effect of human immunodeficiency virus (HIV) on the TB epidemic (1, 11). However, the role of Th2 cells in TB has been controversial, in part because the prototypical Th2 cytokine, IL-4, is highly bioactive and generally produced at very low levels, making its detection problematic. Some work, using indirect markers such as immunoglobulin E or soluble CD30, has suggested that progression to TB is associated with a relative increase in the production of IL-4 or a shift in the Th1/Th2 balance (18). More compellingly, recent work has shown that elevated expression of IL-4 in recall responses by peripheral blood mononuclear cells from health care workers exposed to Mycobacterium tuberculosis in the course of their duties correlated with later development of TB (23).

    The use of highly sensitive, quantitative reverse transcription-PCR (RT-PCR) now allows analysis of IL-4 gene activation directly ex vivo (32), and we have recently used PCR analysis of unstimulated leukocytes to examine cytokine responses in individuals with active or latent M. tuberculosis infection. This study demonstrated that IL-4 was elevated both in latently infected, healthy individuals and in TB patients with acute, progressive tuberculosis but that, significantly, the IL-4 antagonist IL-42 was elevated only in individuals with latent, and not in those with acute, TB (4, 8). We hypothesized that IL-4 production was elevated during the acute phase of infection and that progression to clinical disease was halted at least in part by increased production of the antagonistic splice variant, leading to long-term maintenance of the disease in the latent form. This hypothesis would tie our earlier observations together with data from other longitudinal studies suggesting that an increase in early IL-4 production correlated with a higher risk of later developing TB (23).

    Therefore, we have used RT-PCR to compare the expression of IL-4, IL-42, and IFN- mRNAs in the peripheral blood of close household contacts of sputum-positive TB patients in Ethiopia, a country where TB is endemic. In this environment, and consistent with our earlier findings (4), we found that IL-4 expression was only slightly elevated in household contacts compared to community controls. However, when the household contact group was segregated into groups with or without immunological signs of M. tuberculosis infection (i.e., 6-kDa early secreted antigenic target [ESAT-6] positivity), IL-4 expression was found to be clearly elevated in the ESAT-6-positive group. Moreover, expression of the Th1 cytokine IFN- was lower. These differences were not present when the household contacts were segregated on the basis of general mycobacterial immune reactivity (for example, by purified protein derivative or Ag85A reactivity). Thus, a strong, early response to ESAT-6 in healthy, TB-exposed individuals correlates with relatively lower IFN- expression and higher IL-4 expression, suggesting a mechanism for the previously observed poor prognosis.

    MATERIALS AND METHODS

    Study design. The cohort analyzed is comprised of individuals from 2 years of intake from the Ethiopian arm of an ongoing, multicenter, longitudinal study being carried out in Africa (VACSEL/VACSIS). Healthy household contacts (HHC) (n = 167) of sputum-positive TB patients (TB group) (n = 75) were recruited from TB clinics in the rural towns of Hossana and Butajira, Ethiopia. Community controls (CC) (n = 40) were randomly selected from the same neighborhoods, and recent TB contact was excluded by questionnaire. Active TB was excluded in all healthy participants (HHC and CC) on entry to the study by radiological and clinical examination, sputum microscopy, and culture (where possible) as previously described (4, 7). To be defined as "healthy" household contacts or community controls, the subjects were defined prior to the study as having a normal X-ray result, no symptoms, and negative results in all sputum tests. A small number of contacts were found to be symptomatic and sputum positive. They met the criteria for TB patients and were enrolled in that cohort. A substantial proportion (n = 37) were found to have either X-ray results suggestive of earlier TB (as assessed by the presiding clinician) or characteristic symptoms (generally cough/fever) or both. This group was assumed to have early-stage TB, but as they were neither sputum positive nor asymptomatic, they did not meet the inclusion criteria for any clinical group laid down for the study and thus are not included in this analysis. Blood samples were obtained from all donors at entry into the study. Tuberculin skin test results are not available, as the test is regarded as unreliable (roughly 60% of all adults are positive at the 10-mm level in this region [unpublished data]) and is neither recommended by local health authorities nor routinely performed. All participants were screened for HIV by repeated enzyme-linked immunosorbent assay (ELISA), and samples from HIV-positive individuals were excluded from the analysis. Pre- and posttest counseling was offered to all participants. Only adults (15 to 60 years of age) who had given written consent were included in the study, and this work was performed under a study protocol approved by the institutional and national ethical review boards.

    Quantitative RT-PCR. Unstimulated leukocytes were lysed immediately after drawing of blood with the RNEASY blood RNA system (QIAGEN, Dusseldorf, Germany) according to the manufacturer's instructions. The mRNA was transcribed into cDNA, using the Omniscript reverse transcription kit (QIAGEN, Dusseldorf, Germany) with oligo(dT) primers according to the manufacturer's instructions; the concentration was calculated from the optical density using a GeneQuant spectrophotometer (Amersham Biosciences, Amersham, United Kingdom); and the sample was stored at –20°C until use. PCR was carried out in a total volume of 50 μl with 1 μg of cDNA, using the HotStarTaq Master Mix kit (QIAGEN, Dusseldorf, Germany) according to the manufacturer's instructions. Primers were designed to span introns so that amplification from genomic DNA should not occur, and this was confirmed by comparing the results from PCR of RNA preparations and the cDNA that was prepared from it. A negative (no-template) control was also included in all PCR assays to test for contamination of reagents. PCR products were visualized by running on 1% agarose (Nusieve; FMC, Rockland, ME) gels containing SBYR green (Molecular Probes, Eugene, OR) at 1:10,000 (5 μl in a 50-ml gel), normalized against the -actin housekeeping gene, and quantitated against standard curves using the same primers but based on standardized samples containing known copy numbers of cDNA, as previously described (32). The PCR conditions and primers used were designed for the project at University College, London, and the number of cycles was optimized for each cytokine as previously described (4). The fluorescence of bands in the gel under UV transillumination was read using a 12-bit charge-coupled-device camera (Sensicam; UVP, San Gabriel, CA) and the data analyzed using the supplied Labworks software.

    IFN- ELISA. In vitro restimulation of peripheral blood mononuclear cells with either purified protein derivative (20 μg/ml), Ag85A (2 μg/ml), or ESAT-6 (2 μg/ml) was carried out as previously described (7). The supernatants were harvested at day 5 after stimulation and the levels of IFN- assayed in duplicate culture supernatants by using a commercial double-sandwich ELISA kit, in accordance with the manufacturer's instructions (Mabtech; AB, Sweden). The mean level in unstimulated cultures was 98 pg/ml for Ethiopian subjects, with a standard deviation of 59 pg/ml. The sensitivity of the assay was 1 to 9 pg/ml.

    Statistical analysis. The data obtained are presented as percent expression of the target gene compared to the -actin housekeeping gene and are shown as means ± standard deviations. Since analysis of variance showed wide differences, comparisons between groups were assessed by the Kruskal-Wallis and Dunnett's multiple-comparison test. The t test was used for analyses within groups. In all instances, a P value of <0.05 was considered significant.

    RESULTS

    Assessment of cytokine production by different cohorts by using semiquantitative RT-PCR. Initially, production of IL-4, IL-42, and IFN- by leukocytes from healthy household contacts (n = 167) was compared to that by index cases (TB group) (n = 75) and community controls (n = 40). Cells from the peripheral blood of each group were lysed without in vitro culture to analyze the responses ex vivo, and mRNA was extracted and reverse transcribed into cDNA. This cDNA was used for all subsequent analyses. As seen in Fig. 1a, both the HHC and TB groups expressed higher levels of IL-4 message than the CC group. This is consistent with recently published results (8) which also found elevated IL-4 in HHC compared to CC. However, the TB and HHC groups were essentially identical when compared for IL-4 expression (Fig. 1a) even though the HHC group was, by definition, asymptomatic. As expected, based on previous reports (4, 8), when we analyzed the samples for the expression of the IL-4 antagonist IL-42, we found that mean levels of message for this cytokine were elevated in the CC group compared to both the TB patients and their household contacts (Fig. 1b). A similar analysis was conducted for IFN- (Fig. 1c). The community control group expressed marginally higher levels of IFN- than the HHC or TB group, but this difference was not significant. Though the trend is the same, this is slightly different from our earlier findings (4), which we attribute to the much larger numbers in this study.

    Analysis of cytokine expression in healthy household contacts segregated by antigen recognition. While the clinical status of the TB patients in this study is clear (all were symptomatic, identified by X-ray and smear results, and/or culture positive for M. tuberculosis), the HHC and CC groups are potentially much more heterogeneous, as they may include subclinically or latently infected subjects, together with uninfected individuals. We have already described (4) a more detailed analysis of immune responses in CC segregated into infected and uninfected subgroups based on their recognition of the M. tuberculosis virulence factor ESAT-6 (which has proven to be a reliable marker of prior infection with TB) (16, 27, 35). Here, we randomly selected one-third of the HHC and performed the same, more detailed analysis. As a control, we also segregated the same groups on the basis of response to the common mycobacterial antigen Ag85A (which is both strongly immunogenic and present in a highly conserved form in most, if not all, species of mycobacteria) (9). Recognition of the antigens was determined by IFN- ELISA after in vitro stimulation, a method which has proven robust in many earlier studies (7, 22, 35, 36, 38). Since study participants in this part of Africa typically produce low levels of IFN- response detectable by ELISA without stimulation (and which is therefore not antigen specific), a positive, antigen-specific reaction in the ELISA was defined as more than 3 standard deviations above the mean value for unstimulated cultures (275 pg/ml). Using this cutoff, 23% of the HHC group were classed as high responders to ESAT-6 and therefore identified as definitely infected. For Ag85A, 21% of the HHC group were classified as high responders (i.e., with a response significantly above the mean ± 3 standard deviations for unstimulated cultures). Interestingly, there was only minor overlap between these two high-responder groups, indicating that we are not merely identifying those with a strong response to all mycobacterial antigens. This is congruent with earlier published studies, which indicate that approximately 20 to 30% of close TB contacts make a strong response to ESAT-6 (7, 36).

    In the HHC group, the IFN- mRNA level (from unstimulated blood) was significantly higher in the group that did not respond strongly to ESAT-6, whereas the response was not different when HHC were segregated on the basis of Ag85A (Fig. 2a and b). This is particularly interesting, since it implies that the lower level of IFN- expression is not due to lower immune reactivity but is specifically associated with a strong response to the ESAT-6 antigen and therefore presumably with M. tuberculosis infection. When we also performed the same type of analysis for expression of IL-4, we obtained results polarized in the exact opposite direction. Those individuals who responded most strongly to ESAT-6 also had constitutively higher levels of mRNA for IL-4, while segregation on the basis of recognition of Ag85A did not differentiate the groups (Fig. 3a and b).

    Finally, we performed the same analysis for IL-42. Consistent with the results shown in Fig. 1 and those presented in our recent publication (4), IL-42 levels were generally low in the HHC group, and there was no significant difference between high- and low-responder cohorts, whether segregated on the basis of recognition of the M. tuberculosis virulence factor ESAT-6 or the common antigen, Ag85A (Fig. 4a and b).

    Taken in total, these findings thus suggest that the ratio of IL-4 to IFN- is elevated both in patients with active TB and in HHC with high ESAT-6-specific immune responses. It is the latter group who, based on previous data, are those HHC most likely to have an active, if currently asymptomatic, infection (7).

    DISCUSSION

    The hypothesis that an altered Th1/Th2 response characterizes progress towards clinical tuberculosis has been controversial. Although reduced IFN- production in advanced cases of TB has often been reported (7, 36), the difficulty of measuring the prototypical Th2 cytokine IL-4 has meant that the case for an increased Th2 response in vivo has been made using indirect markers (18). Recently, however, more sensitive methods such as intracellular fluorescence-activated cell sorter analysis or RT-PCR have suggested that IL-4 expression is increased in TB patients and that the level of IL-4 gene expression correlates with both disease severity (12, 13, 31, 33, 34) and poor prognosis (5, 23). This does not prove, of course, that the increased Th2 response is responsible for the development of TB; it could be an attempt to limit the pathological nature of the inflammatory response against a spreading M. tuberculosis infection or could be involved in the modulation of the granulomatous response. Nonetheless, the ability to detect elevated IL-4 levels well in advance of the appearance of symptoms (23) certainly suggests an active role.

    We have also previously reported that elevated responses to the M. tuberculosis virulence factor ESAT-6 in healthy, recently exposed individuals were associated with a higher risk of subsequent clinical illness (7). This is consistent with animal studies showing a clear association between immune reactivity to ESAT-6, bacterial load, and pathology (17, 37; Andersen et al., submitted for publication) and with clinical studies indicating that the degree of ESAT-6 reactivity correlates well with the intensity of exposure/infectious load (10, 16) and therefore with risk.

    Based on these studies, we hypothesized that increased responsiveness to ESAT-6 in asymptomatic recent TB contacts might correlate with elevated levels of IL-4, thereby reconciling all of the available published data. We therefore used RT-PCR to monitor IL-4, its antagonistic splice variant IL-42, and the prototypic Th1 cytokine IFN- in household contacts of recently identified infectious TB patients. The initial analysis indicated that compared to those in CC, mean levels of IL-4 mRNA were elevated in both the TB and the HHC groups, all of whom could be assumed to have had recent or ongoing exposure to M. tuberculosis (Fig. 1a). Expression of IFN- was not significantly different between the groups (Fig. 1c), while elevated levels of IL-42, as previously reported, were restricted to the latently infected group (4).

    However, the HHC group is highly heterogeneous, and it is possible that not all contacts are infected with M. tuberculosis, notwithstanding their recent exposure to the bacteria. We therefore segregated the HHC into two groups: those strongly reactive to ESAT-6 and therefore presumptively infected with M. tuberculosis and those with lower immune responses to this antigen. This analysis confirmed the initial hypothesis, in that elevated IL-4 expression and decreased IFN- expression were strongly associated with strong recognition of ESAT-6 (and therefore presumably with recent, subclinical infection as indicated by Ordway and colleagues [23]). That it is not a simple case of higher responsiveness in these individuals is shown by the fact that no such association was found with antigens that are not TB specific, such as Ag85A (Fig. 2 and 3).

    Based on these results, we can now propose a simple model, which is consistent with all of the published data. There is a growing body of data from animal studies (including from primates) linking the magnitude of the T-cell response to ESAT-6 after M. tuberculosis infection with bacterial load and with the severity of subsequent disease (17, 37). The finding from clinical studies linking the magnitude of the ESAT-6 response to the degree of exposure and prognosis suggests that this pattern applies to human M. tuberculosis infection also (7, 10, 16). However, this conclusion is applicable only to acute infection; cured TB patients can retain a strong ESAT-6 response for decades (38). In addition, many individuals in communities where TB is endemic display significant ESAT-6 responsiveness without the concomitant elevation in risk of TB seen with primary exposure, presumably due to latent infection (perhaps more accurately described as long-term nonprogression to disease) (15). Thus, the ESAT-6-driven response assayed by in vitro restimulation of cells from people with long-term infection probably also includes a memory component that may not be visible using RT-PCR or during acute infection. However, there is good agreement that the initial response to M. tuberculosis infection, in nearly all cases, is a strong cell-mediated immunity response characterized by elevated IFN- production, which is necessary for protection (2, 3, 14).

    We now propose to extend this model by suggesting that in the majority of individuals who are exposed to M. tuberculosis, the early (Th1-dominated) immune response prevents or limits the establishment of infection. These individuals do not develop a high bacterial load and so remain unresponsive or weakly responsive to ESAT-6 (much like vaccinated and protected primates [17]). However, some exposed individuals are unable to prevent progressive infection, which is characterized by the induction of strong responses to ESAT-6 and also by increasing IL-4 production and decreasing IFN- production as they progress to disease. Nonetheless, even among those unable to resist acute infection, it is well known that a significant proportion of TB cases will spontaneously resolve, even in the absence of intervention, resulting in latent infection (19, 24). In many cases, particularly in developing countries, these may not even be recognized as TB cases if the period of clinical illness is not prolonged. We suggest that these individuals do not lose the T-cell responses established during infection, retaining both strong ESAT-6 memory responses and slightly elevated IL-4 production. Instead, they produce an increased level of IL-42 to compensate for the IL-4 and recover their capacity to produce IFN-. This is a simplistic model; it does not address the nature of the cells producing these cytokines, whether CD4 or CD8, T-helper, or regulatory T cells, and we currently lack sufficient data to speculate meaningfully on this (though studies are ongoing). Nor does it (yet) address other cytokines involved in the Th1/Th2 balance. Nonetheless, it is consistent with the published literature from both animal and clinical studies and may provide a useful framework for planning or interpreting further work.

    Such a conclusion is consistent with a growing number of recent publications suggesting that alterations in the balance of IL-4 and IL-42 are associated with different clinical manifestations in a variety of diseases. In an atopic response such as asthma, the ratio of IL-4 and IL-42 is biased towards IL-4 by nearly 3 orders of magnitude compared to the results for TB patients (30), while the Th1-mediated pathology in Helicobacter pylori infection (25) and the induction of fibrosis in systemic sclerosis (29) are both associated with a decreased IL-4/IL-42 ratio. The pathways by which mycobacteria induce or suppress IFN- production are now being mapped (21, 26), and there is some evidence that inhibition of cell-mediated immunity and induction of IL-4 by the bacterium itself may play a role in virulence (20, 28). However, little is known about the ability of M. tuberculosis to induce IL-4 in vivo, and even less is known about the induction and role of IL-42. Our data suggest that studying these factors will be of crucial importance for understanding the pathology of TB.

    ACKNOWLEDGMENTS

    We thank Ato Alemayehu Kifle, Sister Misrak Sisay, Sister Askale Maru, and Fekadu Lemma for excellent technical assistance. Hosanna Hospital, the Southern Regional Health Bureau, and the Ethiopian Science and Technology Commission are gratefully acknowledged for their assistance with this project.

    This work was supported by the European Commission through EU contract ICA4-CT-1999-10005.

    The VACSEL study group also includes Helen Fletcher, University College, London, United Kingdom (until 2003); Chifumbe Chintu, Gina Mulundu, and Peter Mwaba, University of Zambia School of Medicine, Lusaka, Zambia; and K. P. W. J. McAdam (until 2003), Patrick Owiafe, David Warndorff (2001), Christian Lienhardt (until 2001), R. Brookes, and Phillip Hill (from 2001), MRC, Gambia.

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