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Immune parameters differentiating active from latent tuberculosis infection in humans
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IMMUNOLOGICAL ASPECTS
Q2 Q1
Immune parameters differentiating active from latent tuberculosis infection in humans Ji Yeon Lee a, Young Won Jung b, Ina Jeong a, Joon-Sung Joh a, Soo Yeon Sim c, Boram Choi c, Hyeon-Gun Jee c, Dong-Gyun Lim c, * a b c
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, National Medical Center, Seoul 100-799, Republic of Korea Jung-gu Community Health Center, Seoul 132-713, Republic of Korea Center for Chronic Diseases, Research Institute, National Medical Center, Seoul 100-799, Republic of Korea
a r t i c l e i n f o
s u m m a r y
Article history: Received 18 April 2015 Received in revised form 17 July 2015 Accepted 4 August 2015
Tuberculosis remains a highly prevalent infectious disease worldwide. Identification of the immune parameters that differentiate active disease from latent infection will facilitate the development of efficient control measures as well as new diagnostic modalities for tuberculosis. Here, we investigated the cytokine production profiles of monocytes and CD4þ T lymphocytes upon encountering mycobacterial antigens. In addition, cytokines and lipid mediators with immune-modulating activities were examined in plasma samples ex vivo. Comparison of these parameters in active tuberculosis patients and healthy subjects with latent infection revealed that, active tuberculosis was associated with diminished Th1-type cytokine secretion from CD4þ T cells and less augmented inflammatory cytokine secretion from monocytes induced by IFN-g than that in latent tuberculosis infection. In addition, a higher plasma concentration of lipoxin A4 and lower ratio of prostaglandin E2 to lipoxin A4 were observed in active cases than in latent infections. These findings have implications for preparing new therapeutic strategies and for differential diagnosis of the two types of tuberculosis infection. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Tuberculosis Cytokine Eicosanoids CD4þ T cells Monocytes
1. Introduction Successful control of infectious diseases requires effective diagnostic methods, therapeutic regimens, and preventive vaccines. Unfortunately, none of these requirements have been fulfilled for tuberculosis (TB) yet, allowing TB to remain a major cause of mortality and morbidity worldwide. A clear understanding of the mechanisms of pathogenic and protective immune responses against Mycobacterium tuberculosis (Mtb) infection will need to precede new measures to control this infectious disease. Previous animal studies have shown that the Mtb bacilli invade via the respiratory tract and are taken up by phagocytic cells, particularly alveolar macrophages [1]. Depending on their functional status, macrophages may efficiently restrict and/or kill the Abbreviations: ATB, active tuberculosis; LTBI, latent tuberculosis infection; Mtb, Mycobacterium tuberculosis; TB, tuberculosis; MTSA, Mycobacterium tuberculosis soluble antigens; PGE2, prostaglandin E2; LXA4, lipoxin A4. * Corresponding author. Center for Chronic Diseases, Research Institute, National Medical Center, 245, Euljiro, Jung-gu, Seoul 100-799, Republic of Korea. Tel.: þ82 2 2276 2300; fax: þ82 2 2276 2319. E-mail address: [email protected] (D.-G. Lim).
pathogen, and recruit innate immune cells from circulation and activate them via secretion of chemokines and cytokines [2]. Thus, at an early stage of Mtb infection, macrophages play a central role in determining the outcome of the infection. After delayed priming by dendritic cells, CD4þ T lymphocytes are known to actively participate in the immune response to Mtb infection, by secreting a diverse spectrum of cytokines [3]. IFN-g is considered to play a major role in protective immunity by promoting the antimicrobial activity of macrophages/monocytes [4], whereas IL-4, IL-10, and TGF-b counter-regulate the protective immunity [2]. This is illustrated well by the observation that C57BL/6 mice, whose T cells preferentially produce IFN-g in response to mycobacterial antigens, are resistant to mycobacterial infection, whereas Balb/c mice, whose T cells highly produce IL-4, do not demonstrate protective immunity after BCG vaccination [5]. In addition to cytokines, a class of lipid mediators with immune-modulatory activity has been recently shown to play an important role in determining the outcome of Mtb infection. Mice deficient in prostaglandin E2 (PGE2) synthase show greater susceptibility to Mtb infection, whereas mice deficient in 5-lipoxygenase (an enzyme generating lipoxin A4; LXA4) are more resistant [6,7].
http://dx.doi.org/10.1016/j.tube.2015.08.003 1472-9792/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lee JY, et al., Immune parameters differentiating active from latent tuberculosis infection in humans, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.08.003
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In humans, only a few immune components have been shown to be important for the control of tuberculosis. The increased rate of primary TB in patients with a congenital defect in the IFN-g signaling pathway, and the reactivation of active TB (ATB) from latent TB infection (LTBI) in subjects who were receiving anti-TNF-a therapy illustrate well the critical roles of IFN-g and TNF-a in the control of TB [8e10]. Moreover, the high prevalence of TB in AIDS patients suggests the importance of CD4þ T cells in preventing disease progression [11]. In addition to these clinical observations, many ex vivo studies have been performed to explore the relation between immune responses and protection and pathogenesis in human tuberculosis [12,13]. Nevertheless, little is known about the host factors that determine why some individuals are protected while others go on to develop disease. Here, we obtained information on the immunologic correlates of protection and pathogenesis in Mtb infection. On the assumption that a protective immune response is at work in subjects with LTBI and that a pathogenic immune response is operative in ATB patients, we investigated whether there were differences in the cytokine production profiles of monocytes and CD4þ T cells that allow a distinction between LTBI and ATB. In addition, we also compared plasma concentrations of cytokines and eicosanoids between LTBI and ATB cases to determine their value as differential biomarkers. 2. Materials and methods 2.1. Study subjects Twenty-four patients with active pulmonary TB and 25 healthy subjects with LTBI were recruited from the National Medical Center and the Jung-gu Community Health Center (Seoul, Korea). Pulmonary TB was diagnosed by positive sputum smears for acid-fast bacilli and/or Mtb positive cultures, and chest radiography. Blood samples were obtained before or within the first 2 weeks of anti-TB medication. Healthy subjects with LTBI were defined by a positive tuberculin skin test (15 subjects) or positive interferon-g release assay (10 subjects) in the absence of clinical symptoms and chest radiographic abnormalities. All participants in this study were negative for HIV infection. The average age was 48 years (range 28e75) for the ATB group and 48 years (range 23e59) for the LTBI group. The gender composition was 15 males and 9 females for the ATB group, and 14 males and 11 females for the LTBI group. All participants provided written informed consent and protocols were approved by the Ethics Committee of National Medical Center (IRB no. H-1208/022-002; 23 August 2012).
purified by negative selection using a magnetic activated cell sorting (MACS) CD4þ T cell isolation kit (Miltenyi Biotec). Cells were cultured in RPMI 1640 medium, supplemented with 2 mM glutamine, 2 mM non-essential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, penicillin (50 U/mL), streptomycin (50 mg/mL) (medium and culture supplements from Invitrogen, Grand Island, NY), and 10% (v/v) human serum (Biowest, Nuaille, France). Monocytes were cultured in the presence of either IFN-g (20 ng/ mL; R&D Systems, Minneapolis, MN) or MTSA (10 mg/mL) alone, or both. After 20e24 h of culture, the culture supernatants were harvested and stored at 80 C. For antigenic stimulation of CD4þ T cells, monocytes were pre-pulsed for 3 h with either ESAT-6/CFP10 (1 mg/mL of each) or PPD (10 mg/mL) and then cocultured with CD4þ T cells at a cell ratio of 1:4. In some cultures, anti-CD3/antiCD28 monoclonal antibodies (mAbs) (1 mg/mL of each; both from BD Biosciences, San Diego, CA) were added to the coculture without antigen-pulsing of monocytes. After 5 days of incubation, culture supernatants were collected, stored at 80 C, and used for cytokine measurements. 2.4. Cytokine and eicosanoid measurements The concentrations of all cytokines except plasma IL-1b were quantified in culture supernatants or plasma using Bio-Plex Multiplex Immunoassay Systems (Bio-Rad Laboratories, Hercules, CA), according to the manufacturer's instructions. Plasma IL-1b was measured using a commercial ELISA kit (R&D Systems). Plasma levels of PGE2 (Cayman Chemical, Ann Arbor, MI) and LXA4 (Oxford Biomedical Research, Oxford, MI) were assessed using EIA kits following the manufacturers' instructions. 2.5. Statistical analysis Statistical analysis was performed using Prism V5.04 software (GraphPad, San Diego, USA). The nonparametric ManneWhitney U test was used to compare LTBI and ATB groups. The diagnostic values of analytes were assessed using a receiver operating characteristic (ROC) analysis that defined the sensitivity and specificity of the diagnostic approach. Correlation analysis was carried out using the Spearman test. Differences were considered statistically significant at P 0.05. 3. Results 3.1. Cytokine production ability of monocytes
2.2. Mycobacterial antigens M. tuberculosis H37Rv soluble antigens (MTSA), prepared as described previously [14], recombinant early secreted antigenic target 6 (ESAT-6), and culture filtrate protein 10 (CFP-10) were provided by Dr. Sang-Nae Cho (Yonsei University, Seoul, Korea). PPD (RT50) was obtained from Statens Serum Institute (Copenhagen, Denmark). 2.3. Isolation and in vitro culture of cells Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by density gradient centrifugation using FicollePaque solution (GE Healthcare, Uppsala, Sweden). CD14þ monocytes were purified by positive selection using CD14 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's instructions. From the remaining cells after isolation of CD14þ monocytes, CD4þ T cells were
Monocytes/macrophages play a pivotal role in the first line of defense against Mtb infection [15]. We examined if there was any difference in the cytokine-producing ability of monocytes between ATB patients and LTBI subjects. In response to MTSA, monocytes from LTBI subjects produced a higher amount of IL-10 but similar amounts of IL-1, IL-6, and TNF-a, than that in the ATB patients. Upon stimulation with MTSA in the presence of IFN-g, representing T cell help, higher amounts of IL-1, IL-6, and IL-10 production were observed in monocytes from LTBI than that from ATB (Figure 1). These data indicate that, when encountering Mtb components, the inflammatory cytokine-producing ability of monocytes could not be fully augmented by IFN-g in ATB compared to LTBI, while the IL10-producing ability is decreased regardless of the presence of IFNg. However, due to the considerable overlap in individual values of cytokine production from monocytes between these two groups, it was not possible to assign the patients' clinical status correctly using these parameters.
Please cite this article in press as: Lee JY, et al., Immune parameters differentiating active from latent tuberculosis infection in humans, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.08.003
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Figure 1. Cytokine production from monocytes. CD14þ monocytes were stimulated with MTSA in the presence or absence of IFN-g for 20e24 h, and the amount of secreted cytokines was measured from culture supernatants. Each dot represents the values obtained from individual subjects (n ¼ 23 for each group), and horizontal bars indicate the average values. Groups were compared using the ManneWhitney U test. *P < 0.05, **P < 0.005, ***P < 0.0005.
3.2. Cytokine secretion from coculture of CD4þ T cells and monocytes Next, we investigated the production of 6 different cytokines from CD4þ T cells, another central player in protective immunity against Mtb infection, in response to mycobacterial antigens. When CD4þ T cells were cocultured with autologous monocytes prepulsed with Mtb-specific antigens (ESAT-6 & CFP-10), they produced mainly Th1-type of cytokines, regardless if they were from ATB or LTBI. However, the amount of secreted IFN-g and TNF-a was not significantly different between LTBI subjects and ATB patients (Figure 2). When stimulated with PPD, CD4þ T cells cocultured with monocytes showed a similar pattern of secretion, but with enhanced cytokine production compared to the stimulation with ESAT-6 & CFP-10. TNF-a production was significantly higher in LTBI than that in ATB upon stimulation with PPD. The secretion of all other cytokines, IL-17, GM-CSF, IL-4 and IL-10, was not different between LTBI and ATB groups in response to either ESAT-6 & CFP-10 or PPD.
3.3. Plasma levels of cytokines and eicosanoids We further examined the cytokine levels in plasma samples. In contrast to the differential cellular production of cytokines upon antigenic stimulation, there was no difference in IL-1, TNF-a, IFN-g, and IL-10 levels in plasma samples between the ATB and LTBI groups (Figure 3). In addition to cytokines, small lipid mediators like eicosanoids are known to participate in host defense against Mtb infection as immunomodulators [6,7]. When we measured the plasma concentrations of LXA4 and PGE2, we found that the levels of both eicosanoids, especially LXA4, were significantly elevated in ATB compared to LTBI (Figure 4A). Based on the previous report that
PGE2 contributes to protection while LXA4 is involved in pathogenesis in TB [6], our PGE2 finding was unexpected. It is of note that the elevation in LXA4 levels was closely associated with a parallel increase in PGE2 levels in an individual patient (Figure 4B). Thus, when we analyzed the ratio of PGE2 to LXA4, it was revealed that the PGE2/LXA4 ratio was significantly lower in ATB patients compared with LTBI subjects (Figure 4C). The ROC curve analysis demonstrated that the plasma level of LXA4 or the PGE2/LXA4 ratio could efficiently differentiate ATB from LTBI (AUC: 0.9267 and 0.9292, respectively) (Figure 4D).
4. Discussion Understanding the immune responses that underlie the protection from or progression of disease is important to developing protocols for the efficient management and prevention of TB. Our comparative study between LTBI and ATB revealed that ATB was associated with the decreased inflammatory cytokine-producing ability of IFN-g-primed monocytes and the reduced Th1 type cytokine-production of CD4þ T cells in response to mycobacterial antigens. Furthermore, a high plasma concentration of LXA4 was closely linked to ATB, while an increased PGE2/LXA4 ratio was linked to LTBI. These findings could have implications for the preparation of new therapeutic modalities and the differential diagnosis of the two types of TB infection. Because monocytes/macrophages and CD4þ T cells were previously revealed to be major effector cells in protecting the host against Mtb infection [15], we investigated whether there were any functional differences in these cells between LTBI and ATB cases by measuring their cytokine secretion. Our data suggested that disease progression after infection with Mtb is associated with some defect in receiving T cell help, but not in the intrinsic ability of monocytes
Please cite this article in press as: Lee JY, et al., Immune parameters differentiating active from latent tuberculosis infection in humans, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.08.003
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Figure 2. Cytokine production from coculture of CD4þ T cells and monocytes. CD4þ T cells were cocultured with autologous monocytes pre-pulsed with mycobacterial antigens or anti-CD3/anti-CD28 mAbs for 5 days, and the amount of secreted cytokines was measured from culture supernatants. Each dot represents the values obtained from individual subjects (n ¼ 23 for each group), and horizontal bars indicate the average values. Groups were compared using the ManneWhitney U test. *P < 0.05.
to secret inflammatory cytokines. The defect in augmenting cellular function by IFN-g has been previously reported in macrophages from patients with ATB, in terms of the restriction of Mtb replication [16]. Nevertheless, it is still possible that this defect might just reflect the refractory phase of monocytes, which occurred from prior in vivo activation in ATB patients. In terms of the differential production of IL-10 from monocytes of LTBI subjects and ATB patients, conflicting results have been reported by other investigators using distinct experimental procedures [17,18]. In our specific experimental model, contrary to our expectation, the secretion of the immunoregulatory cytokine IL-10 from monocytes was lower in
Figure 3. Plasma levels of cytokines. Each dot represents the value obtained from an individual subject (n ¼ 20 for each group), and horizontal bars indicate the average values.
ATB than in LTBI, regardless of the presence of T cell help. This could be a consequence of the host reaction to prevent collateral tissue damage by a large amount of inflammatory cytokines in LTBI [19]. IFN-g and TNF-a are known to engage in protective immunity against Mtb infection by activating effector mechanisms of monocytes/macrophage, and by maintaining the integrity of granulomas [20,21]. Moreover, previous comparison studies indicated that the production of these two cytokine, especially IFN-g, from mycobacterial antigen-stimulated PBMCs or whole T cells was increased in LTBI subjects compared with ATB patients [12,17,22e24]. Our study with cocultures of CD4þ T cells and monocytes also showed a similar result even if the difference in TNF-a and IFN-g production between the two groups is distinctive and marginal, respectively. While IFN-g is produced by T lymphocytes but not by monocytes, TNF-a is produced by both cell types. According to our study, the compromised TNF-a production in ATB observed in our coculture study was likely to be from CD4þ T cells but not from monocytes, since monocyte TNF-a secretion was similar in LTBI and ATB patients regardless of IFN-g presence. Given that TNF-a, as well as IFN-g, is produced by the Th1 subset of the CD4þ T cell population and that the secretion of both cytokines was somewhat diminished in ATB, functional defects of CD4þ T cells may exist in the Th1 subset of CD4þ T cells in ATB. As previously reported [22,24], other subsets of CD4þ T cells, like Th2 and Th17, seemed to be intact in ATB, which was supported by our finding that there was no difference in IL-4 and IL-17 secretion between ATB and LTBI. Nonetheless, it was not possible to differentiate ATB from LTBI by quantifying secreted levels of these cytokines from CD4þ T cells cocultured with monocytes, because the cytokine levels between these two groups on an individual basis were largely overlapping. This overlap might come from the following possibilities: i) the level of cytokine production of CD4þ T cells and monocytes is not the sole factor that determines the clinical outcome of mycobacterial infection, ii) or alternatively, a valuable parameter is confounded by irrelevant ones in a simple measurement of secreted cytokines. According to the latter possibility, the cytokine-producing profile of individual
Please cite this article in press as: Lee JY, et al., Immune parameters differentiating active from latent tuberculosis infection in humans, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.08.003
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Figure 4. Plasma levels of PGE2 and LXA4. (A) Each dot represents the value obtained from an individual subject (n ¼ 25 and 24 for LTBI and ATB groups, respectively), and horizontal bars indicate the average values. (B) Correlation between the plasma levels of LXA4 and PGE2 across LTBI and ATB patients is shown. Spearman's rank correlation coefficient, the corresponding P value, and the line of best fit are shown. (C) The plasma PGE2/LXA4 ratio was higher in LTBI subjects than that in ATB patients. (D) Receiver Operating Characteristic (ROC) curve analysis was used to evaluate the differentiating capacity of the plasma LXA4 level or PGE2/LXA4 ratio between the LTBI and ATB groups. AUC, area under the curve.
CD4þ T cells rather than the amount of secreted cytokines from a whole population of cells could be an immunological measure for differential diagnosis of ATB and LTBI. Recent studies have actually shown that increase in the percentage of Mtb-specific CD4þ TNF-aonly-secreting T cells, especially with effector phenotype, was associated with ATB, thus making this parameter a promising biomarker to distinguish ATB from LTBI [25,26]. Serum or plasma is the most convenient sample for clinical diagnosis. Although a differential secretion in cytokines was detected in culture supernatants of stimulated cells between ATB and LTBI, similar findings were not reproduced in plasma samples in our study. The involvement of dilution and/or noise factors in plasma likely contributed to this discordance. So far, none of the cytokines or chemokines except IP-10 has been identified as a promising biomarker to discriminate ATB from LTBI in unmanipulated plasma samples [27e29]. Recent animal studies have indicated that the balance between LXA4 and PGE2 plays a major role in determining the outcome of Mtb infection. While PGE2 contributes to the control of Mtb infection by inducing apoptosis of infected macrophages with the subsequent inhibition of infected Mtb replication, LXA4 is associated with disease susceptibility by inducing necrosis of macrophages and by negatively regulating the protective Th1 responses against mycobacterial infection [6,7]. In line with the known negative role of LXA4 in TB, most ATB patients contained higher concentrations of LXA4 in their plasma than the LTBI subjects. However, in contrast to our expectations but similar to recent results obtained by other investigators [30,31], the increased PGE2 concentrations were also observed in ATB patients compared with LTBI subjects. Considering that both PGE2 and LXA4 are generated from lipid metabolism of the plasma membrane, the rate of which is greatly influenced by the level of inflammation [32,33], the high PGE2 in ATB might be due to the strong inflammatory milieu elicited by actively replicating Mtb. This possibility is supported by our additional finding that plasma concentrations of both types of eicosanoids in ATB patients showed a positive correlation with one
another. In spite of the lower levels of PGE2 in LTBI than in ATB, the ratio of PGE2 to LXA4 was higher in LTBI than in ATB, as previously reported [31]. Thus, disease activity in tuberculosis is closely associated with the ratio of PGE2 to LXA4, but not with the absolute concentration of plasma PGE2. For application, the ROC curve analysis indicated that the ratio of PGE2 to LXA4, as well as the plasma level of LXA4 alone, could be used as promising diagnostic biomarkers to differentiate LTBI from ATB. Further studies are required to verify the feasibility of their use in large populations. In conclusion, we observed Th1-type cytokine secretion from CD4þ T cells and IFN-ginduced augmentation of inflammatory cytokine secretion from monocytes was diminished in ATB. Moreover, as the plasma LXA4 concentration and PGE2/LXA4 ratio are substantially different between the LTBI and ATB groups, these parameters have potential value as diagnostic biomarkers in differentiating LTBI from ATB. Acknowledgments We thank Dr. Sang-Nae Cho (Yonsei University, Seoul, Korea) for providing mycobacterial antigens. Funding: This study was supported by Medical Science Research Program funded by National Medical Center Research Institute [NMC2012-MS-07]. Competing interests: interest.
The authors have no conflicts of
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[19] Higgins DM, Sanchez-Campillo J, Rosas-Taraco AG, Lee EJ, Orme IM, GonzalezJuarrero M. Lack of IL-10 alters inflammatory and immune responses during pulmonary Mycobacterium tuberculosis infection. Tuberculosis 2009;89: 149e57. [20] Flynn JL, Chan J, Triebold KJ, Dalton DK, Stewart TA, Bloom BR. An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection. J Exp Med 1993;178:2249e54. [21] Kindler V, Sappino AP, Grau GE, Piguet PF, Vassalli P. The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection. Cell 1989;56:731e40. [22] Zhang M, Lin Y, Iyer DV, Gong J, Abrams JS, Barnes PF. T-cell cytokine responses in human infection with Mycobacterium tuberculosis. Infect Immun 1995;63:3231e4. [23] Riano F, Arroyo L, paris S, Rojas M, Friggen AH, van Meijgaarden KE, et al. T cell responses to DosR and Rpf proteins in actively and latently infected individuals from Colombia. Tuberculosis 2012;92:148e59. [24] Marin ND, Paris SC, Rojas M, Garcia LF. Functional profile of CD4þ and CD8þ T cells in latently infected individuals and patients with active TB. Tuberculosis 2013;93:155e66. [25] Harari A, Rozot V, Bellutti Enders F, Perreau M, Stalder JM, Nicod LP, et al. Dominant TNF-aþ Mycobacterium tuberculosis-specific CD4þ T cell responses discriminate between latent infection and active disease. Nat Med 2011;17: 372e6. [26] Pollock KM, Whitworth HS, Montamat-Sicotte DJ, Grass L, Cooke GS, Kapembwa MS, et al. T-cell immunophenotyping distinguishes active from latent tuberculosis. J Infect Dis 2013;208:952e68. [27] Djoba Siawaya JF, Chegou NN, van den Heuvel MM, Diacon AH, Beyers N, van Helden P, et al. Differential cytokine/chemokines and KL-6 profiles in patients with different forms of tuberculosis. Cytokine 2009;47(2):132e6. [28] Yu Y, Zhang Y, Hu S, Jin D, Chen X, Jin Q, et al. Different patterns of cytokines and chemokines combined with IFN-gamma production reflect Mycobacterium tuberculosis infection and disease. PLoS One 2012;7:e44944. [29] Mihret A, Bekele Y, Bobosha K, Kidd M, Aseffa A, Howe R, et al. Plasma cytokines and chemokines differentiate between active disease and non-active tuberculosis infection. J Infect 2013;66:357e65. [30] Shu CC, Wu MF, Hsu CL, Huang CT, Wang JY, Hsieh SL, et al. Apoptosis-associated biomarkers in tuberculosis: promising for diagnosis and prognosis prediction. BMC Infect Dis 2013;13:45. [31] Mayer-Barber KD, Andrade BB, Oland SD, Amaral EP, Barber DL, Gonzales J, et al. Host-directed therapy of tuberculosis based on interleukin-1 and type I interferon crosstalk. Nature 2014;511:99e103. [32] Levy BD, Clish CB, Schmidt B, Gronert K, Serhan CN. Lipid mediator class switching during acute inflammation: signals in resolution. Nat Immunol 2001;2:612e9. [33] Chan MM, Moore AR. Resolution of inflammation in murine autoimmune arthritis is disrupted by cyclooxygenase-2 inhibition and restored by prostaglandin E2-mediated lipoxin A4 production. J Immunol 2010;184:6418e26.
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Contents lists available at ScienceDirect
Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube
IMMUNOLOGICAL ASPECTS
Q2 Q1
Immune parameters differentiating active from latent tuberculosis infection in humans Ji Yeon Lee a, Young Won Jung b, Ina Jeong a, Joon-Sung Joh a, Soo Yeon Sim c, Boram Choi c, Hyeon-Gun Jee c, Dong-Gyun Lim c, * a b c
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, National Medical Center, Seoul 100-799, Republic of Korea Jung-gu Community Health Center, Seoul 132-713, Republic of Korea Center for Chronic Diseases, Research Institute, National Medical Center, Seoul 100-799, Republic of Korea
a r t i c l e i n f o
s u m m a r y
Article history: Received 18 April 2015 Received in revised form 17 July 2015 Accepted 4 August 2015
Tuberculosis remains a highly prevalent infectious disease worldwide. Identification of the immune parameters that differentiate active disease from latent infection will facilitate the development of efficient control measures as well as new diagnostic modalities for tuberculosis. Here, we investigated the cytokine production profiles of monocytes and CD4þ T lymphocytes upon encountering mycobacterial antigens. In addition, cytokines and lipid mediators with immune-modulating activities were examined in plasma samples ex vivo. Comparison of these parameters in active tuberculosis patients and healthy subjects with latent infection revealed that, active tuberculosis was associated with diminished Th1-type cytokine secretion from CD4þ T cells and less augmented inflammatory cytokine secretion from monocytes induced by IFN-g than that in latent tuberculosis infection. In addition, a higher plasma concentration of lipoxin A4 and lower ratio of prostaglandin E2 to lipoxin A4 were observed in active cases than in latent infections. These findings have implications for preparing new therapeutic strategies and for differential diagnosis of the two types of tuberculosis infection. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Tuberculosis Cytokine Eicosanoids CD4þ T cells Monocytes
1. Introduction Successful control of infectious diseases requires effective diagnostic methods, therapeutic regimens, and preventive vaccines. Unfortunately, none of these requirements have been fulfilled for tuberculosis (TB) yet, allowing TB to remain a major cause of mortality and morbidity worldwide. A clear understanding of the mechanisms of pathogenic and protective immune responses against Mycobacterium tuberculosis (Mtb) infection will need to precede new measures to control this infectious disease. Previous animal studies have shown that the Mtb bacilli invade via the respiratory tract and are taken up by phagocytic cells, particularly alveolar macrophages [1]. Depending on their functional status, macrophages may efficiently restrict and/or kill the Abbreviations: ATB, active tuberculosis; LTBI, latent tuberculosis infection; Mtb, Mycobacterium tuberculosis; TB, tuberculosis; MTSA, Mycobacterium tuberculosis soluble antigens; PGE2, prostaglandin E2; LXA4, lipoxin A4. * Corresponding author. Center for Chronic Diseases, Research Institute, National Medical Center, 245, Euljiro, Jung-gu, Seoul 100-799, Republic of Korea. Tel.: þ82 2 2276 2300; fax: þ82 2 2276 2319. E-mail address: [email protected] (D.-G. Lim).
pathogen, and recruit innate immune cells from circulation and activate them via secretion of chemokines and cytokines [2]. Thus, at an early stage of Mtb infection, macrophages play a central role in determining the outcome of the infection. After delayed priming by dendritic cells, CD4þ T lymphocytes are known to actively participate in the immune response to Mtb infection, by secreting a diverse spectrum of cytokines [3]. IFN-g is considered to play a major role in protective immunity by promoting the antimicrobial activity of macrophages/monocytes [4], whereas IL-4, IL-10, and TGF-b counter-regulate the protective immunity [2]. This is illustrated well by the observation that C57BL/6 mice, whose T cells preferentially produce IFN-g in response to mycobacterial antigens, are resistant to mycobacterial infection, whereas Balb/c mice, whose T cells highly produce IL-4, do not demonstrate protective immunity after BCG vaccination [5]. In addition to cytokines, a class of lipid mediators with immune-modulatory activity has been recently shown to play an important role in determining the outcome of Mtb infection. Mice deficient in prostaglandin E2 (PGE2) synthase show greater susceptibility to Mtb infection, whereas mice deficient in 5-lipoxygenase (an enzyme generating lipoxin A4; LXA4) are more resistant [6,7].
http://dx.doi.org/10.1016/j.tube.2015.08.003 1472-9792/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lee JY, et al., Immune parameters differentiating active from latent tuberculosis infection in humans, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.08.003
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In humans, only a few immune components have been shown to be important for the control of tuberculosis. The increased rate of primary TB in patients with a congenital defect in the IFN-g signaling pathway, and the reactivation of active TB (ATB) from latent TB infection (LTBI) in subjects who were receiving anti-TNF-a therapy illustrate well the critical roles of IFN-g and TNF-a in the control of TB [8e10]. Moreover, the high prevalence of TB in AIDS patients suggests the importance of CD4þ T cells in preventing disease progression [11]. In addition to these clinical observations, many ex vivo studies have been performed to explore the relation between immune responses and protection and pathogenesis in human tuberculosis [12,13]. Nevertheless, little is known about the host factors that determine why some individuals are protected while others go on to develop disease. Here, we obtained information on the immunologic correlates of protection and pathogenesis in Mtb infection. On the assumption that a protective immune response is at work in subjects with LTBI and that a pathogenic immune response is operative in ATB patients, we investigated whether there were differences in the cytokine production profiles of monocytes and CD4þ T cells that allow a distinction between LTBI and ATB. In addition, we also compared plasma concentrations of cytokines and eicosanoids between LTBI and ATB cases to determine their value as differential biomarkers. 2. Materials and methods 2.1. Study subjects Twenty-four patients with active pulmonary TB and 25 healthy subjects with LTBI were recruited from the National Medical Center and the Jung-gu Community Health Center (Seoul, Korea). Pulmonary TB was diagnosed by positive sputum smears for acid-fast bacilli and/or Mtb positive cultures, and chest radiography. Blood samples were obtained before or within the first 2 weeks of anti-TB medication. Healthy subjects with LTBI were defined by a positive tuberculin skin test (15 subjects) or positive interferon-g release assay (10 subjects) in the absence of clinical symptoms and chest radiographic abnormalities. All participants in this study were negative for HIV infection. The average age was 48 years (range 28e75) for the ATB group and 48 years (range 23e59) for the LTBI group. The gender composition was 15 males and 9 females for the ATB group, and 14 males and 11 females for the LTBI group. All participants provided written informed consent and protocols were approved by the Ethics Committee of National Medical Center (IRB no. H-1208/022-002; 23 August 2012).
purified by negative selection using a magnetic activated cell sorting (MACS) CD4þ T cell isolation kit (Miltenyi Biotec). Cells were cultured in RPMI 1640 medium, supplemented with 2 mM glutamine, 2 mM non-essential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, penicillin (50 U/mL), streptomycin (50 mg/mL) (medium and culture supplements from Invitrogen, Grand Island, NY), and 10% (v/v) human serum (Biowest, Nuaille, France). Monocytes were cultured in the presence of either IFN-g (20 ng/ mL; R&D Systems, Minneapolis, MN) or MTSA (10 mg/mL) alone, or both. After 20e24 h of culture, the culture supernatants were harvested and stored at 80 C. For antigenic stimulation of CD4þ T cells, monocytes were pre-pulsed for 3 h with either ESAT-6/CFP10 (1 mg/mL of each) or PPD (10 mg/mL) and then cocultured with CD4þ T cells at a cell ratio of 1:4. In some cultures, anti-CD3/antiCD28 monoclonal antibodies (mAbs) (1 mg/mL of each; both from BD Biosciences, San Diego, CA) were added to the coculture without antigen-pulsing of monocytes. After 5 days of incubation, culture supernatants were collected, stored at 80 C, and used for cytokine measurements. 2.4. Cytokine and eicosanoid measurements The concentrations of all cytokines except plasma IL-1b were quantified in culture supernatants or plasma using Bio-Plex Multiplex Immunoassay Systems (Bio-Rad Laboratories, Hercules, CA), according to the manufacturer's instructions. Plasma IL-1b was measured using a commercial ELISA kit (R&D Systems). Plasma levels of PGE2 (Cayman Chemical, Ann Arbor, MI) and LXA4 (Oxford Biomedical Research, Oxford, MI) were assessed using EIA kits following the manufacturers' instructions. 2.5. Statistical analysis Statistical analysis was performed using Prism V5.04 software (GraphPad, San Diego, USA). The nonparametric ManneWhitney U test was used to compare LTBI and ATB groups. The diagnostic values of analytes were assessed using a receiver operating characteristic (ROC) analysis that defined the sensitivity and specificity of the diagnostic approach. Correlation analysis was carried out using the Spearman test. Differences were considered statistically significant at P 0.05. 3. Results 3.1. Cytokine production ability of monocytes
2.2. Mycobacterial antigens M. tuberculosis H37Rv soluble antigens (MTSA), prepared as described previously [14], recombinant early secreted antigenic target 6 (ESAT-6), and culture filtrate protein 10 (CFP-10) were provided by Dr. Sang-Nae Cho (Yonsei University, Seoul, Korea). PPD (RT50) was obtained from Statens Serum Institute (Copenhagen, Denmark). 2.3. Isolation and in vitro culture of cells Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by density gradient centrifugation using FicollePaque solution (GE Healthcare, Uppsala, Sweden). CD14þ monocytes were purified by positive selection using CD14 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's instructions. From the remaining cells after isolation of CD14þ monocytes, CD4þ T cells were
Monocytes/macrophages play a pivotal role in the first line of defense against Mtb infection [15]. We examined if there was any difference in the cytokine-producing ability of monocytes between ATB patients and LTBI subjects. In response to MTSA, monocytes from LTBI subjects produced a higher amount of IL-10 but similar amounts of IL-1, IL-6, and TNF-a, than that in the ATB patients. Upon stimulation with MTSA in the presence of IFN-g, representing T cell help, higher amounts of IL-1, IL-6, and IL-10 production were observed in monocytes from LTBI than that from ATB (Figure 1). These data indicate that, when encountering Mtb components, the inflammatory cytokine-producing ability of monocytes could not be fully augmented by IFN-g in ATB compared to LTBI, while the IL10-producing ability is decreased regardless of the presence of IFNg. However, due to the considerable overlap in individual values of cytokine production from monocytes between these two groups, it was not possible to assign the patients' clinical status correctly using these parameters.
Please cite this article in press as: Lee JY, et al., Immune parameters differentiating active from latent tuberculosis infection in humans, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.08.003
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Figure 1. Cytokine production from monocytes. CD14þ monocytes were stimulated with MTSA in the presence or absence of IFN-g for 20e24 h, and the amount of secreted cytokines was measured from culture supernatants. Each dot represents the values obtained from individual subjects (n ¼ 23 for each group), and horizontal bars indicate the average values. Groups were compared using the ManneWhitney U test. *P < 0.05, **P < 0.005, ***P < 0.0005.
3.2. Cytokine secretion from coculture of CD4þ T cells and monocytes Next, we investigated the production of 6 different cytokines from CD4þ T cells, another central player in protective immunity against Mtb infection, in response to mycobacterial antigens. When CD4þ T cells were cocultured with autologous monocytes prepulsed with Mtb-specific antigens (ESAT-6 & CFP-10), they produced mainly Th1-type of cytokines, regardless if they were from ATB or LTBI. However, the amount of secreted IFN-g and TNF-a was not significantly different between LTBI subjects and ATB patients (Figure 2). When stimulated with PPD, CD4þ T cells cocultured with monocytes showed a similar pattern of secretion, but with enhanced cytokine production compared to the stimulation with ESAT-6 & CFP-10. TNF-a production was significantly higher in LTBI than that in ATB upon stimulation with PPD. The secretion of all other cytokines, IL-17, GM-CSF, IL-4 and IL-10, was not different between LTBI and ATB groups in response to either ESAT-6 & CFP-10 or PPD.
3.3. Plasma levels of cytokines and eicosanoids We further examined the cytokine levels in plasma samples. In contrast to the differential cellular production of cytokines upon antigenic stimulation, there was no difference in IL-1, TNF-a, IFN-g, and IL-10 levels in plasma samples between the ATB and LTBI groups (Figure 3). In addition to cytokines, small lipid mediators like eicosanoids are known to participate in host defense against Mtb infection as immunomodulators [6,7]. When we measured the plasma concentrations of LXA4 and PGE2, we found that the levels of both eicosanoids, especially LXA4, were significantly elevated in ATB compared to LTBI (Figure 4A). Based on the previous report that
PGE2 contributes to protection while LXA4 is involved in pathogenesis in TB [6], our PGE2 finding was unexpected. It is of note that the elevation in LXA4 levels was closely associated with a parallel increase in PGE2 levels in an individual patient (Figure 4B). Thus, when we analyzed the ratio of PGE2 to LXA4, it was revealed that the PGE2/LXA4 ratio was significantly lower in ATB patients compared with LTBI subjects (Figure 4C). The ROC curve analysis demonstrated that the plasma level of LXA4 or the PGE2/LXA4 ratio could efficiently differentiate ATB from LTBI (AUC: 0.9267 and 0.9292, respectively) (Figure 4D).
4. Discussion Understanding the immune responses that underlie the protection from or progression of disease is important to developing protocols for the efficient management and prevention of TB. Our comparative study between LTBI and ATB revealed that ATB was associated with the decreased inflammatory cytokine-producing ability of IFN-g-primed monocytes and the reduced Th1 type cytokine-production of CD4þ T cells in response to mycobacterial antigens. Furthermore, a high plasma concentration of LXA4 was closely linked to ATB, while an increased PGE2/LXA4 ratio was linked to LTBI. These findings could have implications for the preparation of new therapeutic modalities and the differential diagnosis of the two types of TB infection. Because monocytes/macrophages and CD4þ T cells were previously revealed to be major effector cells in protecting the host against Mtb infection [15], we investigated whether there were any functional differences in these cells between LTBI and ATB cases by measuring their cytokine secretion. Our data suggested that disease progression after infection with Mtb is associated with some defect in receiving T cell help, but not in the intrinsic ability of monocytes
Please cite this article in press as: Lee JY, et al., Immune parameters differentiating active from latent tuberculosis infection in humans, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.08.003
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Figure 2. Cytokine production from coculture of CD4þ T cells and monocytes. CD4þ T cells were cocultured with autologous monocytes pre-pulsed with mycobacterial antigens or anti-CD3/anti-CD28 mAbs for 5 days, and the amount of secreted cytokines was measured from culture supernatants. Each dot represents the values obtained from individual subjects (n ¼ 23 for each group), and horizontal bars indicate the average values. Groups were compared using the ManneWhitney U test. *P < 0.05.
to secret inflammatory cytokines. The defect in augmenting cellular function by IFN-g has been previously reported in macrophages from patients with ATB, in terms of the restriction of Mtb replication [16]. Nevertheless, it is still possible that this defect might just reflect the refractory phase of monocytes, which occurred from prior in vivo activation in ATB patients. In terms of the differential production of IL-10 from monocytes of LTBI subjects and ATB patients, conflicting results have been reported by other investigators using distinct experimental procedures [17,18]. In our specific experimental model, contrary to our expectation, the secretion of the immunoregulatory cytokine IL-10 from monocytes was lower in
Figure 3. Plasma levels of cytokines. Each dot represents the value obtained from an individual subject (n ¼ 20 for each group), and horizontal bars indicate the average values.
ATB than in LTBI, regardless of the presence of T cell help. This could be a consequence of the host reaction to prevent collateral tissue damage by a large amount of inflammatory cytokines in LTBI [19]. IFN-g and TNF-a are known to engage in protective immunity against Mtb infection by activating effector mechanisms of monocytes/macrophage, and by maintaining the integrity of granulomas [20,21]. Moreover, previous comparison studies indicated that the production of these two cytokine, especially IFN-g, from mycobacterial antigen-stimulated PBMCs or whole T cells was increased in LTBI subjects compared with ATB patients [12,17,22e24]. Our study with cocultures of CD4þ T cells and monocytes also showed a similar result even if the difference in TNF-a and IFN-g production between the two groups is distinctive and marginal, respectively. While IFN-g is produced by T lymphocytes but not by monocytes, TNF-a is produced by both cell types. According to our study, the compromised TNF-a production in ATB observed in our coculture study was likely to be from CD4þ T cells but not from monocytes, since monocyte TNF-a secretion was similar in LTBI and ATB patients regardless of IFN-g presence. Given that TNF-a, as well as IFN-g, is produced by the Th1 subset of the CD4þ T cell population and that the secretion of both cytokines was somewhat diminished in ATB, functional defects of CD4þ T cells may exist in the Th1 subset of CD4þ T cells in ATB. As previously reported [22,24], other subsets of CD4þ T cells, like Th2 and Th17, seemed to be intact in ATB, which was supported by our finding that there was no difference in IL-4 and IL-17 secretion between ATB and LTBI. Nonetheless, it was not possible to differentiate ATB from LTBI by quantifying secreted levels of these cytokines from CD4þ T cells cocultured with monocytes, because the cytokine levels between these two groups on an individual basis were largely overlapping. This overlap might come from the following possibilities: i) the level of cytokine production of CD4þ T cells and monocytes is not the sole factor that determines the clinical outcome of mycobacterial infection, ii) or alternatively, a valuable parameter is confounded by irrelevant ones in a simple measurement of secreted cytokines. According to the latter possibility, the cytokine-producing profile of individual
Please cite this article in press as: Lee JY, et al., Immune parameters differentiating active from latent tuberculosis infection in humans, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.08.003
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Figure 4. Plasma levels of PGE2 and LXA4. (A) Each dot represents the value obtained from an individual subject (n ¼ 25 and 24 for LTBI and ATB groups, respectively), and horizontal bars indicate the average values. (B) Correlation between the plasma levels of LXA4 and PGE2 across LTBI and ATB patients is shown. Spearman's rank correlation coefficient, the corresponding P value, and the line of best fit are shown. (C) The plasma PGE2/LXA4 ratio was higher in LTBI subjects than that in ATB patients. (D) Receiver Operating Characteristic (ROC) curve analysis was used to evaluate the differentiating capacity of the plasma LXA4 level or PGE2/LXA4 ratio between the LTBI and ATB groups. AUC, area under the curve.
CD4þ T cells rather than the amount of secreted cytokines from a whole population of cells could be an immunological measure for differential diagnosis of ATB and LTBI. Recent studies have actually shown that increase in the percentage of Mtb-specific CD4þ TNF-aonly-secreting T cells, especially with effector phenotype, was associated with ATB, thus making this parameter a promising biomarker to distinguish ATB from LTBI [25,26]. Serum or plasma is the most convenient sample for clinical diagnosis. Although a differential secretion in cytokines was detected in culture supernatants of stimulated cells between ATB and LTBI, similar findings were not reproduced in plasma samples in our study. The involvement of dilution and/or noise factors in plasma likely contributed to this discordance. So far, none of the cytokines or chemokines except IP-10 has been identified as a promising biomarker to discriminate ATB from LTBI in unmanipulated plasma samples [27e29]. Recent animal studies have indicated that the balance between LXA4 and PGE2 plays a major role in determining the outcome of Mtb infection. While PGE2 contributes to the control of Mtb infection by inducing apoptosis of infected macrophages with the subsequent inhibition of infected Mtb replication, LXA4 is associated with disease susceptibility by inducing necrosis of macrophages and by negatively regulating the protective Th1 responses against mycobacterial infection [6,7]. In line with the known negative role of LXA4 in TB, most ATB patients contained higher concentrations of LXA4 in their plasma than the LTBI subjects. However, in contrast to our expectations but similar to recent results obtained by other investigators [30,31], the increased PGE2 concentrations were also observed in ATB patients compared with LTBI subjects. Considering that both PGE2 and LXA4 are generated from lipid metabolism of the plasma membrane, the rate of which is greatly influenced by the level of inflammation [32,33], the high PGE2 in ATB might be due to the strong inflammatory milieu elicited by actively replicating Mtb. This possibility is supported by our additional finding that plasma concentrations of both types of eicosanoids in ATB patients showed a positive correlation with one
another. In spite of the lower levels of PGE2 in LTBI than in ATB, the ratio of PGE2 to LXA4 was higher in LTBI than in ATB, as previously reported [31]. Thus, disease activity in tuberculosis is closely associated with the ratio of PGE2 to LXA4, but not with the absolute concentration of plasma PGE2. For application, the ROC curve analysis indicated that the ratio of PGE2 to LXA4, as well as the plasma level of LXA4 alone, could be used as promising diagnostic biomarkers to differentiate LTBI from ATB. Further studies are required to verify the feasibility of their use in large populations. In conclusion, we observed Th1-type cytokine secretion from CD4þ T cells and IFN-ginduced augmentation of inflammatory cytokine secretion from monocytes was diminished in ATB. Moreover, as the plasma LXA4 concentration and PGE2/LXA4 ratio are substantially different between the LTBI and ATB groups, these parameters have potential value as diagnostic biomarkers in differentiating LTBI from ATB. Acknowledgments We thank Dr. Sang-Nae Cho (Yonsei University, Seoul, Korea) for providing mycobacterial antigens. Funding: This study was supported by Medical Science Research Program funded by National Medical Center Research Institute [NMC2012-MS-07]. Competing interests: interest.
The authors have no conflicts of
Ethical approval: This study was approved by the Ethics Committee of National Medical Center, Seoul, Korea. References [1] Schlesinger LS. Entry of Mycobacterium tuberculosis into mononuclear phagocytes. Curr Top Microbiol Immunol 1996;215:71e96.
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