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Increased intra- and extracellular granzyme expression in patients with tuberculosis
Tuberculosis xxx (2015) 1e6
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Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube
IMMUNOLOGICAL ASPECTS
Increased intra- and extracellular granzyme expression in patients with tuberculosis M. Isabel Garcia-Laorden a, b, *, Dana C. Blok a, b, Liesbeth M. Kager a, b, Arie J. Hoogendijk a, b, Gerard J. van Mierlo c, Ivar O. Lede a, d, Wahid Rahman e, Rumana Afroz e, Aniruddha Ghose e, Caroline E. Visser a, d, Abu Shahed Md Zahed e, Md Anwar Husain f, Khan Mashrequl Alam f, Pravat Chandra Barua g, Mahtabuddin Hassan e, Ahmed Hossain h, 1, Md Abu Tayab i, Nick Day j, Arjen M. Dondorp j, Alex F. de Vos a, b, Tom van der Poll a, b, k a
Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands b Center for Experimental and Molecular Medicine (CEMM), Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands c Department of Immunopathology, Sanquin Research, Plesmanlaan 125, 1066CX Amsterdam, The Netherlands d Department of Microbiology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands e Department of Internal Medicine, Chittagong Medical College & Hospital (CMCH), Chittagong, Bangladesh f Department of Microbiology, Chittagong Medical College & Hospital (CMCH), Chittagong, Bangladesh g National TB Control & Leprosy Elimination Program, Dhaka, Bangladesh h Chest Disease Clinic Chittagong (CDCC), Anderkilla, Chittagong, Bangladesh i Chittagong General Hospital, Arderkilla, Chittagong, Bangladesh j Mahidol Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, 3/F 60th Anniversary Chalermprakiat Building, 420/6 Rajvithi Road, 10400 Bangkok, Thailand k Division of Infectious Diseases, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
a r t i c l e i n f o
s u m m a r y
Article history: Received 4 March 2015 Accepted 31 May 2015
Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. Granzymes (gzms) are proteases mainly found in cytotoxic lymphocytes, but also extracellularly. While the role of gzms in target cell death has been widely characterized, considerable evidence points towards broader roles related to infectious and inflammatory responses. To investigate the expression of the gzms in TB, intracellular gzms A, B and K were measured by flow cytometry in lymphocyte populations from peripheral blood mononuclear cells from 18 TB patients and 12 healthy donors from Bangladesh, and extracellular levels of gzmA and B were measured in serum from 58 TB patients and 31 healthy controls. TB patients showed increased expression of gzmA in CD8þ T, CD4þ T and CD56þ T, but not NK, cells, and of gzmB in CD8þ T cells, when compared to controls. GzmK expression was not altered in TB patients in any lymphocyte subset. The extracellular levels of gzmA and, to a lesser extent, of gzmB, were increased in TB patients, but did not correlate with intracellular gzm expression in lymphocyte subsets. Our results reveal enhanced intra- and extracellular expression of gzmA and B in patients with pulmonary TB, suggesting that gzms are part of the host response to tuberculosis. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Tuberculosis Granzymes T lymphocytes Cytokines
* Corresponding author. Academic Medical Center, Center for Experimental and Molecular Medicine, Meibergdreef 9, Room G2-130, 1105 AZ Amsterdam, The Netherlands. Tel.: þ31 20 5665910; fax: þ31 20 6977192. E-mail addresses: [email protected] (M.I. Garcia-Laorden), [email protected] (D.C. Blok), [email protected] (L.M. Kager), [email protected] (A.J. Hoogendijk), [email protected] (G.J. van Mierlo), [email protected] (I.O. Lede), [email protected] (W. Rahman), [email protected] (R. Afroz), [email protected] (A. Ghose), [email protected] (C.E. Visser), [email protected] (N. Day), [email protected] (A.M. Dondorp), [email protected] (A.F. de Vos), [email protected] (T. van der Poll). 1 Dr. Hossain passed away during the course of the study. http://dx.doi.org/10.1016/j.tube.2015.05.016 1472-9792/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Garcia-Laorden MI, et al., Increased intra- and extracellular granzyme expression in patients with tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.05.016
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M.I. Garcia-Laorden et al. / Tuberculosis xxx (2015) 1e6
1. Introduction Tuberculosis (TB) is caused by infection with Mycobacterium (M.) tuberculosis and typically affects the lungs. This disease constitutes a serious health problem worldwide, with around onethird of the world's population infected and an estimated 9 million new cases and 1.5 million deaths in 2013 [1]. The majority of TB cases occur in developing countries, with Bangladesh being one of the most affected. Immunity against M. tuberculosis depends on a wide range of innate and adaptive immune responses mainly driven by macrophages and different T cell subsets [2]. Granzymes (gzms) are a family of serine proteases found in granules of cytotoxic lymphocytes. In humans five different gzms (A, B, H, K and M) have been identified, of which the expression is generally restricted to cells of the lymphoid lineage [3]. GzmA and gzmB, the most abundant gzms, are expressed constitutively in several cell types including cytotoxic T lymphocytes (CTL), natural killer (NK) cells, NKT cells, and gd T cell receptor (TCR) cells [4,5]. GzmK is mainly expressed by CTL [5]. Expression of gzms has also been observed in other cells, including non-lymphoid cells, at least after stimulation [6,7], and even lung parenchymal cells [8]. Multiple studies have described the role of gzms in eliminating infected, neoplasic or foreign cells. While there is a general consensus regarding the role of gzmB in cell death, the physiological relevance of the cytotoxicity produced by gzmA and other gzms remains controversial [3]. Considerable evidence suggests that the effects of gzms are diverse and go beyond cytotoxicity. Plasma levels of gzmA and B have been found elevated in patients with parasitic, viral and bacterial diseases [9,6], severe sepsis [10,11] as well as in induced human endotoxemia [12], whilst induction of gzmA and B secretion has been shown after stimulation of whole blood with bacteria [12]. Extracellular gzms have been implicated in several processes, including induction of proinflammatory cytokine release [7,13]. Studies showing that gzmA and gzmB deficient mice are relatively protected against endotoxin-induced shock further suggest a role for gzms in the regulation of inflammatory responses [14,15]. Some studies have addressed the expression of gzms A and/or B after stimulation with M. tuberculosis, while others have studied their extracellular levels or their expression by a specific lymphocyte population in TB patients, hinting at increased gzm expression after stimulation or compared to controls [16e22]. The present study was designed to explore the intracellular expression of gzms A, B and K by diverse lymphocyte populations as well as the extracellular expression of gzms A and B in patients with active pulmonary TB compared to healthy individuals. 2. Materials and methods 2.1. Study subjects This survey was conducted as an additional study of a previous investigation evaluating activation of the coagulation system in patients with pulmonary TB, of which the results have been published [23]. The current study included 61 adult patients with pulmonary TB recruited prospectively in the Tuberculosis Clinic of Chittagong General Hospital and the Chittagong Medical College & Hospital, Chittagong, Bangladesh. Pulmonary TB was considered confirmed when at least two out of three sputum samples collected on two consecutive days, including an early morning sample, tested positive on Ziehl-Neelsen (ZN)-staining. TB-positivity was confirmed by polymerase chain reaction on M. tuberculosis (GeneXpert, Cepheid, Solna, Sweden) in the Laboratory of Microbiology in the Academic Medical Center in Amsterdam, the Netherlands. Patients with concomitant disease or a clinical condition which could interfere with the conduct of the study were excluded. Thirty
two healthy blood donors were recruited from the Chittagong Medical College & Hospital and served as controls. Controls and patients were recruited between September and November 2010. The study was approved by the National Research Ethics Committee (NREC), Bangladesh Medical Research Council, Bangladesh and the Oxford Tropical Research Ethics Committee, University of Oxford, Oxford, UK (OXTREC 35-09). Written informed consent was obtained from all study subjects or next-of-kin by a native Bengali speaker. 2.2. Assays Serum and plasma samples were obtained from patients and controls and were kept at 80 C. Levels of gzmA and B were measured in serum by ELISA using reagents from Sanquin (Amsterdam, The Netherlands) as previously described [24]. Levels of Interleukin (IL)-6, chemokine (CeXeC motif) ligand (CXCL) 8, interferon gamma-induced protein (IP) 10, soluble IL-2 receptor subunit-a (sIL-2Ra), soluble intercellular adhesion molecule (sICAM)-1, soluble tumor necrosis factor receptor (sTNFR)-1 and sTNFR-2, IL-12, interferon (IFN)-g and tumor necrosis factor (TNF)a were measured in EDTA plasma by a multiplex assay (Luminex, Austin, TX, USA) using reagents from Bio-Rad (Veenendaal, The Netherlands). C-reactive protein (CRP) was measured in heparinised plasma samples with the C-Reactive Protein Gen.3 test kit (Roche Diagnostics, Mannheim, Germany), an immunoturbidimetric method, on the Hitachi Modular P-800 module (Hitachi, Hitachinaka, Japan). 2.3. Flow cytometry Peripheral blood mononuclear cells (PBMCs) were isolated from blood harvested in Cell Preparation Tubes (Becton Dickinson, Franklin Lakes, NJ, USA) according to the manufacturer's instructions, from 18 TB patients and 12 healthy controls (of which 15 patients and 11 controls had also serum data) and viable frozen in RPMI 1640 (Life Technologies) medium with 20% FCS (Lonza, Basel, Switzerland) and 20% DMSO with a mr. Frosty freezing container (Thermo Scientific, Waltham, MA, USA), first placed overnight in a 20 C freezer and subsequently in the gas phase of a liquid nitrogen container. For flow cytometry, cryopreserved cells were carefully thawed and washed, and 2e5 105 cells/well were incubated with monoclonal antibodies against CD3 (AF700), CD4 (PerCP-Cy5.5), CD56 (APC) (all from BD Pharmingen, Breda, The Netherlands) and CD8 (PE-Cy7; Biolegend, San Diego, CA, USA), at 4 C for 25 min in the dark. For the intracellular staining, cells were fixed for 20 min in Cytofix/Cytoperm (BD Bioscience, , CA, USA) at 4 C in the dark and then washed with Perm/ San Jose Wash buffer. Subsequently, the cells were resuspended in Perm/ wash buffer containing the antibodies against gzmA (PE; BD Pharmingen), gzmB (PE-CF594; BD Horizon) and gzmK (FITC; Santa Cruz Biotechnology, Dallas, TX, USA). The samples were analyzed by flow cytometry with a FACSCanto (BD Bioscience). The FlowJo software (Tree Star Inc, Ashland, OR, USA) was used to analyze the data. Lymphocytes were gated in the forward scatter versus side scatter dot plot. Cells were selected as CD3þ (T cells), or as CD3þCD4þ, CD3þCD8þ, CD3þCD56þ and CD3CD56þ (NK cells), and expression of gzms was analyzed in these populations. The results are expressed as percentage of cells of the specific population expressing the corresponding gzm and as the median intensity of fluorescence (MFI). Alternatively, cells were selected as positive for each gzm and the percentage of the above mentioned lymphocyte populations were analyzed within the gzmþ lymphocytes.
Please cite this article in press as: Garcia-Laorden MI, et al., Increased intra- and extracellular granzyme expression in patients with tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.05.016
M.I. Garcia-Laorden et al. / Tuberculosis xxx (2015) 1e6
2.4. Statistical analysis Data in the figures are expressed as box and whiskers showing the smallest observation, lower quartile, median, upper quartile and largest observation, or as individual data points. Data in the tables are expressed as medians with interquartile ranges or as single values. Comparisons between groups were performed using the ManneWhitney U test. Correlations were calculated using the Spearman's rho test. Analyses were done using GraphPad Prism version 5.01 (San Diego, CA, USA) and SPSS version 15.0 (Chicago, Ill, USA). P-values <0.05 were considered statistically significant. 3. Results 3.1. Patients and controls characteristics The characteristics of patients and controls included in the present study are provided in Table 1. 3.2. Intracellular expression of granzymes A, B and K by lymphocyte populations We sought to evaluate the intracellular expression of the gzms by different lymphocyte populations and the potential differences in this expression between patients and controls. For this purpose, we first analyzed the proportion of these lymphocyte populations present in both groups of individuals (Table 2). Patients with TB had similar proportions of CD8þ T, NK and CD56þ T cells, and lower CD4þ T cell numbers, when compared with healthy controls. The total number of gzm positive cells did not differ significantly between patients and controls. Then we analyzed which lymphocytes were expressing each gzm (Figure 1 and Supplementary Figure 1). In both patients and controls gzmA and gzmB were primarily expressed by NK and CD8þ T cells, while gzmK was mainly expressed by CD8þ T cells. In TB patients there was a slight increase in the percentage of CD56þ T cells expressing gzmA and K (P < 0.05 versus controls). Figure 2 shows the expression of each gzm within different lymphocyte populations, expressed as the percentage of gzmþ cells (Figure 2AeD) and the MFI of these cells (Figure 2EeH) (see Supplementary Figure 2 for gating strategy). Patients with TB demonstrated an increase in the percentage of CD8þ T, CD4þ T and CD56þ T cells expressing gzmA, as well as higher MFI of gzmA in these cells, when compared to controls. TB patients also had a significant increase in the percentage and the MFI of CD8þ T cells expressing gzmB. GzmK expression did not differ between groups. Positive correlations existed between the percentage of lymphocyte populations expressing gzmA versus gzmB in patients: CD8þ T cells (r ¼ 0.83, P < 0.0001), CD4þ T cells (r ¼ 0.69, P ¼ 0.002), CD56þ T cells (r ¼ 0.50, P ¼ 0.036) and NK cells (r ¼ 0.50, P ¼ 0.033).
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Table 2 Proportion of lymphocyte populations and granzymes in patients with pulmonary tuberculosis and controls.
CD8þ T cells CD4þ T cells CD56þ T cells NK cells gzmAþ cells gzmBþ cells gzmKþ cells
Controls, N ¼ 12
Patients, N ¼ 18
17.2 22.3 3.9 13.3 28.0 29.9 8.3
19.0 12.9 6.4 12.7 34.7 34.6 6.9
(14.0e27.3) (19.2e25.6) (3.1e6.8) (9.9e14.5) (21.8e38.1) (26.0e35.6) (6.9e12.9)
(15.2e23.7) (10.4e18.0)** (4.1e8.2) (10.6e17.8) (22.6e42.6) (28.7e45.6) (5.1e9.3)
Values are calculated as percentage of total lymphocytes and presented as median percentage (interquartile range). NK: CD3CD56þ, gzm: granzyme. **P < 0.01 determined by ManneWhitney U test when compared to controls.
In controls this correlation was only observed in CD4þ T cells (r ¼ 0.76, P ¼ 0.005). To determine whether the correlation between cells expressing gzmA and gzmB was due to co-expression in the same cells, we analyzed the individual and concurrent expression of gzmA and gzmB in CD8þ T, CD56þ T and NK cells (only around 11% of CD4þ T cells were positive for any gzm). Most cells positive for gzmA or B, co-expressed both gzms in both patients and controls, although in CD8þ T cells less in controls than in patients (Supplementary Figure 3AeF). 3.3. Circulating levels of granzyme A and B To get insight into the extracellular role of gzms, serum levels of gzmA and B were measured in controls and patients. The concentrations of gzmB and, to a larger extent, of gzmA, were increased in TB patients (Figure 3). The serum levels of gzmA and gzmB did not correlate with their respective intracellular expression in either patients or controls (data not shown). There was also not correlation when taking into account each lymphocyte population. 3.4. Association of circulating levels of granzyme A and B and relevant disease biomarkers We next investigated whether circulating levels of gzms correlated with the plasma concentrations of established biomarkers of TB. For this we measured IL-6, CXCL8 and IP-10 (which are relevant in active TB) as well as sIL-2Ra, sTNFR-1, sTNFR-2 and CRP (which correlate with the extent of disease in TB [25]) in plasma of TB patients and healthy controls. As expected [25] and reported previously by our group [23], the plasma levels of all biomarkers were elevated in patients versus controls (all P < 0.0001; Supplementary Table 1). Levels of IFN-g, IL-12 and TNF-a remained below the limit of detection in most individuals (data not shown).
Table 1 Characteristics of patients with pulmonary tuberculosis and controls. Extracellular expression study
Intracellular expression study
Patients, N ¼ 58
Controls, N ¼ 31
Patients, N ¼ 18
Controls, N ¼ 12
Demographics Age (years) Gender (male) HIV-positive
32.7 ± 12.3 42 (72.4) 1 (1.7)
30.0 ± 5.7 23 (74.2) 0 (0)
28.7 ± 9.1 11 (61.1) 0
29.3 ± 5.7 10 (83.3) 0
Symptoms Fever Productive cough Weight loss
58 (100) 54 (93.1) 42 (72.4)
0 (0) 1 (3.2) 0 (0)
18 (100) 15 (83.3) 12 (66.7)
0 0 0
Values are number of individuals (%) except for age, which are mean ± standard deviation.
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Figure 1. Lymphocyte populations expressing granzymes A, B and K in patients with pulmonary tuberculosis and controls. Distribution of the gzmA, B and K positive cells among the different lymphocyte populations, expressed as percentage of total of each gzmþ lymphocytes. NK: CD3CD56þ, gzm: granzyme. N ¼ 12 controls and 18 patients. Data are boxand-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation. *P < 0.05 determined by ManneWhitney U test.
Figure 2. Expression of granzymes A, B and K by lymphocyte populations in patients with pulmonary tuberculosis and controls. Percentage of lymphocyte population expressing each gzm (AeD) and MFI of the intracellular expression of each granzyme in the lymphocyte populations (EeH). MFI values are expressed with Log10 scale. NK: CD3CD56þ, MFI: median fluorescence intensity, gzm: granzyme. N ¼ 12 controls and 18 patients. Data are box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation. *P < 0.05, **P < 0.01 determined by ManneWhitney U test.
We found positive weak correlations between gzmA levels with the concentrations of IL-6, CXCL8, IP-10, sIL-2Ra, sTNFR-1 and CRP in patients (Table 3). GzmB levels correlated positively with CXCL8, IP-10 and sIL-2Ra. Most of these associations were not found in the control group (data not shown). 4. Discussion In the present study we investigated the expression of gzms A, B and K in different lymphocyte populations in PBMCs as well as the serum levels of gzmA and B in patients with active pulmonary TB and healthy individuals. To our knowledge, this is the first study analyzing the intracellular expression of these three gzms in diverse lymphocyte populations of TB patients. The current study showed an increase in the expression of gzmA and B in T cells (mainly CD8þ T cells) from TB patients, but no differences in either gzmA or gzmB expression in NK cells nor in the expression of gzmK in any subgroup of lymphocytes. The analysis of the extracellular levels of gzmA and B also showed an increase of both gzms in patients relative to controls. We first aimed to identify the lymphocyte populations producing each gzm as well as the potential differences between TB patients and healthy individuals. As expected [5], both gzmA and B were mainly produced by NK and CD8þ T cells, whilst the main producers of gzmK were CD8þ T cells. This cellular distribution of gzm expression did not differ significantly between patients and controls. Since the number of NK cells in blood is relatively low
compared to T lymphocytes, while both cell types are quantitatively similar sources of gzms, likely a very high percentage of NK cells are gzmþ (and the same would be true for CD56þ T cells). This was indeed observed when we studied the proportion of each lymphocyte population expressing each gzm. This analysis produced additional relevant results when we compared such proportions between patients and controls: we observed a significant increase in the percentage of T lymphocytes subsets expressing gzmA (CD8þ T, CD4þ T and CD56þ T cells) and gzmB (CD8þ T cells) in TB patients. These increases were also found in the MFI, indicating that there are not only more T lymphocytes expressing gzms, but that the gzmþ cells are also expressing more gzm. Thus, T lymphocyte expression of gzmA and B is increased considerably in TB, but, although NK cells are big producers of gzmA and B, TB does not result in an increase of their gzm production. Rao et al. [20] also found no differences in the expression of gzmA and B in NK cells when comparing HIV negative TB patients with controls. A possible explanation for the apparent absence of stimulation of the expression of these gzms in NK cells during TB is a matter of timing. NK cells are basically innate immune cells, whilst T lymphocytes are essentially adaptive immune cells; possibly, at the moment the patients had developed symptoms and were recruited, the time for a relevant role of the NK cells had passed. In the same study of Rao et al. [20] they found that, after stimulation with M. tuberculosis or IL-15 þ IL-12, the percentage of NK cells expressing gzmB (but not gzmA) increased in both controls and TB patients. In this sense, the expression of gzmB in NK CD45ROþ (memory NK cells) from pleural
Please cite this article in press as: Garcia-Laorden MI, et al., Increased intra- and extracellular granzyme expression in patients with tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.05.016
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Figure 3. Circulating levels of granzyme A and B in patients with pulmonary tuberculosis and controls. Serum levels of gzmA and gzmB in patients (N ¼ 58) and controls (N ¼ 31). Each circle represents an individual, with horizontal lines showing medians. Dotted lines represent the lower detection limit. *P < 0.05, ***P < 0.001 determined by ManneWhitney U test.
Table 3 Correlations of circulating levels of granzyme A and B with cytokines levels in patients with pulmonary tuberculosis. gzmA
IL-6 CXCL8 IP-10 sIL-2Ra sTNFR-1 sTNFR-2 sICAM-1 CRP gzmB
gzmB
r
P
r
P
0.42 0.46 0.38 0.38 0.27 0.28 0.20 0.32 0.24
0.001 0.0003 0.004 0.004 0.040 0.033 0.127 0.016 0.075
0.22 0.36 0.36 0.33 0.20 0.26 0.23 0.14
0.094 0.006 0.006 0.011 0.135 0.051 0.097 0.306
IL: interleukin; CXCL8: chemokine (CeXeC motif) ligand 8; IP-10: Interferon gamma-induced protein 10; sIL-2Ra: soluble interleukin-2 receptor a; sTNFR-1,-2: soluble tumor necrosis factor receptor-1 and -2; sICAM-1: soluble intercellular adhesion molecule-1; CRP: C-reactive protein; gzm: granzyme. Correlations were calculated using the Spearman's test. r: Spearman's rho coefficient of correlation. N ¼ 58.
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fluid from patients increased after stimulation with IL-12 [21]. All this seems to indicate that NK cells express high amounts of gzmA and B constitutively, that the stimulation of NK cells to produce more gzms could be limited in time, and that the NK expression of gzmB is more prone to be stimulated than gzmA. On the other hand, it has been previously observed that the percentage of M. tuberculosis specific CD8þ T cells positive for gzmA and B is higher in active than in latent TB [22]. That would support the idea that the stimulation of CD8þ T cells to produce more gzmA and B depends on the development of active disease. However, contradictory results were recently published, showing down-regulated expression of gzmB in CD8þ T cells from active TB patients compared to latent TB and healthy controls [26]. Our results showed similar expression of gzmK in patients and controls in all lymphocyte populations studied, indicating that TB does not trigger its expression. To our knowledge, this is the first survey studying gzmK in the context of TB. When we analyzed the correlation between the percentage of lymphocyte populations expressing gzmA and gzmB, we found a strong correlation in CD8þ T cells from patients, but not from controls, suggesting that CD8þ T cells had been stimulated to produce both gzms simultaneously. This was confirmed by our finding of co-expression of both gzms in the same CD8þ T cells, which was much higher in patients than in controls. On the other hand, since a high percentage of NK cells express gzmA as well as gzmB, it is not surprising that we found co-expression of both gzms in these cells. Nevertheless, this co-expression was similar in patients and controls, and the correlation between these gzms was modest in NK cells from patients and inexistent in controls. This is consistent with the absence of increased expression found in these cells. For years, it has been thought that the effect of gzms almost exclusively involved target cell death. However, more and more evidence not only points to broader roles, but also questions whether all gzms are cytotoxic in vivo. Diverse non-cytotoxic and even non-lymphoid cells expressing gzms have been described, as well as multiple extracellular gzms substrates not related with cell death [3,6,7]. Moreover, gzms have been found extracellularly in plasma and serum as well as in other bodily fluids, with low levels in healthy individuals and higher levels related to some infectious and inflammatory diseases [6,7]. In the present study we have measured the serum levels of gzmA and B and, as observed in the analysis of the intracellular expression, it showed an increase of gzmB and, to a larger extent, of gzmA, in patients compared to controls. A previous report described higher levels of gzmB at diagnosis in TB patients with a slow response to treatment than in community controls [19], although another study failed to find differences in the levels of gzmB between patients and controls, but found higher levels of the gzm in patients with greater cavity sizes [27]. Although evidence support a purposeful release of gzms in absence of cytotoxicity, the origin of extracellular gzms is not clear. In an attempt to discern a possible source for the circulating gzms, we analyzed the correlation between the extra- and intracellular expression of these gzms, but we did not find significant results for any of the lymphocyte populations studied. Extracellular gzmA can be produced by g9d2 T cells by a non-cytotoxic mechanism and then soluble gzmA can induce M. tuberculosis infected macrophages to produce TNF-a which leads, by itself and/or in combination with other factors, to intracellular mycobacterial growth inhibition [28]. gd T cells tend to accumulate in mucosal epithelial tissues, as the lung [28], however, they do not comprise a high proportion of human lymphocytes. It is not unreasonable to think that a similar role exists for gzms released by other lymphocytes. Several studies have involved extracellular gzms in the production, release and/or processing of proinflammatory cytokines, such as IL-6, IL-8, TNF-a, IL-1b and IL-1a [7,13]. We analyzed the correlation between the
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circulating levels of gzmA and B and the levels of several TB biomarkers, and we found some positive weak correlations (mainly with IL-8, IP-10 and sIL2Ra), suggesting that plasma gzm levels are indicative of the extent of inflammation. In conclusion, the results of the present study show enhanced intracellular expression of gzmA and B, but not K, by T lymphocytes, as well as raised extracellular levels, in patients with active pulmonary TB. The increase is more pronounced for gzmA than for gzmB. Further research is warranted to determine the functional role of gzms in host defense against M. tuberculosis. Acknowledgments We are grateful to the patients and the healthy volunteers for their trust and cooperation. We would also like to thank Mr. Sahid Ullah, Ms. Runia and Mr. Pradip Sen for performing and scoring the Ziehl-Neelsen stainings, Ms. Rupali and Mr. Yusuf for allowing us to work in the tuberculosis laboratory in the General Hospital, Anderkilla, Chittagong, Montri Ridjaibun, Nuttapol Panachuenwongsakul and Thatsanun Ngernseng for their help with the patient database, and Barbara Dierdorp and Tamara Dekker for performing the cytokine and chemokine measurements. In addition, we are grateful to the staff of Chittagong General Hospital and Chittagong Medical College & Hospital for their continuing support. Funding: This research was partially supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme. The study sponsor did not have any role in the study design, in the collection, analysis and interpretation of data, in the writing of the manuscript, or in the decision to submit the manuscript for publication. Competing interests: Ethical approval:
None declared. Not required.
Appendix A. Supplementary material Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.tube.2015.05.016. References [1] WHO. Global tuberculosis report. 2014. http://www.who.int/tb/publications/ global_report/en/. [2] O'Garra A, Redford PS, McNab FW, Bloom CI, Wilkinson RJ, Berry MP. The immune response in tuberculosis. Annu Rev Immunol 2013;31:475e527. [3] Susanto O, Trapani JA, Brasacchio D. Controversies in granzyme biology. Tissue Antigens 2012;80:477e87. [4] Grossman WJ, Verbsky JW, Tollefsen BL, Kemper C, Atkinson JP, Ley TJ. Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells. Blood 2004;104:2840e8. [5] Bratke K, Kuepper M, Bade B, Virchow Jr JC, Luttmann W. Differential expression of human granzymes A, B, and K in natural killer cells and during CD8þ T cell differentiation in peripheral blood. Eur J Immunol 2005;35: 2608e16. [6] Anthony DA, Andrews DM, Watt SV, Trapani JA, Smyth MJ. Functional dissection of the granzyme family: cell death and inflammation. Immunol Rev 2010;235:73e92.
[7] Joeckel LT, Bird PI. Are all granzymes cytotoxic in vivo? Biol Chem 2014;395: 181e202. [8] Vernooy JH, Moller GM, van Suylen RJ, van Spijk MP, Cloots RH, Hoet PH, et al. Increased granzyme A expression in type II pneumocytes of patients with severe chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2007;175:464e72. [9] Buzza MS, Bird PI. Extracellular granzymes: current perspectives. Biol Chem 2006;387:827e37. [10] Zeerleder S, Hack CE, Caliezi C, van MG, Eerenberg-Belmer A, Wolbink A, Wuillenmin WA. Activated cytotoxic T cells and NK cells in severe sepsis and septic shock and their role in multiple organ dysfunction. Clin Immunol 2005;116:158e65. [11] Napoli AM, Fast LD, Gardiner F, Nevola M, Machan JT. Increased granzyme levels in cytotoxic T lymphocytes are associated with disease severity in emergency department patients with severe sepsis. Shock 2012;37:257e62. [12] Lauw FN, Simpson AJ, Hack CE, Prins JM, Wolbink AM, van Deventer SJ, et al. Soluble granzymes are released during human endotoxemia and in patients with severe infection due to gram-negative bacteria. J Infect Dis 2000;182: 206e13. [13] Wensink AC, Hack CE, Bovenschen N. Granzymes regulate proinflammatory cytokine responses. J Immunol 2015;194:491e7. [14] Metkar SS, Menaa C, Pardo J, Wang B, Wallich R, Freudenberg M, et al. Human and mouse granzyme A induce a proinflammatory cytokine response. Immunity 2008;29:720e33. [15] Anthony DA, Andrews DM, Chow M, Watt SV, House C, Akira S, et al. A role for granzyme M in TLR4-driven inflammation and endotoxicosis. J Immunol 2010;185:1794e803. [16] Canaday DH, Wilkinson RJ, Li Q, Harding CV, Silver RF, Boom WH. CD4(þ) and CD8(þ) T cells kill intracellular Mycobacterium tuberculosis by a perforin and Fas/Fas ligand-independent mechanism. J Immunol 2001;167:2734e42. [17] Toossi Z, Mayanja-Kizza H, Kanost A, Edmonds K, McHugh M, Hirsch C. Protective responses in tuberculosis: induction of genes for interferon-gamma and cytotoxicity by Mycobacterium tuberculosis and during human tuberculosis. Scand J Immunol 2004;60:299e306. [18] Walch M, Rampini SK, Stoeckli I, Latinovic-Golic S, Dumrese C, Sundstrom H, et al. Involvement of CD252 (CD134L) and IL-2 in the expression of cytotoxic proteins in bacterial- or viral-activated human T cells. J Immunol 2009;182: 7569e79. [19] Brahmbhatt S, Black GF, Carroll NM, Beyers N, Salker F, Kidd M, et al. Immune markers measured before treatment predict outcome of intensive phase tuberculosis therapy. Clin Exp Immunol 2006;146:243e52. [20] Rao PV, Ramanavelan S, Rajasekaran S, Raja A. Natural-killer cell-derived cytolytic molecules in HIV-associated pulmonary tuberculosis-role of exogenous interleukins. J Clin Immunol 2010;30:393e401. [21] Fu X, Liu Y, Li L, Li Q, Qiao D, Wang H, et al. Human natural killer cells expressing the memory-associated marker CD45RO from tuberculous pleurisy respond more strongly and rapidly than CD45RO natural killer cells following stimulation with interleukin-12. Immunology 2011;134:41e9. [22] Rozot V, Vigano S, Mazza-Stalder J, Idrizi E, Day CL, Perreau M, et al. Mycobacterium tuberculosis-specific CD8þ T cells are functionally and phenotypically different between latent infection and active disease. Eur J Immunol 2013;43:1568e77. [23] Kager LM, Blok DC, Lede IO, Rahman W, Afroz R, Bresser P, et al. Pulmonary tuberculosis induces a systemic hypercoagulable state. J Infect 2015;70: 324e34. pii: S0163-4453(14)00313-2. C, van Deuren M, van der [24] Hermsen CC, Konijnenberg Y, Mulder L, Loe Meer JW, et al. Circulating concentrations of soluble granzyme A and B increase during natural and experimental Plasmodium falciparum infections. Clin Exp Immunol 2003;132:467e72. [25] Walzl G, Ronacher K, Hanekom W, Scriba TJ, Zumla A. Immunological biomarkers of tuberculosis. Nat Rev Immunol 2011;11:343e54. [26] Silva BD, Trentini MM, da Costa AC, Kipnis A, Junqueira-Kipnis AP. Different phenotypes of CD8þ T cells associated with bacterial load in active tuberculosis. Immunol Lett 2014;160:23e32. [27] Djoba Siawaya JF, Bapela NB, Ronacher K, Veenstra H, Kidd M, Gie R, et al. Immune parameters as markers of tuberculosis extent of disease and early prediction of anti-tuberculosis chemotherapy response. J Infect 2008;56: 340e7. [28] Spencer CT, Abate G, Sakala IG, Xia M, Truscott SM, Eickhoff CS, et al. Granzyme A produced by g(9)d(2) T cells induces human macrophages to inhibit growth of an intracellular pathogen. PLoS Pathog 2013;9:e1003119.
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Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube
IMMUNOLOGICAL ASPECTS
Increased intra- and extracellular granzyme expression in patients with tuberculosis M. Isabel Garcia-Laorden a, b, *, Dana C. Blok a, b, Liesbeth M. Kager a, b, Arie J. Hoogendijk a, b, Gerard J. van Mierlo c, Ivar O. Lede a, d, Wahid Rahman e, Rumana Afroz e, Aniruddha Ghose e, Caroline E. Visser a, d, Abu Shahed Md Zahed e, Md Anwar Husain f, Khan Mashrequl Alam f, Pravat Chandra Barua g, Mahtabuddin Hassan e, Ahmed Hossain h, 1, Md Abu Tayab i, Nick Day j, Arjen M. Dondorp j, Alex F. de Vos a, b, Tom van der Poll a, b, k a
Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands b Center for Experimental and Molecular Medicine (CEMM), Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands c Department of Immunopathology, Sanquin Research, Plesmanlaan 125, 1066CX Amsterdam, The Netherlands d Department of Microbiology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands e Department of Internal Medicine, Chittagong Medical College & Hospital (CMCH), Chittagong, Bangladesh f Department of Microbiology, Chittagong Medical College & Hospital (CMCH), Chittagong, Bangladesh g National TB Control & Leprosy Elimination Program, Dhaka, Bangladesh h Chest Disease Clinic Chittagong (CDCC), Anderkilla, Chittagong, Bangladesh i Chittagong General Hospital, Arderkilla, Chittagong, Bangladesh j Mahidol Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, 3/F 60th Anniversary Chalermprakiat Building, 420/6 Rajvithi Road, 10400 Bangkok, Thailand k Division of Infectious Diseases, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
a r t i c l e i n f o
s u m m a r y
Article history: Received 4 March 2015 Accepted 31 May 2015
Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. Granzymes (gzms) are proteases mainly found in cytotoxic lymphocytes, but also extracellularly. While the role of gzms in target cell death has been widely characterized, considerable evidence points towards broader roles related to infectious and inflammatory responses. To investigate the expression of the gzms in TB, intracellular gzms A, B and K were measured by flow cytometry in lymphocyte populations from peripheral blood mononuclear cells from 18 TB patients and 12 healthy donors from Bangladesh, and extracellular levels of gzmA and B were measured in serum from 58 TB patients and 31 healthy controls. TB patients showed increased expression of gzmA in CD8þ T, CD4þ T and CD56þ T, but not NK, cells, and of gzmB in CD8þ T cells, when compared to controls. GzmK expression was not altered in TB patients in any lymphocyte subset. The extracellular levels of gzmA and, to a lesser extent, of gzmB, were increased in TB patients, but did not correlate with intracellular gzm expression in lymphocyte subsets. Our results reveal enhanced intra- and extracellular expression of gzmA and B in patients with pulmonary TB, suggesting that gzms are part of the host response to tuberculosis. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Tuberculosis Granzymes T lymphocytes Cytokines
* Corresponding author. Academic Medical Center, Center for Experimental and Molecular Medicine, Meibergdreef 9, Room G2-130, 1105 AZ Amsterdam, The Netherlands. Tel.: þ31 20 5665910; fax: þ31 20 6977192. E-mail addresses: [email protected] (M.I. Garcia-Laorden), [email protected] (D.C. Blok), [email protected] (L.M. Kager), [email protected] (A.J. Hoogendijk), [email protected] (G.J. van Mierlo), [email protected] (I.O. Lede), [email protected] (W. Rahman), [email protected] (R. Afroz), [email protected] (A. Ghose), [email protected] (C.E. Visser), [email protected] (N. Day), [email protected] (A.M. Dondorp), [email protected] (A.F. de Vos), [email protected] (T. van der Poll). 1 Dr. Hossain passed away during the course of the study. http://dx.doi.org/10.1016/j.tube.2015.05.016 1472-9792/© 2015 Elsevier Ltd. All rights reserved.
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1. Introduction Tuberculosis (TB) is caused by infection with Mycobacterium (M.) tuberculosis and typically affects the lungs. This disease constitutes a serious health problem worldwide, with around onethird of the world's population infected and an estimated 9 million new cases and 1.5 million deaths in 2013 [1]. The majority of TB cases occur in developing countries, with Bangladesh being one of the most affected. Immunity against M. tuberculosis depends on a wide range of innate and adaptive immune responses mainly driven by macrophages and different T cell subsets [2]. Granzymes (gzms) are a family of serine proteases found in granules of cytotoxic lymphocytes. In humans five different gzms (A, B, H, K and M) have been identified, of which the expression is generally restricted to cells of the lymphoid lineage [3]. GzmA and gzmB, the most abundant gzms, are expressed constitutively in several cell types including cytotoxic T lymphocytes (CTL), natural killer (NK) cells, NKT cells, and gd T cell receptor (TCR) cells [4,5]. GzmK is mainly expressed by CTL [5]. Expression of gzms has also been observed in other cells, including non-lymphoid cells, at least after stimulation [6,7], and even lung parenchymal cells [8]. Multiple studies have described the role of gzms in eliminating infected, neoplasic or foreign cells. While there is a general consensus regarding the role of gzmB in cell death, the physiological relevance of the cytotoxicity produced by gzmA and other gzms remains controversial [3]. Considerable evidence suggests that the effects of gzms are diverse and go beyond cytotoxicity. Plasma levels of gzmA and B have been found elevated in patients with parasitic, viral and bacterial diseases [9,6], severe sepsis [10,11] as well as in induced human endotoxemia [12], whilst induction of gzmA and B secretion has been shown after stimulation of whole blood with bacteria [12]. Extracellular gzms have been implicated in several processes, including induction of proinflammatory cytokine release [7,13]. Studies showing that gzmA and gzmB deficient mice are relatively protected against endotoxin-induced shock further suggest a role for gzms in the regulation of inflammatory responses [14,15]. Some studies have addressed the expression of gzms A and/or B after stimulation with M. tuberculosis, while others have studied their extracellular levels or their expression by a specific lymphocyte population in TB patients, hinting at increased gzm expression after stimulation or compared to controls [16e22]. The present study was designed to explore the intracellular expression of gzms A, B and K by diverse lymphocyte populations as well as the extracellular expression of gzms A and B in patients with active pulmonary TB compared to healthy individuals. 2. Materials and methods 2.1. Study subjects This survey was conducted as an additional study of a previous investigation evaluating activation of the coagulation system in patients with pulmonary TB, of which the results have been published [23]. The current study included 61 adult patients with pulmonary TB recruited prospectively in the Tuberculosis Clinic of Chittagong General Hospital and the Chittagong Medical College & Hospital, Chittagong, Bangladesh. Pulmonary TB was considered confirmed when at least two out of three sputum samples collected on two consecutive days, including an early morning sample, tested positive on Ziehl-Neelsen (ZN)-staining. TB-positivity was confirmed by polymerase chain reaction on M. tuberculosis (GeneXpert, Cepheid, Solna, Sweden) in the Laboratory of Microbiology in the Academic Medical Center in Amsterdam, the Netherlands. Patients with concomitant disease or a clinical condition which could interfere with the conduct of the study were excluded. Thirty
two healthy blood donors were recruited from the Chittagong Medical College & Hospital and served as controls. Controls and patients were recruited between September and November 2010. The study was approved by the National Research Ethics Committee (NREC), Bangladesh Medical Research Council, Bangladesh and the Oxford Tropical Research Ethics Committee, University of Oxford, Oxford, UK (OXTREC 35-09). Written informed consent was obtained from all study subjects or next-of-kin by a native Bengali speaker. 2.2. Assays Serum and plasma samples were obtained from patients and controls and were kept at 80 C. Levels of gzmA and B were measured in serum by ELISA using reagents from Sanquin (Amsterdam, The Netherlands) as previously described [24]. Levels of Interleukin (IL)-6, chemokine (CeXeC motif) ligand (CXCL) 8, interferon gamma-induced protein (IP) 10, soluble IL-2 receptor subunit-a (sIL-2Ra), soluble intercellular adhesion molecule (sICAM)-1, soluble tumor necrosis factor receptor (sTNFR)-1 and sTNFR-2, IL-12, interferon (IFN)-g and tumor necrosis factor (TNF)a were measured in EDTA plasma by a multiplex assay (Luminex, Austin, TX, USA) using reagents from Bio-Rad (Veenendaal, The Netherlands). C-reactive protein (CRP) was measured in heparinised plasma samples with the C-Reactive Protein Gen.3 test kit (Roche Diagnostics, Mannheim, Germany), an immunoturbidimetric method, on the Hitachi Modular P-800 module (Hitachi, Hitachinaka, Japan). 2.3. Flow cytometry Peripheral blood mononuclear cells (PBMCs) were isolated from blood harvested in Cell Preparation Tubes (Becton Dickinson, Franklin Lakes, NJ, USA) according to the manufacturer's instructions, from 18 TB patients and 12 healthy controls (of which 15 patients and 11 controls had also serum data) and viable frozen in RPMI 1640 (Life Technologies) medium with 20% FCS (Lonza, Basel, Switzerland) and 20% DMSO with a mr. Frosty freezing container (Thermo Scientific, Waltham, MA, USA), first placed overnight in a 20 C freezer and subsequently in the gas phase of a liquid nitrogen container. For flow cytometry, cryopreserved cells were carefully thawed and washed, and 2e5 105 cells/well were incubated with monoclonal antibodies against CD3 (AF700), CD4 (PerCP-Cy5.5), CD56 (APC) (all from BD Pharmingen, Breda, The Netherlands) and CD8 (PE-Cy7; Biolegend, San Diego, CA, USA), at 4 C for 25 min in the dark. For the intracellular staining, cells were fixed for 20 min in Cytofix/Cytoperm (BD Bioscience, , CA, USA) at 4 C in the dark and then washed with Perm/ San Jose Wash buffer. Subsequently, the cells were resuspended in Perm/ wash buffer containing the antibodies against gzmA (PE; BD Pharmingen), gzmB (PE-CF594; BD Horizon) and gzmK (FITC; Santa Cruz Biotechnology, Dallas, TX, USA). The samples were analyzed by flow cytometry with a FACSCanto (BD Bioscience). The FlowJo software (Tree Star Inc, Ashland, OR, USA) was used to analyze the data. Lymphocytes were gated in the forward scatter versus side scatter dot plot. Cells were selected as CD3þ (T cells), or as CD3þCD4þ, CD3þCD8þ, CD3þCD56þ and CD3CD56þ (NK cells), and expression of gzms was analyzed in these populations. The results are expressed as percentage of cells of the specific population expressing the corresponding gzm and as the median intensity of fluorescence (MFI). Alternatively, cells were selected as positive for each gzm and the percentage of the above mentioned lymphocyte populations were analyzed within the gzmþ lymphocytes.
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2.4. Statistical analysis Data in the figures are expressed as box and whiskers showing the smallest observation, lower quartile, median, upper quartile and largest observation, or as individual data points. Data in the tables are expressed as medians with interquartile ranges or as single values. Comparisons between groups were performed using the ManneWhitney U test. Correlations were calculated using the Spearman's rho test. Analyses were done using GraphPad Prism version 5.01 (San Diego, CA, USA) and SPSS version 15.0 (Chicago, Ill, USA). P-values <0.05 were considered statistically significant. 3. Results 3.1. Patients and controls characteristics The characteristics of patients and controls included in the present study are provided in Table 1. 3.2. Intracellular expression of granzymes A, B and K by lymphocyte populations We sought to evaluate the intracellular expression of the gzms by different lymphocyte populations and the potential differences in this expression between patients and controls. For this purpose, we first analyzed the proportion of these lymphocyte populations present in both groups of individuals (Table 2). Patients with TB had similar proportions of CD8þ T, NK and CD56þ T cells, and lower CD4þ T cell numbers, when compared with healthy controls. The total number of gzm positive cells did not differ significantly between patients and controls. Then we analyzed which lymphocytes were expressing each gzm (Figure 1 and Supplementary Figure 1). In both patients and controls gzmA and gzmB were primarily expressed by NK and CD8þ T cells, while gzmK was mainly expressed by CD8þ T cells. In TB patients there was a slight increase in the percentage of CD56þ T cells expressing gzmA and K (P < 0.05 versus controls). Figure 2 shows the expression of each gzm within different lymphocyte populations, expressed as the percentage of gzmþ cells (Figure 2AeD) and the MFI of these cells (Figure 2EeH) (see Supplementary Figure 2 for gating strategy). Patients with TB demonstrated an increase in the percentage of CD8þ T, CD4þ T and CD56þ T cells expressing gzmA, as well as higher MFI of gzmA in these cells, when compared to controls. TB patients also had a significant increase in the percentage and the MFI of CD8þ T cells expressing gzmB. GzmK expression did not differ between groups. Positive correlations existed between the percentage of lymphocyte populations expressing gzmA versus gzmB in patients: CD8þ T cells (r ¼ 0.83, P < 0.0001), CD4þ T cells (r ¼ 0.69, P ¼ 0.002), CD56þ T cells (r ¼ 0.50, P ¼ 0.036) and NK cells (r ¼ 0.50, P ¼ 0.033).
3
Table 2 Proportion of lymphocyte populations and granzymes in patients with pulmonary tuberculosis and controls.
CD8þ T cells CD4þ T cells CD56þ T cells NK cells gzmAþ cells gzmBþ cells gzmKþ cells
Controls, N ¼ 12
Patients, N ¼ 18
17.2 22.3 3.9 13.3 28.0 29.9 8.3
19.0 12.9 6.4 12.7 34.7 34.6 6.9
(14.0e27.3) (19.2e25.6) (3.1e6.8) (9.9e14.5) (21.8e38.1) (26.0e35.6) (6.9e12.9)
(15.2e23.7) (10.4e18.0)** (4.1e8.2) (10.6e17.8) (22.6e42.6) (28.7e45.6) (5.1e9.3)
Values are calculated as percentage of total lymphocytes and presented as median percentage (interquartile range). NK: CD3CD56þ, gzm: granzyme. **P < 0.01 determined by ManneWhitney U test when compared to controls.
In controls this correlation was only observed in CD4þ T cells (r ¼ 0.76, P ¼ 0.005). To determine whether the correlation between cells expressing gzmA and gzmB was due to co-expression in the same cells, we analyzed the individual and concurrent expression of gzmA and gzmB in CD8þ T, CD56þ T and NK cells (only around 11% of CD4þ T cells were positive for any gzm). Most cells positive for gzmA or B, co-expressed both gzms in both patients and controls, although in CD8þ T cells less in controls than in patients (Supplementary Figure 3AeF). 3.3. Circulating levels of granzyme A and B To get insight into the extracellular role of gzms, serum levels of gzmA and B were measured in controls and patients. The concentrations of gzmB and, to a larger extent, of gzmA, were increased in TB patients (Figure 3). The serum levels of gzmA and gzmB did not correlate with their respective intracellular expression in either patients or controls (data not shown). There was also not correlation when taking into account each lymphocyte population. 3.4. Association of circulating levels of granzyme A and B and relevant disease biomarkers We next investigated whether circulating levels of gzms correlated with the plasma concentrations of established biomarkers of TB. For this we measured IL-6, CXCL8 and IP-10 (which are relevant in active TB) as well as sIL-2Ra, sTNFR-1, sTNFR-2 and CRP (which correlate with the extent of disease in TB [25]) in plasma of TB patients and healthy controls. As expected [25] and reported previously by our group [23], the plasma levels of all biomarkers were elevated in patients versus controls (all P < 0.0001; Supplementary Table 1). Levels of IFN-g, IL-12 and TNF-a remained below the limit of detection in most individuals (data not shown).
Table 1 Characteristics of patients with pulmonary tuberculosis and controls. Extracellular expression study
Intracellular expression study
Patients, N ¼ 58
Controls, N ¼ 31
Patients, N ¼ 18
Controls, N ¼ 12
Demographics Age (years) Gender (male) HIV-positive
32.7 ± 12.3 42 (72.4) 1 (1.7)
30.0 ± 5.7 23 (74.2) 0 (0)
28.7 ± 9.1 11 (61.1) 0
29.3 ± 5.7 10 (83.3) 0
Symptoms Fever Productive cough Weight loss
58 (100) 54 (93.1) 42 (72.4)
0 (0) 1 (3.2) 0 (0)
18 (100) 15 (83.3) 12 (66.7)
0 0 0
Values are number of individuals (%) except for age, which are mean ± standard deviation.
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Figure 1. Lymphocyte populations expressing granzymes A, B and K in patients with pulmonary tuberculosis and controls. Distribution of the gzmA, B and K positive cells among the different lymphocyte populations, expressed as percentage of total of each gzmþ lymphocytes. NK: CD3CD56þ, gzm: granzyme. N ¼ 12 controls and 18 patients. Data are boxand-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation. *P < 0.05 determined by ManneWhitney U test.
Figure 2. Expression of granzymes A, B and K by lymphocyte populations in patients with pulmonary tuberculosis and controls. Percentage of lymphocyte population expressing each gzm (AeD) and MFI of the intracellular expression of each granzyme in the lymphocyte populations (EeH). MFI values are expressed with Log10 scale. NK: CD3CD56þ, MFI: median fluorescence intensity, gzm: granzyme. N ¼ 12 controls and 18 patients. Data are box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation. *P < 0.05, **P < 0.01 determined by ManneWhitney U test.
We found positive weak correlations between gzmA levels with the concentrations of IL-6, CXCL8, IP-10, sIL-2Ra, sTNFR-1 and CRP in patients (Table 3). GzmB levels correlated positively with CXCL8, IP-10 and sIL-2Ra. Most of these associations were not found in the control group (data not shown). 4. Discussion In the present study we investigated the expression of gzms A, B and K in different lymphocyte populations in PBMCs as well as the serum levels of gzmA and B in patients with active pulmonary TB and healthy individuals. To our knowledge, this is the first study analyzing the intracellular expression of these three gzms in diverse lymphocyte populations of TB patients. The current study showed an increase in the expression of gzmA and B in T cells (mainly CD8þ T cells) from TB patients, but no differences in either gzmA or gzmB expression in NK cells nor in the expression of gzmK in any subgroup of lymphocytes. The analysis of the extracellular levels of gzmA and B also showed an increase of both gzms in patients relative to controls. We first aimed to identify the lymphocyte populations producing each gzm as well as the potential differences between TB patients and healthy individuals. As expected [5], both gzmA and B were mainly produced by NK and CD8þ T cells, whilst the main producers of gzmK were CD8þ T cells. This cellular distribution of gzm expression did not differ significantly between patients and controls. Since the number of NK cells in blood is relatively low
compared to T lymphocytes, while both cell types are quantitatively similar sources of gzms, likely a very high percentage of NK cells are gzmþ (and the same would be true for CD56þ T cells). This was indeed observed when we studied the proportion of each lymphocyte population expressing each gzm. This analysis produced additional relevant results when we compared such proportions between patients and controls: we observed a significant increase in the percentage of T lymphocytes subsets expressing gzmA (CD8þ T, CD4þ T and CD56þ T cells) and gzmB (CD8þ T cells) in TB patients. These increases were also found in the MFI, indicating that there are not only more T lymphocytes expressing gzms, but that the gzmþ cells are also expressing more gzm. Thus, T lymphocyte expression of gzmA and B is increased considerably in TB, but, although NK cells are big producers of gzmA and B, TB does not result in an increase of their gzm production. Rao et al. [20] also found no differences in the expression of gzmA and B in NK cells when comparing HIV negative TB patients with controls. A possible explanation for the apparent absence of stimulation of the expression of these gzms in NK cells during TB is a matter of timing. NK cells are basically innate immune cells, whilst T lymphocytes are essentially adaptive immune cells; possibly, at the moment the patients had developed symptoms and were recruited, the time for a relevant role of the NK cells had passed. In the same study of Rao et al. [20] they found that, after stimulation with M. tuberculosis or IL-15 þ IL-12, the percentage of NK cells expressing gzmB (but not gzmA) increased in both controls and TB patients. In this sense, the expression of gzmB in NK CD45ROþ (memory NK cells) from pleural
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Figure 3. Circulating levels of granzyme A and B in patients with pulmonary tuberculosis and controls. Serum levels of gzmA and gzmB in patients (N ¼ 58) and controls (N ¼ 31). Each circle represents an individual, with horizontal lines showing medians. Dotted lines represent the lower detection limit. *P < 0.05, ***P < 0.001 determined by ManneWhitney U test.
Table 3 Correlations of circulating levels of granzyme A and B with cytokines levels in patients with pulmonary tuberculosis. gzmA
IL-6 CXCL8 IP-10 sIL-2Ra sTNFR-1 sTNFR-2 sICAM-1 CRP gzmB
gzmB
r
P
r
P
0.42 0.46 0.38 0.38 0.27 0.28 0.20 0.32 0.24
0.001 0.0003 0.004 0.004 0.040 0.033 0.127 0.016 0.075
0.22 0.36 0.36 0.33 0.20 0.26 0.23 0.14
0.094 0.006 0.006 0.011 0.135 0.051 0.097 0.306
IL: interleukin; CXCL8: chemokine (CeXeC motif) ligand 8; IP-10: Interferon gamma-induced protein 10; sIL-2Ra: soluble interleukin-2 receptor a; sTNFR-1,-2: soluble tumor necrosis factor receptor-1 and -2; sICAM-1: soluble intercellular adhesion molecule-1; CRP: C-reactive protein; gzm: granzyme. Correlations were calculated using the Spearman's test. r: Spearman's rho coefficient of correlation. N ¼ 58.
5
fluid from patients increased after stimulation with IL-12 [21]. All this seems to indicate that NK cells express high amounts of gzmA and B constitutively, that the stimulation of NK cells to produce more gzms could be limited in time, and that the NK expression of gzmB is more prone to be stimulated than gzmA. On the other hand, it has been previously observed that the percentage of M. tuberculosis specific CD8þ T cells positive for gzmA and B is higher in active than in latent TB [22]. That would support the idea that the stimulation of CD8þ T cells to produce more gzmA and B depends on the development of active disease. However, contradictory results were recently published, showing down-regulated expression of gzmB in CD8þ T cells from active TB patients compared to latent TB and healthy controls [26]. Our results showed similar expression of gzmK in patients and controls in all lymphocyte populations studied, indicating that TB does not trigger its expression. To our knowledge, this is the first survey studying gzmK in the context of TB. When we analyzed the correlation between the percentage of lymphocyte populations expressing gzmA and gzmB, we found a strong correlation in CD8þ T cells from patients, but not from controls, suggesting that CD8þ T cells had been stimulated to produce both gzms simultaneously. This was confirmed by our finding of co-expression of both gzms in the same CD8þ T cells, which was much higher in patients than in controls. On the other hand, since a high percentage of NK cells express gzmA as well as gzmB, it is not surprising that we found co-expression of both gzms in these cells. Nevertheless, this co-expression was similar in patients and controls, and the correlation between these gzms was modest in NK cells from patients and inexistent in controls. This is consistent with the absence of increased expression found in these cells. For years, it has been thought that the effect of gzms almost exclusively involved target cell death. However, more and more evidence not only points to broader roles, but also questions whether all gzms are cytotoxic in vivo. Diverse non-cytotoxic and even non-lymphoid cells expressing gzms have been described, as well as multiple extracellular gzms substrates not related with cell death [3,6,7]. Moreover, gzms have been found extracellularly in plasma and serum as well as in other bodily fluids, with low levels in healthy individuals and higher levels related to some infectious and inflammatory diseases [6,7]. In the present study we have measured the serum levels of gzmA and B and, as observed in the analysis of the intracellular expression, it showed an increase of gzmB and, to a larger extent, of gzmA, in patients compared to controls. A previous report described higher levels of gzmB at diagnosis in TB patients with a slow response to treatment than in community controls [19], although another study failed to find differences in the levels of gzmB between patients and controls, but found higher levels of the gzm in patients with greater cavity sizes [27]. Although evidence support a purposeful release of gzms in absence of cytotoxicity, the origin of extracellular gzms is not clear. In an attempt to discern a possible source for the circulating gzms, we analyzed the correlation between the extra- and intracellular expression of these gzms, but we did not find significant results for any of the lymphocyte populations studied. Extracellular gzmA can be produced by g9d2 T cells by a non-cytotoxic mechanism and then soluble gzmA can induce M. tuberculosis infected macrophages to produce TNF-a which leads, by itself and/or in combination with other factors, to intracellular mycobacterial growth inhibition [28]. gd T cells tend to accumulate in mucosal epithelial tissues, as the lung [28], however, they do not comprise a high proportion of human lymphocytes. It is not unreasonable to think that a similar role exists for gzms released by other lymphocytes. Several studies have involved extracellular gzms in the production, release and/or processing of proinflammatory cytokines, such as IL-6, IL-8, TNF-a, IL-1b and IL-1a [7,13]. We analyzed the correlation between the
Please cite this article in press as: Garcia-Laorden MI, et al., Increased intra- and extracellular granzyme expression in patients with tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.05.016
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M.I. Garcia-Laorden et al. / Tuberculosis xxx (2015) 1e6
circulating levels of gzmA and B and the levels of several TB biomarkers, and we found some positive weak correlations (mainly with IL-8, IP-10 and sIL2Ra), suggesting that plasma gzm levels are indicative of the extent of inflammation. In conclusion, the results of the present study show enhanced intracellular expression of gzmA and B, but not K, by T lymphocytes, as well as raised extracellular levels, in patients with active pulmonary TB. The increase is more pronounced for gzmA than for gzmB. Further research is warranted to determine the functional role of gzms in host defense against M. tuberculosis. Acknowledgments We are grateful to the patients and the healthy volunteers for their trust and cooperation. We would also like to thank Mr. Sahid Ullah, Ms. Runia and Mr. Pradip Sen for performing and scoring the Ziehl-Neelsen stainings, Ms. Rupali and Mr. Yusuf for allowing us to work in the tuberculosis laboratory in the General Hospital, Anderkilla, Chittagong, Montri Ridjaibun, Nuttapol Panachuenwongsakul and Thatsanun Ngernseng for their help with the patient database, and Barbara Dierdorp and Tamara Dekker for performing the cytokine and chemokine measurements. In addition, we are grateful to the staff of Chittagong General Hospital and Chittagong Medical College & Hospital for their continuing support. Funding: This research was partially supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme. The study sponsor did not have any role in the study design, in the collection, analysis and interpretation of data, in the writing of the manuscript, or in the decision to submit the manuscript for publication. Competing interests: Ethical approval:
None declared. Not required.
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Please cite this article in press as: Garcia-Laorden MI, et al., Increased intra- and extracellular granzyme expression in patients with tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.05.016