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TLR2 and TLR4 expression on the immune cells of tuberculous pleural fluid
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Immunology Letters 117 (2008) 26–34
TLR2 and TLR4 expression on the immune cells of tuberculous pleural fluid Prabha C., Rajashree P., Sulochana D. Das ∗ Department of Immunology, Tuberculosis Research Centre (ICMR), Mayor V.R. Ramanathan Road, Chetput, Chennai 600031, India Received 4 October 2007; received in revised form 1 November 2007; accepted 4 November 2007 Available online 3 December 2007
Abstract Toll-like receptors (TLRs) play an important role in mediating the down stream signaling of immune response in tuberculosis. The predominance of Th1 response in tuberculous pleurisy prompted us to study the expression profiles of TLR2 and TLR4 on different immune cells and on subsets of T cells obtained from the site of infection. Our results showed that TLR2 was up-regulated on the monocytes from pleural fluid indicating a prominent role for this receptor in anti-tuberculous immunity. Notably, TLR2 and TLR4 expression were also enhanced on IFN-␥ secreting CD4+ T cells. However, their expression was down-regulated on activated and IL-4 secreting CD4+ T cells from the site of infection indicating that TLR expression is differentially modulated on the different subsets of T cells depending on their activation status and cytokine expression. The down-regulation of both TLRs on the natural regulatory T cells despite their higher number at the site of infection might be a mechanism to maintain their suppressive activity. © 2008 Published by Elsevier B.V. Keywords: Tuberculosis pleuritis; TLR2; TLR4; Regulatory T cells
1. Introduction Toll-like receptors (TLR) are pattern recognition receptors that recognize pathogen-associated molecular patterns and functions as critical mediators of proinflammatory cytokine response. Recent reports stand out as an evidence for the role of these receptors, principally TLR2 and TLR4 in tuberculosis (TB) immunity. These receptors appear to be the major mediator of Mycobacterium tuberculosis (MTB)-induced cellular activation [1,2]. Studies on TLR2 and TLR4 knock out mice have shown conflicting data. One of the studies has demonstrated that TLR2 and TLR4 are important for defense against infection with MTB but not essential for the pathology of infection [3]. While another study reported TLR4 signaling is required to mount a protective response during chronic MTB infection and in elimination of mycobacteria [4]. On the contrary, it was also reported that TLR4 is not required for the immunity against TB [5,6]. Thus infection with whole MTB bacilli evokes a
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Corresponding author. Tel.: +91 44 28369753; fax: +91 44 28362528. E-mail address: [email protected] (D.D. Sulochana).
0165-2478/$ – see front matter © 2008 Published by Elsevier B.V. doi:10.1016/j.imlet.2007.11.002
more complex activation pattern involving at least TLR2 and TLR4 leading to differential activation of antibacterial effector pathways. Analysis of human tissue expression of TLRs reveals that the lung is the major site for TLR mRNA expression [7]. Specifically TLR2 expression is up-regulated in alveolar macrophages and alveolar epithelial cell type II. There are very few studies available on TLR expression at the site of infection in TB. A study on tuberculous lymphadenitis demonstrates TLR2 expression on the cells of monocytes/macrophage lineage within granulomas [8]. A more detailed study on the association of TLR expression and IL-4 in the granuloma of TB patients has been reported [9]. Selective up-regulation of TLR, especially a splice variant of TLR1, on the leucocytes of TB patients has been evidenced [10]. There are accumulating evidences for the co-stimulatory functions of TLRs on T cells. It has been shown that TLR2 ligands co-stimulate the production of cytokines and proliferation of human CD4+ T cells. There are few reports on the significant alterations in T cell TLR levels in infectious diseases. Although the impact of altered T cell TLR expression on the pathology or immune defense is yet to be elucidated, it is certainly of
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immunological interest to study whether the TLR expression is altered in different subsets of T cells, especially in a disease like tuberculosis. However no studies are available till-date on the expression profile of TLR2 and TLR4 on the various subsets of CD4+ T cells from the site of infection in tuberculosis. The current study addresses this issue and reports the alterations in TLR2 and TLR4 expression on different subsets of CD4+ T cells using tuberculous pleuritis (TP) as a model. Since the immune response in TP is predominated by Th1 response, one can presume a role for TLRs, as these receptors in general induce Th1 response. Studying the modulations in the expression of these receptors on various immune cells at the site of infection may throw light on the importance of these receptors in protective immunity against TB. 2. Subjects and methods 2.1. Patients Blood and pleural fluid (PF) samples were collected from 15 subjects who were suspected to have tuberculous pleuritis based on the clinical symptoms and sputum. The patients were recruited at the Institute for Thoracic Medicine and Government General Hospital, Chennai. A postero-anterior view of the chest is taken with the X-ray in which the tracheal position, extent of effusion, evidence of fibrosis and parenchymal lesion, evidence of lymph nodes and calcification were looked for. Once the diagnosis of the PF was confirmed by chest X-ray, PF aspiration was done. The PF was sent for analysis to Government General Hospital, Chennai, which included the cytology, AFB staining, protein and sugar to ascertain the exudative nature of the effusion based on light’s criteria and to diagnose TB. The blood biochemistry, which included urea, sugar, creatinine and serum total proteins were also measured for these patients. PCR was carried out for pleural fluid using primers for the MTB-specific insertion element IS6110. Of the total pleuritis patients, 12 were diagnosed to have TB based on smear, culture and PCR positivity (IS6110-specific) of sputum or PF together with the clinical picture of chest Xray. These patients were positive by at least two of the above criteria, together with the clinical symptoms and were recruited for this study. The mean age of the study subjects was 32 years and these subjects were sero-negative for HIV. These patients did not have any previous history of tuberculosis. Samples were collected before the start of the treatment. Informed consent was obtained from each patient and the study was approved by relevant ethical review committee. 2.2. Isolation of mononuclear cells Pleural fluid was obtained from the patient by needle aspiration and 10–20 ml of blood was collected in a 50 ml tube (Falcon, USA), containing heparin (10 IU of heparin for 1 ml of blood) and processed within 1 h of drawing. The peripheral blood mononuclear cells (PBMC) and the pleural fluid mononuclear cells (PFMC) were isolated by Ficoll-Hypaque (Amersham Pharmacia) density gradient cen-
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trifugation. The cells were washed once in 1× Hank’s balanced salt solution (HBSS) (Whittaker) followed by washing with 1× phosphate-buffered saline (PBS). A final re-suspension was made in 1× PBS with 1% fetal calf serum (FCS), to the required concentration and the samples were processed for flow cytometry. 2.3. Immuno-fluorescent antibodies used To characterize the immune cell architecture in blood and pleural fluid, a panel consisting of unstained cells, isotype control (mouse IgG conjugated to FITC and PE, and T cells/B cells marker (CD3-FITC/CD-19PE), helper T cells/cytotoxic T cells marker (CD3-FITC/CD4-PE or CD8-PE) and T cells/NK cells markers (CD3-FITC/CD16+56-PE) were chosen. To assess the TLR2 and TLR4 expression on various immune cells the following combinations were used: TLR2/4-FITC with PE-conjugated with anti-CD4, −CD8, −CD19, −CD16+56 and −CD14. To assess the TLR expression on activated CD4+ T cells, antibodies of TLR2/4-FITC and CD4-PE were used in combination with the PerCP-tagged CD25 and CD69 and PECy5-tagged CD27 antibodies. To characterize natural regulatory T cells and TLR expression on these cells, the purified CD4+ cells were stained with TLR2/4-FITC, FoxP3-PE and CD25-PerCP. To measure the percentage of Th1 and Th2 cells expressing TLRs, the cells were stained with TLR2/4-FITC, IFN-␥/IL-4-PE and CD4PECy5. All the antibodies were purchased from BD biosciences (USA) except antibodies for FoxP3 and TLRs which were purchased from e-bioscience (San Diego, CA) and Imgenex (San Diego, CA), respectively. 2.4. Purification of CD4+ T cells The CD4+ T cells were isolated from fresh PBMC and PFMC by positive selection using CD4-conjugated magnetic microbeads and purified using MACS column according to the manufacturer’s instruction (Miltenyi Biotech, Bergisch Galdbach, Germany). The resulting cell population was >95% pure as assessed by flow cytometric analysis using anti-CD3 and CD4 antibodies. These cells were subsequently stained with antibodies against CD25, FoxP3 and TLR2/4 to enumerate the regulatory T cell population and TLR expression on these cells. 2.5. Flow cytometry Since the mRNA quantification not always represents the functional protein due to the post-translational modification mechanisms, we utilized flow cytometry to assess TLR proteins using reliable monoclonal antibodies. Also contamination/presence of other cells is not an issue in flow cytometry due to the possibility of using different combination of antibodies unlike quantitative real-time PCRs that demand high degree of purity of the isolated cell populations. The cells were stained for various surface molecules with the respective antibodies as per surface staining protocol. Briefly, 100 l of the cell suspension containing 0.5–1 × 106
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cells were washed with PBS and were resuspended in 100 l of staining buffer (1× PBS with 1% FCS) containing fluorescence-conjugated monoclonal antibodies at the concentration determined by titration for optimal staining (usually <1 g/1 × 106 cells corresponding to 2–4 l of the antibody solution). The cells were incubated in ice for 20–30 min and were washed with 1× PBS. These cells were fixed and either acquired directly or subjected to intracellular staining.
Table 1 Immune cell architecture of blood and pleural fluid in tuberculous pleuritis
PBMC PFMC
CD4
CD8
B cells
NK cells
Monocytes
29 ± 4.1 51 ± 4.4*
27 ± 4.1 24 ± 4.4
5.2 ± 1.2 6.8 ± 1.4
21 ± 13.5 9 ± 5.6*
7.1 ± 2.6 4.2 ± 0.7*
The mean percentage of various immune cells was assessed in PBMC and PFMC of patients with tuberculous pleuritis (N = 12) by flow cytometry. The values in the table represent the mean percentage of cells ± standard deviation. * Represents p < 0.05 when PFMC are compared with PBMC.
2.6. Intracellular staining For the intracellular staining of IFN-␥ and IL-4, the cells were fixed, permeabilized and stained according to the protocol of intracellular staining kit from BD biosciences. Since the initial standardization experiments showed that prior incubation with brefeldin A did not improve the percentage positive cell population of ex vivo IFN-␥ and IL-4, direct staining protocol was followed without brefeldin A incubation. Intracellular staining for FoxP3 was carried out following manufacturer’s instruction (ebioscience, San Diego, CA). Cells were acquired within 24 h of staining on FACS Calibur (Becton Dickinson, USA) and the analyses were done using CellQuest Pro software. For each sample, a total of 30,000 events within the gate were acquired. An unstained sample or negative isotype immunoglobulin-stained control and single-stained samples for each conjugated dye were used for compensation settings.
3.2. CD4+ T cell subsets from blood and pleural fluid
The significance of the observed differences was calculated using Mann–Whitney U-test. The correlation analysis was done using Pearsons correlation method. The analysis of the data was done using SPSS software version 13.0 and the p-value of less than 0.05 was considered to be significant.
The activation markers (CD25, CD27 and CD69) that have a prominent role in infectious diseases were studied on CD4+ T cells to understand the activation status of these cells. We did not observe any significant difference in expression levels of CD69 on CD4+ T cells obtained from PBMC and PFMC. Interestingly, the expression of CD25 and CD27 was significantly up-regulated on the CD4+ T cells of PFMC compared to PBMC indicating that most of the cells in PFMC are activated (Table 2). Although the results presented here includes total CD27+ cells, further analysis showed that this population was predominantly CD27high sub-type. Characterization of IFN-␥ and IL-4 secretion by CD4+ T cells showed significant increase only in IFN-␥ secreting CD4+ T cells in PFMC confirming their predominance at the site of infection (Table 2). Out of 12, 3 patients did not show detectable amount of IL-4 in the pleural cells and hence are not included for further analysis of TLR expression on IL-4 secreting CD4+ T cells. Since CD4+ T cells in the PF showed enhanced expression of CD25, the study was extended to find whether these CD4+ CD25+ T cells represent the expansion of regulatory T cell population in pleural fluid. And our results showed significant increase in natural Tregs at the site of infection, i.e. in PFMC (Table 2).
3. Results
3.3. TLR expression on immune cells
3.1. Immune architecture in blood and pleural fluid
The expression of TLR2 and TLR4 was studied on various immune cells of blood and PF. The expression of TLR2 was highest on monocytes, followed by B cells, CD16+56+ cells, and T cells (Fig. 1A). Interestingly, the expression of TLR2 was significantly increased on monocytes but decreased on B cells and CD16+56+ cells of PFMC compared to PBMC. Both CD4+ and CD8+ T cells of PBMC and PFMC did not show any difference in their TLR2 expression.
2.7. Statistical analysis
The immunological architecture of blood and pleural fluid in these study patients is listed in Table 1. The T cell profile was similar to the already reported profile from our previous study [11]. In addition, we also observed a significant decrease in the percentage of NK cells and monocytes in PFMC compared to PBMC.
Table 2 Characterization of CD4+ T cells from blood and pleural fluid in tuberculous pleuritis
PBMC PFMC
CD25 (N = 12)
CD27 (N = 12)
CD69 (N = 12)
IFN-␥ (N = 12)
IL-4 (N = 9)
Natural Tregs (N = 12)
4 ± 1.4 8 ± 1.6*
34 ± 11 57 ± 9*
6±2 7±2
4.30 ± 1.5 12.56 ± 2.6*
2.84 ± 1.2 4.02 ± 1.3
16.9 ± 3 22.8 ± 4*
The table represents the mean percentage of cells expressing CD25, CD27, CD69, IFN-␥, IL-4 and the natural regulatory T cells (Natural Tregs) among CD4+ T cell population from PBMC and PFMC of patients with tuberculous pleuritis. The values in the table represent the mean percentage of cells ± standard deviation. * Represents p < 0.05 when PFMC are compared with PBMC.
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Fig. 1. TLR2 and TLR4 expression on immune cells in tuberculous pleuritis. The mean percentage of various immune cells viz. CD4+ T cells, CD8+ T cells, B cells, CD16+56+ cells and monocytes expressing TLR2 (A) and TLR4 (B) was assessed in PBMC and PFMC of patients with TP (N = 12). The expression of the receptor was determined by flow cytometry using anti-TLR antibodies in combination with the antibodies for cell-surface markers. Results represent the mean ± S.E.M. values. *Represents p < 0.05 when PBMC are compared with PFMC. Representative scatter plots are shown in C.
The expression pattern of TLR4 was similar to that of TLR2 with maximum expression on monocytes. The only difference was CD16+56+ cells of PFMC expressed higher amount of TLR4 than that of PBMC (Fig. 1B). Though the expression levels of TLR4 were different on different immune cells, there was no difference between PBMC and PFMC TLR4 expression on T cells, B cells and monocytes. A representative scatter plot is shown in Fig. 1C.
3.4. TLR expression on subsets of CD4+ T cells In TP, CD4+ T cells predominate at the site of infection. To find whether the TLR expression is dependent on the activation status of T-helper cells and to find the association of IFN-␥ and IL-4 expression with TLRs, the expression levels of TLR2 and TLR4 were assessed in different subsets of CD4+ T cells.
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Fig. 2. TLR2 and TLR4 expression on subsets of CD4+ T cells in tuberculous pleuritis. The figure represents the mean percentage of CD25+ , CD27+ and CD69+ T-helper cell subsets expressing TLR2 and TLR4 (N = 12). The expression of the receptor was determined by flow cytometry using anti-TLR antibodies in combination with the antibodies for activation markers present on cell surface (CD25, CD27 and CD69).
Interestingly, TLR2 expression levels were down-regulated on activated CD4+ T cells derived from PFMC when compared with PBMC as shown in Fig. 2. This down-regulation was not dependent on the activation stage, whether early or late. Similarly, TLR4 expression levels were also down-regulated in activated T cells (CD25+ and CD27+ ) of PFMC but not in their early stage of activation (CD69+ ) (Figs. 2 and 3B). As shown in Fig. 3, the expression of TLR2 and TLR4 was significantly increased in IFN-␥ secreting CD4+ T cells of PFMC when compared with PBMC (p < 0.01). On the other hand, the expression of both the TLRs was significantly decreased on IL4 secreting CD4+ T cells of PFMC. Notably, the expression of TLR2 and TLR4 was significantly higher on IL-4+ CD4+ T cells than IFN-␥+ CD4+ T cells of PBMC (p < 0.01). However, this difference was not observed for PFMC. Representative scatter plots are shown in Fig. 3B. 3.5. TLR expression on natural Tregs The pattern of TLR2 and TLR4 expression on natural Tregs (CD4+ CD25+ FoxP3+ cells) of PBMC and PFMC is depicted in Fig. 4A and B. Surprisingly, both these receptors were downregulated on Tregs derived from the site of infection, i.e. from PFMC. The percentage of Tregs expressing TLR2 and TLR4 were almost similar in both blood and PF. 4. Discussion The immune response in tuberculous pleuritis (TP) is predominated by Th1 response. One can presume a role for TLRs in TP as these receptors in general, induce Th1 response. The expression profile of TLRs observed on various immune cells of blood and pleural fluid in this study agrees with the already reported mRNA expression profile of TLRs in blood cells and lungs, respectively [12]. Further we studied the expression pro-
files of TLR2 and TLR4 on different subsets of T cells obtained from the site of infection, i.e. pleural fluid. Our results on the activation status of CD4+ T cells in TP patients showed that only a small percentage of these cells were in the very early stage of activation in both blood and PF as observed by CD69 expression. The CD4+ T cells in PF showed enhanced expression of CD25 and expansion of natural Tregs. This observation concords with the recent report by Guyot-Revol et al. demonstrating the higher frequency of natural Tregs in PF of TP patients [13]. However, they have not reported the proportion of CD4+ CD25+ T cells expressing FoxP3, which was addressed in the current study by the use of monoclonal antibody against FoxP3. In humans, CD27 expression increases transiently with activation and is subsequently down-regulated on effector T cells after several rounds of cell division. This loss of CD27 correlates with high levels of effector function [14]. The increased CD27 expression on CD4+ T cells of PFMC indicates that these cells are in the activated state but yet to be differentiated into complete effector cells. Whether this increase is due to the upregulated expression of CD70 (ligand of CD27) needs further study. In a recent report, it was shown that in tuberculous lung tissue from mice, an abundance of CD27low lymphocytes was achieved by their preferential migration to the lung and by their local maturation following stimulation with mycobacterial antigens [15]. This is in contrast to the observation of this study from humans where most of the CD4+ T cells in the pleural fluid were CD27high . Further the author had demonstrated that the accumulation of CD27low CD4+ T cells in the lungs correlates with protection against fatal TB infection. If this observation applies to human TB, then the cause for the predominance of CD27high population at the disease site, in the protective immune response model of TB, needs further investigation. Purified mycobacterial antigens preferentially interact with TLR2 while infection with whole bacilli evokes a more complex activation pattern involving TLR2 and TLR4 leading to antimycobacterial effector pathways [16]. TP is a pauci-bacillary disease involving DTH response more to mycobacterial antigens than the bacilli per se. Hence the principal role for TLR2 than TLR4 can be speculated in this disease. Accordingly, TLR2 but not TLR4 expression was up-regulated on monocytes of PFMC when compared with PBMC, corroborating the involvement of this receptor in immunity at the site of infection. This supports the previous reports emphasizing the important role of TLR2 than TLR4 in anti-mycobacterial immunity [5,1,17]. Challenging of NK cells with the agonists of TLR2 was shown to enhance the pro-inflammatory cytokine production, especially IFN-␥, leading to anti-bacterial immunity [18]. The reason for the down-regulation of TLR2 on CD16+56+ cells despite the higher IFN-␥ levels observed in PFMC could not be explained in the present study. Interestingly, down-regulation of TLR2 was compensated by the up-regulation of TLR4 on these cells at the site of infection. Together, the present observation provides a clue for the possible role of TLR4 on NK cells in anti-mycobacterial immunity, especially at the site of infection in humans. In a report by O’Connor et al., it was demonstrated that almost all human CD56dim NK cells expressed TLR4 whilst CD56bright NK cells did not express TLR4 [19].
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Fig. 3. TLR2 and TLR4 expression on IFN-␥+ and IL-4+ CD4+ T-cells in tuberculous pleuritis. The boxplot represents the median, 25/75 percentile of IFN-␥+ (N = 12) and IL-4+ CD4+ T cells (N = 9) expressing TLR2 and TLR4 (A). The expression of the receptor was determined by flow cytometry using anti-TLR antibodies in combination with the antibodies for intracellular molecules (IFN-␥ and IL-4). *Represents p < 0.05 when PBMC are compared with PFMC. B represents TLR expression as determined by flow cytometry.
In contrary, our analysis of TLR2, -4 and-9 expression on NK cell subsets showed increased expression of these receptors on CD56bright NK cells (Supriya et al., communicated). There are controversial reports on the functionality of TLR4 on NK cells [18]. Hence, whether the observed increase in TLR4 expres-
sion on CD16+56+ cells despite lower NK cell percentage in PFMC is of any functional importance is now being studied. Further dissection of CD16+56+ cells into NK cells and NKT cells and their TLR expression is the focus of our future studies.
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Fig. 4. TLR2 and TLR4 expression on natural regulatory T cells in tuberculous pleuritis. The figure represents the mean percentage of natural regulatory Tregs expressing TLR2 and TLR4 from the blood and pleural fluid of 12 TP patients (A) and a representative FACS scatter plot (B). The expression of the receptor was determined by flow cytometry using anti-TLR antibodies in combination with the antibodies for CD25 and FoxP3. Results represent the mean ± S.E.M. values. *Represents p < 0.05 when PBMC are compared with PFMC.
The enhanced expression of TLR2 and TLR4 on IFN-␥ secreting CD4+ T cells is not surprising as the triggering of TLR expression by IFN-␥ is well documented [20]. An earlier report states that the induction of TLR expression on T cells is TCR activation-dependent [21]. Once induced, TLR2 signaling further enhances the T cell functions. Our observations agree with
this, as the T cells of PFMC are in the activated state and the functions of T cells, like IFN-␥ secretion is also enhanced. However, it was surprising to observe that the TLR2 and TLR4 were down-regulated in the activated CD4+ T cells obtained from the site of infection when compared to periphery. A similar observation of down-regulation of TLR on T cells was made in patients
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with filariasis [22]. The authors have proposed that this diminished expression and function of T cell TLR is a mechanism underlying T cell immune tolerance in lymphatic filariasis. This hypothesis may be extended to TP in the current study. However, the TLR expression should be further dissected on memory T cells and on T cells in the late activation stage to have an exact scenario of TLR2 and 4 expressions and to understand its functions. It has been reported that IL-4 down-regulates TLR2 expression and impairs signaling via TLR2 [23] Hence, downregulation of TLRs on Th2 cells at the site of infection might lead to the loss of co-stimulatory function of TLRs in these cells that in turn may block the development of Th2 cells further. It is to be noted that this down-regulation happens only at the site of infection and not in the periphery. On the other hand, immune response favors higher expression of TLRs by IFN-␥ secreting CD4+ T cells at the site of infection, a mechanism to increase the co-stimulatory effect of TLRs in these cells leading to perpetuation and activation of long-term T cell memory required for anti-tuberculosis immunity. In this study, we have not assessed the co-expression of TLR2 and TLR4. However, a correlation analysis was performed for these two receptors on various cell types and it showed a significant positive correlation for all immune cell types of PFMC except in NK cells (data not shown). The maximum positive correlation was seen for CD4+ T cells (r = 0.935). The positive correlation was also observed both in IFN-␥ and IL-4 secreting CD4+ T cells of PFMC. However, in PBMC, significant correlations could not be demonstrated except in CD4+ T cells and monocytes. These observations support the previous report by Fenhalls et al. demonstrating a significant association between the expression of TLR2 and TLR4 in the granulomas of tuberculosis patients suggesting a possible coordinated role of these receptors at the site of infection in tuberculosis [9]. Expression of TLRs on Tregs has been reported by few studies in mice [24,25]. The presence of TLRs on Tregs raises an intriguing possibility that the TLR triggering or down-regulation on Tregs might be involved in the immune inhibitory pathways. It was observed that both TLR2 and TLR 4 were down-regulated on the natural Tregs from the site of infection. Previous reports suggested that TLR2 agonists strongly enhanced the proliferation of TCR-triggered Tregs, rendering them transiently non-suppressive [26]. Thus the down-regulation of this receptor on Tregs despite their higher number at the site of infection might be a mechanism to maintain the suppressive activity of Tregs. This is in accordance with the recent study which has showed that the Treg cells derived from PF inhibited MTB-specific immunity [27]. In another study, increased expression of TLR4 on human CD25+ CD4+ Treg compared to CD25− CD4+ T cells and direct effects of highly purified LPS on the FoxP3 expression in Treg has been demonstrated [28]. This concords with the observations of the present study as Tregs express higher amount of TLR2 and TLR4 than the other subsets of CD4+ T cells. Thus the results of the present study suggest that TLRs may play an important role in adaptive immunity by directly enhancing antigen-specific Th1 cell function. Further characterization of the TLR functions on T cells and exploration of the causes for
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their modulations on different subsets of T cells would provide insight into the mechanism of T cell TLR mediated immune response to mycobacteria. Acknowledgements Prabha C. and Rajashree P., render their thanks to Indian Council of Medical Research (ICMR) for providing Senior Research fellowships. The kind help of Dr. Ramprasad Matsa, Madras Medical College and Dr. Gokula Krishnan, Institute of Thoracic Medicine, in collecting the pleural samples is sincerely acknowledged. Authors are grateful to Mr. S. Anbalagan for his technical assistance in flow cytometry. This work received partial support from the NIH through NIAID/TRC ICER programme. References [1] Means TK, Wang S, Lien E, Yoshimura A, Golenbock DT, Fenton MJ. Human toll-like receptors mediate cellular activation by Mycobacterium tuberculosis. J Immunol 1999;163:3920–7. [2] Underhill DM, Ozinsky A, Smith KD, Aderem A. Toll-like receptor2 mediates mycobacteria-induced proinflammatory signaling in macrophages. Proc Natl Acad Sci USA 1999;96:14459–63. [3] Reiling N, Holscher C, Fehrenbach A, Kroger S, Kirschning CJ, Goyert S, et al. Cutting edge: toll-like receptor (TLR)2- and TLR4-mediated pathogen recognition in resistance to airborne infection with Mycobacterium tuberculosis. J Immunol 2002;169:3480–4. [4] Abel B, Thieblemont N, Quesniaux VJ, Brown N, Mpagi J, Miyake K, et al. Toll-like receptor 4 expression is required to control chronic Mycobacterium tuberculosis infection in mice. J Immunol 2002;169:3155–62. [5] Kamath AB, Alt J, Debbabi H, Behar SM. Toll-like receptor 4-defective C3H/HeJ mice are not more susceptible than other C3H substrains to infection with Mycobacterium tuberculosis. Infect Immun 2003;71:4112–8. [6] Shim TS, Turner OC, Orme IM. Toll-like receptor 4 plays no role in susceptibility of mice to Mycobacterium tuberculosis infection. Tuberculosis (Edinb) 2003;83:367–71. [7] Zarember KA, Godowski PJ. Tissue expression of human toll-like receptors and differential regulation of toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol 2002;168:554–61. [8] Thoma-Uszynski S, Stenger S, Takeuchi O, Ochoa MT, Engele M, Sieling PA, et al. Induction of direct antimicrobial activity through mammalian toll-like receptors. Science 2001;291:1544–7. [9] Fenhalls G, Squires GR, Stevens-Muller L, Bezuidenhout J, Amphlett G, Duncan K, et al. Associations between toll-like receptors and interleukin4 in the lungs of patients with tuberculosis. Am J Respir Cell Mol Biol 2003;29:28–38. [10] Chang JS, Huggett JF, Dheda K, Kim LU, Zumla A, Rook GA. Myobacterium tuberculosis induces selective up-regulation of TLRs in the mononuclear leukocytes of patients with active pulmonary tuberculosis. J Immunol 2006;176:3010–8. [11] Jalapathy KV, Prabha C, Das SD. Correlates of protective immune response in tuberculous pleuritis. FEMS Immunol Med Microbiol 2004;40:139–45. [12] Hornung V, Rothenfusser S, Britsch S, Krug A, Jahrsdorfer B, Giese T, et al. Quantitative expression of toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol 2002;168:4531–7. [13] Guyot-Revol V, Innes JA, Hackforth S, Hinks T, Lalvani A. Regulatory T cells are expanded in blood and disease sites in patients with tuberculosis. Am J Respir Crit Care Med 2006;173:803–10. [14] Hendriks J, Xiao Y, Borst J. CD27 promotes survival of activated T cells and complements CD28 in generation and establishment of the effector T cell pool. J Exp Med 2003;198:1369–80.
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[22] Babu S, Blauvelt CP, Kumaraswami V, Nutman TB. Cutting edge: diminished T cell TLR expression and function modulates the immune response in human filarial infection. J Immunol 2006;176:3885–9. [23] Krutzik SR, Ochoa MT, Sieling PA, Uematsu S, Ng YW, Legaspi A, et al. Activation and regulation of toll-like receptors 2 and 1 in human leprosy. Nat Med 2003;9:525–32. [24] Sutmuller RP, den Brok MH, Kramer M, Bennink EJ, Toonen LW, Kullberg BJ, et al. Toll-like receptor 2 controls expansion and function of regulatory T cells. J Clin Invest 2006;116:485–94. [25] Liu H, Komai-Koma M, Xu D, Liew FY. Toll-like receptor 2 signaling modulates the functions of CD4+ CD25+ regulatory T cells. Proc Natl Acad Sci USA 2006;103:7048–53. [26] Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR, et al. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 2006;311:1770–3. [27] Chen X, Zhou B, Li M, Deng Q, Wu X, Le X, et al. CD4(+)CD25(+)FoxP3(+) regulatory T cells suppress Mycobacterium tuberculosis immunity in patients with active disease. Clin Immunol 2007;123:50–9. [28] Lewkowicz P, Lewkowicz N, Sasiak A, Tchorzewski H. Lipopolysaccharide-activated CD4+CD25+ T regulatory cells inhibit neutrophil function and promote their apoptosis and death. J Immunol 2006;177:7155–63.
Immunology Letters 117 (2008) 26–34
TLR2 and TLR4 expression on the immune cells of tuberculous pleural fluid Prabha C., Rajashree P., Sulochana D. Das ∗ Department of Immunology, Tuberculosis Research Centre (ICMR), Mayor V.R. Ramanathan Road, Chetput, Chennai 600031, India Received 4 October 2007; received in revised form 1 November 2007; accepted 4 November 2007 Available online 3 December 2007
Abstract Toll-like receptors (TLRs) play an important role in mediating the down stream signaling of immune response in tuberculosis. The predominance of Th1 response in tuberculous pleurisy prompted us to study the expression profiles of TLR2 and TLR4 on different immune cells and on subsets of T cells obtained from the site of infection. Our results showed that TLR2 was up-regulated on the monocytes from pleural fluid indicating a prominent role for this receptor in anti-tuberculous immunity. Notably, TLR2 and TLR4 expression were also enhanced on IFN-␥ secreting CD4+ T cells. However, their expression was down-regulated on activated and IL-4 secreting CD4+ T cells from the site of infection indicating that TLR expression is differentially modulated on the different subsets of T cells depending on their activation status and cytokine expression. The down-regulation of both TLRs on the natural regulatory T cells despite their higher number at the site of infection might be a mechanism to maintain their suppressive activity. © 2008 Published by Elsevier B.V. Keywords: Tuberculosis pleuritis; TLR2; TLR4; Regulatory T cells
1. Introduction Toll-like receptors (TLR) are pattern recognition receptors that recognize pathogen-associated molecular patterns and functions as critical mediators of proinflammatory cytokine response. Recent reports stand out as an evidence for the role of these receptors, principally TLR2 and TLR4 in tuberculosis (TB) immunity. These receptors appear to be the major mediator of Mycobacterium tuberculosis (MTB)-induced cellular activation [1,2]. Studies on TLR2 and TLR4 knock out mice have shown conflicting data. One of the studies has demonstrated that TLR2 and TLR4 are important for defense against infection with MTB but not essential for the pathology of infection [3]. While another study reported TLR4 signaling is required to mount a protective response during chronic MTB infection and in elimination of mycobacteria [4]. On the contrary, it was also reported that TLR4 is not required for the immunity against TB [5,6]. Thus infection with whole MTB bacilli evokes a
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Corresponding author. Tel.: +91 44 28369753; fax: +91 44 28362528. E-mail address: [email protected] (D.D. Sulochana).
0165-2478/$ – see front matter © 2008 Published by Elsevier B.V. doi:10.1016/j.imlet.2007.11.002
more complex activation pattern involving at least TLR2 and TLR4 leading to differential activation of antibacterial effector pathways. Analysis of human tissue expression of TLRs reveals that the lung is the major site for TLR mRNA expression [7]. Specifically TLR2 expression is up-regulated in alveolar macrophages and alveolar epithelial cell type II. There are very few studies available on TLR expression at the site of infection in TB. A study on tuberculous lymphadenitis demonstrates TLR2 expression on the cells of monocytes/macrophage lineage within granulomas [8]. A more detailed study on the association of TLR expression and IL-4 in the granuloma of TB patients has been reported [9]. Selective up-regulation of TLR, especially a splice variant of TLR1, on the leucocytes of TB patients has been evidenced [10]. There are accumulating evidences for the co-stimulatory functions of TLRs on T cells. It has been shown that TLR2 ligands co-stimulate the production of cytokines and proliferation of human CD4+ T cells. There are few reports on the significant alterations in T cell TLR levels in infectious diseases. Although the impact of altered T cell TLR expression on the pathology or immune defense is yet to be elucidated, it is certainly of
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immunological interest to study whether the TLR expression is altered in different subsets of T cells, especially in a disease like tuberculosis. However no studies are available till-date on the expression profile of TLR2 and TLR4 on the various subsets of CD4+ T cells from the site of infection in tuberculosis. The current study addresses this issue and reports the alterations in TLR2 and TLR4 expression on different subsets of CD4+ T cells using tuberculous pleuritis (TP) as a model. Since the immune response in TP is predominated by Th1 response, one can presume a role for TLRs, as these receptors in general induce Th1 response. Studying the modulations in the expression of these receptors on various immune cells at the site of infection may throw light on the importance of these receptors in protective immunity against TB. 2. Subjects and methods 2.1. Patients Blood and pleural fluid (PF) samples were collected from 15 subjects who were suspected to have tuberculous pleuritis based on the clinical symptoms and sputum. The patients were recruited at the Institute for Thoracic Medicine and Government General Hospital, Chennai. A postero-anterior view of the chest is taken with the X-ray in which the tracheal position, extent of effusion, evidence of fibrosis and parenchymal lesion, evidence of lymph nodes and calcification were looked for. Once the diagnosis of the PF was confirmed by chest X-ray, PF aspiration was done. The PF was sent for analysis to Government General Hospital, Chennai, which included the cytology, AFB staining, protein and sugar to ascertain the exudative nature of the effusion based on light’s criteria and to diagnose TB. The blood biochemistry, which included urea, sugar, creatinine and serum total proteins were also measured for these patients. PCR was carried out for pleural fluid using primers for the MTB-specific insertion element IS6110. Of the total pleuritis patients, 12 were diagnosed to have TB based on smear, culture and PCR positivity (IS6110-specific) of sputum or PF together with the clinical picture of chest Xray. These patients were positive by at least two of the above criteria, together with the clinical symptoms and were recruited for this study. The mean age of the study subjects was 32 years and these subjects were sero-negative for HIV. These patients did not have any previous history of tuberculosis. Samples were collected before the start of the treatment. Informed consent was obtained from each patient and the study was approved by relevant ethical review committee. 2.2. Isolation of mononuclear cells Pleural fluid was obtained from the patient by needle aspiration and 10–20 ml of blood was collected in a 50 ml tube (Falcon, USA), containing heparin (10 IU of heparin for 1 ml of blood) and processed within 1 h of drawing. The peripheral blood mononuclear cells (PBMC) and the pleural fluid mononuclear cells (PFMC) were isolated by Ficoll-Hypaque (Amersham Pharmacia) density gradient cen-
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trifugation. The cells were washed once in 1× Hank’s balanced salt solution (HBSS) (Whittaker) followed by washing with 1× phosphate-buffered saline (PBS). A final re-suspension was made in 1× PBS with 1% fetal calf serum (FCS), to the required concentration and the samples were processed for flow cytometry. 2.3. Immuno-fluorescent antibodies used To characterize the immune cell architecture in blood and pleural fluid, a panel consisting of unstained cells, isotype control (mouse IgG conjugated to FITC and PE, and T cells/B cells marker (CD3-FITC/CD-19PE), helper T cells/cytotoxic T cells marker (CD3-FITC/CD4-PE or CD8-PE) and T cells/NK cells markers (CD3-FITC/CD16+56-PE) were chosen. To assess the TLR2 and TLR4 expression on various immune cells the following combinations were used: TLR2/4-FITC with PE-conjugated with anti-CD4, −CD8, −CD19, −CD16+56 and −CD14. To assess the TLR expression on activated CD4+ T cells, antibodies of TLR2/4-FITC and CD4-PE were used in combination with the PerCP-tagged CD25 and CD69 and PECy5-tagged CD27 antibodies. To characterize natural regulatory T cells and TLR expression on these cells, the purified CD4+ cells were stained with TLR2/4-FITC, FoxP3-PE and CD25-PerCP. To measure the percentage of Th1 and Th2 cells expressing TLRs, the cells were stained with TLR2/4-FITC, IFN-␥/IL-4-PE and CD4PECy5. All the antibodies were purchased from BD biosciences (USA) except antibodies for FoxP3 and TLRs which were purchased from e-bioscience (San Diego, CA) and Imgenex (San Diego, CA), respectively. 2.4. Purification of CD4+ T cells The CD4+ T cells were isolated from fresh PBMC and PFMC by positive selection using CD4-conjugated magnetic microbeads and purified using MACS column according to the manufacturer’s instruction (Miltenyi Biotech, Bergisch Galdbach, Germany). The resulting cell population was >95% pure as assessed by flow cytometric analysis using anti-CD3 and CD4 antibodies. These cells were subsequently stained with antibodies against CD25, FoxP3 and TLR2/4 to enumerate the regulatory T cell population and TLR expression on these cells. 2.5. Flow cytometry Since the mRNA quantification not always represents the functional protein due to the post-translational modification mechanisms, we utilized flow cytometry to assess TLR proteins using reliable monoclonal antibodies. Also contamination/presence of other cells is not an issue in flow cytometry due to the possibility of using different combination of antibodies unlike quantitative real-time PCRs that demand high degree of purity of the isolated cell populations. The cells were stained for various surface molecules with the respective antibodies as per surface staining protocol. Briefly, 100 l of the cell suspension containing 0.5–1 × 106
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cells were washed with PBS and were resuspended in 100 l of staining buffer (1× PBS with 1% FCS) containing fluorescence-conjugated monoclonal antibodies at the concentration determined by titration for optimal staining (usually <1 g/1 × 106 cells corresponding to 2–4 l of the antibody solution). The cells were incubated in ice for 20–30 min and were washed with 1× PBS. These cells were fixed and either acquired directly or subjected to intracellular staining.
Table 1 Immune cell architecture of blood and pleural fluid in tuberculous pleuritis
PBMC PFMC
CD4
CD8
B cells
NK cells
Monocytes
29 ± 4.1 51 ± 4.4*
27 ± 4.1 24 ± 4.4
5.2 ± 1.2 6.8 ± 1.4
21 ± 13.5 9 ± 5.6*
7.1 ± 2.6 4.2 ± 0.7*
The mean percentage of various immune cells was assessed in PBMC and PFMC of patients with tuberculous pleuritis (N = 12) by flow cytometry. The values in the table represent the mean percentage of cells ± standard deviation. * Represents p < 0.05 when PFMC are compared with PBMC.
2.6. Intracellular staining For the intracellular staining of IFN-␥ and IL-4, the cells were fixed, permeabilized and stained according to the protocol of intracellular staining kit from BD biosciences. Since the initial standardization experiments showed that prior incubation with brefeldin A did not improve the percentage positive cell population of ex vivo IFN-␥ and IL-4, direct staining protocol was followed without brefeldin A incubation. Intracellular staining for FoxP3 was carried out following manufacturer’s instruction (ebioscience, San Diego, CA). Cells were acquired within 24 h of staining on FACS Calibur (Becton Dickinson, USA) and the analyses were done using CellQuest Pro software. For each sample, a total of 30,000 events within the gate were acquired. An unstained sample or negative isotype immunoglobulin-stained control and single-stained samples for each conjugated dye were used for compensation settings.
3.2. CD4+ T cell subsets from blood and pleural fluid
The significance of the observed differences was calculated using Mann–Whitney U-test. The correlation analysis was done using Pearsons correlation method. The analysis of the data was done using SPSS software version 13.0 and the p-value of less than 0.05 was considered to be significant.
The activation markers (CD25, CD27 and CD69) that have a prominent role in infectious diseases were studied on CD4+ T cells to understand the activation status of these cells. We did not observe any significant difference in expression levels of CD69 on CD4+ T cells obtained from PBMC and PFMC. Interestingly, the expression of CD25 and CD27 was significantly up-regulated on the CD4+ T cells of PFMC compared to PBMC indicating that most of the cells in PFMC are activated (Table 2). Although the results presented here includes total CD27+ cells, further analysis showed that this population was predominantly CD27high sub-type. Characterization of IFN-␥ and IL-4 secretion by CD4+ T cells showed significant increase only in IFN-␥ secreting CD4+ T cells in PFMC confirming their predominance at the site of infection (Table 2). Out of 12, 3 patients did not show detectable amount of IL-4 in the pleural cells and hence are not included for further analysis of TLR expression on IL-4 secreting CD4+ T cells. Since CD4+ T cells in the PF showed enhanced expression of CD25, the study was extended to find whether these CD4+ CD25+ T cells represent the expansion of regulatory T cell population in pleural fluid. And our results showed significant increase in natural Tregs at the site of infection, i.e. in PFMC (Table 2).
3. Results
3.3. TLR expression on immune cells
3.1. Immune architecture in blood and pleural fluid
The expression of TLR2 and TLR4 was studied on various immune cells of blood and PF. The expression of TLR2 was highest on monocytes, followed by B cells, CD16+56+ cells, and T cells (Fig. 1A). Interestingly, the expression of TLR2 was significantly increased on monocytes but decreased on B cells and CD16+56+ cells of PFMC compared to PBMC. Both CD4+ and CD8+ T cells of PBMC and PFMC did not show any difference in their TLR2 expression.
2.7. Statistical analysis
The immunological architecture of blood and pleural fluid in these study patients is listed in Table 1. The T cell profile was similar to the already reported profile from our previous study [11]. In addition, we also observed a significant decrease in the percentage of NK cells and monocytes in PFMC compared to PBMC.
Table 2 Characterization of CD4+ T cells from blood and pleural fluid in tuberculous pleuritis
PBMC PFMC
CD25 (N = 12)
CD27 (N = 12)
CD69 (N = 12)
IFN-␥ (N = 12)
IL-4 (N = 9)
Natural Tregs (N = 12)
4 ± 1.4 8 ± 1.6*
34 ± 11 57 ± 9*
6±2 7±2
4.30 ± 1.5 12.56 ± 2.6*
2.84 ± 1.2 4.02 ± 1.3
16.9 ± 3 22.8 ± 4*
The table represents the mean percentage of cells expressing CD25, CD27, CD69, IFN-␥, IL-4 and the natural regulatory T cells (Natural Tregs) among CD4+ T cell population from PBMC and PFMC of patients with tuberculous pleuritis. The values in the table represent the mean percentage of cells ± standard deviation. * Represents p < 0.05 when PFMC are compared with PBMC.
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Fig. 1. TLR2 and TLR4 expression on immune cells in tuberculous pleuritis. The mean percentage of various immune cells viz. CD4+ T cells, CD8+ T cells, B cells, CD16+56+ cells and monocytes expressing TLR2 (A) and TLR4 (B) was assessed in PBMC and PFMC of patients with TP (N = 12). The expression of the receptor was determined by flow cytometry using anti-TLR antibodies in combination with the antibodies for cell-surface markers. Results represent the mean ± S.E.M. values. *Represents p < 0.05 when PBMC are compared with PFMC. Representative scatter plots are shown in C.
The expression pattern of TLR4 was similar to that of TLR2 with maximum expression on monocytes. The only difference was CD16+56+ cells of PFMC expressed higher amount of TLR4 than that of PBMC (Fig. 1B). Though the expression levels of TLR4 were different on different immune cells, there was no difference between PBMC and PFMC TLR4 expression on T cells, B cells and monocytes. A representative scatter plot is shown in Fig. 1C.
3.4. TLR expression on subsets of CD4+ T cells In TP, CD4+ T cells predominate at the site of infection. To find whether the TLR expression is dependent on the activation status of T-helper cells and to find the association of IFN-␥ and IL-4 expression with TLRs, the expression levels of TLR2 and TLR4 were assessed in different subsets of CD4+ T cells.
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Fig. 2. TLR2 and TLR4 expression on subsets of CD4+ T cells in tuberculous pleuritis. The figure represents the mean percentage of CD25+ , CD27+ and CD69+ T-helper cell subsets expressing TLR2 and TLR4 (N = 12). The expression of the receptor was determined by flow cytometry using anti-TLR antibodies in combination with the antibodies for activation markers present on cell surface (CD25, CD27 and CD69).
Interestingly, TLR2 expression levels were down-regulated on activated CD4+ T cells derived from PFMC when compared with PBMC as shown in Fig. 2. This down-regulation was not dependent on the activation stage, whether early or late. Similarly, TLR4 expression levels were also down-regulated in activated T cells (CD25+ and CD27+ ) of PFMC but not in their early stage of activation (CD69+ ) (Figs. 2 and 3B). As shown in Fig. 3, the expression of TLR2 and TLR4 was significantly increased in IFN-␥ secreting CD4+ T cells of PFMC when compared with PBMC (p < 0.01). On the other hand, the expression of both the TLRs was significantly decreased on IL4 secreting CD4+ T cells of PFMC. Notably, the expression of TLR2 and TLR4 was significantly higher on IL-4+ CD4+ T cells than IFN-␥+ CD4+ T cells of PBMC (p < 0.01). However, this difference was not observed for PFMC. Representative scatter plots are shown in Fig. 3B. 3.5. TLR expression on natural Tregs The pattern of TLR2 and TLR4 expression on natural Tregs (CD4+ CD25+ FoxP3+ cells) of PBMC and PFMC is depicted in Fig. 4A and B. Surprisingly, both these receptors were downregulated on Tregs derived from the site of infection, i.e. from PFMC. The percentage of Tregs expressing TLR2 and TLR4 were almost similar in both blood and PF. 4. Discussion The immune response in tuberculous pleuritis (TP) is predominated by Th1 response. One can presume a role for TLRs in TP as these receptors in general, induce Th1 response. The expression profile of TLRs observed on various immune cells of blood and pleural fluid in this study agrees with the already reported mRNA expression profile of TLRs in blood cells and lungs, respectively [12]. Further we studied the expression pro-
files of TLR2 and TLR4 on different subsets of T cells obtained from the site of infection, i.e. pleural fluid. Our results on the activation status of CD4+ T cells in TP patients showed that only a small percentage of these cells were in the very early stage of activation in both blood and PF as observed by CD69 expression. The CD4+ T cells in PF showed enhanced expression of CD25 and expansion of natural Tregs. This observation concords with the recent report by Guyot-Revol et al. demonstrating the higher frequency of natural Tregs in PF of TP patients [13]. However, they have not reported the proportion of CD4+ CD25+ T cells expressing FoxP3, which was addressed in the current study by the use of monoclonal antibody against FoxP3. In humans, CD27 expression increases transiently with activation and is subsequently down-regulated on effector T cells after several rounds of cell division. This loss of CD27 correlates with high levels of effector function [14]. The increased CD27 expression on CD4+ T cells of PFMC indicates that these cells are in the activated state but yet to be differentiated into complete effector cells. Whether this increase is due to the upregulated expression of CD70 (ligand of CD27) needs further study. In a recent report, it was shown that in tuberculous lung tissue from mice, an abundance of CD27low lymphocytes was achieved by their preferential migration to the lung and by their local maturation following stimulation with mycobacterial antigens [15]. This is in contrast to the observation of this study from humans where most of the CD4+ T cells in the pleural fluid were CD27high . Further the author had demonstrated that the accumulation of CD27low CD4+ T cells in the lungs correlates with protection against fatal TB infection. If this observation applies to human TB, then the cause for the predominance of CD27high population at the disease site, in the protective immune response model of TB, needs further investigation. Purified mycobacterial antigens preferentially interact with TLR2 while infection with whole bacilli evokes a more complex activation pattern involving TLR2 and TLR4 leading to antimycobacterial effector pathways [16]. TP is a pauci-bacillary disease involving DTH response more to mycobacterial antigens than the bacilli per se. Hence the principal role for TLR2 than TLR4 can be speculated in this disease. Accordingly, TLR2 but not TLR4 expression was up-regulated on monocytes of PFMC when compared with PBMC, corroborating the involvement of this receptor in immunity at the site of infection. This supports the previous reports emphasizing the important role of TLR2 than TLR4 in anti-mycobacterial immunity [5,1,17]. Challenging of NK cells with the agonists of TLR2 was shown to enhance the pro-inflammatory cytokine production, especially IFN-␥, leading to anti-bacterial immunity [18]. The reason for the down-regulation of TLR2 on CD16+56+ cells despite the higher IFN-␥ levels observed in PFMC could not be explained in the present study. Interestingly, down-regulation of TLR2 was compensated by the up-regulation of TLR4 on these cells at the site of infection. Together, the present observation provides a clue for the possible role of TLR4 on NK cells in anti-mycobacterial immunity, especially at the site of infection in humans. In a report by O’Connor et al., it was demonstrated that almost all human CD56dim NK cells expressed TLR4 whilst CD56bright NK cells did not express TLR4 [19].
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Fig. 3. TLR2 and TLR4 expression on IFN-␥+ and IL-4+ CD4+ T-cells in tuberculous pleuritis. The boxplot represents the median, 25/75 percentile of IFN-␥+ (N = 12) and IL-4+ CD4+ T cells (N = 9) expressing TLR2 and TLR4 (A). The expression of the receptor was determined by flow cytometry using anti-TLR antibodies in combination with the antibodies for intracellular molecules (IFN-␥ and IL-4). *Represents p < 0.05 when PBMC are compared with PFMC. B represents TLR expression as determined by flow cytometry.
In contrary, our analysis of TLR2, -4 and-9 expression on NK cell subsets showed increased expression of these receptors on CD56bright NK cells (Supriya et al., communicated). There are controversial reports on the functionality of TLR4 on NK cells [18]. Hence, whether the observed increase in TLR4 expres-
sion on CD16+56+ cells despite lower NK cell percentage in PFMC is of any functional importance is now being studied. Further dissection of CD16+56+ cells into NK cells and NKT cells and their TLR expression is the focus of our future studies.
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Fig. 4. TLR2 and TLR4 expression on natural regulatory T cells in tuberculous pleuritis. The figure represents the mean percentage of natural regulatory Tregs expressing TLR2 and TLR4 from the blood and pleural fluid of 12 TP patients (A) and a representative FACS scatter plot (B). The expression of the receptor was determined by flow cytometry using anti-TLR antibodies in combination with the antibodies for CD25 and FoxP3. Results represent the mean ± S.E.M. values. *Represents p < 0.05 when PBMC are compared with PFMC.
The enhanced expression of TLR2 and TLR4 on IFN-␥ secreting CD4+ T cells is not surprising as the triggering of TLR expression by IFN-␥ is well documented [20]. An earlier report states that the induction of TLR expression on T cells is TCR activation-dependent [21]. Once induced, TLR2 signaling further enhances the T cell functions. Our observations agree with
this, as the T cells of PFMC are in the activated state and the functions of T cells, like IFN-␥ secretion is also enhanced. However, it was surprising to observe that the TLR2 and TLR4 were down-regulated in the activated CD4+ T cells obtained from the site of infection when compared to periphery. A similar observation of down-regulation of TLR on T cells was made in patients
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with filariasis [22]. The authors have proposed that this diminished expression and function of T cell TLR is a mechanism underlying T cell immune tolerance in lymphatic filariasis. This hypothesis may be extended to TP in the current study. However, the TLR expression should be further dissected on memory T cells and on T cells in the late activation stage to have an exact scenario of TLR2 and 4 expressions and to understand its functions. It has been reported that IL-4 down-regulates TLR2 expression and impairs signaling via TLR2 [23] Hence, downregulation of TLRs on Th2 cells at the site of infection might lead to the loss of co-stimulatory function of TLRs in these cells that in turn may block the development of Th2 cells further. It is to be noted that this down-regulation happens only at the site of infection and not in the periphery. On the other hand, immune response favors higher expression of TLRs by IFN-␥ secreting CD4+ T cells at the site of infection, a mechanism to increase the co-stimulatory effect of TLRs in these cells leading to perpetuation and activation of long-term T cell memory required for anti-tuberculosis immunity. In this study, we have not assessed the co-expression of TLR2 and TLR4. However, a correlation analysis was performed for these two receptors on various cell types and it showed a significant positive correlation for all immune cell types of PFMC except in NK cells (data not shown). The maximum positive correlation was seen for CD4+ T cells (r = 0.935). The positive correlation was also observed both in IFN-␥ and IL-4 secreting CD4+ T cells of PFMC. However, in PBMC, significant correlations could not be demonstrated except in CD4+ T cells and monocytes. These observations support the previous report by Fenhalls et al. demonstrating a significant association between the expression of TLR2 and TLR4 in the granulomas of tuberculosis patients suggesting a possible coordinated role of these receptors at the site of infection in tuberculosis [9]. Expression of TLRs on Tregs has been reported by few studies in mice [24,25]. The presence of TLRs on Tregs raises an intriguing possibility that the TLR triggering or down-regulation on Tregs might be involved in the immune inhibitory pathways. It was observed that both TLR2 and TLR 4 were down-regulated on the natural Tregs from the site of infection. Previous reports suggested that TLR2 agonists strongly enhanced the proliferation of TCR-triggered Tregs, rendering them transiently non-suppressive [26]. Thus the down-regulation of this receptor on Tregs despite their higher number at the site of infection might be a mechanism to maintain the suppressive activity of Tregs. This is in accordance with the recent study which has showed that the Treg cells derived from PF inhibited MTB-specific immunity [27]. In another study, increased expression of TLR4 on human CD25+ CD4+ Treg compared to CD25− CD4+ T cells and direct effects of highly purified LPS on the FoxP3 expression in Treg has been demonstrated [28]. This concords with the observations of the present study as Tregs express higher amount of TLR2 and TLR4 than the other subsets of CD4+ T cells. Thus the results of the present study suggest that TLRs may play an important role in adaptive immunity by directly enhancing antigen-specific Th1 cell function. Further characterization of the TLR functions on T cells and exploration of the causes for
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their modulations on different subsets of T cells would provide insight into the mechanism of T cell TLR mediated immune response to mycobacteria. Acknowledgements Prabha C. and Rajashree P., render their thanks to Indian Council of Medical Research (ICMR) for providing Senior Research fellowships. The kind help of Dr. Ramprasad Matsa, Madras Medical College and Dr. Gokula Krishnan, Institute of Thoracic Medicine, in collecting the pleural samples is sincerely acknowledged. Authors are grateful to Mr. S. Anbalagan for his technical assistance in flow cytometry. This work received partial support from the NIH through NIAID/TRC ICER programme. References [1] Means TK, Wang S, Lien E, Yoshimura A, Golenbock DT, Fenton MJ. Human toll-like receptors mediate cellular activation by Mycobacterium tuberculosis. J Immunol 1999;163:3920–7. [2] Underhill DM, Ozinsky A, Smith KD, Aderem A. Toll-like receptor2 mediates mycobacteria-induced proinflammatory signaling in macrophages. Proc Natl Acad Sci USA 1999;96:14459–63. [3] Reiling N, Holscher C, Fehrenbach A, Kroger S, Kirschning CJ, Goyert S, et al. Cutting edge: toll-like receptor (TLR)2- and TLR4-mediated pathogen recognition in resistance to airborne infection with Mycobacterium tuberculosis. J Immunol 2002;169:3480–4. [4] Abel B, Thieblemont N, Quesniaux VJ, Brown N, Mpagi J, Miyake K, et al. Toll-like receptor 4 expression is required to control chronic Mycobacterium tuberculosis infection in mice. J Immunol 2002;169:3155–62. [5] Kamath AB, Alt J, Debbabi H, Behar SM. Toll-like receptor 4-defective C3H/HeJ mice are not more susceptible than other C3H substrains to infection with Mycobacterium tuberculosis. Infect Immun 2003;71:4112–8. [6] Shim TS, Turner OC, Orme IM. Toll-like receptor 4 plays no role in susceptibility of mice to Mycobacterium tuberculosis infection. Tuberculosis (Edinb) 2003;83:367–71. [7] Zarember KA, Godowski PJ. Tissue expression of human toll-like receptors and differential regulation of toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol 2002;168:554–61. [8] Thoma-Uszynski S, Stenger S, Takeuchi O, Ochoa MT, Engele M, Sieling PA, et al. Induction of direct antimicrobial activity through mammalian toll-like receptors. Science 2001;291:1544–7. [9] Fenhalls G, Squires GR, Stevens-Muller L, Bezuidenhout J, Amphlett G, Duncan K, et al. Associations between toll-like receptors and interleukin4 in the lungs of patients with tuberculosis. Am J Respir Cell Mol Biol 2003;29:28–38. [10] Chang JS, Huggett JF, Dheda K, Kim LU, Zumla A, Rook GA. Myobacterium tuberculosis induces selective up-regulation of TLRs in the mononuclear leukocytes of patients with active pulmonary tuberculosis. J Immunol 2006;176:3010–8. [11] Jalapathy KV, Prabha C, Das SD. Correlates of protective immune response in tuberculous pleuritis. FEMS Immunol Med Microbiol 2004;40:139–45. [12] Hornung V, Rothenfusser S, Britsch S, Krug A, Jahrsdorfer B, Giese T, et al. Quantitative expression of toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol 2002;168:4531–7. [13] Guyot-Revol V, Innes JA, Hackforth S, Hinks T, Lalvani A. Regulatory T cells are expanded in blood and disease sites in patients with tuberculosis. Am J Respir Crit Care Med 2006;173:803–10. [14] Hendriks J, Xiao Y, Borst J. CD27 promotes survival of activated T cells and complements CD28 in generation and establishment of the effector T cell pool. J Exp Med 2003;198:1369–80.
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