Rationale: IL-22–producing helper T cells (Th22 cells) have been reported to be involved in tuberculosis infection. However, differentiation and immune regulation of Th22 cells in tuberculous pleural effusion (TPE) remain unknown.
Objectives: To elucidate the mechanism by which Th22 cells differentiate and recruit into the pleural space.
Methods: The distribution and phenotypic features of Th22 cells in both TPE and blood were determined. The impacts of proinflammatory cytokines and antigen presentation by pleural mesothelial cells (PMCs) on Th22-cell differentiation were explored. The chemoattractant activity of chemokines produced by PMCs for Th22 cells was observed.
Measurements and Main Results: Th22 cells were significantly higher in TPE than in blood. IL-1β, IL-6, and/or tumor necrosis factor-α promoted Th22-cell differentiation from CD4+ T cells. It was found that PMCs expressed CCL20, CCL22, and CCL27, and that TPE and PMC supernatants were chemotactic for Th22 cells. This activity was partly blocked by anti-CCL20, anti-CCL22, and anti-CCL27 antibodies. IL-22 and IL-17 significantly improved PMC wound healing. Moreover, PMCs were able to stimulate CD4+ T-cell proliferation and Th22-cell differentiation by presenting tuberculosis-specific antigen.
Conclusions: The overrepresentation of Th22 cells in TPE may be due to pleural cytokines and to PMC-produced chemokines. Our data suggest a collaborative loop between PMCs and Th22 cells in TPE. In particular, PMCs were able to function as antigen-presenting cells to stimulate CD4+ T-cell proliferation and Th22-cell differentiation.
Although IL-22–producing helper T (Th22) cells have been reported to be involved in human tuberculosis, the mechanisms by which Th22 cells differentiate and are recruited into the pleural space are unknown.
Pleural mesothelial cells stimulate CD4+ T-cell proliferation and Th22 cell differentiation in response to tuberculosis antigen. Our results suggest that the accumulation of Th22 cells in tuberculous pleural effusion may be due to the increased local proinflammatory cytokines and to pleural mesothelial cell–produced chemokines.
Naive CD4+ T cells can develop into various helper T (Th) subsets with different cytokine profiles and play a discriminative role in translating antigen-specific immune responses into tissue functions or immunopathology. Cellular immunity, particularly of CD4+ T cells, IFN-γ and tumor necrosis factor (TNF)-α, has a central role in the control of and protection against Mycobacterium tuberculosis (MTB) infection (1). Tuberculous pleural effusion (TPE) is caused by a severe delayed-type hypersensitivity reaction in response to the rupture of a subpleural focus of MTB infection. An accumulation of lymphocytes, especially CD4+ T cells, in TPE has been well documented (2). More and more data have demonstrated that several Th subsets, such as Th1 cells (3), Th17 cells (4), regulatory T cells (5), and so on, are involved in the pathogenesis of TPE. Therefore, TPE is an ideal model for studying the cellular immunity of Th subsets in MTB infection.
The pleural mesothelium is a metabolically active monolayer of cells covering the chest wall and lungs. Pleural mesothelial cells (PMCs) are the most common cells of the pleural space that not only encounter invading microbes, but also subsequently initiate and propagate an inflammatory reaction by coordinating the other kinds of inflammatory cells (6). Actually, early studies have demonstrated that PMCs facilitate monocyte transmigration across the pleural mesothelium during MTB infection (7).
IL-22 is a member of the IL-10 cytokine family and is produced primarily by Th17 T cells (8). The relationship between IL-22 and IL-17, as well as between IL-17 and IFN-γ, is of particular interest, and their expression is often linked to proinflammatory processes. IL-22 exerts functions similar to those of IL-17, both contributing to the control of extracellular bacterial infection (9). However, IL-22 can also be produced by non–Th17-cell types independently of IL-17 production (10, 11). Furthermore, IL-22 has specific functions, such as induction of wound-healing responses and tissue repair protecting from myocarditis (12) or liver disease (13), where IL-17 is not implicated.
It was well known that Th17 and Th1 cells are involved in MTB infection (14, 15). It has been reported that like Th17 and Th1 cells, IL-22–producing helper T (Th22) cells may also contribute to the immune response against MTB infection (16–18). In the present study, we investigated the distribution of Th22 cells in relation to Th17 and Th1 cells in TPE, the phenotypic characteristics of Th22 cells, the possible mechanism of differentiation and recruitment of Th22 cells into pleural space, and the capabilities of PMCs to function as antigen-presenting cells to stimulate Th22-cell differentiation in response to MTB antigens.
See the online supplement for an extended version of Methods.
The study protocol was approved by our institutional review boards for human studies, and informed consent was obtained from all subjects. Twenty-eight patients (see Table E1 in the online supplement) were proven to have TPE, as evidenced by growth of MTB from pleural fluid or by demonstration of granulomatous pleurisy on closed pleural biopsy specimen in the absence of any evidence of other granulomatous diseases. The methods of collecting and processing TPE and blood samples are described in the online supplement.
The expression of markers on T cells from TPE and blood, and the expression of molecules HLA-DR, CD80, CD86, IL-22 receptor (IL-22R), IL-17R, as well IFN-γR1 on PMCs, were determined by flow cytometry as previously described (19), and the antibody panel is described in the online supplement.
Methods for isolating naive CD4+ T cells and PMCs are described in the online supplement.
The expression of chemokines CCL20, CCL22, and CCL27 on PMCs isolated from TPE was identified by immunofluorescence staining and confocal imaging, and the methods are described in the online supplement.
The concentrations of CCL20, CCL22, and CCL27 in TPE and sera were measured with ELISA kits according to the instructions provided by manufacturer.
Differentiation of Th22 cells from naive CD4+ T cells in vitro was determined in the presence of one or more proinflammatory cytokines, as previously described (19) with slight modifications, and the cytokine panel is described in the online supplement.
Th22-cell in vitro chemotaxis assays were performed as previously described (19) with slight modifications (see the online supplement).
The in vitro experiments, involving an injury model and long-term culture of PMCs, were designed to evaluate the effects of IL-22, IL-17, and IFN-γ on the growth of PMCs in wound healing and late-stage repair. The methods are described in the online supplement.
The methods for determining CD4+ T-cell proliferation and Th22-, Th17-, and Th1-cell differentiation stimulated by antigen presentation by PMCs are described in the online supplement.
Data are expressed as means ± SEM (unless otherwise indicated in the figure legends). For a description of statistical methods see the extended version of Methods in the online supplement.
Because Th22 cells are always linked with Th17 cells (20) and may also be linked with Th1 cells (21), we first investigated the distribution of Th22 cells in relation to Th17 and Th1 cells in TPE. Flow cytometry was performed on mononuclear cells from TPE and peripheral blood with gating on CD3+ and CD8− T cells (Figure 1A). We noted that most IL-22–producing T cells were CD4+ T cells; however, they could also be present within CD8+ T cells to a lesser extent. IL-22+, IL-17+, and IFN-γ+ CD4+ T cells were found in both TPE and blood, and some CD4+ T cells were IL-22+IL-17+ and IL-22+IFN-γ+, especially in TPE (Figure 1B).
As shown in Figure 1C, percentages of Th22 cells represented the higher values in TPE (4.4 ± 0.3%), showing a significant increase in comparison with those in the corresponding blood (0.9 ± 0.1%, n = 28; Wilcoxon signed-rank test, P < 0.001). Similarly, significant increases in both Th17 and Th1 cells were observed in TPE (2.7 ± 0.1 and 40.2 ± 2.0%, respectively) compared with those in blood (0.7 ± 0.1 and 9.3 ± 0.7%, respectively; n = 28; both P < 0.001). It was noted that numbers of pleural Th22 cells were correlated positively with numbers of Th17 and Th1 cells, respectively (both P < 0.001; Figure 1D). As shown in Table E2, neither age nor sex affected the distribution of Th22, Th17, and Th1 cells in TPE.
We also noted that IL-22+IL-17+ and IL-22+IFN-γ+ CD4+ T cells were significantly higher in TPE (1.5 ± 0.1 and 1.5 ± 0.2%, respectively) than in corresponding blood (0.3 ± 0.1 and 0.4 ± 0.1%, respectively; both P < 0.001).
We observed that most Th22 cells expressed high levels of CD45RO in TPE and blood (85.5 ± 1.6 vs. 70.0 ± 1.3%, respectively; n = 28; P < 0.001), low levels of CD45RA (7.3 ± 0.4 vs. 8.6 ± 0.5%, respectively; P = 0.018), and low levels of CD62L (5.6 ± 0.4 vs. 9.5 ± 0.5%, respectively; P < 0.001), indicating that they were effector memory cells, especially those in TPE (Figure 2). Also as shown in Figure 2, pleural Th22 cells expressed a medium level of chemokine receptor-7 (CCR7, 55.5 ± 1.6%), which was much higher compared with blood Th22 cells (8.0 ± 0.8%; P < 0.001).
Except for CCR7, the expression profiles of the other CCRs on Th22 cells are shown in Figure 3. Overall, pleural Th22 cells expressed high levels of CCR4, CCR6, and CCR10 and low levels of CCR2, CCR3, and CCR5. In addition, much lower expression levels of CCR3, CCR4, CCR5, and CCR6, but not of CCR2 and CCR10, were seen on blood Th22 cells compared with their compartments on pleural Th22 cells.
Because some proinflammatory cytokines, such as IL-1β, IL-6, TNF-α, and IFN-γ, have been reported to be elevated in TPE (22–24), we evaluated the contribution of these cytokines to the differentiation of Th22 cells. With IL-2–containing medium providing a baseline for comparison, IL-1β, IL-6, and TNF-α, but not IFN-γ, were able to promote the differentiation of pleural Th22 cells from naive CD4+ T cells (Figure 4A). As expected, the combinations of IL-1β plus IL-6, IL-1β plus TNF-α, IL-6 plus TNF-α, and IL-1β plus IL-6 plus TNF-α increased the percentages of Th22 cells to even higher extents compared with the corresponding single cytokine. IFN-γ did not affect Th22-cell numbers; on the contrary, it could reduce the increased percentage of Th22 cells stimulated by the other cytokines.
We also found that the previously mentioned proinflammatory cytokines or their combinations could promote differentiation of Th22 cells from blood-derived naive CD4+ T cells in a similar manner (Figure 4A).
To further confirm Th22 differentiation stimulated by proinflammatory cytokines, we cultured carboxyfluorescein succinimidyl ester (CFSE)–labeled CD4+ T cells in medium alone or in the presence of IL-6 plus TNF-α under the previously described conditions. CFSE+ cells were then analyzed by flow cytometry. Figures 4B and 4C present representative flow cytometric dot plots from one of five independent experiments, showing enhanced CD4+ T-cell proliferation and Th22-cell differentiation stimulated by IL-6 plus TNF-α.
Our data showed that concentrations of chemokines CCL20, CCL22, and CCL27 in TPE were much higher than their compartments in serum (all P < 0.001) (Figure 5A). In addition, significant expression of CCL20, CCL22, and CCL27 was observed in all PMCs identified by anti-calretinin monoclonal antibody (mAb), using confocal microscopy (Figure 5B). Taken together with our observation that Th22 cells expressed high level of CCR6, CCR4, and CCR10 (Figure 3), which are ligands for CCL20, CCL22, and CCL27, respectively, we hypothesized that Th22 cells could migrate into the pleural space in response to PMC-produced chemokines. Indeed, both TPE and supernatants of cultured PMCs exerted a potent chemoattractant activity for circulating Th22 cells, whereas anti-CCL20, anti-CCL22, and anti-CCL27 mAbs significantly suppressed Th22-cell chemotaxis (Figure 5C). Therefore, recruitment of Th22 cells into TPE could be induced by PMCs via CCL20–CCR6, CCL22–CCR4, and/or CCL27–CCR10 axes.
As shown in Figures 6A and 6B, substantial expression levels of IL-22R (61.5 ± 4.0%, n = 5), IL-17R (57.4 ± 3.2%), and IFN-γR1 (65.7 ± 5.4%) were observed on PMCs isolated from TPE.
To evaluate the effects of IL-22, IL-17, and IFN-γ on the growth of PMCs in early-stage repair, an in vitro injury model was used. As early as 16 hours after wounding, both IL-22 and IL-17 had substantial and persistent improving effects on PMC layer closure (Figures 6C and 7A). These two cytokines did not significantly affect PMC viability (Figure 7B), and further promoted PMC proliferation (Figures 7C and 7D). Unexpectedly, IFN-γ was noted to be harmful to PMC wound healing during the whole 48-hour culture, and even to decrease PMC viability and proliferation (Figures 6C and 7).
As expected, we also observed that addition of anti−IL-22 or −IL-17 mAb to the TPE supernatants exerted a potent inhibitory activity on PMC wound healing, with stronger activity observed with anti−IL-22 mAb, compatible with findings that the IL-22 concentration was higher than the IL-17 concentration in TPE (18); in contrast, addition of anti–IFN-γ mAb significantly improved PMC wound healing (Figures 6C, 7A, and 7B). Moreover, similar effects were further confirmed by proliferation assay (Figures 7C and 7D).
In addition, in in vitro experiments involving the long-term culture of PMCs, designed to evaluate effects of the previously described cytokines on the growth of PMCs in late-stage repair, both IL-22 and IL-17 could significantly improve long-term restoration of PMCs as represented by the density of cells; in contrast, IFN-γ severely impaired this restoration (Figure 8).
Our data showed that PMCs isolated from TPE (n = 5) expressed more class II MHC protein, HLA-DR, than PMCs from transudative effusion (n = 5) did, and that compared with PMCs from transudative effusion, PMCs from TPE expressed much higher levels of CD80 and CD8, two proteins with important roles as costimulatory signals for T-cell responses (all P < 0.01) (Figures 9A and 9B) (25).
It was found that in the absence of PMCs, the numbers of Ki-67+CD4+ T cells were low even in the presence of antigen 85B (10 μg/ml), a major protein secreted by all Mycobacterium species and belonging to the antigen 85 family; the addition of PMCs isolated from TPE, but not those from transudative effusion, yielded a significant increase in Ki-67+CD4+ T cells even in the absence of antigen 85B, suggesting that PMCs could process antigen to which they were exposed within the TPE environment and present them to CD4+ T cells in in vitro culture (Figure 9C). The addition of exogenous antigen 85B to the PMC–CD4+ T-cell coculture resulted in even greater proliferation, indicating that PMCs were capable of processing antigen in vitro. We also noted that IL-22, IL-17, and IFN-γ each could amplify the enhanced proliferation response of CD4+ T cells induced by antigen presentation by PMCs (Figure 9C).
We cultured CD4+ T cells and PMCs with antigen 85B in the presence or absence of anti-CD80 mAb, anti-CD86 mAb, a combination of both, or CTLA-4Ig. Anti-CD80 and anti-CD86 mAbs alone partially blocked proliferation responses; a combination of both anti-CD80 and anti-CD86 blocking mAbs and CTLA-4Ig yielded even greater inhibition of PMC-elicited T-cell proliferation (Figure 9D).
As expected, PMCs were able to promote differentiation of Th22 (Figure 10A), Th17 (Figure 10C), and Th1 (Figure 10E) cells from naive CD4+ T cells by presenting antigen. We also noted that IL-22 and IL-17 could amplify the enhanced differentiation of Th22, Th17, and Th1 cells induced by antigen presentation by PMCs, and that IFN-γ amplified the enhanced differentiation of Th1 cells, but not Th22 or Th17 cells, induced by antigen presentation by PMCs. In addition, either anti-CD80 or anti-CD86 mAb alone partially suppressed the differentiation of Th22 cells (Figure 10B), Th17 cells (Figure 10D), and Th1 cells (Figure 10E) induced by antigen presentation by PMCs, and the combination of anti-CD80 and anti-CD86 mAbs or CTLA-4Ig, to block both B7-1– and/or B7-2–mediated costimulation.
In the present study, we have confirmed that like Th17 cells (4) and Th1 cells (3), Th22 cells have also been found to be present in TPE (18, 26). Our data have demonstrated that the numbers of Th22, Th17, and Th1 cells represented in TPE were much higher than their compartments in blood. It should be mentioned that the numbers of Th22, Th17, and Th1 cells in TPE found in our study seemed uniformly higher than those reported by Qiao and colleagues (18). We believe the main explanation for these discrepancies was that we assessed Th22, Th17, and Th1 cells with CD4+ T cells stimulated with phorbol myristate acetate plus ionomycin in vitro, whereas Qiao and colleagues detected these helper T cells by using MTB-specific stimulation (18).
The molecular mechanisms underlying the generation and regulation of these helper T subsets, especially Th22 cells, in TPE remain unknown. Our data were not consistent with those reported by Volpe and colleagues (21), which showed that neither IL-1β, IL-6, nor TNF-α induced IL-22 production in vitro from naive CD4+ T cells. We found that IL-1β, IL-6, or TNF-α could significantly promote the differentiation of Th22 cells from naive CD4+ T cells, and that the combination of IL-1β and IL-6, IL-1β and TNF-α, IL-6 and TNF-α, or IL-1β and IL-6 and TNF-α promoted Th22-cell differentiation to even higher extents. In addition, IFN-γ did not affect Th22-cell numbers; on the contrary, it could reduce the increased percentage of Th22 cells stimulated by IL-6 and/or TNF-α. There were two potential explanations for the discrepancies in our study as compared with those in the study by Volpe and colleagues (21). First, the subjects were normal healthy donors in their study whereas our subjects were patients with TPE. It is therefore possible that Th22-cell differentiation might have been affected by MTB infection in our study. Second, other factors, such as the different concentrations of cytokines used in the in vitro culture, could also account for the discrepancies. In interpreting the results of our study, one should consider that Th22-cell differentiation promoted by proinflammatory cytokines in our study was similar to those reported for patients with skin disorders (27).
Most Th22 cells from TPE displayed the phenotype of effector memory cells, indicated by the expression of high levels of CD45RO, as well as low levels of CD45RA and CD62L. The presence of CCR7 expression endows antigen-experienced CD45RA− T cells with the potential for lymph node homing, which is characteristic of central memory cells, whereas the absence of CCR7 expression allows migration to the site of infection, which is typical of effector cells (28). Indeed, we noted that blood Th22 cells expressed a low level of CCR7, enabling them to migrate easily into the pleural space in MTB infection. Interestingly, CCR7 expression was up-regulated significantly on Th22 cells in the TPE environment.
On the other hand, an increase in the numbers of Th22 cells in TPE might also be due to Th22-cell recruitment from the peripheral blood. We noted that all PMCs expressed CCL20, CCL22, and CCL27 (concentrations of soluble CCL20, CCL22, and CCL27 in TPE were much higher than those in serum) and that Th22 cells in both TPE and blood expressed high levels of CCR6, CCR4, and CCR10 on their surface. An in vitro migration assay further confirmed that both TPE and supernatants of cultured PMCs could induce the migration of Th22 cells, and that anti-CCL20, anti-CCL22, and anti-CCL27 mAbs each significantly inhibited the ability of TPE or supernatants to stimulate Th22-cell chemotaxis. Therefore, PMC-produced chemokines might be able to chemoattract Th22 cells into the pleural space in MTB infection.
Although Th22 cells have been found to be increased in TPE, their pathophysiological functions have been poorly defined. Pleural mesothelium has tight intercellular junctions, which promote cell–cell adhesion and contribute to the control of pleural permeability (29). Pleural mesothelial integrity is necessary in barrier maintenance against various pathogens including MTB. It has been reported that in TPE, mycobacteria caused release of vascular endothelial growth factor from PMCs and resulted in protein exudation by altering mesothelial adherent junction proteins (30). When PMCs were stimulated with bacillus Calmette-Guérin in vitro, the expression of intercellular adhesion molecule-1 on PMCs could facilitate monocyte transmigration across the injured pleural mesothelium (7). We thus investigated whether Th22, Th17, or Th1 cells were involved in the regulation of mesothelial membrane repair. Our findings showed that both IL-22 and IL-17 significantly improved wound healing and long-term restoration by PMCs; in contrast, IFN-γ severely impaired this wound healing and restoring.
We were also interested in knowing whether PMCs could promote differentiation of pleural Th22, Th17, and Th1 cells in MTB infection. It has been reported that human peritoneal mesothelial cells are able to present antigen to T cells (31, 32). To our knowledge, the current study is the first to examine whether PMCs function as antigen-presenting cells. If PMCs are to function in presenting antigen to T cells, they need to express HLA-DR and costimulatory molecules. Our current study of PMCs recovered from TPE confirmed that these cells exhibit the above-mentioned requisite features. By coculturing purified naive CD4+ T cells and PMCs, significantly promoted proliferation of CD4+ T cells and differentiation of Th22 cells could be observed even in the absence of exogenous MTB-specific protein antigen; the addition of antigen 85B yielded more intensive proliferation of CD4+ T cells and differentiation of Th22, Th17, and Th1 cells by CD80- and CD86-dependent means. These data suggest that exposure of PMCs to MTB-related antigen is sufficient to allow these PMCs to serve as antigen-presenting cells to T cells, and that PMCs could further process exogenous antigen during in vitro culture.
Because IL-22 and IL-17 improved wound healing and long-term restoration by PMCs, it was not surprising that both cytokines amplified the enhanced proliferation response of CD4+ T cells and differentiation of Th22, Th17, and Th1 cells induced by antigen presentation by PMCs. Although IFN-γ impaired PMC wound healing and restoration, it did enhance CD4+ T-cell proliferation elicited by antigen presentation by PMCs. Interestingly, IFN-γ was capable of amplifying the differentiation of Th1 cells, but not of Th22 or Th17 cells, caused by antigen presentation by PMCs.
In conclusion, our data showed that the numbers of Th22 cells in TPE were significantly increased when compared with their compartments in blood, and that overrepresentation of Th22 cells in TPE may be due to the increased local proinflammatory cytokines and to PMC-produced chemokines. Our data also showed that IL-22 and IL-17 significantly improved PMC wound healing and long-term restoration. Moreover, PMCs were able to function as antigen-presenting cells to stimulate CD4+ T-cell proliferation and the differentiation of Th22 cells, Th17 cells, and Th1 cells in response to MTB antigens by CD80- and CD86-dependent means.
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*These authors contributed equally to the present work.
Supported in part by a grant from the National Science Fund for Distinguished Young Scholars (no. 30925032) of China, in part by a grant from the National Basic Research Program of China (973 Program 2012CB518706), and in part by a grant from the National Natural Science Foundation of China (no. 30872343).
Author Contributions: Y.Z.J., Q.Z., and H.Z.S. designed the study design and the experiments. M.L.Y. and R.H.D. were responsible for flow cytometry and data collection. W.B.Y., X.Z.Z., and B.H. analyzed the data. B.H. and H.Z.S. drafted the manuscript. All authors read, critically revised, and approved the final manuscript.
This article has an online supplement, which is available from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.201107-1198OC on December 23, 2011