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* Laboratory of Mycobacterial Immunity and Pathogenesis, Public Health Research Institute, Newark, NJ 07103;
Health Protection Agency, London, United Kingdom;
Center for AIDS Research, New York University New York, NY 10016; and
National Hansen’s Disease Programs Laboratory Research Branch, Louisiana State University, Baton Rouge, LA 70803
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Naive T cells require two different signals from APCs to become activated. Signal 1 depends on the TCR-recognizing Ag presented by MHC molecules located on the surface of the APC. Signal 2 depends on the production of cytokines and their expression on the surface of the APCs of costimulatory molecules that interact with T cell ligands. The delivery of both signal 1 and signal 2 results in the activation of Ag-specific T cells (3). Signal 1, in the absence of signal 2, induces T cell anergy, and the absence of complete APC activation has been proposed to initiate a Th2 type response (4). Many microbial factors induce APC activation, resulting in cytokine i.e., TNF-
and IL 12, secretion and up-regulation of costimulatory molecules; together, these promote the development of Th1 cells. The early APC-mediated activation of naive T cells by M. leprae might result in Th1 immunity (T-lep) or in Th2-mediated anergy (L-lep), depending on the strength of the cytokine and the costimulatory signals accompanying TCR engagement.
Dendritic cells (DCs) are APC unique in their ability to induce primary T cell activation. This ability, however, depends on the state of DC maturation. Immature DCs (iDC) are highly phagocytic. Upon stimulation of iDCs with microbial products, the iDC surface-expressed TLRs trigger NF-
B and MAPK-mediated DC maturation and activation. Mature DCs become efficient APCs by increasing peptide loading and delivery of MHC molecules to the cell surface. In addition, expression of the costimulatory molecules CD86/B7.2, CD80/B7.1, CD83, and CD40 is up-regulated. Mature DCs migrate from the infected/inflamed tissue and enter lymphatic circulation, presumably directed by up-regulation of surface expression of CCR7, the only chemokine receptor expressed by mature DCs (5). M. leprae activates DCs via TLR2-TLR1 heterodimers, presumably through triacylated lipoproteins like the 19-kDa lipoprotein (6). TLR2 polymorphisms have been linked to increased susceptibility to leprosy (7, 8).
Activation of an appropriate immune response is critical for maintaining well-regulated immunologic homeostasis. For example, chronically activated DCs have been proposed to mediate the presentation of self-nucleoprotein complexes in lupus (9). We hypothesized that M. leprae might subvert the activation of DCs, impacting on the development of normal protective Th1 type immunity. In the present report, we studied the effect of M. leprae on iDC activation and maturation and on DC-mediated primary T cell activation. We compared M. leprae to Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis bacillus Calmette-Guerin (BCG) for their abilities to induce up-regulation of maturation markers, stimulate cytokine production, drive T cell activation, and induce the expression of genes associated with the phenotypic and functional maturation of DCs. We show that, relative to Mtb and BCG, M. leprae does not induce DC maturation but in fact reduces the expression of various activation pathway genes. Interestingly, M. leprae-infected DCs seem to promote both a Th1 and a Th2 cytokine response during a MLR. Thus, early interactions between M. leprae and DCs could be important in determining whether T cell anergy or activation is achieved.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The M. leprae Thai-53 strain was used for these experiments. The mycobacteria had been maintained in programmed serial passage in the footpads of athymic nu/nu mice infected with 5 x 107 freshly harvested M. leprae. Briefly, bacilli were harvested from the footpads 3–4 mo after infection (at mid-log growth) as described previously (10). Bacilli were washed in Middlebrook 7H12 medium and enumerated by direct count according to Shepard’s method (11). The relative viability of M. leprae in the suspension was evaluated using the LIVE/DEAD BacLight bacterial viability kit (Molecular Probes) as recently adapted for M. leprae (10). Pure preparations of bacilli free of mouse footpad tissue were obtained by treating the footpad suspension with 0.1 M NaOH for 5 min followed by neutralization with 0.1 M HCl and three washes with PBS. M. leprae were irradiated by gamma irradiation with 104 grays from a cobalt source (12) and boiled for 10 min in water before use.
M. bovis BCG Pasteur strain (Trudeau Mycobacterial Culture Collection no. 1011) and M. tuberculosis H37Rv Pasteur strain (Trudeau Mycobacterial Culture Collection no. 102) (Mtb) were grown in Middlebrook 7H9 medium (Difco) and kept as frozen stocks until they were used. Stocks were thawed, sonicated, and appropriately diluted for infection. For heat-killed mycobacteria, sonicated stocks were boiled in water for 10 min. All mycobacteria were passed through a 1-ml syringe attached to a 263/8-gauge needle 3–4 times before preparing appropriate dilutions for infection. Unless otherwise stated, mycobacteria were added to leukocytes in culture at a multiplicity of infection (MOI) of five bacilli per cell.
Generation of immature human DCs in vitro
PBMCs were isolated from leukocyte preparations obtained at the New Jersey Blood Center (East Orange, NJ) by density-gradient centrifugation. Monocytes were purified by positive selection using magnetic bead-conjugated anti-CD14 Abs (Miltenyi Biotec). Monocytes were cultured in 5% human serum (GemCell) in RPMI 1640 (Invitrogen Life Technologies) and 10 mM HEPES (Invitrogen Life Technologies) with IL-4 (1 ng/ml) (Endogen) and GM-CSF (0.2 ng/ml) (Endogen) in 6-well plates (Falcon; BD Bioscience) at 3 x 106/well at 37°C in 5% CO2. IL-4 and GM-CSF were replenished on days 2 and 4. On day 6, nonadherent cells were harvested, cultured in 10% human serum in RPMI 1640 (R10) with IL-4 and GM-CSF and plated in 24-well plates (Falcon; BD Biosciences) at 2.5 x 105/well (for phenotyping and to generate target cells for allogeneic reactions) or in a 6-well plate at 2.5 x 106/well (for RNA extraction).
DC maturation in culture
On day 6 of cell culture as described above, iDCs were either treated with medium (R10 plus IL-4/GM-CSF), medium plus a maturation mixture (designated hereafter as MC for maturation "cocktail") consisting of TNF- (10 ng/ml) (Alexis), IL-1
(10 ng/ml) (BD Pharmingen), and PGE2 (1 mg/ml) (Sigma-Aldrich), medium plus mycobacteria, or medium plus mycobacteria plus MC. Cells were cultured under these conditions for 48 h before phenotyping or before the use of DCs as targets for allogeneic reactions. For RNA isolation, cells were cultured for 5 h before harvesting.
DC RNA isolation
RNA was isolated from DC following culture with medium plus IL4/GM-CSF or medium with dead mycobacteria at a MOI of 5. Total cellular RNA was isolated using TriReagent (Molecular Research Center), and 5 µg of RNA from each sample was reverse transcribed into cDNA with Moloney murine leukemia virus reverse transcriptase. The resulting biotinylated cDNA probes were hybridized to a DC-specific, 96-gene cDNA array spotted on the GEArray membranes (SuperArray). The arrays were developed with A CDP-Star chemiluminescence substrate (PerkinElmer) and recorded with x-ray film. The ScanAlyze 2 program (SuperArray) was used for image analysis and the GEArrayAnalyzer (SuperArray) for processing the raw data. Cells from different donors were used for each experiment. The signal from the expression of each gene was normalized to the signal derived from -actin. A 2-fold difference in mRNA expression relative to DC treated with medium plus IL4/GM-CSF was considered significant.
Evaluation of phenotypic DC maturation
The DC phenotype was assessed by flow cytometric analysis of surface-marker expression. On days 6 and 8 of DC culture, cells were harvested and stained in 2% FCS (Gemcell) in PBS (Invitrogen Life Technologies) with fluorescently labeled Abs to CD14, CD25, CD40, CD80, CD83, CD86, CCR-7, HLA-DR, and HLA-ABC (BD Biosciences). Cell death was determined by staining with 7-aminoactinomycin (7-AAD) (BD Biosciences) and dead cells were excluded from analysis. Day 6 and day 8 geometric mean fluorescence intensity (MFI) for each marker was determined by flow cytometry on a FACSCalibur (BD Biosciences) with analysis conducted in CellQuest software (BD Biosciences) and FlowJo software (Tree Star). Day 6 MFI was normalized to 100%, and day 8 MFI was then expressed as a percentage of day 6 MFI.
Evaluation of M. leprae-DC binding
M. leprae-iDC binding was evaluated by flow cytometric analysis. M. leprae was stained with PKH26 (Sigma-Aldrich) at 4 µM at room temperature. Staining was stopped with serum, followed by washing. Stained bacteria were incubated with iDCs at different MOI values (1, 5, 10, 40, 100 for M. leprae-DC) over the course of 4 h. The FL2 MFI was measured at the different time points for the different MOI values.
Mixed lymphocyte reaction
CD3+ cells were isolated from PBMCs with magnetic beads (Miltenyi Biotec). Cells were stained with CFSE (Molecular Probes) at 5 µM for 10 min at 37°C, the staining was stopped with R10, and the cells were incubated for 10 min at room temperature. Lymphocytes were then incubated at different ratios (0.01 and 0.1 DC:T cell) with DCs that had been treated for 2 days with medium plus either IL-4/GM-CSF (iDC) plus MC or BCG (MOI of 5) or Mtb (MOI of 5) or M. leprae (MOI of 5). Coculture went on for 6 days, when cells were harvested, fixed with FACS lysing buffer (BD Biosciences), stained with fluorescently conjugated Abs against CD4 and CD8 (BD Biosciences), and analyzed by flow cytometry on a FACSCalibur (BD Biosciences).
Cytokines
Day 8 DC culture supernatants were harvested and used to evaluate cytokine production with commercial ELISA kits for IL-10, IL-12, and TNF- (Endogen). Following 6 days of DC and T cell coculture (MLR), supernatants were collected from the DC:T cell 0.1 ratio and analyzed with human Th1/Th2 cytokine BD Biosciences cytometric bead array (CBA) kits. Flow cytometric acquisition was done on a FACSCalibur (BD Biosciences), and further analysis was done with BD Biosciences CBA software.
Statistics
A nonparametric Mann-Whitney t test was used to compare means from two different groups. Two-way ANOVA was used to compare differences between groups for the MLR. A value of p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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In vitro cultured human monocytes exposed for >6 days to IL-4 and GM-CSF develop into iDCs. Following the exposure of 6-day iDCs to IL-4/GM-CSF-containing medium for an extra 48 h, the geometric MFI of surface markers of maturation and activation (CD25, CD40, CD80, CD83, CD86, and CCR7) and MHC molecules (HLA-DR and HLA-ABC) among live cells (7-AAD–) was determined by flow cytometry (Fig. 1A). The MFI for these markers varied from 
12 to 340. Addition of MC (IL-1, PGE2, and TNF-) to the IL4/GM-CSF-containing medium from day 6 to day 8 of culture resulted in further up-regulation of various markers (Fig. 1B). This effect was clearly MC dose dependent as shown by the response of cells to titration of MC (Fig. 1, C and D). The results suggest that the assay selected is a sensitive method to compare the effect of different stimuli on iDC maturation.
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We tested the activity of Mtb and BCG in the iDC maturation assay using viable or heat-killed (boiled) bacteria. Monocyte-derived iDCs were infected on day 6 at a MOI of 5 with live or heat-killed BCG or Mtb (Fig. 2). At this MOI the bacilli associated very efficiently with the iDC; all cells had at least one bacterium attached to them as determined by flow cytometric analysis of iDC exposed to PKH26 stained bacilli (data not shown). Both BCG and Mtb infection resulted in similar up-regulation of iDC surface markers of activation and maturation. Phenotypic iDC maturation appeared independent of mycobacterial viability. We therefore used heat-killed mycobacteria for subsequent experiments. In general, the extent of induction of phenotypic maturation by BCG and Mtb was somewhat lower than the response of the cells to MC.
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DC culture is conducted at 37°C; however, M. leprae viability is reduced at this temperature. We explored the possibility of maturing DC and/or activating iDC at 30°C to use viable M. leprae. At 30°C, DC as well as iDC viability and function were suboptimal. We therefore decided to use irradiated and boiled M. leprae for subsequent experiments. To evaluate the effect of M. leprae on iDC maturation, day 6 iDCs from the same blood donors were exposed to M. leprae, BCG, or Mtb in parallel. Exposure of iDCs to M. leprae at an MOI of 5 (Fig. 3B) or 10 (data not shown) did not up-regulate iDC surface markers of maturation. In contrast, under the same conditions and in the same donors, BCG and Mtb were clearly stimulatory (Fig. 3A). In addition, exposure to M. leprae neither enhanced nor inhibited MC-induced iDC maturation (Fig. 3B). This lack of response was noted even when MC was titrated down to 0.5% (data not shown).
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To confirm that the lack of stimulation of surface marker expression was not a result of lack of bacillary association with APCs, we next examined the interaction of M. leprae with the iDCs in culture. iDC-M. leprae association and iDC viability following M. leprae infection at different MOI values (1, 5, 10, 40, 100 for M. leprae-DC) were evaluated over time (Fig. 4). By using fluorescently labeled M. leprae, we determined mycobacteria-iDC association through changes in MFI. Over the course of 4 h, increasing numbers of bacteria were found to be associated with iDC. As MOI values increased, so did bacteria-cell association (Fig. 4A). We determined iDC viability by enumerating the percentage of cells that were stained with 7-AAD at different MOI values and found that up to a MOI of 40, DC viability was not diminished by interaction with M. leprae (Fig. 4F). Consequently, a MOI value of 5 or 10 was determined to be optimal for all experiments. At these MOI values the bacilli were associated with the iDCs without inducing significant host cell death. In addition, an MOI of 5 was determined to induce optimal iDC stimulation by Mtb and BCG.
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The effect of exposure of iDC for 48 h to M. leprae, BCG, or Mtb on cytokine production was evaluated. Both Mtb and BCG induced iDC secretion of IL-12 and TNF- (Fig. 5), with Mtb inducing higher TNF- secretion despite inducing similar or often lower surface expression of maturation markers (Fig. 2). In contrast, M. leprae did not induce DC production of IL-12 or TNF-, and its secretion profile was similar to that for iDCs. None of the three mycobacterial strains or MC induced significant secretion of IL-10 by iDCs.
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We next determined whether the differences in iDC phenotypic maturation and activation affected T cell priming. Day 6 iDCs were treated with BCG, Mtb, or M. leprae for 48 h, after which they were irradiated and used as targets in a MLR (Fig. 6). M. leprae-infected DCs did not induce allogeneic lymphocyte proliferation at the 0.01 or 0.1 DC:T cell ratio; proliferation levels were actually lower than those observed in unstimulated iDC at these E:T ratios. In contrast, Mtb and BCG induced somewhat increased T cell proliferation compared with unstimulated iDCs. MC induced the highest proliferation of both CD4+ and CD8+ T cells (Fig. 6A). The cytokine profile in culture supernatants of the MLRs was evaluated (Fig. 6B). Surprisingly, although the Th2 cytokine (IL-4 and IL-5) response to M. leprae was higher than the response to Mtb or BCG, the Th1 cytokine (IFN-
and TNF-) response to M. leprae was similar to the response induced by the other mycobacteria.
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RNA was isolated from DCs treated with medium (iDC), Mtb, BCG, or M. leprae, and cDNA was synthesized from RNA and used to probe a DC-specific array. We compared the signal for each gene in infected DCs to the signal for uninfected iDCs. Table I shows particular genes for which expression was up-regulated, down-regulated, or unaffected by mycobacterial stimulation. Stimulation with M. leprae did not induce expression of any of the genes involved in DC maturation/activation and, in fact, some genes that were up-regulated by exposure to Mtb and/or BCG were down-regulated by M. leprae. Genes encoding MHC II and costimulator molecules such as CD80 and CD83, as well as IL-12 and TNF-, were induced in DCs exposed to BCG or Mtb but not to M. leprae, confirming the observations made on cell-associated protein (surface marker) and on secreted protein (cytokines). Among the genes down-regulated by M. leprae but up-regulated by Mtb or BCG were IFN- inducible proteins and IL-2. IL-1 and NFB were up-regulated in response to Mtb or BCG, consistent with numerous reports, and down-regulated by M. leprae. CCR7 was the only chemokine receptor expressed by DC. Mtb or BCG induced increased CCR7 expression, whereas M. leprae inhibited it. IFN- was down-regulated by M. leprae but unaffected by Mtb or BCG. Expression of TLR9, which has recently been implicated as playing a role in the host response to mycobacteria (13), was up-regulated by Mtb or BCG but unaffected by M. leprae.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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In contrast to our present study, other investigators have reported M. leprae-stimulated DC activation and maturation, but only at a very high MOI value of >40 (18). This dose-dependent effect might be explained by the possibility that DC-stimulating M. leprae molecules such as major membrane protein II are present in subcellular locations at concentrations that are relatively low (19, 20). It is likely that the effect of very low doses of stimulating factors is offset by inhibitory molecules exposed on the surface of the bacilli, ultimately resulting in a neutral cellular response. Defective IFN- mediated activation has been reported in mouse macrophages heavily infected with live M. leprae (21, 22). Possible inhibitory molecules present in the cell wall of M. leprae include phenolic glycolipid 1 (PGL-1) (23, 24). PGL-1 has been implicated in dampening the allogeneic response caused by M. leprae-infected DCs (18). It has also been implicated in reducing cytokine production in macrophages (25). Our analysis of DC-specific genes expressed in response to mycobacterial stimulation suggests there might be active suppression of maturation/activation pathways, which would be consistent with PGL-1 dampening activation signals. Alternatively, PGE2, which has been implicated in suppressing IFN- mediated activation of M. leprae-infected macrophages (26), could be polarizing DC into DC2 (27). The dampening effect of PGL-1 or PGE2 might eventually be overcome at higher MOI values; the interplay between the activating and suppressing signals could determine the tipping point between the development of Th1- or Th2-type immunity.
Because of the difficulties of working with M. leprae i.e., the inability to grow the mycobacterium in vitro and the lack of an optimal animal model for pathogenesis studies, limited progress has been made in understanding the host-pathogen interactions that lead to the dramatic anergy of M. leprae-specific, cell-mediated immunity in lepromatous patients. Host-pathogen interactions for other mycobacteria, however, are better understood. By comparing cellular responses to well-characterized strains such as M. tuberculosis H37Rv and M. bovis BCG with responses to M. leprae, possible mechanisms that lead to mycobacterial modulation of cellular function can be explored. Various BCG and Mtb molecules that modulate APC activation have been described. One such mycobacterial component that has been extensively studied is a major cell wall component, lipoarabinomannan (LAM) (28). A recent report showed that lipomannan, the biosynthetic LAM precursor, displays strong proinflammatory effects via TLR-2 (29). Mannose-capped LAM (man-LAM) from slow growing pathogenic mycobacteria, in contrast, has been found to inhibit phagosome maturation, apoptosis, and IFN- signaling (30). M. leprae LAM has been reported to be a potent inhibitor of IFN--mediated activation of mouse macrophages (26). Mannooligosacharide caps on LAM have been shown to interact with the mannose receptor and to induce expression of IRAK-M, which interferes with TLR-mediated macrophage activation (31). Man-LAM has also been shown to bind the DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a C-type lectin on DC and macrophages (32), and to interfere with TLR-mediated DC maturation (33). Interestingly, increased DC-SIGN expression in M. leprae-infected DCs has been recently described in L-lep vs T-lep patients (34). This report and a second one (35) showed that DC-SIGN acts as an entry receptor for M. leprae, much the same as has been described for M. tuberculosis (32, 36). Taken together, these data suggest that M. leprae might induce expression of its own receptor, which would provide access to a safe haven and a means to reduce Man-LAM/TLR mediated DC activation. These observations also suggest that the cell wall contains molecules that enhance and others that suppress APC activation and that the ratio of lipomannan to Man-LAM and other lipids such as PGL-1 and the availability of their receptors on DCs may determine the nature of the cellular response to exposure to the microorganism.
In summary, by using different assays that target different manifestations of DC maturation, this report provides evidence that M. leprae actively suppresses APC activation/maturation. This effect is probably an important determinant of T cell-dependent, Ag-specific responsiveness. Improved understanding of the mechanism by which M. leprae both inhibits and enhances early immunologic events that affect acquired immunity should contribute to improved understanding of how immune anergy vs immune activation are regulated in leprosy as well as other human diseases.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by American Leprosy Missions (to J.K.), the Heiser Program for Leprosy and Tuberculosis Research (to R.A.M.), and National Institutes of Health Grant R01AI022616 (to G.K.). 
2 Address correspondence and reprint requests to Dr. Gilla Kaplan, Laboratory of Mycobacterial Immunity and Pathogenesis, Public Health Research Institute, 225 Warren Street, Newark, NJ 07103, E-mail address: kaplan{at}phri.org
3 Abbreviations used in this paper: T-lep, tuberculoid leprosy; 7-AAD, 7-aminoactinomycin D; BCG, bacillus Calmette-Guerin; CBA, cytometric bead array; DC, dendritic cell; DC-SIGN, DC-specific ICAM-3-grabbing nonintegrin; iDC, immature DC; L-lep, lepromatous leprosy; LAM, lipoarabinomannan; man-LAM, mannose-capped LAM; MC, maturation ("cocktail") mixture; MFI, mean fluorescence intensity; MOI, multiplicity of infection; Mtb, Mycobacterium tuberculosis; PGL-1, phenolic glycolipid 1.
Received for publication July 10, 2006. Accepted for publication October 12, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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in systemic lupus erythematosus. Science 294: 1540-1543. interferon in macrophages infected with Mycobacterium leprae. Infect. Immun. 56: 1912-1919. interferon-mediated activation of macrophages. Infect. Immun. 56: 1232-1236. This article has been cited by other articles:
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  L. Lei, B. L. Plattner, and J. M. Hostetter Live Mycobacterium avium subsp. paratuberculosis and a Killed-Bacterium Vaccine Induce Distinct Subcutaneous Granulomas, with Unique Cellular and Cytokine Profiles Clin. Vaccine Immunol., May 1, 2008; 15(5): 783 - 793. [Abstract] [Full Text] [PDF]
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 C. Favali, N. Tavares, J. Clarencio, A. Barral, M. Barral-Netto, and C. Brodskyn Leishmania amazonensis infection impairs differentiation and function of human dendritic cells J. Leukoc. Biol., December 1, 2007; 82(6): 1401 - 1406. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 5 (
), 10 (
), 40 (
), and 100 (•). MFI values of iDC without M. leprae (time = –1 h), at the time of bacterial addition (time = 0), and over the course of 4 h are shown. F, iDC viability is reduced when cells are infected at or above an MOI of 40 for M. leprae-iDC. iDC viability was determined with 7-AAD following 4 h of iDC-M. leprae incubation.
