A role for IL-12 receptor expression and signal transduction in host defense in leprosy.

The generation of cell-mediated immunity against intracellular infection involves the production of IL-12, a critical cytokine required for the development of Th1 responses. The biologic activities of IL-12 are mediated through a specific, high affinity IL-12R composed of an IL-12Rbeta1/IL-12Rbeta2 heterodimer, with the IL-12Rbeta2 chain involved in signaling via Stat4. We investigated IL-12R expression and function in human infectious disease, using the clinical/immunologic spectrum of leprosy as a model. T cells from tuberculoid patients, the resistant form of leprosy, are responsive to IL-12; however, T cells from lepromatous patients, the susceptible form of leprosy, do not respond to IL-12. We found that the IL-12Rbeta2 was more highly expressed in tuberculoid lesions compared with lepromatous lesions. In contrast, IL-12Rbeta1 expression was similar in both tuberculoid and lepromatous lesions. The expression of IL-12Rbeta2 on T cells was up-regulated by Mycobacterium leprae in tuberculoid but not in lepromatous patients. Furthermore, IL-12 induced Stat4 phosphorylation and DNA binding in M. leprae-activated T cells from tuberculoid but not from lepromatous patients. Interestingly, IL-12Rbeta2 in lepromatous patients could be up-regulated by stimulation with M. tuberculosis. These data suggest that Th response to M. leprae determines IL-12Rbeta2 expression and function in host defense in leprosy.     

The HLA system and leprosy in Thailand

Summary To investigate immunogenetics of leprosy, 205 leprosy patients (26 with tuberculoid, 57 with borderline-tuberculoid, 21 with borderline, 31 with borderline-lepromatous, and 70 with lepromatous leprosy) have been typed for HLA antigens, and compared with 183 healthy controls from the same region (Nothern Thailand). There was no significant difference between the overal group of leprosy patients or the three borderline classes and the controls. The two polar forms, tuberculoid and lepromatous leprosy, however, showed significant associations: HLA-A2 is decreased and HLA-Bw17 is increased in tuberculoid leprosy; HLA-B7 is increased in lepromatous leprosy. When both polar forms are compared with each other, HLA-A2 is significantly higher, HLA-Bw40 lower in patients with lepromatous than in those with tuberculoid leprosy. The results are discussed with respect to the different immune responsiveness in the two polar forms of leprosy.     

Signaling lymphocytic activation molecule expression and regulation in human intracellular infection correlate with Th1 cytokine patterns.

Induction of Th1 cytokines, those associated with cell-mediated immunity, is critical for host defense against infection by intracellular pathogens, including mycobacteria. Signaling lymphocytic activation molecule (SLAM, CD150) is a transmembrane protein expressed on lymphocytes that promotes T cell proliferation and IFN-gamma production. The expression and role of SLAM in human infectious disease were investigated using leprosy as a model. We found that SLAM mRNA and protein were more strongly expressed in skin lesions of tuberculoid patients, those with measurable CMI to the pathogen, Mycobacterium leprae, compared with lepromatous patients, who have weak CMI against M. leprae. Peripheral blood T cells from tuberculoid patients showed a striking increase in the level of SLAM expression after stimulation with M. leprae, whereas the expression of SLAM on T cells from lepromatous patients show little change by M. leprae stimulation. Engagement of SLAM by an agonistic mAb up-regulated IFN-gamma production from tuberculoid patients and slightly increased the levels of IFN-gamma in lepromatous patients. In addition, IFN-gamma augmented SLAM expression on M. leprae-stimulated peripheral blood T cells from leprosy patients. Signaling through SLAM after IFN-gamma treatment of Ag-stimulated cells enhanced IFN-gamma production in lepromatous patients to the levels of tuberculoid patients. Our data suggest that the local release of IFN-gamma by M. leprae-activated T cells in tuberculoid leprosy lesions leads to up-regulation of SLAM expression. Ligation of SLAM augments IFN-gamma production in the local microenvironment, creating a positive feedback loop. Failure of T cells from lepromatous leprosy patients to produce IFN-gamma in response to M. leprae contributes to reduced expression of SLAM. Therefore, the activation of SLAM may promote the cell-mediated immune response to intracellular bacterial pathogens.     

Analysis of apoptosis and Bcl-2 expression in polar forms of leprosy

Apoptosis eliminates pathogen-infected cells. Its modulation can influence the course of infections, permitting the survival of intracellular pathogens. In leprosy, which presents several clinical manifestations related to bacillary burden and host immune status, the mechanisms responsible for the persistence of the bacillus are unknown. Few studies have focused on apoptosis over the disease spectrum and as a defense mechanism against Mycobacterium leprae. We evaluated apoptosis using terminal transferase dUTP nick end labeling and the expression of Bcl-2 by immunohistochemistry in skin lesions from 11 tuberculoid and 15 lepromatous leprosy patients. Each specimen was evaluated by determining the number of positive cells in 10 fields at × 400 magnification. We observed a higher number of apoptotic cells in tuberculoid lesions in comparison with lepromatous leprosy (42.5 cells per 10 fields vs. 11.5 cells per 10 fields, P<0.0001). Expression of Bcl-2, conversely, was larger in lepromatous than in tuberculoid samples (172.0 cells per 10 fields vs. 17.7 cells per 10 fields, P<0.0001). These observations suggest modulation of apoptosis in leprosy, primarily in lepromatous patients, for which the decrease in cell death could support M. leprae survival and contribute to the success of infection. Conversely, in tuberculoid patients, apoptosis could contribute to reducing propagation of the bacillus.     

Antibodies against BCG antigen 60 in mycobacterial infection.

A sensitive specific radioimmunoassay was developed to measure antibodies against BCG antigen 60, a prominent antigenic component of BCG bacilli which cross-reacts with similar components in many mycobacterial species including Mycobacterium leprae and M tuberculosis. A lepromatous serum pool had anti-BCG-60 activity with a titre of 10(5) and the tuberculoid pool a titre of 10(4). Testing of individual sera showed striking variations within groups of patients with lepromatous and tuberculoid leprosy. In five of the 20 tuberculoid leprosy sera the anti-BCG-60 activity was above the median for the lepromatous group. The current view that antibody formation against mycobacterial antigens is very low in tuberculoid leprosy thus no longer appears to be tenable. Sera from eight patients with active pulmonary tuberculosis also showed a striking variation in anti-BCG-60 content, and the median value of this group was even higher than in those with lepromatous leprosy.     

In situ identification of cells in human leprosy granulomas with monoclonal antibodies to interleukin 2 and its receptor

Leprosy is a chronic granulomatous disease with an immunologic spectrum in which lepromatous leprosy patients have defective cell-mediated immune responses, in comparison to tuberculoid leprosy patients. Immunoregulatory aspects of this spectrum were investigated by using monoclonal antibodies to interleukin 2 (IL 2), IL 2 receptors (Tac), and T lymphocyte subpopulations with immunoperoxidase techniques on frozen sections of skin biopsy specimens from 10 tuberculoid and 10 lepromatous patients. A comparison of IL 2+ cells revealed markedly fewer IL 2+ cells in lepromatous specimens (lep. 0.028% +/- 0.02 vs tub. 0.46% +/- 0.28, p less than 0.001). These IL 2+ cells were large, exhibited cytoplasmic staining, and on double immunostaining were Leu-4+, Leu-3a+, Leu-2a-, Tac-, and OKT6-, consistent with the fact they are IL 2 producers. Equivalent numbers of Tac+ cells were observed in both lepromatous and tuberculoid granulomas (lep. 1.5% +/- 0.5 vs tub. 2.1% +/- 0.7, p, NS), suggesting that the responder cells are present in both conditions. The tuberculoid granuloma was highly organized, composed of a central core of mature macrophages, Leu-3a+ and Tac+ cells with a surrounding mantle of Leu-2a+, Leu-3a+, IL 2+, Tac+, and OKT6+ cells. In lepromatous granulomas, Leu-2a+, Leu-3a+, Tac+, and rare IL 2+ cells were randomly admixed with bacilli-laden macrophages. The defective cell-mediated immune responses in lepromatous leprosy appears to be associated with diminished IL 2 production and disorganization of the granuloma.     

Factors contributing to impairment of the mixed lymphocyte reaction in leprosy.

Whereas the mixed lymphocyte reaction was essentially normal in inactive lepromatous leprosy and tuberculoid leprosy, it was severely impaired in active lepromatous leprosy. The impairment was found to be contributed by certain unknown factors in their plasma and subnormal reactivity of their T lymphocytes. The plasma derived from active lepromatous leprosy patients depressed the reaction of normal cells and normal plasma enhanced the reaction of active lepromatous lymphocytes. The cellular factor was studied by using a one-way reaction in which one of the two lymphocyte preparations was inactivated with mitomycin C. The impairment of blastogenesis of active lepromatous lymphocytes was partially reversed by substituting inactivated normal cells for similarly treated leprous cells, and conversely the response of normal allogeneic lymphocytes was depressed by substituting inactivated leprous lymphocytes as the stimulator cells.     

A role for CD40-CD40 ligand interactions in the generation of type 1 cytokine responses in human leprosy

The interaction of CD40 ligand (CD40L) expressed by activated T cells with CD40 on macrophages has been shown to be a potent stimulus for the production of IL-12, an obligate signal for generation of Th1 cytokine responses. The expression and interaction of CD40 and CD40L were investigated in human infectious disease using leprosy as a model. CD40 and CD40L mRNA and surface protein expression were predominant in skin lesions of resistant tuberculoid patients compared with the highly susceptible lepromatous group. IL-12 release from PBMC of tuberculoid patients stimulated with Mycobacterium leprae was partially inhibited by mAbs to CD40 or CD40L, correlating with Ag-induced up-regulation of CD40L on T cells. Cognate recognition of M. leprae Ag by a T cell clone derived from a tuberculoid lesion in the context of monocyte APC resulted in CD40L-CD40-dependent production of IL-12. In contrast, M. leprae-induced IL-12 production by PBMC from lepromatous patients was not dependent on CD40L-CD40 ligation, nor was CD40L up-regulated by M. leprae. Furthermore, IL-10, a cytokine predominant in lepromatous lesions, blocked the IFN-gamma up-regulation of CD40 on monocytes. These data suggest that T cell activation in situ by M. leprae in tuberculoid leprosy leads to local up-regulation of CD40L, which stimulates CD40-dependent induction of IL-12 in monocytes. The CD40-CD40L interaction, which is not evident in lepromatous leprosy, probably participates in the cell-mediated immune response to microbial pathogens.     

Suppressor T lymphocytes from lepromatous leprosy skin lesions

The immune response in leprosy forms a spectrum with lepromatous leprosy patients exhibiting specific unresponsiveness to antigens of Mycobacterium leprae. This unresponsiveness is thought to be related to the prevalence of T8-positive lymphocyte in these lepromatous lesions. To analyze the immunoregulatory function of these T8 cells, we developed simple procedures to extract lymphocytes from skin biopsy specimens of patients with leprosy. These lymphocytes were sorted for T8 and T4 positive cells, and cell lines were established by expansion with interleukin 2 (IL 2) and irradiated feeder cells. All T8 positive lines tested were positive for IL 2 receptors and HLA-DR determinants. These lines were additionally assayed for lepromin-induced suppression of the normal peripheral blood lymphocyte Con A proliferative response. Thirteen of 32 lines from six lepromatous patients showed significant suppressor activity, whereas nine lines from six tuberculoid patients and one line from normal peripheral blood failed to show suppression (p less than 0.001). Taken together, the finding of M. leprae-triggered suppressor cells within lepromatous skin lesions may in part explain the M. leprae unresponsiveness of lepromatous leprosy patients.     

Activation of human monocytes in leprosy

In leprosy, the common etiologic agent is the same Mycobacterium leprae, but the clinical manifestations are various, including the tuberculoid and lepromatous types. In tuberculoid type leprosy, macrophages in the granuloma differentiate into epithelioid cells; in the lepromatous type, in contrast, they differentiate into lepra cells containing multiple M. leprae. Thus host factors, which regulate macrophage activities, determine the type of leprosy. To understand such regulation of macrophage activities, we assayed superoxide production, hydrogen peroxide production and glucose consumption in monocytes in vitro. Glucose consumption spontaneously increased, with lymphokine enhancing the consumption rate. Superoxide production increased spontaneously and decreased from the 4th day; lymphokine added on the 4th day supressed the decrease of superoxide production. Hydrogen peroxide production increased until the 3rd day of culture. Twenty-four hour incubation with lymphokine, from day 0 to the 1st day, had no effect on hydrogen peroxide production, while from the 2nd to 3rd day it enhanced such production. Supernatants of lymphocytes incubated with M. leprae were prepared from tuberculoid and lepromatous patients. Tuberculoid supernatant enhanced reactive oxygen production and glucose consumption, while that from lepromatous patients had no remarkable effect on glucose consumption or reactive oxygen production. The range of spontaneous increase and decrease of reactive oxygen production was greater than the regulatory effect of lymphokine on these activities. These data show that rapid provision of new monocytes to the granuloma is one of the important factors in the defense mechanism, that lymphocytes separated from lepromatous patients are not activated in response to M. leprae antigen, and that they do not secrete corresponding lymphokines.     

Increase in TGF-β Secreting CD4+CD25+ FOXP3+ T Regulatory Cells in Anergic Lepromatous Leprosy Patients

Background

Lepromatous leprosy caused by Mycobacterium leprae is associated with antigen specific T cell unresponsiveness/anergy whose underlying mechanisms are not fully defined. We investigated the role of CD25⁺FOXP3⁺ regulatory T cells in both skin lesions and M.leprae stimulated PBMC cultures of 28 each of freshly diagnosed patients with borderline tuberculoid (BT) and lepromatous leprosy (LL) as well as 7 healthy household contacts of leprosy patients and 4 normal skin samples.

Methodology/Principle Findings

Quantitative reverse transcribed PCR (qPCR), immuno-histochemistry/flowcytometry and ELISA were used respectively for gene expression, phenotype characterization and cytokine levels in PBMC culture supernatants. Both skin lesions as well as in vitro antigen stimulated PBMC showed increased percentage/mean fluorescence intensity of cells and higher gene expression for FOXP3⁺, TGF-β in lepromatous (p<0.01) as compared to tuberculoid leprosy patients. CD4⁺CD25⁺FOXP3⁺ T cells (Tregs) were increased in unstimulated basal cultures (p<0.0003) and showed further increase in in vitro antigen but not mitogen (phytohemaglutinin) stimulated PBMC (iTreg) in lepromatous as compared to tuberculoid leprosy patients (p<0.002). iTregs of lepromatous patients showed intracellular TGF-β which was further confirmed by increase in TGF-β in culture supernatants (p<0.003). Furthermore, TGF-β in iTreg cells was associated with phosphorylation of STAT5A. TGF-β was seen in CD25⁺ cells of the CD4⁺ but not that of CD8⁺ T cell lineage in leprosy patients. iTregs did not show intracellular IFN-γ or IL-17 in lepromatous leprosy patients.

Conclusions/Significance

Our results indicate that FOXP3⁺ iTregs with TGF-β may down regulate T cell responses leading to the antigen specific anergy associated with lepromatous leprosy.     

Numerical Changes in T Cell Subsets (Tγ and Tμ) in Leprosy Patients

Eighty-six leprosy patients (49 active lepromatous, 24 inactive lepromatous, 7 borderline, and 6 tuberculoid) and nine healthy controls were examined for numerical changes in T cell subsets (Tγ and Tμ), and complement levels in peripheral blood to determine the roles of T cell subsets and complement in the etiology of leprosy. The percentage and number of Tγ and Tμ cells showed no significant differences among the different clinical groups, but 4 out of 49 active lepromatous, 3 out of 24 inactive lepromatous and 3 out of 7 borderline cases showed a high percentage of Tγ cells. Serum concentrations of C4, C3c, and C3 activator, an important factor in the alternative pathway of complement activation, were not significantly different among the groups. However, C3 activator and C3c concentrations were significantly high in active lepromatous patients complicated by an immune complex disease called “erythema nodosum leprosum” (ENL) compared with ENL-free active lepromatous leprosy.     

Serum Atypical Pseudocholinesterase and Genetic Factors in Leprosy

The levels of pseudocholinesterase and its nature were studied in 390 leprosy patients and 343 control subjects. The levels of the enzyme in both leprosy patients and normal controls were comparable. The study of the nature of the enzyme by determining its dibucaine number showed that a statistically significant number of lepromatous patients had the atypical variety of the enzyme compared with normal controls or tuberculoid patients. Since the presence of the atypical variety of the enzyme is genetically determined it is suggested that genetic factors play an important part in deciding susceptibility to lepromatous leprosy.     

Lepromin-induced suppressor cells in patients with leprosy.

The possibility of an active mechanism of immunologic suppression in leprosy was explored by assessing the in vitro lymphocyte responses of 61 leprosy patients and 30 normal individuals to the mitogen Con A in the presence or absence of Dharmendra lepromin. Lepromin-induced suppression of Con A stimulation was found in 32 of 35 lepromatous patients and 15 of 15 borderline patients, but only 2 of 15 tuberculoid patients and 2 of 30 normal controls. Cell fractionation studies indicated at least two cell populations involved in the in vitro lepromin-induced suppressor activity, adherent cells and T gamma-cells.     

TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells

Leprosy enables investigation of mechanisms by which the innate immune system contributes to host defense against infection, because in one form, the disease progresses, and in the other, the infection is limited. We report that Toll-like receptor (TLR) activation of human monocytes induces rapid differentiation into two distinct subsets: DC-SIGN+ CD16+ macrophages and CD1b+ DC-SIGN- dendritic cells. DC-SIGN+ phagocytic macrophages were expanded by TLR-mediated upregulation of interleukin (IL)-15 and IL-15 receptor. CD1b+ dendritic cells were expanded by TLR-mediated upregulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor, promoted T cell activation and secreted proinflammatory cytokines. Whereas DC-SIGN+ macrophages were detected in lesions and after TLR activation in all leprosy patients, CD1b+ dendritic cells were not detected in lesions or after TLR activation of peripheral monocytes in individuals with the progressive lepromatous form, except during reversal reactions in which bacilli were cleared by T helper type 1 (TH1) responses. In tuberculoid lepromatous lesions, DC-SIGN+ cells were positive for macrophage markers, but negative for dendritic cell markers. Thus, TLR-induced differentiation of monocytes into either macrophages or dendritic cells seems to crucially influence effective host defenses in human infectious disease.     

Phenoloxidase activity in organisms isolated from lepromatous and tuberculoid leprosy

Anaerobic corynebacteria (propionibacteria), isolated in pure culture from the plasma of a case of tuberculoid leprosy and from lepromata of cases of lepromatous leprosy, exhibited phenoloxidase activity such as that shown by Prabhakaran to be associated with leprosy bacilli harvested from patients suffering from leprosy. Several corynebacteria, mycobacteria, and nocardias similarly examined did not produce phenoloxidase.     

Mycobacterium leprae mediated stimulation of macrophages from leprosy patients and hydrogen peroxide production

Macrophages cultured from the peripheral blood of normal individuals, tuberculoid leprosy patients and long-term-treated, bacteriologically negative lepromatous leprosy patients are able to release hydrogen peroxide on stimulation withMycobacterium leprae. Macrophages from lepromatous leprosy patients who are bacteriologically positive produce considerably lower levels of hydrogen peroxide, even though stimulation of these cells withMycobacterium leprae is definitely demonstrable. This differential stimulation of macrophages appears to be largely specific toMycobacterium leprae. There is also a good indication that decreased stimulation of macrophages from positive patients could be due to an after-effect of infection. It is possible that while other factors aid survival ofMycobacterium leprae in the macrophages, hydrogen peroxide may not be as effective in the killing of the bacteria in infected patients as it would be, perhaps, in other infections.     

Subclinical Infection in Leprosy

Lymphocyte transformation has been used to study the immune response to Mycobacterium leprae among contacts and non-contacts of leprosy patients. Of 26 subjects living in a leprosy endemic area for less than two months none responded to M. leprae; 24% of subjects who had lived in an endemic area for more than a year gave a positive response to M. leprae; more than 50% of individuals with occupational contact of leprosy for more than a year responded; and about 50% of contacts of tuberculoid and treated lepromatous patients responded to M. leprae, while only 22% (4/18) of contacts of lepromatous patients treated for less than six months responded.It seems that leprosy is more highly infectious than is indicated by the prevalence of the disease and that a subclinical infection commonly follows exposure to M. leprae. The relatively low response found in contacts of active lepromatous patients suggests that in these contacts “superexposure” to M. leprae can bring about a decrease in host resistance.     

The human CD1-restricted T cell repertoire is limited to cross-reactive antigens: implications for host responses against immunologically related pathogens

The repertoires of CD1- and MHC-restricted T cells are complementary, permitting the immune recognition of both lipid and peptide Ags, respectively. To compare the breadth of the CD1-restricted and MHC-restricted T cell repertoires, we evaluated T cell responses against lipid and peptide Ags of mycobacteria in leprosy, comparing tuberculoid patients, who are able to restrict the pathogen, and lepromatous patients, who have disseminated infection. The striking finding was that in lepromatous leprosy, T cells did not efficiently recognize lipid Ags from the leprosy pathogen, Mycobacterium leprae, or the related species, Mycobacterium tuberculosis, yet were able to efficiently recognize peptide Ags from M. tuberculosis, but not M. leprae. To identify a mechanism for T cell unresponsiveness against mycobacterial lipid Ags in lepromatous patients, we used T cell clones to probe the species specificity of the Ags recognized. We found that the majority of M. leprae-reactive CD1-restricted T cell clones (92%) were cross-reactive for multiple mycobacterial species, whereas the majority of M. leprae-reactive MHC-restricted T cells were species specific (66%), with a limited number of T cell clones cross-reactive (34%) with M. tuberculosis. In comparison with the MHC class II-restricted T cell repertoire, the CD1-restricted T cell repertoire is limited to recognition of cross-reactive Ags, imparting a distinct role in the host response to immunologically related pathogens.