IL-12 and IL-23 modulate plasticity of FoxP3+ regulatory T cells in human Leprosy.

Leprosy is a bacterial disease caused by M. leprae. Its clinical spectrum reflects the host's immune response to the M. leprae and provide an ideal model to investigate the host pathogen interaction and immunological dysregulation. Tregs are high in leprosy patients and responsible for immune suppression of the host by producing IL-10 and TGF-ß cytokines. In leprosy, plasticity of Tregs remain unstudied. This is the first study describing the conversion of Tregs into Th1-like and Th17-like cells using in vitro cytokine therapy in leprosy patients. Peripheral blood mononuclear cells from leprosy patients were isolated and stimulated with M. leprae antigen (MLCwA), rIL-12 and rIL-23 for 48h. Expression of FoxP3 in CD4+CD25+ Tregs, intracellular cytokines IFN-γ, TGF-ß, IL-10 and IL-17 in Tregs cells were evaluated by flow cytometry (FACS) after stimulation. rIL-12 treatment increases the levels of pStat4 in Tregs and IFN-γ production. In the presence of rIL-23, pStat3+ and IL-17A+ cells increase. rIL-12 and r-IL-23 treatment downregulated the FoxP3 expression, IL-10 and TGF-ß production by Tregs and enhances the expression of co-stimulatory molecules (CD80, CD86). In conclusion rIL-12 converts Tregs into IFN-γ producing cells through STAT-4 signaling while rIL-23 converts Tregs into IL-17 producing cells through STAT-3 signaling in leprosy patients. This study may helpful to provide a new avenue to overcome the immunosuprression in leprosy patients using in vitro cytokine.

Activation and cytokine profile of monocyte derived dendritic cells in leprosy: in vitro stimulation by sonicated Mycobacterium leprae induces decreased level of IL-12p70 in lepromatous leprosy.

Dendritic cells (DCs) play a pivotal role in the connection of innate and adaptive immunity of hosts to mycobacterial infection. Studies on the interaction of monocyte-derived DCs (MO-DCs) using Mycobacterium leprae in leprosy patients are rare. The present study demonstrated that the differentiation of MOs to DCs was similar in all forms of leprosy compared to normal healthy individuals. In vitro stimulation of immature MO-DCs with sonicated M. leprae induced variable degrees of DC maturation as determined by the increased expression of HLA-DR, CD40, CD80 and CD86, but not CD83, in all studied groups. The production of different cytokines by the MO-DCs appeared similar in all of the studied groups under similar conditions. However, the production of interleukin (IL)-12p70 by MO-DCs from lepromatous (LL) leprosy patients after in vitro stimulation with M. leprae was lower than tuberculoid leprosy patients and healthy individuals, even after CD40 ligation with CD40 ligand-transfected cells. The present cumulative findings suggest that the MO-DCs of LL patients are generally a weak producer of IL-12p70 despite the moderate activating properties ofM. leprae. These results may explain the poor M. leprae-specific cell-mediated immunity in the LL type of leprosy.

Leprosy and tuberculosis co-infection: clinical and immunological report of two cases and review of the literature.

A review of the records of patients seen between 2004 and 2011 at the Dermatology Clinic of the São Paulo University Medical School showed that only two leprosy patients had been co-infected with tuberculosis (TB). One patient showed a type 1 leprosy reaction during the first 3 months of treatment of pleural TB and in the other patient, pulmonary TB was diagnosed during the first 3 months of treatment of a type 1 leprosy reaction. Both patients showed normal cellular immune response tests, including those of the interferon-gamma (IFN-γ)/interleukin 12 (IL-12) axis. Although both mycobacterial infections are endemic in developing countries like Brazil, the co-infection has hardly been reported in the last decade. There is no suitable explanation for this observation. The reports on the interaction between the two mycobacteria are highly speculative: some studies suggest that leprosy, especially the anergic form, would predispose to TB, whereas other investigations suggested an antagonism between the two diseases.

Enhancing sensitivity of detection of immune responses to Mycobacterium leprae peptides in whole-blood assays.

Although worldwide leprosy prevalence has been reduced considerably following multidrug therapy, new case detection rates remain relatively stable, suggesting that transmission of infection still continues. This calls for new efforts, among which is development of assays that can identify subclinical/early-stage Mycobacterium leprae-infected subjects, a likely source of transmission. Areas in which leprosy is endemic often lack sophisticated laboratories, necessitating development of field-friendly immunodiagnostic tests for leprosy, like short-term whole-blood assays (WBA). In classical, peripheral blood mononuclear cell (PBMC)-based gamma interferon (IFN-gamma) release assays, M. leprae peptides have been shown to discriminate in a more specific fashion than M. leprae proteins between M. leprae-exposed contacts and patients as opposed to healthy controls from the same area of endemicity. However, peptides induced significantly lower levels of IFN-gamma than did proteins, particularly when whole blood was used. Therefore, possibilities of specifically enhancing IFN-gamma production in response to M. leprae peptides in 24-h WBA were sought by addition of various cytokines and antibodies or by mannosylation of peptides. In addition, other cytokines and chemokines were analyzed as potential biomarkers in WBA. We found that only interleukin 12 (IL-12), not other costimulants, increased IFN-gamma production in WBA while maintaining M. leprae peptide specificity, as evidenced by lack of increase of IFN-gamma in control samples stimulated with IL-12 alone. The IL-12-induced increase in IFN-gamma was mainly mediated by CD4+ T cells that did not produce IL-2 or tumor necrosis factor (TNF). Mannosylation further allowed the use of 100-fold-less peptide. Although not statistically significantly, macrophage inflammatory protein 1beta (MIP-1beta) and macrophage c protein 1 (MCP-1) levels specific for M. leprae peptide tended to be increased by IL-12. IP-10 production was also found to be a useful marker of M. leprae peptide responses, but its production was enhanced by IL-12 nonspecifically. We conclude that IFN-gamma-based WBA combined with IL-12 represents a more sensitive and robust assay for measuring reactivity to M. leprae peptides.

Induction of cross-priming of naive CD8+ T lymphocytes by recombinant bacillus Calmette-Guerin that secretes heat shock protein 70-major membrane protein-II fusion protein.

Because Mycobacterium bovis bacillus Calmette-Guérin (BCG) unconvincingly activates human naive CD8(+) T cells, a rBCG (BCG-70M) that secretes a fusion protein comprising BCG-derived heat shock protein (HSP)70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed to potentiate the ability of activating naive CD8(+) T cells through dendritic cells (DC). BCG-70M secreted HSP70-MMP-II fusion protein in vitro, which stimulated DC to produce IL-12p70 through TLR2. BCG-70M-infected DC activated not only memory and naive CD8(+) T cells, but also CD4(+) T cells of both types to produce IFN-gamma. The activation of these naive T cells by BCG-70M was dependent on the MHC and CD86 molecules on BCG-70M-infected DC, and was significantly inhibited by pretreatment of DC with chloroquine. Both brefeldin A and lactacystin significantly inhibited the activation of naive CD8(+) T cells by BCG-70M through DC. Thus, the CD8(+) T cell activation may be induced by cross-presentation of Ags through a TAP- and proteosome-dependent cytosolic pathway. When naive CD8(+) T cells were stimulated by BCG-70M-infected DC in the presence of naive CD4(+) T cells, CD62L(low)CD8(+) T cells and perforin-producing CD8(+) T cells were efficiently produced. MMP-II-reactive CD4(+) and CD8(+) memory T cells were efficiently produced in C57BL/6 mice by infection with BCG-70M. These results indicate that BCG-70M activated DC, CD4(+) T cells, and CD8(+) T cells, and the combination of HSP70 and MMP-II may be useful for inducing better T cell activation.

Impaired maturation and function of dendritic cells by mycobacteria through IL-1beta.

Dendritic cells (DC) are pivotal for initiation and regulation of innate and adaptive immune responses evoked by vaccination and natural infection. After infection, mycobacterial pathogens first encounter monocytes, which produce pro-inflammatory cytokines, including IL-1beta, TNF-alpha and IL-6. The role of these cytokines in DC maturation remains incompletely understood. Here, we show that maturation of DC from monocytes was impaired by pretreatment of monocytes with low doses of IL-1beta. Under these conditions, Mycobacterium leprae-infected DC failed to stimulate antigen-specific T cell responses. Expression of CD86 and CD83 and production of IL-12 in response to lipopolysaccharide and peptidoglycan were diminished. In contrast, these DC functions were not impaired by pretreatment with TNF-alpha, IL-6 or IL-10. When monocytes were infected with M. bovis Bacillus Calmette-Guérin, and subsequently differentiated to DC, the activity of these DC was suppressed as well. Thus, IL-1beta acts at early stages of differentiation of DC and impairs biological functions of DC at later stages. Therefore, production of IL-1beta by mycobacteria-infected antigen-presenting cells counteracts effective stimulation of innate and adaptive immune responses.

Control of human host immunity to mycobacteria.

Infection with Mycobacterium tuberculosis results in disease in 5-10% of exposed individuals, whereas the remainder controls infection effectively. Similar inter-individual differences in disease susceptibility are characteristic features of leprosy, typhoid fever, leishmaniasis and other chronic infectious diseases, including viral infections. Although the outcome of infection is influenced by many factors, it is clear that genetic host factors play an important role in controlling disease susceptibility to intracellular pathogens. Knowledge of the genes involved and their downstream cellular pathways will provide new insights for the design of improved and rationalized strategies to enhance host-resistance, e.g. by vaccination. In addition, this knowledge will aid in identifying better biomarkers of protection and disease, which are essential tools for the monitoring of vaccination and other intervention trials. The recent identification of patients with deleterious mutations in genes that encode major proteins in the type-1 cytokine (IL-12/IL23-IFN-gamma) axis, that suffered from severe infections due to otherwise poorly pathogenic mycobacteria (non-tuberculous mycobacteria (NTM) or M. bovis Bacille Calmette-Guerin (BCG)) or Salmonella species has revealed the major role of this system in innate and adaptive immunity to mycobacteria and salmonellae. Clinical tuberculosis has now been described in a number of patients with IL-12/IL23-IFN-gamma system defects. Moreover, unusual mycobacterial infections were reported in several patients with genetic defects in NEMO, a key regulatory molecule in the NFkappaB pathway. These new findings will be discussed since they provide further insights into the role of type-1 cytokines in immunity to mycobacteria, including M. tuberculosis.

Studies of lipoproteins of Mycobacterium leprae.

The deciphering of the genomic sequence of Mycobacterium leprae has made possible to predict the possible lipoproteins. The consensus sequence at the N-terminal region of the protein, including the cysteine residue to which the lipid moiety gets attached, provides a clue to the search. As such, more than 20 putative lipoproteins have been identified from Mycobacterium leprae genomic sequence. Lipoprotein LpK (Accession no. ML0603) which encodes for 371 amino acid precursor protein, was identified. Expression of the protein, in Escherichia coli revealed a 33 kD protein, and metabolic labeling experiments proved that the protein was lipidated. The purified lipoprotein was found to induce production of IL-12 in human peripheral blood monocytes which may imply that M. leprae LpK is involved in protective immunity against leprosy. Pursuit of such lipoproteins may reveal insights into the pathogenesis of the disease.

NK cells modulate the cytotoxic activity generated by Mycobacterium leprae-hsp65 in leprosy patients: role of IL-18 and IL-13.

Protection against intracellular pathogens such as Mycobacterium leprae is critically dependent on the function of NK cells at early stages of the immune response and on Th1 cells at later stages. In the present report we evaluated the role of IL-18 and IL-13, two cytokines that can influence NK cell activity, in the generation of M. leprae-derived hsp65-cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) of leprosy patients. We demonstrated that IL-18 modulates hsp65-induced CTL generation and collaborates with IL-12 for this effect. In paucibacillary (PB) patients and normal controls (N) depletion of NK cells reduces the cytolytic activity. Under these conditions, IL-12 cannot up-regulate this CTL generation, while, in contrast, IL-18 increases the cytotoxic activity both in the presence or absence of NK cells. IL-13 down-regulates the hsp65-induced CTL generation and counteracts the positive effect of IL-18. The negative effect of IL-13 is observed in the early stages of the response, suggesting that this cytokine affects IFNgamma production by NK cells. mRNA coding for IFNgamma is induced by IL-18 and reduced in the presence of IL-13, when PBMC from N or PB patients are stimulated with hsp65. Neutralization of IL-13 in PBMC from multibacillary (MB) leprosy patients induces the production of IFNgamma protein by lymphocytes. A modulatory role on the generation of hsp65 induced CTL is demonstrated for IL-18 and IL-13 and this effect takes place through the production of IFNgamma.

The combination of plasmid interleukin-12 with a single DNA vaccine is more effective than Mycobacterium bovis (bacille Calmette-Guèrin) in protecting against systemic Mycobacterim avium infection.

Sub-unit vaccines utilizing purified mycobacterial proteins or DNA vaccines induce partial protection against mycobacterial infections. For example, immunization with DNA vaccines expressing the gene for the immunodominant 35000 MW protein, common to Mycobacterium avium and Mycobacterium leprae but absent from the Mycobacterium tuberculosis complex, conferred significant protection against infection with either virulent M. avium or M. leprae in mice. However, the level of protection was equivalent to that obtained with the viable, attenuated vaccine, Mycobacterium bovis, bacille Calmette-Guèrin (BCG). The cytokine, interleukin (IL)-12, is essential for priming naïve CD4+ T lymphocytes to differentiate into interferon-gamma (IFN-gamma)-secreting T cells. We have used a novel self-splicing vector expressing both chains of murine IL-12 to determine if plasmid IL-12 would increase the efficacy of a vaccine expressing the M. avium 35000 MW protein (DNA-Av35). Co-immunization with p2AIL-12 and DNA-Av35 led to a significant increase in the number of antigen-specific IFN-gamma secreting cells and total amount of IFN-gamma released, but a concomitant fall in the antibody response to the 35000 MW protein. This pattern of response was associated with enhanced clearance of M. avium from the liver and spleen of coimmunized mice, and was significantly more effective than BCG or DNA-Av35. alone. Following M. avium challenge there was significant increase in the expansion of the 35000 MW antigen-reactive T cells in the coimmunized mice. Therefore, plasmid-delivered IL-12 acts as an effective adjuvant to increase the protective efficacy of a single DNA vaccine against M. avium infection above that achieved by BCG, and this strategy may improve the efficacy of subunit vaccines against M. leprae and M. tuberculosis.

Mycobacterium bovis bacillus Calmette-Guérin-infected dendritic cells potently activate autologous T cells via a B7 and interleukin-12-dependent mechanism.

Mycobacteria are potent adjuvants, can survive intracellularly and have been safely used for many years as vaccines against tuberculosis and leprosy. They are thus important potential vectors for recombinant vaccines. Many of their adjuvant properties are mediated following phagocytosis by dendritic cells (DC), which are in turn critical for priming naïve T cells. Although the maturation of DC in response to mycobacteria, such as Mycobacterium bovis bacillus Calmette-Guérin (BCG), is well described the subsequent responses of autologous T cells to mycobacterium-infected DC remains uncharacterized. In our experiments DC infected with BCG expressed more co-stimulatory molecules than tumour-necrosis factor-alpha (TNF-alpha) -treated DC and stimulated more potent mixed leucocyte reactions. When autologous T cells were co-cultured with BCG-exposed DC they became highly activated, as determined by display of CD25, CD54 and CD71 on both CD4+ and CD8+ cells. In contrast, the response of T cells to TNF-alpha-matured DC was significantly less. Cytokine production from T cells cultured with BCG-exposed DC was enhanced with elevated secretion of interleukin-2 (IL-2), IL-10 and interferon-gamma (IFN-gamma) and was produced by both CD4+ and CD8+ lymphocytes as determined by intracellular staining. In particular, IFN-gamma secretion was increased from 50 pg/ml to 25 000 pg/ml and IL-10 secretion increased from 20 pg/ml to 300 pg/ml in BCG-exposed DC co-cultures. Blocking antibodies to B7.1 and B7.2 or IL-12 significantly reduced the secretion of IFN-gamma and reductions were also seen in the expression of CD25 and CD71 by CD4+ cells. These data demonstrate that mycobacterially infected DC are particularly potent activators of autologous T cells compared to TNF-alpha-exposed DC and that the resultant T cells are functionally superior.

Evaluation of B7-1 (CD80) and B7-2 (CD86) costimulatory molecules and dendritic cells on the immune response in leprosy.

The cell activation depends on T cell antigen receptor binding to antigen plus MHC and costimulation. The binding of CD28, expressed on the T cell surface to B7 (B7-1 or CD80/B7-2 or CD86) present on the antigen--presenting cells (APCs), determines, in several T cell function models, if activation or anergy follows antigenic stimulation. In leprosy, the role of CD80 and CD86 as costimulatory signal in M. leprae-specific cellular immunity has not yet been defined. We investigated the role of B7-CD28 pathway of T cell activation in the in vitro response to M. leprae, following stimulation in the presence of monocytes or dendritic cells (DCs) as APCs. Monocytes were purified, by cold aggregation, from peripheral blood mononuclear leukocytes (PBMC), isolated from leprosy patients. In order to obtain DCs, the monocytes were cultured in the presence of IL-4 and GM-CSF. T cells were purified from PBMC by negative selection with mABs and C'. The phenotype of the cell populations was monitored by FACS. Lymphoproliferative assays were performed with T cells, in the presence of monocytes or DCs. The cells were stimulated by M. leprae in the presence of anti-CD80 antibody (Ab) and/or anti-CD86 antibody (Ab) (Innogenetics). In some experiments Il-10, Il-12 and anti-Il-12 Ab were also added to the culture. We observed a significantly more efficient APC function for DCs when compared to monocytes in T cell in vitro responses to M. leprae. Regardless of the clinical form of Leprosy, the M. leprae-specific immune response was markedly reduced in the presence of anti-CD86 Ab. Il-12 increase the immune response to M. leprae while IL-10 or anti-IL-12 Ab reduce this response when monocytes or DCs were used as APCs.

Interleukin-12 amplifies the M. leprae hsp65-cytotoxic response in the presence of tumour necrosis factor-alpha and interferon-gamma generating CD56+ effector cells: interleukin-4 downregulates this effect.

Interleukin-12 (IL-12) is a major immunomodulatory cytokine that represents a functional bridge between the early resistance and the subsequent antigen specific adaptive immunity. TNF-alpha and IFN-gamma have an important role in the generation of hsp65 specific cytotoxic T lymphocytes (CTL) that lyse hsp65-pulsed autologous macrophages (hsp65 CTL). Since a positive feedback mechanism between TNF-alpha, IFN-gamma and IL-12 has been described, we undertook to evaluate the role of IL-12 on the hsp65 CTL generation in leprosy patients. Our results show that the presence of IL-12 during the first 24 h of the in vitro antigen stimulation amplifies the hsp65 cytotoxic response whenever both IFN-gamma and TNF-alpha are present. The addition of these three cytokines (CKs) was able to abrogate the inhibitory effect of IL-10 on hsp65 CTL in cells from paucibacillary patients (PB) but not that of IL-4 in PB and normal controls (N). Both IL-12 or anti IL-4 enhanced the cytotoxic activity in cells from multibacillary patients (MB). Anti IL-4 upregulated the binding of IFN-gamma and did not modify that of TNF-alpha so the low CTL activity could be as a result of IL-4 by a decrease of the IFN-gamma binding on MB cells. Cells from those MB patients taking thalidomide (MB-T) did neither bind IFN-gamma nor TNF-alpha even when antigen or anti-IL-4 were added, demonstrating that thalidomide inhibits either the in vitro binding or receptor expression of both TNF-alpha and IFN-gamma. Development of CD56 effector cells during the hsp65 stimulation was observed in PB and N by the addition of IL-12 plus TNF-alpha and IFN-gamma, while in MB and MB-T anti IL-4 was also required. So, the inhibitory effect of IL-4 on either production of IFN-gamma, TNF-alpha and/or IL-12 or their receptors could be the mechanism underlying the lack of the hsp65 CTL generation in cells from MB.

IL-18 promotes type 1 cytokine production from NK cells and T cells in human intracellular infection.

We investigated the role of IL-18 in leprosy, a disease characterized by polar cytokine responses that correlate with clinical disease. In vivo, IL-18 mRNA expression was higher in lesions from resistant tuberculoid as compared with susceptible lepromatous patients, and, in vitro, monocytes produced IL-18 in response to Mycobacterium leprae. rIL-18 augmented M. leprae-induced IFN-gamma in tuberculoid patients, but not lepromatous patients, while IL-4 production was not induced by IL-18. Anti-IL-12 partially inhibited M. leprae-induced release of IFN-gamma in the presence of IL-18, suggesting a combined effect of IL-12 and IL-18 in promoting M. leprae-specific type 1 responses. IL-18 enhanced M. leprae-induced IFN-gamma production rapidly (24 h) by NK cells and in a more sustained manner (5 days) by T cells. Finally, IL-18 directly induced IFN-gamma production from mycobacteria-reactive T cell clones. These results suggest that IL-18 induces type 1 cytokine responses in the host defense against intracellular infection.

The possible role of interleukin (IL)-12 and interferon-gamma-inducing factor/IL-18 in protection against experimental Mycobacterium leprae infection in mice.

Cell-mediated immunity participates in host defense against mycobacterial infection. Both interleukin 12 (IL-12) and interferon-gamma-inducing factor (IGIF/IL-18), produced mainly by macrophages, play a critical role in expression of cell-mediated immunity. To investigate the role of IL-12 and IGIF/IL-18 in vivo, we examined cytokine profile, bacterial growth, and the potential benefit of cytokine therapy in susceptible and resistant mice infected with Mycobacterium leprae. The early expression of IL-12 p40 and IGIF/IL-18 at the site of inoculation was found in resistant mice 3-72 h after the infection, but not in susceptible mice. Both strains of mice did not show expression of IFN-gamma and IL-4. IL-12 administration resulted in a significant reduction of bacterial counts in mice with established M. leprae infection. The results imply that susceptible mice exhibit decreased expression of type 1 helper T (Th1) response without reciprocal increased Th2 response and show responsiveness to exogenous IL-12. IL-12 therapy may be a possible rationale for treatment of M. leprae infection.

IL-2 and IL-12 act in synergy to overcome antigen-specific T cell unresponsiveness in mycobacterial disease.

IL-12 secretion by APC is critical for the development of protective Th1-type responses in mycobacterial (Mycobacterium avium and Mycobacterium tuberculosis) infections in mice. We have studied the role of IL-12 and IL-2 in the generation of Mycobacterium leprae-specific T cell responses in humans. Leprosy patients were defined as low/nonresponders or high responders based on the level of T cell proliferation in M. leprae-stimulated PBMC. In high responders, M. leprae-induced proliferation was markedly suppressed by neutralizing anti-IL-12 mAb (inhibition 55 +/- 6%). Neutralization of IL-2 activity resulted in an inhibition of 77 +/- 4%. Given the importance of endogenous IL-2 and IL-12 in M. leprae-induced responses, we investigated the ability of rIL-2 and rIL-12 to reverse T cell unresponsiveness in low/nonresponder patients. Interestingly, rIL-12 and rIL-2 strongly synergized in restoring both M. leprae-specific T cell proliferation and IFN-gamma secretion almost completely to the level of responder patients. A similar synergy between rIL-2 and rIL-12 was also observed in high responders when suboptimal M. leprae concentrations were used for T cell stimulation. Our data demonstrate a crucial role for endogenous IL-12 and IL-2 in M. leprae-induced T cell activation. Most importantly, we show that rIL-2 and rIL-12 act in synergy to overcome Ag-specific Th1 cell unresponsiveness. These findings may be applicable to the design of antimicrobial and antitumor vaccines.