Enhanced IL-6 and IL-12B Gene Expression After SARS-CoV-2 Infection in Leprosy Patients May Increase the Risk of Neural Damage.

Experts have called attention to the possible negative impact of the coronavirus disease 2019 (COVID-19)-related cytokine storm syndrome on the progression of leprosy-related disabilities. We assessed the frequency of reactional states in patients co-infected with Mycobacterium leprae and severe acute respiratory syndrome (SARS) coronavirus (CoV) 2 (SARS-CoV-2). We consecutively included patients during the first peak of the COVID-19 epidemic in Brazil and analyzed the expressions of genes encoding interleukin (IL)-1ß, IL-6, IL-8, IL-10, IL-12A, IL-12B, and tumor necrosis factor-α in peripheral blood mononuclear cells. We included 64 leprosy patients and 50 controls. Twelve of the leprosy patients and 14 of the controls had been diagnosed with COVID-19. Co-infection was associated with increased IL-6 (P = 0.043) and IL-12B (P = 0.017) expression. The median disability grades were higher for leprosy/COVID-19 patients; however, the difference was not significant (P = 0.194). Patients co-infected with M. leprae and SARS-CoV-2 may experience a higher-grade proinflammatory state.

Nutritional status and immune response in murine experimental Jorge Lobo's disease.

There are no studies investigating the role of nutritional status and immunity associated with Jorge Lobo's disease. The objective of this study was to evaluate the effects of protein-calorie malnutrition on the immune response of BALB/c mice inoculated with Lacazia loboi. In this study,the animals were divided into four groups: G1: inoculated with restricted diet, G2: not inoculated with restricted diet, G3: inoculated with regular diet, G4: not inoculated with regular diet. The animals of groups G1 and G2 were submitted to malnutrition for 20 days and once installed the animals were inoculated intradermally into the footpad. After 4 months, they were euthanised for the isolation of peritoneal lavage cells and removal of the footpad. The production of IL-2, IL-4, IL-10, IL-12, IFN-γ, TNF-α, H2 O2 and nitric oxide (NO) was evaluated in the peritoneal lavage cells. The footpad was evaluated regarding the size of macroscopic lesions, number of fungi and viability index. The results showed that the infection did not exert great influence on the body weight of the mice and previous malnutrition was an unfavourable factor for viability index, number of fungi, macroscopic lesion size in the footpad and production of H2 O2 , NO, IL-12, IL-10 and IFN-γ, suggesting that malnutrition significantly altered fungal activity and peritoneal cells. The results suggest considerable interaction between nutrition and immunity in Jorge Lobo's disease.

Liposomal-lupane system as alternative chemotherapy against cutaneous leishmaniasis: macrophage as target cell.

Leishmania amazonensis causes human diseases that range from self-healing to diffusion cutaneous lesions. The chemotherapy of leishmaniasis requires long-term treatment and has been based on the use of pentavalent antimonials. Liposomes have been used as antileishmanial drug carries and have adjuvant activity in vaccines against several microorganisms, representing an important option to the development of new therapeutics for the disease. In this study, we developed a liposomal formulation containing lupane [3ß,6ß,16ß-trihydroxylup-20(29)-ene], isolated from fruits of Combretum leprosum with pharmacological properties as antinociceptive, anti-inflammatory, antiulcerogenic and antileishmanial activities. The aim of the present study was to evaluate the efficacy of liposomal-lupane in L. amazonensis-infection model. Liposomes were prepared by the extrusion method with DPPC, DPPS and cholesterol at 5:1:4 weight ratio. The lupane (2 mg/mL) was added to the lipid mixture, solubilized in chloroform and dried under nitrogen flow. The activity of liposomal-lupane was conducted in vitro with mouse peritoneal infected macrophages. Furthermore, mice were infected in the right hind footpad with 10(5) stationary growth phase of L. amazonensis promastigotes. After 6 weeks, animals were treated with liposomal-lupane for 15 days by intraperitoneal injection. The evolution of disease was monitored weekly by measuring footpad thickness with a caliper. Three days after the treatment, peritoneal macrophages were collected, plated and production of the cytokines IL-10 and IL-12 was evaluated in supernatants of the cultures after 24 h. The results indicate that the liposomal system containing lupane achieved here is a promising tool to confer antileishmanial activity to infected macrophages.

A lipopeptide facilitate induction of Mycobacterium leprae killing in host cells.

Little is known of the direct microbicidal activity of T cells in leprosy, so a lipopeptide consisting of the N-terminal 13 amino acids lipopeptide (LipoK) of a 33-kD lipoprotein of Mycobacterium leprae, was synthesized. LipoK activated M. leprae infected human dendritic cells (DCs) to induce the production of IL-12. These activated DCs stimulated autologous CD4+ or CD8+ T cells towards type 1 immune response by inducing interferon-gamma secretion. T cell proliferation was also evident from the CFSE labeling of target CD4+ or CD8+ T cells. The direct microbicidal activity of T cells in the control of M. leprae multiplication is not well understood. The present study showed significant production of granulysin, granzyme B and perforin from these activated CD4+ and CD8+ T cells when stimulated with LipoK activated, M. leprae infected DCs. Assessment of the viability of M. leprae in DCs indicated LipoK mediated T cell-dependent killing of M. leprae. Remarkably, granulysin as well as granzyme B could directly kill M. leprae in vitro. Our results provide evidence that LipoK could facilitate M. leprae killing through the production of effector molecules granulysin and granzyme B in T cells.

Immunostimulatory activity of major membrane protein II from Mycobacterium tuberculosis.

Previously, we observed that both major membrane protein II of Mycobacterium leprae (MMP-ML) and its fusion with M. bovis BCG (BCG)-derived heat shock protein 70 (HSP70) (Fusion-ML) are immunogenic and that recombinant BCG secreting either of these proteins effectively inhibits the multiplication of M. leprae in mice. Here, we purified M. tuberculosis-derived major membrane protein II (MMP-MTB) and its fusion with HSP70 (Fusion-MTB) in a lipopolysaccharide-free condition and evaluated their immunostimulatory abilities. Both MMP-MTB and Fusion-MTB activated monocyte-derived dendritic cells (DC) in terms of phenotype and interleukin-12 (IL-12) production, but Fusion-MTB more efficiently activated them than MMP-MTB did. The IL-12 production was a consequence of the ligation of those recombinant proteins with Toll-like receptor 2. The M. tuberculosis-derived and M. leprae-derived recombinant proteins activated naïve T cells of both CD4 and CD8 subsets, but M. tuberculosis-derived proteins were superior to M. leprae-derived proteins and fusion proteins were superior to MMP, regardless of the origin of the protein. Memory-type CD4(+) T cells obtained from BCG-vaccinated healthy individuals seem to be primed with MMP-MTB by the vaccination, and both M. tuberculosis-derived recombinant proteins produced perforin-producing CD8(+) T cells from memory-type CD8(+) T cells. Further, infection of DC and macrophages with M. tuberculosis H37Ra and H37Rv induced the expression of MMP on their surface. These results indicate that M. tuberculosis-derived MMP, as a sole protein or as part of a fusion protein, may be useful for developing new vaccinating agents against tuberculosis.

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.

Systemic suppression of interferon-gamma responses in Buruli ulcer patients resolves after surgical excision of the lesions caused by the extracellular pathogen Mycobacterium ulcerans.

Buruli ulcer (BU), caused by Mycobacterium ulcerans, is the third most common mycobacterial infection in immunocompetent humans besides tuberculosis and leprosy. We have compared by ex vivo enzyme-linked immunospot analysis interferon-gamma (IFN-gamma) responses in peripheral blood mononuclear cells (PBMC) from BU patients, household contacts, and individuals living in an adjacent M. ulcerans nonendemic region. PBMC were stimulated with purified protein derivative (PPD) and nonmycobacterial antigens such as reconstituted influenza virus particles and isopentenyl-pyrophosphate. With all three antigens, the number of IFN-gamma spot-forming units was reduced significantly in BU patients compared with the controls from a nonendemic area. This demonstrates for the first time that M. ulcerans infection-associated systemic reduction in IFN-gamma responses is not confined to stimulation with live or dead mycobacteria and their products but extends to other antigens. Interleukin (IL)-12 secretion by PPD-stimulated PBMC was not reduced in BU patients, indicating that reduction in IFN-gamma responses was not caused by diminished IL-12 production. Several months after surgical excision of BU lesions, IFN-gamma responses of BU patients against all antigens used for stimulation recovered significantly, indicating that the measured systemic immunosuppression was not the consequence of a genetic defect in T cell function predisposing for BU but is rather related to the presence of M. ulcerans bacteria.

Immunomodulation and protection induced by DNA-hsp65 vaccination in an animal model of arthritis.

We described a prophylactic and therapeutic effect of a DNA vaccine encoding the Mycobacterium leprae 65-kDa heat shock protein (DNA-hsp65) in experimental murine tuberculosis. However, high homology of the vaccine to the corresponding mammalian hsp60, together with the CpG motifs in the plasmidial vector, could trigger or exacerbate an autoimmune disease. In the present study, we evaluate the potential of DNA-hsp65 vaccination to induce or modulate arthritis in mice genetically selected for acute inflammatory reaction (AIR), either maximal (AIRmax) or minimal (AIRmin). Mice immunized with DNA-hsp65 or injected with the corresponding DNA vector (DNAv) developed no arthritis, whereas pristane injection resulted in arthritis in 62% of AIRmax mice and 7.3% of AIRmin mice. Administered after pristane, DNA-hsp65 downregulated arthritis induction in AIRmax animals. Levels of interleukin (IL)-12 were significantly lower in mice receiving pristane plus DNA-hsp65 or DNAv than in mice receiving pristane alone. However, when mice previously injected with pristane were inoculated with DNA-hsp65 or DNAv, the protective effect was significantly correlated with lower IL-6 and IL-12 levels and higher IL-10 levels. Our results strongly suggest that DNA-hsp65 has no arthritogenic potential and is actually protective against experimentally induced arthritis in mice.

Polymorphism of the 5' flanking region of the IL-12 receptor beta2 gene partially determines the clinical types of leprosy through impaired transcriptional activity.

BACKGROUND: Individual differences in T cell responsiveness to interleukin 12 (IL-12), resulting from inherited factors, may be responsible for differences in the intensity of cell mediated immune (CMI) responses in patients with leprosy, a disease with a wide clinical spectrum. AIM: Polymorphisms in the 5' flanking region of the IL12RB2 gene were analysed to determine potential immunogenetic factors affecting CMI responses, using leprosy as a model. METHODS: Polymorphisms in the 5' flanking region of IL12RB2 were examined using direct sequencing techniques, and allele frequencies between patients with lepromatous leprosy and patients with tuberculoid leprosy were compared. The effect of these single nucleotide polymorphisms (SNPs) on IL12RB2 expression was estimated using the dual luciferase reporter gene assay in Jurkat T cells. RESULTS: Several SNPs, including -1035A>G, -1023A>G, -650delG, and -465A>G, were detected within the 5' flanking region of IL12RB2. The frequency of haplotype 1 (-1035A, -1023A, -650G, -464A) was high in the general Japanese population, but was significantly lower in lepromatous patients compared with tuberculoid patients and healthy controls. Reporter gene assays using Jurkat T cells revealed that all haplotypes carrying one or more SNP exhibited a lower transcriptional activity compared with haplotype 1. CONCLUSION: SNPs within the 5' flanking region of IL12RB2 affect the degree of expression of this gene and may be implicated in individual differences in CMI responsiveness to mycobacterial antigens, leading to lepromatous or tuberculoid leprosy.

Monocytes of distinct clinical types of leprosy are differentially activated by cross-linking class II HLA molecules to secrete IL-12.

Leprosy is characterized by a wide spectrum of clinical features depending on the individual differences in Th1-type immunity. The objective of this study was to evaluate whether monocyte activation by stimulus via class II HLA molecules would be correlated with the differences in cellular immune responses among diverse clinical forms of leprosy. IL-1beta and IL-12 productivity in monocyte preparations obtained from PBMCs was estimated in patients with lepromatous- and tuberculoid-type leprosy. We found that monocytes from lepromatous patients produced significantly higher (about 4-fold higher) amounts of IL-12 as compared to in patients with tuberculoid type of leprosy when class II HLA molecules were cross-linked with anti-HLA class II antibodies, whereas almost equal amounts of IL-1beta were produced from each monocyte preparation by stimulus via class II HLA molecules regardless of the clinical form of leprosy. These results suggest that monocyte activation differs between lepromatous and tuberculoid patients in terms of IL-12 secretion, which might be related to individual differences in the cellular immune responses according to the clinical type of leprosy.

Tissue-specific down-regulation of RIPK 2 in Mycobacterium leprae-infected nu/nu mice.

RIPK 2 is adapter molecule in the signal pathway involved in Toll-like receptors. However, there has been no reported association between receptor-interacting serine/threonine kinase 2 (RIPK 2) expression and the infectious diseases involving mycobacterial infection. This study found that its expression was down-regulated in the footpads and skin but was up-regulated in the liver of Mycobacterium leprae-infected nu/nu mice compared with those of the M. leprae non-infected nu/nu mice. It was observed that the interlukin-12p40 and interferon-gamma genes involved in the susceptibility of M. leprae were down-regulated in the skin but were up-regulated in the liver. Overall, this suggests that regulation of RIPK 2 expression is tissue-specifically associated with M. leprae infection.

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.

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.

Therapy of tuberculosis in mice by DNA vaccination.

Mycobacterium tuberculosis continues to kill about 3 million people every year, more than any other single infectious agent. This is attributed primarily to an inadequate immune response towards infecting bacteria, which suffer growth inhibition rather than death and subsequently multiply catastrophically. Although the bacillus Calmette-Guerin (BCG) vaccine is widely used, it has major limitations as a preventative measure. In addition, effective treatment requires that patients take large doses of antibacterial drug combinations for at least 6 months after diagnosis, which is difficult to achieve in many parts of the world and is further restricted by the emergence of multidrug-resistant strains of M. tuberculosis. In these circumstances, immunotherapy to boost the efficiency of the immune system in infected patients could be a valuable adjunct to antibacterial chemotherapy. Here we show in mice that DNA vaccines, initially designed to prevent infection, can also have a pronounced therapeutic action. In heavily infected mice, DNA vaccinations can switch the immune response from one that is relatively inefficient and gives bacterial stasis to one that kills bacteria. Application of such immunotherapy in conjunction with conventional chemotherapeutic antibacterial drugs might result in faster or more certain cure of the disease in humans.

Cytokine patterns at the site of mycobacterial infection.

Distinct patterns of T cell cytokine production have been shown to influence the outcome of infection in mouse models and humans. Th1 or Type 1 cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) are generally associated with resistance to infection, whereas Th2 or Type 2 cytokines, IL-4 and IL-10 are associated with progressive disease. Leprosy is a useful model for studying the role of cytokines in modulating T cell responses in human infectious disease. Infection by Mycobacterium leprae results in disease manifestations that encompass an immunological spectrum. Tuberculoid patients are able to restrict the growth of the pathogen and mount strong T cell responses to M. leprae. In contrast, lepromatous patients manifest disseminated infection and their T cells weakly respond to M. leprae. We have found that tuberculoid leprosy lesions have a predominance of CD4+ T cells producing the Type 1 cytokine pattern. Secondly, IL-12 mRNA was expressed at 10-fold higher levels in tuberculoid lesions as compared to lepromatous lesions and that IL-12 promotes the selective expansion of the Type 1 cytokine producing cells. In contrast, lepromatous lesions contain CD8+ IL-4-producing cells that suppress antigen-specific T cell responses and promote the outgrowth of additional suppressor T cells. IL-10, also expressed at higher levels in lepromatous as compared to tuberculoid lesions, was found to be produced by macrophages, effectively inhibiting cytokine production and macrophage activity.