The cellular architecture of the antimicrobial response network in human leprosy granulomas.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.

Iron and vitamin D intake on a diet are able to modify the in vitro immune response to Mycobacterium leprae.

BACKGROUND: The impact of nutrient availability on the survival of Mycobacterium leprae and the development of leprosy remains largely unknown. Iron is essential for the survival and replication of pathogens, while vitamin D has been involved with pathogen elimination and immunoregulation. OBJECTIVES: We evaluated the influence of dietary iron and vitamin D supplementation and restriction on the inflammatory response of mouse immune cells in vitro. METHODS: After 30 days of standard or modified diets, peritoneal cells and splenocytes were stimulated with the alive microorganisms and sonicated antigens of M. leprae, respectively. The production of inflammatory cytokines, reactive oxygen species, and cell proliferation were evaluated. FINDINGS: In peritoneal cells, vitamin D supplementation and iron restriction reduced the production of IL-6 and TNF in response to M. leprae, while splenocytes presented a reduction in TNF production under the same conditions. Lower levels of IFN-γ and TNF were observed in both iron-supplemented and iron-deficient splenocytes. Besides, iron supplementation also reduced the production of IL-6 and IL-10. No changes in the production of reactive oxygen species or in cell proliferation were observed related to different diets. MAIN CONCLUSIONS: Taken together, these data point to an interference of the status of these nutrients on the interaction between the host and M. leprae, with the potential to interfere with the progression of leprosy. Our results highlight the impact of nutritional aspects on this neglected disease, which is significantly associated with unfavourable social conditions.

Correlates of immune exacerbations in leprosy.

Leprosy is still a considerable health threat in pockets of several low and middle income countries worldwide where intense transmission is witnessed, and often results in irreversible disabilities and deformities due to delayed- or misdiagnosis. Early detection of leprosy represents a substantial hurdle in present-day leprosy health care. The dearth of timely diagnosis has, however, particularly severe consequences in the case of inflammatory episodes, designated leprosy reactions, which represent the major cause of leprosy-associated irreversible neuropathy. There is currently no accurate, routine diagnostic test to reliably detect leprosy reactions, or to predict which patients will develop these immunological exacerbations. Identification of host biomarkers for leprosy reactions, particularly if correlating with early onset prior to development of clinical symptoms, will allow timely interventions that contribute to decreased morbidity. Development of a point-of-care (POC) test based on such correlates would be a definite game changer in leprosy health care. In this review, proteomic-, transcriptomic and metabolomic research strategies aiming at identification of host biomarker-based correlates of leprosy reactions are discussed, next to external factors associated with occurrence of these episodes. The vast diversity in research strategies combined with the variability in patient- and control cohorts argues for harmonisation of biomarker discovery studies with geographically overarching study sites. This will improve identification of specific correlates associated with risk of these damaging inflammatory episodes in leprosy and subsequent application to rapid field tests.

A distinct double positive IL-17A+/F+ T helper 17 cells induced inflammation leads to IL17 producing neutrophils in Type 1 reaction of leprosy patients.

Type 1 reactions (T1R) an inflammatory condition, of local skin patches in 30-40% leprosy patients during the course of MDT. IL-17A and IL-17F play an important role in regulating skin inflammation through neutrophils. In the present study, we have analyzed 18 of each T1R and Non-reactions (NR) patients through flow cytometry and qPCR. Interestingly we found that, CD3+CD4+ gated IL-17A+IL-17F+ cells were significantly high in T1R in both MLSA stimulated PBMCs and skin lesions as compared to NR leprosy patients. Hierarchical clustering analysis of gene expression showed that CXCL6, CXCL5, CCL20, CCL7, MMP13 and IL-17RB expression were significantly associated with IL-17A and IL-17F expression (Spearman r2 = 0.77 to 0.98), neutrophils and monocyte markers respectively. In this study, the inflammation noted in lesions of T1R is a different phenotype of Th17 which produce double positive IL-17A+IL17F+ and also contributes IL-17 producing neutrophils and thus would be useful for monitoring, diagnosis and treatment response before reactions episodes.

Leprosy surveillance study in a highly endemic Brazilian area using leprosy specific serologic tests and IFNγ whole blood assay.

This surveillance study evaluated leprosy-serologic tests and the IFNγ whole-blood-assay/WBA as adjunct diagnostic tools. Previously diagnosed leprosy index cases, intradomiciliary, peridomiciliary contacts from a Brazilian endemic area were enrolled during domiciliary visits. Physical evaluation was performed by trained nurses and leprosy diagnosis confirmed by expert dermatologist. ELISA detected IgM anti-PGL-I, IgG anti-LID-1, and IgM/IgG anti-ND-O-LID antibodies. Heparinized WBA plasma stimulated with LID-1, 46f + LID-1, ML0276 + LID-1 (24 h, 37 °C, 5% CO2) was tested for human IFNγ (QuantiFERON®-TB Gold/QFT-G; Qiagen). The survey included 1731 participants: 44 leprosy index cases, 64 intradomiciliary, 1623 peridomiciliary contacts. Women represented 57.7%, median age was 32 years, 72.2% had BCG scar. Leprosy prevalence was higher in intradomiciliary (8.57%) versus peridomiciliary contacts (0.67%), p < 0.001. Among 23 suspects, five leprosy cases were confirmed: 4 multibacillary/MB and 1 paucibacillary/PB. Leprosy incidence was 0.30%: 1.56% in intradomiciliary versus 0.25% in peridomiciliary (p = 0.028). Seropositivity rates were 1.9% to PGL-I, 4.9% to LID-1, and 1.0% to ND-O-LID. LID-1 positivity was higher in all groups; incident cases were LID-1 seropositive. ND-O-LID positivity was higher in intra- versus peridomiciliary contacts (p = 0.022). IFNγ WBA (40 index cases, 19 suspects, 35 intradomiciliary, 74 peridomiciliary contacts) showed higher LID-1/WBA positivity in peridomiciliary contacts (p > 0.05); significant differences among groups were seen with 46f + LID-1 but 0276 + LID-1 induced higher IFNγ levels. Incident cases were LID-1 seropositive, while IFNγ-WBA had marginal diagnostic application. As seropositivity indicates exposed individuals at higher risk of disease development, the utility of serologic screening for surveillance and prophylactic measures remains to be demonstrated.

Expression of NLRP3 inflammasome in leprosy indicates immune evasion of Mycobacterium leprae.

BACKGROUND: Leprosy is an infectious-contagious disease caused by Mycobacterium leprae that remain endemic in 105 countries. This neglected disease has a wide range of clinical and histopathological manifestations that are related to the host inflammatory and immune responses. More recently, the inflammasome has assumed a relevant role in the inflammatory response against microbiological agents. However, the involvement of inflammasome in leprosy remains poorly understood. OBJECTIVES: The aim is to associate biomarkers of inflammasome with the different immunopathological forms of leprosy. METHODS: We performed an observational, cross-sectional, and comparative study of the immunophenotypic expression of inflammasome-associated proteins in immunopathological forms of leprosy of 99 skin lesion samples by immunohistochemistry. The intensity and percentage of NLRP3, Caspase-1, Caspases-4/5, interleukin-1ß and interleukin-18 immunoreactivities in the inflammatory infiltrate of skin biopsies were evaluated. FINDINGS: Strong expression of NLRP3 and inflammatory Caspases-4/5 were observed in lepromatous leprosy (lepromatous pole). In addition, were observed low expression of caspase-1, interleukin-1ß, and interleukin-18 in tuberculoid and lepromatous leprosy. The interpolar or borderline form showed immunophenotype predominantly similar to the lepromatous pole. MAIN CONCLUSIONS: Our results demonstrate that the NLRP3 inflammasome is inactive in leprosy, suggesting immune evasion of M. leprae.

Experimental Modeling of Leprosy in BALB/c, BALB/c Nude, CBA, and C57BL/6ТNF-/- Mice.

Leprosy was modeled in an experiment on BALB/c, BALB/cNude, CBA, and C57BL/6ТNF-/- mice using three Mycobacterium leprae strains obtained from patients with a diagnosis of A30 according to ICD-10 from different regions of the Russian Federation. Proliferation of M. leprae of the used strains showed a temporal-quantitative dependence on the used mouse line. CBA and BALB/cNude mice were optimal for strain R and BALB/c and BALB/cNude lines were optimal for strain I. BALB/cNude mice infected with strain I had low lifespan. M. leprae strain M showed low proliferation activity in BALB/cNude and C57BL/6ТNF-/- mice.

Autophagy Is an Innate Mechanism Associated with Leprosy Polarization.

Leprosy is a chronic infectious disease that may present different clinical forms according to the immune response of the host. Levels of IFN-γ are significantly raised in paucibacillary tuberculoid (T-lep) when compared with multibacillary lepromatous (L-lep) patients. IFN-γ primes macrophages for inflammatory activation and induces the autophagy antimicrobial mechanism. The involvement of autophagy in the immune response against Mycobacterium leprae remains unexplored. Here, we demonstrated by different autophagic assays that LC3-positive autophagosomes were predominantly observed in T-lep when compared with L-lep lesions and skin-derived macrophages. Accumulation of the autophagic receptors SQSTM1/p62 and NBR1, expression of lysosomal antimicrobial peptides and colocalization analysis of autolysosomes revealed an impairment of the autophagic flux in L-lep cells, which was restored by IFN-γ or rapamycin treatment. Autophagy PCR array gene-expression analysis revealed a significantly upregulation of autophagy genes (BECN1, GPSM3, ATG14, APOL1, and TPR) in T-lep cells. Furthermore, an upregulation of autophagy genes (TPR, GFI1B and GNAI3) as well as LC3 levels was observed in cells of L-lep patients that developed type 1 reaction (T1R) episodes, an acute inflammatory condition associated with increased IFN-γ levels. Finally, we observed increased BCL2 expression in L-lep cells that could be responsible for the blockage of BECN1-mediated autophagy. In addition, in vitro studies demonstrated that dead, but not live M. leprae can induce autophagy in primary and lineage human monocytes, and that live mycobacteria can reduce the autophagy activation triggered by dead mycobacteria, suggesting that M. leprae may hamper the autophagic machinery as an immune escape mechanism. Together, these results indicate that autophagy is an important innate mechanism associated with the M. leprae control in skin macrophages.

Murine experimental leprosy: Evaluation of immune response by analysis of peritoneal lavage cells and footpad histopathology.

This study evaluated the immune response of nude and BALB/c mice inoculated in the footpads (FP) with Mycobacterium leprae after 3, 5 and 8 months. At each timepoint peritoneal cells, peripheral blood, FP and popliteal lymph nodes (PLN) were collected. Peritoneal cell cultures were performed to measure the H2 O2 , O2- , NO, IL-2, IL-4, IL-10, IL-12, IFN-γ and TNF levels. Serum levels of anti-PGL-I antibodies were also quantified. The results showed that the infection was progressive in nude mice with bacterial multiplication, development of macroscopic lesions in the FP and presence of bacilli in the PLN at 8 months. In BALB/c mice, the infection reached a plateau of bacillary multiplication at 5 months and regressed at 8 months. Histopathological analysis of FP revealed a mononuclear inflammatory infiltrate with a large number of neutrophils at 5 months, with a higher number in nude mice. At 8 months, the number of neutrophils decreased and the infiltrate was predominantly mononuclear in both mouse strains. There was no H2 O2, O2- , IL-2, IL-4, IL-10 and IFN-γ production in the course of infection in nude mice; however, in BALB/c, O2- and IL-12 production was higher at 5 months and NO, IFN-γ and TNF production was higher at 8 months when there was a decrease in the number of bacilli. The level of anti-PGL-I antibodies was higher in BALB/c mice. Thus, nude and BALB/c mice can be used as experimental models for the study of various aspects of leprosy.

Diet and nutrition: An important risk factor in leprosy.

Leprosy, once considered as poor man's disease may cause severe neurological complications and physical disabilities. Classification of leprosy depends upon the cell mediated and humoral immune responses of the host, from tuberculoid to lepromatous stage. Current therapy to prevent the disease is not only very lengthy but also consists of expensive multiple antibiotics in combination. Treatment and the duration depend on the bacillary loads, from six months in paucibacillary to a year in multibacillary leprosy. Although as per WHO recommendations, these antibiotics are freely available but still out of reach to patients of many rural areas of the world. In this review, we have focused on the nutritional aspect during the multi-drug therapy of leprosy along with the role of nutrition, particularly malnutrition, on susceptibility of Mycobacterium leprae and development of clinical symptoms. We further discussed the diet plan for the patients and how diet plans can affect the immune responses during the disease.

Performance of serological tests PGL1 and NDO-LID in the diagnosis of leprosy in a reference Center in Brazil.

BACKGROUND: Early detection of leprosy and multidrug therapy are crucial to achieve zero transmission and zero grade II incapacities goals of World Health Organization. Leprosy is difficult to diagnose because clinical forms vary and there are no gold standard methods to guide clinicians. The serological rapid tests aid the clinical diagnosis and are available for field use. They are easy to perform, do not require special equipment or refrigeration and are cheaper than the molecular tests. METHODS: We evaluated the performance of two rapid serological tests (PGL1 and NDO-LID) in the discrimination of leprosy cases from healthy individuals at the Alfredo da Matta Foundation, a reference center for the disease in Manaus, Amazonas, Brazil. PGL1 and NDO-LID rapid tests are capable of detecting specific antibodies of M. leprae, IgM and IgM/IgG, respectively. A total of 530 healthy subjects and 171 patients (50 with paucibacillary and 121 multibacillary leprosy) were included in the study. RESULTS: Among the paucibacillary leprosy patients, the sensitivity was 34.0 and 32.0% for the NDO-LID and PGL1, respectively. In multibacillary leprosy patients, the NDO-LID sensitivity was 73.6% and the PGL1 was 81.0%. Serological tests demonstrated specificities of 75.9% for PGL-1 and 81.7% for NDO-LID. The positive predictive value (PPV), negative predictive value (NPV) and accuracy in multibacillary patients were 47.9, 93.1, and 80.2% respectively for the NDO-LID, and 43.4, 94.6 76.8% for PGL1. CONCLUSIONS: The tests showed limited capacity in the diagnosis of the disease, however, the high negative predictive value of the tests indicates a greater chance of true negatives in this group favoring exclusion of leprosy. This characteristic of the ML flow test is important in aiding clinical Diagnosis, especially in a region endemic to the disease and with other confounding skin conditions.

Social and environmental conditions related to Mycobacterium leprae infection in children and adolescents from three leprosy endemic regions of Colombia.

BACKGROUND: Leprosy is is still considered a public health issue and in Colombia 7-10% of new cases are found in children, indicating both active transmission and social inequality. We hypothesized that circulating antibodies against Natural Octyl Disaccharide-Leprosy IDRI Diagnostic (NDO-LID) (a combination of Mycobacterium leprae antigens) could reveal the social and environmental aspects associated with higher frequencies of M. leprae infection among children and adolescents in Colombia. METHODS: An observational cross-sectional study was conducted involving sampling from 82 children and adolescents (younger than 18 years of age) who had household contact with index leprosy patients diagnosed in the last 5 years. Data were analyzed through bivariate analysis made by applying a Pearson x2 test for qualitative variables, while quantitative variables, depending on their distribution, were analyzed using either a Student's t-test or Mann-Whitney U test. Multivariate analysis was performed using a multiple regression and binomial logistic approach. RESULTS: A bivariate analysis demonstrated that antibody titers against NDO-LID were significantly greater in children and adolescents with a low socioeconomic status that had: lived in vulnerable areas of the UAChR shared region; eaten armadillo meat; exposure of over 10 years to an index case and; not received BCG immunization. Moreover, a multivariate analysis showed that residing in the UAChR region has a strong association with a greater possibility of M. leprae infection. CONCLUSIONS: M. leprae transmission persists among young Colombians, and this is associated with social and environmental conditions. An intensification of efforts to identify new leprosy cases in vulnerable and forgotten populations where M. leprae transmission continues therefore appears necessary.

Transcription factors STAT-4, STAT-6 and CREB regulate Th1/Th2 response in leprosy patients: effect of M. leprae antigens.

BACKGROUND: Leprosy is an ideal human disease to study T cell regulation as patients show correlation between cytokine skewed Th1-Th2 responses and clinical forms of the disease. The Role of transcription factors on the modulation of Th1 and Th2 responses by M. leprae antigens has not been adequately studied. In the present study, we studied the effect of M. leprae antigens on transcription factors STAT-4, STAT-6 and CREB and their correlation with Th1/Th2 cell mediated immune responses in leprosy. METHODS: Leprosy patients of both categories of tuberculoid leprosy (BT/TT) and lepromatous leprosy (BL/LL) were selected from the OPD of NJ1L & OMD, (ICMR), Agra and healthy individuals (H) were chosen from the staff and students working in the institute. Peripheral blood mononuclear cells (PBMCs) of the study subjects were stimulated with M. leprae antigens (WCL, MLSA, and PGL-1). Sandwich ELISA was done in the culture supernatants of healthy and leprosy patients to detect IL-4, IL-10 and IFN-γ. Further, expression of IFN-γ and IL-4 and activation of STAT4, STAT6 and CREB transcription factors in CD4+ T cell with or without stimulation of M. leprae antigens was investigated by flow cytometry. RESULTS: Lepromatous leprosy patients showed significantly lower IFN-γ and higher IL-4 levels in culture supernatant and significantly low expression of IFN-γ and higher expression of IL-4 by CD4+ T cells than healthy individuals with or without antigenic stimulation. Antigenic stimulation significantly increased IL-10 in BL/LL patients but not in BT/TT patients or healthy individuals. PGL-1 stimulation led to significantly higher activation of STAT-6 in BT/TT and BL/LL patients in comparison to healthy individuals. All the three antigens led to activation of CREB in healthy and BT/TT patients but not in BL/LL patients. CONCLUSION: Our findings show that M. leprae antigens differentially modulate activation of T cell transcription factors STAT-4/STAT-6 and CREB. These transcription factors are well known to regulate Th1 and Th2 mediated immune response which in turn could play vital role in the clinical manifestations of leprosy. These observations may help to determine how these T cell transcription factors affect the development of immune dysfunction and whether these new pathways have a role in immunomodulation in intracellular diseases like leprosy and TB.

S100A12 Is Part of the Antimicrobial Network against Mycobacterium leprae in Human Macrophages.

Triggering antimicrobial mechanisms in macrophages infected with intracellular pathogens, such as mycobacteria, is critical to host defense against the infection. To uncover the unique and shared antimicrobial networks induced by the innate and adaptive immune systems, gene expression profiles generated by RNA sequencing (RNAseq) from human monocyte-derived macrophages (MDMs) activated with TLR2/1 ligand (TLR2/1L) or IFN-γ were analyzed. Weighed gene correlation network analysis identified modules of genes strongly correlated with TLR2/1L or IFN-γ that were linked by the "defense response" gene ontology term. The common TLR2/1L and IFN-γ inducible human macrophage host defense network contained 16 antimicrobial response genes, including S100A12, which was one of the most highly induced genes by TLR2/1L. There is limited information on the role of S100A12 in infectious disease, leading us to test the hypothesis that S100A12 contributes to host defense against mycobacterial infection in humans. We show that S100A12 is sufficient to directly kill Mycobacterium tuberculosis and Mycobacterium leprae. We also demonstrate that S100A12 is required for TLR2/1L and IFN-γ induced antimicrobial activity against M. leprae in infected macrophages. At the site of disease in leprosy, we found that S100A12 was more strongly expressed in skin lesions from tuberculoid leprosy (T-lep), the self-limiting form of the disease, compared to lepromatous leprosy (L-lep), the progressive form of the disease. These data suggest that S100A12 is part of an innate and adaptive inducible antimicrobial network that contributes to host defense against mycobacteria in infected macrophages.

Phenotypic and functional features of innate and adaptive immunity as putative biomarkers for clinical status and leprosy reactions.

The aim of this study was to identify phenotypic and functional biomarkers associated with distinct clinical status of leprosy or leprosy reactions. The study included tuberculoid/borderline (BB/BT/T) and lepromatous (BL/L) leprosy poles as well as Type-1 and Type-2 leprosy reactions along with healthy controls (NI). A range of peripheral blood biomarkers of innate (neutrophils - NEU and monocytes - MON) and adaptive immunity (CD4+ and CD8+ T-cells) were evaluated ex vivo and upon in vitro stimuli with M. leprae antigen. Data analysis allowed the selection of NEUTLR4+ (ex vivo) and CD4+IL-10+ (in vitro) as universal biomarkers increased in all leprosy patients and those exhibiting leprosy reactions. A range of biomarkers were commonly found in both poles of leprosy patients, including decreased levels of MONTGF-ß+ (ex vivo) and increased levels of MONTNF-α+, CD4+TGF-ß+, CD8+TLR2+, CD8+TNF-α+, CD8+IL-4+ and CD8+TGF-ß+ (in vitro). Noteworthy was that MONHLA-DR+ (ex vivo) and CD8+IL-10+ (in vitro) were particularly found in BL/L patients. Leprosy patients with Type-1 reaction exhibited a larger list of altered biomarkers, mainly involving activation markers (TLR2, TLR4, HLA-DR and DAF-2T) in NEU and MON along with CD4+ and CD8+ cells. In summary, this study provided insights about immunological features of leprosy poles and leprosy reactional episodes with putative applicability, including novel biomarkers for complementary diagnosis and future therapeutic approaches in clinical studies.

Mast cell heterogeneity and anti-inflammatory annexin A1 expression in leprosy skin lesions.

Mast cells (MCs) have important immunoregulatory roles in skin inflammation. Annexin A1 (ANXA1) is an endogenous anti-inflammatory protein that can be expressed by mast cells, neutrophils, eosinophils, monocytes, epithelial and T cells. This study investigated MCs heterogeneity and ANXA1 expression in human dermatoses with special emphasis in leprosy. Sixty one skin biopsies from 2 groups were investigated: 40 newly diagnosed untreated leprosy patients (18 reaction-free, 11 type 1 reaction/T1R, 11 type 2 reaction/T2R); 21 patients with other dermatoses. Tryptase/try+ and chymase/chy + phenotypic markers and toluidine blue stained intact/degranulated MC counts/mm2 were evaluated. Try+/chy+ MCs and ANXA1 were identified by streptavidin-biotin-peroxidase immunostaining and density was reported. In leprosy, degranulated MCs outnumbered intact ones regardless of the leprosy form (from tuberculoid/TT to lepromatous/LL), leprosy reactions (reactional/reaction-free) and type of reaction (T1R/T2R). Compared to other dermatoses, leprosy skin lesions showed lower numbers of degranulated and intact MCs. Try+ MCs outnumbered chy+ in leprosy lesions (reaction-free/reactional, particularly in T2R), but not in other dermatoses. Compared to other dermatoses, ANXA1 expression, which is also expressed in mast cells, was higher in the epidermis of leprosy skin lesions, independently of reactional episode. In leprosy, higher MC degranulation and differential expression of try+/chy+ subsets independent of leprosy type and reaction suggest that the Mycobacterium leprae infection itself dictates the inflammatory MCs activation in skin lesions. Higher expression of ANXA1 in leprosy suggests its potential anti-inflammatory role to maintain homeostasis preventing tissue and nerve damage.

Whole blood profiling of leprosy type 1(reversal) reactions highlights prominence of innate immune response genes.

BACKGROUND: The major factors contributing for nerve damage and permanent disabilities in leprosy are type 1 or reversal reactions (RR) and type 2 or erythema nodosum leprosum (ENL). Gene profiling of leprosy reactions have shown that different pathways are activated during the course of reactions, which is consistent with the exacerbated immune response exhibited by these patients. METHODS: We used qPCR to screen a panel of 90 genes related to the immune response in leprosy in RNA-derived peripheral leukocytes of patients with (N = 94) and without leprosy reactions (N = 57) in order to define expression signatures correlated to RR or ENL. RESULTS: Our results show that there is a marked signature for RR in the blood, comprising genes mostly related to the innate immune responses, including type I IFN components, autophagy, parkins and Toll like receptors. On the other hand, only Parkin was differentially expressed in the ENL group. CONCLUSIONS: The data put together corroborates previous work that brings evidence that an acute uncontrolled exacerbated immune response designed to contain the spread of M. leprae antigens might be cause of RR pathogenesis. Identifying a blood profile useful to predict leprosy reactions prior to its development might help to reduce the morbidity associated to this disabling disease.

Immunohistochemical characterization of the M4 macrophage population in leprosy skin lesions.

BACKGROUND: Since macrophages are one of the major cell types involved in the Mycobacterium leprae immune response, roles of the M1 and M2 macrophage subpopulations have been well defined. However, the role of M4 macrophages in leprosy or other infectious diseases caused by mycobacteria has not yet been clearly characterized. This study aimed to investigate the presence and potential role of M4 macrophages in the immunopathology of leprosy. METHODS: We analyzed the presence of M4 macrophage markers (CD68, MRP8, MMP7, IL-6, and TNF-α) in 33 leprosy skin lesion samples from 18 patients with tuberculoid leprosy and 15 with lepromatous leprosy by immunohistochemistry. RESULTS: The M4 phenotype was more strongly expressed in patients with the lepromatous form of the disease, indicating that this subpopulation is less effective in the elimination of the bacillus and consequently is associated with the evolution to one of the multibacillary clinical forms of infection. CONCLUSION: M4 macrophages are one of the cell types involved in the microbial response to M. leprae and probably are less effective in controlling bacillus replication, contributing to the evolution to the lepromatous form of the disease.

DNA Sensing via TLR-9 Constitutes a Major Innate Immunity Pathway Activated during Erythema Nodosum Leprosum.

The chronic course of lepromatous leprosy may be interrupted by acute inflammatory episodes known as erythema nodosum leprosum (ENL). Despite its being a major cause of peripheral nerve damage in leprosy patients, the immunopathogenesis of ENL remains ill-defined. Recognized by distinct families of germline-encoded pattern recognition receptors, endogenous and pathogen-derived nucleic acids are highly immunostimulatory molecules that play a major role in the host defense against infections, autoimmunity, and autoinflammation. The aim of this work was to investigate whether DNA sensing via TLR-9 constitutes a major inflammatory pathway during ENL. Flow cytometry and immunohistochemistry analysis showed significantly higher TLR-9 expression in ENL when compared with nonreactional lepromatous patients, both locally in the skin lesions and in circulating mononuclear cells. The levels of endogenous and pathogen-derived TLR-9 ligands in the circulation of ENL patients were also higher. Furthermore, PBMCs isolated from the ENL patients secreted higher levels of TNF, IL-6, and IL-1ß in response to a TLR-9 agonist than those of the nonreactional patients and healthy individuals. Finally, E6446, a TLR-9 synthetic antagonist, was able to significantly inhibit the secretion of proinflammatory cytokines by ENL PBMCs in response to Mycobacterium leprae lysate. Our data strongly indicate that DNA sensing via TLR-9 constitutes a major innate immunity pathway involved in the pathogenesis and evolution of ENL. Thus, the use of TLR-9 antagonists emerges as a potential alternative to more effectively treat ENL aiming to prevent the development of nerve injuries and deformities in leprosy.

Utility of immunoglobulin isotypes against LID-1 and NDO-LID for, particularly IgG1, confirming the diagnosis of multibacillary leprosy.

BACKGROUND Leprosy remains a health problem in many countries, with difficulties in diagnosis resulting in delayed treatment and more severe disabilities. Antibodies against several Mycobacterium leprae antigens have, however, shown value as diagnostic and/or prognostic markers. OBJECTIVES The objective of this study was to evaluate serum immunoglobulin (Ig) IgM and IgG subclass reactivity against three M. leprae specific antigens: NDO-HSA, a conjugate formed by natural octyl disaccharide bound to human serum albumin; LID-1, the fusion protein product of the ml0405 and ml2331 genes; and NDO-LID, a combination of LID-1 and NDO. METHODS Sera from healthy controls, paucibacillary (PB) and multibacillary (MB) leprosy patients, and their respective household contacts, were evaluated for the presence of antigen-specific IgM, IgG, and IgG subclass antibodies by enzyme-linked immunosorbent assay (ELISA). The sensitivity and specificity of each ELISA were evaluated by receiver operating characteristic (ROC) curve analysis. FINDINGS Our data confirm that serum IgM antibodies against NDO-HSA and IgG antibodies against LID-1, as well as IgG/M antibodies against NDO-LID, are markedly increased in MB patients. For the first time, our data reveal a selective increase in IgG1 and IgG3 antibodies against LID-1 and NDO-LID in MB patients, demonstrating that these antibody isotypes are suitable for differentiation between MB and PB patients. ROC curve analysis indicates an improved capacity for diagnosing MB leprosy patients using the detection of IgG antibodies, particularly the IgG1 isotype, specific to LID-1 and NDO-LID over the performance levels attained with NDO-HSA. CONCLUSIONS Our findings indicate that serological tests based on the detection of antigen-specific IgG1 antibodies are a useful tool to differentiate MB from PB patients, and indicate the enhanced performance of the LID-1 and NDO-LID antigens in the serodiagnosis of leprosy.