T-Regulatory Cells in Erythema Nodosum Leprosum: An Immunohistochemical and Image Morphometric Study.

ABSTRACT: Erythema nodosum leprosum (ENL) occurs as an immune-inflammatory complication of multibacillary leprosy (MBL), precipitated by an interaction between the host, bacilli, and the environment. This complication often causes significant morbidity due to systemic involvement and needs to be treated aggressively. T-regulatory cells (T-regs) are the immunomodulatory subset of T cells that are hypothesized to play a role in ENL. We have performed immunohistochemistry for FoxP3 (T-reg), CD3 (pan-T), CD4 (helper T), and CD8 (cytotoxic T) on 50 biopsy-proven cases of ENL along with 84 biopsy-proven cases of paucibacillary leprosy (PBL) (n = 49) and MBL (n = 35). Image morphometry was applied to objectively assess the relative preponderance of these subsets of T cells. The area fraction of T-regs showed a trend of reduction from PBL to MBL to ENL (P = 0.068), whereas the FoxP3:CD3 (T-reg: pan-T) ratio showed a significant reduction across these groups (P = 0.023). However, there was no significant difference of T-regs or FoxP3:CD3 ratio between MBL and ENL. The T-regs showed a significant positive correlation (P = 0.007) with the cytotoxic T cells in the skin biopsy. The presence of dermal eosinophils in ENL showed a trend association with the FoxP3:CD3 ratio (P = 0.05). Various histopathological parameters including epidermal spongiosis, dermal stromal edema, dermal ill-formed granuloma, and the presence of bacilli within the endothelium and vascular smooth muscle correlated with various T-cell subsets. Our study, one of the largest on this topic, objectively assessed the role of T-regs in the spectrum of leprosy. Nevertheless, the precipitation of ENL from MBL is probably not associated with the T-reg subset alone.

Oct-6 transcriptional factor a possible biomarker for leprosy diagnosis.

Leprosy is an infectious disease caused by Mycobacterium leprae that affects the skin and nerves. The nerve damage in leprosy may be related to alterations in transcriptional factors, such as Krox-20, Oct-6, Sox-10. Thirty skin biopsies in leprosy patients and 15 non-leprosy skin biopsies were evaluated using RT-qPCR to assess Krox-20, Oct-6, and Sox-10 and these data was related with S-100 immunohistochemistry. Changes in gene expression were observed in the skin and dermal nerves of leprosy patients in Oct-6 and Sox-10. When comparing Oct-6 with S-100 IHC as diagnostic tests for leprosy, Oct-6 showed a sensitivity of 73.3%, and specificity of 100%, while S-100 IHC showed a sensitivity of 96.6% and specificity of 100%. Our data suggest Oct-6 could be an auxiliary biomarker specific to detecting changes in dermal nerves in leprosy and thus useful to health workers and pathologists with no expertise to observe nerve injuries in leprosy.

Image Morphometric Analysis of B Cells and Plasma Cells in Erythema Nodosum Leprosum With Clinicopathological Correlation.

ABSTRACT: Erythema nodosum leprosum (ENL) occurs as an immunological complication of multibacillary leprosy (MBL). The pathogenesis of ENL is long considered to be a T-cell-mediated process. The role of B cells and plasma cells in ENL is not well described in the literature. Therefore, we investigated the B-cell and plasma cell infiltrates in the skin biopsies of biopsy-proven cases of ENL by immunohistochemistry and image morphometry and compared the result with paucibacillary leprosy and MBL. Moreover, we sought a correlation of the B-cell and plasma cell infiltrates with different clinical, hematological, histopathological, and bacteriological parameters as well as the T-cell subsets in the skin biopsies. Our study highlighted a significant reduction in the number of B cells from paucibacillary leprosy to MBL to ENL, although there was no significant variation in the plasma cell infiltrate. The plasma cell infiltrate correlated with absolute neutrophilia in the blood and the presence of eosinophils in the ENL lesions. Both B cells and plasma cells positively correlated with CD4-positive T-helper cells and the CD8-positive cytotoxic T cells. Besides, the B cells also correlated positively with the CD3-positive pan T cells in the biopsy and negatively correlated with the T-regulatory:T-cell ratio. Our results suggested the role of B cells and plasma cells even at the tissue level in the pathobiogenesis of ENL.

Association of IL-10 Gene Polymorphism With IL-10 Secretion by CD4 and T Regulatory Cells in Human Leprosy.

Leprosy is a chronic bacterial disease caused by Mycobacterium leprae. Cytokines are known to play vital role as a peacekeeper during inflammatory and other immunocompromised conditions such as leprosy. This study has tried to bridge the gap of information on cytokine gene polymorphisms and its potential role in the pathogenesis of leprosy. Interleukin-10 (IL-10) is an immunosuppressive cytokine, found to be elevated in leprosy that accounted for the suppression of host's immune system by regulating the functions of other immune cells. T helper cells and T regulatory (Tregs) cells are the major source of IL-10 in lepromatous leprosy patients. In this study, we have documented the association of IL-10 cytokine gene polymorphism with the disease progression. A total of 132 lepromatous leprosy patients and 120 healthy controls were analyzed for IL-10 cytokine gene polymorphisms using PCR-SSP assay and flow cytometry was used to analyze IL-10 secretion by CD4 and Tregs in various genotype of leprosy patients. The frequencies of IL-10 (-819) TT and IL-10 (-1082) GG genotypes were significantly higher in leprosy patients as compared to healthy controls. This observation advocates that these genotypes were associated with the susceptibility and development of the disease. In addition, flow cytometry analysis demonstrated an increased number of IL-10 producing CD4 and Treg cells in IL-10 (819) TT genotype compared to CT and CC genotypes. These observations were further supported by immunohistochemical studies. Therefore, we can conclude that IL-10 cytokine gene polymorphisms by affecting its production can determine the predilection and progression of leprosy in the study population.

Expression of pigment epithelium-derived factor in psoriasis, verrucae, squamous cell carcinoma and normal skin: An immunohistochemical study.

BACKGROUND: Preservation of homeostasis status in the skin needs an equilibrium of keratinocyte proliferation, differentiation, necrosis and apoptosis. Disturbance of these regulatory mechanisms may lead to keratinocyte neoplastic and hyperproliferative diseases. Pigment epithelium-derived factor is a glycoprotein that is endogenously produced in different tissues and has a variety of biological effects in different diseases. OBJECTIVE: To evaluate the keratinocyte expression of pigment epithelium-derived factor in normal skin and three epidermal hyperproliferative diseases, namely, psoriasis, verrucae and squamous cell carcinoma. METHODS: This study included skin biopsy samples from 80 participants who were divided into four equal groups; each containing 20 samples. The first group included skin biopsies from normal skin, the second group from psoriatic lesions, the third group from verruca vulgaris and the fourth group from squamous cell carcinoma. All tissue samples were stained with hematoxylin and eosin stain and later immunohistochemically for pigment epithelium-derived factor expression. RESULTS: Scores of pigment epithelium-derived factor expression were lower in squamous cell carcinoma and verruca and psoriasis than normal skin with a significant difference (P = 0.04). In addition, the pattern of pigment epithelium-derived factor expression was mainly cytoplasmic in normal skin with a significant difference with that seen in psoriasis, squamous cell carcinoma and verruca vulgaris (P = 0.001). CONCLUSION: Pigment epithelium-derived factor may play a role in keratinocyte differentiation.

M2-Polarized Macrophages Determine Human Cutaneous Lesions in Lacaziosis.

Lacaziosis is a cutaneous chronic mycosis caused by Lacazia loboi. Macrophages are important cells in the host immune response in fungal infections. The macrophage population exhibits strong plasticity that varies according to the stimuli in the microenvironment of lesions M1 profile promotes a Th1 pattern of cytokines and a microbicidal function and M2 is related to Th2 cytokines and immunomodulatory response. We investigated the population of M1 and M2 polarized macrophages in human cutaneous lesions. A total of 27 biopsies from human lesions were submitted to an immunohistochemistry protocol using antibodies to detect M1 and M2 macrophages (Arginase-1, CD163, iNOS, RBP-J and cMAF). We could observe high number of cells expressing Arginase1, CD163 and c-MAF that correspond to elements of the M2 profile of macrophage, over iNOS and RBP-J (elements of the M1 profile). The results suggest a predominant phenotype of M2 macrophages, which have an immunomodulatory role and probably contributing to chronicity of Lacaziosis.

Notch Signaling Pathway Expression in the Skin of Leprosy Patients: Association With Skin and Neural Damage.

Introduction: Leprosy is an infectious disease caused by Mycobacterium leprae, a debilitating disease that affects the skin and peripheral nerves. It is possible that tissue changes during infection with leprosy are related to alterations in the activity of the Notch signaling pathway, an innate signaling pathway in the physiology of the skin and peripheral nerves. Methods: This is a descriptive observational study. Thirty skin biopsies from leprosy patients and 15 from individuals with no history of this disease were evaluated. In these samples, gene expressions of cellular components associated with the Notch signaling pathway, Hes-1, Hey-1, Runx-1 Jagged-1, Notch-1, and Numb, were evaluated using q-PCR, and protein expression was evaluated using immunohistochemistry of Runx-1 and Hes-1. Results: Changes were observed in the transcription of Notch signaling pathway components; Hes-1 was downregulated and Runx-1 upregulated in the skin of infected patients. These results were confirmed by immunohistochemistry, where reduction of Hes-1 expression was found in the epidermis, eccrine glands, and hair follicles. Increased expression of Runx-1 was found in inflammatory cells in the dermis of infected patients; however, it is not related to tissue changes. With these results, a multivariate analysis was performed to determine the causes of transcription factor Hes-1 reduction. It was concluded that tissue inflammation was the main cause. Conclusions: The tissue changes found in the skin of infected patients could be associated with a reduction in the expression of Hes-1, a situation that would promote the survival and proliferation of M. leprae in this tissue.

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.

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-1β 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-1β, 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.

Fibrosis: a distinguishing feature in the pathology of neural leprosy.

BACKGROUND: Fibrosis in the peripheral nerve is the end stage of leprous neuropathy and the cause of the resulting permanent neural function impairments. Preventive measures to avoid this irreversible pathological state are a relief strategy for leprosy sufferers. OBJECTIVES: The present study describes the frequency of fibrosis along with its characterisation and pathogenic development. METHODS: Six-hundred-and-thirteen nerve samples were sorted from 278 neural leprosy (NL) and 335 non-leprosy neuropathy patients (ON). The total number of samples was histologically examined by routine staining methods (haematoxylin-eosin, Wade staining and Gomori's trichrome) and fibrosis was evaluated via semi-quantitative estimation. FINDINGS: Fibrosis was most frequent in the NL group (33% against 0.4% in ON) while fibrosis in association with endoneurial microfasciculation was found in 38 (41.3%) of the NL samples in the examination of semithin sections. Pericytic activation in the perivascular environment was confirmed to be the source of the fibroblasts and perineurial cells delimiting microfascicles. End-stage fibrosis in leprosy displays an arrangement of microfascicles devoid of neural components (i.e., Schwann cells and axons) lined by an intermediate phenotype of fibroblastic-perineurial cells filled with bundles of collagen fibres. MAIN CONCLUSIONS: The present study underscores that fibrosis is frequently the severe end stage of neural leprosy NL pathogeny after analysing the notably distinct development of fibrosis within the neural environment.

Does histoid leprosy represent a locally hyperimmune variant of lepromatous leprosy?

BACKGROUND: Histoid leprosy is a subtype of leprosy with distinct clinical presentation and histologic features. It accounts for <4% of leprosy cases. The exact location of histoid leprosy along the immune spectrum and its relation to other subtypes is unclear. AIM: To evaluate the local immune phenomenon which define histoid leprosy. DESIGN: Parallel skin biopsies from histoid lesions and from unremarkable skin in patients with histoid leprosy were evaluated and the histologic findings compared. METHODS: Formalin fixed, paraffin embedded tissue sections from lesional and non-lesional biopsies were assessed for changes in epidermis and dermis; type and extent of infiltrate, presence or absence of pseudocapsule and associated reactions. Bacillary index was evaluated using Wade Fite stain for lepra bacilli. RESULTS: Amongst 208 leprosy cases, six cases of histoid leprosy were identified (2.88%). The cases showed presence of nodules, patches and plaques overlying clinically unremarkable skin. Fourteen skin biopsies were evaluated of which the lesional biopsies showed equal proportion of fusocellular, fusocellular epithelioid and fusocellular-vacuolated histology. A greater circumscription was noted in lesional biopsies; however the cellular content of the infiltrate was similar in lesional and non-lesional biopsies. A case of erythema nodosum leprosum in histoid leprosy was also seen. CONCLUSIONS: Ours is the first study comparing normal and lesional skin in histoid leprosy. Though the histoid lesions appear to have a derivation from lepromatous leprosy, the local histologic and clinical alterations may be a result of heightened local immunity or reactive local modifying factors.