Mycobacterium leprae Induces Neutrophilic Degranulation and Low-Density Neutrophil Generation During Erythema Nodosum Leprosum.

Erythema Nodosum Leprosum (ENL) is a recurrent acute inflammatory complication of leprosy affecting up to 50% of all Borderline Lepromatous and Lepromatous Leprosy (BL/LL) patients. Although ENL is described as an immune reaction mediated by neutrophils, studies demonstrating the direct role of neutrophils in ENL are still rare. One subpopulation of low-density neutrophils (LDNs), present within the fraction of peripheral blood mononuclear cells (PBMC), has been associated with the pathogenesis and severity of diseases like sepsis, lupus, and tuberculosis. We herein analyzed LDNs and high-density neutrophils (HDNs) in terms of frequency, phenotype, and morphology. Serum levels of MMP-9 (a neutrophilic degranulation marker) were evaluated by ELISA; and LDNs were generated in vitro by stimulating healthy-donor, whole-blood cultures. PBMC layers of ENL patients presented segmented/hypersegmented cells that were morphologically compatible with neutrophils. Immunofluorescence analyses identified LDNs in ENL. Flow cytometry confirmed the elevated frequency of circulating LDNs (CD14-CD15+) in ENL patients compared to healthy donors and nonreactional Borderline Tuberculoid (BT) patients. Moreover, flow cytometry analyses revealed that ENL LDNs had a neutrophilic-activated phenotype. ENL patients under thalidomide treatment presented similar frequency of LDNs as observed before treatment but its activation status was lower. In addition, Mycobacterium leprae induced in vitro generation of LDNs in whole blood in a dose-dependent fashion; and TGF-ß, an inhibitor of neutrophilic degranulation, prevented LDNs generation. MMP-9 serum levels of BL/LL patients with or without ENL correlated with LDNs frequency at the same time that ultrastructural observations of ENL LDNs showed suggestive signs of degranulation. Together, our data provide new insights into the knowledge and understanding of the pathogenesis of ENL while enriching the role of neutrophils in leprosy.

Neutrophils in Leprosy.

Leprosy is an infectious disease caused by the intracellular bacillus Mycobacterium leprae that mainly affects the skin and peripheral nerves. One of the most intriguing aspects of leprosy is the diversity of its clinical forms. Paucibacillary patients are characterized as having less than five skin lesions and rare bacilli while the lesions in multibacillary patients are disseminated with voluminous bacilli. The chronic course of leprosy is often interrupted by acute episodes of an inflammatory immunological response classified as either reversal reaction or erythema nodosum leprosum (ENL). Although ENL is considered a neutrophilic immune-complex mediated condition, little is known about the direct role of neutrophils in ENL and leprosy disease overall. Recent studies have shown a renewed interest in neutrophilic biology. One of the most interesting recent discoveries was that the neutrophilic population is not homogeneous. Neutrophilic polarization leads to divergent phenotypes (e.g., a pro- and antitumor profile) that are dynamic subpopulations with distinct phenotypical and functional abilities. Moreover, there is emerging evidence indicating that neutrophils expressing CD64 favor systemic inflammation during ENL. In the present review, neutrophilic involvement in leprosy is discussed with a particular focus on ENL and the potential of neutrophils as clinical biomarkers and therapeutic targets.

Envolvimento da via IL-10/IL-10R na resposta imunológica durante o curso da infecção pelo Mycobacterium leprae / Involvement of the IL-10 / IL-10R pathway in the immune response during the course of Mycobacterium leprae infection

A interleucina-10 (IL-10) possui papel chave no controle das respostas inflamatórias. A atividade biológica da IL-10 é mediada pela ligação da IL-10 com o receptor heterodimérico composto pelas cadeias IL-10R1 e IL-10R2. Estudos anteriores demonstram que neutrófilos circulantes de doadores sadios expressam constitutivamente a cadeia IL-10R2, porém não expressam IL-10R1, e dessa forma não respondem a IL-10 in vitro. A expressão das duas cadeias é essencial para a responsividade a IL-10. Neutrófilos circulantes de pacientes sépticos expressam o receptor IL-10R1 e essa expressão é corroborada com a responsividade à IL-10. O eritema nodoso hansênico (ENH) é uma complicação imunológica grave da hanseníase multibacilar que apresenta aspectos semelhantes à sepse, como leucocitose neutrofílica, febre, mal-estar e inflamação sistêmica. O ENH altera o curso crônico da hanseníase, pois acelera os danos nos nervos periféricos, resultando em incapacidades físicas permanentes. Um dos aspectos marcantes desta reação é a presença de neutrófilos na derme profunda e no tecido subcutâneo nas lesões. Dessa forma, o ENH tem sido caracterizado como uma condição imunomediada por neutrófilos. O objetivo deste trabalho foi investigar o potencial da via da IL-10 na regulação da produção de citocinas produzidas por neutrófilos durante o episódio de ENH.

Nesse estudo, reportamos que neutrófilos de sangue periférico de pacientes acometidos pelo ENH expressam altos níveis de IL-10R1. Além disso, demonstramos que esses mesmos pacientes, quando tratados com a talidomida, possuem níveis ainda mais elevados de IL-10R1 na superfície de neutrófilos circulantes. Observamos também que neutrófilos presentes nas lesões de pele de pacientes acometidos pelo ENH expressam IL-10R1. Ensaios ex vivo revelaram que a IL-10 foi capaz de inibir a produção espontânea de TNF de neutrófilos de pacientes com ENH, e induzir a produção de TGF-1. Por outro lado, neutrófilos de pacientes controle não-reacionais não produzem espontanêamete TNF, porém, assim como neutrófilos de pacientes com ENH, produzem TGF-ß1. Em seguida, demonstramos que o M. leprae foi capaz de induzir a produção de IL-8, IL-1ß, IL-6, MIP-1/CCL4, TGF- e IL-1RA em neutrófilos de doadores sadios in vitro, e que a adição de IL-10 reduziu a produção dessas citocinas, com exceção de TGF-1 que aumentou. Nossas conclusões demonstraram a capacidade do M. leprae em induzir a expressão de IL-10R1 na superfície de neutrófilos sadios. Coletivamente, nossos dados confirmam que neutrófilos participam da inflamação sistêmica associada ao ENH por meio da produção de citocinas. Esses resultados contribuem para o melhor entendimento dos mecanismos patogênicos e apontam para a via da IL-10 como alvo molecular terapêutico e diagnóstico. (AU)

Expression of CD64 on Circulating Neutrophils Favoring Systemic Inflammatory Status in Erythema Nodosum Leprosum.

Erythema Nodosum Leprosum (ENL) is an immune reaction in leprosy that aggravates the patient´s clinical condition. ENL presents systemic symptoms of an acute infectious syndrome with high leukocytosis and intense malaise clinically similar to sepsis. The treatment of ENL patients requires immunosuppression and thus needs to be early and efficient to prevent both disabilities and permanent nerve damage. Some patients experience multiple episodes of ENL and prolonged use of immunosuppressive drugs may lead to serious adverse effects. Thalidomide treatment is extremely effective at ameliorating ENL symptoms. Several mechanisms have been proposed to explain the efficacy of thalidomide in ENL, including the inhibition of TNF production. Given its teratogenicity, thalidomide is prohibitive for women of childbearing age. A rational search for molecular targets during ENL episodes is essential to better understand the disease mechanisms involved, which may also lead to the discovery of new drugs and diagnostic tests. Previous studies have demonstrated that IFN-γ and GM-CSF, involved in the induction of CD64 expression, increase during ENL. The aim of the present study was to investigate CD64 expression during ENL and whether thalidomide treatment modulated its expression. Leprosy patients were allocated to one of five groups: (1) Lepromatous leprosy, (2) Borderline leprosy, (3) Reversal reaction, (4) ENL, and (5) ENL 7 days after thalidomide treatment. The present study demonstrated that CD64 mRNA and protein were expressed in ENL lesions and that thalidomide treatment reduced CD64 expression and neutrophil infiltrates-a hallmark of ENL. We also showed that ENL blood neutrophils exclusively expressed CD64 on the cell surface and that thalidomide diminished overall expression. Patient classification based on clinical symptoms found that severe ENL presented high levels of neutrophil CD64. Collectively, these data revealed that ENL neutrophils express CD64, presumably contributing to the immunopathogenesis of the disease.