T-cell regulation in Erythema Nodosum Leprosum

Abstract

Leprosy is a disease caused by Mycobacterium leprae where the clinical spectrum correlates with the patient immune response. Erythema Nodosum Leprosum (ENL) is an immune-mediated inflammatory complication, which causes significant morbidity in affected leprosy patients. The underlying cause of ENL is not conclusively known. However, immune-complexes and cell-mediated immunity have been suggested in the pathogenesis of ENL. The aim of this study was to investigate the regulatory T-cells in patients with ENL. Forty-six untreated patients with ENL and 31 non-reactional lepromatous leprosy (LL) patient controls visiting ALERT Hospital, Ethiopia were enrolled to the study. Blood samples were obtained before, during and after prednisolone treatment of ENL cases. Peripheral blood mononuclear cells (PBMCs) were isolated and used for immunophenotyping of regulatory T-cells by flow cytometry. Five markers: CD3, CD4 or CD8, CD25, CD27 and FoxP3 were used to define CD4+ and CD8+ regulatory T-cells. Clinical and histopathological data were obtained as supplementary information. All patients had been followed for 28 weeks. Patients with ENL reactions had a lower percentage of CD4+ regulatory T-cells (1.7%) than LL patient controls (3.8%) at diagnosis of ENL before treatment. After treatment, the percentage of CD4+regulatory T-cells was not significantly different between the two groups. The percentage of CD8+ regulatory T-cells was not significantly different in ENL and LL controls before and after treatment. Furthermore, patients with ENL had higher percentage of CD4+ T-ells and CD4+/CD8+ T-cells ratio than LL patient controls before treatment. The expression of CD25 on CD4+ and CD8+ T-cells was not significantly different in ENL and LL controls suggesting that CD25 expression is not associated with ENL reactions while FoxP3 expression on CD4+ T-cells was significantly lower in patients with ENL than in LL controls. We also found that prednisolone treatment of patients with ENL reactions suppresses CD4+ T-cell but not CD8+ T-cell frequencies. Hence, ENL is associated with lower levels of T regulatory cells and higher CD4+/CD8+ T-cell ratio. We suggest that this loss of regulation is one of the causes of ENL.

In situ T regulatory cells and Th17 cytokines in paired samples of leprosy type 1 and type 2 reactions

“…T reg expression in leprosy reactions has been described in distinct body compartments either in situ T reg in biopsies of skin lesions or circulating systemic T reg in unstimulated fresh and in fresh or thawed stimulated cells [ 14 , 16 – 18 , 24 ]. Additionally, different antigen preparations have been used for culture stimulation (ML sonicate, PHA) [ 16 , 20 ]. More importantly, the different compositions of control groups used in the studies clearly impact comparisons.…”

“…Leprosy reactions have been compared to non-reactional leprosy with similar clinical forms, non-reactional leprosy with all clinical forms, exposed contacts or even healthy individuals. Study groups have included different sample sizes, naïve and MDT treated patients and T2R treated with prednisolone [ 14 , 16 , 17 , 19 , 20 , 23 ]. Although each methodological approach and study design may have advantages and disadvantages, it’s possible that intra-individual paired reaction-free/reactional lesions, as shown in this study, illustrate better the impact of leprosy reaction upon the expression of different T cell populations and cytokines in skin lesions.…”

“…However, Foxp3 gene expression by quantitative PCR was increased in stimulated PBMC from T1R and T2Rcompared to patients with stable disease. A recent case-control study using flow cytometry and PBMCs in patients with T2R (n = 46) compared to matched non-reactional LL patient controls (n = 31) showed lower rate of CD4+ T reg cells before prednisolone treatment [ 20 ]. A retrospective immunostaining study on 20 leprosy cases (TT, BT, BL, LL, BB-T1R, BT-T1R and T2R) showed higher expression of T reg cells in patients affected by T1R (n = 3) compared with patients affected by T2R (n = 3) and reaction-free patients [ 14 ].…”

“…These episodes are responsible for irreversible nerve damage representing the major cause of permanent physical disabilities and deformities [ 13 ]. Contradictory results and interpretations have been reported about the role of T reg cells in the immunopathogenesis of leprosy reactions [ 14 – 20 ]. Several relevant methodological differences and more importantly, the use of different compositions of comparative control groups limit comparisons among these studies.…”

The Effects of Prednisolone Treatment on Cytokine Expression in Patients with Erythema Nodosum Leprosum Reactions

“…Peripheral blood mononuclear cells were separated by density gradient centrifugation at 800 × g for 25 min on Ficoll–Hypaque (Histopaque, Sigma Aldrich, UK) as described earlier ( 33 , 34 ). Cells were washed three times in sterile 1× phosphate-buffered saline (Sigma Aldrich ® , UK) and re-suspended with 1 mL of Roswell Park Memorial Institute (RPMI medium 1640 (1×) + GlutaMAX™ + Pen-Strip GBICO™, Life technologies™, UK).…”