Helen Ferreira

High Matrix Metalloproteinase Production Correlates with Immune Activation and Leukocyte Migration in Leprosy Reactional Lesions

ABSTRACT Gelatinases A and B (matrix metalloproteinase 2 [MMP-2] and MMP-9, respectively) can induce basal membrane breakdown and leukocyte migration, but their role in leprosy skin inflammation remains unclear. In this study, we analyzed clinical specimens from leprosy patients taken from stable, untreated skin lesions and during reactional episodes (reversal reaction [RR] and erythema nodosum leprosum [ENL]). The participation of MMPs in disease was suggested by (i) increased MMP mRNA expression levels in skin biopsy specimens correlating with the expression of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), (ii) the detection of the MMP protein and enzymatic activity within the inflammatory infiltrate, (iii) increased MMP levels in patient sera, and (iv) the in vitro induction of MMP-9 by Mycobacterium leprae and/or TNF-α. It was observed that IFN-γ, TNF-α, MMP-2, and MMP-9 mRNA levels were higher in tuberculoid than lepromatous lesions. In contrast, interleukin-10 and tissue inhibitor of MMP (TIMP-1) message were not differentially modulated. These data correlated with the detection of the MMP protein evidenced by immunohistochemistry and confocal microscopy. When RR and ENL lesions were analyzed, an increase in TNF-α, MMP-2, and MMP-9, but not TIMP-1, mRNA levels was observed together with stronger MMP activity (zymography/in situ zymography). Moreover, following in vitro stimulation of peripheral blood cells, M. leprae induced the expression of MMP-9 (mRNA and protein) in cultured cells. Overall, the present data demonstrate an enhanced MMP/TIMP-1 ratio in the inflammatory states of leprosy and point to potential mechanisms for tissue damage. These results pave the way toward the application of new therapeutic interventions for leprosy reactions.

Effect of Apoptotic Cell Recognition on Macrophage Polarization and Mycobacterial Persistence

ABSTRACT Intracellular Mycobacterium leprae infection modifies host macrophage programming, creating a protective niche for bacterial survival. The milieu regulating cellular apoptosis in the tissue plays an important role in defining susceptible and/or resistant phenotypes. A higher density of apoptotic cells has been demonstrated in paucibacillary leprosy lesions than in multibacillary ones. However, the effect of apoptotic cell removal on M. leprae-stimulated cells has yet to be fully elucidated. In this study, we investigated whether apoptotic cell removal (efferocytosis) induces different phenotypes in proinflammatory (Mϕ1) and anti-inflammatory (Mϕ2) macrophages in the presence of M. leprae. We stimulated Mϕ1 and Mϕ2 cells with M. leprae in the presence or absence of apoptotic cells and subsequently evaluated the M. leprae uptake, cell phenotype, and cytokine pattern in the supernatants. In the presence of M. leprae and apoptotic cells, Mϕ1 macrophages changed their phenotype to resemble the Mϕ2 phenotype, displaying increased CD163 and SRA-I expression as well as higher phagocytic capacity. Efferocytosis increased M. leprae survival in Mϕ1 cells, accompanied by reduced interleukin-15 (IL-15) and IL-6 levels and increased transforming growth factor beta (TGF-β) and IL-10 secretion. Mϕ1 cells primed with M. leprae in the presence of apoptotic cells induced the secretion of Th2 cytokines IL-4 and IL-13 in autologous T cells compared with cultures stimulated with M. leprae or apoptotic cells alone. Efferocytosis did not alter the Mϕ2 cell phenotype or cytokine secretion profile, except for TGF-β. Based on these data, we suggest that, in paucibacillary leprosy patients, efferocytosis contributes to mycobacterial persistence by increasing the Mϕ2 population and sustaining the infection.

Increased Langerhans cell accumulation after mycobacterial stimuli.

Aims:u2002 To evaluate the role of Langerhans cells (LCs) in the local activation of leprosy lesions. LCs, acting as tolerance inducers and immune stimuli, are dendritic cells recently implicated in cutaneous homeostasis. The role of LCs in the defence against mycobacterial infection remains poorly understood.

Long-term culture of multibacillary leprosy macrophages isolated from skin lesions: a new model to study Mycobacterium leprae-human cell interaction.

Backgroundu2002 Leprosy is characterized by a disease spectrum having two polar clinical forms dependent on the presence or not of cell‐mediated immunity. In the tuberculoid forms, granuloma‐activated macrophages kill Mycobacterium leprae in conjunction with a Th1 response while, in multibacillary (MB) lesions, M. leprae nonactivated macrophages infiltrate the nerves and internal organs together with a Th2 response. The functional properties and activation pathways of macrophages isolated from patients with MB leprosy remain only partially understood.

Genotyping of Mycobacterium leprae from Brazilian leprosy patients suggests the occurrence of reinfection or of bacterial population shift during disease relapse

We performed genotyping of Mycobacterium leprae present in skin biopsy samples that were collected during the first and the second disease occurrences from eight leprosy patients, seven of whom were diagnosed as suffering from disease relapse. Sequence analysis of part of the M. leprae rpoB, folP1, gyrB and gyrA genes did not show genetic change that supported the presence of drug-resistant bacilli. However, we observed a synonymous nucleotide change at position 297 of gyrA among five of these patients, one presenting C to T (CgyrAT) and four presenting T to C (TgyrAC) at this position. Additional genotyping by analysis of the four short tandem repeats GAA, GTA9, AT17 and TA18 showed that the gyrA single nucleotide polymorphism change was accompanied by a change in short tandem repeat genotype. Our data suggest that leprosy relapse in these patients, living in an area endemic for leprosy, could be caused by M. leprae with a genotype different from the one that caused initial disease.

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.

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.

Indoleamine 2,3-dioxygenase and iron are required for Mycobacterium leprae survival

Our previous study has demonstrated that IL-10 may modulate both indoleamine 2,3-dioxygenase (IDO) and CD163 expression in lepromatous leprosy (LL) cells, favoring Mycobacterium leprae persistence through induction of regulatory pathways and iron storage. Here, we observed that in LL lesion cells there is an increase in the expression of proteins involved in iron metabolism such as hemoglobin (Hb), haptoglobin, heme oxygenase 1 and transferrin receptor 1 (TfR1) when compared to tuberculoid leprosy (BT) cells. We also found increased iron deposits and diminished expression of the iron exporter ferroportin 1 in LL lesion cells. Hemin, but not FeSO4 stimulation, was able to enhance M. leprae viability by a mechanism that involves IDO. Analysis of cell phenotype in lesions demonstrated a predominance of M2 markers in LL when compared with BT lesion cells. A positive correlation between CD163 and PPARG with the bacillary index (BI) was observed. In contrast, TNF, STAT1 and CSF2 presented a negative correlation with the BI. In summary, this study demonstrates that iron may regulate IDO expression by a mechanism that involves IL-10, which may contribute for the predominance of M2-like phenotype in LL lesions that favors the phagocytosis and maintenance of M. leprae in host cells.

CXCL10, MCP-1, and Other Immunologic Markers Involved in Neural Leprosy

Nerve damage in leprosy can be directly induced by Mycobacterium leprae in the early stages of infection, however, immunomediated mechanisms add gravity to the impairment of neural function in symptomatic periods of the disease. This study investigated the immunohistochemical expression of immunomarkers involved in the pathogenic mechanisms of leprosy nerve damage. These markers selected were CXCL10, CCL2 chemokines and immunomarkers as CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, and metalloproteinases 2 and 9 (MMP2 and MMP9) occurring in nerve biopsy specimens collected from leprosy (23) and nonleprosy patients (5) suffering peripheral neuropathy. CXCL10, CCL2, MMP2, and MMP9 immunoreactivities were found in the leprosy nerves but not in nonleprosy samples. Immunolabeling was predominantly found in recruited macrophages and Schwann cells composing the inflammatory cellular population in the leprosy-affected nerves. The immunohistochemical expression of all the markers, but CXCL10, was associated with fibrosis, however, only CCL2 was, independently from the others, associated with this excessive deposit of extracellular matrix. No difference in the frequency of the immunolabeling was detected between the AFB+ and AFB− leprosy subgroups of nerve, exception made to some statistical trend to difference in regard to CD68− and HLA-DR+ cells in the AFB− nerves exhibiting epithelioid granuloma. MMP9 expression associated with fibrosis is consistent with previous results of research group. The findings conveys the idea that CCL2 and CXCL10 chemokines at least in advanced stages of leprosy nerve lesions are not determinant for the establishment of AFB+ or AFB− leprosy lesions, however, CCL2 is associated with macrophage recruitment and fibrosis.

An attempt to improve pure neural leprosy diagnosis using immunohistochemistry tests in peripheral nerve biopsy specimens.

The diagnosis of pure neural leprosy (PNL) is based on clinical and laboratory data, including the histopathology of nerve biopsy specimens and detection of Mycobacterium leprae DNA by polymerase chain reaction (PCR). Given that histopathologic examination and PCR methods may not be sufficient to confirm the diagnosis, immunolabeling of lipoarabinomanan (LAM) and/or phenolic glycolipid 1 (PGL-1) M. leprae wall components was utilized in the present investigation in an attempt to detect any vestigial presence of M. leprae in acid-fast bacilli (AFB)− nerve samples. Twenty-three PNL nerve samples (6 AFB+ and 17 AFB−PCR+) were cryosectioned and subjected to LAM and PGL-1 immunohistochemical staining by immunoperoxidase. Five nonleprosy nerve samples were used as controls. The 6 AFB+ samples showed LAM/PGL-1 immunoreactivity. Among the 17 AFB− samples, 8 revealed LAM and/or PGL-1 immunoreactivity. In 17 AFB−PCR+ patients, just 7 yielded LAM+ and/or PGL-1+ nerve results. In the PNL cases, the detection of immunolabeled LAM and PGL-1 in the nerve samples would have contributed to an enhanced diagnostic efficiency in the absence of molecular diagnostic facilities.