Autophagy-Associated IL-15 Production Is Involved in the Pathogenesis of Leprosy Type 1 Reaction.

Leprosy reactional episodes are acute inflammatory events that may occur during the clinical course of the disease. Type 1 reaction (T1R) is associated with an increase in neural damage, and the understanding of the molecular pathways related to T1R onset is pivotal for the development of strategies that may effectively control the reaction. Interferon-gamma (IFN-γ) is a key cytokine associated with T1R onset and is also associated with autophagy induction. Here, we evaluated the modulation of the autophagy pathway in Mycobacterium leprae-stimulated cells in the presence or absence of IFN-γ. We observed that IFN-γ treatment promoted autophagy activation and increased the expression of genes related to the formation of phagosomes, autophagy regulation and function, or lysosomal pathways in M. leprae-stimulated cells. IFN-γ increased interleukin (IL)-15 secretion in M. leprae-stimulated THP-1 cells in a process associated with autophagy activation. We also observed higher IL15 gene expression in multibacillary (MB) patients who later developed T1R during clinical follow-up when compared to MB patients who did not develop the episode. By overlapping gene expression patterns, we observed 13 common elements shared between T1R skin lesion cells and THP-1 cells stimulated with both M. leprae and IFN-γ. Among these genes, the autophagy regulator Translocated Promoter Region, Nuclear Basket Protein (TPR) was significantly increased in T1R cells when compared with non-reactional MB cells. Overall, our results indicate that IFN-γ may induce a TPR-mediated autophagy transcriptional program in M. leprae-stimulated cells similar to that observed in skin cells during T1R by a pathway that involves IL-15 production, suggesting the involvement of this cytokine in the pathogenesis of T1R.

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.

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.

Subversion of Schwann Cell Glucose Metabolism by Mycobacterium leprae.

Mycobacterium leprae, the intracellular etiological agent of leprosy, infects Schwann promoting irreversible physical disabilities and deformities. These cells are responsible for myelination and maintenance of axonal energy metabolism through export of metabolites, such as lactate and pyruvate. In the present work, we observed that infected Schwann cells increase glucose uptake with a concomitant increase in glucose-6-phosphate dehydrogenase (G6PDH) activity, the key enzyme of the oxidative pentose pathway. We also observed a mitochondria shutdown in infected cells and mitochondrial swelling in pure neural leprosy nerves. The classic Warburg effect described in macrophages infected by Mycobacterium avium was not observed in our model, which presented a drastic reduction in lactate generation and release by infected Schwann cells. This effect was followed by a decrease in lactate dehydrogenase isoform M (LDH-M) activity and an increase in cellular protection against hydrogen peroxide insult in a pentose phosphate pathway and GSH-dependent manner. M. leprae infection success was also dependent of the glutathione antioxidant system and its main reducing power source, the pentose pathway, as demonstrated by a 50 and 70% drop in intracellular viability after treatment with the GSH synthesis inhibitor buthionine sulfoximine, and aminonicotinamide (6-ANAM), an inhibitor of G6PDH 6-ANAM, respectively. We concluded that M. leprae could modulate host cell glucose metabolism to increase the cellular reducing power generation, facilitating glutathione regeneration and consequently free-radical control. The impact of this regulation in leprosy neuropathy is discussed.

Subversion of Schwann cell glucose metabolism by Mycobacterium leprae

Mycobacterium leprae, the intracellular etiological agent of leprosy, infects Schwann promoting irreversible physical disabilities and deformities. These cells are responsible for myelination and maintenance of axonal energy metabolism through export of metabolites, such as lactate and pyruvate. In the present work, we observed that infected Schwann cells increase glucose uptake with a concomitant increase in glucose-6-phosphate dehydrogenase (G6PDH) activity, the key enzyme of the oxidative pentose pathway. We also observed a mitochondria shutdown in infected cells and mitochondrial swelling in pure neural leprosy nerves. The classic Warburg effect described in macrophages infected by Mycobacterium avium was not observed in our model, which presented a drastic reduction in lactate generation and release by infected Schwann cells. This effect was followed by a decrease in lactate dehydrogenase isoform M (LDH-M) activity and an increase in cellular protection against hydrogen peroxide insult in a pentose phosphate pathway and GSH-dependent manner. M. leprae infection success was also dependent of the glutathione antioxidant system and its main reducing power source, the pentose pathway, as demonstrated by a 50 and 70% drop in intracellular viability after treatment with the GSH synthesis inhibitor buthionine sulfoximine, and aminonicotinamide (6-ANAM), an inhibitor of G6PDH 6-ANAM, respectively. We concluded that M. leprae could modulate host cell glucose metabolism to increase the cellular reducing power generation, facilitating glutathione regeneration and consequently free-radical control. The impact of this regulation in leprosy neuropathy is discussed.

Interaction of Mycobacterium leprae with the HaCaT human keratinocyte cell line: new frontiers in the cellular immunology of leprosy.

Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae affecting the skin and peripheral nerves. Despite M. leprae invasion of the skin and keratinocytes importance in innate immunity, the interaction of these cells in vitro during M. leprae infection is poorly understood. Conventional and fluorescence optical microscopy, transmission electronic microscopy, flow cytometry and ELISA were used to study the in vitro interaction of M. leprae with the HaCaT human keratinocyte cell line. Keratinocytes uptake of M. leprae is described, and modulation of the surface expression of CD80 and CD209, cathelicidin expression and TNF-α and IL-1ß production of human keratinocytes are compared with dendritic cells and macrophages during M. leprae interaction. This study demonstrated that M. leprae interaction with human keratinocytes enhanced expression of cathelicidin and greatly increased TNF-α production. The highest spontaneous expression of cathelicidin was by dendritic cells which are less susceptible to M. leprae infection. In contrast, keratinocytes displayed low spontaneous cathelicidin expression and were more susceptible to M. leprae infection than dendritic cells. The results show, for the first time, an active role for keratinocytes during infection by irradiated whole cells of M. leprae and the effect of vitamin D on this process. They also suggest that therapies which target cathelicidin modulation may provide novel approaches for treatment of leprosy.

Histopathological examination of nerve samples from pure neural leprosy patients: obtaining maximum information to improve diagnostic efficiency.

Nerve biopsy examination is an important auxiliary procedure for diagnosing pure neural leprosy (PNL). When acid-fast bacilli (AFB) are not detected in the nerve sample, the value of other nonspecific histological alterations should be considered along with pertinent clinical, electroneuromyographical and laboratory data (the detection of Mycobacterium leprae DNA with polymerase chain reaction and the detection of serum anti-phenolic glycolipid 1 antibodies) to support a possible or probable PNL diagnosis. Three hundred forty nerve samples [144 from PNL patients and 196 from patients with non-leprosy peripheral neuropathies (NLN)] were examined. Both AFB-negative and AFB-positive PNL samples had more frequent histopathological alterations (epithelioid granulomas, mononuclear infiltrates, fibrosis, perineurial and subperineurial oedema and decreased numbers of myelinated fibres) than the NLN group. Multivariate analysis revealed that independently, mononuclear infiltrate and perineurial fibrosis were more common in the PNL group and were able to correctly classify AFB-negative PNL samples. These results indicate that even in the absence of AFB, these histopathological nerve alterations may justify a PNL diagnosis when observed in conjunction with pertinent clinical, epidemiological and laboratory data.

Histopathological examination of nerve samples from pure neural leprosy patients: obtaining maximum information to improve diagnostic efficiency

Nerve biopsy examination is an important auxiliary procedure for diagnosing pure neural leprosy (PNL). When acid-fast bacilli (AFB) are not detected in the nerve sample, the value of other nonspecific histological alterations should be considered along with pertinent clinical, electroneuromyographical and laboratory data (the detection of Mycobacterium leprae DNA with polymerase chain reaction and the detection of serum anti-phenolic glycolipid 1 antibodies) to support a possible or probable PNL diagnosis. Three hundred forty nerve samples [144 from PNL patients and 196 from patients with non-leprosy peripheral neuropathies (NLN)] were examined. Both AFB-negative and AFB-positive PNL samples had more frequent histopathological alterations (epithelioid granulomas, mononuclear infiltrates, fibrosis, perineurial and subperineurial oedema and decreased numbers of myelinated fibres) than the NLN group. Multivariate analysis revealed that independently, mononuclear infiltrate and perineurial fibrosis were more common in the PNL group and were able to correctly classify AFB-negative PNL samples. These results indicate that even in the absence of AFB, these histopathological nerve alterations may justify a PNL diagnosis when observed in conjunction with pertinent clinical, epidemiological and laboratory data.

Microfasciculation: a morphological pattern in leprosy nerve damage.

AIMS: To study Microfasciculation, a perineurial response found in neuropathies, emphasizing its frequency, detailed morphological characteristics and biological significance in pure neural leprosy (PNL), post-treatment leprosy neuropathy (PTLN) and non-leprosy neuropathies (NLN). METHODS AND RESULTS: Morphological characteristics of microfascicles were examined via histological staining methods, immunohistochemical expression of neural markers and transmission electronmicroscopy. The detection of microfasciculation in 18 nerve biopsy specimens [12 PNL, six PTLN but not in the NLN group, was associated strongly with perineurial damage and the presence of a multibacillary inflammatory process in the nerves, particularly in the perineurium. Immunoreactivity to anti-S100 protein, anti-neurofilament, anti-nerve growth receptor and anti-myelin basic protein immunoreactivity was found within microfascicles. Ultrastructural examination of three biopsies showed that fibroblast-perineurial cells were devoid of basement membrane despite perineurial-like NGFr immunoreactivity. Morphological evidence demonstrated that multipotent pericytes from inflammation-activated microvessels could be the origin of fibroblast-perineurial cells. CONCLUSIONS: A microfasciculation pattern was found in 10% of leprosy-affected nerves. The microfascicles were composed predominantly of unmyelinated fibres and denervated Schwann cells (SCs) surrounded by fibroblast-perineurial cells. This pattern was found more frequently in leprosy nerves with acid-fast bacilli (AFB) and perineurial damage while undergoing an inflammatory process. Further experimental studies are necessary to elucidate microfascicle formation.

DOPA-stained melanocytes in the macular lesions of early leprosy

The mechanism of association of hypopigmentation and sensorial loss in a leprosy macular lesions has not been clarified yet. The biopsy of a macular lesion on the medial face of the right forearm of a fourteen-year old male leprosy patient was submitted to DOPA-staining for melanocytes, which is specific for the melanocytic tyrosinase enzyme and it is a proper method for identifying and couting these cells in the skin. A contralateral specimen of the same patient went through the same procedure as a control experiment. The specimen from the macular lesion showed a higher number of DOPA-stained melanocytes than the control fragment. Dermal melanocytes were present in high amounts in the abnormal specimen. Increased expression of tyrosinase by melanocytes in the macular lesions may reflect a positive feed-back stimulus represented by the lack of substance tyrosine, which may in turn be utilized by the myocobacterial agent. Ultrastructural study of the normal and pathological specimens showed no significant differences in the morphological appearance of melanocytes and their melanosomes. These results suggests that the utilization of phenolic compound by the Mycobacterium leprae may be involved in the mechanism of hypopigmentation. A higher number of cases will be necessary to confirm this hypothesis.