Histomorphometric analysis of the lung of Swiss mice treated with a fibrinolytic protease.

Fibrinolytic enzymes are considered promising alternative in the treatment of cardiovascular diseases by preventing fibrin clots. A protease from Mucor subtilissimus UCP 1262 was obtained by solid state fermentation and purified by ion exchange chromatography. The purified extract was administered at an acute dose of 2000 mg/mL to evaluate its toxic effects to the lungs of mice. After 14 days of treatment, a histomorphometric study was performed by the type 1 and 2 pneumocyte count and the evaluation of the lung area. As result, the experimental group showed a significant decrease of type 2 pneumocyte and although a decrease in the alveolar area was observed in relation to the control group, no significant pulmonary toxicity, emphysema, and fibrosis characteristics were detected. The in vitro tests suggest possible clinical applications for the enzyme.

Mycobacterium leprae intracellular survival relies on cholesterol accumulation in infected macrophages: a potential target for new drugs for leprosy treatment.

We recently showed that Mycobacterium leprae (ML) is able to induce lipid droplet formation in infected macrophages. We herein confirm that cholesterol (Cho) is one of the host lipid molecules that accumulate in ML-infected macrophages and investigate the effects of ML on cellular Cho metabolism responsible for its accumulation. The expression levels of LDL receptors (LDL-R, CD36, SRA-1, SR-B1, and LRP-1) and enzymes involved in Cho biosynthesis were investigated by qRT-PCR and/or Western blot and shown to be higher in lepromatous leprosy (LL) tissues when compared to borderline tuberculoid (BT) lesions. Moreover, higher levels of the active form of the sterol regulatory element-binding protein (SREBP) transcriptional factors, key regulators of the biosynthesis and uptake of cellular Cho, were found in LL skin biopsies. Functional in vitro assays confirmed the higher capacity of ML-infected macrophages to synthesize Cho and sequester exogenous LDL-Cho. Notably, Cho colocalized to ML-containing phagosomes, and Cho metabolism impairment, through either de novo synthesis inhibition by statins or depletion of exogenous Cho, decreased intracellular bacterial survival. These findings highlight the importance of metabolic integration between the host and bacteria to leprosy pathophysiology, opening new avenues for novel therapeutic strategies to leprosy.

CD163 favors Mycobacterium leprae survival and persistence by promoting anti-inflammatory pathways in lepromatous macrophages.

Lepromatous macrophages possess a regulatory phenotype that contributes to the immunosuppression observed in leprosy. CD163, a scavenger receptor that recognizes hemoglobin-haptoglobin complexes, is expressed at higher levels in lepromatous cells, although its functional role in leprosy is not yet established. We herein demonstrate that human lepromatous lesions are microenvironments rich in IDO⁺CD163⁺. Cells isolated from these lesions were CD68⁺IDO⁺CD163⁺ while higher levels of sCD163 in lepromatous sera positively correlated with IL-10 levels and IDO activity. Different Myco-bacterium leprae (ML) concentrations in healthy monocytes likewise revealed a positive correlation between increased concentrations of the mycobacteria and IDO, CD209, and CD163 expression. The regulatory phenotype in ML-stimulated monocytes was accompanied by increased TNF, IL-10, and TGF-ß levels whereas IL-10 blockade reduced ML-induced CD163 expression. The CD163 blockade reduced ML uptake in human monocytes. ML uptake was higher in HEK293 cells transfected with the cDNA for CD163 than in untransfected cells. Simultaneously, increased CD163 expression in lepromatous cells seemed to be dependent on ML uptake, and contributed to augmented iron storage in lepromatous macrophages. Altogether, these results suggest that ML-induced CD163 expression modulates the host cell phenotype to create a favorable environment for myco-bacterial entry and survival.

High matrix metalloproteinase production correlates with immune activation and leukocyte migration in leprosy reactional lesions.

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-gamma) and tumor necrosis factor alpha (TNF-alpha), (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-alpha. It was observed that IFN-gamma, TNF-alpha, 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-alpha, 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.