S.C. Lai

Induction of matrix metalloproteinase-9 in murine eosinophilic meningitis caused by Angiostrongylus cantonensis

Abstract Matrix metalloproteinases (MMP) have been implicated in the pathogenesis of various inflammatory diseases of the central nervous system. In the present study, a gelatinase was found to be induced in parasitic meningitis caused, in mice, by Angiostrongylus cantonensis. The enzyme had a molecular weight of about 94 kDa, showed maximal activity between pH 6 and pH 8, and was clearly inhibited by EDTA and 1,10-phenanthroline but not by leupeptin or phenylmethanesulphonyl fluoride. When samples of cerebrospinal fluid from the mice with meningitis were blotted with specific antiserum against gelatinase B (MMP-9), a 94-kDa immunopositive band was observed, indicating that the induced gelatinase was MMP-9. In the A. cantonensis-infected mice, immuno-histochemistry demonstrated MMP-9 within the endothelial cells lining the vascular spaces of the brain and in the leucocytes that were found, in aggregates, in the subarachnoid space. Leucocytes may play an important role in the pathogenisis of inflammatory disorders of the central nervous system.

Efficacy of curcumin therapy against Angiostrongylus cantonensis -induced eosinophilic meningitis

Angiostrongylus cantonensis can invade the central nervous system, leading to human eosinophilic meningitis or eosinophilic meningoencephalitis. Curcumin is a natural product which has the effects of anti-inflammation, anti-oxidation and anti-carcinogensis, while the administration of curcumin has been reported to possibly relieve the symptoms of meningitis. The present study tested the potential efficacy of curcumin in A. cantonensis-induced eosinophilic meningitis of BALB/c mice. Assay indicators for the therapeutic effect included the larvicidal effect, eosinophil counts and matrix metalloproteinase-9 (MMP-9) activity in angiostrongyliasis. Eosinophils were mildly reduced in treatment groups compared with infected-untreated mice. However, there were no significant differences in larvicidal effects or MMP-9 activity. This study suggests that anti-inflammatory treatment with curcumin alone has low efficacy, but the treatment does not interfere with MMP-9 expression and is not useful for larvicidal effects. The possible reasons include low curcumin across the blood-brain barrier and also those larvae that survive stimulate MMP-9 production, which promotes blood-brain barrier damage, with leukocytes then crossing the blood-brain barrier to cause meningitis. Further studies will be required to test these possibilities.

Induced eosinophilia and proliferation in Angiostrongylus cantonensis-infected mouse brain are associated with the induction of JAK/STAT1, IAP/NF-kappaB and MEKK1/JNK signals.

Eosinophilic meningitis or meningoencephalitis caused by Angiostrongylus cantonensis is endemic to the Pacific area of Asia, especially Taiwan, Thailand, and Japan. Although eosinophilia is an important clinical manifestation of A. cantonensis infection, the role of eosinophils in the progress of the infection remains to be elucidated. In this experiment, we show that A. cantonensis-induced eosinophilia and inflammation might lead to the induction of IAP/NF-kappaB, JAK/STAT1 and MEKK1/JNK signals. The phosphorylation levels of JAK and JNK, STAT1, IAP, NF-kappaB and MEKK1 protein products were significantly increased after 12 days or 15 days of A. cantonensis infection. However, no significant differences in MAPKs such as Raf, MEK-1, ERK1/2 and p38 expression were found between control and infected mice. The activation potency of JAK/STAT1, IAP/NF-kappaB and MEKK1/JNK started increasing on day 3, with significant induction on day 12 or day 15 after A. cantonensis infection. Consistent results were noted in the pathological observations, including eosinophilia, leukocyte infiltration, granulomatous reactions, and time responses in the brain tissues of infected mice. These data suggest that the development of brain injury by eosinophilia of A. cantonensis infection is associated with activation of JAK/STAT1 signals by cytokines, and/or activation of MEKK1/JNK by oxidant stress, and/or activation of NF-kappaB by increasing IAP expression.

Matrix metalloproteinase-2 and -9 in the granulomatous fibrosis of rats infected with Angiostrongylus cantonensis

Abstract The histomorphology of granuloma formation and gelatinase production were investigated in the brains, hearts, lungs and livers of Sprague–Dawley rats infected with Angiostrongylus cantonensis. The relationships between two gelatinases and granulomatous fibrosis were explored, following infection of each rat with 60 infective larvae of the nematode. Worm recovery from the brain was maximal on day 15 post-inoculation whereas peak recovery from the lungs was maximal 75 days later, on day 90. The granulomatous reactions and fibrosis were marked in the lungs but only mild, if present at all, in the brain, heart and liver. Gelatin zymography revealed that matrix metalloproteinase2 (MMP-2) was present, at all time-points, in the heart and lungs, although only in the lungs was there partial conversion of the 72-kDa pro-enzyme to the 64-kDa active form during granulomatous fibrosis. The activity of the MMP-9 pro-enzyme was significantly higher at the time-points when granuloma formation was observed than at other times. Immuno-histochemistry revealed MMP-2 and MMP-9 within the lung granulomas, around infiltrating leucocytes and the epithelial cells of the alveoli. As the granulomatous fibrosis appeared to be strongly associated with MMP-2 and MMP-9, these enzymes may be useful markers in the lungs of rats infected with A. cantonensis.

Matrix metalloproteinase-2 and -9 lead to fibronectin degradation in astroglia infected with Toxoplasma gondii.

Toxoplasma gondii is a zoonotic parasite and its infection in human can induce toxoplasmic encephalitis in immune disorders. In this study, astroglia were infected with the TS-4 strain of T. gondii tachyzoite in vitro to investigate the changes of matrix metalloproteinase (MMP)-2, MMP-9 and their substrate fibronectin. MMP-2 and MMP-9 were significantly increased at 1 h, 6 h and 12 h post-infection (PI) in the cell homogenates, and increased at 6 h, 12 h, 24 h and 48 h PI in the cell-cultured supernatants. Fibronectin degradation also occurred at the same time points. In addition, immunocytochemistry showed that MMP-2 and MMP-9 localized in the cytoplasm, and confocal scanning laser microscopy revealed co-labeled patterns of MMP-2 and MMP-9 with fibronectin. MMP-2 and MMP-9 interacted with fibronectin, respectively. These results suggest that MMP-2 and MMP-9 induction from astroglia may contribute to extracellular matrix (ECM) degradation occurring in toxoplasmosis. Thus, we hypothesize that MMP-2 and MMP-9 cleave fibronectin and may contribute to the astroglia reaction and leukocyte migration to the sites of T. gondii replication during toxoplasmic encephalitis.

Induction of matrix metalloproteinase-2 and -9 via Erk1/2-NF-κB pathway in human astroglia infected with Toxoplasma gondii.

Matrix metalloproteinase (MMP)-2 and MMP-9 can cleave fibronectin, allowing leukocyte migration to the site of Toxoplasma gondii infection during toxoplasmic encephalitis. The aim of this study was to investigate the association between extracellular signal-regulated kinase (Erk)1/2-nuclear factor (NF)-κB pathway and MMP-2/-9 expression in astroglia infected with T. gondii tachyzoite in vitro. Our results showed that phosphorylated (p)-Erk1/2 transiently increased 1h post-infection (PI) and p-NF-κB significantly increased from 1h PI to 12h PI in cell homogenates. NF-κB was bound directly to oligonucleotides containing putative NF-κB binding sites for the MMP-9 promoter. Additionally, expression of p-NF-κB, MMP-2, and MMP-9 was significantly decreased by MG132, an indirect NF-κB inhibitor. Treatment with PD98059, an Erk kinase inhibitor, efficiently reduced p-Erk1/2, p-NF-κB, MMP-2, and MMP-9 expression. These results suggest that suppression of the Erk1/2-NF-κB signaling pathway causes reductions in MMP-2 and MMP-9 activities in astroglia response to T. gondii infection. Thus, inhibiting this signaling intermediate involved in MMP-2 and MMP-9 expression may be a potential method for controlling inflammatory development of T. gondii-induced encephalitis.

Matrix metalloproteinase-9 leads to blood–brain barrier leakage in mice with eosinophilic meningoencephalitis caused by Angiostrongylus cantonensis

Blood-brain barrier (BBB) disruption is associated with tight junction protein degradation, basal membrane disruption, and astrocyte damage. This study aims to investigate the role of matrix metalloproteinase (MMP)-9 in BBB disruption during Angiostrongylus cantonensis infection. We used mice infected with A. cantonensis, in which parasite-induced eosinophilia and inflammation might induce MMP-9 elevation. MMP-9 could cause claudin-5 degradation in endothelium tight junction, collagen type IV degradation in basal membranes, and S100B degradation in astrocytes of wild-type mice. BBB permeability was significantly attenuated in MMP-9 knockout mice than in wild-type mice in angiostrongyliasis meningoencephalitis. Immune cell aggregates were also more attenuated in the brains of MMP-9 knockout mice than in the brains of wild-type mice. Results suggest that MMP-9 activities are significant in BBB disruption in angiostrongyliasis meningoencephalitis. This study improves understanding of molecular mechanisms that underlie brain invasion by A. cantonensis, which is a key step in the pathogenesis of meningoencephalitis, and can offer a new strategy to reduce mortality.

Comparative efficacies of albendazole and the Chinese herbal medicine long-dan-xie-gan-tan, used alone or in combination, in the treatment of experimental eosinophilic meningitis induced by Angiostrongylus cantonensis

Abstract Angiostrongylus cantonensis, the rat lungworm, is the principal cause of human eosinophilic meningitis or meningoencephalitis world-wide. In the present study, the efficacies of early-stage treatment with the Chinese herbal medicine long-dan-xie-gan-tan (LDXGT) and albendazole, used alone or in combination, were evaluated in BALB/c mice with A. cantonensis-induced dysfunction of the blood–central-nervous-system barrier and eosinophilic meningo-encephalitis. Indicators of the therapeutic effect included worm recovery, histopathological scores for the meningitis, assays of tissue-type plasminogen activator (PA), urokinase-type PA and matrix metalloproteinase-9 (MMP-9) in the brain, the ratio between albumin concentrations in the cerebrospinal fluid (CSF) and serum, and counts of eosinophils in the CSF. Combined treatment with albendazole and LDXGT gave better results than monotherapy based on either drug, apparently inhibiting eosinophilic meningitis via antagonists of the PA/MMP-9 system. LDXGT may have a therapeutic role in reducing inflammatory reaction in the subarachnoid space. Monotherapy with such an anti-inflammatory drug may relieve the symptoms of mild infection and the hosts immune responses to A. cantonensis larvae. In severe infection, however, co-therapy with an anthelmintic (to kill the larvae) and an anti-inflammatory agent (to provide symptomatic relief) is probably a better approach. The therapeutic strategy should be tailored to the severity of the illness and the numbers of eosinophils in the CSF.

Alterations of myelin proteins in inflammatory demyelination of BALB/c mice caused by Angiostrongylus cantonensis.

Angiostrongylus cantonensis causes eosinophilic meningitis or meningoencephalitis, yet little is known about demyelination caused by this parasite. To define the course of demyelination caused by A. cantonensis, we analyzed the expression of myelin proteins including myelin-associated glycoprotein (MAG), myelin basic protein (MBP), myelin-associated oligodendrocytic basic protein (MOBP), and proteolipid protein (PLP) in brain and cerebrospinal fluid (CSF)-like fluid of infected and uninfected BALB/c mice. In A. cantonensis-infected mice, the expression of MAG, MBP, MOBP, and PLP mRNAs in brain tissue was decreased, while expression of the corresponding proteins was significantly increased in CSF-like fluid. Light microscopy revealed perivascular infiltrates in the brain during meningoencephalitis, suggesting that the cause of demyelination in angiostrongyliasis was immune system attack on the oligodendrocytic myelin sheath and subsequent release of myelin proteins into the CSF. Thus, intracerebral myelin breakdown in angiostrongyliasis may be a response to inflammatory mediators and the cause of increased myelin proteins in the CSF-like fluid.