TPL-2 restricts Ccl24-dependent immunity to Heligmosomoides polygyrus.

TPL-2 (COT, MAP3K8) kinase activates the MEK1/2-ERK1/2 MAPK signaling pathway in innate immune responses following TLR, TNFR1 and IL-1R stimulation. TPL-2 contributes to type-1/Th17-mediated autoimmunity and control of intracellular pathogens. We recently demonstrated TPL-2 reduces severe airway allergy to house dust mite by negatively regulating type-2 responses. In the present study, we found that TPL-2 deficiency resulted in resistance to Heligmosomoides polygyrus infection, with accelerated worm expulsion, reduced fecal egg burden and reduced worm fitness. Using co-housing experiments, we found resistance to infection in TPL-2 deficient mice (Map3k8-/-) was independent of microbiota alterations in H. polygyrus infected WT and Map3k8-/-mice. Additionally, our data demonstrated immunity to H. polygyrus infection in TPL-2 deficient mice was not due to dysregulated type-2 immune responses. Genome-wide analysis of intestinal tissue from infected TPL-2-deficient mice identified elevated expression of genes involved in chemotaxis and homing of leukocytes and cells, including Ccl24 and alternatively activated genes. Indeed, Map3k8-/-mice had a significant influx of eosinophils, neutrophils, monocytes and Il4GFP+ T cells. Conditional knockout experiments demonstrated that specific deletion of TPL-2 in CD11c+ cells, but not Villin+ epithelial cells, LysM+ myeloid cells or CD4+ T cells, led to accelerated resistance to H. polygyrus. In line with a central role of CD11c+ cells, CD11c+ CD11b+ cells isolated from TPL-2-deficient mice had elevated Ccl24. Finally, Ccl24 neutralization in TPL-2 deficient mice significantly decreased the expression of Arg1, Retnla, Chil3 and Ear11 correlating with a loss of resistance to H. polygyrus. These observations suggest that TPL-2-regulated Ccl24 in CD11c+CD11b+ cells prevents accelerated type-2 mediated immunity to H. polygyrus. Collectively, this study identifies a previously unappreciated role for TPL-2 controlling immune responses to H. polygyrus infection by restricting Ccl24 production.

Angiostrongylus cantonensis cathepsin B-like protease (Ac-cathB-1) is involved in host gut penetration.

Although the global spread of the emerging zoonosis, human angiostrongyliasis, has attracted increasing attention, understanding of specific gene function has been impeded by the inaccessibility of genetic manipulation of the pathogen nematode causing this disease, Angiostrongylus cantonensis. Many parasitic proteases play key roles in host-parasite interactions, but those of A. cantonensis are always expressed as the inactive form in prokaryotic expression systems, thereby impeding functional studies. Hence, a lentiviral system that drives secreted expression of target genes fused to a Myc-His tag was used to obtain recombinant Ac-cathB-1 with biological activity. Although this class of proteases was always reported to function in nutrition and immune evasion in parasitic nematodes, recombinant Ac-cathB-1 was capable of hydrolysis of fibronectin and laminin as well as the extracellular matrix of IEC-6 monolayer, so that the intercellular space of the IEC-6 monolayer increased 5.15 times as compared to the control, while the shape of the adherent cells partly rounded up. This suggests a probable role for this protease in intestinal epithelial penetration. The inhibition of Ac-cathB-1 enzymatic activity with antiserum partly suppressed larval penetration ability in the isolated intestine. Thus, an effective system for heterologous expression of parasite proteases is presented for studying gene function in A. cantonensis; and Ac-cathB-1 was related to larval penetration ability in the host small intestine.

Th2-specific immunity and function of peripheral T cells is regulated by the p56Lck Src homology 3 domain.

T cell activation and effector function is essential for robust immunity. Ag TCR signals are known to regulate T lymphocyte differentiation, but the mechanisms involved in this regulation remain unclear. Recent work has demonstrated that the Src family protein tyrosine kinase p56Lck specifically links TCR signaling to activation of the MAPK pathway through the function of its Src homology 3 (SH3) domain. The MAPK pathway is involved in T cell activation and has previously been implicated in Th2 immunity. We have used Lck SH3 mutant knockin mice (LckW97A) to investigate the potential role of this regulatory mechanism in T lymphocyte activation and effector function. Our results demonstrate that Lck SH3 domain function regulates activation of T lymphocytes as indicated by reduced IL-2 production, CD69 induction, and proliferation of LckW97A T cells following TCR stimulation. Biochemical studies confirm that activation of the MAPK pathway is selectively altered following TCR ligation in LckW97A T lymphocytes. Phospho-ERK induction is reduced, but phospho-phospholipase Cgamma1 induction and calcium mobilization are largely unaffected. Immunization with DNP-keyhole limpet hemocyanin, heat-killed Brucella abortus, or infection with Nippostrongylus brasiliensis demonstrates selectively impaired Th2 immunity with reduced serum levels of IgG1, IgE, and IL-4. In vitro studies show that LckW97A T cells can differentiate into Th2-type cells, but they form IFN-gamma-producing cells under conditions that normally favor Th2 development. These data indicate that the Lck SH3 domain controls T lymphocyte activation by regulating MAPK pathway induction and demonstrate a novel role for Lck in the regulation of Th2-type immunity.

Oxidative stress in mice infected with Angiostrongylus cantonensis coincides with enhanced glutathione-dependent enzymes activity.

This study aimed to estimate reactive oxygen species (ROS) production, antioxidants activity, and biomarkers level of oxidative damage to protein and DNA in the cerebrospinal fluid (CSF) of C57BL/6 mice infected with Angiostrongylus cantonensis. The mean ROS concentration in the CSF of infected mice increased gradually, and the increase in ROS in CSF became statistical significance at days 12-30 post-infection compared to that before infection (P<0.001), and then ROS returned to normal level at day 45 after infection. In parallel with the increase in ROS in the CSF, infected mice showed similar of changes in reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST) as that in ROS in the CSF. GSH, GR, GPx, and GST in the CSF of infected mice were all significantly higher than they were before infection during days 12-30 post-infection. However, protein carbonyl content and 8-hydroxy-2'-deoxyguanosine, biomarkers of oxidative damage to protein and DNA, respectively, were also significantly higher in the CSF of infected mice during this period. These results suggest that oxidative stress occur in the cells of central nervous system of mice infected with A. cantonensis during days 12-30 after infection due to ROS overproduction in CSF despite the increase in antioxidants during this period.

5-lipoxygenase and cyclooxygenase-2 in porcine parasitic bronchopneumonia: immunohistochemical and biochemical investigations.

Eicosanoids are products of arachidonic acid metabolism and have numerous biological roles. The present study aimed to investigate the role of 5-lipoxygenase (5-LOX)- and cyclooxygenase-2 (COX-2)- dependent enzymatic pathways in the pathogenesis of porcine parasitic bronchopneumonia caused by Metastrongylus spp. Pulmonary tissue samples from healthy control and parasitized pigs were processed for histopathological, immunohistochemical and biochemical investigations. In control animals, immunohistochemistry demonstrated that 5-LOX and COX-2 expression was almost exclusively limited to the bronchiolar epithelial cells. Parasitized pigs had greater 5-LOX- and COX-2- specific immunoreactivity, involving a wide range of cell types within foci of granulomatous and eosinophilic bronchopneumonia. Biochemical investigations demonstrated the presence of 5-LOX (and the related product Leukotriene B(4)) and COX-2 (and the related product prostaglandin E(2); PGE(2)) in all tissues under study. COX-2 activity and PGE(2) concentration were significantly higher in diseased lungs compared with normal healthy controls. These findings demonstrate that 5-LOX and COX-2 are differentially expressed in normal versus lungworm-infected lungs and therefore suggest that both biochemical pathways are likely to be involved in the pathogenesis of porcine parasitic bronchopneumonia.

Nippostrongylus brasiliensis infection leads to the development of emphysema associated with the induction of alternatively activated macrophages.

Chronic obstructive pulmonary disease (COPD) is the 5(th) most prevalent disease worldwide leading to severe morbidity and mortality in developed countries. The disease is strongly associated with smoking, and can be characterized by progressive and irreversible deterioration in lung function and destruction of the lung parenchyma. We show here that infection with the hookworm Nippostrongylus brasiliensis results in deterioration in lung function, destruction of alveoli and long-term airways hyperresponsiveness, consistent with COPD and emphysema. N. brasiliensis infection leads to chronic low level hemorrhaging in the lung and the presence of hemosiderin-laden macrophages in the absence of an overt inflammatory infiltrate. Microarray analysis of gene expression in diseased lungs and quantitative RT-PCR analysis of purified macrophages revealed a state of prolonged tissue injury and the presence of alternatively activated macrophages producing MMP-12. Taken together, these data show that lung tissue damage caused by hookworm infection can result in the development of COPD and emphysema.

Angiostrongylus cantonensis: induction of urokinase-type PA and degradation of type IV collagen in rat lung granulomatous fibrosis.

Granuloma formation and subsequent fibrosis around Angiostrongylus cantonensis larvae in the lungs were induced experimentally in Sprague-Dawley strain rats. Casein zymogram analysis demonstrated that urokinase-type plasminogen activator (uPA) activity was increased during lung inflammation and fibrosis. Granulomatous fibrosis, type IV collagen degradation and activation of uPA occur simultaneously. Furthermore, the present study demonstrated that collagen avidly binds uPA. Immunohistochemical observations showed localization of uPA within the infiltrating leucocytes. We propose that uPA may participate in A. cantonensis-induced granulomatous fibrosis.

Matrix metalloproteinase-2, -9 and -13 are involved in fibronectin degradation of rat lung granulomatous fibrosis caused by Angiostrongylus cantonensis.

Pulmonary granuloma formation and fibrosis were experimentally induced in Sprague-Dawley strain rats by Angiostrongylus cantonensis. Increased protein levels of matrix metalloproteinase (MMP)-2, -9, -13 and the imbalance between these enzymes and metalloproteinase inhibitors, tissue inhibitors of MMPs (TIMP-1 and -2), occur during granulomatous fibrosis. Activation of proteolytic enzymes (MMP-2, -9 and -13) and fibronectin degradation occur simultaneously. Furthermore, the present study demonstrated that fibronectin avidly binds MMP-2, -9 or -13. Immunohistochemical observations also showed the localization of MMP-13, TIMP-1 and -2 within the infiltrating leucocytes. These results suggest that MMP-2, -9 and -13 may participate in the fibronectin degradation of A. cantonensis-induced granulomatous fibrosis.

Biochemical and pathological evaluation of albendazole/thalidomide co-therapy against eosinophilic meningitis or meningoencephalitis induced by Angiostrongylus cantonensis.

OBJECTIVES: To evaluate the curative effect of albendazole/thalidomide co-therapy on eosinophilic meningitis in BALB/c mice caused by Angiostrongylus cantonensis. METHODS: Male mice were infected with 50 A. cantonensis larvae and treated with albendazole (5, 10 or 20 mg/kg per day) alone, thalidomide (25, 50 or 100 mg/kg per day) alone, or a combination of albendazole (10 mg/kg per day) and thalidomide (50 mg/kg per day) for 7 consecutive days on days 5, 10 and 15 post-inoculation (PI), respectively. RESULTS: Indicators used to measure this effect included: (i) worm recovery; (ii) histopathological score of meningitis; (iii) eosinophil counts; (iv) level of pro-inflammatory cytokines, such as tumour necrosis factor-alpha, interleukin-1beta and interleukin-5; (v) activity of enzymes, such as tissue-type plasminogen activator, urokinase-type plasminogen activator and matrix metalloproteinase-9; and (vi) CSF/serum albumin ratio. The results showed that albendazole/thalidomide co-therapy significantly decreased (P < 0.05) these factors when treatment was initiated on days 5 or 10 PI compared with treatment initiated on day 15 PI. CONCLUSIONS: The timing of medication use is important and is closely related to the anthelmintic efficacy of a drug. For a given dosage, earlier medication use is more effective. This novel approach to treating parasitic meningitis may suggest other new methods of treatment.

Induction of tumour necrosis factor, interleukin-1beta and matrix metalloproteinases in pulmonary fibrosis of rats infected with Angiostrongylus cantonensis.

In angiostrongyliasis, chronic parasite-induced granuloma formation can lead to tissue destruction and fibrosis. Here, the histomorphology of granulomatous fibrosis and proteinase production in the lungs of Angiostrongylus cantonensis-infected Sprague-Dawley rats were investigated. The relationship between metalloproteinases and granulomatous fibrosis was investigated following infection of each rat with 60 infective larvae. Granulomata and fibrosis were marked in the lungs of rats on day 180 post-inoculation. Reverse transcriptase polymerase chain reaction of lung mRNA showed an up-expression of proinflammatory cytokine including tumour necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta). According to Western blot analysis, matrix metalloproteinase-2 (MMP-2) proenzyme was presented in the lungs of uninfected and infected rats, and partial conversion of 72 kDa proenzyme to the 64 kDa active form occurred in infected rats. In addition, increased protein levels of MMP-9 and MMP-13 were detected in infected lungs, but were undetectable in controls. The results suggest that TNF-alpha, IL-1beta, MMP-2, -9, and -13 may be associated with the granulomatous fibrosis.

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

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.

Villus epithelial injury induced by infection with the nematode Nippostrongylus brasiliensis is associated with upregulation of granzyme B.

Intestinal parasite infections induce thymus-dependent villus atrophy, but the effector mechanisms directly responsible for the development of villus atrophy are not thoroughly understood. In this study, we analyzed the expression of cytotoxic factors or ligands in athymic nude rnu/rnu rats and their littermate euthymic rnu/+ rats infected with the nematode Nippostrongylus brasiliensis. Morphometric analyses showed that partial villus atrophy developed 10 days after infection in euthymic but not in athymic rats, whereas crypt hyperplasia occurred in both types of animal. Reverse transcription-polymerase chain reaction analyses of the isolated jejunal epithelial fraction showed that the development of villus atrophy in euthymic rats was positively correlated with an increase of granzyme B transcript levels but not with Fas ligand or tumor necrosis factor-alpha expression. In addition, the number of granzyme B-immunoreactive cells was increased significantly in euthymic rat villus epithelium and the propria mucosa after infection. The CD8+ cell number did not change significantly. Collectively, these findings showed that significant increases in the number of cells that express the cytotoxic factor granzyme B occur in the nematode-infected small intestine of immunocompetent hosts. The type of cells that express granzyme B and their role in the progression of enteropathy remain to be elucidated.

Activation of caspases in intestinal villus epithelial cells of normal and nematode infected rats.

BACKGROUND: Small intestinal epithelial cells (IEC) show apoptosis in physiological turnover of cells and in certain inflammatory diseases. AIMS: To investigate the role of caspases in the progression of IEC apoptosis in vivo. METHODS: IEC were separated along the villus-crypt axis from the jejunum of normal and Nippostrongylus brasiliensis infected rats at 4 degrees C. Caspases were examined by a fluorometric assay method, histochemistry, and immunoblotting. RESULTS: Villus cell rich IEC from normal rats exhibited a high level of caspase-3-like activity whereas activities of caspase-1, -8, and -9 were negligible. Immunoblotting analysis of villus cell rich IEC revealed partial cleavage of procaspase-3 into a 17 kDa molecule as well as cleavage of a caspase-3 substrate, poly(ADP-ribose) polymerase (PARP), whereas in crypt cell rich IEC, caspase-3 cleavage was less significant. Caspase-3 activity was also observed histochemically in villus epithelium on frozen sections of the normal small intestine. IEC prepared at 4 degrees C did not reveal nuclear degradation whereas subsequent incubation in a suspension at 37 degrees C induced intense nuclear degradation within one hour in accordance with increases in active caspase-3. This apoptosis was partially suppressed by the caspase inhibitor Z-VAD-fmk. Nematode infected animals showed villus atrophy together with significant increases in levels of caspase-3 in IEC but not of caspase-1, -8, or -9. CONCLUSION: Caspase-3 may have an important role in the physiological replacement of IEC as well as in progression of IEC apoptosis induced by nematode infection.

Cloning, expression, and properties of a nonneuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis.

We have isolated a full-length cDNA encoding an acetylcholinesterase secreted by the nematode parasite Nippostrongylus brasiliensis. The predicted protein is truncated in comparison with acetylcholinesterases from other organisms such that the carboxyl terminus aligns closely to the end of the catalytic domain of the vertebrate enzymes. The residues in the catalytic triad are conserved, as are the six cysteines which form the three intramolecular disulfide bonds. Three of the fourteen aromatic residues which line the active site gorge in the Torpedo enzyme are substituted by nonaromatic residues, corresponding to Tyr-70 (Thr), Trp-279 (Asn), and Phe-288 (Met). High level expression was obtained via secretion from Pichia pastoris. The purified enzyme behaved as a monomeric hydrophilic species. Although of invertebrate origin and possessing the above substitutions in the active site gorge residues, the enzyme efficiently hydrolyzed acetylthiocholine and showed minimal activity against butyrylthiocholine. It displayed excess substrate inhibition with acetylthiocholine at concentrations over 2. 5 mM and was highly sensitive to both active site and "peripheral" site inhibitors. Northern blot analysis indicated a progressive increase in mRNA for AChE B in parasites isolated from 6 days postinfection.

Cloning of the cDNAs for mast-cell chymases from the jejunum of Mongolian gerbils, Meriones unguiculatus, and their sequence similarities with chymases expressed in the connective-tissue mast cells of mice and rats.

By using the combination of reverse-transcription CR and rapid amplification of cDNA ends methods, two distinct cDNAs encoding mast-cell proteases (chymases; MCPs), designated as gMCP-1 and -2, were successfully cloned and sequenced from the jejunum of Mongolian gerbil, Meriones unguiculatus, infected with Nippostrongylus brasiliensis. On the basis of a comparison of the deduced amino acid sequences with those of known rodent mast-cell chymases, gMCP-1 was found to be highly similar to mouse mast-cell protease (mMCP)-4 and rat mast-cell protease (rMCP)-1, while gMCP-2 was similar to mMCP-5 and rMCP-3. Alghough mMCP-4 and -5 and rMCP-1 and -3 were restrictedly or mainly expressed in connective-tissue mast cells and serosal mast cells, the gMCP-1 and -2 genes were mainly transcribed in the jejunal mucosa and to a lesser extent in the skin and tongue. Moreover, kinetic study after infection revealed that the amounts of the gMCP-1 and -2 mRNAs in jejunum paralleled well the degree of intestinal mastocytosis. The expression of gMCP-1 and -2 in mucosal mast cells of gerbil jejunum was also confirmed by in situ hybridization. Since a tryptase, another type of MCP, was also expressed in mucosal mast cells of gerbils but not in those of mice and rats, the expression of MPCs in mucosal mast cells of gerbils is different from those of mice and rats. The Mongolian gerbil would be a useful model with which to investigate the physiopathological role of MCPs.

Cloning of the cDNA encoding a novel rat mast-cell proteinase, rMCP-3, and its expression in comparison with other rat mast-cell proteinases.

A cDNA encoding a novel rat mast-cell proteinase (MCP) named rMCP-3 was successfully cloned and sequenced from the peritoneal cells of Lewis rats infected with the intestinal nematode Nippostrongylus brasiliensis by using the combination of reverse transcription-PCR and rapid-amplification-of-cDNA-ends ('RACE') methods. The cDNA was 979 bp long and included a 741 bp open reading frame. When the deduced amino acid sequence was compared with those of other known mast-cell proteinases, rMCP-3 was considered to be translated as a preproenzyme with a 19-amino-acid signal peptide, a two-amino-acid activation peptide and a 226-amino-acid mature enzyme. The amino acid identity in the mature enzyme was 52.9% and 55.1% with rMCP-1 and rMCP-2 respectively. The rMCP-3 mRNA was not detected in the peritoneal cells of mast-cell-deficient Ws/Ws rats, though it was strongly detected in those of littermate +/+ and Lewis rats, indicating the mast-cell origin of rMCP-3 In addition to being present in peritoneal mast cells, the rMCP-3 mRNA was strongly detected in the skin, tongue, and RBL2H3 rat basophilic leukaemia cells and weakly in the jejunum of N. brasiliensis-infected rats by RNA blot analysis using a rMCP-3 gene-specific probe. By reverse transcription-PCR, the rMCP-3 mRNA was also detected in the lung. While the expression of rMCP-1 and rMCP-2 are clearly restricted in connective-tissue mast cells and mucosal mast cells respectively, rMCP-3 was widely expressed in both types of mast cells with a predominance in connective-tissue mast cells.

Altered expression of mast cell proteases in the rat. Quantitative and immunohistochemical analysis of the distribution of rat mast cell proteases I and II during helminth infection.

Expression of mast cell granule protease is regulated in a tissue-specific fashion in the rat. The granule chymases rat mast cell proteases I and II (RMCP I and II) predominate in non-mucosal and mucosal sites, respectively. Intestinal mastocytosis, a T cell-mediated phenomenon associated with enteric nematodiasis, is accompanied by massive local expression of RMCP II and by release of this protease systemically into blood. The present observations, where both RMCP I and II have been quantified by ELISA and immunolocalized by paired fluorescence, show that the expression of both proteases in parasitized rats is profoundly altered at sites distant from infection. Thus, RMCP II-containing cells are recruited to liver and thymus, and in the thymus there is a > 2-fold increase in concentration of RMCP I. The latter protease is depleted from bone marrow and mesenteric lymph node early during infection, but concentrations of RMCP I in trachea/larynx, lung, and skeletal and cardiac muscle are increased. Increased mast cell counts in intestine, lung and liver are highly correlated with tissue concentrations of RMCP II.