RESUMEN
BACKGROUND: The abomasal helminth Teladorsagia circumcincta is one of the most economically important parasites affecting sheep in temperate regions. Infection is particularly detrimental to lambs, in which it can cause pronounced morbidity and severe production losses. Due to the spreading resistance of this parasite to all classes of anthelmintic drugs, teladorsagiosis is having an increasingly severe impact on the sheep industry with significant implications for sheep welfare. Protective immunity develops slowly, wanes rapidly and does not appear to be as effective in young lambs. To investigate the development of immunity to T. circumcincta in sheep and lambs, we used cytokine transcript profiling to examine differences in the abomasal mucosa and gastric lymph node of naïve and previously infected sheep and lambs following challenge. RESULTS: The results of these experiments demonstrated that the abomasal mucosa is a major source of cytokines during abomasal helminth infection. A local Th2-type cytokine response was observed in the abomasal mucosa and gastric lymph node of the previously infected sheep and lambs when compared with those of the naïve during the early stages of infection. In contrast, a pro-inflammatory component more was evident in the abomasal mucosa and gastric lymph node of the naïve sheep when compared with those of the previously infected, which was not observed in the lambs. CONCLUSIONS: The greater levels of Th2-type cytokine transcripts in both the abomasum and gastric lymph node of the previously infected compared with naïve sheep and lambs emphasises the importance of these mechanisms in the immune response to T. circumcincta infection. Younger lambs appear to be able to generate similar Th2-type responses in the abomasum suggesting that the increased morbidity and apparent lack of resistance in younger lambs following continuous or repeated exposure to T. circumcincta is unlikely to be due to a lack of appropriate Th2-type cytokine production.
Asunto(s)
Citocinas/fisiología , Ostertagia , Ostertagiasis/veterinaria , Enfermedades de las Ovejas/parasitología , Abomaso/parasitología , Animales , Animales Recién Nacidos/inmunología , Animales Recién Nacidos/parasitología , Animales Recién Nacidos/fisiología , Recuento de Células/veterinaria , Citocinas/biosíntesis , Mastocitos/fisiología , Ostertagiasis/inmunología , Ovinos/parasitología , Enfermedades de las Ovejas/inmunología , Transcripción GenéticaRESUMEN
Continual low-level exposure of sheep to the helminth Teladorsagia circumcincta elicits a temporary protective immunity, where factors in the immune abomasal mucosa prevent penetration of infective larvae, but which is essentially lost within 6 weeks of cessation of parasite challenge. Here, a proteomic approach was used to identify proteins that are differentially regulated in immune compared to naïve sheep, as potential key mediators of immunity. Six naïve sheep and 12 sheep trickle-infected with T. circumcincta were treated with anthelmintic, and the naïve (control) and 6 immune sheep were killed 7 days later. The remaining 6 sheep (immune waning) were killed 42 days after anthelmintic treatment. Abomasal tissue samples were subjected to 2D-gel electrophoresis and densitometric analysis. Selected spots (n=73) were identified by peptide mass fingerprinting and confirmatory Western blotting was carried out for 10 proteins. Spots selectively up-regulated in immune versus control, but not immune waning versus control sheep, included galectin-15 and thioredoxin, which were confirmed by Western blotting. In immune sheep, serum albumin was significantly down-regulated and albumin proteolytic cleavage fragments were increased compared to controls. Unexpectedly, albumin mRNA was relatively highly expressed in control mucosa, down-regulated in immune, and was immunolocalized to mucus-producing epithelial cells. Thus we have identified differential expression of a number of proteins following T. circumcincta trickle infection that may play a role in host protection and inhibition of parasite establishment.
Asunto(s)
Abomaso/metabolismo , Proteínas del Helminto/metabolismo , Enfermedades de las Ovejas/metabolismo , Trichostrongyloidea , Tricostrongiloidiasis/veterinaria , Abomaso/efectos de los fármacos , Abomaso/inmunología , Inmunidad Adaptativa , Animales , Antihelmínticos/administración & dosificación , Western Blotting , Electroforesis en Gel Bidimensional , Galectinas/genética , Galectinas/metabolismo , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/inmunología , Mucosa Gástrica/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Proteínas del Helminto/genética , Proteómica , ARN Mensajero/metabolismo , Albúmina Sérica/genética , Albúmina Sérica/metabolismo , Ovinos , Enfermedades de las Ovejas/tratamiento farmacológico , Enfermedades de las Ovejas/inmunología , Oveja Doméstica , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Tricostrongiloidiasis/tratamiento farmacológico , Tricostrongiloidiasis/inmunología , Tricostrongiloidiasis/metabolismoRESUMEN
BACKGROUND: C57BL/6J mice possess a single intelectin (Itln) gene on chromosome 1. The function of intelectins is not well understood, but roles have been postulated in insulin sensitivity, bacterial recognition, intestinal lactoferrin uptake and response to parasites and allergens. In contrast to C57BL/6J mice, there is evidence for expansion of the Itln locus in other strains and at least one additional mouse Itln gene product has been described. The aim of this study was to sequence and characterise the Itln locus in the 129S7 strain, to determine the nature of the chromosomal expansion and to inform possible future gene deletion strategies. RESULTS: Six 129S7 BAC clones were sequenced and assembled to generate 600 kbp of chromosomal sequence, including the entire Itln locus of approximately 500 kbp. The locus contained six distinct Itln genes, two CD244 genes and several Itln- and CD244-related pseudogenes. It was approximately 433 kbp larger than the corresponding C57BL/6J locus. The expansion of the Itln locus appears to have occurred through multiple duplications of a segment consisting of a full-length Itln gene, a CD244 (pseudo)gene and an Itln pseudogene fragment. Strong evidence for tissue-specific distribution of Itln variants was found, indicating that Itln duplication contributes more than a simple gene dosage effect. CONCLUSIONS: We have characterised the Itln locus in 129S7 mice to reveal six Itln genes with distinct sequence and expression characteristics. Since C57BL/6J mice possess only a single Itln gene, this is likely to contribute to functional differences between C57BL/6J and other mouse strains.
Asunto(s)
Dosificación de Gen , Sitios Genéticos , Lectinas/genética , Animales , Antígenos CD/genética , Secuencia de Bases , Sitios de Unión , Cromosomas Artificiales Bacterianos , Cromosomas de los Mamíferos/genética , Evolución Molecular , Biblioteca de Genes , Genómica , Proteínas de Homeodominio/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Filogenia , Regiones Promotoras Genéticas , Seudogenes , Receptores Inmunológicos/genética , Duplicaciones Segmentarias en el Genoma , Análisis de Secuencia de ADN , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Factores de Transcripción/metabolismoRESUMEN
Infection of sheep with the gastric nematode Teladorsagia circumcincta results in distinct Th2-type changes in the mucosa, including mucous neck cell and mast cell hyperplasia, eosinophilia, recruitment of IgA/IgE producing cells and neutrophils, altered T-cell subsets and mucosal hypertrophy. To address the protective mechanisms generated in animals on previous exposure to this parasite, gene expression profiling was carried out using samples of abomasal mucosa collected pre- and post- challenge from animals of differing immune status, using an experimental model of T. circumcincta infection. Recently developed ovine cDNA arrays were used to compare the abomasal responses of sheep immunised by trickle infection with worm-naïve sheep, following a single oral challenge of 50 000 T. circumcincta L3. Key changes were validated using qRT-PCR techniques. Immune animals demonstrated highly significant increases in levels of transcripts normally associated with cytotoxicity such as granulysin and granzymes A, B and H, as well as mucous-cell derived transcripts, predominantly calcium-activated chloride channel 1 (CLCA1). Challenge infection also induced up-regulation of transcripts potentially involved in initiating or modulating the immune response, such as heat shock proteins, complement factors and the chemokine CCL2. In contrast, there was marked infection-associated down-regulation of gene expression of members of the gastric lysozyme family. The changes in gene expression levels described here may reflect roles in direct anti-parasitic effects, immuno-modulation or tissue repair.
Asunto(s)
Abomaso/metabolismo , Regulación de la Expresión Génica , Mucosa Intestinal/metabolismo , Enfermedades de las Ovejas/genética , Trichostrongyloidea/fisiología , Tricostrongiloidiasis/veterinaria , Abomaso/parasitología , Animales , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica/veterinaria , Mucosa Intestinal/parasitología , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , Análisis de Secuencia de ADN/veterinaria , Ovinos , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/parasitología , Tricostrongiloidiasis/inmunología , Tricostrongiloidiasis/metabolismo , Tricostrongiloidiasis/parasitologíaRESUMEN
Intelectins (Itlns) are lectins with potential roles in innate immunity, capable of binding bacteria via galactofuranose residues. Itlns also function as intestinal receptors for the antimicrobial glycoprotein lactoferrin (Lf). Since Lf binds strongly to enterohemorrhagic Escherichia coli O157:H7 (EHEC), we aimed to determine the expression of Lf receptor in terminal rectum, the site of predilection of EHEC in cattle. We sequenced two bovine intelectins (Itln1 and Itln2) and showed that both were expressed in abomasum and rectum, but expression appeared minimal in the jejunum. There was significantly higher expression of Itln2 in terminal rather than proximal rectum. Lactoferrin was expressed in all samples examined. Thus, we have demonstrated two novel bovine Itlns and shown that they are expressed along with Lf in the gastrointestinal tract, where they may interact with microbial pathogens.
Asunto(s)
Enfermedades de los Bovinos/inmunología , Escherichia coli Enterohemorrágica/inmunología , Infecciones por Escherichia coli/veterinaria , Enfermedades Intestinales/veterinaria , Receptores de Superficie Celular/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Enfermedades de los Bovinos/genética , Enfermedades de los Bovinos/microbiología , ADN Complementario/genética , Escherichia coli Enterohemorrágica/genética , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Enfermedades Intestinales/inmunología , Enfermedades Intestinales/microbiología , Datos de Secuencia Molecular , ARN/química , ARN/genética , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Alineación de SecuenciaRESUMEN
A novel intelectin molecule designated sheep intelectin 2 (sITLN2) was detected in sheep abomasal mucosa. The full sequence shared 76-83% homology with other mammalian intelectins. Intelectins are mucus-associated proteins that have been shown to be up-regulated in gastrointestinal nematode infections in rodents and in human asthma. Expression of sheep abomasal ITLN2 mRNA was significantly up-regulated on day 10 post-challenge of worm-free sheep with Teladorsagia circumcincta and at day 2 in previously infected, immune sheep. Increased expression of ITLN protein following challenge was confirmed by Western blot and was immunolocalised to the mucous neck cells of the abomasal mucosa. Infection with T. circumcincta was also associated with increased levels of abomasal transcripts encoding sheep mast cell protease-1, ovine galectin-14 and IL4, which collectively suggested a Th2 type response. Intelectin may play an important role in the mucosal response to gastrointestinal nematode infections in ruminants.
Asunto(s)
Abomaso/inmunología , Galectinas/metabolismo , Parasitosis Intestinales/inmunología , Infecciones por Nematodos/inmunología , Enfermedades de las Ovejas/parasitología , Regulación hacia Arriba , Abomaso/parasitología , Animales , Secuencia de Bases , Western Blotting/métodos , Quimasas/genética , Quimasas/metabolismo , Femenino , Galectinas/genética , Mucosa Gástrica/metabolismo , Mucosa Gástrica/parasitología , Interacciones Huésped-Parásitos , Interleucina-4/genética , Interleucina-4/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , OvinosRESUMEN
Phospholipase A2 (PLA2) enzymes play a central role in the initiation, propagation and resolution of inflammation. Here, we describe de novo expression of group IVC PLA2 (PLA2g4c) within the intestinal epithelium of Trichinella spiralis parasitised mice. This mouse mast cell protease-1 sensitive, calcium-independent PLA2 is not detectable in the jejunal epithelium of uninfected mice but becomes highly expressed within the epithelial compartment within days of nematode establishment. We propose that epithelial PLA2g4c accounts for the increased lysophospholipase activity observed during intestinal nematodiasis and that it plays a major role in the inflammatory response to nematodes.
Asunto(s)
Fosfolipasas A2 Grupo IV/genética , Parasitosis Intestinales/enzimología , Mucosa Intestinal/enzimología , Trichinella spiralis/fisiología , Triquinelosis/enzimología , Animales , Quimasas/metabolismo , Expresión Génica , Fosfolipasas A2 Grupo IV/metabolismo , Inflamación , Yeyuno , Ratones , Ratones Endogámicos BALB CRESUMEN
Upregulation of intelectin (ITLN) transcript and protein has previously been shown in intestinal nematode infections of resistant mice strains with immunolocalisation of protein to goblet cells and paneth cells. In man, intelectin expression has been shown in respiratory tract epithelium, with upregulation occurring in bronchoalveolar lavage fluid of asthmatic individuals. This study describes the expression of intelectin in the respiratory tract of sheep and the immunolocalisation to goblet cells using a novel affinity-purified chicken anti-intelectin peptide antibody. Furthermore we show that when sheep tracheal explants were cultured for 48 h+/- recombinant sheep IL-4, sheep ITLN transcripts were upregulated compared with controls. Putative roles for intelectin have included an antibacterial role and an alteration of the character of mucus. Our data suggest ITLNs may play an important role in the mucosal response in allergy and parasitic infections.
Asunto(s)
Células Caliciformes/efectos de los fármacos , Células Caliciformes/metabolismo , Interleucina-4/farmacología , Lectinas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/metabolismo , Línea Celular Tumoral , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Ratones Endogámicos BALB C , Ovinos , Células Th2/efectos de los fármacos , Células Th2/fisiología , Tráquea/citologíaRESUMEN
Sheep intelectin1 and sheep intelectin3 (sITLN1 and sITLN3) were cloned and sequenced. The amino acid sequences of sITLN1 and sITLN3 shared 86% and 91% homology with the previously cloned sheep intelectin2 (sITLN2), respectively. Expression of sITLN1 and sITLN3 transcript was demonstrated in abomasum, lung, colon and gastric lymph node, terminal rectum, skin, jejunum, mesenteric lymph node, ileal peyer's patches, brain, kidney, liver, spleen, skin, ear pinna, heart and ovary in normal sheep tissues. sITLN2 transcript expression was restricted to the abomasal mucosa in normal sheep tissues. Using a non selective chicken anti-intelectin antibody, tissue intelectin protein was demonstrated in mucus neck cells in the abomasum, mucus cells in the colon, free mucus in ileum, goblet cells in the lung, small intestinal epithelium and brush border, epidermal layer of the skin and skin sebaceous glands. The expression of the three sITLN transcripts was examined in two nematode infections in sheep known to induce a Th2 response; a Teladorsagia circumcincta challenge infection model and a Dictyocaulus filaria natural infection. The three sITLN were absent in unchallenged naïve lambs and present in the abomasal mucosa of both naïve and immune lambs following T. circumcincta challenge infection. Upregulation of sITLN2 and sITLN3 was shown in sheep lung following D. filaria natural infection. Intelectins may play an important role in the mucosal response to nematode infections in ruminants.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Lectinas/metabolismo , Infecciones por Nematodos/veterinaria , Enfermedades de las Ovejas/metabolismo , Animales , Clonación Molecular , Infecciones por Nematodos/metabolismo , Ovinos , Enfermedades de las Ovejas/parasitologíaRESUMEN
Infection of mice with the nematode Trichinella spiralis triggers recruitment and differentiation of intraepithelial intestinal mucosal mast cells expressing mouse mast cell protease 1 (Mcpt-1), which contributes to expulsion of the parasite. Expression of Mcpt-1 is transforming growth factor (TGF)-beta1-dependent in vitro. TGF-beta1, which is secreted within tissues as a biologically inactive complex with latency-associated peptide, requires extracellular modification to become functionally active. The integrin-alpha(nu)beta(6) mediates local activation of TGF-beta(1) in association with epithelia. Using T. spiralis-infected beta(6)(-/-) mice, we show accumulation of mucosal mast cells in the lamina propria of the small intestine with minimal recruitment into the epithelial compartment. This was accompanied by a coordinate reduction in expression of both Mcpt-1 and -2 in the jejunum and increased tryptase expression, whereas Mcpt-9 became completely undetectable. In contrast, the cytokine stem cell factor, a regulator of mast cell differentiation and survival, was significantly up-regulated in T. spiralis-infected beta(6)(-/-) mice compared with infected beta(6)(+/+) controls. Despite these changes, beta(6)(-/-) mice still appeared to expel the worms normally. We postulate that compromised TGF-beta(1) activation within the gastrointestinal epithelial compartment is a major, but not the only, contributing factor to the observed changes in mucosal mast cell protease and epithelial cytokine expression in beta(6)(-/-) mice.
Asunto(s)
Antígenos de Neoplasias/genética , Quimasas/metabolismo , Integrinas/genética , Mucosa Intestinal/inmunología , Mastocitos/enzimología , Factor de Crecimiento Transformador beta1/metabolismo , Trichinella spiralis , Triquinelosis/inmunología , Animales , Médula Ósea/inmunología , Quimasas/análisis , Quimasas/genética , Colon/inmunología , Citocinas/genética , Citocinas/metabolismo , Oído , Yeyuno/inmunología , Mastocitos/inmunología , Ratones , Ratones Mutantes , Estómago/inmunologíaRESUMEN
The granule-derived mouse mast cell proteases-1 and -2 (mMCP-1 and -2) colocalize in similar quantities in mucosal mast cells but micrograms of mMCP-1 compared with nanograms of mMCP-2 are detected in peripheral blood during intestinal nematode infection. This differential systemic response was investigated both in vitro and in vivo. Bone marrow-derived mucosal mast cell homologs released similar quantities of mMCP-1 and-2 concomitantly with beta-hexosaminidase in response to calcium ionophore ( approximately 60% release) or IgE/DNP (25% release). In contrast, serum from mice sensitized by infection with Nippostrongylus brasiliensis 10 days earlier contained >1500-fold more mMCP-1 (10,130 +/- 1,609 ng/ml) than mMCP-2 (6.4 +/- 1 ng/ml), but, in gut lumen, the difference was approximately 8-fold. After OVA sensitization, >600-fold more mMCP-1 (7,861 +/- 2,209 ng/ml) than mMCP-2 (12.8 +/- 4.7 ng/ml) was present in blood 1 h after challenge, but, in gut lumen, there were relatively comparable levels of mMCP-1 and -2. To estimate the rates of systemic accumulation and clearance, 10 microg of mMCP-1 or -2 was injected i.p. Plasma levels of injected mMCP-2 peaked (1%) at 15 min then declined, whereas levels of mMCP-1 were maximal (approximately 25%) at 3 h. Inactivation of mMCP-1 with PMSF before injection resulted in mMCP-2-like kinetics, but inhibition of mMCP-1 by serum gave kinetics similar to that of native mMCP-1. mMCP-1 isolated from serum is complexed with serpins and we conclude that both the accumulation and the longevity of mMCP-1 in blood is due to complex formation, protecting it from a pathway that rapidly clears mMCP-2, which is unable to form complexes with serpins.
Asunto(s)
Anafilaxia/enzimología , Anafilaxia/inmunología , Mastocitos/enzimología , Mastocitos/inmunología , Serina Endopeptidasas/metabolismo , Serpinas/metabolismo , Anafilaxia/fisiopatología , Animales , Degranulación de la Célula/inmunología , Células Cultivadas , Quimasas , Inmunización , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Cinética , Mastocitos/fisiología , Ratones , Ratones Endogámicos BALB C , Complejos Multiproteicos , Nippostrongylus/inmunología , Nippostrongylus/patogenicidad , Ovalbúmina/inmunología , Serina Endopeptidasas/sangre , Serina Endopeptidasas/química , Infecciones por Strongylida/enzimología , Infecciones por Strongylida/inmunologíaRESUMEN
Mast cells migrate to the mucosal epithelium during intestinal nematode infections in mice, where they express abundant mucosal mast cell-specific proteases, mouse mast cell protease-1 and -2 (MCPT1 and MCPT2). Expression of these proteases is strictly controlled by transforming growth factor-beta1 (TGF-beta1) in the epithelium. In vitro homologues of mucosal mast cells are generated by culturing bone marrow-derived mast cells (BMMC) in the presence of TGF-beta1. We examined the proteome of BMMC cultured either in the presence of TGF-beta1 (n = 5) or of a neutralising anti-TGF-beta1 antibody (n = 5). Cell extracts were examined by 2-DE, and changes in expression levels of protein spots were determined by densitometry. Spots of interest were identified by tryptic peptide mapping. In addition to the up-regulation of MCPT1 and MCPT2, which accounted for approximately 40% of all soluble protein in the TGF-beta1 treated cells, MCPT7 was modestly up-regulated by TGF-beta1, and calnexin was up-regulated fivefold. A 7.6-fold down-regulation of galectin-1 was verified by Western blotting and FACS analysis. Galectin-1 is located on the cell surface where it mediates cellular adhesion to basement membranes. Regulation of its expression by TGF-beta1 may be of relevance to mast cell adhesion within the epithelium.
Asunto(s)
Biomarcadores/metabolismo , Células de la Médula Ósea/metabolismo , Mastocitos/metabolismo , Membrana Mucosa/metabolismo , Proteoma , Factor de Crecimiento Transformador beta/farmacología , Animales , Western Blotting , Células de la Médula Ósea/citología , Células Cultivadas , Electroforesis en Gel Bidimensional , Citometría de Flujo , Masculino , Mastocitos/citología , Ratones , Ratones Endogámicos BALB C , Membrana Mucosa/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1RESUMEN
Host immune responses to commensal flora and enteric pathogens are known to influence gene expression in the intestinal epithelium. Although the Cdx family of caudal-related transcription factors represents critical regulators of gene expression in the intestinal epithelium, the effect of intestinal immune responses on Cdx expression and function has not been determined. We have shown that bacterial colonization and Th2 immune stimulation by intestinal nematode infection induce expression of the intestinal goblet cell-specific gene RELM beta. In this study, we investigated the transcriptional regulation of resistin-like molecule/found in inflammatory zone (RELM/FIZZ, RELM beta) and its isoforms RELM alpha and RELM gamma to ascertain the role of Cdx in modifying intestinal gene expression associated with innate and adaptive immune responses. Analysis of the RELM beta promoter showed that Cdx2 plays a critical role in basal gene activation in vitro. This was confirmed in vivo using transgenic mice, where ectopic gastric and hepatic expression of Cdx2 induces expression of RELM beta, but not RELM alpha or RELM gamma, exclusively in the stomach. Although there was no quantitative change in colonic Cdx2 mRNA expression, protein distribution, or phosphorylation of Cdx2, bacterial colonization induced expression of RELM beta, but not RELM alpha or RELM gamma. In contrast, parasitic nematode infections activated colonic expression of all three RELM isoforms without alteration in Cdx2 expression. These results demonstrated that Cdx2 participates in directing intestine-specific expression of RELM beta in the presence of commensal bacteria and that adaptive Th2 immune responses to intestinal nematode infections can activate intestinal goblet cell-specific gene expression independent of Cdx2.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteínas de Homeodominio/fisiología , Hormonas Ectópicas/biosíntesis , Inmunidad Mucosa/fisiología , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Factores de Transcripción/fisiología , Animales , Factor de Transcripción CDX2 , Línea Celular Tumoral , Mucosa Gástrica/metabolismo , Humanos , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Infecciones por Nematodos , Factor de Crecimiento Nervioso/biosíntesis , Isoformas de Proteínas , Proteínas , Activación TranscripcionalRESUMEN
Infection with the intestinal nematode Trichinella spiralis induces profound, but stereotypic pathological changes to the epithelium, which are common to many nematode infections. This study describes changes in jejunal epithelial protein expression that reflect these stereotypic responses. Adult male BALB/c mice were infected with T. spiralis, and groups (n = 4) examined on day 14/15 (time of worm rejection) were compared with uninfected controls (n = 4). Jejunal epithelium was harvested and extracted for two-dimensional gel electrophoresis. Tryptic peptide mass fingerprinting was used to create a reference map consisting of a total of 52 landmark spots. Of these, 16 were observed to change in intensity during infection. The changes observed at day 14/15 were of relevance to such mechanisms as lipid utilization and transport (increase in triacylglycerol lipase, and reduction in intestinal fatty acid binding protein) and innate immunity (appearance of intelectin-2). As a result, candidate molecules have been identified for further focused studies on their role in the host response to intestinal nematode infection.
Asunto(s)
Epitelio/metabolismo , Yeyuno/metabolismo , Metabolismo de los Lípidos , Proteoma , Trichinella spiralis/metabolismo , Animales , Electroforesis en Gel Bidimensional , Epitelio/parasitología , Epitelio/patología , Inmunidad Innata/inmunología , Inmunidad Innata/fisiología , Yeyuno/parasitología , Yeyuno/patología , Ratones , Mapeo PeptídicoRESUMEN
The immunoregulatory cytokine transforming growth factor (TGF)-beta(1) is secreted as a biologically inactive complex with latency-associated peptide, which must be modified by local factors to expose the functionally active cytokine. The epithelial integrin alpha(v)beta(6) mediates local activation of TGF-beta(1) in the lung and beta(6)(-/-) mice exhibit exaggerated pulmonary inflammation, but their response to inflammatory stimuli in the gut has not been investigated. We found that both beta(6) and TGF-beta(1) are constitutively expressed in the jejunal epithelial compartment in uninfected mice and during infection with the intestinal nematode Nippostrongylus brasiliensis. We also present data showing that beta(6)(-/-) mice are seriously compromised in their ability to mount a mucosal mast cell response after infection, and there is a significant reduction in the expression and systemic release of the granule chymase, mouse mast cell protease-1. Because in vitro expression of this chymase is regulated by TGF-beta(1), these data indicate that in the absence of alpha(v)beta(6) epithelially expressed TGF-beta(1) may not be activated, with a consequent absence of expression of mouse mast cell protease-1 and down-regulation of the mucosal mast cell response.
Asunto(s)
Antígenos de Neoplasias , Integrinas/biosíntesis , Mastocitos/metabolismo , Mastocitos/parasitología , Serina Endopeptidasas/biosíntesis , Infecciones por Strongylida/metabolismo , Infecciones por Strongylida/patología , Animales , Quimasas , Regulación hacia Abajo , Humanos , Hiperplasia , Integrinas/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitología , Mucosa Intestinal/patología , Ratones , Ratones Transgénicos , Nippostrongylus , Serina Endopeptidasas/genética , Infecciones por Strongylida/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND & AIMS: The relationship between intestinal pathology and immune expulsion of gastrointestinal nematodes remains controversial. Immune expulsion of gastrointestinal helminth parasites is usually associated with Th2 responses, but the effector mechanisms directly responsible for parasite loss have not been elucidated. Mast cell hyperplasia is a hallmark of infection with gastrointestinal nematodes, in particular Trichinella spiralis. Although the precise mechanism by which mast cells induce expulsion of these parasites has not been elucidated, it has been proposed that mast cell mediators, including cytokines and granule chymases, act to create an environment inhospitable to the parasite, part of this being the induction of intestinal inflammation. Therefore, the aims of this study were to dissect the role of mast cells and mast cell proteases in the induction of parasite-induced enteropathy. METHODS: Mast cell-deficient W/Wv and mouse mast cell protease-1 (mMCP-1)-deficient mice were infected with T. spiralis, and parasite expulsion, enteropathy, and Th2 responses were determined. RESULTS: Expulsion of the parasite was delayed in both strains of mice compared with wild-type controls; additionally, in both cases, the enteropathy was significantly ameliorated. Although Th2 responses were significantly reduced in mast cell-deficient W/Wv mice, those from mMCP-1-deficient mice were similar to wild-type mice. Additionally, levels of TNF-alpha and nitric oxide were significantly reduced in both W/Wv and mMCP-1 deficient mice. CONCLUSIONS: These results imply that mast cells may contribute to the induction of protective Th2 responses and, importantly, that the intestinal inflammation associated with gastrointestinal helminths is partly mediated by mMCP-1.
Asunto(s)
Parasitosis Intestinales/inmunología , Serina Endopeptidasas/inmunología , Trichinella spiralis/inmunología , Triquinelosis/inmunología , Animales , Quimasas , Interacciones Huésped-Parásitos/inmunología , Parasitosis Intestinales/etiología , Ratones , Serina Endopeptidasas/efectos adversos , Células Th2/inmunología , Triquinelosis/complicacionesRESUMEN
Infection of mice with the nematode parasite Nippostrongylus brasiliensis results in a well characterized intestinal mastocytosis with intraepithelial migration of mucosal mast cells (MMC). The molecules mediating this response are unknown. We examined expression of several putative mast cell chemoattractants in intestinal epithelium following N. brasiliensis infection. Expression of the chemokines monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 alpha (MIP-1alpha), RANTES (regulated on activation normal T-cell expressed and secreted), fractalkine, and thymocyte expressed chemokine (TECK); and the cytokines stem cell factor (SCF) and transforming growth factor beta1 (TGFbeta1), was constitutive and no alteration was detected following infection. MCP-1 expression was also constitutive but at much lower levels and increased expression was detected on days 7 and 14 postinfection. Expression of MCP-1 in whole jejunum was at much higher levels than in epithelium. Constitutive expression of MCP-1, MIP-1alpha and TGFbeta1 was also detected in cultured bone marrow-derived homologues of MMC. In an intestinal epithelial cell line (CMT-93), there was constitutive expression of SCF, TGFalpha1, fractalkine and MCP-1. The results show that, in vivo, epithelium is a potentially important source of mast cell chemoattractants.
Asunto(s)
Quimiocinas/metabolismo , Citocinas/metabolismo , Mucosa Intestinal/inmunología , Nippostrongylus/fisiología , Infecciones por Strongylida/inmunología , Animales , Línea Celular , Células Cultivadas , Mucosa Intestinal/parasitología , Mucosa Intestinal/ultraestructura , Yeyuno/inmunología , Yeyuno/ultraestructura , Masculino , Ratones , Ratones Endogámicos BALB C , Recuento de Huevos de Parásitos , Homología de Secuencia , Infecciones por Strongylida/parasitología , Infecciones por Strongylida/patologíaRESUMEN
Infection with intestinal nematodes induces profound pathological changes to the gut that are associated with eventual parasite expulsion. We have applied expression profiling as an initial screening process with oligonucleotide microarrays (Affymetrix MG-U74AV2 gene chips) and time course kinetics to investigate gene transcription triggered by the intraepithelial nematode Trichinella spiralis in jejunal epithelium from BALB/c mice. Of the 4,114 genes detected, 2,617 were present in all uninfected and T. spiralis-infected replicates, 8% of which were notably upregulated, whereas 12% were downregulated at the time of worm expulsion (day 14 postinfection). Upregulation of goblet cell mucin gene transcripts intestinal mucin gene 3 (MUC3), calcium chloride channel 5 (CLCA5), and goblet cell gene 4 (GOB4) is consistent with enhanced production and alteration of mucus, whereas a 60- to 70-fold upregulation of transcripts for mast cell proteases 1 and 2 (MCPT-1 and -2) is consistent with intraepithelial mucosal mast cell recruitment. Importantly, there was novel expression of sialyltransferase 4C (SIAT4C), small proline-rich protein 2A (SPRR2A), and resistin-like molecule beta (RELMbeta) on day 14 postinfection. In contrast, DNase I and regenerating protein 3 (REG3) transcripts were substantially downregulated. Time course analyses revealed early (within 48 h of infection) induction of Siat4c, Sprr2A, and Relmbeta and later (within 120 h) induction of Mcpt-1 and -2. The findings demonstrate early innate responses and later inflammatory changes within the epithelium. The early epithelial responses may be associated both with repair (Sprr2A) and with the development of innate immunity (Siat4c and Relmbeta).
Asunto(s)
Células Epiteliales/metabolismo , Células Epiteliales/parasitología , Perfilación de la Expresión Génica , Inflamación/genética , Yeyuno/citología , Trichinella spiralis/fisiología , Triquinelosis/genética , Animales , Antioxidantes/metabolismo , Citoesqueleto/genética , Citoesqueleto/parasitología , Células Epiteliales/citología , Células Epiteliales/enzimología , Femenino , Regulación de la Expresión Génica , Glutatión/metabolismo , Células Caliciformes/metabolismo , Células Caliciformes/parasitología , Inmunidad/genética , Inflamación/parasitología , Yeyuno/enzimología , Yeyuno/metabolismo , Yeyuno/parasitología , Masculino , Mastocitos/metabolismo , Mastocitos/parasitología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Mucinas/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos , Células de Paneth/metabolismo , Células de Paneth/parasitología , Uniones Estrechas/genética , Uniones Estrechas/parasitología , Transcripción Genética/genética , Triquinelosis/enzimología , Triquinelosis/metabolismoRESUMEN
Infection of mice with the nematode parasite Trichinella spiralis induces changes in the proteome of the jejunal epithelium, including substantial up-regulation of a novel variant of interlectin. In this study we sequence this novel lectin, termed intelectin-2, and compare expression levels during T. spiralis infection of resistant (BALB/c) with susceptible (C57BL/10) mouse strains. Intelectin-2 was cloned and sequenced from BALB/c mRNA extracted on day 14 of infection, and was found to have 91% amino acid identity with intelectin (within our study termed intelectin-1). Intelectin-2 transcripts were up-regulated early (day 3) during infection with T. spiralis in BALB/c mice, suggesting an innate response, and levels remained high through to day 14 (time of parasite rejection). Immunohistochemistry of jejunal sections with a rabbit polyclonal Ab to Xenopus laevis 35-kDa cortical granule lectin (XL35; 68% identity with intelectin-2) followed a similar pattern, with intense labeling of goblet and Paneth cells at day 14. However, intelectin-2 transcripts and protein were absent, and immunohistochemistry negative when C57BL/10 mice were infected with T. spiralis. Genomic PCR and Southern blotting confirmed that the intelectin-2 gene is absent from the C57BL/10 genome. The presence of intelectin-2 in resistant BALB/c mice, its absence from the susceptible C57BL/10 strain and the kinetics of its up-regulation during T. spiralis infection suggest that this novel lectin may serve a protective role in the innate immune response to parasite infection.
Asunto(s)
Células Caliciformes/metabolismo , Inmunidad Innata/genética , Lectinas/fisiología , Ratones Endogámicos BALB C/genética , Ratones Endogámicos C57BL/genética , Células de Paneth/metabolismo , Trichinella spiralis/inmunología , Triquinelosis/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Clonación Molecular , Femenino , Eliminación de Gen , Regulación de la Expresión Génica , Lectinas/biosíntesis , Lectinas/química , Lectinas/genética , Masculino , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Conejos , Alineación de Secuencia , Homología de Secuencia , Especificidad de la Especie , Transcripción Genética , Triquinelosis/genética , Triquinelosis/parasitología , Xenopus laevis/inmunologíaRESUMEN
Peak intestinal mucosal mast cell (MMC) recruitment coincides with expulsion of Trichinella spiralis, at a time when the majority of the MMCs are located within the epithelium in BALB/c mice. Although expression of integrin-alpha(E)beta(7) by MMCs has not been formally demonstrated, it has been proposed as a potential mechanism to account for the predominantly intraepithelial location of MMCs during nematode infection. Co-expression of integrin-alpha(E)beta(7) and the MMC chymase mouse mast cell protease-1, by mouse bone marrow-derived mast cells, is strictly regulated by transforming growth factor (TGF)-beta(1). However, TGF-beta(1) is secreted as part of a latent complex in vivo and subsequent extracellular modification is required to render it biologically active. We now show, for the first time, that intraepithelial MMCs express integrin-alpha(E)beta(7) in Trichinella-infected BALB/c and S129 mice. In S129 mice that lack the gene for the integrin-beta(6) subunit and, as consequence, do not express the epithelial integrin-alpha(v)beta(6), integrin-alpha(E) expression is virtually abolished and recruitment of MMCs into the intestinal epithelium is dramatically reduced despite significant overall augmentation of the MMC population. Because a major function of integrin-alpha(v)beta(6) is to activate latent TGF-beta(1,) these findings strongly support a role for TGF-beta(1) in both the recruitment and differentiation of murine MMCs during nematode infection.