RESUMEN
Background: Immediate and delayed-type hypersensitivity reactions to pet-borne allergens are common in atopic diseases. In atopic dermatitis (AD), controversy surrounds the contribution to the disease of cross-reactivity to self-proteins. Human cystatin A and the cat allergen Fel d 3 belong to the cystatins, an evolutionary conserved protein family. The objective of the present study was to assess crossreactivity between mammalian cystatins and to analyze T-cell responses to cystatin in AD patients sensitized to pet dander. Methods: cDNA coding for dog cystatin was cloned from dog skin. Sera from 245 patients with IgE-mediated sensitization to cat and dog dander were tested for IgE binding to recombinantly expressed feline, canine, and human cystatin. Of these, 141 were also diagnosed with AD. Results: Cystatin-specific IgE was detected in 36 patients (14.7%), of whom 19 were considerably affected by AD. Within the AD patients, 9 had measurable IgE against all 3 cystatins. Cystatin-sensitized AD patients did not differ from noncystatin-sensitized patients in terms of disease severity, age, or total IgE levels. T-cell cytokine measurements showed elevated IL-4 levels after stimulation with feline and human cystatin. Conclusion: The humoral response suggests that in addition to Fel d 3, the homologous protein from dog might play a role in allergy. Furthermore, human cystatin appears to be capable of driving a type 2 immune response in sensitized AD patients and may therefore be considered a so-called autoallergen, as proposed for other evolutionary conserved proteins. (AU)
Antecedentes: Las reacciones de hipersensibilidad de tipo inmediato y retardado a los alérgenos que están en las mascotas son comunes en las enfermedades atópicas. En este estudio, en pacientes con dermatitis atopica (DA), se analiza la reactividad cruzada con las autoproteínas y su contribución a la enfermedad. Tanto la cistatina A humana como el alérgeno felino Fel d 3 pertenecen a la familia de las cistatinas, una familia de proteínas conservadas evolutivamente. El objetivo del presente estudio fue evaluar la reactividad cruzada entre las cistatinas de mamíferos y analizar la respuestas de las células T a la cistatina en pacientes con DA sensibilizados a la caspa de las mascotas. Métodos: El ADNc que codifica la cistatina de perro se clonó a partir de piel de perro. Se analizaron sueros de 245 pacientes con sensibilización por IgE a la caspa de gato y perro para determinar la unión de IgE a cistatina felina, canina y humana expresada de forma recombinante, respectivamente. De estos 245 pacientes, 141 fueron diagnosticados de DA. Resultados: Se detectó IgE específica frente a cistatina en el 14,7% (36) de los pacientes, de los cuales 19 padecían DA. Dentro de los pacientes con DA, 9 tenían IgE medible contra las tres cistatinas. Los pacientes con DA sensibilizados frente a cistatina no difirieron de los pacientes no sensibilizados con cistatina en términos de gravedad de la enfermedad, edad o niveles totales de IgE. El análisis de citocinas de células T reveló niveles elevados de IL-4 después de la estimulación con cistatina felina y humana. Conclusión: La respuesta humoral sugiere que, además de Fel d 3, la proteína homóloga de perro también podría desempeñar un papel en la alergia. Además, la cistatina humana parece ser capaz de promover una respuesta inmune de tipo 2 en pacientes con DA sensibilizados y, por lo tanto, puede considerarse un autoalérgeno, como se ha propuesto para otras proteínas conservadas evolutivamente. (AU)
Asunto(s)
Humanos , Animales , Gatos , Perros , Dermatitis Atópica/etiología , Mascotas , Reactividad Cruzada , Cistatinas/inmunología , Linfocitos T/inmunología , Dermatitis Atópica/inmunología , Ensayo de Inmunoadsorción Enzimática , Electroforesis en Gel de PoliacrilamidaRESUMEN
OBJECTIVES: Trichinella spiralis is a zoonotic parasite with a complex parasitic life cycle and exposed to animals or humans by infectious meat. To control transmissions of T. spiralis through the food chain to humans, sensitive and selective multihost sera-diagnosis is urgent needed for monitoring T. spiralis exposure. METHODS: A competition enzyme-linked immunosorbent assay (cELISA) for T. spiralis infection diagnosis in multihost sera was developed based on recombinant cystatin-like protein (rCLP-cELISA) as well as monoclonal antibodies. The sensitivity and accuracy of the rCLP-cELISA were quantified using swine (n = 1316), mice (n = 189) and human (n = 157) serum samples. T. spiralis-antibody targeting test ability of the rCLP-cELISA in swine (n = 22) and human (n = 36), instead of other parasites or viruses antibodies, was evaluated. RESULTS: The rCLP-cELISA showed high agreement with commercial ELISA kits in field swine sera assessed by Cohen's kappa value (κ = 0.7963). And it showed 100% specificity in human trichinellosis detection with sensitivity of 96.49%, no cross-reaction with other parasite or virus infections, and high positive detection rate of 87.5% in low-dose infected swine. Besides, the rCLP-cELISA exhibited potential in the detection of T. spiralis, T. nelsoni and Trichinella T8 infections. CONCLUSIONS: The rCLP-cELISA can be used for T. spiralis-associated antibody test in multihost sera.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Cistatinas/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Enfermedades de los Porcinos/sangre , Trichinella spiralis/aislamiento & purificación , Triquinelosis/sangre , Animales , Cistatinas/genética , Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Humanos , Ratones , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Porcinos , Enfermedades de los Porcinos/parasitología , Trichinella spiralis/genética , Trichinella spiralis/inmunología , Triquinelosis/parasitologíaRESUMEN
Trichinella spiralis can modulate host immune responses to retain a suitable environment for its long-term survival. Incidentally, the parasite elicits regulatory effects through immunomodulatory molecule release, which can suppress host inflammation and may be used for the treatment of unrelated inflammatory diseases in someday. Here we identified and characterized a novel T. spiralis cystatin (TsCstN), which inhibits inflammation mediated by LPS-treated macrophages.Proteins contained in the excretory-secretory (ES) product of muscle-stage T. spiralis (ES-L1) were fractionated, and each was treated with mouse bone marrow-derived macrophages (mBMDMs) before LPS stimulation. The fractions that exhibited high immunomodulatory property by decreasing pro-inflammatory cytokines or increasing anti-inflammatory cytokines were identified by mass spectrometry. Incidentally, the conserved hypothetical protein (Tsp_04814) was selected for further characterization as it presented the most significant MS score. An annotation of Tsp_04814 using protein structural homology comparison suggested that it has high structural similarity to human cystatin E/M (TM score 0.690). The recombinant T. spiralis novel cystatin (rTsCstN) was expressed in Escherichia coli at a molecular weight of approximately 13 kDa. Mouse anti-rTsCstN polyclonal antibody (pAb) could detect native TsCstN in crude worm antigens (CWA) and ES-L1 and be predominantly localized in the stichosome and subcuticular cells. rTsCstN inhibited cysteine proteases in vitro, especially cathepsin L, at an optimal pH of 6. Besides, rTsCstN could be internalized into mBMDMs, which were mostly distributed in the cytoplasm and lysosome both before and after LPS stimulation. To evaluate the rTsCstN immunomodulatory properties on mBMDMs, rTsCstN was incubated with mBMDM before LPS stimulation; this demonstrated that rTsCstN suppressed pro-inflammatory cytokine production and MHC class II expression.T. spiralis L1-derived TsCstN was characterized as a novel cysteine protease inhibitor. The protein elicits an anti-inflammatory property by suppressing pro-inflammatory cytokines and interfering with the antigen presentation process through depletion of MHC class II expression.
Asunto(s)
Antígenos Helmínticos/inmunología , Cistatinas/inmunología , Citocinas/inmunología , Macrófagos/inmunología , Trichinella spiralis , Animales , Medios de Cultivo Condicionados/farmacología , Cistatinas/genética , Inhibidores de Cisteína Proteinasa , Inflamación/inducido químicamente , Inflamación/inmunología , Larva , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB CRESUMEN
Tick saliva contains immunosuppressants which are important to obtain a blood meal and enhance the infectivity of tick-borne pathogens. In Japan, Ixodes persulcatus is a major vector for Lyme borreliosis pathogens, such as Borrelia garinii, as well as for those causing relapsing fever, such as B. miyamotoi. To date, little information is available on bioactive salivary molecules, produced by this tick. Thus, in this study, we identified two proteins, I. persulcatus derived sialostatin L1 (Ip-sL1) and sL2 (Ip-sL2), as orthologs of I. scapularis derived sL1 and sL2. cDNA clones of Ip-sL1 and Ip-sL2 shared a high identity with sequences of sL1 and sL2 isolated from the salivary glands of I. scapularis. Semi-quantitative PCR revealed that Ip-sL1 and Ip-sL2 were expressed in the salivary glands throughout the life of the tick. In addition, Ip-sL1 and Ip-sL2 were expressed even before the ticks started feeding, and their expression continued during blood feeding. Recombinant Ip-sL1 and Ip-sL2 were developed to characterize the proteins via biological and immunological analyses. These analyses revealed that both Ip-sL1 and Ip-sL2â¯had inhibitory effects on cathepsins L and S. Ip-sL1 and Ip-sL2 inhibited the production of IP-10, TNFα, and IL-6 by LPS-stimulated bone-marrow-derived dendritic cells (BMDCs). Additionally, Ip-sL1 significantly impaired BMDC maturation. Taken together, these results suggest that Ip-sL1 and Ip-sL2 confer immunosuppressive functions and appear to be involved in the transmission of pathogens by suppressing host immune responses, such as cytokine production and dendritic cell maturation. Therefore, further studies are warranted to investigate the immunosuppressive functions of Ip-sL1 and Ip-sL2 in detail to clarify their involvement in pathogen transmission via I. persulcatus.
Asunto(s)
Proteínas de Artrópodos/inmunología , Cistatinas/inmunología , Inmunidad Innata/fisiología , Ixodes/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Femenino , Ratones , Ratones Endogámicos BALB C , Filogenia , Alineación de SecuenciaRESUMEN
Severe helminth infections are negatively associated to allergic diseases like asthma; therefore, the immunomodulatory properties of parasite-derived components have been analyzed, raising the possibility of their use as anti-inflammatory molecules. We evaluated the immunomodulatory properties of Ascaris lumbricoides recombinant cysteine protease inhibitor (rAl-CPI) in a mouse model of allergic airway inflammation induced by the house dust mite (HDM) Blomia tropicalis and its effects on human monocyte-derived dendritic cells (HmoDCs). The B. tropicalis sensitized/challenged mice developed extensive cellular airway inflammatory response, which was significantly reduced upon treatment with rAl-CPI prior to B. tropicalis sensitization, affecting particularly the perivascular/peribronchial infiltrate cells, eosinophils/neutrophils, and goblet cells. A significant decrease of Th2 cytokines, total, and specific IgE antibodies was observed in rAl-CPI treated mice. The antibody response was biased to IgG, mainly IgG2a. Administration of rAl-CPI-alone and rAl-CPI before mite sensitization were associated with a significant increase of regulatory T cells (Tregs) in spleen and elevated IL-10 levels in BAL and splenocytes culture supernatants, which was partially affected by anti-IL10 receptor use. In vitro, rAl-CPI showed a modulatory effect on HmoDCs, lowering the expression of HLA-DR, CD83, and CD86, while inducing IL-10 and IL-6 production. This suggests an inhibition of HmoDC maturation and a possible link with the inhibition of the allergic response observed in the murine model.
Asunto(s)
Ascaris lumbricoides/inmunología , Cistatinas/inmunología , Hipersensibilidad/inmunología , Inflamación/inmunología , Alérgenos/inmunología , Animales , Asma/inmunología , Citocinas/inmunología , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Eosinófilos/inmunología , Femenino , Humanos , Inmunoglobulina G/inmunología , Interleucina-10/inmunología , Ratones , Ratones Endogámicos BALB C , Neutrófilos/inmunología , Pyroglyphidae/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
Infection with canine heartworm (Dirofilaria immitis), spread via mosquito vectors, causes coughing, asthma, pneumonia, and bronchitis in humans and other animals. The disease is especially severe and often fatal in dogs and represents a serious threat to public health worldwide. Cysteine protease inhibitors (CPIs), also known as cystatins, are major immunomodulators of the host immune response during nematode infections. Herein, we cloned and expressed the cystatin Di-CPI from D. immitis. Sequence analysis revealed two specific cystatin-like domains, a Q-x-V-x-G motif, and a SND motif. Phylogenetic analysis indicates that Di-CPI is a member of the second subgroup of nematode type II cystatins. Probing of D. immitis total proteins with anti-rDi-CPI polyclonal antibody revealed a weak signal, and immunofluorescence-based histochemical analysis showed that native Di-CPI is mainly localized in the cuticle of male and female worms and the gut of male worms. Treatment of canine peripheral blood mononuclear cells (PMBCs) with recombinant Di-CPI induced a Th2-type immune response characterized by high expression of the anti-inflammatory factor interleukin-10. Proliferation assays showed that Di-CPI inhibits the proliferation of canine PMBCs by 15%. Together, the results indicate that Di-CPI might be related to cellular hyporesponsiveness in dirofilariasis and may help D. immitis to evade the host immune system.
Asunto(s)
Clonación Molecular/efectos de los fármacos , Cistatinas/genética , Cistatinas/metabolismo , Dirofilaria immitis/enzimología , Análisis de Secuencia de ADN/métodos , Animales , Anticuerpos/metabolismo , Células Cultivadas , Cistatinas/química , Cistatinas/inmunología , Dirofilaria immitis/genética , Dirofilaria immitis/inmunología , Perros , Femenino , Tracto Gastrointestinal/metabolismo , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Proteínas del Helminto/metabolismo , Evasión Inmune , Interleucina-10/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Masculino , Filogenia , Dominios Proteicos , Conejos , Distribución TisularRESUMEN
Helminthiasis may ameliorate inflammatory diseases, such as inflammatory bowel disease and asthma. Information about immunomodulators from Ascaris lumbricoides is scarce, but could be important considering the co-evolutionary relationships between helminths and humans. We evaluated the immunomodulatory effects of a recombinant cystatin from A. lumbricoides on an acute model of dextran sodium sulphate (DSS)-induced colitis in mice. From an A. lumbricoides cDNA library, we obtained a recombinant cystatin (rAl-CPI). Protease activity inhibition was demonstrated on cathepsin B and papain. Immunomodulatory effects were evaluated at two intraperitoneal doses (0.5 and 0.25 µg/G) on mice with DSS-induced colitis. Body weight, colon length, Disease Activity Index (DAI), histological inflammation score, myeloperoxidase (MPO) activity, gene expression of cytokines and cytokines levels in colon tissue were analysed. Treatment with rAl-CPI significantly reduced DAI, MPO activity and inflammation score without toxic effects. Also, IL-10 and TGF-B gene overexpression was observed in rAl-CPI-treated group compared to DSS-exposed control and healthy mice. Furthermore, a reduction in IL-6 and TNF-A expression was found, and this was confirmed by the levels of these cytokines in colonic tissue. In conclusion, rAl-CPI reduces inflammation in a mouse model of DSS-induced colitis, probably by increasing the expression of anti-inflammatory cytokines and reducing pro-inflammatory ones.
Asunto(s)
Colitis/inmunología , Colitis/terapia , Cistatinas/administración & dosificación , Inmunosupresores/administración & dosificación , Animales , Ascaris lumbricoides/genética , Colitis/inducido químicamente , Colitis/patología , Colon/metabolismo , Cistatinas/genética , Cistatinas/inmunología , Citocinas/análisis , Sulfato de Dextran , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunosupresores/inmunología , Inflamación/patología , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéuticoRESUMEN
In the present study a thiol proteinase inhibitor was isolated from buffalo kidney making use of ammonium sulphate precipitation and gel filtration chromatography on Sephacryl S-100HR column. Purified inhibitor is homogeneous as it displayed a single band in gel electrophoresis both under reducing and non-reducing environment and is of 65KDa as revealed by gel filtration and SDS PAGE. Kinetic studies revealed the presence of reversible accompanied with competitive mode of inhibition; showing maximum efficacy against papain (Ki=2.90×10-4). It was maximally active at pH 8.0 and was stable for a period of 30, 60 and 90 days at 37, 4 and -20°C respectively. Immunological studies confirmed its purity of epitopes as a single precipitin line is obtained in immunodiffusion. N-terminal analysis revealed that it shared a good homology with mouse kidney cystatin as well as with Human Cys C and Cys E thereby advocating its use as a model for various human oriented studies which targets how the kidney cystatin level varies in accordance with various drugs that are currently being used as a target for variety of diseases.
Asunto(s)
Cistatinas/química , Riñón/química , Papaína/química , Inhibidores de Proteasas/química , Compuestos de Sulfhidrilo/química , Secuencia de Aminoácidos , Animales , Bromelaínas/antagonistas & inhibidores , Bromelaínas/química , Búfalos , Cistatinas/inmunología , Cistatinas/aislamiento & purificación , Ficaína/antagonistas & inhibidores , Ficaína/química , Humanos , Concentración de Iones de Hidrógeno , Riñón/inmunología , Cinética , Ratones , Peso Molecular , Papaína/antagonistas & inhibidores , Inhibidores de Proteasas/inmunología , Inhibidores de Proteasas/aislamiento & purificación , Estabilidad Proteica , Alineación de SecuenciaRESUMEN
This study describes the isolation and purification of a phytocystatin from seeds of Brassica juncea (Indian mustard; cultivar RoAgro 5444), which is an important oilseed crop both agriculturally and economically. The protein was purified by gel filtration chromatography with 24.3% yield and 204-fold purification, and visualised by 2D gel electrophoresis. The 18.1 kDa mustard cystatin was highly specific for cysteine proteinases. The plant cystatin inhibited cathepsin B, confirming its role in conferring pest resistance. The inhibitor was highly stable over a pH range of 3-10 and retained significant inhibitory potential up to 70 °C. The stoichiometry of its interaction with papain, determined by isothermal calorimetry, suggests a 1:1 complex. Secondary structural elements calculated by far-UV circular dichroism (CD) spectroscopy show an 18.8% α-helical and 21% ß-sheet structure. The protein was a non-competitive inhibitor of thiol proteinases. The Stokes radius and frictional co-efficient were used to describe the shape and size of the protein. Homology modelling and docking studies proposed a prototype illustrating the Brassica phytocystatin mediated papain inhibition. Molecular dynamics (MD) study revealed the excellent stability of the papain-phytocystatin complex during a simulation for 100 ns. Detailed results identify the mustard cystatin as an important member of the phytocystatin family.
Asunto(s)
Cistatinas/química , Cistatinas/metabolismo , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/metabolismo , Planta de la Mostaza/metabolismo , Animales , Formación de Anticuerpos , Cromatografía en Gel , Simulación por Computador , Cistatinas/inmunología , Cistatinas/aislamiento & purificación , Inhibidores de Cisteína Proteinasa/inmunología , Inhibidores de Cisteína Proteinasa/aislamiento & purificación , Inmunoglobulina G/inmunología , Cinética , Masculino , Modelos Moleculares , Simulación de Dinámica Molecular , Planta de la Mostaza/crecimiento & desarrollo , Papaína/metabolismo , Proteínas de Plantas/inmunología , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , ConejosRESUMEN
Respiratory Syncytial Virus (RSV) is a major pathogen causing low respiratory tract disease (bronchiolitis), primarily in infants. Helminthic infections may alter host immune responses to both helminths and to unrelated immune triggers. For example, we have previously shown that filarial cystatin (AvCystatin/Av17) ameliorates allergic airway inflammation. However, helminthic immunomodulators have so far not been tested in virus-induced disease. We now report that AvCystatin prevents Th2-based immunopathology in vaccine-enhanced RSV lung inflammation, a murine model for bronchiolitis. AvCystatin ablated eosinophil influx, reducing both weight loss and neutrophil recruitment without impairing anti-viral immune responses. AvCystatin also protected mice from excessive inflammation following primary RSV infection, significantly reducing neutrophil influx and cytokine production in the airways. Interestingly, we found that AvCystatin induced an influx of CD4+ FoxP3+ interleukin-10-producing T cells in the airway and lungs, correlating with immunoprotection, and the corresponding cells could also be induced by adoptive transfer of AvCystatin-primed F4/80+ macrophages. Thus, AvCystatin ameliorates enhanced RSV pathology without increasing susceptibility to, or persistence of, viral infection and warrants further investigation as a possible therapy for virus-induced airway disease.
Asunto(s)
Cistatinas/inmunología , Proteínas del Helminto/inmunología , Inflamación/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/inmunología , Linfocitos T Reguladores/inmunología , Animales , Bronquiolitis/complicaciones , Bronquiolitis/inmunología , Bronquiolitis/prevención & control , Línea Celular Tumoral , Cistatinas/farmacología , Modelos Animales de Enfermedad , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Eosinófilos/metabolismo , Citometría de Flujo , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Proteínas del Helminto/farmacología , Humanos , Factores Inmunológicos/farmacología , Inflamación/complicaciones , Inflamación/prevención & control , Interleucina-10/inmunología , Interleucina-10/metabolismo , Ratones , Infecciones por Virus Sincitial Respiratorio/complicaciones , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/fisiología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismoRESUMEN
Recombinant SjCystatin (rSjCystatin), a recombinant protein of Schistosoma japonicum cystatin, has been reported to have an effect on immunoregulation mediated by IL-10 induction. Rheumatoid arthritis (RA) is a common autoimmune inflammatory arthropathy, and recombinant immune-modulating drugs for RA treatment are under development. We aimed to study the putative immune regulation of rSjCystatin and its prophylactic/therapeutic effects on murine collagen-induced arthritis (CIA). CIA was induced in DBA/1 mice by inoculation with bovine collagen II (CII). rSjCystatin was administered prior or post development of CIA. The severity of CIA was assessed using established clinical and histopathological scoring systems. The incidence was also determined. The CII-specific antibodies in sera and cytokines in splenocyte culture supernatants were measured by ELISA. Th1/Th2/Th17 cells and Tregs development in splenocytes were monitored by flow cytometry. The inflammatory mediators in the diseased joint were semiquantitated by qPCR. Prophylactic injection of rSjCystatin attenuated paw clinical scores, incidence, and histopathology scores of joints in CIA mice. The arthritis-alleviative effects were closely associated with the augmentation of IL-4, IL-10, and collagen-specific IgG1, and with the distinct reduction of IFN-γ, collagen-specific IgG2a, and the marked decrease of proinflammatory cytokines IL-6, IL-17, and TNF-α and RANKL. The data indicate that rSjCystatin may prevent cartilage destruction and inflammation of joints in CIA mice. The effects are related to the inhibitory modulation of Th1 and Th17 and upregulation of Tregs and Th2 via a shift of cytokines profiling from Th1 to Th2 response.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/inmunología , Colágeno Tipo II/efectos adversos , Cistatinas/administración & dosificación , Proteínas del Helminto/administración & dosificación , Schistosoma japonicum/inmunología , Animales , Artritis Reumatoide/genética , Bovinos , Colágeno Tipo II/inmunología , Cistatinas/inmunología , Proteínas del Helminto/inmunología , Humanos , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Interleucina-6/inmunología , Masculino , Ratones , Ratones Endogámicos DBA , Schistosoma japonicum/química , Células Th17/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Eosinophils are now recognized as multifunctional leukocytes that provide critical homeostatic signals to maintain other immune cells and aid tissue repair. Paradoxically, eosinophils also express an armory of granule-localized toxins and hydrolases believed to contribute to pathology in inflammatory disease. How eosinophils deliver their supporting functions while avoiding self-inflicted injury is poorly understood. We have demonstrated that cystatin F (CF) is a critical survival factor for eosinophils. Eosinophils from CF null mice had reduced lifespan, reduced granularity, and disturbed granule morphology. In vitro, cysteine protease inhibitors restored granularity, demonstrating that control of cysteine protease activity by CF is critical for normal eosinophil development. CF null mice showed reduced pulmonary pathology in a model of allergic lung inflammation but also reduced ability to combat infection by the nematode Brugia malayi. These data identify CF as a "cytoprotectant" that promotes eosinophil survival and function by ensuring granule integrity. VIDEO ABSTRACT.
Asunto(s)
Brugia Malayi/inmunología , Supervivencia Celular/inmunología , Cistatinas/genética , Cistatinas/inmunología , Gránulos Citoplasmáticos/metabolismo , Eosinófilos/inmunología , Filariasis/inmunología , Animales , Supervivencia Celular/genética , Células Cultivadas , Proteasas de Cisteína/metabolismo , Filariasis/parasitología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/inmunologíaRESUMEN
Tick saliva contains a number of effector molecules that inhibit host immunity and facilitate pathogen transmission. How tick proteins regulate immune signaling, however, is incompletely understood. Here, we describe that loop 2 of sialostatin L2, an anti-inflammatory tick protein, binds to annexin A2 and impairs the formation of the NLRC4 inflammasome during infection with the rickettsial agent Anaplasma phagocytophilum Macrophages deficient in annexin A2 secreted significantly smaller amounts of interleukin-1ß (IL-1ß) and IL-18 and had a defect in NLRC4 inflammasome oligomerization and caspase-1 activation. Accordingly, Annexin a2-deficient mice were more susceptible to A. phagocytophilum infection and showed splenomegaly, thrombocytopenia, and monocytopenia. Providing translational support to our findings, better binding of annexin A2 to sialostatin L2 in sera from 21 out of 23 infected patients than in sera from control individuals was also demonstrated. Overall, we establish a unique mode of inflammasome evasion by a pathogen, centered on a blood-feeding arthropod.
Asunto(s)
Anaplasma phagocytophilum/inmunología , Anexina A2/inmunología , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas de Unión al Calcio/inmunología , Cistatinas/inmunología , Ehrlichiosis/microbiología , Evasión Inmune , Secuencia de Aminoácidos , Anaplasma phagocytophilum/genética , Animales , Anexina A2/química , Anexina A2/genética , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Vectores Arácnidos/química , Vectores Arácnidos/genética , Vectores Arácnidos/inmunología , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Caspasa 1/genética , Caspasa 1/inmunología , Caspasas/genética , Caspasas/inmunología , Caspasas Iniciadoras , Cistatinas/química , Cistatinas/genética , Ehrlichiosis/inmunología , Ehrlichiosis/patología , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Humanos , Inflamasomas/genética , Inflamasomas/inmunología , Interleucina-18/genética , Interleucina-18/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Ixodes/química , Ixodes/genética , Ixodes/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Modelos Moleculares , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Transducción de SeñalRESUMEN
Cystatin F, a member of the family II cystatins, plays important roles in immune response-related processes through inhibiting specific enzyme targets. In this study, a cystatin F homologue, LycCysF, was identified and characterized from large yellow croaker (Larimichthys crocea). The deduced LycCysF protein exhibits a typical structural feature of type II cystatins, including three evolutionally conserved motifs, Gly(35), QVVRG(79-83) and PW(130-131). Tissue expression analysis showed that LycCysF mRNA was expressed in all tissues examined, albeit at different levels. Recombinant LycCysF (rLycCysF) produced in Pichia pastoris could inhibit the activity of multiple cysteine proteases, including papain, legumain and recombinant large yellow croaker cathepsin B, L and S. Moreover, rLycCysF could inhibit the Ii chain processing by recombinant cathepsin S in vitro. These data suggest that LycCysF may participate in regulation of cathepsins and MHC-II associated Ii chain processing. In addition, mammalian cystatin F is produced as an inactive dimer, becoming activated by proteolysis in the endo/lysosome of immune cells and then exerts its function of regulating downstream proteases activity. However, the N-terminal extension and two additional cysteine residues responsible for dimer formation are absent in LycCysF and cystatin F from other fish species, reptiles and Aves, indicating that these proteins can not form dimer and may regulate the proteases activity via an alternate pathway distinct from mammalian cystatin F. To our knowledge, this is the first report on molecular characteristics of a teleost cystatin F and its role in Ii chain processing.
Asunto(s)
Cistatinas , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas de Peces , Perciformes , Aeromonas hydrophila , Animales , Presentación de Antígeno , Encéfalo/metabolismo , Cistatinas/química , Cistatinas/genética , Cistatinas/inmunología , Proteasas de Cisteína/metabolismo , ADN Complementario/genética , Enfermedades de los Peces/genética , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Genes MHC Clase II/inmunología , Branquias/metabolismo , Infecciones por Bacterias Gramnegativas/genética , Infecciones por Bacterias Gramnegativas/veterinaria , Mucosa Intestinal/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Músculos/metabolismo , Perciformes/genética , Perciformes/inmunología , Perciformes/metabolismo , ARN Mensajero/sangre , ARN Mensajero/metabolismo , Piel/metabolismo , Bazo/metabolismoRESUMEN
Freshly isolated human primary NK cells induce preferential lysis of Oral Squamous Carcinoma Stem Cells (OSCSCs) when compared to differentiated Oral Squamous Carcinoma Cells (OSCCs), while anti-CD16 antibody and monocytes induce functional split anergy in primary NK cells by decreasing the cytotoxic function of NK cells and increasing the release of IFN-γ. Since NK92 cells have relatively lower levels of cytotoxicity when compared to primary NK cells, and have the ability to increase secretion of regulatory cytokines IL-10 and IL-6, we used these cells as a model of NK cell anergy to identify and to study the upstream regulators of anergy. We demonstrate in this paper that the levels of truncated monomeric cystatin F, which is known to inhibit the functions of cathepsins C and H, is significantly elevated in NK92 cells and in anergized primary NK cells. Furthermore, cystatin F co-localizes with cathepsins C and H in the lysosomal/endosomal vesicles of NK cells. Accordingly, the mature forms of aminopeptidases cathepsins C and H, which regulate the activation of effector granzymes in NK cells, are significantly decreased, whereas the levels of pro-cathepsin C enzyme is increased in anergized NK cells after triggering of the CD16 receptor. In addition, the levels of granzyme B is significantly decreased in anti-CD16mAb and target cell anergized primary NK cells and NK92 cells. Our study provides the cellular and molecular mechanisms by which target cells may utilize to inhibit the cytotoxic function of NK cells.
Asunto(s)
Biomarcadores de Tumor/inmunología , Catepsina C/inmunología , Catepsina H/inmunología , Cistatinas/inmunología , Células Asesinas Naturales/inmunología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Anergia Clonal , Humanos , Células Asesinas Naturales/citologíaRESUMEN
Coevolution of ticks and the vertebrate immune system has led to the development of immunosuppressive molecules that prevent immediate response of skin-resident immune cells to quickly fend off the parasite. In this article, we demonstrate that the tick-derived immunosuppressor sialostatin L restrains IL-9 production by mast cells, whereas degranulation and IL-6 expression are both unaffected. In addition, the expression of IL-1ß and IRF4 is strongly reduced in the presence of sialostatin L. Correspondingly, IRF4- or IL-1R-deficient mast cells exhibit a strong impairment in IL-9 production, demonstrating the importance of IRF4 and IL-1 in the regulation of the Il9 locus in mast cells. Furthermore, IRF4 binds to the promoters of Il1b and Il9, suggesting that sialostatin L suppresses mast cell-derived IL-9 preferentially by inhibiting IRF4. In an experimental asthma model, mast cell-specific deficiency in IRF4 or administration of sialostatin L results in a strong reduction in asthma symptoms, demonstrating the immunosuppressive potency of tick-derived molecules.
Asunto(s)
Cistatinas/farmacología , Inmunidad Innata/efectos de los fármacos , Inmunosupresores/farmacología , Factores Reguladores del Interferón/inmunología , Interleucina-9/inmunología , Mastocitos/efectos de los fármacos , Animales , Asma/genética , Asma/inmunología , Asma/patología , Sitios de Unión , Degranulación de la Célula/inmunología , Cistatinas/inmunología , Regulación de la Expresión Génica , Interacciones Huésped-Parásitos/inmunología , Factores Reguladores del Interferón/deficiencia , Factores Reguladores del Interferón/genética , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Interleucina-9/antagonistas & inhibidores , Interleucina-9/genética , Mastocitos/inmunología , Mastocitos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras Genéticas , Unión Proteica , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/inmunología , Transducción de Señal , Transcripción GenéticaRESUMEN
BACKGROUND: Cystatins are a group of cysteine protease inhibitors responsible for physiological proteolysis regulation and present in a wide range of organisms. Studies about this class of inhibitors in parasites have contributed to clarify their roles in important physiological processes, like blood digestion and modulation of host immune response during blood feeding. Thus, cystatins are a subject of research on the development of new parasite control methods. Additionally, the characterization of proteins shared by different parasite species represents a valuable strategy to find potential targets in multi-species control methods. However, cystatin functions in ticks remain undetermined, especially in Rhipicephalus microplus and Ixodes ovatus, two species that affect livestock and human health, respectively. METHODS: Here we report the inhibitory profile of two R. microplus (BrBmcys2b and BrBmcys2c) and one I. ovatus (JpIocys2a) cystatins to commercial cathepsins B, C, and L. The presence of native cystatins in R. microplus tissues was analyzed using sera against recombinant BrBmcys2b and BrBmcys2c. Also, a peptide from JpIocys2a was synthesized for rabbit immunization, and this serum was used to analyze the cross antigenicity between R. microplus and I. ovatus cystatins. RESULTS: Enzymatic inhibition profile of tick cystatins shows a distinct modulation for cathepsins related to tick blood digestion and evasion of host immune response. Furthermore, BrBmcys2b was detected in saliva and different tissues along tick stages, while BrBmcys2c was detected mainly in gut from partially engorged R. microplus females, demonstrating a distinct pattern of cystatin expression, secretion and traffic between tick tissues. Moreover, phylogenetic analysis suggests that JpIocys2a belongs to the group of tick gut secreted cystatins. Finally, cross-antigenicity assays revealed that antibodies against the JpIocys2a peptide recognize native and recombinant R. microplus cystatins. CONCLUSION: The presence of these proteins in different tissues and their ability to differentially inhibit cathepsins suggest distinct roles for JpIocys2a, BrBmcys2b, and BrBmcys2c in blood digestion, egg and larvae development, and modulation of host immune response in tick physiology. The cross-antigenicity between native and recombinant cystatins supports further experiments using JpIocys2a, BrBmcys2b, and BrBmcys2c as vaccine antigens.
Asunto(s)
Cistatinas/inmunología , Inmunización/veterinaria , Ixodes/inmunología , Rhipicephalus/inmunología , Secuencia de Aminoácidos , Animales , Sangre , Cricetinae , Reacciones Cruzadas , Digestión , Femenino , Interacciones Huésped-Parásitos , Humanos , Ixodes/genética , Ixodes/fisiología , Masculino , Datos de Secuencia Molecular , Filogenia , Conejos , Proteínas Recombinantes , Rhipicephalus/genética , Rhipicephalus/fisiología , Alineación de SecuenciaRESUMEN
BACKGROUND: The intestinal phase is the early invasion stage of Trichinella spiralis (T. spiralis), in which muscle larvae invade intestine epithelial cells and then develop into adult worms to breed newborn larvae. Thus, intestinal infective larvae are first exposed to the immune system of the host, and antigens from the worms may be the earliest marker in the diagnosis of trichinellosis and may contribute to vaccine development to prevent Trichinella infections in pigs. METHODS: A cDNA library of intestinal infective larvae of T. spiralis at 6 hours post infection (p.i.) was constructed and immunoscreened using serum collected from pigs that were infected with T. spiralis at 26 days p.i. T. spiralis cystatin-like protein (Ts-CLP) gene encoding a 45.9 kDa protein was cloned and expressed in Escherichia coli. The rabbit antisera were generated and used to determine the location of Ts-CLP in the parasite. Transcription levels of Ts-CLP in different developmental stages of T. spiralis were observed by RT-PCR. The potential application of recombinant Ts-CLP in diagnosis against T. spiralis infection was tested by ELISA. The immune protection of recombinant Ts-CLP protein against T. spiralis infection was evaluated in mice. RESULTS: Thirty-three positive clones were selected from cDNA library, among which 20 clones encoded the same novel cystatin-like protein (Ts-CLP). Immunolocalisation and real-time quantitative PCR revealed that native Ts-CLP was localised primarily to ß-stichocytes and that the Ts-clp gene was transcribed and expressed in all developmental stages of T. spiralis. The recombinant protein rTs-CLP was recognised by pig antiserum as early as 15 days p.i., and could induce protective immunity in mice, with a 61.21% reduction in the number of muscle larvae. CONCLUSIONS: These data preliminarily suggested that Ts-CLP may play an important role in the early infection of T. spiralis and that recombinant Ts-CLP protein is a candidate antigen for diagnosis and vaccine development in Trichinella infections.
Asunto(s)
Antígenos Helmínticos/inmunología , Cistatinas/inmunología , Enfermedades de los Porcinos/parasitología , Trichinella spiralis/inmunología , Trichinella spiralis/patogenicidad , Triquinelosis/veterinaria , Animales , Antígenos Helmínticos/genética , Cistatinas/genética , Femenino , Ratones , Ratones Endogámicos BALB C , Conejos , Ratas , Ratas Wistar , Porcinos , Enfermedades de los Porcinos/inmunología , Trichinella spiralis/genética , Trichinella spiralis/crecimiento & desarrollo , Triquinelosis/inmunología , Triquinelosis/parasitología , VirulenciaRESUMEN
Epidemiological surveys have demonstrated that helminth infections are negatively related to atopic diseases, including asthma. Defining and characterising specific helminth molecules that have excellent immunomodulatory capacities as potential therapeutics for the treatment or prophylaxis of allergic manifestations are of great interest. AcCystatin, a cystatin protease inhibitor of Angiostrongylus cantonensis, is a homologue of other nematode cystatins with immunoregulatory properties. Here, we aim to determine the effects of AcCystatin on an ovalbumin/aluminium hydroxide (OVA/Al[OH]3)-induced rat model of asthma. Wistar rats were randomly divided into four groups, including a control group, an OVA/Al[OH]3-induced asthma group, a group receiving AcCystatin immunisation prior to OVA/Al[OH]3-induced asthma and a group receiving AcCystatin treatment after OVA/Al[OH]3-induced asthma. The numbers of eosinophils, basophils, neutrophils, lymphocytes and monocytes in the peripheral blood and of eosinophils in the bronchoalveolar lavage fluid (BALF) were counted for each animal. The expression levels of the cytokines interferon-γ, interleukin (IL) 4, IL-5, IL-6, IL-10, IL17A and tumour necrosis factor receptor-α in BALF, of OVA-specific immunoglobulin E in BALF and serum and of the chemokines eotaxin-1, eotaxin-2, eotaxin-3, MCP-1 and MCP-3 in lung tissue were measured. In addition, the degree of peribronchial and perivascular inflammation and the intensity of goblet cell metaplasia were qualitatively evaluated. The sensitised/challenged rats developed an extensive cell inflammatory response of the airways. AcCystatin administration significantly reduced the cellular infiltrate in the perivascular and peribronchial lung tissues and reduced both goblet mucous production and eosinophil infiltration. The rats that were treated with AcCystatin before or after sensitisation with OVA showed significant decreases in eotaxin-1, eotaxin-3 and MCP-1 expression in the lung tissue. The production of IL-4, IL-5, IL-6 and IL-17A and of OVA-specific IgE antibodies was also significantly reduced in AcCystatin-treated rats compared with untreated asthmatic rats. The AcCystatin treatment was associated with a significant increase in IL-10 levels. Our present findings provide the first demonstration that AcCystatin is an effective agent in the prevention and treatment of the airway inflammation associated with asthma.