RESUMO
Aging is linked to greater susceptibility to chronic inflammatory diseases, several of which, including periodontitis, involve neutrophil-mediated tissue injury. Here we found that aging-associated periodontitis was accompanied by lower expression of Del-1, an endogenous inhibitor of neutrophil adhesion dependent on the integrin LFA-1, and by reciprocal higher expression of interleukin 17 (IL-17). Consistent with that, IL-17 inhibited gingival endothelial cell expression of Del-1, thereby promoting LFA-1-dependent recruitment of neutrophils. Young Del-1-deficient mice developed spontaneous periodontitis that featured excessive neutrophil infiltration and IL-17 expression; disease was prevented in mice doubly deficient in Del-1 and LFA-1 or in Del-1 and the IL-17 receptor. Locally administered Del-1 inhibited IL-17 production, neutrophil accumulation and bone loss. Therefore, Del-1 suppressed LFA-1-dependent recruitment of neutrophils and IL-17-triggered inflammatory pathology and may thus be a promising therapeutic agent for inflammatory diseases.
Assuntos
Perda do Osso Alveolar/imunologia , Proteínas de Transporte/metabolismo , Interleucina-17/antagonistas & inibidores , Interleucina-17/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Periodontite/metabolismo , Envelhecimento/imunologia , Animais , Proteínas de Ligação ao Cálcio , Proteínas de Transporte/imunologia , Proteínas de Transporte/farmacologia , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Feminino , Integrinas/antagonistas & inibidores , Integrinas/imunologia , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-17/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Atrofia Periodontal/imunologia , Atrofia Periodontal/metabolismo , Periodontite/imunologia , Periodontite/terapia , Receptores de Interleucina-17/deficiência , Receptores de Interleucina-17/metabolismoRESUMO
Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity.
Assuntos
Cistatinas , Ixodes , Animais , Cistatinas Salivares/química , Peptídeo Hidrolases/metabolismo , Cisteína/metabolismo , Cistatinas/farmacologia , Ixodes/química , Vertebrados , Catepsinas/metabolismo , Endopeptidases/metabolismoRESUMO
Pancreatic adenocarcinoma is one of the leading causes of cancer-related deaths, and its therapy remains a challenge. Our proposed therapeutic approach is based on the intratumoral injections of mannan-BAM, toll-like receptor ligands, and anti-CD40 antibody (thus termed MBTA therapy), and has shown promising results in the elimination of subcutaneous murine melanoma, pheochromocytoma, colon carcinoma, and smaller pancreatic adenocarcinoma (Panc02). Here, we tested the short- and long-term effects of MBTA therapy in established subcutaneous Panc02 tumors two times larger than in previous study and bilateral Panc02 models as well as the roles of CD4+ and CD8+ T lymphocytes in this therapy. The MBTA therapy resulted in eradication of 67% of Panc02 tumors with the development of long-term memory as evidenced by the rejection of Panc02 cells after subcutaneous and intracranial transplantations. The initial Panc02 tumor elimination is not dependent on the presence of CD4+ T lymphocytes, although these cells seem to be important in long-term survival and resistance against tumor retransplantation. The resistance was revealed to be antigen-specific due to its inability to reject B16-F10 melanoma cells. In the bilateral Panc02 model, MBTA therapy manifested a lower therapeutic response. Despite numerous combinations of MBTA therapy with other therapeutic approaches, our results show that only simultaneous application of MBTA therapy into both tumors has potential for the treatment of the bilateral Panc02 model.
Assuntos
Adenocarcinoma/patologia , Antígenos CD40/antagonistas & inibidores , Imidazóis/farmacologia , Lipopolissacarídeos/farmacologia , Mananas/farmacologia , Neoplasias Pancreáticas/patologia , Poli I-C/farmacologia , Ácidos Teicoicos/farmacologia , Adenocarcinoma/imunologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Imunoterapia , Ligantes , Camundongos , Neoplasias Pancreáticas/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Receptores Toll-Like , Neoplasias PancreáticasRESUMO
In Type 1 Diabetes Mellitus (T1DM), leukocyte infiltration of the pancreatic islets and the resulting immune-mediated destruction of beta cells precede hyperglycemia and clinical disease symptoms. In this context, the role of the pancreatic endothelium as a barrier for autoimmunity- and inflammation-related destruction of the islets is not well studied. Here, we identified Robo4, expressed on endothelial cells, as a regulator of pancreatic vascular endothelial permeability during autoimmune diabetes. Circulating levels of Robo4 were upregulated in mice subjected to the Multiple Low-Dose Streptozotocin (MLDS) model of diabetes. Upon MLDS induction, Robo4-deficiency resulted in increased pancreatic vascular permeability, leukocyte infiltration to the islets and islet apoptosis, associated with reduced insulin levels and faster diabetes development. On the contrary, in vivo administration of Slit2 in mice modestly delayed the emergence of hyperglycaemia and ameliorated islet inflammation in MLDS-induced diabetes. Thus, Robo4-mediated endothelial barrier integrity reduces insulitis and islet destruction in autoimmune diabetes. Our findings highlight the importance of the endothelium as gatekeeper of pancreatic inflammation during T1DM development and may pave the way for novel Robo4-related therapeutic approaches for autoimmune diabetes.
Assuntos
Permeabilidade Capilar , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Células Endoteliais/metabolismo , Células Secretoras de Insulina/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Apoptose , Linhagem Celular , Células Cultivadas , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/patologia , Células Endoteliais/patologia , Humanos , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/sangue , Receptores de Superfície Celular/genéticaRESUMO
'Omics' technologies have facilitated the identification of hundreds to thousands of tick molecules that mediate tick feeding and play a role in the transmission of tick-borne diseases. Deep sequencing methodologies have played a key role in this knowledge accumulation, profoundly facilitating the study of the biology of disease vectors lacking reference genomes. For example, the nucleotide sequences of the entire set of tick salivary effectors, the so-called tick 'sialome', now contain at least one order of magnitude more transcript sequences compared to similar projects based on Sanger sequencing. Tick feeding is a complex and dynamic process, and while the dynamic 'sialome' is thought to mediate tick feeding success, exactly how transcriptome dynamics relate to tick-host-pathogen interactions is still largely unknown. The identification and, importantly, the functional analysis of the tick 'sialome' is expected to shed light on this 'black box'. This information will be crucial for developing strategies to block pathogen transmission, not only for anti-tick vaccine development but also the discovery and development of new, pharmacologically active compounds for human diseases.
Assuntos
Proteômica , Glândulas Salivares/fisiologia , Carrapatos/fisiologia , Transcriptoma/fisiologia , Animais , Genoma/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Carrapatos/genéticaRESUMO
The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.
Assuntos
Proteínas Sanguíneas/metabolismo , Cistatinas/metabolismo , Sistema Digestório/metabolismo , Ixodes/metabolismo , Carrapatos/metabolismo , Sequência de Aminoácidos , Animais , Catepsina L/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , ProteóliseRESUMO
Protease inhibitors (PIs) are ubiquitous regulatory proteins present in all kingdoms. They play crucial tasks in controlling biological processes directed by proteases which, if not tightly regulated, can damage the host organism. PIs can be classified according to their targeted proteases or their mechanism of action. The functions of many PIs have now been characterized and are showing clinical relevance for the treatment of human diseases such as arthritis, hepatitis, cancer, AIDS, and cardiovascular diseases, amongst others. Other PIs have potential use in agriculture as insecticides, anti-fungal, and antibacterial agents. PIs from tick salivary glands are special due to their pharmacological properties and their high specificity, selectivity, and affinity to their target proteases at the tick-host interface. In this review, we discuss the structure and function of PIs in general and those PI superfamilies abundant in tick salivary glands to illustrate their possible practical applications. In doing so, we describe tick salivary PIs that are showing promise as drug candidates, highlighting the most promising ones tested in vivo and which are now progressing to preclinical and clinical trials.
Assuntos
Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/uso terapêutico , Saliva/metabolismo , Animais , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Humanos , Saliva/química , Glândulas Salivares/metabolismo , Carrapatos/metabolismo , Transcriptoma/genéticaRESUMO
Tick saliva is a rich source of antihemostatic, anti-inflammatory, and immunomodulatory molecules that actively help the tick to finish its blood meal. Moreover, these molecules facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-8, a salivary serpin from the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Iripin-8 displayed blood-meal-induced mRNA expression that peaked in nymphs and the salivary glands of adult females. Iripin-8 inhibited multiple proteases involved in blood coagulation and blocked the intrinsic and common pathways of the coagulation cascade in vitro. Moreover, Iripin-8 inhibited erythrocyte lysis by complement, and Iripin-8 knockdown by RNA interference in tick nymphs delayed the feeding time. Finally, we resolved the crystal structure of Iripin-8 at 1.89 Å resolution to reveal an unusually long and rigid reactive center loop that is conserved in several tick species. The P1 Arg residue is held in place distant from the serpin body by a conserved poly-Pro element on the P' side. Several PEG molecules bind to Iripin-8, including one in a deep cavity, perhaps indicating the presence of a small-molecule binding site. This is the first crystal structure of a tick serpin in the native state, and Iripin-8 is a tick serpin with a conserved reactive center loop that possesses antihemostatic activity that may mediate interference with host innate immunity.
Assuntos
Coagulação Sanguínea/fisiologia , Ativação do Complemento/fisiologia , Ixodes/metabolismo , Serpinas/metabolismo , Animais , Proteínas de Artrópodes/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Ativação do Complemento/efeitos dos fármacos , Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Eritrócitos/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Ixodes/enzimologia , Ixodes/genética , Doença de Lyme , Ninfa , Saliva/química , Glândulas Salivares/metabolismo , Serpinas/ultraestruturaRESUMO
To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Šresolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.
Assuntos
Proteínas de Artrópodes/farmacologia , Cistatinas/farmacologia , Imunossupressores/farmacologia , Cistatinas Salivares/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Cristalografia por Raios X , Cistatinas/classificação , Cistatinas/genética , Citocinas/metabolismo , Compostos de Epóxi/metabolismo , Feminino , Imunossupressores/química , Imunossupressores/metabolismo , Ixodes/química , Ixodes/genética , Ixodes/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Filogenia , Proteólise/efeitos dos fármacos , Cistatinas Salivares/química , Cistatinas Salivares/genética , Homologia de Sequência de Aminoácidos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
BACKGROUND: Using killed microorganisms or their parts to stimulate immunity for cancer treatment dates back to the end of 19th century. Since then, it undergone considerable development. Our novel approach binds ligands to the tumor cell surface, which stimulates tumor phagocytosis. The therapeutic effect is further amplified by simultaneous application of agonists of Toll-like receptors. We searched for ligands that induce both a strong therapeutic effect and are safe for humans. METHODS: B16-F10 murine melanoma model was used. For the stimulation of phagocytosis, mannan or N-formyl-methionyl-leucyl-phenylalanine, was covalently bound to tumor cells or attached using hydrophobic anchor. The following agonists of Toll-like receptors were studied: monophosphoryl lipid A (MPLA), imiquimod (R-837), resiquimod (R-848), poly(I:C), and heat killed Listeria monocytogenes. RESULTS: R-848 proved to be the most suitable Toll-like receptor agonist for our novel immunotherapeutic approach. In combination with covalently bound mannan, R-848 significantly reduced tumor growth. Adding poly(I:C) and L. monocytogenes resulted in complete recovery in 83% of mice and in their protection from the re-transplantation of melanoma cells. CONCLUSION: An efficient cancer treatment results from the combination of Toll-like receptor agonists and phagocytosis stimulating ligands bound to the tumor cells.
Assuntos
Imunidade Inata , Imunoterapia , Neoplasias/imunologia , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Imidazóis/farmacologia , Imunoterapia/métodos , Ligantes , Mananas/imunologia , Melanoma Experimental , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagocitose , Poli I-C/imunologia , Explosão Respiratória/imunologia , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismoRESUMO
Th17 cells constitute a subset of CD4(+) T lymphocytes that play a crucial role in protection against extracellular bacteria and fungi. They are also associated with tissue injury in autoimmune and inflammatory diseases. Here, we report that serpin from the tick Ixodes ricinus, IRS-2, inhibits Th17 differentiation by impairment of the interleukin-6 (IL-6)/STAT-3 signaling pathway. Following activation, mature dendritic cells produce an array of cytokines, including the pleiotropic cytokine IL-6, which triggers the IL-6 signaling pathway. The major transcription factor activated by IL-6 is STAT-3. We show that IRS-2 selectively inhibits production of IL-6 in dendritic cells stimulated with Borrelia spirochetes, which leads to attenuated STAT-3 phosphorylation and finally to impaired Th17 differentiation. The results presented extend the knowledge about the effect of tick salivary serpins on innate immunity cells and their function in driving adaptive immune responses.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Interleucina-6/antagonistas & inibidores , Fator de Transcrição STAT3/antagonistas & inibidores , Serpinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Animais , Borrelia/imunologia , Células Dendríticas/fisiologia , Feminino , Interleucina-6/metabolismo , Ixodes , Camundongos Endogâmicos C57BL , Fator de Transcrição STAT3/metabolismo , Células Th17/fisiologiaRESUMO
Platelet aggregation and acute inflammation are key processes in vertebrate defense to a skin injury. Recent studies uncovered the mediation of 2 serine proteases, cathepsin G and chymase, in both mechanisms. Working with a mouse model of acute inflammation, we revealed that an exogenous salivary protein of Ixodes ricinus, the vector of Lyme disease pathogens in Europe, extensively inhibits edema formation and influx of neutrophils in the inflamed tissue. We named this tick salivary gland secreted effector as I ricinus serpin-2 (IRS-2), and we show that it primarily inhibits cathepsin G and chymase, while in higher molar excess, it affects thrombin activity as well. The inhibitory specificity was explained using the crystal structure, determined at a resolution of 1.8 Å. Moreover, we disclosed the ability of IRS-2 to inhibit cathepsin G-induced and thrombin-induced platelet aggregation. For the first time, an ectoparasite protein is shown to exhibit such pharmacological effects and target specificity. The stringent specificity and biological activities of IRS-2 combined with the knowledge of its structure can be the basis for the development of future pharmaceutical applications.
Assuntos
Catepsina G/imunologia , Quimases/imunologia , Inflamação/imunologia , Proteínas de Insetos/imunologia , Ixodes/genética , Serpinas/imunologia , Sequência de Aminoácidos , Animais , Catepsina G/metabolismo , Quimases/metabolismo , Cristalização , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Inflamação/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Ixodes/imunologia , Ixodes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Agregação Plaquetária/genética , Agregação Plaquetária/imunologia , Estrutura Quaternária de Proteína , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/imunologia , Proteínas e Peptídeos Salivares/metabolismo , Análise de Sequência de Proteína , Serpinas/genética , Serpinas/metabolismoRESUMO
Serpins are widely distributed and functionally diverse inhibitors of serine proteases. Ticks secrete serpins with anti-coagulation, anti-inflammatory, and immunomodulatory activities via their saliva into the feeding cavity to modulate host's hemostatic and immune reaction initiated by the insertion of tick's mouthparts into skin. The suppression of the host's immune response not only allows ticks to feed on a host for several days but also creates favorable conditions for the transmission of tick-borne pathogens. Herein we present the functional and structural characterization of Iripin-1 (Ixodes ricinus serpin-1), whose expression was detected in the salivary glands of the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Of 16 selected serine proteases, Iripin-1 inhibited primarily trypsin and further exhibited weaker inhibitory activity against kallikrein, matriptase, and plasmin. In the mouse model of acute peritonitis, Iripin-1 enhanced the production of the anti-inflammatory cytokine IL-10 and chemokines involved in neutrophil and monocyte recruitment, including MCP-1/CCL2, a potent histamine-releasing factor. Despite increased chemokine levels, the migration of neutrophils and monocytes to inflamed peritoneal cavities was significantly attenuated following Iripin-1 administration. Based on the results of in vitro experiments, immune cell recruitment might be inhibited due to Iripin-1-mediated reduction of the expression of chemokine receptors in neutrophils and adhesion molecules in endothelial cells. Decreased activity of serine proteases in the presence of Iripin-1 could further impede cell migration to the site of inflammation. Finally, we determined the tertiary structure of native Iripin-1 at 2.10 Å resolution by employing the X-ray crystallography technique. In conclusion, our data indicate that Iripin-1 facilitates I. ricinus feeding by attenuating the host's inflammatory response at the tick attachment site.
Assuntos
Ixodes , Serpinas , Camundongos , Animais , Serpinas/metabolismo , Células Endoteliais/metabolismo , Ixodes/metabolismo , Quimiocinas , Monócitos/metabolismo , Tripsina , Anti-Inflamatórios/farmacologiaRESUMO
Iripin-4, one of the many salivary serpins from Ixodes ricinus ticks with an as-yet unexplained function, crystallized in two different structural conformations, namely the native partially relaxed state and the cleaved serpin. The native structure was solved at a resolution of 2.3â Å and the structure of the cleaved conformation was solved at 2.0â Å resolution. Furthermore, structural changes were observed when the reactive-centre loop transitioned from the native conformation to the cleaved conformation. In addition to this finding, it was confirmed that Glu341 represents a primary substrate-recognition site for the inhibitory mechanism. The presence of glutamate instead of the typical arginine in the P1 recognition site of all structurally characterized I. ricinus serpins (PDB entries 7b2t, 7pmu and 7ahp), except for the tyrosine in the P1 site of Iripin-2 (formerly IRS-2; PDB entry 3nda), would explain the absence of inhibition of the tested proteases that cleave their substrate after arginine. Further research on Iripin-4 should focus on functional analysis of this interesting serpin.
Assuntos
Ixodes , Serpinas , Animais , Serpinas/química , Conformação Proteica , Modelos Moleculares , ArgininaRESUMO
Despite constant advances in cancer research, the treatment of pancreatic adenocarcinoma remains extremely challenging. The intratumoral immunotherapy approach that was developed by our research group and was based on a combination of mannan-BAM, TLR ligands, and anti-CD40 antibody (MBTA) showed promising therapeutic effects in various murine tumor models, including a pancreatic adenocarcinoma model (Panc02). However, the efficacy of MBTA therapy in the Panc02 model was negatively correlated with tumor size at the time of therapy initiation. Here, we aimed to further improve the outcome of MBTA therapy in the Panc02 model using the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON). The combination of intratumoral MBTA therapy and intraperitoneal administration of DON resulted in the complete elimination of advanced Panc02 subcutaneous tumors (140.8 ± 46.8 mm3) in 50% of treated animals and was followed by development of long-term immune memory. In the bilateral Panc02 subcutaneous tumor model, we observed a significant reduction in tumor growth in both tumors as well as prolonged survival of treated animals. The appropriate timing and method of administration of DON were also addressed to maximize its therapeutic effects and minimize its side effects. In summary, our findings demonstrate that the intraperitoneal application of DON significantly improves the efficacy of intratumoral MBTA therapy in both advanced and bilateral Panc02 subcutaneous tumor murine models.
Assuntos
Adenocarcinoma , Neoplasias Pancreáticas , Animais , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Glutamina/uso terapêutico , Adenocarcinoma/tratamento farmacológico , Imunoterapia/métodos , Linhagem Celular Tumoral , Neoplasias PancreáticasRESUMO
BACKGROUND: Alpha-Gal syndrome (AGS) is a tick-borne food allergy caused by IgE antibodies against the glycan galactose-alpha-1,3-galactose (α-Gal) present in glycoproteins and glycolipids from mammalian meat. To advance in the diagnosis and treatment of AGS, further research is needed to unravel the molecular and immune mechanisms underlying this syndrome. The objective of this study is the characterization of tick salivary components and proteins with and without α-Gal modifications involved in modulating human immune response against this carbohydrate. METHODS: Protein and α-Gal content were determined in tick saliva components, and proteins were identified by proteomics analysis of tick saliva fractions. Pathophysiological changes were recorded in the zebrafish (Danio rerio) model after exposure to distinct Ixodes ricinus tick salivary components. Serum samples were collected from zebrafish at day 8 of exposure to determine anti-α-Gal, anti-glycan, and anti-tick saliva protein IgM antibody titers by enzyme-linked immunosorbent assay (ELISA). RESULTS: Zebrafish treated with tick saliva and saliva protein fractions combined with non-protein fractions demonstrated significantly higher incidence of hemorrhagic type allergic reactions, abnormal behavioral patterns, or mortality when compared to the phosphate-buffered saline (PBS)-treated control group. The main tick salivary proteins identified in these fractions with possible functional implication in AGS were the secreted protein B7P208-salivary antigen p23 and metalloproteases. Anti-α-Gal and anti-tick salivary gland IgM antibody titers were significantly higher in distinct saliva protein fractions and deglycosylated saliva group when compared with PBS-treated controls. Anti-glycan antibodies showed group-related profiles. CONCLUSIONS: Results support the hypothesis that tick salivary biomolecules with and without α-Gal modifications are involved in modulating immune response against this carbohydrate.
Assuntos
Hipersensibilidade Alimentar , Ixodes , Picadas de Carrapatos , Animais , Humanos , Peixe-Zebra/metabolismo , Saliva , Galactose , Imunoglobulina E , Hipersensibilidade Alimentar/etiologia , Proteínas de Artrópodes , Imunoglobulina M , MamíferosRESUMO
Tick saliva has been extensively studied in the context of tick-host interactions because it is involved in host homeostasis modulation and microbial pathogen transmission to the host. Accumulated knowledge about the tick saliva composition at the molecular level has revealed that serine protease inhibitors play a key role in the tick-host interaction. Serpins are one highly expressed group of protease inhibitors in tick salivary glands, their expression can be induced during tick blood-feeding, and they have many biological functions at the tick-host interface. Indeed, tick serpins have an important role in inhibiting host hemostatic processes and in the modulation of the innate and adaptive immune responses of their vertebrate hosts. Tick serpins have also been studied as potential candidates for therapeutic use and vaccine development. In this review, we critically summarize the current state of knowledge about the biological role of tick serpins in shaping tick-host interactions with emphasis on the mechanisms by which they modulate host immunity. Their potential use in drug and vaccine development is also discussed.
Assuntos
Serpinas , Carrapatos , Animais , Saliva/metabolismo , Glândulas Salivares/metabolismo , Inibidores de Serina Proteinase/fisiologia , Serpinas/metabolismo , Carrapatos/metabolismoRESUMO
Immunotherapy has become an essential component in cancer treatment. However, the majority of solid metastatic cancers, such as pheochromocytoma, are resistant to this approach. Therefore, understanding immune cell composition in primary and distant metastatic tumors is important for therapeutic intervention and diagnostics. Combined mannan-BAM, TLR ligand, and anti-CD40 antibody-based intratumoral immunotherapy (MBTA therapy) previously resulted in the complete eradication of murine subcutaneous pheochromocytoma and demonstrated a systemic antitumor immune response in a metastatic model. Here, we further evaluated this systemic effect using a bilateral pheochromocytoma model, performing MBTA therapy through injection into the primary tumor and using distant (non-injected) tumors to monitor size changes and detailed immune cell infiltration. MBTA therapy suppressed the growth of not only injected but also distal tumors and prolonged MBTA-treated mice survival. Our flow cytometry analysis showed that MBTA therapy led to increased recruitment of innate and adaptive immune cells in both tumors and the spleen. Moreover, adoptive CD4+ T cell transfer from successfully MBTA-treated mice (i.e., subcutaneous pheochromocytoma) demonstrates the importance of these cells in long-term immunological memory. In summary, this study unravels further details on the systemic effect of MBTA therapy and its use for tumor and metastasis reduction or even elimination.
RESUMO
Iripin-5 is the main Ixodes ricinus salivary serpin, which acts as a modulator of host defence mechanisms by impairing neutrophil migration, suppressing nitric oxide production by macrophages and altering complement functions. Iripin-5 influences host immunity and shows high expression in the salivary glands. Here, the crystal structure of Iripin-5 in the most thermodynamically stable state of serpins is described. In the reactive-centre loop, the main substrate-recognition site of Iripin-5 is likely to be represented by Arg342, which implies the targeting of trypsin-like proteases. Furthermore, a computational structural analysis of selected Iripin-5-protease complexes together with interface analysis revealed the most probable residues of Iripin-5 involved in complex formation.
Assuntos
Anti-Inflamatórios , Inibidores Enzimáticos , Ixodes/metabolismo , Serpinas , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Células Cultivadas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Eritrócitos , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos , Coelhos , Serpinas/química , Serpinas/isolamento & purificaçãoRESUMO
Tick saliva is a rich source of pharmacologically and immunologically active molecules. These salivary components are indispensable for successful blood feeding on vertebrate hosts and are believed to facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-3, a protein expressed in the salivary glands of the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Belonging to the serpin superfamily of protease inhibitors, Iripin-3 strongly inhibited the proteolytic activity of serine proteases kallikrein and matriptase. In an in vitro setup, Iripin-3 was capable of modulating the adaptive immune response as evidenced by reduced survival of mouse splenocytes, impaired proliferation of CD4+ T lymphocytes, suppression of the T helper type 1 immune response, and induction of regulatory T cell differentiation. Apart from altering acquired immunity, Iripin-3 also inhibited the extrinsic blood coagulation pathway and reduced the production of pro-inflammatory cytokine interleukin-6 by lipopolysaccharide-stimulated bone marrow-derived macrophages. In addition to its functional characterization, we present the crystal structure of cleaved Iripin-3 at 1.95 Å resolution. Iripin-3 proved to be a pluripotent salivary serpin with immunomodulatory and anti-hemostatic properties that could facilitate tick feeding via the suppression of host anti-tick defenses. Physiological relevance of Iripin-3 activities observed in vitro needs to be supported by appropriate in vivo experiments.