RESUMEN
Cystatin F (CstF) is a protease inhibitor of cysteine cathepsins, including those involved in activating the perforin/granzyme cytotoxic pathways. It is targeted at the endolysosomal pathway but can also be secreted to the extracellular milieu or endocytosed by bystander cells. CstF was shown to be significantly increased in tuberculous pleurisy, and during HIV coinfection, pleural fluids display high viral loads. In human macrophages, our previous results revealed a strong upregulation of CstF in phagocytes activated by interferon γ or after infection with Mycobacterium tuberculosis (Mtb). CstF manipulation using RNA silencing led to increased proteolytic activity of lysosomal cathepsins, improving Mtb intracellular killing. In the present work, we investigate the impact of CstF depletion in macrophages during the coinfection of Mtb-infected phagocytes with lymphocytes infected with HIV. The results indicate that decreasing the CstF released by phagocytes increases the major pro-granzyme convertase cathepsin C of cytotoxic immune cells from peripheral blood-derived lymphocytes. Consequently, an observed augmentation of the granzyme B cytolytic activity leads to a significant reduction in viral replication in HIV-infected CD4+ T-lymphocytes. Ultimately, this knowledge can be crucial for developing new therapeutic approaches to control both pathogens based on manipulating CstF.
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Catepsina C , Coinfección , Granzimas , Infecciones por VIH , Macrófagos , Mycobacterium tuberculosis , Humanos , Granzimas/metabolismo , Granzimas/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/inmunología , Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/virología , Coinfección/microbiología , Catepsina C/metabolismo , Catepsina C/genética , Cistatinas/metabolismo , Cistatinas/genética , Tuberculosis/metabolismo , Tuberculosis/inmunología , Tuberculosis/microbiología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , VIH-1/fisiología , Biomarcadores de TumorRESUMEN
OBJECTIVE: To investigate the protective effect of recombinant Schistosoma japonicum cystatin (rSj-Cys) against acute liver injury induced by lipopolysaccharide (LPS) and D-GalN in mice. METHODS: Adult male C57BL/6J mice with or without LPS/D-GaIN-induced acute liver injury were given intraperitoneal injections of rSj-Cys or PBS 30 min after modeling (n=18), and serum and liver tissues samples were collected from 8 mice in each group 6 h after modeling. The survival of the remaining 10 mice in each group within 24 h was observed. Serum levels of ALT, AST, TNF-α and IL-6 of the mice were measured, and liver pathologies was observed with HE staining. The hepatic expressions of macrophage marker CD68, Bax, Bcl-2 and endoplasmic reticulum stress (ERS)-related proteins were detected using immunohistochemistry or immunoblotting, and TUNEL staining was used to detect hepatocyte apoptosis. RESULTS: The survival rates of PBS- and rSj-Cys-treated mouse models of acute liver injury were 30% and 80% at 12 h and were 10% and 60% at 24 h after modeling, respectively; no death occurred in the two control groups within 24 h. The mouse models showed significantly increased serum levels of AST, ALT, IL-6 and TNF-α and serious liver pathologies with increased hepatic expressions of CD68 and Bax, lowered expression of Bcl-2, increased hepatocyte apoptosis, and up-regulated expressions of ERS-related signaling pathway proteins GRP78, CHOP and NF-κB p-p65. Treatment of the mouse models significantly lowered the levels of AST, ALT, IL-6 and TNF-α, alleviated liver pathologies, reduced hepatic expressions of CD68, Bax, GRP78, CHOP and NF-κB p-p65, and enhanced the expression of Bcl-2. In the normal control mice, rSj-Cys injection did not produce any significant changes in these parameters compared with PBS. CONCLUSION: rSj-Cys alleviates LPS/D-GalN-induced acute liver injury in mice by suppressing ERS, attenuating inflammation and inhibiting hepatocyte apoptosis.
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Apoptosis , Cistatinas , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Hepatocitos , Inflamación , Ratones Endogámicos C57BL , Schistosoma japonicum , Animales , Ratones , Estrés del Retículo Endoplásmico/efectos de los fármacos , Apoptosis/efectos de los fármacos , Masculino , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Cistatinas/farmacología , Hígado/patología , Hígado/metabolismo , Lipopolisacáridos , Factor de Necrosis Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Proteínas Recombinantes/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Galactosamina , Antígenos CD/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Molécula CD68RESUMEN
OBJECTIVE: In this in vivo proof-of-concept study, acquired pellicle engineering was implemented to promote alterations in the protein composition of the acquired enamel pellicle (AEP) and the bacterial composition of the dental biofilm after treatment with Sugarcane cystatin (CaneCPI-5). DESIGN: After prophylaxis, 10 volunteers rinsed (10 mL, 1 min) with the following solutions: 1) deionized water (H2O- negative control or 2) 0.1 mg/mL CaneCPI-5. The AEP and biofilm were formed along 2 or 3 h, respectively. The AEP was collected with electrode filter papers soaked in 3 % citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. The biofilm microbiome was collected with a dental curette. The DNA was extracted, amplified, and analyzed by 16S-rRNA Next Generation Sequencing (NGS). RESULTS: Treatment with CaneCPI-5 increased several proteins with antimicrobial, acid-resistance, affinity for hydroxyapatite, structural and calcium binding properties, such as Cysteine-rich-3 (6-fold-p = 0.03), Cystatin-B (5.5-fold-p < 0.01), Neutrophil-defensin 1 (4.7-fold-p < 0.01), Mucin (3.9-fold-p < 0.01), Immunoglobulin-heavy-constant (3.8-fold-p < 0.01) and Lactotransferrin (2.8-fold-p < 0.01). Microbiome revealed that several commensal bacteria had their abundance increased after rinsing with CaneCPI-5, such as Corynebacterium and Neisseria, while Streptococcus and Prevotella nigrescens were decreased. The results indicate the efficiency of CaneCPI-5 in promoting beneficial changes in the AEP and biofilm, making this phytocystatin a potential target for incorporation into dental products. CONCLUSION: Cane demonstrated the capability to alter the protein composition of the acquired enamel pellicle (AEP) and the initial colonizers of the biofilm, enhancing the presence of proteins and bacteria crucial for dental protection.
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Biopelículas , Película Dental , Proteómica , Película Dental/microbiología , Humanos , Microbiota , Masculino , Adulto , ARN Ribosómico 16S , Femenino , Cistatinas , Prueba de Estudio ConceptualRESUMEN
BACKGROUND: Paraquat (PQ) is a widely used herbicide that poisons human by accident or intentional ingestion. PQ poisoning causes systemic inflammatory response syndrome (SIRS) resulting in acute lung injury (ALI) with an extremely high mortality rate. Blood trematode Schistosoma japonicum-produced cystatin (Sj-Cys) is a strong immunomodulatory protein that has been experimentally used to treat inflammation related diseases. In this study, Sj-Cys recombinant protein (rSj-Cys) was used to treat PQ-induced lung injury and the immunological mechanism underlying the therapeutic effect was investigated. METHODS: PQ-induced acute lung injury mouse model was established by intraperitoneally injection of 20â¯mg/kg of paraquat. The poisoned mice were treated with rSj-Cys and the survival rate was observed up to 7 days compared with the group without treatment. The pathological changes of PQ-induced lung injury were observed by examining the histochemical sections of affected lung tissue and the wet to dry ratio of lung as a parameter for inflammation and edema. The levels of the inflammation related cytokines IL-6 and TNF-α and regulatory cytokines IL-10 and TGF-ß were measured in sera and in affected lung tissue using ELISA and their mRNA levels in lung tissue using RT-PCR. The macrophages expressing iNOS were determined as M1 and those expressing Arg-1 as M2 macrophages. The effect of rSj-Cys on the transformation of inflammatory M1 to regulatory M2 macrophages was measured in affected lung tissue in vivo (EKISA and RT-PCR) and in MH-S cell line in vitro (flow cytometry). The expression levels of TLR2 and MyD88 in affected lung tissue were also measured to determine their role in the therapy of rSj-Cys on PQ-induced lung injury. RESULT: We identified that treatment with rSj-Cys significantly improved the survival rate of mice with PQ-induced lung injury from 30â¯% (untreated) to 80â¯%, reduced the pathological damage of poisoning lung tissue, associated with significantly reduced levels of proinflammatory cytokines (IL-6 from 1490 to 590â¯pg/ml, TNF-α from 260 to 150â¯pg/ml) and increased regulatory cytokines (IL-10 from360 to 550â¯pg/ml, and TGF-ß from 220 to 410â¯pg/ml) in both sera (proteins) and affected lung tissue (proteins and mRNAs). The polarization of macrophages from M1to M2 type was found to be involved in the therapeutic effect of rSj-Cys on the PQ-induced acute lung injury, possibly through inhibiting TLR2/MyD88 signaling pathway. CONCLUSIONS: Our study demonstrated the therapeutic effect of rSj-Cys on PQ poisoning caused acute lung injury by inducing M2 macrophage polarization through inhibiting TLR2/MyD88 signaling pathway. The finding in this study provides an alternative approach for the treatment of PQ poisoning and other inflammatory diseases.
Asunto(s)
Lesión Pulmonar Aguda , Cistatinas , Paraquat , Schistosoma japonicum , Animales , Paraquat/toxicidad , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/patología , Lesión Pulmonar Aguda/tratamiento farmacológico , Ratones , Herbicidas/toxicidad , Macrófagos/efectos de los fármacos , Pulmón/patología , Pulmón/efectos de los fármacos , Masculino , Citocinas/metabolismo , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: Cystatin F is a secreted lysosomal cysteine protease inhibitor that has been implicated in affecting the severity of demyelination and enhancing remyelination in pre-clinical models of immune-mediated demyelination. How cystatin F impacts neurologic disease severity following viral infection of the central nervous system (CNS) has not been well characterized and was the focus of this study. We used cystatin F null-mutant mice (Cst7-/-) with a well-established model of murine coronavirus-induced neurologic disease to evaluate the contributions of cystatin F in host defense, demyelination and remyelination. METHODS: Wildtype controls and Cst7-/- mice were intracranially (i.c.) infected with a sublethal dose of the neurotropic JHM strain of mouse hepatitis virus (JHMV), with disease progression and survival monitored daily. Viral plaque assays and qPCR were used to assess viral levels in CNS. Immune cell infiltration into the CNS and immune cell activation were determined by flow cytometry and 10X genomics chromium 3' single cell RNA sequencing (scRNA-seq). Spinal cord demyelination was determined by luxol fast blue (LFB) and Hematoxylin/Eosin (H&E) staining and axonal damage assessed by immunohistochemical staining for SMI-32. Remyelination was evaluated by electron microscopy (EM) and calculation of g-ratios. RESULTS: JHMV-infected Cst7-/- mice were able to control viral replication within the CNS, indicating that cystatin F is not essential for an effective Th1 anti-viral immune response. Infiltration of T cells into the spinal cords of JHMV-infected Cst7-/- mice was increased compared to infected controls, and this correlated with increased axonal damage and demyelination associated with impaired remyelination. Single-cell RNA-seq of CD45 + cells enriched from spinal cords of infected Cst7-/- and control mice revealed enhanced expression of transcripts encoding T cell chemoattractants, Cxcl9 and Cxcl10, combined with elevated expression of interferon-g (Ifng) and perforin (Prf1) transcripts in CD8 + T cells from Cst7-/- mice compared to controls. CONCLUSIONS: Cystatin F is not required for immune-mediated control of JHMV replication within the CNS. However, JHMV-infected Cst7-/- mice exhibited more severe clinical disease associated with increased demyelination and impaired remyelination. The increase in disease severity was associated with elevated expression of T cell chemoattractant chemokines, concurrent with increased neuroinflammation. These findings support the idea that cystatin F influences expression of proinflammatory gene expression impacting neuroinflammation, T cell activation and/or glia cell responses ultimately impacting neuroinflammation and neurologic disease.
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Infecciones por Coronavirus , Cistatinas , Enfermedades Desmielinizantes , Ratones Noqueados , Virus de la Hepatitis Murina , Animales , Ratones , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/virología , Enfermedades Desmielinizantes/inmunología , Virus de la Hepatitis Murina/patogenicidad , Cistatinas/genética , Cistatinas/metabolismo , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/patología , Ratones Endogámicos C57BL , Enfermedades Neuroinflamatorias/inmunología , Enfermedades Neuroinflamatorias/patología , Enfermedades Neuroinflamatorias/metabolismoRESUMEN
BACKGROUND: Cystatin is a protease inhibitor that also regulates genes expression linked to inflammation and plays a role in defense and regulation. METHODS AND RESULTS: Cystatin 10 (Smcys10) was cloned from Scophthalmus maximus and encodes a 145 amino acid polypeptide. The results of qRT-PCR showed that Smcys10 exhibited tissue-specific expression patterns, and its expression was significantly higher in the skin than in other tissues. The expression level of Smcys10 was significantly different in the skin, gill, head kidney, spleen and macrophages after Vibrio anguillarum infection, indicating that Smcys10 may play an important role in resistance to V. anguillarum infection. The recombinant Smcys10 protein showed binding and agglutinating activity in a Ca2+-dependent manner against bacteria. rSmcys10 treatment upregulated the expression of IL-10, TNF-α and TGF-ß in macrophages of turbot and hindered the release of lactate dehydrogenase (LDH) from macrophages after V. anguillarum infection, which confirmed that rSmcys10 reduced the damage to macrophages by V. anguillarum. The NF-κB pathway was suppressed by Smcys10, as demonstrated by dual-luciferase analysis. CONCLUSIONS: These results indicated that Smcys10 is involved in the host antibacterial immune response.
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Cistatinas , Enfermedades de los Peces , Proteínas de Peces , Peces Planos , Macrófagos , Vibrio , Animales , Peces Planos/inmunología , Peces Planos/genética , Peces Planos/metabolismo , Vibrio/patogenicidad , Cistatinas/genética , Cistatinas/metabolismo , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Proteínas de Peces/inmunología , Macrófagos/metabolismo , Macrófagos/inmunología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/genética , Enfermedades de los Peces/microbiología , Vibriosis/inmunología , Vibriosis/veterinaria , Vibriosis/genética , FN-kappa B/metabolismo , Clonación Molecular/métodos , Regulación de la Expresión GénicaRESUMEN
BACKGROUND: Understanding the molecular mechanisms of Alzheimer's disease (AD) has important clinical implications for guiding therapy. Impaired amyloid beta (Aß) clearance is critical in the pathogenesis of sporadic AD, and blood monocytes play an important role in Aß clearance in the periphery. However, the mechanism underlying the defective phagocytosis of Aß by monocytes in AD remains unclear. METHODS: Initially, we collected whole blood samples from sporadic AD patients and isolated the monocytes for RNA sequencing analysis. By establishing APP/PS1 transgenic model mice with monocyte-specific cystatin F overexpression, we assessed the influence of monocyte-derived cystatin F on AD development. We further used a nondenaturing gel to identify the structure of the secreted cystatin F in plasma. Flow cytometry, enzyme-linked immunosorbent assays and laser scanning confocal microscopy were used to analyse the internalization of Aß by monocytes. Pull down assays, bimolecular fluorescence complementation assays and total internal reflection fluorescence microscopy were used to determine the interactions and potential interactional amino acids between the cystatin F protein and Aß. Finally, the cystatin F protein was purified and injected via the tail vein into 5XFAD mice to assess AD pathology. RESULTS: Our results demonstrated that the expression of the cystatin F protein was specifically increased in the monocytes of AD patients. Monocyte-derived cystatin F increased Aß deposition and exacerbated cognitive deficits in APP/PS1 mice. Furthermore, secreted cystatin F in the plasma of AD patients has a dimeric structure that is closely related to clinical signs of AD. Moreover, we noted that the cystatin F dimer blocks the phagocytosis of Aß by monocytes. Mechanistically, the cystatin F dimer physically interacts with Aß to inhibit its recognition and internalization by monocytes through certain amino acid interactions between the cystatin F dimer and Aß. We found that high levels of the cystatin F dimer protein in blood contributed to amyloid pathology and cognitive deficits as a risk factor in 5XFAD mice. CONCLUSIONS: Our findings highlight that the cystatin F dimer plays a crucial role in regulating Aß metabolism via its peripheral clearance pathway, providing us with a potential biomarker for diagnosis and potential target for therapeutic intervention.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Ratones Transgénicos , Monocitos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Monocitos/metabolismo , Ratones , Humanos , Péptidos beta-Amiloides/metabolismo , Masculino , Femenino , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/patología , Anciano , Cistatinas/metabolismo , Cistatinas/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Anciano de 80 o más Años , Ratones Endogámicos C57BLRESUMEN
Background: Ascaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation. Methods: moDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry. Results: Compared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI. Conclusion: Al-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.
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Cistatinas , Monocitos , Humanos , Animales , Ratones , Ascaris lumbricoides , Ácido Mevalónico/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Diferenciación Celular , Citocinas/metabolismo , Inflamación/metabolismo , Inmunidad , Células Dendríticas , ARN/metabolismoRESUMEN
BACKGROUND: Ticks, which are obligate blood-feeding parasites, transmit a wide range of pathogens during their hematophagic process. Certain enzymes and macromolecules play a crucial role in inhibition of several tick physiological processes, including digestion and reproduction. In the present study, genes encoding type 2 cystatin were cloned and characterized from Haemaphysalis doenitzi, and the potential role of cystatin in tick control was further assessed. RESULTS: Two cystatin genes, HDcyst-1 and HDcyst-2, were successfully cloned from the tick H. doenitzi. Their open reading frames are 390 and 426 base pairs, and the number of coding amino acids are 129 and 141, respectively. In the midgut, salivary glands, Malpighian tubules and ovaries of ticks, the relative expression of HDcyst-1 was higher in the midgut and Malpighian tubules, and HDcyst-2 was higher in the salivary glands of H. doenitzi, respectively. Lipopolysaccharide (LPS) injection and low-temperature stress elevated cystatin expression in ticks. Enzyme-linked immunosorbent assay showed that both rHDcyst-1 and rHDcyst-2 protein vaccines increased antibody levels in immunized rabbits. A vaccination trial in rabbits infected with H. doenitzi showed that both recombinant cystatin proteins significantly reduced tick engorgement weights and egg mass weight, in particular, rHDcyst-1 significantly prolonged tick engorgement time by 1 day and reduced egg hatching rates by 16.9%. In total, rHDcyst-1 and rHDcyst-2 protein vaccinations provided 64.1% and 51.8% protection to adult female ticks, respectively. CONCLUSION: This is the first report on the immunological characterization of the cystatin protein and sequencing of the cystatin gene in H. doenitzi. Cystatin proteins are promising antigens that have the potential to be used as vaccines for infestation of H. doenitzi control. © 2024 Society of Chemical Industry.
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Proteínas de Artrópodos , Frío , Cistatinas , Ixodidae , Vacunas , Animales , Cistatinas/genética , Conejos , Femenino , Vacunas/inmunología , Ixodidae/inmunología , Ixodidae/fisiología , Ixodidae/genética , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Proteínas de Artrópodos/inmunología , Estrés Fisiológico , Lipopolisacáridos/farmacología , Secuencia de AminoácidosRESUMEN
Cystatins comprise a vast superfamily of evolutionary conserved proteins, predominantly recognized for their roles as endogenous inhibitors by regulating the activity of cysteine proteases. Emerging lines of research evidence also provides insight into their alternative roles in a spectrum of biological and pathological processes, including neurodegenerative disorders, tumor progression, inflammatory diseases, and immune response. Nowadays, various type-1 cystatins (stefins) have been demonstrated among a variety of discovered vertebrate groups, while little is known about the related homologue in cephalochordate amphioxus, which are repositioned at the base of the chordate phylum. In the present study, a single type-1 cystatin homologue in Branchiostoma japonicum was first successfully cloned and designated as Bjcystatin-1. The deduced Bjcystatin-1 protein is structurally characterized by the presence of typical wedge-shaped cystatin features, including the 'QxVxG' and 'Px' motif, as well as the conserved N-terminal glycine residue. Phylogenomic analyses utilizing different cystatin counterparts affirmed the close evolutionary relationship of Bjcystatin-1 and type-1 cystatin homologue. Bjcystatin-1 was predominantly expressed in the gills and hind-gut in a tissue-specific pattern, and its expression was remarkably up-regulated in response to challenge with bacteria or their signature molecules LPS and LTA, suggesting the involvement in immune response. Additionally, the recombinant Bjcystatin-1 (rBjcystatin-1) protein showed significant inhibitory activity towards papain and binding ability to LPS and LTA, indicating its hypothesized role as a pattern recognition receptor in immune response. Subcellular localization results also showed that Bjcystatin-1 was located in the cytoplasm and nucleus, and its overexpression could attenuate the activation of LPS-induced nuclear transcription factors NF-κB. Taken together, our study suggests that amphioxus Bjcystatin-1 acts as a dual role in protease inhibitor and an immunocompetent factor, providing new insights into the immune defense effect of type-1 cystatin in amphioxus.
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Cistatinas , Anfioxos , Animales , Lipopolisacáridos , Cistatinas/genética , Evolución Biológica , Factores de TranscripciónRESUMEN
PURPOSE: Although less than one-third of anti-nuclear antibody (ANA) positive patients with oJIA develop uveitis, ANA positivity is still the most well-known marker for assessing the risk of uveitis in oligoarticular JIA (oJIA). Therefore, novel biomarkers are needed to better assess the risk of developing uveitis. For this purpose, we performed a comparative tear proteome analysis of uveitis patients to reveal the identity of differentially regulated proteins. DESIGN: Tear samples were collected using the Schirmer strips in 7 oJIA and 7 oJIA patients with uveitis (oJIA-U). All oJIA-U patients had developed bilateral anterior uveitis and were inactive and topical treatment-free. METHODS: The nHPLC LC-MS/MS system was used for protein identification and label-free proteome comparisons. The PANTHER and STRING analyses were carried out using UniProt accession numbers of the identified proteins. RESULTS: Patient characteristics, e.g., age, gender, disease duration, and treatments were similar. For protein identification, three different databases were searched. Twenty-two, 147, and 258 database searches, respectively. Of these, 15 were common to all three proteome databases. Of these 15 proteins, 10 proteins were upregulated, and 2 were downregulated, based on the twofold regulation criteria. The upregulated proteins were, namely, cystatin-S, secretoglobin family 1D member, opiorphin prepropeptide, mammaglobin-B, lysozyme C, mesothelin, immunoglobulin kappa constant, extracellular glycoprotein lacritin, beta-2-microglobulin, and immunoglobulin J chain. The downregulated proteins were dermcidin and prolactin-inducible protein. Among the differentially regulated proteins, cystatin-S was the most regulated protein with an 18-fold upregulation ratio in tear samples from uveitis patients. CONCLUSION: Here, the identities and regulation ratios of several proteins were revealed when tear samples from uveitis patients were compared to patients without uveitis. These proteins are putative biomarkers for assessing uveitis risk and require further attention.
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Artritis Juvenil , Cistatinas , Uveítis , Humanos , Artritis Juvenil/complicaciones , Artritis Juvenil/diagnóstico , Proteoma , Cromatografía Liquida , Espectrometría de Masas en Tándem , BiomarcadoresRESUMEN
Cellular senescence, a hallmark of aging, results in a senescence-associated secretory phenotype (SASP) with an increased production of proinflammatory cytokines, growth factors, and proteases. Evidence from nonhuman models demonstrates that SASP contributes to tissue dysfunction and pathological effects of aging. However, there are relatively few human studies on the relationship between SASP and aging-related health outcomes. Proteins from the SASP Atlas were measured in plasma using aptamer-based proteomics (SomaLogic). Regression models were used to identify SASP protein associations with aging-related traits representing multiple aspects of physiology in 1 201 participants from 2 human cohort studies (BLSA/GESTALT and InCHIANTI). Traits examined were fasting glucose, C-reactive protein, interleukin-6, alkaline phosphatase, blood urea nitrogen, albumin, red blood cell distribution width, waist circumference, systolic and diastolic blood pressure, gait speed, and grip strength. Study results were combined with a fixed-effect inverse-variance weighted meta-analysis. In the meta-analysis, 28 of 77 SASP proteins were significantly associated with age. Of the 28 age-associated SASP proteins, 18 were significantly associated with 1 or more clinical traits, and 7 SASP proteins were significantly associated with 3 or more traits. Growth/differentiation factor 15, Insulin-like growth factor-binding protein 2, and Cystatin-C showed significant associations with inflammatory markers and measures of physical function (grip strength or gait speed). These results support the relevance of SASP proteins to human aging, identify specific traits that are potentially affected by SASP, and prioritize specific SASP proteins for their utility as biomarkers of human aging.
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Cistatinas , Fenotipo Secretor Asociado a la Senescencia , Humanos , Factor 15 de Diferenciación de Crecimiento/metabolismo , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina , Proteómica , Envejecimiento/metabolismo , Senescencia Celular/fisiología , Fenotipo , Cistatinas/metabolismoRESUMEN
Ticks are blood-sucking ectoparasites that secrete immunomodulatory substances in saliva to hosts during engorging. Cystatins, a tick salivary protein and natural inhibitor of Cathepsins, are attracting growing interest globally because of the immunosuppressive activities and the feasibility as an antigen for developing anti-tick vaccines. This review outlines the classification and the structure of tick Cystatins, and focuses on the anti-inflammatory effects and molecular mechanisms. Tick Cystatins can be divided into four families based on structures and cystatin 1 and cystatin 2 are the most abundant. They are injected into hosts during blood feeding and effectively mitigate the host inflammatory response. Mechanically, tick Cystatins exert anti-inflammatory properties through the inhibition of TLR-NF-κb, JAK-STAT and p38 MAPK signaling pathways. Further investigations are crucial to confirm the reduction of inflammation in other cell types like neutrophils and mast cells, and fully elucidate the underlying mechanism (like the structural mechanism) to make Cystatin a potential candidate for the development of novel anti-inflammation agents.
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Cistatinas , Garrapatas , Humanos , Animales , Garrapatas/fisiología , Saliva , Antiinflamatorios/farmacologíaRESUMEN
BACKGROUND: High fluoride exposure is increasingly discussed attributing to kidney injury as a causative factor. Depending on geochemistry, differential fluoride levels in drinking water are identified in different regions in Sri Lanka. However, the levels of fluoride exposure, and associations with kidney health has not been adequately studied in Sri Lanka, particularly in pediatric communities. Hence, the present study aimed to assess fluoride exposure in selected pediatric communities in the dry, wet and intermediate climatic zones in Sri Lanka, along with an assessment of renal health using urinary Cystatin-C (uCys-C), and albumin-creatinine ratio (uACR). METHODS: We conducted a cross-sectional study with school students in selected education zones representing dry (N = 331), wet (N = 152), and intermediate (N = 292) climatic zones in Sri Lanka. Fluoride contents in urine and drinking water were assessed as measures of fluoride exposure. RESULTS: The median (interquartile distance) urinary fluoride levels of participants in the dry, wet and intermediate zones were 1.63(1.04-2.85), 1.29(0.85-2.21), and 1.07(0.61-1.98) mg/gCr while the fluoride contents of drinking water samples were 1.76(1.36-2.30), 0.25(0.18-0.37), and 0.43(0.26-0.63) ppm respectively with significant differences among the three groups. Median uCys-C level (ng/mgCr) of the participants in intermediate zone [30.26(8.49-71.44)] was significantly low (p < 0.05) compared to that of the participants in dry zone [56.19(7.08-211.8)], and wet zone [66.29(30.43-125.20)]. The incidences of elevated uCys-C levels above reference intervals in participants of dry zone (47.7%), and wet zone (50.0%) were significantly high (p < 0.001) compared to the intermediate zone (26.4%). CONCLUSION: Relatively high fluoride exposure is likely in dry and wet zone communities compared to the intermediate zone along with significantly higher incidence of uCys-C levels above reference intervals in study groups with higher fluoride exposure. However, to conclude a clear link between fluoride exposure and kidney health we need in-depth studies.
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Cistatinas , Agua Potable , Insuficiencia Renal Crónica , Humanos , Niño , Fluoruros/análisis , Creatinina , Agua Potable/análisis , Sri Lanka/epidemiología , Estudios Transversales , Riñón/química , Albúminas , Insuficiencia Renal Crónica/epidemiologíaRESUMEN
Microglial endolysosomal (dys)function is strongly implicated in neurodegenerative disease. Transcriptomic studies show that a microglial state characterised by a set of genes involved in endolysosomal function is induced in both mouse Alzheimer's disease (AD) models and human AD brain, and that the emergence of this state is emphasised in females. Cst7 (encoding cystatin F) is among the most highly upregulated genes in these microglia. However, despite such striking and robust upregulation, the function of Cst7 in neurodegenerative disease is not understood. Here, we crossed Cst7-/- mice with the AppNL-G-F mouse to test the role of Cst7 in a model of amyloid-driven AD. Surprisingly, we found that Cst7 plays a sexually dimorphic role regulating microglia in this model. In females, Cst7-/-AppNL-G-F microglia had greater endolysosomal gene expression, lysosomal burden, and amyloid beta (Aß) burden in vivo and were more phagocytic in vitro. However, in males, Cst7-/-AppNL-G-F microglia were less inflammatory and had a reduction in lysosomal burden but had no change in Aß burden. Overall, our study reveals functional roles for one of the most commonly upregulated genes in microglia across disease models, and the sex-specific profiles of Cst7-/--altered microglial disease phenotypes. More broadly, the findings raise important implications for AD including crucial questions on sexual dimorphism in neurodegenerative disease and the interplay between endolysosomal and inflammatory pathways in AD pathology.
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Enfermedad de Alzheimer , Cistatinas , Enfermedades Neurodegenerativas , Animales , Femenino , Humanos , Masculino , Ratones , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Cistatinas/metabolismo , Modelos Animales de Enfermedad , Ratones Transgénicos , Microglía/metabolismo , Enfermedades Neurodegenerativas/patologíaRESUMEN
Cystatin F, a cysteine peptidase inhibitor, is a potent modulator of NK cytotoxicity. By inhibiting granule-mediated cytotoxicity pathway, cystatin F induces formation of non-functional NK cell stage, called split-anergy. We show that N-glycosylation determines the localization and cellular function of cystatin F. Cystatin F mostly exhibited high-mannose glycosylation in U-937 cells, both high-mannose and complex glycosylation in NK-92 and primary NKs, and predominantly complex glycosylation in super-charged NKs. Manipulating N-glycosylation with kifunensine increased high-mannose glycosylation of cystatin F and lysosome localisation, which decreased cathepsin C activity and reduced NK cytotoxicity. Mannose-6-phosphate could significantly reduce the internalization of extracellular cystatin F. By comparing NK cells with different cytotoxic potentials, we found that high-mannose cystatin F was strongly associated with lysosomes and cathepsin C in NK-92 cell line. In contrast, in highly cytotoxic super-charged NKs, cystatin F with complex glycosylation was associated with the secretory pathway and less prone to inhibit cathepsin C. Modulating glycosylation to alter cystatin F localisation could increase the cytotoxicity of NK cells, thereby enhancing their therapeutic potential for treating cancer patients.
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Antineoplásicos , Cistatinas , Humanos , Glicosilación , Manosa , Catepsina C/metabolismo , Células Asesinas Naturales/metabolismoRESUMEN
Subolesin is a conserved molecule in both hard and soft ticks and is considered as an effective candidate molecule for the development of anti-tick vaccine. Previous studies have reported the role of subolesin in blood feeding, reproduction, development, and gene expression in hard ticks. However, studies addressing the role of subolesin in soft ticks are limited. In this study, we report that subolesin is not only important in soft tick Ornithodoros turicata americanus blood feeding but also in the regulation of innate immune gene expression in these ticks. We identified and characterized several putative innate immune genes including Toll, Lysozyme precursor (Lp), fibrinogen-domain containing protein (FDP), cystatin and ML-domain containing protein (MLD) in O. turicata americanus ticks. Quantitative real-time polymerase chain reaction analysis revealed the expression of these genes in both O. turicata americanus salivary glands and midgut and in all developmental stages of these soft ticks. Significantly increased expression of fdp was noted in salivary glands and midgut upon O. turicata americanus blood feeding. Furthermore, RNAi-mediated knockdown of O. turicata americanus subolesin expression affected blood feeding and innate immune gene expression in these ticks. Significant downregulation of toll, lp, fdp, cystatin, and mld transcripts was evident in sub-dsRNA-treated ticks when compared to the levels noted in mock-dsRNA-treated control. Collectively, our study not only reports identification and characterization of various innate immune genes in O. turicata americanus ticks but also provides evidence on the role of subolesin in blood feeding and innate immune gene expression in these medically important ticks.
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Argasidae , Cistatinas , Ornithodoros , Vacunas , Animales , Ornithodoros/genética , Vacunas/genética , Expresión Génica , Cistatinas/genética , Inmunidad InnataRESUMEN
Cystatins encode a high functional variability not only because of their ability to inhibit different classes of proteases but also because of their propensity to form oligomers and amyloid fibrils. Phytocystatins, essential regulators of protease activity in plants, specifically inhibit papain-like cysteine proteases (PLCPs) and legumains through two distinct cystatin domains. Mammalian cystatins can form amyloid fibrils; however, the potential for amyloid fibril formation of phytocystatins remains unknown. In this study, we demonstrate that Arabidopsis thaliana phytocystatin 6 (AtCYT6) exists as a mixture of monomeric, dimeric, and oligomeric forms in solution. Noncovalent oligomerization was facilitated by the N-terminal cystatin domain, while covalent dimerization occurred through disulfide bond formation in the interdomain linker. The noncovalent dimeric form of AtCYT6 retained activity against its target proteases, papain and legumain, albeit with reduced inhibitory potency. Additionally, we observed the formation of amyloid fibrils by AtCYT6 under acidic pH conditions and upon heating. The amyloidogenic potential could be attributed to the AtCYT6's N-terminal domain (AtCYT6-NTD). Importantly, AtCYT6 amyloid fibrils harbored inhibitory activities against both papain and legumain. These findings shed light on the oligomerization and amyloidogenic behavior of AtCYT6, expanding our understanding of phytocystatin biology and its potential functional implications for plant protease regulation.
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Arabidopsis , Cistatinas , Animales , Papaína/química , Amiloide/química , Cistatinas/química , Cistatinas/farmacología , Péptido Hidrolasas , MamíferosRESUMEN
OBJECTIVE: To evaluate the protective effect of recombinant Schistosoma japonicum cystatin (rSj-Cys) against acute kidney injury induced by acute liver failure and unravel the underlying mechanism, so as to provide insights into the clinical therapy of acute kidney injury. METHODS: Twenty-four male C57BL/6J mice at ages of 6 to 8 weeks were randomly divided into the normal control group, rSj-Cys control group, lipopolysaccharide (LPS)/D-galactosamine (D-GaIN) model group and LPS/D-GaIN + rSj-Cys treatment group, of 6 mice each group. Mice in the LPS/D-GaIN group and LPS/D-GaIN + rSj-Cys group were intraperitoneally injected with LPS (10 µg/kg) and D-GaIN (700 mg/kg), and mice in the LPS/D-GaIN + rSj-Cys group were additionally administered with rSj-Cys (1.25 mg/kg) by intraperitoneal injection 30 min post-modeling, while mice in the rSj-Cys group were intraperitoneally injected with rSj-Cys (1.25 mg/kg), and mice in the normal control group were injected with the normal volume of PBS. All mice were sacrificed 6 h post-modeling, and mouse serum and kidney samples were collected. Serum creatinine (Cr) and urea nitrogen (BUN) levels were measured, and the pathological changes of mouse kidney specimens were examined using hematoxylin-eosin (HE) staining. Serum tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were detected using enzyme-linked immunosorbent assay (ELISA), and the expression of inflammatory factors and pyroptosis-related proteins was quantified in mouse kidney specimens using immunohistochemistry. In addition, the expression of pyroptosis-related proteins and nuclear factor-kappa B (NF-κB) signaling pathway-associated proteins was determined in mouse kidney specimens using Western blotting assay. RESULTS: HE staining showed no remarkable abnormality in the mouse kidney structure in the normal control group and the rSj-Cys control group, and renal tubular injury was found in LPS/D-GaIN group, while the renal tubular injury was alleviated in LPS/D-GaIN+rSj-Cys treatment group. There were significant differences in serum levels of Cr (F = 46.33, P < 0.001), BUN (F = 128.60, P < 0.001), TNF-α (F = 102.00, P < 0.001) and IL-6 (F = 202.10, P < 0.001) among the four groups, and lower serum Cr [(85.35 ± 32.05) µmol/L], BUN [(11.90 ± 2.76) mmol/L], TNF-α [(158.27 ± 15.83) pg/mL] and IL-6 levels [(56.72 ± 4.37) pg/mL] were detected in the in LPS/D-GaIN + rSj-Cys group than in the LPS/D-GaIN group (all P values < 0.01). Immunohistochemical staining detected significant differences in TNF-α (F = 24.16, P < 0.001) and IL-10 (F = 15.07, P < 0.01) expression among the four groups, and lower TNF-α [(106.50 ± 16.57)%] and higher IL-10 expression [(91.83 ± 5.23)%] was detected in the LPS/D-GaIN + rSj-Cys group than in the LPS/D-GaIN group (both P values < 0.01). Western blotting and immunohistochemistry detected significant differences in the protein expression of pyroptosis-related proteins NOD-like receptor thermal protein domain associated protein 3 (NLRP3) (F = 24.57 and 30.72, both P values < 0.001), IL-1ß (F = 19.24 and 22.59, both P values < 0.001) and IL-18 (F = 16.60 and 19.30, both P values < 0.001) in kidney samples among the four groups, and lower NLRP3, IL-1ß and IL-18 expression was quantified in the LPS/D-GaIN + rSj-Cys treatment group than in the LPS/D-GaIN group (P values < 0.05). In addition, there were significant differences in the protein expression of NF-κB signaling pathway-associated proteins p-NF-κB p-P65/NF-κB p65 (F = 71.88, P < 0.001), Toll-like receptor (TLR)-4 (F = 45.49, P < 0.001) and p-IκB/IκB (F = 60.87, P < 0.001) in mouse kidney samples among the four groups, and lower expression of three NF-κB signaling pathway-associated proteins was determined in the LPS/D-GaIN + rSj-Cys treatment group than in the LPS/D-GaIN group (all P values < 0.01). CONCLUSIONS: rSj-Cys may present a protective effect against acute kidney injury caused by acute liver failure through inhibiting inflammation and pyroptosis and downregulating the NF-κB signaling pathway.
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Lesión Renal Aguda , Cistatinas , Fallo Hepático Agudo , Schistosoma japonicum , Ratones , Masculino , Animales , Interleucina-10 , Factor de Necrosis Tumoral alfa/genética , FN-kappa B/metabolismo , FN-kappa B/uso terapéutico , Interleucina-18/uso terapéutico , Schistosoma japonicum/metabolismo , Interleucina-6/uso terapéutico , Lipopolisacáridos/uso terapéutico , Proteína con Dominio Pirina 3 de la Familia NLR , Ratones Endogámicos C57BL , Lesión Renal Aguda/tratamiento farmacológico , Cistatinas/uso terapéuticoRESUMEN
BACKGROUND: Accumulating evidence indicates that type II cystatin (CST) genes play a pivotal role in several tumor pathological processes, thereby affecting all stages of tumorigenesis and tumor development. However, the prognostic and predictive value of type II CST genes in GC has not yet been investigated. METHODS: The present study evaluated the expression and prognostic value of type II CST genes in GC by using The Cancer Genome Atlas (TCGA) database and the Kaplan-Meier plotter (KM plotter) online database. The type II CST genes related to the prognosis of GC were then screened out. We then validated the expression and prognostic value of these genes by immunohistochemistry. We also used Database for Annotation, Visualization, and Integrated Discovery (DAVID), Gene Multiple Association Network Integration Algorithm (GeneMANIA), Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), nomogram, genome-wide co-expression analysis, and other bioinformatics tools to analyze the value of type II CST genes in GC and the underlying mechanism. RESULTS: The data from the TCGA database and the KM plotter online database showed that high expression of CST2 and CST4 was associated with the overall survival (OS) of patients with GC. The immunohistochemical expression analysis showed that patients with high expression of CST4 in GC tissues have a shorter OS than those with low expression of CST4 (HR = 1.85,95%CI: 1.13-3.03, P = 0.015). Multivariate Cox regression analysis confirmed that the high expression level of CST4 was an independent prognostic risk factor for OS. CONCLUSIONS: Our findings suggest that CST4 could serve as a tumor marker that affects the prognosis of GC and could be considered as a potential therapeutic target for GC.