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1.
Gene ; 761: 145036, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32777525

RESUMO

Lupinus albus γ-conglutin is proposed to positively affect glucose metabolism through inhibition of hepatic glucose production and insulin-mimetic activity; however, the action mechanism is not entirely known. Besides, most studies had focused on its effect on molecular targets directly related to glucose metabolism, and few studies have investigated how γ-conglutin may affect the liver gene expression or if it plays a role in other metabolic processes. Therefore, we investigated the influence of γ-conglutin on the liver transcriptome of streptozotocin-induced diabetic rats using DNA microarrays, ontological analyses, and quantitative PCR. Of the 22,000 genes evaluated, 803 and 173 were downregulated and upregulated, respectively. The ontological analyses of the differentially expressed genes revealed that among others, the mitochondria, microtubules, cytoskeleton, and oxidoreductase activity terms were enriched, implying a possible role of γ-conglutin on autophagy. To corroborate the microarray results, we selected and quantified, by PCR, the expression of two genes associated with autophagy (Atg7 and Snx18) and found their expression augmented two and threefold, respectively; indicating a higher autophagy activity in animals treated with γ-conglutin. Although complementary studies are required, our findings indicate for the first time that the hypoglycaemic effects of γ-conglutin may involve an autophagy induction mechanism, a pivotal process for the preservation of cell physiology and glucose homeostasis.


Assuntos
Colectinas/farmacologia , Lupinus/metabolismo , Soroglobulinas/farmacologia , Transcriptoma/genética , Animais , Glicemia/metabolismo , Colectinas/metabolismo , Colectinas/fisiologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Fígado/patologia , Lupinus/genética , Masculino , Proteínas de Plantas/genética , Ratos , Ratos Wistar , Sementes/metabolismo , Soroglobulinas/metabolismo , Soroglobulinas/fisiologia
2.
J Immunol ; 204(6): 1598-1606, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-32041782

RESUMO

C1q/TNF-related protein (CTRP) 6 is a member of the CTRP protein family associated with the regulation of cellular and endocrine processes. CTRP6 contains collagen and globular structures, resembling the pattern recognition molecules (PRMs) of the classical and lectin complement pathways. We expressed human CTRP6 in Chinese hamster ovary cells and investigated the binding to different putative ligands (acetylated BSA [AcBSA], zymosan, mannan, and LPS from Escherichia coli and Salmonella as well as to the monosaccharides l-fucose, d-mannose, N-acetylglucosamine, N-acetylgalactosamine, and galactose). Furthermore, we investigated the binding of CTRP6 to various Gram-negative bacteria as well as PRMs and enzymes of the lectin complement pathway. We found that CTRP6 bound to AcBSA and to a lesser extent to zymosan. Using EDTA as chelating agent, we observed an increased binding to AcBSA, zymosan and the two strains of LPS. We detected no binding to mannan and BSA. We identified l-fucose as a ligand for CTRP6 and that it bound to certain enteroaggregative Escherichia coli and Pseudomonas aeruginosa isolates, whereas to other bacterial isolates, no binding was observed. CTRP6 did not appear to interact directly with the activating enzymes of the lectin pathway; however, we could show the specific recruitment of collectin-11 and subsequent initiation of the complement cascade through deposition of C4. In conclusion, our results demonstrate the binding of CTRP6 to a variety of microbial and endogenous ligands identifying CTRP6 as a novel human lectin and PRM of importance for complement recognition and innate immunity.


Assuntos
Antígenos de Bactérias/metabolismo , Colágeno/metabolismo , Colectinas/metabolismo , Complemento C4/metabolismo , Lectina de Ligação a Manose da Via do Complemento/imunologia , Animais , Antígenos de Bactérias/imunologia , Células CHO , Colágeno/genética , Colágeno/isolamento & purificação , Ativação do Complemento , Cricetulus , Escherichia coli/imunologia , Escherichia coli/metabolismo , Ligantes , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
3.
J Immunol ; 204(7): 1919-1928, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32094208

RESUMO

The complement system constitutes an important part of the innate immune system. The collectins and the ficolins are soluble pattern recognition molecules that contribute to complement activation via the lectin pathway. During previous experiments with ficolin-2 and ficolin-3, we have observed that the molecules may interact. We therefore hypothesized the existence of stable ficolin-2/-3 heterocomplexes. We could demonstrate ficolin-2/-3 heterocomplexes in normal human serum and plasma by ELISA using Abs specific for ficolin-2 and ficolin-3. The formation of heteromeric protein complexes were validated by coimmunoprecipitation and Western blot analysis. When recombinant ficolin-2 and recombinant ficolin-3 were mixed, no complexes were formed. However, when coexpressing ficolin-2 and ficolin-3 in Chinese hamster ovary cells, we could detect ficolin-2/-3 heterocomplexes in the supernatant. Furthermore, we measured concentration of the ficolin-2/-3 heterocomplexes in arbitrary units in 94 healthy individuals. We also established the relationship between the concentrations of ficolin-2, ficolin-3, and the ficolin-2/-3 heterocomplexes. We observed that the concentration of the ficolin-2/-3 heterocomplex correlated significantly with ficolin-2 (ρ: 0.24, p < 0.018) and ficolin-3 concentrations (ρ: 0.46, p < 0.0001). In conclusion, we describe a novel protein complex between ficolin-2 and ficolin-3 present in serum and plasma, which might be of additional biological relevance apart from the native ficolin-2 and ficolin-3 molecules.


Assuntos
Lectinas/sangue , Animais , Células CHO , Linhagem Celular , Colectinas/metabolismo , Ativação do Complemento/fisiologia , Lectina de Ligação a Manose da Via do Complemento/fisiologia , Proteínas do Sistema Complemento/metabolismo , Cricetulus , Humanos , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Camundongos
4.
Dev Comp Immunol ; 102: 103486, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31473265

RESUMO

The complement system is a crucial component of the innate immune system that links innate and adaptive immunity. CL-11, a protein similar to Mannose-binding lectin (MBL), plays significant role in the innate immune system in mammals and fish, serving as an initiator of the lectin pathway of complement activation. In this study, a CL-11 homolog (TfCol-11) was identified in roughskin sculpin (Trachidermus fasciatus), and its expression and role in immune responses were characterized. The open reading frame of TfCol-11 is 795 bp long, encoding a 264 amino acid polypeptide. The deduced amino acid sequence of this protein is highly homologous to sequences in other teleosts, and is similar to vertebrate CL-11, containing a canonical collagen-like region, a carbohydrate recognition domain, and a neck region. Recombinant TfCol-11 purified from Escherichia coli(E.coli) was able to bind to different microbes in a Ca2+-independent manner. Meanwhile, a 993 bp-long of partial MASP cDNA with a 96 bp 5' untranslated region (UTR) was also cloned from roughskin sculpin, containing 299 amino acids and consisting of three domains (CUB-EGF-CUB). qRT-PCR indicated that TfCol-11 and MASP mRNAs were predominately co-expressed in the liver. The temporal expression of TfCol-11 and MASP were both drastically up-regulated in the liver, skin, and blood by LPS challenge. Recombinant TfCol-11 purified from E.coli BL21(DE3) was able to agglutinate some bacteria in a Ca2+-dependent manner. In addition, an in vitro pull-down experiment demonstrated that TfCol-11 was able to bind to MASP, and in vivo experiments showed that TfCol-11 was associated with increased membrane attack complex (MAC) levels. It is therefore possible that TfCol-11 may plays a role in activating the complement system and in the defense against invading microorganisms in roughskin sculpin.


Assuntos
Colectinas/metabolismo , Ativação do Complemento , Proteínas de Peixes/metabolismo , Perciformes/imunologia , Testes de Aglutinação , Sequência de Aminoácidos , Animais , Sequência de Bases , Colectinas/química , Colectinas/genética , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Imunidade Inata , Serina Proteases Associadas a Proteína de Ligação a Manose/química , Serina Proteases Associadas a Proteína de Ligação a Manose/genética , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Fases de Leitura Aberta , Filogenia , Domínios Proteicos , Alinhamento de Sequência , Distribuição Tecidual
5.
J Biol Chem ; 294(45): 17155-17165, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31562242

RESUMO

Bovine conglutinin is an immune protein that is involved in host resistance to microbes and parasites and interacts with complement component iC3b, agglutinates erythrocytes, and neutralizes influenza A virus. Here, we determined the high-resolution (0.97-1.46 Å) crystal structures with and without bound ligand of a recombinant fragment of conglutinin's C-terminal carbohydrate-recognition domain (CRD). The structures disclosed that the high-affinity ligand N-acetyl-d-glucosamine (GlcNAc) binds in the collectin CRD calcium site by interacting with the O3' and O4' hydroxyls alongside additional specific interactions of the N-acetyl group oxygen and nitrogen with Lys-343 and Asp-320, respectively. These residues, unique to conglutinin and differing both in sequence and in location from those in other collectins, result in specific, high-affinity binding for GlcNAc. The binding pocket flanking residue Val-339, unlike the equivalent Arg-343 in the homologous human surfactant protein D, is sufficiently small to allow conglutinin Lys-343 access to the bound ligand, whereas Asp-320 lies in an extended loop proximal to the ligand-binding site and bounded at both ends by conserved residues that coordinate to both calcium and ligand. This loop becomes ordered on ligand binding. The electron density revealed both α and ß anomers of GlcNAc, consistent with the added α/ßGlcNAc mixture. Crystals soaked with α1-2 mannobiose, a putative component of iC3b, reported to bind to conglutinin, failed to reveal bound ligand, suggesting a requirement for presentation of mannobiose as part of an extended physiological ligand. These results reveal a highly specific GlcNAc-binding pocket in conglutinin and a novel collectin mode of carbohydrate recognition.


Assuntos
Acetilglucosamina/metabolismo , Colectinas/química , Colectinas/metabolismo , Soroglobulinas/química , Soroglobulinas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica
6.
J Immunol Res ; 2019: 9164202, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482100

RESUMO

Collectin-12 (collectin placenta 1, CL-P1, or CL-12) is a newly identified pattern recognition molecule of the innate immune system. Recent evidences show that CL-12 plays important roles not only in innate immune protection against certain clinically important pathogens but also in scavenging of host molecules, leukocyte recruitment, and cancer metastasis. Furthermore, CL-12 has been shown to be associated with the pathogenesis of human diseases such as Alzheimer's disease and multiple sclerosis lesion development. Therefore, the functional consequence of CL-12 remains intriguing and awaits further elucidation. However, available protocols for the purification of recombinant CL-12 with high purity are laborious and inefficient and hamper further functional studies. Here, we report a simple, rapid, and efficient solution to obtain biologically active CL-12 with high purity. We established stable transfected Flp-In™-CHO cells expressing the recombinant CL-12 extracellular domain in high amounts. Recombinant CL-12 was purified from cell culture supernatants using a 3-step rapid purification procedure utilizing disposable affinity and ion exchange minicolumns. Purified recombinant CL-12 adopted an oligomeric structure with monomers, dimers, and trimers and retained its binding capacity towards the A. fumigatus strain that has been described before. Furthermore, we demonstrated the opsonic properties towards eight clinical isolates of A. fumigatus strains and diverse clinically important fungal pathogens. Purified recombinant CL-12 revealed a differential binding capacity towards selected fungal pathogens in vitro. In conclusion, we demonstrate a rapid and efficient purification solution for further biochemical and functional characterization of CL-12 and reveal opsonic properties of CL-12 towards diverse fungal pathogens.


Assuntos
Aspergillus fumigatus/imunologia , Colectinas/isolamento & purificação , Proteínas Opsonizantes/isolamento & purificação , Receptores Depuradores/isolamento & purificação , Animais , Aspergillus fumigatus/metabolismo , Células CHO , Colectinas/genética , Colectinas/metabolismo , Colectinas/farmacologia , Cricetulus , Humanos , Proteínas Opsonizantes/genética , Proteínas Opsonizantes/metabolismo , Proteínas Opsonizantes/farmacologia , Receptores Depuradores/genética , Receptores Depuradores/metabolismo
7.
Clin Appl Thromb Hemost ; 25: 1076029618821189, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30808212

RESUMO

In sepsis, systemic coagulation activation frequently causes disseminated intravascular coagulation (DIC), and the uncontrolled activation of the complement system can induce multiple organ dysfunction and poor prognosis. This study aimed to examine the association of DIC with levels of collectin kidney 1 (CL-K1), a novel collectin of the complement system, and mannose-binding lectin (MBL), a classical-type collectin in patients with sepsis. We collected blood samples prospectively from adult patients with sepsis admitted to the intensive care unit (ICU) from day 1 (admission) to day 5. The CL-K1 and MBL levels were measured by enzyme-linked immunosorbent assay, and DIC was diagnosed by using a scoring algorithm. The correlation of CL-K1 and MBL levels with other coagulation markers was analyzed. There were 37 patients with DIC (DIC group) and 15 without DIC (non-DIC group). Compared to the non-DIC group, the DIC group had more severe conditions and higher mortality. During the 5 days after ICU admission, plasma CL-K1 levels were similar between the groups, but plasma MBL levels were significantly lower in the DIC group. Plasma CL-K1 levels were weakly correlated with prothrombin time, activated partial thromboplastin time, and antithrombin levels; plasma MBL levels were weakly correlated with fibrin/fibrinogen degradation product levels and DIC score. In conclusion, during the first 5 days of ICU admission, plasma CL-K1 levels were similar between the DIC and non-DIC groups. However, plasma MBL levels were lower in the DIC group compared to the non-DIC group, and the significance of this difference grew gradually over time.


Assuntos
Biomarcadores/sangue , Colectinas/metabolismo , Coagulação Intravascular Disseminada/sangue , Lectina de Ligação a Manose/metabolismo , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
8.
Front Immunol ; 9: 2238, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30323815

RESUMO

Collectin-11 (CL-11) is a pattern recognition molecule of the lectin pathway of complement with diverse functions spanning from host defense to embryonic development. CL-11 is found in the circulation in heterocomplexes with the homologous collectin-10 (CL-10). Abnormal CL-11 plasma levels are associated with the presence of disseminated intravascular coagulation, urinary schistosomiasis, and congenital disorders. Although there has been a marked development in the characterization of CL-11 there is still a scarcity of clinical tools for its analysis. Thus, we generated monoclonal antibodies and developed a quantitative ELISA to measure CL-11 in the circulation. The antibodies were screened against recombinant CL-11 and validated by ELISA and immunoprecipitation of serum and plasma. The best candidates were pairwise compared to develop a quantitative ELISA. The assay was validated regarding its sensitivity, reproducibility, and dilution linearity, demonstrating a satisfactory variability over a working range of 0.29-18.75 ng/ml. The mean plasma concentration of CL-11 in healthy controls was determined to be 289.4 ng/ml (range 143.2-459.4 ng/ml), highly correlated to the levels of CL/10/11 complexes (r = 0.729). Plasma CL-11 and CL-10/11 co-migrated in size exclusion chromatography as two major complexes of ~400 and >600 kDa. Furthermore, we observed a significant decrease at admission in CL-11 plasma levels in patients admitted to intensive care with systemic inflammatory response syndrome. By using the in-house antibodies and recombinant CL-11, we found that CL-11 can bind to zymosan independently of calcium by a separate site from the carbohydrate-binding region. Finally, we showed that CL-11/MASP-2 complexes trigger C4b deposition on zymosan. In conclusion, we have developed a specific and sensitive ELISA to investigate the ever-expanding roles of CL-11 in health and disease and shown a novel interaction between CL-11 and zymosan.


Assuntos
Colectinas/sangue , Colectinas/química , Ensaio de Imunoadsorção Enzimática/métodos , Síndrome de Resposta Inflamatória Sistêmica/sangue , Análise de Variância , Animais , Anticorpos Monoclonais/imunologia , Células CHO , Cromatografia em Gel , Colectinas/imunologia , Colectinas/metabolismo , Complemento C4/metabolismo , Cricetulus , Congelamento , Humanos , Lectinas/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Ligação Proteica , Estatísticas não Paramétricas , Zimosan/química
9.
Front Immunol ; 9: 1998, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30233589

RESUMO

Different families of endogenous lectins use complementary defense strategies against pathogens. They may recognize non-self glycans typically found on pathogens and/or host glycans. The collectin and galectin families are prominent examples of these two lectin categories. Collectins are C-type lectins that contain a carbohydrate recognition domain and a collagen-like domain. Members of this group include surfactant protein A (SP-A) and D (SP-D), secreted by the alveolar epithelium to the alveolar fluid. Lung collectins bind to several microorganisms, which results in pathogen aggregation and/or killing, and enhances phagocytosis of pathogens by alveolar macrophages. Moreover, SP-A and SP-D influence macrophage responses, contributing to resolution of inflammation, and SP-A is essential for tissue-repair functions of macrophages. Galectins also function by interacting directly with pathogens or by modulating the immune system in response to the infection. Direct binding may result in enhanced or impaired infection of target cells, or can have microbicidal effects. Immunomodulatory effects of galectins include recruitment of immune cells to the site of infection, promotion of neutrophil function, and stimulation of the bactericidal activity of infected macrophages. Moreover, intracellular galectins can serve as danger receptors, promoting autophagy of the invading pathogen. This review will focus on the role of collectins and galectins in pathogen clearance and immune response activation in infectious diseases of the respiratory system.


Assuntos
Colectinas/metabolismo , Galectinas/metabolismo , Inflamação/imunologia , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Mucosa Respiratória/imunologia , Infecções Respiratórias/imunologia , Animais , Autofagia , Humanos , Imunidade Inata , Imunomodulação , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Cicatrização
10.
Front Immunol ; 9: 2023, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30237800

RESUMO

The complement system is a dynamic subset of the innate immune system, playing roles in host defense, clearance of immune complexes and cell debris, and priming the adaptive immune response. Over the last 40 years our understanding of the complement system has evolved from identifying its presence and recognizing its role in the blood to now focusing on understanding the role of local complement synthesis in health and disease. In particular, the local synthesis of complement was found to have an involvement in mediating ischaemic injury, including following transplantation. Recent work on elucidating the triggers of local complement synthesis and activation in renal tissue have led to the finding that Collectin-11 (CL-11) engages with L-fucose at the site of ischaemic stress, namely at the surface of the proximal tubular epithelial cells. What remains unknown is the precise structure of the damage-associated ligand that participates in CL-11 binding and subsequent complement activation. In this article, we will discuss our hypothesis regarding the role of CL-11 as an integral tissue-based pattern recognition molecule which we postulate has a significant contributory role in complement-mediated ischaemic injury.


Assuntos
Colectinas/metabolismo , Células Epiteliais/fisiologia , Isquemia/imunologia , Transplante de Rim , Rim/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Animais , Ativação do Complemento , Proteínas do Sistema Complemento/metabolismo , Humanos , Rim/patologia
11.
Mol Immunol ; 103: 21-34, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30189385

RESUMO

Collectin-K1 (CL-K1), a multifunctional Ca2+-dependent lectin, is able to bind carbohydrates on pathogens and inhibit infection by direct neutralization, agglutination, opsonization and killing, which plays an important role in innate immunity. In this study, a CL-K1 homolog (OnCL-K1) was identified from Nile tilapia (Oreochromis niloticus) and characterized at expression and agglutination functional levels. The open reading frame of OnCL-K1 is 720 bp of nucleotide sequence encoding a polypeptide of 239 amino acids. The deduced amino acid sequence has two characteristic structures, containing a collagen-like region and a carbohydrate recognition domain. Expression analysis revealed that the OnCL-K1 was highly expressed in the liver, and widely exhibited in other tissues including kidney, intestine and spleen. In addition, the OnCL-K1 expression was significantly up-regulated in spleen and anterior kidney following challenges with a Gram-positive bacterial pathogen (Streptococcus agalactiae) and a Gram-negative bacterial pathogen (Aeromonas hydrophila). The up-regulation of OnCL-K1 expression was also demonstrated in hepatocytes and monocytes/macrophages in vitro stimulation with S. agalactiae and A. hydrophila. Recombinant OnCL-K1 protein was able to agglutinate both S. agalactiae and A. hydrophila in vitro, and participate in the regulation of inflammatory, migration reaction and promote the phagocytosis by monocytes/macrophages. Taken together, the results of this study indicated that OnCL-K1, possessing apparent agglutination, opsonization and killing ability to bacterial pathogens and participating in the regulation mechanisms of the non-specific cellular immune, might be involved in host defense of innate immunity against bacterial infection in Nile tilapia.


Assuntos
Ciclídeos/imunologia , Colectinas/imunologia , Proteínas de Peixes/imunologia , Imunidade Inata/imunologia , Aeromonas hydrophila/imunologia , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Ciclídeos/genética , Ciclídeos/microbiologia , Colectinas/genética , Colectinas/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/genética , Homologia de Sequência de Aminoácidos , Streptococcus agalactiae/imunologia , Streptococcus agalactiae/fisiologia
12.
Front Immunol ; 9: 1757, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30108587

RESUMO

Collectin liver 1 (CL-L1, alias collectin 10) and collectin kidney 1 (CL-K1, alias collectin 11) are oligomeric pattern recognition molecules associated with the complement system, and mutations in either of their genes may lead to deficiency and developmental defects. The two collectins are reportedly localized and synthesized in the liver, kidneys, and adrenals, and can be found in the circulation as heteromeric complexes (CL-LK), which upon binding to microbial high mannose-like glycoconjugates activates the complement system via the lectin activation pathway. The tissue distribution of homo- vs. heteromeric CL-L1 and -K1 complexes, the mechanism of heteromeric complex formation and in which tissues this occurs, is hitherto incompletely described. We have by immunohistochemistry using monoclonal antibodies addressed the precise cellular localization of the two collectins in the main human tissues. We find that the two collectins have widespread and almost identical tissue distribution with a high expression in epithelial cells in endo-/exocrine secretory tissues and mucosa. There is also accordance between localization of mRNA transcripts and detection of proteins, showing that local synthesis likely is responsible for peripheral localization and eventual formation of the CL-LK complexes. The functional implications of the high expression in endo-/exocrine secretory tissue and mucosa is unknown but might be associated with the activity of MASP-3, which has a similar pattern of expression and is known to potentiate the activity of the alternative complement activation pathway.


Assuntos
Colectinas/genética , Epitélio/metabolismo , Membrana Mucosa/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Colectinas/imunologia , Colectinas/metabolismo , Ativação do Complemento/efeitos dos fármacos , Ativação do Complemento/imunologia , Glândulas Endócrinas/metabolismo , Células Epiteliais/metabolismo , Glândulas Exócrinas/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Rim/metabolismo , Fígado/metabolismo , Ligação Proteica
13.
J Immunol Methods ; 457: 30-32, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29625075

RESUMO

A sensitive assay for the functional activity of complement Factor I is described. This is based on its third proteolytic clip whereby Factor I cleaves cell-bound iC3b to cell-bound C3dg and soluble C3c, thereby abolishing conglutination of the cells. Factor H is required as a co-factor for Factor I activity. Because of the low affinity of iC3b for Factor H, the assay needs to be performed at low ionic strength. This assay is easier to perform than those based on the conversion of C3b to iC3b (the first two Factor I clips), there being no need for the unstable intermediate EAC142 or for purified C3.


Assuntos
Complemento C3b/metabolismo , Fator I do Complemento/metabolismo , Imunoensaio/métodos , Colectinas/análise , Colectinas/metabolismo , Fator H do Complemento/metabolismo , Fator I do Complemento/análise , Humanos , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise , Sensibilidade e Especificidade , Soroglobulinas/análise , Soroglobulinas/metabolismo
14.
Sci Rep ; 8(1): 3821, 2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29491476

RESUMO

Tissue stroma is known to be important in regulating Hp-mediated inflammation, but its interaction with Hp and dendritic cells (DCs) remains to be determined. To this end, the potential crosstalk between H. pylori (Hp) infected gastric stromal cells (Hp-GSCs) and DCs was investigated. Primary GSCs from cancerous and adjacent normal tissues were generated from gastric cancer patients, and monocyte-derived DCs were obtained from healthy individuals. Levels of cytokines and prostaglandin E2 (PGE2) were measured by ELISA, and C-type lectin expression in GSCs was assessed by flow cytometry and immunohistochemistry. In a trans-well co-culture system, significantly upregulated DC-derived IL-23 expression was found when DCs were co-cultured with Hp-infected GSCs (Hp-GSCs). Further, PGE2 from Hp-GSCs was discovered to possess the priming effect, which could be inhibited by anti-COLEC12 (Collectin subfamily member 12) Abs, COLEC12 knockdown or when alpha3-fucosyltransferase-null (futB; HP0651) strain of Hp was used. Also, the expression of COLEC12 was co-localized with CD90+ stromal cells in cancerous tissues. Hp-GSCs-conditioned DCs were able to induce the expression of IL-17 from CD4+ T cells, which could be inhibited by IL-23-neutralizing Abs. These results suggested the importance of COLEC12 as a receptor involved in Hp-stromal cell interaction and its subsequent conditioning effect on DCs.


Assuntos
Colectinas/metabolismo , Dinoprostona/metabolismo , Helicobacter pylori/fisiologia , Imunidade Inata , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Receptores Depuradores/metabolismo , Neoplasias Gástricas/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-23/metabolismo , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Células Estromais/metabolismo , Células Estromais/microbiologia , Células Estromais/patologia , Células Th17/imunologia
15.
Dev Comp Immunol ; 84: 230-240, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29481905

RESUMO

Collectins, a subfamily of the C-type lectins, are able to bind non-self glycoconjugates on the surface of microorganisms and inhibit infection by direct neutralization, agglutination and/or opsonization, which play important roles in innate immunity. In this study, a CL11X1-like collectin (OnCL11X1) was identified from Nile tilapia (Oreochromis niloticus) and characterized at expression and agglutination functional levels. The open reading frame of OnCL11X1 is 840 bp of nucleotide sequence encoding polypeptides of 279 amino acids. The deduced amino acid sequence is highly homology to teleost and similar to mammalian CL11X1, containing a canonical collagen-like region, a carbohydrate recognition domain and a neck region. Expression analysis revealed that the OnCL11X1 was highly expressed in the liver, and widely exhibited in other tissues including kidney, intestines and spleen. In addition, the OnCL11X1 expression was significantly up-regulated in spleen and anterior kidney following challenges with a Gram-positive bacterial pathogen (Streptococcus agalactiae) and a Gram-negative bacterial pathogen (Aeromonas hydrophila). The up-regulation of OnCL11X1 expression was also demonstrated in hepatocytes and macrophages in vitro stimulation with S. agalactiae and A. hydrophila. Recombinant OnCL11X1 protein was able to agglutinate both S. agalactiae and A. hydrophila in vitro and promote the phagocytosis by macrophages. Taken together, the results of this study indicated that OnCL11X1, possessing apparent agglutination and opsonization ability to bacterial pathogens, might be involved in host defense against bacterial infection in Nile tilapia.


Assuntos
Aeromonas hydrophila/imunologia , Ciclídeos/imunologia , Colectinas/metabolismo , Proteínas de Peixes/metabolismo , Infecções por Bactérias Gram-Negativas/imunologia , Fígado/fisiologia , Macrófagos/fisiologia , Infecções Estreptocócicas/imunologia , Streptococcus agalactiae/imunologia , Aglutinação , Animais , Células Cultivadas , Clonagem Molecular , Colectinas/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Imunidade Inata
16.
Front Immunol ; 9: 3159, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30804949

RESUMO

Bovine conglutinin, the first animal collectin to be discovered, is structurally very similar to Surfactant Protein D (SP-D). SP-D is known to interact with Mycobacterium tuberculosis, and the closely-related M. bovis, the causative agent of bovine tuberculosis. We speculated that due to the overall similarities between conglutinin and SP-D, conglutinin is likely to have a protective influence in bovine tuberculosis. We set out to investigate the role of conglutinin in host-pathogen interaction during mycobacterial infection. We show here that a recombinant truncated form of conglutinin (rfBC), composed of the neck and C-type lectin domains, binds specifically and in a dose-dependent manner to the model organism Mycobacterium bovis BCG. rfBC showed a significant direct bacteriostatic effect on the growth of M. bovis BCG in culture. In addition, rfBC inhibited the uptake of M. bovis BCG by THP-1 macrophages (human monocyte lineage cell line) and suppressed the subsequent pro-inflammatory response. Conglutinin is well-known as a binder of the complement activation product, iC3b. rfBC was also able to inhibit the uptake of complement-coated M. bovis BCG by THP-1 macrophages, whilst modulating the pro-inflammatory response. It is likely that rfBC inhibits the phagocytosis of mycobacteria by two distinct mechanisms: firstly, rfBC interferes with mannose receptor-mediated uptake by masking lipoarabinomannan (LAM) on the mycobacterial surface. Secondly, since conglutinin binds iC3b, it can interfere with complement receptor-mediated uptake via CR3 and CR4, by masking interactions with iC3b deposited on the mycobacterial surface. rfBC was also able to modulate the downstream pro-inflammatory response in THP-1 cells, which is important for mobilizing the adaptive immune response, facilitating containment of mycobacterial infection. In conclusion, we show that conglutinin possesses complement-dependent and complement-independent anti-mycobacterial activities, interfering with both known mechanisms of mycobacterial uptake by macrophages. As mycobacteria are specialized intracellular pathogens, conglutinin may inhibit M. bovis and M. tuberculosis from establishing an intracellular niche within macrophages, and thus, negatively affect the long-term survival of the pathogen in the host.


Assuntos
Colectinas/imunologia , Proteínas do Sistema Complemento/imunologia , Mycobacterium bovis/imunologia , Soroglobulinas/imunologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/microbiologia , Animais , Biomarcadores , Bovinos , Colectinas/metabolismo , Proteínas do Sistema Complemento/metabolismo , Citocinas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Fagocitose/imunologia , Soroglobulinas/metabolismo , Células THP-1 , Tuberculose Bovina/metabolismo
17.
Int Urol Nephrol ; 50(4): 695-703, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29071557

RESUMO

The innate immune system serves as the frontline defense against invading pathogens and initiates an inflammatory response to microorganisms. Collectins are C-type lectins that are structurally characterized by a collagen-like sequence and a carbohydrate recognition domain. Moreover, they are widely expressed throughout the body and are involved in the innate immunity against a variety of pathogens, regulating inflammation, and protecting the lungs from pathogens. Recently, two classical collectins, surfactant protein A (SP-A) and surfactant protein D (SP-D), as well as novel collectin 11, were found present in urinary tract tissues. They are increasingly recognized as key players in activating the humoral arm of innate immunity and host defense in urinary tract and kidney diseases, although their biological features, functions, and mechanisms in this regard remain largely unclear. In this review, we aim to integrate results reported by ourselves and others to summarize and gain a better understanding of the functions of collectins (SP-A, SP-D, and collectin 11) in urinary tract and kidney diseases.


Assuntos
Colectinas/imunologia , Rim/patologia , Infecções Urinárias/imunologia , Sistema Urinário/imunologia , Lesão Renal Aguda/imunologia , Lesão Renal Aguda/metabolismo , Aterosclerose/imunologia , Aterosclerose/metabolismo , Colectinas/metabolismo , Fibrose , Humanos , Imunidade Inata , Proteína A Associada a Surfactante Pulmonar/imunologia , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/imunologia , Proteína D Associada a Surfactante Pulmonar/metabolismo , Sistema Urinário/metabolismo , Infecções Urinárias/metabolismo
18.
J Am Soc Nephrol ; 29(1): 168-181, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29142050

RESUMO

Collectin-11 is a recently described soluble C-type lectin, a pattern recognition molecule of the innate immune system that has distinct roles in host defense, embryonic development, and acute inflammation. However, little is known regarding the role of collectin-11 in tissue fibrosis. Here, we investigated collectin-11 in the context of renal ischemia-reperfusion injury. Compared with wild-type littermate controls, Collec11 deficient (CL-11-/- ) mice had significantly reduced renal functional impairment, tubular injury, renal leukocyte infiltration, renal tissue inflammation/fibrogenesis, and collagen deposition in the kidneys after renal ischemia-reperfusion injury. In vitro, recombinant collectin-11 potently promoted leukocyte migration and renal fibroblast proliferation in a carbohydrate-dependent manner. Additionally, compared with wild-type kidney grafts, CL-11-/-mice kidney grafts displayed significantly reduced tubular injury and collagen deposition after syngeneic kidney transplant. Our findings demonstrate a pathogenic role for collectin-11 in the development of tubulointerstitial fibrosis and suggest that local collectin-11 promotes this fibrosis through effects on leukocyte chemotaxis and renal fibroblast proliferation. This insight into the pathogenesis of tubulointerstitial fibrosis may have implications for CKD mediated by other causes as well.


Assuntos
Proliferação de Células/efeitos dos fármacos , Quimiotaxia de Leucócito/genética , Colectinas/genética , Colectinas/farmacologia , Túbulos Renais/patologia , Nefrite/genética , Aloenxertos/patologia , Animais , Quimiotaxia de Leucócito/efeitos dos fármacos , Colágeno/metabolismo , Colectinas/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/fisiologia , Fibrose , Transplante de Rim , Túbulos Renais/metabolismo , Masculino , Camundongos , Camundongos Knockout , Nefrite/etiologia , Nefrite/patologia , Nefrite/fisiopatologia , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/patologia
19.
Semin Immunopathol ; 40(1): 75-85, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28894916

RESUMO

Pattern recognition molecules are sensors for the innate immune system and trigger a number of pathophysiological functions after interaction with the corresponding ligands on microorganisms or altered mammalian cells. Of those pattern recognition molecules used by the complement system, collagen-like lectins (collectins) are an important subcomponent. Whereas the best known of these collectins, mannose-binding lectin, largely occurs as a circulating protein following production by hepatocytes, the most recently described collectins exhibit strong local biosynthesis. This local production and release of soluble collectin molecules appear to serve local tissue functions at extravascular sites, including a developmental function. In this article, we focus on the characteristics of collectin-11 (CL-11 or CL-K1), whose ubiquitous expression and multiple activities likely reflect a wide biological relevance. Collectin-11 appears to behave as an acute phase protein whose production associated with metabolic and physical stress results in locally targeted inflammation and tissue cell death. Early results indicate the importance of fucosylated ligand marking the injured cells targeted by collectin-11, and we suggest that further characterisation of this and related ligands will lead to better understanding of pathophysiological significance and exploitation for clinical benefit.


Assuntos
Colectinas/química , Colectinas/metabolismo , Suscetibilidade a Doenças , Lectinas/química , Lectinas/metabolismo , Animais , Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Suscetibilidade a Doenças/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade
20.
Sci Rep ; 7(1): 14625, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29116192

RESUMO

Age-related macular degeneration (AMD) is a major cause of blindness and is associated with complement dysregulation. The disease is a potential target for stem cell therapy but success is likely to be limited by the inflammatory response. We investigated the innate immune properties of human induced-pluripotent stem cell (iPSC)-derived RPE cells, particularly with regard to the complement pathway. We focused on collectin-11 (CL-11), a pattern recognition molecule that can trigger complement activation in renal epithelial tissue. We found evidence of constitutive and hypoxia-induced expression of CL-11 in iPS-RPE cells, and in the extracellular fluid. Complement activation on the cell surface occurred in conjunction with CL-11 binding. CL-11 has been shown to activate inflammatory responses through recognition of L-fucose, which we confirmed by showing that fucosidase-treated cells, largely, failed to activate complement. The presence of CL-11 in healthy murine and human retinal tissues confirmed the biological relevance of CL-11. Our data describe a new trigger mechanism of complement activation that could be important in disease pathogenesis and therapeutic interventions.


Assuntos
Colectinas/metabolismo , Ativação do Complemento/imunologia , Complemento C3/metabolismo , Hipóxia/fisiopatologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Epitélio Pigmentado da Retina/fisiopatologia , Animais , Células Cultivadas , Complemento C3/imunologia , Olho/citologia , Olho/fisiopatologia , Fucose/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Estresse Oxidativo , Epitélio Pigmentado da Retina/citologia
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