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1.
PLoS Pathog ; 17(6): e1009655, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34125873

RESUMO

Microbial pathogens bind host complement regulatory proteins to evade the immune system. The bacterial pathogen Neisseria meningitidis, or meningococcus, binds several complement regulators, including human Factor H (FH). FH binding protein (FHbp) is a component of two licensed meningococcal vaccines and in mice FHbp elicits antibodies that inhibit binding of FH to FHbp, which defeat the bacterial evasion mechanism. However, humans vaccinated with FHbp develop antibodies that enhance binding of FH to the bacteria, which could limit the effectiveness of the vaccines. In the present study, we show that two vaccine-elicited antibody fragments (Fabs) isolated from different human subjects increase binding of complement FH to meningococcal FHbp by ELISA. The two Fabs have different effects on the kinetics of FH binding to immobilized FHbp as measured by surface plasmon resonance. The 1.7- and 2.0-Å resolution X-ray crystal structures of the Fabs in complexes with FHbp illustrate that the two Fabs bind to similar epitopes on the amino-terminal domain of FHbp, adjacent to the FH binding site. Superposition models of ternary complexes of each Fab with FHbp and FH show that there is likely minimal contact between the Fabs and FH. Collectively, the structures reveal that the Fabs enhance binding of FH to FHbp by altering the conformations and mobilities of two loops adjacent to the FH binding site of FHbp. In addition, the 1.5 Å-resolution structure of one of the isolated Fabs defines the structural rearrangements associated with binding to FHbp. The FH-enhancing human Fabs, which are mirrored in the human polyclonal antibody responses, have important implications for tuning the effectiveness of FHbp-based vaccines.


Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Fator H do Complemento/imunologia , Vacinas Meningocócicas/imunologia , Anticorpos Antibacterianos/metabolismo , Fator H do Complemento/metabolismo , Humanos , Infecções Meningocócicas/prevenção & controle , Neisseria meningitidis , Receptores de Complemento/imunologia , Receptores de Complemento/metabolismo , Fatores de Virulência/imunologia
2.
PLoS One ; 16(6): e0252577, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34133431

RESUMO

Exosomes are a class of extracellular vesicles (EVs) that are mediators of normal intercellular communication, but exosomes are also used by tumor cells to promote oncogenesis and metastasis. Complement factor H (CFH) protects host cells from attack and destruction by the alternative pathway of complement-dependent cytotoxicity (CDC). Here we show that CFH can protect exosomes from complement-mediated lysis and phagocytosis. CFH was found to be associated with EVs from a variety of tumor cell lines as well as EVs isolated from the plasma of patients with metastatic non-small cell lung cancer. Higher levels of CFH-containing EVs correlated with higher metastatic potential of cell lines. GT103, a previously described antibody to CFH that preferentially causes CDC of tumor cells, was used to probe the susceptibility of tumor cell-derived exosomes to destruction. Exosomes were purified from EVs using CD63 beads. Incubation of GT103 with tumor cell-derived exosomes triggered exosome lysis primarily by the classical complement pathway as well as antibody-dependent exosome phagocytosis by macrophages. These results imply that GT103-mediated exosome destruction can be triggered by antibody Fc-C1q interaction (in the case of lysis), and antibody-Fc receptor interactions (in the case of phagocytosis). Thus, this work demonstrates CFH is expressed on tumor cell derived exosomes, can protect them from complement lysis and phagocytosis, and that an anti-CFH antibody can be used to target tumor-derived exosomes for exosome destruction via innate immune mechanisms. These findings suggest that a therapeutic CFH antibody has the potential to inhibit tumor progression and reduce metastasis promoted by exosomes.


Assuntos
Fator H do Complemento/metabolismo , Exossomos/metabolismo , Macrófagos/imunologia , Fagocitose/fisiologia , Anticorpos/imunologia , Anticorpos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Fator H do Complemento/imunologia , Humanos , Imunidade Inata , Leucócitos Mononucleares/citologia , Neoplasias Pulmonares/patologia , Macrófagos/citologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Estadiamento de Neoplasias , Receptores de Complemento/metabolismo , Células Tumorais Cultivadas
3.
Nat Immunol ; 22(6): 757-768, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34031614

RESUMO

Maturation of B cells within germinal centers (GCs) generates diversified B cell pools and high-affinity B cell antigen receptors (BCRs) for pathogen clearance. Increased receptor affinity is achieved by iterative cycles of T cell-dependent, affinity-based B cell positive selection and clonal expansion by mechanisms hitherto incompletely understood. Here we found that, as part of a physiologic program, GC B cells repressed expression of decay-accelerating factor (DAF/CD55) and other complement C3 convertase regulators via BCL6, but increased the expression of C5b-9 inhibitor CD59. These changes permitted C3 cleavage on GC B cell surfaces without the formation of membrane attack complex and activated C3a- and C5a-receptor signals required for positive selection. Genetic disruption of this pathway in antigen-activated B cells by conditional transgenic DAF overexpression or deletion of C3a and C5a receptors limited the activation of mechanistic target of rapamycin (mTOR) in response to BCR-CD40 signaling, causing premature GC collapse and impaired affinity maturation. These results reveal that coordinated shifts in complement regulation within the GC provide crucial signals underlying GC B cell positive selection.


Assuntos
Linfócitos B/imunologia , Ativação do Complemento , Complemento C3a/metabolismo , Complemento C5a/metabolismo , Centro Germinativo/imunologia , Animais , Animais Geneticamente Modificados , Linfócitos B/metabolismo , Antígenos CD55/genética , Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Linhagem Celular Tumoral , Hematopoiese Clonal/imunologia , Centro Germinativo/citologia , Centro Germinativo/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Tonsila Palatina/citologia , Tonsila Palatina/patologia , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/metabolismo
4.
Front Immunol ; 12: 662164, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995387

RESUMO

The ß2-integrin receptor family has a broad spectrum of physiological functions ranging from leukocyte adhesion, cell migration, activation, and communication to the phagocytic uptake of cells and particles. Among the members of this family, complement receptor 3 (CR3; CD11b/CD18, Mac-1, αMß2) is particularly promiscuous in its functional profile and ligand selectivity. There are close to 100 reported structurally unrelated ligands for CR3, and while many ligands appear to cluster at the αMI domain, molecular details about binding modes remain largely elusive. The versatility of CR3 is reflected in its functional portfolio, which includes prominent roles in the removal of invaders and cell debris, induction of tolerance and synaptic pruning, and involvement in the pathogenesis of numerous autoimmune and chronic inflammatory pathologies. While CR3 is an interesting therapeutic target for immune modulation due to these known pathophysiological associations, drug development efforts are limited by concerns of potential interference with host defense functions and, most importantly, an insufficient molecular understanding of the interplay between ligand binding and functional impact. Here, we provide a systematic summary of the various interaction partners of CR3 with a focus on binding mechanisms and functional implications. We also discuss the roles of CR3 as an immune receptor in health and disease, as an activation marker in research and diagnostics, and as a therapeutic target.


Assuntos
Receptores de Complemento/imunologia , Receptores de Complemento/metabolismo , Animais , Movimento Celular , Desenvolvimento de Medicamentos , Humanos , Integrinas/imunologia , Leucócitos/metabolismo , Ligantes , Camundongos , Neutrófilos/imunologia , Receptores de Complemento/classificação
5.
J Biol Chem ; 296: 100801, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019878

RESUMO

Phagocytosis plays diverse roles in biology, but our understanding of the purpose, interplay, and cell signaling mechanisms associated with different modes of phagocytosis is limited, without being able to capture and visualize each step in this rapid process from the beginning to end. A new study by Walbaum et al. uses stunning time-lapse 3D imaging of the engulfment of erythrocytes by macrophages via sinking, ruffling, and cup formation, unequivocally confirming a visionary 44-year-old theory derived from still electron microscopy photos that phagocytosis mediated by complement receptor CR3 occurs via a sinking mechanism and antibody-mediated phagocytosis occurs via phagocytic cup formation. The article also challenges the dogma, showing that phagocytic cup formation is not unique to antibody receptor phagocytosis, rather CR3 plays a complex role in different modes of phagocytosis. For example, inhibition of antibody-mediated phagocytosis leads to a compensatory upregulation of CR3-mediated sinking phagocytosis. These findings animate, in vivid colors, processes previously only captured as stills, exposing interactions between different phagocytic mechanisms and altering our basic understanding of this important process.


Assuntos
Fagócitos/metabolismo , Receptores de Complemento/metabolismo , Receptores de IgG/metabolismo , Animais , Proteínas do Sistema Complemento/fisiologia , Fagocitose/fisiologia
6.
Cell Death Dis ; 12(6): 526, 2021 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-34023853

RESUMO

Thymic atrophy in sepsis is a critical disadvantage because it induces immunosuppression and increases the mortality rate as the disease progresses. However, the exact mechanism of thymic atrophy has not been fully elucidated. In this study, we discovered a novel role for VSIG4-positive peritoneal macrophages (V4(+) cells) as the principal cells that induce thymic atrophy and thymocyte apoptosis. In CLP-induced mice, V4(+) cells were activated after ingestion of invading microbes, and the majority of these cells migrated into the thymus. Furthermore, these cells underwent a phenotypic shift from V4(+) to V4(-) and from MHC II(low) to MHC II(+). In coculture with thymocytes, V4(+) cells mainly induced apoptosis in DP thymocytes via the secretion of TNF-α. However, there was little effect on CD4 or CD8 SP and DN thymocytes. V4(-) cells showed low levels of activity compared to V4(+) cells. Thymic atrophy in CLP-induced V4(KO) mice was much less severe than that in CLP-induced wild-type mice. In addition, V4(KO) peritoneal macrophages also showed similar activity to V4(-) cells. Taken together, the current study demonstrates that V4(+) cells play important roles in inducing immunosuppression via thymic atrophy in the context of severe infection. These data also suggest that controlling the function of V4(+) cells may play a crucial role in the development of new therapies to prevent thymocyte apoptosis in sepsis.


Assuntos
Macrófagos Peritoneais/fisiologia , Receptores de Complemento/metabolismo , Sepse/patologia , Timócitos/fisiologia , Animais , Apoptose/genética , Ceco/patologia , Ceco/cirurgia , Modelos Animais de Doenças , Feminino , Ligadura , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Punções , Receptores de Complemento/genética , Sepse/genética , Sepse/metabolismo , Timócitos/metabolismo , Timócitos/patologia , Fator de Necrose Tumoral alfa/metabolismo
7.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799879

RESUMO

The development of nanocarriers (NC) for biomedical applications has gained large interest due to their potential to co-deliver drugs in a cell-type-targeting manner. However, depending on their surface characteristics, NC accumulate serum factors, termed protein corona, which may affect their cellular binding. We have previously shown that NC coated with carbohydrates to enable biocompatibility triggered the lectin-dependent complement pathway, resulting in enhanced binding to B cells via complement receptor (CR)1/2. Here we show that such NC also engaged all types of splenic leukocytes known to express CR3 at a high rate when NC were pre-incubated with native mouse serum resulting in complement opsonization. By focusing on dendritic cells (DC) as an important antigen-presenting cell type, we show that CR3 was essential for binding/uptake of complement-opsonized NC, whereas CR4, which in mouse is specifically expressed by DC, played no role. Further, a minor B cell subpopulation (B-1), which is important for first-line pathogen responses, and co-expressed CR1/2 and CR3, in general, engaged NC to a much higher extent than normal B cells. Here, we identified CR-1/2 as necessary for binding of complement-opsonized NC, whereas CR3 was dispensable. Interestingly, the binding of complement-opsonized NC to both DC and B-1 cells affected the expression of activation markers. Our findings may have important implications for the design of nano-vaccines against infectious diseases, which codeliver pathogen-specific protein antigen and adjuvant, aimed to induce a broad adaptive cellular and humoral immune response by inducing cytotoxic T lymphocytes that kill infected cells and pathogen-neutralizing antibodies, respectively. Decoration of nano-vaccines either with carbohydrates to trigger complement activation in vivo or with active complement may result in concomitant targeting of DC and B cells and thereby may strongly enhance the extent of dual cellular/humoral immune responses.


Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Antígeno CD11b/imunologia , Proteínas do Sistema Complemento/imunologia , Células Dendríticas/imunologia , Receptores de Complemento/imunologia , Animais , Subpopulações de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Células Cultivadas , Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Células Dendríticas/metabolismo , Dextranos/química , Portadores de Fármacos/química , Humanos , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nanopartículas/química , Proteínas Opsonizantes/imunologia , Proteínas Opsonizantes/metabolismo , Fagocitose/imunologia , Receptores de Complemento/metabolismo
8.
Front Immunol ; 12: 660194, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33868311

RESUMO

Inflammation is a common denominator of diseases. The complement system, an intrinsic part of the innate immune system, is a key driver of inflammation in numerous disorders. Recently, a family of proteins has been suggested to be of vital importance in conditions characterized by complement dysregulation: the human Factor H (FH) family. This group of proteins consists of FH, Factor H-like protein 1 and five Factor H-related proteins. The FH family has been linked to infectious, vascular, eye, kidney and autoimmune diseases. In contrast to FH, the functions of the other highly homologous proteins are largely unknown and, hence, their role in the different disease-specific pathogenic mechanisms remains elusive. In this perspective review, we address the major challenges ahead in this emerging area, including 1) the controversies about the functional roles of the FH protein family, 2) the discrepancies in quantification of the FH protein family, 3) the unmet needs for validated tools and 4) limitations of animal models. Next, we also discuss the opportunities that exist for the immunology community. A strong multidisciplinary approach is required to solve these obstacles and is only possible through interdisciplinary collaboration between biologists, chemists, geneticists and physicians. We position this review in light of our own perspective, as principal investigators of the SciFiMed Consortium, a consortium aiming to create a comprehensive analytical system for the quantitative and functional assessment of the entire FH protein family.


Assuntos
Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Inflamação/imunologia , Degeneração Macular/imunologia , Receptores de Complemento/imunologia , Animais , Fator H do Complemento/análise , Fator H do Complemento/imunologia , Fator H do Complemento/metabolismo , Proteínas do Sistema Complemento/análise , Proteínas do Sistema Complemento/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Inflamação/sangue , Inflamação/metabolismo , Degeneração Macular/metabolismo , Receptores de Complemento/metabolismo , Reprodutibilidade dos Testes
9.
Bioengineered ; 12(1): 1391-1402, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33904378

RESUMO

Rab18 and V-set and immunoglobulin domain-containing 4 (VSIG4) were reportedly implicated in the malignant progression of glioma. In this study, their relationship was further explored, accompanied by the investigation into their effects on the sensitivity of temozolomide (TMZ). The proliferation and apoptosis of U87-MG and U251-MG were detected after Rab18 silencing through CCK8 assay and flow cytometry, respectively. The interaction between Rab18 and VSIG4 was predicted through database and verified by immunoprecipitation assay. The suspicion that whether the sensitivity of glioma to temozolomide was affected by the Rab18-VSIG4 interaction was explored through CCK8 assay. We observed decreased proliferation and increased apoptosis and TMZ sensitivity in U87-MG and U251-MG treated by siRNA-Rab18. Not only was the interaction predicted using database, but also it was confirmed by IP assay. Intriguingly, VSIG4 overexpression effectively reversed above biological process and TMZ sensitivity caused by Rab18 silencing. To conclude, the Rab18-VSIG4 interaction was implicated in the proliferation and apoptosis of glioma, as well as TMZ sensitivity. Targeting the interaction between Rab18 and VSIG4 may help exploit new therapies to enhance TMZ sensitivity for treating patients with glioma.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Glioma/metabolismo , Receptores de Complemento/metabolismo , Temozolomida/farmacologia , Proteínas rab de Ligação ao GTP/metabolismo , Antineoplásicos Alquilantes/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Glioma/genética , Humanos , Receptores de Complemento/genética , Proteínas rab de Ligação ao GTP/genética
10.
Front Immunol ; 12: 655753, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33912182

RESUMO

Follicular dendritic cells (FDCs) are rare and enigmatic cells that mainly reside in germinal centers (GCs). They are capable of capturing immune complexes, via their Fc (FcRs) and complement receptors (CRs) and storing them for long periods in non-degradative vesicles. Presentation of ICs on FDCs to B cells is believed to drive affinity maturation. CR1 and CR2 are expressed on B cells and FDCs. Cr2 knock out (KO) mice, lacking both receptors, have impaired antibody and GC responses. Utilizing a novel ImageJ macro to analyze confocal fluorescence microscopy images of spleen sections, we here investigate how FDCs in wild type (WT) and Cr2 KO mice behave during the first two weeks after immunization with sheep red blood cells (SRBC). Mice were immunized with SRBC i.v. and spleen and serum samples harvested at various time points. As expected, antibody and GC responses in Cr2 KO mice were impaired in comparison to WT mice. Fewer FDCs were identified in Cr2 KO mice, and these exhibited differential localization and organization in comparison to WT mice. WT FDCs were primarily located within GCs at the light zone/dark zone border. FDCs from WT but not Cr2 KO mice were actively dispersed in GCs, i.e. tended to move away from each other, presumably to increase their surface area for B cell interaction. FDCs from Cr2 KO mice were more often found on follicles outside of the GCs and those within the GCs were closer to the periphery in comparison to WT FDCs. Expression of CR1 and CR2, FcγRIIB, and FcµR increased in FDCs from WT mice during the course of immunization. The results suggest that decreased ability to capture ICs by FDCs lacking CR1 and CR2 may not be the only explanation for the impaired GC and antibody responses in Cr2 KO mice. Poor FDC organization in GCs and failure to increase receptor expression after immunization may further contribute to the inefficient immune responses observed.


Assuntos
Células Dendríticas Foliculares/imunologia , Células Dendríticas Foliculares/metabolismo , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Imagem Molecular , Receptores de Complemento 3d/metabolismo , Receptores de Complemento/metabolismo , Animais , Formação de Anticorpos , Complexo Antígeno-Anticorpo/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores , Feminino , Imunofluorescência , Imunofenotipagem , Masculino , Camundongos , Camundongos Knockout , Receptores Fc/metabolismo , Baço
11.
Brain Behav Immun ; 95: 310-320, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33838249

RESUMO

Complement pathway over-activation has been implicated in a variety of neurological diseases. However, the signaling pathways governing astrocytic complement activation in Parkinson's disease (PD) are poorly understood. Kir6.1, a pore-forming subunit of ATP-sensitive potassium (K-ATP) channel, is prominently expressed in astrocytes and exhibits anti-inflammatory effects. Therefore, we hypothesize that Kir6.1/K-ATP channel may regulate astrocytic complement activation in the pathogenesis of PD. In this study, astrocytic Kir6.1 knockout (KO) mice were used to examine the effect of astrocytic Kir6.1/K-ATP channel on astrocytic complement activation triggered by the lipopolysaccharide (LPS). Here, we found that astrocytic Kir6.1 KO mice showed more dopaminergic neuron loss and more astrocyte reactivity in substantia nigra compacta than controls. We also found that astrocytic Kir6.1 KO increased the expression of complement C3 in astrocytes in LPS-induced mouse model of PD. Mechanistically, astrocytic Kir6.1 KO promoted astroglial NF-κB activation to elicit extracellular release of C3, which in turn interacted with neuronal C3aR to induce neuron death. Blocking complement function by NF-κB inhibitor or C3aR antagonist rescued the aggravated neuron death induced by Kir6.1 KO. Collectively, our findings reveal that astrocytic Kir6.1/K-ATP channel prevents neurodegeneration in PD via astrocyte-neuron cross talk through NF-κB/C3/C3aR signaling and suggest that targeting astroglial Kir6.1/K-ATP channel-NF-κB-C3-neuronal C3aR signaling represents a novel therapeutic strategy for PD.


Assuntos
Astrócitos , Canais KATP/genética , Doença de Parkinson , Animais , Complemento C3/metabolismo , Deleção de Genes , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios , Receptores de Complemento/metabolismo
12.
Front Immunol ; 12: 628062, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33746964

RESUMO

Complement component 3 fragment C3a is an anaphylatoxin involved in promoting cellular responses important in immune response and host defense. Its receptor (C3a receptor, C3aR) is distributed on the plasma membrane; however, lysosomal localization in immune cells has been reported. Oxidative stress increases intracellular reactive oxygen species (ROS), and ROS activate complement signaling in immune cells and metabolic reprogramming. Here we tested oxidative stress and intracellular complement in mitochondrial dysfunction in RPE cells using high resolution live-cell imaging, and metabolism analysis in isolated mitochondria using Seahorse technology. While C3aR levels were unaffected by oxidative stress, its cell membrane levels decreased and mitochondrial (mt) localization increased. Trafficking was dependent on endocytosis, utilizing endosomal-to-mitochondrial cargo transfer. H2O2-treatment also increased C3a-mtC3aR co-localization dose-dependently. In isolated mitochondria from H2O2-treated cells C3a increased mitochondrial Ca2+ uptake, that could be inhibited by C3aR antagonism (SB290157), mitochondrial Ca2+ uniporter blocker (Ru360), and Gαi-protein inhibition (pertussis toxin, PTX); and inhibited mitochondrial repiration in an SB290157- and PTX-dependent manner. Specifically, mtC3aR activation inhibited state III ADP-driven respiration and maximal respiratory capacity. Mitochondria from control cells did not respond to C3a. Furthermore, transmitochondrial cybrid ARPE-19 cells harboring J haplogroup mitochondria that confer risk for age-related macular degeneration, showed high levels of mtC3aR and reduced ATP production upon C3a stimulation. Our findings suggest that oxidative stress increases mtC3aR, leading to altered mitochondrial calcium uptake and ATP production. These studies will have important implication in our understanding on the balance of extra- and intracellular complement signaling in controlling cellular health and dysfunction.


Assuntos
Metabolismo Energético , Células Epiteliais/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Receptores de Complemento/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Linhagem Celular , Respiração Celular , Endocitose , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Humanos , Peróxido de Hidrogênio/toxicidade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Estresse Oxidativo/efeitos dos fármacos , Transporte Proteico , Receptores de Complemento/genética , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/imunologia
13.
Front Immunol ; 12: 580594, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33767691

RESUMO

The zoonotic intracellular bacterium Chlamydia psittaci causes life-threatening pneumonia in humans. During mouse lung infection, complement factor C3 and the anaphylatoxin C3a augment protection against C. psittaci by a so far unknown mechanism. To clarify how complement contributes to the early, innate and the late, specific immune response and resulting protection, this study addresses the amount of C3, the timing when its presence is required as well as the anaphylatoxin receptor(s) mediating its effects and the complement-dependent migration of dendritic cells. Challenge experiments with C. psittaci on various complement KO mice were combined with transient decomplementation by pharmacological treatment, as well as the analysis of in vivo dendritic cells migration. Our findings reveal that a plasma concentration of C3 close to wildtype levels was required to achieve full protection. The diminished levels of C3 of heterozygote C3+/- mice permitted already relative effective protection and improved survival as compared to C3-/- mice, but overall recovery of these animals was delayed. Complement was in particular required during the first days of infection. However, additionally, it seems to support protection at later stages. Migration of CD103+ dendritic cells from the infected lung to the draining lymph node-as prerequisite of antigen presentation-depended on C3 and C3aR and/or C5aR. Our results provide unique mechanistic insight in various aspects of complement-dependent immune responses under almost identical, rather physiological experimental conditions. Our study contributes to an improved understanding of the role of complement, and C3a in particular, in infections by intracellular bacteria.


Assuntos
Movimento Celular/imunologia , Infecções por Chlamydiaceae/imunologia , Chlamydophila psittaci/imunologia , Complemento C3a/imunologia , Células Dendríticas/imunologia , Pulmão/imunologia , Anafilatoxinas/imunologia , Anafilatoxinas/metabolismo , Animais , Linhagem Celular , Infecções por Chlamydiaceae/metabolismo , Infecções por Chlamydiaceae/microbiologia , Chlamydophila psittaci/fisiologia , Ativação do Complemento/imunologia , Complemento C3a/genética , Complemento C3a/metabolismo , Células Dendríticas/citologia , Células Dendríticas/microbiologia , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Complemento/genética , Receptores de Complemento/imunologia , Receptores de Complemento/metabolismo , Transdução de Sinais/imunologia , Análise de Sobrevida
14.
Med Sci Monit ; 27: e927977, 2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33539329

RESUMO

BACKGROUND This study was designed to explore the incompletely investigated role of the complement component 3a receptor 1 (C3AR1) in the prognosis of stomach adenocarcinomas (STAD). MATERIAL AND METHODS Using bioinformatic methods, we systematically determined the expression and prognosis value of C3AR1 in various cancers by using the TIMER (Tumor Immune Estimation Resource) database, UALCAN platform, GEPIA (Gene Expression Profiling Interactive Analysis) server, and the OncoLnc tool. The biological processes influenced by C3AR1 were determined using the GSEA (Gene Set Enrichment Analysis) software (Copyright 2004-2020 Broad Institute, Inc., Massachusetts Institute of Technology, and Regents of the University of California). The correlation between C3AR1 expression and the immune-infiltrating cells as well as the correlation analysis between C3AR1 expression and the corresponding immune-marker sets were conducted using the TIMER and GEPIA databases. RESULTS The expression of C3AR1 was significantly (P<0.001) differentially expressed on several tumor types, while its prognosis value could only be determined on STAD, with a high expression of C3AR1 closely correlated with a poor prognosis. The GSEA analysis revealed that the differential expression of C3AR1 profoundly affected the immune-related biological processes. The expression of C3AR1 was strongly and positively correlated with the infiltration of monocytes, tumor-associated macrophages, M2 macrophages, dendritic cells, and exhausted T cells. CONCLUSIONS Our results have revealed that a high expression of C3AR1 is positively correlated with a poor prognosis and increased tumor-immune infiltration. C3AR1 can promote the polarization of M2 macrophages and T cell exhaustion, leading to the immune escape of STAD. These findings suggest that C3AR1 could be used as a prognostic and immune-infiltration marker in the pathogenesis of STAD.


Assuntos
Receptores de Complemento/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Biomarcadores Tumorais/genética , Biologia Computacional/métodos , Bases de Dados Genéticas , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Prognóstico , Receptores de Complemento/metabolismo , Transcriptoma/genética
15.
J Innate Immun ; 13(3): 164-178, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33445177

RESUMO

To study the molecular interplay between TLRs and complement representing ancient danger-sensing mechanisms, we examined the regulation of the C3a/anaphylatoxin C3a receptor (C3aR) axis in normal human epidermal keratinocytes (NHEKs) by treatment with different TLR ligands. Protein staining followed by flow cytometry revealed highly constitutive intracellular expression levels of the C3aR in NHEKs. Stimulation with Poly I:C up-regulated C3aR mRNA and intra- and extracellular expression in NHEKs which showed functional relevance by up-regulating CXCL10 and down-regulating C3 expression in response to C3a. mRNA and protein levels of C3 and protease cathepsin L (CTSL) that can cleave C3 were up-regulated by the TLR3 ligand Poly I:C. Enhanced intracellular expression levels of the biologically active C3 fragment (C3a), in response to TLR3 stimulation were also detectable in NHEKs. Cathelicidin antimicrobial peptide LL-37 potentiated Poly I:C-induced C3aR, C3, and CTSL up-regulation. In conclusion, we point to a role of TLR3 to promote up-regulation of C3aR, C3, and CTSL expression levels and generation of C3a. Our data provide evidence that local generation and activation of complement components as described for T cells or myeloid cells represent a scenario which may take place in a similar way in NHEKs.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Complemento C3a/metabolismo , Células Epidérmicas/imunologia , Queratinócitos/imunologia , Receptores de Complemento/metabolismo , Receptor 3 Toll-Like/metabolismo , Catepsina L/metabolismo , Células Cultivadas , Quimiocina CXCL10/metabolismo , Ativação do Complemento , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Poli I-C/imunologia , Cultura Primária de Células
16.
J Invest Dermatol ; 141(2): 404-414.e6, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32682912

RESUMO

Nonmelanoma skin cancer such as cutaneous squamous cell carcinoma (cSCC) is the most common form of cancer and can occur as a consequence of DNA damage to the epithelium by UVR or chemical carcinogens. There is growing evidence that the complement system is involved in cancer immune surveillance; however, its role in cSCC remains unclear. Here, we show that complement genes are expressed in tissue from patients with cSCC, and C3 activation fragments are present in cSCC biopsies, indicating complement activation. Using a range of complement-deficient mice in a two-stage mouse model of chemically-induced cSCC, where a subclinical dose of 7,12-dimethylbenz[a]anthracene causes oncogenic mutations in epithelial cells and 12-O-tetradecanoylphorbol-13-acetate promotes the outgrowth of these cells, we found that C3-deficient mice displayed a significantly reduced tumor burden, whereas an opposite phenotype was observed in mice lacking C5aR1, C5aR2, and C3a receptor. In addition, in mice unable to form the membrane attack complex, the tumor progression was unaltered. C3 deficiency did not affect the cancer response to 7,12-dimethylbenz[a]anthracene treatment alone but reduced the epidermal hyperplasia during 12-O-tetradecanoylphorbol-13-acetate-induced inflammation. Collectively, these data indicate that C3 drives tumorigenesis during chronic skin inflammation, independently of the downstream generation of C5a or membrane attack complex.


Assuntos
Carcinoma de Células Escamosas/imunologia , Complemento C3/metabolismo , Neoplasias Experimentais/imunologia , Neoplasias Cutâneas/imunologia , 9,10-Dimetil-1,2-benzantraceno/administração & dosagem , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Carcinógenos/administração & dosagem , Carcinógenos/toxicidade , Carcinoma de Células Escamosas/induzido quimicamente , Carcinoma de Células Escamosas/patologia , Ativação do Complemento/genética , Ativação do Complemento/imunologia , Complemento C3/genética , Complemento C5/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neoplasias Experimentais/sangue , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/patologia , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Pele/efeitos dos fármacos , Pele/imunologia , Pele/patologia , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/patologia , Evasão Tumoral
17.
J Clin Invest ; 131(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-32990682

RESUMO

Dysfunction of immune and vascular systems has been implicated in aging and Alzheimer disease; however, their interrelatedness remains poorly understood. The complement pathway is a well-established regulator of innate immunity in the brain. Here, we report robust age-dependent increases in vascular inflammation, peripheral lymphocyte infiltration, and blood-brain barrier (BBB) permeability. These phenotypes were subdued by global inactivation and by endothelial cell-specific ablation of C3ar1. Using an in vitro model of the BBB, we identified intracellular Ca2+ as a downstream effector of C3a/C3aR signaling and a functional mediator of vascular endothelial cadherin junction and barrier integrity. Endothelial C3ar1 inactivation also dampened microglia reactivity and improved hippocampal and cortical volumes in the aging brain, demonstrating a crosstalk between brain vasculature dysfunction and immune cell activation and neurodegeneration. Further, prominent C3aR-dependent vascular inflammation was also observed in a tau-transgenic mouse model. Our studies suggest that heightened C3a/C3aR signaling through endothelial cells promotes vascular inflammation and BBB dysfunction and contributes to overall neuroinflammation in aging and neurodegenerative disease.


Assuntos
Envelhecimento/metabolismo , Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar , Endotélio Vascular/metabolismo , Receptores de Complemento/metabolismo , Vasculite/metabolismo , Envelhecimento/genética , Envelhecimento/patologia , Animais , Barreira Hematoencefálica/patologia , Complemento C3a/genética , Complemento C3a/metabolismo , Endotélio Vascular/patologia , Camundongos , Camundongos Knockout , Receptores de Complemento/genética , Vasculite/genética , Vasculite/patologia
18.
Gastroenterology ; 160(3): 863-874, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33152356

RESUMO

BACKGROUND & AIMS: Liver CRIg+ (complement receptor of the immunoglobulin superfamily) macrophages play a critical role in filtering bacteria and their products from circulation. Translocation of microbiota-derived products from an impaired gut barrier contributes to the development of obesity-associated tissue inflammation and insulin resistance. However, the critical role of CRIg+ macrophages in clearing microbiota-derived products from the bloodstream in the context of obesity is largely unknown. METHODS: We performed studies with CRIg-/-, C3-/-, cGAS-/-, and their wild-type littermate mice. The CRIg+ macrophage population and bacterial DNA abundance were examined in both mouse and human liver by either flow cytometric or immunohistochemistry analysis. Gut microbial DNA-containing extracellular vesicles (mEVs) were adoptively transferred into CRIg-/-, C3-/-, or wild-type mice, and tissue inflammation and insulin sensitivity were measured in these mice. After coculture with gut mEVs, cellular insulin responses and cGAS/STING-mediated inflammatory responses were evaluated. RESULTS: Gut mEVs can reach metabolic tissues in obesity. Liver CRIg+ macrophages efficiently clear mEVs from the bloodstream through a C3-dependent opsonization mechanism, whereas obesity elicits a marked reduction in the CRIg+ macrophage population. Depletion of CRIg+ cells results in the spread of mEVs into distant metabolic tissues, subsequently exacerbating tissue inflammation and metabolic disorders. Additionally, in vitro treatment of obese mEVs directly triggers inflammation and insulin resistance of insulin target cells. Depletion of microbial DNA blunts the pathogenic effects of intestinal EVs. Furthermore, the cGAS/STING pathway is crucial for microbial DNA-mediated inflammatory responses. CONCLUSIONS: Deficiency of CRIg+ macrophages and leakage of intestinal EVs containing microbial DNA contribute to the development of obesity-associated tissue inflammation and metabolic diseases.


Assuntos
Microbioma Gastrointestinal/imunologia , Hepatite/imunologia , Resistência à Insulina/imunologia , Macrófagos do Fígado/imunologia , Obesidade/complicações , Animais , Complemento C3/genética , DNA Bacteriano/imunologia , DNA Bacteriano/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Microbioma Gastrointestinal/genética , Hepatite/microbiologia , Hepatite/patologia , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Macrófagos do Fígado/metabolismo , Fígado/citologia , Fígado/imunologia , Fígado/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Nucleotidiltransferases/metabolismo , Obesidade/sangue , Obesidade/imunologia , Receptores de Complemento/metabolismo , Transdução de Sinais/imunologia
19.
Adv Mater ; 33(3): e2006160, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33296121

RESUMO

Nanomedicines that target specific blood cells represent an emerging strategy to improve drug biodistribution. However, the protein corona usually disrupts nanomedicine targeting to cells and tissues. Herein, instead of exploring synthetic methods to mitigate the impact of the protein corona, its natural interactions with blood cells are leveraged and turn the protein corona into an active ingredient in treating lung inflammation. It is discovered that molecularly engineered liposomes with inverse phosphocholine lipids rapidly enrich complement fragment iC3b by "voluntary opsonization," which triggers neutrophil hijacking through complement receptor 3 phagocytosis. This neutrophil targeting is cell-state dependent as only those activated by acute inflammation display efficient neutrophil reconstruction. The liposome-loaded neutrophils migrate across the alveolar-capillary barrier, accumulate in the inflamed lung parenchyma within hours, and release their payloads to kill the bacteria. This work shows that, in addition to biological cells, the protein corona can be a new platform for active and precision nanomedicine.


Assuntos
Nanomedicina/métodos , Proteínas Opsonizantes/metabolismo , Medicina de Precisão/métodos , Engenharia , Inflamação/imunologia , Inflamação/terapia , Neutrófilos/imunologia , Receptores de Complemento/metabolismo
20.
Mol Med Rep ; 22(6): 4629-4636, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33173973

RESUMO

Inflammation is one of the fundamental processes in numerous diseases. Cluster of differentiation (CD) 93, a glycoprotein, has been reported to be associated with a number of these diseases. There are reports indicating that a high plasma level of CD93 is associated with adverse events in ischaemic heart disease. Additionally, there are reports indicating different cardiovascular risks between different single nucleotide polymorphisms (SNPs) of CD93. Therefore, the present study aimed to determine whether the plasma concentration of CD93 and polymorphism of rs2749812 in CD93 were associated with clinical conditions and mortality in an elderly population. In 470 healthy elderly community­living individuals a novel clinical examination involving echocardiography and blood sampling was performed. The population was followed for 6.7 years. Plasma levels of CD93 and SNP analyses of rs2749812 of CD93 using PCR methodology were used. During the follow­up period, 106 (22.6%) all­cause and 61 (13.0%) cardiovascular deaths were registered. Those with the highest plasma concentration had markedly higher all­cause mortality. Evaluating the A/A, A/G and G/G genotypes, the G/G group exhibited significantly higher cardiovascular mortality (P=0.026), and an almost two­fold increased risk in a multivariate Cox regression model compared with the A/G genotype. Evaluation of subgroups with respect to sex, diabetes and hypertension revealed markedly increased cardiovascular risk in the G/G genotype in all subgroups. All results persisted in the multiple models used. In the present study, the glycoprotein CD93 was demonstrated to have prognostic cardiovascular information, with increased risk for those with a high plasma concentration. Furthermore, the G/G genotype of rs2749812 of CD93 has a significantly higher cardiovascular risk, as demonstrated here, and could therefore be regarded as a possible cardiovascular risk biomarker that might in the future be used to offer optimised cardiovascular patient handling. However, this was a small study, and more research is required.


Assuntos
Doenças Cardiovasculares/genética , Glicoproteínas de Membrana/genética , Receptores de Complemento/genética , Idoso , Biomarcadores/sangue , Doenças Cardiovasculares/mortalidade , Sistema Cardiovascular , Estudos de Casos e Controles , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/mortalidade , Feminino , Seguimentos , Variação Genética/genética , Genótipo , Humanos , Inflamação/sangue , Masculino , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/metabolismo , Plasma , Polimorfismo de Nucleotídeo Único/genética , Modelos de Riscos Proporcionais , Receptores de Complemento/sangue , Receptores de Complemento/metabolismo
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