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1.
Jpn J Ophthalmol ; 66(3): 326-334, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35397057

RESUMO

PURPOSE: To investigate the localized expression of C1q/tumor necrosis factor related protein (CTRP) 6 in human age-related macular degeneration (AMD) retinal tissues. EXPERIMENTAL STUDY DESIGN: 4 AMD and 3 non-AMD whole eyes of Caucasian donors were used. Eyecups were excised at Eye Bank CorneaGen, Inc. METHODS: To elucidate the effects of CTRP6, C3b was measured by an enzyme-linked immunosorbent-like assay. CFB versus CTRP6 competitive binding assay was applied to clarify the inhibition by CTRP6 of C3bBb complex formation. The cornea, iris, lens, and vitreous were removed and the eyes were cut into a posterior eye-cup including the retina, choroid, and sclera. Six-µm-thick serial sections of frozen samples underwent hematoxylin-eosin (HE) staining and indirect immunohistochemical staining using primary antibodies, anti-CTRP6, -CTRP5, -CTRP10, -Complement factor H (CFH) and -Clusterin (CLU). Results The two in vitro studies confirmed that CTRP6 has an inhibitory effect on alternative pathways of complement (APC) function and that the molecular target of CTRP6 is the inhibition of the formation of C3bBb. Localized expression for CTRP6 and CFH was found in the drusen of the AMD eyes, both associated with APC inhibition, CLU associated with membrane-attack complex (MAC) inhibition, and CTRP5 associated with retinal degeneration. CONCLUSION: The localized expression of CTRP6 in the drusen of AMD eyes may open a new insight into the possible involvement of APC regulatory factors in the pathogenesis of AMD, together with the known CFH so far analyzed solely as an APC inhibitor.


Assuntos
Degeneração Macular , Corioide/patologia , Colágeno , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Humanos , Fatores Imunológicos , Degeneração Macular/diagnóstico , Retina/patologia
2.
Curr Eye Res ; 47(7): 1087-1093, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35282732

RESUMO

PURPOSE: GEM103 is a recombinantly produced full-length version of the human complement factor H (CFH) under clinical investigation for treatment of age-related macular degeneration (AMD) in individuals carrying an AMD risk-associated genetic variant of CFH. This study aimed to investigate the complement pathway-related functions of GEM103 in comparison with those of native human CFH. METHODS: Key biological activities of GEM103 and human serum-derived CFH (sdCFH) were compared using four independent functional assays. Assays of C3b binding and C3 convertase decay-accelerating activity (DAA) were performed by surface plasmon resonance (SPR). Cofactor activity (CA) was measured using 8-anilinonaphthalene-1-sulfonic acid as a fluorescent probe of C3b integrity. The abilities of GEM103 and sdCFH to protect sheep erythrocytes from hemolysis by CFH-depleted normal human serum were assessed colorimetrically. RESULTS: In multiple SPR-based assays of C3b binding and DAA, the performance of GEM103 was consistently comparable to that of sdCFH across a range of matching concentrations. The EC50 ± SD in the fluorescence-based fluid-phase CA assay was 0.21 ± 0.06 µM for GEM103 compared to 0.20 ± 0.09 µM for sdCFH. In hemolysis assays, the EC50 value of 0.33 ± 0.16 µM for GEM103 versus 0.46 ± 0.06 µM for sdCFH were not significantly different (p = 0.81). CONCLUSIONS: GEM103, a recombinant CFH developed by Gemini Therapeutics, shows activity profiles comparable to sdCFH in all complement-related assays employed in this study, suggesting that GEM103 is equivalent to the native glycoprotein in terms of its in vitro functional activity. These results support further study of GEM103 as a potential therapy for AMD.


Assuntos
Fator H do Complemento , Degeneração Macular , Animais , Antivirais , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Hemólise , Humanos , Degeneração Macular/tratamento farmacológico , Degeneração Macular/genética , Degeneração Macular/metabolismo , Polimorfismo de Nucleotídeo Único , Ovinos
3.
Commun Biol ; 5(1): 152, 2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35194132

RESUMO

The complement system constitutes the innate defense against pathogens. Its dysregulation leads to diseases and is a critical determinant in many viral infections, e.g., COVID-19. Factor H (FH) is the main regulator of the alternative pathway of complement activation and could be a therapy to restore homeostasis. However, recombinant FH is not available. Engineered FH versions may be alternative therapeutics. Here, we designed a synthetic protein, MFHR13, as a multitarget complement regulator. It combines the dimerization and C5-regulatory domains of human FH-related protein 1 (FHR1) with the C3-regulatory and cell surface recognition domains of human FH, including SCR 13. In summary, the fusion protein MFHR13 comprises SCRs FHR11-2:FH1-4:FH13:FH19-20. It protects sheep erythrocytes from complement attack exhibiting 26 and 4-fold the regulatory activity of eculizumab and human FH, respectively. Furthermore, we demonstrate that MFHR13 and FHR1 bind to all proteins forming the membrane attack complex, which contributes to the mechanistic understanding of FHR1. We consider MFHR13 a promising candidate as therapeutic for complement-associated diseases.


Assuntos
Proteínas Sanguíneas/metabolismo , Ativação do Complemento , Fator H do Complemento/metabolismo , Proteínas do Sistema Complemento/metabolismo , Eritrócitos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Bryopsida/genética , Bryopsida/metabolismo , COVID-19/epidemiologia , COVID-19/metabolismo , COVID-19/virologia , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Humanos , Modelos Moleculares , Pandemias/prevenção & controle , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , SARS-CoV-2/fisiologia , Ovinos
4.
J Immunol ; 208(5): 1232-1247, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35110419

RESUMO

The ß protein from group B Streptococcus (GBS) is a ∼132-kDa, cell-surface exposed molecule that binds to multiple host-derived ligands, including complement factor H (FH). Many details regarding this interaction and its significance to immune evasion by GBS remain unclear. In this study, we identified a three-helix bundle domain within the C-terminal half of the B75KN region of ß as the major FH-binding determinant and determined its crystal structure at 2.5 Å resolution. Analysis of this structure suggested a role in FH binding for a loop region connecting helices α1 and α2, which we confirmed by mutagenesis and direct binding studies. Using a combination of protein cross-linking and mass spectrometry, we observed that B75KN bound to complement control protein (CCP)3 and CCP4 domains of FH. Although this binding site lies within a complement regulatory region of FH, we determined that FH bound by ß retained its decay acceleration and cofactor activities. Heterologous expression of ß by Lactococcus lactis resulted in recruitment of FH to the bacterial surface and a significant reduction of C3b deposition following exposure to human serum. Surprisingly, we found that FH binding by ß was not required for bacterial resistance to phagocytosis by neutrophils or killing of bacteria by whole human blood. However, loss of the B75KN region significantly diminished bacterial survival in both assays. Although our results show that FH recruited to the bacterial surface through a high-affinity interaction maintains key complement-regulatory functions, they raise questions about the importance of FH binding to immune evasion by GBS as a whole.


Assuntos
Proteínas de Bactérias/metabolismo , Evasão da Resposta Imune/imunologia , Proteínas de Membrana/metabolismo , Streptococcus agalactiae/imunologia , Sítios de Ligação/fisiologia , Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Humanos , Neutrófilos/imunologia , Ligação Proteica/imunologia , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/patologia
5.
J Immunol Res ; 2022: 1737419, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35097132

RESUMO

Complement factor H (FH) is a key regulator of the alternative pathway of complement, in man and mouse. Earlier, our studies revealed that the absence of FH causes the C57BL6 mouse to become susceptible to chronic serum sickness (CSS) along with an increase in the renal infiltration of macrophages compared to controls. To understand if the increased recruitment of macrophages (Mϕs) to the kidney was driving inflammation and propagating injury, we examined the effect of Mϕ depletion with clodronate in FH knockout mice with CSS. Eight-week-old FHKO mice were treated with apoferritin (4 mg/mouse) for 5 wks and with either vehicle (PBS) or clodronate (50 mg/kg ip, 3 times/wk for the last 3 weeks). The administration of clodronate decreased monocytes and Mϕs in the kidneys by >80%. Kidney function assessed by BUN and albumin remained closer to normal on depletion of Mϕs. Clodronate treatment prevented the alteration in cytokines, TNFα and IL-6, and increase in gene expression of connective tissue growth factor (CTGF), TGFß-1, matrix metalloproteinase-9 (MMP9), fibronectin, laminin, and collagen in FHKO mice with CSS (P < 0.05). Clodronate treatment led to relative protection from immune complex- (IC-) mediated disease pathology during CSS as assessed by the significantly reduced glomerular pathology (GN) and extracellular matrix. Our results suggest that complement activation is one of the mechanism that regulates the macrophage landscape and thereby fibrosis. The exact mechanism remains to be deciphered. In brief, our data shows that Mϕs play a critical role in FH-dependent ICGN and Mϕ depletion reduces disease progression.


Assuntos
Glomerulonefrite/imunologia , Doenças do Complexo Imune/imunologia , Rim/metabolismo , Macrófagos/imunologia , Animais , Apoferritinas/administração & dosagem , Movimento Celular , Ácido Clodrônico/administração & dosagem , Fator H do Complemento/metabolismo , Progressão da Doença , Fibrose , Rim/imunologia , Rim/patologia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
6.
Cell Rep ; 38(1): 110183, 2022 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-34986357

RESUMO

Candida albicans is a commensal of the urogenital tract and the predominant cause of vulvovaginal candidiasis (VVC). Factors that increase circulatory estrogen levels such as pregnancy, the use of oral contraceptives, and hormone replacement therapy predispose women to VVC, but the reasons for this are largely unknown. Here, we investigate how adaptation of C. albicans to estrogen impacts the fungal host-pathogen interaction. Estrogen promotes fungal virulence by enabling C. albicans to avoid the actions of the innate immune system. Estrogen-induced innate immune evasion is mediated via inhibition of opsonophagocytosis through enhanced acquisition of the human complement regulatory protein, Factor H, on the fungal cell surface. Estrogen-induced accumulation of Factor H is dependent on the fungal cell surface protein Gpd2. The discovery of this hormone-sensing pathway might pave the way in explaining gender biases associated with fungal infections and may provide an alternative approach to improving women's health.


Assuntos
Candida albicans/imunologia , Candidíase Vulvovaginal/patologia , Via Alternativa do Complemento/imunologia , Estrogênios/metabolismo , Evasão da Resposta Imune/imunologia , Fagocitose/imunologia , Candida albicans/patogenicidade , Fator H do Complemento/metabolismo , Feminino , Glicerol-3-Fosfato Desidrogenase (NAD+)/metabolismo , Humanos , Imunidade Inata/imunologia , Progesterona/metabolismo , Virulência/imunologia
7.
Stem Cell Res ; 60: 102669, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35063911

RESUMO

Age-related macular degeneration (AMD) is a major cause of vision loss among the elderly in the Western world. AMD is multifactorial eye disease with a strong genetic contribution. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from peripheral blood mononuclear cells of three patients with AMD carrying rare variants in the complement factor H (CFH) gene. These cell lines were generated for cellular studies investigating the disease mechanisms and developing therapeutic interventions for AMD.


Assuntos
Células-Tronco Pluripotentes Induzidas , Degeneração Macular , Idoso , Linhagem Celular , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/metabolismo , Degeneração Macular/genética , Degeneração Macular/metabolismo , Polimorfismo de Nucleotídeo Único
8.
Exp Biol Med (Maywood) ; 247(2): 77-86, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34775843

RESUMO

Age-related macular degeneration is the leading cause of blindness in the elderly. The Y402H polymorphism in complement factor H promotes disease-like pathogenesis, and a Cfh+/- murine model can replicate this phenotype, but only after two years. We reasoned that by combining CFH deficiency with cigarette smoke exposure, we might be able to accelerate disease progression to facilitate preclinical research in this disease. Wild-type and Cfh+/- mice were exposed to nose-only cigarette smoke for three months. Retinal tissue morphology and visual function were evaluated by optical coherence tomography, fundus photography and autofluorescence, and electroretinogram. Retinal pigment epithelial cell phenotype and ultrastructure were evaluated by immunofluorescence staining and transmission electron microscopy. Cfh+/- smoking mice showed a dome-like protruding lesion at the ellipsoid zone (drusen-like deposition), many retinal hyper-autofluorescence spots, and a marked decrease in A- and B-wave amplitudes. Compared with non-smoking mice, wild-type and Cfh+/- smoking mice showed sub-retinal pigment epithelium complement protein 3 deposition, activation of microglia, metabolic waste accumulation, and impairment of tight junctions. Microglia cells migrated into the photoreceptor outer segment layer in Cfh+/- smoking mice showed increased activation. Our results suggest that exposing Cfh+/- mice to smoking leads to earlier onset of age-related macular degeneration than in other animal models, which may facilitate preclinical research into the pathophysiology and treatment of this disease.


Assuntos
Fator H do Complemento/deficiência , Doenças da Deficiência Hereditária de Complemento/metabolismo , Nefropatias/metabolismo , Degeneração Macular/metabolismo , Fumar/metabolismo , Animais , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Modelos Animais de Doenças , Doenças da Deficiência Hereditária de Complemento/genética , Nefropatias/genética , Degeneração Macular/etiologia , Degeneração Macular/genética , Camundongos , Camundongos Knockout , Fumar/efeitos adversos , Fumar/genética
9.
BMC Immunol ; 22(1): 75, 2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34876020

RESUMO

BACKGROUND: The aim of the study was to investigate the clinical relevance of IgM deposition in patients with lupus nephritis (LN) in a large cohort. RESULTS: 217 patients with renal biopsy-proven active LN were enrolled. The associations between glomerular IgM deposition and clinicopathological parameters were further analyzed. IgM deposition was positively correlated with glomerular C1q and C3 deposition moderately (r = 0.436, P < 0.001; r = 0.408, P < 0.001, respectively), and inversely correlated with plasma levels of C3 and CFH mildly (r = - 0.138, P = 0.043; r = - 0.147, P = 0.037, respectively). By multivariate analysis, we found that glomerular IgM deposition independently contributed to glomerular C3 deposition in patients with LN (OR = 2.002, 95% CI 1.295-3.094, P = 0.002). In addition, we also found that patients with IgM 0-2+ had similar plasma CFH levels, but in patients with IgM3+-4+, plasma CFH levels were significantly lower (300.4 ± 155.8 µg/mL vs. 429.9 ± 187.5 µg/mL, P < 0.001). Furthermore, patients with high density of glomerular IgM and low levels of CFH had heavier proteinuria, higher serum creatinine and lower plasma C3 levels (5.7 ± 3.1 g/d vs. 4.7 ± 3.5 g/d, P = 0.037; 150.1 ± 121.0 µmol/L vs. 105.6 ± 97.1 µmol/L, P = 0.005; 0.3 ± 0.2 µg/L vs. 0.4 ± 0.2 µg/L, P = 0.04, respectively), comparing with those with low density of glomerular IgM and low levels of CFH. CONCLUSIONS: Our results suggested the involvement of glomerular deposited IgM in complement activation and renal injury in LN.


Assuntos
Complemento C1q/metabolismo , Complemento C3/metabolismo , Imunoglobulina M/metabolismo , Glomérulos Renais/metabolismo , Nefrite Lúpica/imunologia , Adulto , Estudos de Coortes , Ativação do Complemento , Fator H do Complemento/metabolismo , Creatinina/sangue , Feminino , Humanos , Glomérulos Renais/patologia , Masculino , Proteinúria , Adulto Jovem
10.
Nat Commun ; 12(1): 7293, 2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34911940

RESUMO

Age-related Macular Degeneration (AMD), a blinding eye disease, is characterized by pathological protein- and lipid-rich drusen deposits underneath the retinal pigment epithelium (RPE) and atrophy of the RPE monolayer in advanced disease stages - leading to photoreceptor cell death and vision loss. Currently, there are no drugs that stop drusen formation or RPE atrophy in AMD. Here we provide an iPSC-RPE AMD model that recapitulates drusen and RPE atrophy. Drusen deposition is dependent on AMD-risk-allele CFH(H/H) and anaphylatoxin triggered alternate complement signaling via the activation of NF-κB and downregulation of autophagy pathways. Through high-throughput screening we identify two drugs, L-745,870, a dopamine receptor antagonist, and aminocaproic acid, a protease inhibitor that reduce drusen deposits and restore RPE epithelial phenotype in anaphylatoxin challenged iPSC-RPE with or without the CFH(H/H) genotype. This comprehensive iPSC-RPE model replicates key AMD phenotypes, provides molecular insight into the role of CFH(H/H) risk-allele in AMD, and discovers two candidate drugs to treat AMD.


Assuntos
Ácido Aminocaproico/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Degeneração Macular/tratamento farmacológico , Piridinas/farmacologia , Pirróis/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Alelos , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Degeneração Macular/genética , Degeneração Macular/metabolismo , Modelos Biológicos , Fenótipo , Epitélio Pigmentado da Retina/metabolismo
11.
Front Immunol ; 12: 769242, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34819935

RESUMO

Complement Factor H-Related 3 (FHR-3) is a major regulator of the complement system, which is associated with different diseases, such as age-related macular degeneration (AMD). However, the non-canonical local, cellular functions of FHR-3 remained poorly understood. Here, we report that FHR-3 bound to oxidative stress epitopes and competed with FH for interaction. Furthermore, FHR-3 was internalized by viable RPE cells and modulated time-dependently complement component (C3, FB) and receptor (C3aR, CR3) expression of human RPE cells. Independently of any external blood-derived proteins, complement activation products were detected. Anaphylatoxin C3a was visualized in treated cells and showed a translocation from the cytoplasm to the cell membrane after FHR-3 exposure. Subsequently, FHR-3 induced a RPE cell dependent pro-inflammatory microenvironment. Inflammasome NLRP3 activation and pro-inflammatory cytokine secretion of IL-1ß, IL-18, IL-6 and TNF-α were induced after FHR-3-RPE interaction. Our previously published monoclonal anti-FHR-3 antibody, which was chimerized to reduce immunogenicity, RETC-2-ximab, ameliorated the effect of FHR-3 on ARPE-19 cells. Our studies suggest FHR-3 as an exogenous trigger molecule for the RPE cell "complosome" and as a putative target for a therapeutic approach for associated degenerative diseases.


Assuntos
Proteínas Sanguíneas/imunologia , Ativação do Complemento/imunologia , Fator H do Complemento/imunologia , Células Epiteliais/imunologia , Epitélio Pigmentado da Retina/citologia , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Linhagem Celular , Ativação do Complemento/genética , Complemento C3/genética , Complemento C3/imunologia , Complemento C3/metabolismo , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/imunologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Células HEK293 , Humanos , Inflamassomos/genética , Inflamassomos/imunologia , Inflamassomos/metabolismo , Antígeno de Macrófago 1/genética , Antígeno de Macrófago 1/imunologia , Antígeno de Macrófago 1/metabolismo , Degeneração Macular/genética , Degeneração Macular/imunologia , Degeneração Macular/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Polimorfismo de Nucleotídeo Único/imunologia
12.
Front Immunol ; 12: 660852, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34671340

RESUMO

Besides being a key effector arm of innate immunity, a plethora of non-canonical functions of complement has recently been emerging. Factor H (FH), the main regulator of the alternative pathway of complement activation, has been reported to bind to various immune cells and regulate their functions, beyond its role in modulating complement activation. In this study we investigated the effect of FH, its alternative splice product FH-like protein 1 (FHL-1), the FH-related (FHR) proteins FHR-1 and FHR-5, and the recently developed artificial complement inhibitor mini-FH, on two key innate immune cells, monocytes and neutrophilic granulocytes. We found that, similar to FH, the other factor H family proteins FHL-1, FHR-1 and FHR-5, as well as the recombinant mini-FH, are able to bind to both monocytes and neutrophils. As a functional outcome, immobilized FH and FHR-1 inhibited PMA-induced NET formation, but increased the adherence and IL-8 production of neutrophils. FHL-1 increased only the adherence of the cells, while FHR-5 was ineffective in altering these functions. The adherence of monocytes was increased on FH, recombinant mini-FH and FHL-1 covered surfaces and, except for FHL-1, the same molecules also enhanced secretion of the inflammatory cytokines IL-1ß and TNFα. When monocytes were stimulated with LPS in the presence of immobilized FH family proteins, FH, FHL-1 and mini-FH enhanced whereas FHR-1 and FHR-5 decreased the secretion of TNFα; FHL-1 and mini-FH also enhanced IL-10 release compared to the effect of LPS alone. Our results reveal heterogeneous effects of FH and FH family members on monocytes and neutrophils, altering key features involved in pathogen killing, and also demonstrate that FH-based complement inhibitors, such as mini-FH, may have effects beyond their function of inhibiting complement activation. Thus, our data provide new insight into the non-canonical functions of FH, FHL-1, FHR-1 and FHR-5 that might be exploited during protection against infections and in vaccine development.


Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas do Sistema Complemento/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Monócitos/metabolismo , Proteínas Musculares/metabolismo , Neutrófilos/metabolismo , Animais , Proteínas Sanguíneas/genética , Forma Celular , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Inativadores do Complemento/farmacologia , Proteínas do Sistema Complemento/genética , Citocinas/metabolismo , Armadilhas Extracelulares/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Proteínas Musculares/genética , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fenótipo , Células Sf9 , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Células U937
13.
Front Immunol ; 12: 676662, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34489931

RESUMO

Complement dysregulation is characteristic of the renal diseases atypical hemolytic uremic syndrome (aHUS) and complement component 3 glomerulopathy (C3G). Complement regulatory protein Factor H (FH) inhibits complement activity, whereas FH-related proteins (FHRs) lack a complement regulatory domain. FH and FHRs compete for binding to host cell glycans, in particular heparan sulfates (HS). HS is a glycosaminoglycan with an immense structural variability, where distinct sulfation patterns mediate specific binding of proteins. Mutations in FH, FHRs, or an altered glomerular HS structure may disturb the FH : FHRs balance on glomerular endothelial cells, thereby leading to complement activation and the subsequent development of aHUS/C3G. In this study, we aimed to identify specific HS structures that could specifically compete off FHRs from HS glycocalyx (HSGlx), without interfering with FH binding. FH/FHR binding to human conditionally immortalized glomerular endothelial cells (ciGEnCs) and HSGlx purified from ciGEnC glycocalyx was assessed. HS modifications important for FH/FHR binding to HSGlx were analyzed using selectively desulfated heparins in competition with purified HSGlx. We further assessed effects of heparinoids on FHR1- and FHR5-mediated C3b deposition on ciGEnCs. In the presence of C3b, binding of FH, FHR1 and FHR5 to ciGEnCs was significantly increased, whereas binding of FHR2 was minimal. FHR1 and 5 competitively inhibited FH binding to HSGlx, leading to alternative pathway dysregulation. FHR1 and FHR5 binding was primarily mediated by N-sulfation while FH binding depended on N-, 2-O- and 6-O-sulfation. Addition of 2-O-desulfated heparin significantly reduced FHR1- and FHR5-mediated C3b deposition on ciGEnCs. We identify 2-O-desulfated heparin derivatives as potential therapeutics for C3G and other diseases with dysregulated complement.


Assuntos
Síndrome Hemolítico-Urêmica Atípica/sangue , Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Proteínas do Sistema Complemento/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Células Cultivadas , Ativação do Complemento , Células Endoteliais/metabolismo , Heparina/análogos & derivados , Heparina/farmacologia , Humanos , Glomérulos Renais/metabolismo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
14.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502105

RESUMO

The human brain and central nervous system (CNS) harbor a select sub-group of potentially pathogenic microRNAs (miRNAs), including a well-characterized NF-kB-sensitive Homo sapiens microRNA hsa-miRNA-146a-5p (miRNA-146a). miRNA-146a is significantly over-expressed in progressive and often lethal viral- and prion-mediated and related neurological syndromes associated with progressive inflammatory neurodegeneration. These include ~18 different viral-induced encephalopathies for which data are available, at least ~10 known prion diseases (PrD) of animals and humans, Alzheimer's disease (AD) and other sporadic and progressive age-related neurological disorders. Despite the apparent lack of nucleic acids in prions, both DNA- and RNA-containing viruses along with prions significantly induce miRNA-146a in the infected host, but whether this represents part of the host's adaptive immunity, innate-immune response or a mechanism to enable the invading prion or virus a successful infection is not well understood. Current findings suggest an early and highly interactive role for miRNA-146a: (i) as a major small noncoding RNA (sncRNA) regulator of innate-immune responses and inflammatory signaling in cells of the human brain and CNS; (ii) as a critical component of the complement system and immune-related neurological dysfunction; (iii) as an inducible sncRNA of the brain and CNS that lies at a critical intersection of several important neurobiological adaptive immune response processes with highly interactive associations involving complement factor H (CFH), Toll-like receptor pathways, the innate-immunity, cytokine production, apoptosis and neural cell decline; and (iv) as a potential biomarker for viral infection, TSE and AD and other neurological diseases in both animals and humans. In this report, we review the recent data supporting the idea that miRNA-146a may represent a novel and unique sncRNA-based biomarker for inflammatory neurodegeneration in multiple species. This paper further reviews the current state of knowledge regarding the nature and mechanism of miRNA-146a in viral and prion infection of the human brain and CNS with reference to AD wherever possible.


Assuntos
Encéfalo/patologia , Viroses do Sistema Nervoso Central/imunologia , Regulação da Expressão Gênica/imunologia , MicroRNAs/metabolismo , Doenças Priônicas/imunologia , Apoptose/genética , Apoptose/imunologia , Biomarcadores/análise , Biomarcadores/metabolismo , Encéfalo/imunologia , Encéfalo/virologia , Viroses do Sistema Nervoso Central/diagnóstico , Viroses do Sistema Nervoso Central/genética , Viroses do Sistema Nervoso Central/virologia , Fator H do Complemento/metabolismo , Citocinas/metabolismo , Humanos , MicroRNAs/análise , MicroRNAs/genética , NF-kappa B/metabolismo , Doenças Priônicas/diagnóstico , Doenças Priônicas/genética , Doenças Priônicas/patologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptores Toll-Like/metabolismo
15.
Avian Dis ; 65(2): 261-268, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34412457

RESUMO

The resistance to serum complement-mediated killing is a vital virulence property of microbial pathogens. Complement factor H (FH) is a key negative regulator of the complement alternative pathway (AP) that prevents formation and accelerates the decay of AP C3 convertase and acts as a cofactor in the inactivation of C3b. Pathogens can recruit host FH through their surface proteins to escape the clearance of the complement system. Riemerella anatipestifer could also evade the complement system attack to achieve host infection, but the mechanism is still unclear. In this study, the R. anatipestifer proteins that could interact with FH in host serum were screened and analyzed, and the functions were determined. Affinity chromatography with a Ni-nitrilotriacetic acid Sefinose column and mass spectrometry identified three outer membrane proteins (Omp) of R. anatipestifer, Omp54, Omp53, and Omp24, as potential FH-binding proteins. We then successfully conducted the prokaryotic expression and polyclonal antibody preparation of three candidate proteins. Indirect immunofluorescence assay showed that three candidate proteins were all present in R. anatipestifer. The affinity blotting assay, anti-serum-inhibiting assay, and serum bactericidal assay presented evidence that Omp24 could bind FH. Moreover, FH bound to Omp24 was associated with resistance to the alternative pathway and functional for R. anatipestifer survival in the normal duck serum. These results suggested that R. anatipestifer Omp24 was a FH-binding protein and the interaction with FH blocked the alternative pathway. Recruitment of complement regulatory proteins may facilitate better R. anatipestifer resistance to this vital line of host defense.


Artículo regular­El factor H del complemento de pato se une a la proteína de la membrana externa Omp24 de Riemerella anatipestifer La resistencia a la destrucción mediada por el complemento sérico es una propiedad vital para la virulencia de los patógenos microbianos. El factor de complemento H (FH) es un regulador negativo clave de la vía alterna del complemento (AP) que previene la formación y acelera la descomposición de la C3 convertasa de la vía alterna y actúa como cofactor en la inactivación de C3b. Los patógenos pueden reclutar factor H del huésped a través de sus proteínas de superficie para escapar de la destrucción por el sistema del complemento. Riemerella anatipestifer también pudo evadir el ataque del sistema del complemento para lograr la infección del huésped, pero el mecanismo aún no está claro. En este estudio, se seleccionaron y analizaron las proteínas de R. anatipestifer que podrían interactuar con el factor H en el suero del huésped y se determinaron las funciones. La cromatografía de afinidad con una columna de sefinosa de Ni-NTA y la espectrometría de masas identificaron tres proteínas de la membrana externa de R. anatipestifer, Omp54, Omp53 y Omp24, como posibles proteínas que se unen al factor H. Posteriormente, se llevó a cabo con éxito la expresión procariota y la preparación de anticuerpos policlonales de las tres proteínas candidatas. El ensayo de inmunofluorescencia indirecta mostró que las tres proteínas candidatas estaban presentes en R. anatipestifer. El ensayo de transferencia para afinidad, el ensayo anti-inhibidor del suero y el ensayo bactericida sérico presentaron evidencia de que la proteína Omp24 podría unirse al factor H. Además, el factor H unido a la proteína Omp24 se asoció con resistencia a la vía alterna y funcional para la supervivencia de R. anatipestifer en el suero de pato normal. Estos resultados sugirieron que la proteína Omp24 de R. anatipestifer era una proteína de unión al factor H y que la interacción con este factor bloqueaba la vía alterna del complemento. El reclutamiento de proteínas reguladoras del complemento puede facilitar una mejor resistencia de R. anatipestifer a esta línea vital de defensa del huésped.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Fator H do Complemento/metabolismo , Riemerella/metabolismo , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/química , Cromatografia de Afinidade/veterinária , Patos , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Camundongos , Coelhos , Riemerella/imunologia
17.
Clin Immunol ; 229: 108794, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34245915

RESUMO

C3 glomerulopathy (C3G) is a rare renal disease characterized by predominant glomerular C3 staining. Complement alternative pathway dysregulation due to inherited complement defects is associated with C3G. To identify novel C3G-related genes, we screened 86 genes in the complement, coagulation and endothelial systems in 35 C3G patients by targeted genomic enrichment and massively parallel sequencing. Surprisingly, the most frequently mutated gene was VWF. Patients with VWF variants had significantly higher proteinuria levels, higher crescent formation and lower factor H (FH) levels. We further selected two VWF variants to transiently express the von Willebrand factor (vWF) protein, we found that vWF expression from the c.1519A > G variant was significantly reduced. In vitro results further indicated that vWF could regulate complement activation, as it could bind to FH and C3b, act as a cofactor for factor I-mediated cleavage of C3b. Thus, we speculated that vWF might be involved in the pathogenesis of C3G.


Assuntos
Complemento C3/metabolismo , Glomerulonefrite Membranoproliferativa/genética , Glomerulonefrite/genética , Fator de von Willebrand/genética , Adolescente , Adulto , Estudos de Casos e Controles , China , Estudos de Coortes , Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Via Alternativa do Complemento , Feminino , Variação Genética , Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Glomerulonefrite Membranoproliferativa/imunologia , Glomerulonefrite Membranoproliferativa/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Técnicas In Vitro , Glomérulos Renais/imunologia , Glomérulos Renais/patologia , Masculino , Pessoa de Meia-Idade , Modelos Imunológicos , Simulação de Dinâmica Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Adulto Jovem , Fator de von Willebrand/química , Fator de von Willebrand/metabolismo
18.
Vet Microbiol ; 259: 109157, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34197978

RESUMO

Salmonella enterica serovar Typhimurium utilizes a series of strategies to evade host innate immune defenses, including the serum complement system. Many microbial pathogens have evolved the ability to bind the complement regulatory protein factor H (FH) through their surface factor H-binding proteins (FHBPs) to circumvent the complement-mediated bactericidal effect. However, the roles of FHBPs in Salmonella pathogenesis are not well understood. In this study, we demonstrated that the survival of S. Typhimurium in human serum was decreased in a time and concentration dependent manner. Pre-incubation with FH attenuated the sensitivity of S. Typhimurium strain χ3761 to complement-mediated serum killing, suggesting FH binding enhance survival in serum. We aimed to identify novel S. Typhimurium FHBPs and characterize their biological functions. Here, six potential FHBPs were identified by two-dimensional (2D)-Far-western blot, and three of them were further confirmed to bind FH by Far-western blot and dot blot. We found that deletion of ompC (ΔompC) significantly inhibited the survival of S. Typhimurium strain χ3761 in human serum. Our results indicated that the ompC mutation does not affect χ3761 adhesion to HeLa cells. Furthermore, a mice infection model showed that deletion of ompC had no significant effect on the histopathological lesions or viability compared with the wild-type strain χ3761. In summary, these results suggested that OmpC is an important FHBP, but not a critical virulence factor of S. Typhimurium.


Assuntos
Aderência Bacteriana/genética , Fator H do Complemento/metabolismo , Porinas/metabolismo , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Fator H do Complemento/genética , Feminino , Células HeLa , Humanos , Camundongos Endogâmicos BALB C , Porinas/genética , Salmonelose Animal , Salmonella typhimurium/química , Salmonella typhimurium/genética , Fatores de Virulência/genética
19.
PLoS Pathog ; 17(7): e1009801, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34324600

RESUMO

Pathogens possess the ability to adapt and survive in some host species but not in others-an ecological trait known as host tropism. Transmitted through ticks and carried mainly by mammals and birds, the Lyme disease (LD) bacterium is a well-suited model to study such tropism. Three main causative agents of LD, Borrelia burgdorferi, B. afzelii, and B. garinii, vary in host ranges through mechanisms eluding characterization. By feeding ticks infected with different Borrelia species, utilizing feeding chambers and live mice and quail, we found species-level differences in bacterial transmission. These differences localize on the tick blood meal, and specifically complement, a defense in vertebrate blood, and a polymorphic bacterial protein, CspA, which inactivates complement by binding to a host complement inhibitor, Factor H (FH). CspA selectively confers bacterial transmission to vertebrates that produce FH capable of allele-specific recognition. CspA is the only member of the Pfam54 gene family to exhibit host-specific FH-binding. Phylogenetic analyses revealed convergent evolution as the driver of such uniqueness, and that FH-binding likely emerged during the last glacial maximum. Our results identify a determinant of host tropism in Lyme disease infection, thus defining an evolutionary mechanism that shapes host-pathogen associations.


Assuntos
Proteínas de Bactérias/genética , Borrelia burgdorferi/crescimento & desenvolvimento , Doença de Lyme/imunologia , Doença de Lyme/transmissão , Tropismo Viral/fisiologia , Animais , Proteínas de Bactérias/metabolismo , Evolução Biológica , Borrelia burgdorferi/genética , Borrelia burgdorferi/imunologia , Fator H do Complemento/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Evasão da Resposta Imune/fisiologia , Camundongos , Codorniz , Especificidade da Espécie , Carrapatos
20.
Blood ; 138(22): 2185-2201, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34189567

RESUMO

Atypical hemolytic uremic syndrome (aHUS) is a life-threatening thrombotic microangiopathy that can progress, when untreated, to end-stage renal disease. Most frequently, aHUS is caused by complement dysregulation due to pathogenic variants in genes that encode complement components and regulators. Among these genes, the factor H (FH) gene, CFH, presents with the highest frequency (15% to 20%) of variants and is associated with the poorest prognosis. Correct classification of CFH variants as pathogenic or benign is essential to clinical care but remains challenging owing to the dearth of functional studies. As a result, significant numbers of variants are reported as variants of uncertain significance. To address this knowledge gap, we expressed and functionally characterized 105 aHUS-associated FH variants. All FH variants were categorized as pathogenic or benign and, for each, we fully documented the nature of the pathogenicity. Twenty-six previously characterized FH variants were used as controls to validate and confirm the robustness of the functional assays used. Of the remaining 79 uncharacterized variants, only 29 (36.7%) alter FH expression or function in vitro and, therefore, are proposed to be pathogenic. We show that rarity in control databases is not informative for variant classification, and we identify important limitations in applying prediction algorithms to FH variants. Based on structural and functional data, we suggest ways to circumvent these difficulties and, thereby, improve variant classification. Our work highlights the need for functional assays to interpret FH variants accurately if clinical care of patients with aHUS is to be individualized and optimized.


Assuntos
Síndrome Hemolítico-Urêmica Atípica/genética , Fator H do Complemento/genética , Síndrome Hemolítico-Urêmica Atípica/metabolismo , Síndrome Hemolítico-Urêmica Atípica/patologia , Fator H do Complemento/química , Fator H do Complemento/metabolismo , Expressão Gênica , Predisposição Genética para Doença , Variação Genética , Humanos , Modelos Moleculares , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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