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1.
Ann Rheum Dis ; 82(6): 848-856, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36801813

RESUMO

OBJECTIVES: Granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) are autoimmune vasculitides associated with antineutrophil cytoplasm antibodies that target proteinase 3 (PR3) or myeloperoxidase (MPO) found within neutrophils and monocytes. Granulomas are exclusively found in GPA and form around multinucleated giant cells (MGCs), at sites of microabscesses, containing apoptotic and necrotic neutrophils. Since patients with GPA have augmented neutrophil PR3 expression, and PR3-expressing apoptotic cells frustrate macrophage phagocytosis and cellular clearance, we investigated the role of PR3 in stimulating giant cell and granuloma formation. METHODS: We stimulated purified monocytes and whole peripheral blood mononuclear cells (PBMCs) from patients with GPA, patients with MPA or healthy controls with PR3 or MPO and visualised MGC and granuloma-like structure formation using light, confocal and electron microscopy, as well as measuring the cell cytokine production. We investigated the expression of PR3 binding partners on monocytes and tested the impact of their inhibition. Finally, we injected zebrafish with PR3 and characterised granuloma formation in a novel animal model. RESULTS: In vitro, PR3 promoted monocyte-derived MGC formation using cells from patients with GPA but not from patients with MPA, and this was dependent on soluble interleukin 6 (IL-6), as well as monocyte MAC-1 and protease-activated receptor-2, found to be overexpressed in the cells of patients with GPA. PBMCs stimulated by PR3 formed granuloma-like structures with central MGC surrounded by T cells. This effect of PR3 was confirmed in vivo using zebrafish and was inhibited by niclosamide, a IL-6-STAT3 pathway inhibitor. CONCLUSIONS: These data provide a mechanistic basis for granuloma formation in GPA and a rationale for novel therapeutic approaches.


Assuntos
Granulomatose com Poliangiite , Poliangiite Microscópica , Animais , Mieloblastina , Granulomatose com Poliangiite/tratamento farmacológico , Peixe-Zebra , Interleucina-6 , Leucócitos Mononucleares , Anticorpos Anticitoplasma de Neutrófilos , Granuloma/complicações , Células Gigantes , Peroxidase
2.
Nat Commun ; 13(1): 6385, 2022 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-36302784

RESUMO

Neutrophils play essential anti-microbial and inflammatory roles in host defense, however, their activities require tight regulation as dysfunction often leads to detrimental inflammatory and autoimmune diseases. Here we show that the adhesion molecule GPR97 allosterically activates CD177-associated membrane proteinase 3 (mPR3), and in conjugation with several protein interaction partners leads to neutrophil activation in humans. Crystallographic and deletion analysis of the GPR97 extracellular region identified two independent mPR3-binding domains. Mechanistically, the efficient binding and activation of mPR3 by GPR97 requires the macromolecular CD177/GPR97/PAR2/CD16b complex and induces the activation of PAR2, a G protein-coupled receptor known for its function in inflammation. Triggering PAR2 by the upstream complex leads to strong inflammatory activation, prompting anti-microbial activities and endothelial dysfunction. The role of the complex in pathologic inflammation is underscored by the finding that both GPR97 and mPR3 are upregulated on the surface of disease-associated neutrophils. In summary, we identify a PAR2 activation mechanism that directs neutrophil activation, and thus inflammation. The PR3/CD177/GPR97/PAR2/CD16b protein complex, therefore, represents a potential therapeutic target for neutrophil-mediated inflammatory diseases.


Assuntos
Ativação de Neutrófilo , Neutrófilos , Receptor PAR-2 , Receptores Acoplados a Proteínas G , Humanos , Inflamação/patologia , Mieloblastina/metabolismo , Ativação de Neutrófilo/fisiologia , Fagocitose , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo
3.
FASEB J ; 34(1): 1107-1121, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914708

RESUMO

The nucleolus is best known for its cellular role in regulating ribosome production and growth. More recently, an unanticipated role for the nucleolus in innate immunity has recently emerged whereby downregulation of fibrillarin and nucleolar contraction confers pathogen resistance across taxa. The mechanism of this downregulation, however, remains obscure. Here we report that rather than fibrillarin itself being the proximal factor in this pathway, the key player is a fibrillarin-stabilizing deubiquitinylase USP-33. This was discovered by a candidate-gene search of Caenorhabditis elegans in which CED-3 caspase was revealed to execute targeted cleavage of USP-33, thus destabilizing fibrillarin. We also showed that cep-1 and ced-3 mutant worms altered nucleolar size and decreased antimicrobial peptide gene, spp-1, expression rendering susceptibility to bacterial infection. These phenotypes were reversed by usp-33 knockdown, thus linking the CEP-1-CED-3-USP-33 pathway with nucleolar control and resistance to bacterial infection in worms. Parallel experiments with the human analogs of caspases and USP36 revealed similar roles in coordinating these two processes. In summary, our work outlined a conserved cascade that connects cell death signaling to nucleolar control and innate immune response.


Assuntos
Infecções Bacterianas/metabolismo , Caenorhabditis elegans/microbiologia , Nucléolo Celular/metabolismo , Enzimas Desubiquitinantes/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismo , Animais , Apoptose , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Infecções por Pseudomonas , Interferência de RNA , Infecções Estafilocócicas , Estaurosporina/farmacologia , Ubiquitina Tiolesterase/metabolismo
4.
Front Immunol ; 9: 2830, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30559745

RESUMO

The adhesion family of G protein-coupled receptors (aGPCRs) comprises 33 members in human, several of which are distinctly expressed and functionally involved in polymorphonuclear cells (PMNs). As former work indicated the possible presence of the aGPCR GPR97 in granulocytes, we studied its cellular distribution, molecular structure, signal transduction, and biological function in PMNs. RNA sequencing and mass-spectrometry revealed abundant RNA and protein expression of ADGRG3/GPR97 in granulocyte precursors and terminally differentiated neutrophilic, eosinophilic, and basophilic granulocytes. Using a newly generated GPR97-specific monoclonal antibody, we confirmed that endogenous GPR97 is a proteolytically processed, dichotomous, N-glycosylated receptor. GPR97 was detected in tissue-infiltrating PMNs and upregulated during systemic inflammation. Antibody ligation of GPR97 increased neutrophil reactive oxygen species production and proteolytic enzyme activity, which is accompanied by an increase in mitogen-activated protein kinases and IκBα phosphorylation. In-depth analysis of the GPR97 signaling cascade revealed a possible switch from basal Gαs/cAMP-mediated signal transduction to a Gαi-induced reduction in cAMP levels upon mutation-induced activation of the receptor, in combination with an increase in downstream effectors of Gßγ, such as SRE and NF-κB. Finally, ligation of GPR97 increased the bacteria uptake and killing activity of neutrophils. We conclude that the specific presence of GPR97 regulates antimicrobial activity in human granulocytes.


Assuntos
Anti-Infecciosos/metabolismo , Granulócitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Anticorpos Monoclonais/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Eosinófilos/metabolismo , Humanos , Inflamação/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Neutrófilos/metabolismo , Fosforilação/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia
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