RESUMO
Adalimumab (ADA) is the only Food and Drug Administrationâapproved treatment for moderate-to-severe hidradenitis suppurativa, whereas etanercept and certolizumab-pegol have been shown to be ineffective, suggesting that the mechanism of action of ADA is distinct in hidradenitis suppurativa and may contribute to improved wound healing. Given that macrophages (MÏs) play pivotal roles throughout the wound healing process, an in vitro MÏ differentiation assay was carried out to assess the impact of TNFâanti-TNF complexes on these cells. TNFâADA complexes exhibited stronger inhibitory effects on inflammatory MÏ differentiation. Moreover, RNA sequencing revealed several unique wound healing profiles for TNFâADAâtreated inflammatory MÏs, which were not observed for those treated with either TNFâetanercept or TNFâcertolizumab-pegol complexes, including the inhibition of the matrix metalloproteinase (MMP) pathway. In addition, ADA administration was found to significantly reduce the levels of inflammatory MMP-1 and MMP-9 while promoting wound-healing MMP-13 and tissue inhibitor of metalloproteinases 2 levels in the circulation of the patients with hidradenitis suppurativa who responded to treatment. Our in vitro findings show that TNFâADAâtreated inflammatory MÏs exhibit a distinct profile resembling wound healing. Moreover, ADA not only differentially regulates MMP expression in patients with hidradenitis suppurativa responding to the therapy but also potentially induces a transition to a profile suggestive of wound healing.
Assuntos
Adalimumab/farmacologia , Hidradenite Supurativa/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Metaloproteinases da Matriz/fisiologia , Inibidores do Fator de Necrose Tumoral/farmacologia , Cicatrização/efeitos dos fármacos , Adalimumab/uso terapêutico , Diferenciação Celular/efeitos dos fármacos , Certolizumab Pegol/farmacologia , Etanercepte/farmacologia , Hidradenite Supurativa/fisiopatologia , Humanos , Macrófagos/citologia , Inibidores de Metaloproteinases de Matriz/farmacologiaRESUMO
B cell acquisition and presentation of specific autoantigens (auto-Ags) are thought to play an important and complex role in autoimmunity development. We previously identified scavenger receptor A (SR-A) as an early target in altering B cell-mediated autoimmunity. SR-A is highly expressed on professional antigen-presenting cells such as macrophages (MΦs) and dendritic cells (DCs). In this study, we demonstrate that SR-A is responsible for controlling B cell interactions with DCs/MΦs to promote Ag transfer from B cells to DCs/MΦs. We established a high-throughput ELISA-based screen to identify novel SR-A inhibitors, the specificity of which was determined by dose dependence and Biacore surface plasmon resonance testing. We identified small molecule inhibitors (SMIs) able to reduce SR-A-mediated Ag transfer in human cells. In particular, the SMIs prevented SR-A-positive cells from accumulating/loading Ag over time. Furthermore, we determined that one SMI, sennoside B, can reduce SR-A-mediated capture of B cells. Finally, SMI-mediated decreases in Ag transfer or accumulation reduced T cell proliferation in vitro and in vivo. These observations demonstrate that B cell-DC/MΦ interactions are conducive to promoting Ag trafficking between these cell types via SR-A. Inhibitors of SR-A may provide a novel therapeutic strategy in ameliorating autoimmune disease development.
Assuntos
Antígenos/metabolismo , Receptores Depuradores Classe A/antagonistas & inibidores , Receptores Depuradores Classe A/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antraquinonas/farmacologia , Antígenos/imunologia , Autoantígenos/imunologia , Autoantígenos/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Extrato de Senna , Senosídeos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de TempoRESUMO
B lymphocytes can function independently as efficient APCs. However, our previous studies demonstrate that both dendritic cells and macrophages are necessary to propagate immune responses initiated by B cell APCs. This finding led us to identify a process in mice whereby Ag-specific B cells transfer Ag to other APCs. In this study, we report the ability and mechanism by which human B lymphocytes can transfer BCR-captured Ag to macrophages. The transfer of Ag involves direct contact between the two cells followed by the capture of B cell-derived membrane and/or intracellular components by the macrophage. These events are abrogated by blocking scavenger receptor A, a receptor involved in the exchange of membrane between APCs. Macrophages acquire greater amounts of Ag in the presence of specific B cells than in their absence. This mechanism allows B cells to amplify or edit the immune response to specific Ag by transferring BCR-captured Ag to other professional APCs, thereby increasing the frequency of its presentation. Ag transfer may perpetuate chronic autoimmune responses to specific self-proteins and help explain the efficacy of B cell-directed therapies in human disease.
Assuntos
Apresentação de Antígeno/imunologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Comunicação Celular/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Receptores Depuradores/fisiologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Membrana Celular/imunologia , Membrana Celular/metabolismo , Técnicas de Cocultura , Citosol/imunologia , Citosol/metabolismo , Humanos , Transporte Proteico/imunologia , Receptores Depuradores/classificação , Transdução de Sinais/imunologiaRESUMO
B cells play an active role in directing immunity against specific proteins in part because of their capacity to sequester antigen via B cell receptor (BCR). Our prior findings indicate that B cells can initiate an immune response in vivo to self proteins independent of other antigen-presenting cells (APC). However, these studies also demonstrated that both dendritic cells and macrophages are important in the ongoing immune response. The present work illustrates a mechanism by which antigen acquired by B cells through BCR is specifically transferred to other APC, in particular, macrophages. The transfer of antigen is dependent on the specificity of BCR and requires direct contact between the cells, but does not require MHC compatibility between the cells and is independent of the activation state of macrophages. Antigen transfer is functional, in that macrophages, which received B cell derived-antigen, can activate CD4 T cells. Overall, these results define a novel mechanism by which B cells can focus immunity toward a specific antigen and transfer the ability to activate CD4 T cells to other APC.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos B/imunologia , Comunicação Celular/imunologia , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Animais , Autoimunidade , Linfócitos T CD4-Positivos/imunologia , Citometria de Fluxo , Cooperação Linfocítica/imunologia , Camundongos , Microscopia de Fluorescência , Microscopia de Fluorescência por Excitação MultifotônicaRESUMO
A hallmark of the immune system is the ability to ignore self-antigens. In attempts to bypass normal immune tolerance, a post-translational protein modification was introduced into self-antigens to break T and B cell tolerance. We demonstrate that immune tolerance is bypassed by immunization with a post-translationally modified melanoma antigen. In particular, the conversion of an aspartic acid to an isoaspartic acid within the melanoma antigen tyrosinase-related protein (TRP)-2 peptide-(181-188) makes the otherwise immunologically ignored TRP-2 antigen immunogenic. Tetramer analysis of iso-Asp TRP-2 peptide-immunized mice demonstrated that CD8+ T cells not only recognized the isoaspartyl TRP-2 peptide but also the native TRP-2 peptide. These CD8+ T cells functioned as cytotoxic T lymphocytes, as they effectively lysed TRP-2 peptide-pulsed targets both in vitro and in vivo. Potentially, post-translational protein modification can be utilized to trigger strong immune responses to either tumor proteins or potentially weakly immunogenic pathogens.
Assuntos
Linfócitos B/imunologia , Ácido Isoaspártico/metabolismo , Processamento de Proteína Pós-Traducional , Linfócitos T/imunologia , Vacinação , Animais , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Técnicas In Vitro , Ácido Isoaspártico/química , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Estrutura MolecularRESUMO
The regulation of the multifunctional calcium/calmodulin dependent protein kinase II (CaMKII) by serine/threonine protein phosphatases has been extensively studied in neuronal cells; however, this regulation has not been investigated previously in fibroblasts. We cloned a cDNA from SV40-transformed human fibroblasts that shares 80% homology to a rat calcium/calmodulin-dependent protein kinase phosphatase that encodes a PPM1F protein. By using extracts from transfected cells, PPM1F, but not a mutant (R326A) in the conserved catalytic domain, was found to dephosphorylate in vitro a peptide corresponding to the auto-inhibitory region of CaMKII. Further analyses demonstrated that PPM1F specifically dephosphorylates the phospho-Thr-286 in autophosphorylated CaMKII substrate and thus deactivates the CaMKII in vitro. Coimmunoprecipitation of CaMKII with PPM1F indicates that the two proteins can interact intracellularly. Binding of PPM1F to CaMKII involves multiple regions and is not dependent on intact phosphatase activity. Furthermore, overexpression of PPM1F in fibroblasts caused a reduction in the CaMKII-specific phosphorylation of the known substrate vimentin(Ser-82) following induction of the endogenous CaM kinase. These results identify PPM1F as a CaM kinase phosphatase within fibroblasts, although it may have additional functions intracellularly since it has been presented elsewhere as POPX2 and hFEM-2. We conclude that PPM1F, possibly together with the other previously described protein phosphatases PP1 and PP2A, can regulate the activity of CaMKII. Moreover, because PPM1F dephosphorylates the critical autophosphorylation site of CaMKII, we propose that this phosphatase plays a key role in the regulation of the kinase intracellularly.