RESUMO
MALT1 forms part of a central signaling node downstream of immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, across a broad range of immune cell subsets, and regulates NF-κB driven transcriptional responses via dual scaffolding-protease activity. Allosteric inhibition of MALT1 activity has demonstrated benefit in animal models of inflammation. However, development of MALT1 inhibitors to treat autoimmune and inflammatory diseases (A&ID) has been hindered by reports linking MALT1 inhibition and genetic loss-of-function to reductions in regulatory T-cell (Treg) numbers and development of auto-inflammatory syndromes. Using an allosteric MALT1 inhibitor, we investigated the consequence of pharmacological inhibition of MALT1 on proinflammatory cells compared to regulatory T-cells. Consistent with its known role in ITAM-driven responses, MALT1 inhibition suppressed proinflammatory cytokine production from activated human T-cells and monocyte-derived macrophages, and attenuated B-cell proliferation. Oral administration of a MALT1 inhibitor reduced disease severity and synovial cytokine production in a rat collagen-induced arthritis model. Interestingly, reduction in splenic Treg numbers was less pronounced in the context of inflammation compared with naïve animals. Additionally, in the context of the disease model, we observed an uncoupling of anti-inflammatory effects of MALT1 inhibition from Treg reduction, with lower systemic concentrations of inhibitor needed to reduce disease severity compared to that required to reduce Treg numbers. MALT1 inhibition did not affect suppressive function of human Tregs in vitro. These data indicate that anti-inflammatory efficacy can be achieved with MALT1 inhibition without impacting the number or function of Tregs, further supporting the potential of MALT1 inhibition in the treatment of autoimmune disease.
Assuntos
Doenças Autoimunes , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Linfócitos T Reguladores , Animais , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/patologia , Citocinas/genética , Inflamação , Ativação Linfocitária , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , NF-kappa B , Ratos , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
OBJECTIVE: Punch biopsy, a standard diagnostic procedure for patients with cutaneous lupus erythematosus (CLE) carries an infection risk, is invasive, uncomfortable and potentially scarring, and impedes patient recruitment in clinical trials. Non-invasive tape sampling is an alternative that could enable serial evaluation of specific lesions. This cross-sectional pilot research study evaluated the use of a non-invasive adhesive tape device to collect messenger RNA (mRNA) from the skin surface of participants with CLE and healthy volunteers (HVs) and investigated its feasibility to detect biologically meaningful differences between samples collected from participants with CLE and samples from HVs. METHODS: Affected and unaffected skin tape samples and simultaneous punch biopsies were collected from 10 participants with CLE. Unaffected skin tape and punch biopsies were collected from 10 HVs. Paired samples were tested using quantitative PCR for a candidate immune gene panel and semi-quantitative immunohistochemistry for hallmark CLE proteins. RESULTS: mRNA collected using the tape device was of sufficient quality for amplification of 94 candidate immune genes. Among these, we found an interferon (IFN)-dominant gene cluster that differentiated CLE-affected from HV (23-fold change; p<0.001) and CLE-unaffected skin (sevenfold change; p=0.002), respectively. We found a CLE-associated gene cluster that differentiated CLE-affected from HV (fourfold change; p=0.005) and CLE-unaffected skin (fourfold change; p=0.012), respectively. Spearman's correlation between per cent area myxovirus 1 protein immunoreactivity and IFN-dominant mRNA gene cluster expression was highly significant (dermis, rho=0.86, p<0.001). In total, skin tape-derived RNA expression comprising both IFN-dominant and CLE-associated gene clusters correlated with per cent area immunoreactivity of some hallmark CLE-associated proteins in punch biopsies from the same lesions. CONCLUSIONS: A non-invasive tape RNA collection technique is a potential tool for repeated skin biomarker measures throughout a clinical trial.
Assuntos
Lúpus Eritematoso Cutâneo , Estudos Transversais , Feminino , Voluntários Saudáveis , Humanos , Masculino , RNA , PeleRESUMO
Hyperphosphorylated tau protein is a pathological hallmark of numerous neurodegenerative diseases and the level of tau pathology is correlated with the degree of cognitive impairment. Tau hyper-phosphorylation is thought to be an early initiating event in the cascade leading to tau toxicity and neuronal death. Inhibition of tau phosphorylation therefore represents an attractive therapeutic strategy. However, the widespread expression of most kinases and promiscuity of their substrates, along with poor selectivity of most kinase inhibitors, have resulted in systemic toxicities that have limited the advancement of tau kinase inhibitors into the clinic. We therefore focused on the CNS-specific tau kinase, TTBK1, and investigated whether selective inhibition of this kinase could represent a viable approach to targeting tau phosphorylation in disease. In the current study, we demonstrate that TTBK1 regulates tau phosphorylation using overexpression or knockdown of this kinase in heterologous cells and primary neurons. Importantly, we find that TTBK1-specific phosphorylation of tau leads to a loss of normal protein function including a decrease in tau-tubulin binding and deficits in tubulin polymerization. We then describe the use of a novel, selective small molecule antagonist, BIIB-TTBK1i, to study the acute effects of TTBK1 inhibition on tau phosphorylation in vivo. We demonstrate substantial lowering of tau phosphorylation at multiple sites implicated in disease, suggesting that TTBK1 inhibitors may represent an exciting new approach in the search for neurodegenerative disease therapies.
Assuntos
Doenças do Sistema Nervoso Central/enzimologia , Doenças do Sistema Nervoso Central/patologia , Sistema Nervoso Central/enzimologia , Sistema Nervoso Central/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas tau/metabolismo , Animais , Células Cultivadas , Masculino , Camundongos Endogâmicos C57BL , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Especificidade de Órgãos , Fosforilação/efeitos dos fármacos , Polimerização , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Tubulina (Proteína)/metabolismoRESUMO
A population pharmacokinetic/pharmacodynamic (popPK/PD) model for BIIB059 (anti-blood dendritic cell antigen 2 [anti-BDCA2]), a humanized immunoglobulin G1 monoclonal antibody currently under development for the treatment of SLE and CLE, is presented. BIIB059 binds BDCA2, a plasmacytoid dendritic cell (pDC)-specific receptor that inhibits the production of IFN-I and other inflammatory mediators when ligated. Phase 1 PK and PD data of healthy adult volunteers (HV, n = 87) and SLE subjects (n = 22) were utilized for the development of the popPK/PD model. The data included single and multiple dosing of intravenous and subcutaneous BIIB059. BDCA2 internalization (PD marker) was measured for all subjects by monitoring reduction of BDCA2 on pDC cell surface and used for development of the popPD model. A two-compartment popPK model with linear plus non-linear elimination was found to best describe BIIB059 PK. BDCA2 levels were best captured using an indirect response model with stimulation of the elimination of BDCA2. Clearance in SLE subjects was 25% higher compared to HV (6.87 vs 5.52 mL/h). Bodyweight was identified as only other covariate on clearance and central volume. The estimates of EC50 and Emax were 0.35 µg/mL and 8.92, respectively. No difference in EC50 and Emax was observed between SLE and HV. The popPK/PD model described the data accurately, as evaluated by pcVPCs and bootstrap. The presented popPK/PD model for BIIB059 provides valuable insight into the dynamics and dose-response relationship of BIIB059 for the treatment of SLE and CLE and was used to guide dose selection for the Phase 2 clinical study (NCT02847598).
Assuntos
Anticorpos Monoclonais Humanizados/farmacocinética , Lectinas Tipo C/antagonistas & inibidores , Lúpus Eritematoso Cutâneo/tratamento farmacológico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Glicoproteínas de Membrana/antagonistas & inibidores , Modelos Biológicos , Receptores Imunológicos/antagonistas & inibidores , Administração Intravenosa , Adulto , Idoso , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Área Sob a Curva , Disponibilidade Biológica , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Meia-Vida , Humanos , Injeções Subcutâneas , Lectinas Tipo C/imunologia , Lúpus Eritematoso Cutâneo/sangue , Lúpus Eritematoso Cutâneo/imunologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Glicoproteínas de Membrana/imunologia , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Receptores Imunológicos/imunologiaRESUMO
Objective: Plasmacytoid dendritic cells (pDCs) are a major source of Type-I Interferon (IFN-I), a key driver in cutaneous lupus erythematosus (CLE). Currently evaluated in Phase II clinical trial, 24F4A (BIIB059) is an antibody targeting BDCA2, an inhibitory receptor expressed on pDCs. Given that Hydroxychloroquine (HCQ), a widely-used CLE therapy, and 24F4A are both able to inhibit pDC-derived IFN-I production; this study aimed to determine whether 24F4A would show an additional inhibitory effect on pDC response after ex vivo or in vivo treatment with HCQ. Methods: The effect of 24F4A on pDC-derived IFNα was measured from peripheral blood mononuclear cells (PBMC) either from healthy donors in presence or absence of HCQ or from CLE patients clinically exposed to various levels of HCQ. TLR7, TLR7/8, and TLR9 agonists (ssRNA, R848, and CpG-A) were used for pDC stimulation. Results: PDCs were the only producers of IFNα in response to CpG-A, R848, and ssRNA stimulation in PBMC cultures. CLE patients with higher levels of blood HCQ showed lower ex vivo pDC responses to CpG-A, but not R848 or ssRNA. In contrast, 24F4A reduced the amount of IFNα produced by pDCs from CLE patients in response to all TLR agonists, irrespective of the blood HCQ level. Conclusion: Our findings reveal that clinically-relevant HCQ concentrations partially inhibit the pDC response to TLR9 and weakly affect the response to TLR7/8 stimulation. 24F4A robustly inhibits pDC responses even in the presence of HCQ, highlighting its unique potential to disrupt pDC disease relevant biology, which could provide additional therapeutic benefit for CLE patients.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Células Dendríticas/efeitos dos fármacos , Hidroxicloroquina/uso terapêutico , Interferon-alfa/metabolismo , Lectinas Tipo C/metabolismo , Lúpus Eritematoso Cutâneo/tratamento farmacológico , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Adulto , Células Dendríticas/metabolismo , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Cutâneo/metabolismo , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores Toll-Like/metabolismoRESUMO
BACKGROUND: Plasmacytoid DCs (pDC) produce large amounts of type I IFN (IFN-I), cytokines convincingly linked to systemic lupus erythematosus (SLE) pathogenesis. BIIB059 is a humanized mAb that binds blood DC antigen 2 (BDCA2), a pDC-specific receptor that inhibits the production of IFN-I and other inflammatory mediators when ligated. A first-in-human study was conducted to assess safety, tolerability, and pharmacokinetic (PK) and pharmacodynamic (PD) effects of single BIIB059 doses in healthy volunteers (HV) and patients with SLE with active cutaneous disease as well as proof of biological activity and preliminary clinical response in the SLE cohort. METHODS: A randomized, double-blind, placebo-controlled clinical trial was conducted in HV (n = 54) and patients with SLE (n = 12). All subjects were monitored for adverse events. Serum BIIB059 concentrations, BDCA2 levels on pDCs, and IFN-responsive biomarkers in whole blood and skin biopsies were measured. Skin disease activity was determined using the Cutaneous Lupus Erythematosus Disease Area and Severity Index Activity (CLASI-A). RESULTS: Single doses of BIIB059 were associated with favorable safety and PK profiles. BIIB059 administration led to BDCA2 internalization on pDCs, which correlated with circulating BIIB059 levels. BIIB059 administration in patients with SLE decreased expression of IFN response genes in blood, normalized MxA expression, reduced immune infiltrates in skin lesions, and decreased CLASI-A score. CONCLUSIONS: Single doses of BIIB059 were associated with favorable safety and PK/PD profiles and robust target engagement and biological activity, supporting further development of BIIB059 in SLE. The data suggest that targeting pDCs may be beneficial for patients with SLE, especially those with cutaneous manifestations. TRIAL REGISTRATION: ClinicalTrials.gov NCT02106897. FUNDING: Biogen Inc.
Assuntos
Anticorpos Monoclonais , Lectinas Tipo C/antagonistas & inibidores , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Glicoproteínas de Membrana/antagonistas & inibidores , Receptores Imunológicos/antagonistas & inibidores , Dermatopatias/tratamento farmacológico , Pele/imunologia , Adolescente , Adulto , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Células Dendríticas/imunologia , Células Dendríticas/patologia , Método Duplo-Cego , Feminino , Humanos , Lectinas Tipo C/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Masculino , Glicoproteínas de Membrana/imunologia , Pessoa de Meia-Idade , Plasmócitos/imunologia , Plasmócitos/patologia , Receptores Imunológicos/imunologia , Pele/patologia , Dermatopatias/imunologia , Dermatopatias/patologiaRESUMO
BIIB059 is a novel humanized monoclonal antibody (mAb) that is currently under development for the treatment of Systemic Lupus Erythematosus and Cutaneous Lupus Erythematosus. BIIB059 is targeted against the blood dendritic cell antigen 2 (BDCA2), a receptor exclusively expressed on the surface of plasmacytoid dendritic cells (pDCs). Herein, we utilized pre-clinical pharmacokinetic (PK) and pharmacodynamic (PD) data to develop a non-human primate (NHP) model and to address whether the NHP model can be successfully scaled to predict the human PK/PD. In particular, PK data from 17 cynomolgus monkeys were utilized for PK model development, wherein BIIB059 was administered intravenously (1 and 10 mg/kg single-dosing and 5 mg/kg multiple-dosing) or subcutaneously (0.2 and 7.5 mg/kg single-dosing). Additionally, PD data (BDCA2 receptor density on pDCs) from 6 cynomolgus monkeys were used for the development of the PD model. The developed NHP two-compartment PK model, linked with an indirect response PD model, was subsequently scaled to humans by combining traditional allometric PK scaling with sensitivity-analysis-driven scaling of the PD. The scaled PK/PD model was then used to simulate the human PK/PD for different dose levels. When clinical data from the BIIB059 Phase I study became available, they were used to evaluate the predictability of the scaled PK/PD model and the model simulations were in agreement with the clinical data. Therefore, the presented approach is suggested to be employed in scaling pre-clinical mAb models to support the selection of safe first-in-human doses and, more broadly, the prediction of PK/PD in the clinic.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Lectinas Tipo C/antagonistas & inibidores , Glicoproteínas de Membrana/antagonistas & inibidores , Modelos Biológicos , Receptores Imunológicos/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Peso Corporal , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Humanos , Imunoglobulina G , Lúpus Eritematoso Cutâneo/tratamento farmacológico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Macaca fascicularisRESUMO
Mouse models lupus nephritis (LN) have provided important insights into disease pathogenesis, although none have been able to recapitulate all features of the human disease. Using comprehensive longitudinal analyses, we characterized a novel accelerated mouse model of lupus using pristane treatment in SNF1 (SWR X NZB F1) lupus prone mice (pristane-SNF1 mice). Pristane treatment in SNF1 mice accelerated the onset and progression of proteinuria, autoantibody production, immune complex deposition and development of renal lesions. At week 14, the pristane-SNF1 model recapitulated kidney disease parameters and molecular signatures seen in spontaneous disease in 36 week-old SNF1 mice and in a traditional IFNα-accelerated NZB X NZW F1 (BWF1) model. Blood transcriptome analysis revealed interferon, plasma cell, neutrophil, T-cell and protein synthesis signatures in the pristane-SNF1 model, all known to be present in the human disease. The pristane-SNF1 model appears to be particularly useful for preclinical research, robustly exhibiting many characteristics reminiscent of human disease. These include i) a stronger upregulation of the cytosolic nucleic acid sensing pathway, which is thought to be key component of the pathogenesis of the human disease, and ii) more prominent kidney interstitial inflammation and fibrosis, which have been both associated with poor prognosis in human LN. To our knowledge, this is the only accelerated model of LN that exhibits a robust tubulointerstitial inflammatory and fibrosis response. Taken together our data show that the pristane-SNF1 model is a novel accelerated model of LN with key features similar to human disease.
Assuntos
Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Nefrite Lúpica/patologia , Terpenos/farmacologia , Animais , Autoanticorpos/biossíntese , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Fibrose , Glomerulonefrite/induzido quimicamente , Glomerulonefrite/complicações , Humanos , Hipergamaglobulinemia/induzido quimicamente , Hipergamaglobulinemia/complicações , Inflamação/induzido quimicamente , Inflamação/complicações , Nefrite Lúpica/complicações , Nefrite Lúpica/imunologia , Nefrite Lúpica/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Accumulation of inflammatory cells in different renal compartments is a hallmark of progressive kidney diseases including glomerulonephritis (GN). Lymphotoxin ß receptor (LTßR) signaling is crucial for the formation of lymphoid tissue, and inhibition of LTßR signaling has ameliorated several non-renal inflammatory models. Therefore, we tested whether LTßR signaling could also have a role in renal injury. Renal biopsies from patients with GN were found to express both LTα and LTß ligands, as well as LTßR. The LTßR protein and mRNA were localized to tubular epithelial cells, parietal epithelial cells, crescents, and cells of the glomerular tuft, whereas LTß was found on lymphocytes and tubular epithelial cells. Human tubular epithelial cells, mesangial cells, and mouse parietal epithelial cells expressed both LTα and LTß mRNA upon stimulation with TNF in vitro. Several chemokine mRNAs and proteins were expressed in response to LTßR signaling. Importantly, in a murine lupus model, LTßR blockade improved renal function without the reduction of serum autoantibody titers or glomerular immune complex deposition. Thus, a preclinical mouse model and human studies strongly suggest that LTßR signaling is involved in renal injury and may be a suitable therapeutic target in renal diseases.
Assuntos
Glomerulonefrite por IGA/metabolismo , Nefrite Lúpica/metabolismo , Receptor beta de Linfotoxina/antagonistas & inibidores , Receptor beta de Linfotoxina/metabolismo , RNA Mensageiro/análise , Transdução de Sinais , Adulto , Animais , Linhagem Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Modelos Animais de Doenças , Células Epiteliais/química , Células Epiteliais/metabolismo , Feminino , Glomerulonefrite por IGA/genética , Humanos , Imunoglobulinas/farmacologia , Glomérulos Renais/química , Glomérulos Renais/patologia , Túbulos Renais/química , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Ligantes , Nefrite Lúpica/genética , Linfócitos/química , Receptor beta de Linfotoxina/análise , Receptor beta de Linfotoxina/genética , Linfotoxina-alfa/análise , Linfotoxina-alfa/genética , Linfotoxina-alfa/metabolismo , Linfotoxina-beta/análise , Linfotoxina-beta/genética , Linfotoxina-beta/metabolismo , Masculino , Células Mesangiais/metabolismo , Camundongos , Pessoa de Meia-Idade , Transdução de Sinais/efeitos dos fármacos , TranscriptomaRESUMO
Type I interferons (IFN-I) are implicated in the pathogenesis of systemic lupus erythematosus (SLE). In SLE, immune complexes bind to the CD32a (FcγRIIa) receptor on the surface of plasmacytoid dendritic cells (pDCs) and stimulate the secretion of IFN-I from pDCs. BDCA2 is a pDC-specific receptor that, when engaged, inhibits the production of IFN-I in human pDCs. BDCA2 engagement, therefore, represents an attractive therapeutic target for inhibiting pDC-derived IFN-I and may be an effective therapy for the treatment of SLE. In this study, we show that 24F4A, a humanized monoclonal antibody (mAb) against BDCA2, engages BDCA2 and leads to its internalization and the consequent inhibition of TLR-induced IFN-I by pDCs in vitro using blood from both healthy and SLE donors. These effects were confirmed in vivo using a single injection of 24F4A in cynomolgus monkeys. 24F4A also inhibited pDC activation by SLE-associated immune complexes (IC). In addition to the inhibitory effect of 24F4A through engagement of BDCA2, the Fc region of 24F4A was critical for potent inhibition of IC-induced IFN-I production through internalization of CD32a. This study highlights the novel therapeutic potential of an effector-competent anti-BDCA2 mAb that demonstrates a dual mechanism to dampen pDC responses for enhanced clinical efficacy in SLE.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Células Dendríticas/imunologia , Lectinas Tipo C/antagonistas & inibidores , Glicoproteínas de Membrana/antagonistas & inibidores , Plasmócitos/imunologia , Receptores de IgG/imunologia , Receptores Imunológicos/antagonistas & inibidores , Animais , Anticorpos Monoclonais Murinos/farmacologia , Células Dendríticas/citologia , Feminino , Humanos , Lectinas Tipo C/imunologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmócitos/citologia , Receptores Imunológicos/imunologiaRESUMO
The C-terminus of the voltage-gated calcium channel Cav1.2 encodes a transcription factor, the calcium channel associated transcriptional regulator (CCAT), that regulates neurite extension and inhibits Cav1.2 expression. The mechanisms by which CCAT is generated in neurons and myocytes are poorly understood. Here we show that CCAT is produced by activation of a cryptic promoter in exon 46 of CACNA1C, the gene that encodes CaV1.2. Expression of CCAT is independent of Cav1.2 expression in neuroblastoma cells, in mice, and in human neurons derived from induced pluripotent stem cells (iPSCs), providing strong evidence that CCAT is not generated by cleavage of CaV1.2. Analysis of the transcriptional start sites in CACNA1C and immune-blotting for channel proteins indicate that multiple proteins are generated from the 3' end of the CACNA1C gene. This study provides new insights into the regulation of CACNA1C, and provides an example of how exonic promoters contribute to the complexity of mammalian genomes.
Assuntos
Canais de Cálcio Tipo L/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Animais , Northern Blotting , Western Blotting , Encéfalo/embriologia , Encéfalo/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Éxons/genética , Humanos , Imunoprecipitação , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Ratos , Fatores de Transcrição/genética , Sítio de Iniciação de TranscriçãoRESUMO
Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding and stabilizing multiple intracellular proteins that have roles in cell activation and proliferation. Many Hsp90 client proteins in tumor cells are mutated or overexpressed oncogenic proteins driving cancer cell growth, leading to the acceptance of Hsp90 as a potential therapeutic target for cancer. Because several signal transduction molecules that are dependent on Hsp90 function are also involved in activation of innate and adaptive cells of the immune system, we investigated the mechanism by which inhibiting Hsp90 leads to therapeutic efficacy in rodent models of inflammation and autoimmunity. EC144, a synthetic Hsp90 inhibitor, blocked LPS-induced TLR4 signaling in RAW 264.7 cells by inhibiting activation of ERK1/2, MEK1/2, JNK, and p38 MAPK but not NF-κB. Ex vivo LPS-stimulated CD11b(+) peritoneal exudate cells from EC144-treated mice were blocked from phosphorylating tumor progression locus 2, MEK1/2, and ERK1/2. Consequently, EC144-treated mice were resistant to LPS administration and had suppressed systemic TNF-α release. Inhibiting Hsp90 also blocked in vitro CD4(+) T cell proliferation in mouse and human MLRs. In vivo, semitherapeutic administration of EC144 blocked disease development in rat collagen-induced arthritis by suppressing the inflammatory response. In a mouse collagen-induced arthritis model, EC144 also suppressed disease development, which correlated with a suppressed Ag-specific Ab response and a block in activation of Ag-specific CD4(+) T cells. Our results describe mechanisms by which blocking Hsp90 function may be applicable to treatment of autoimmune diseases involving inflammation and activation of the adaptive immune response.
Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Doenças Autoimunes/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Imunidade Inata/efeitos dos fármacos , Imunossupressores/farmacologia , Mediadores da Inflamação/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Cristalografia por Raios X , Modelos Animais de Doenças , Feminino , Humanos , Imunossupressores/química , Imunossupressores/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação/síntese química , Mediadores da Inflamação/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Pirimidinas/síntese química , Pirimidinas/uso terapêutico , Pirróis/síntese química , Pirróis/uso terapêutico , RatosRESUMO
Fear can be acquired vicariously through social observation of others suffering from aversive stimuli. We found that mice (observers) developed freezing behavior by observing other mice (demonstrators) receive repetitive foot shocks. Observers had higher fear responses when demonstrators were socially related to themselves, such as siblings or mating partners. Inactivation of anterior cingulate cortex (ACC) and parafascicular or mediodorsal thalamic nuclei, which comprise the medial pain system representing pain affection, substantially impaired this observational fear learning, whereas inactivation of sensory thalamic nuclei had no effect. The ACC neuronal activities were increased and synchronized with those of the lateral amygdala at theta rhythm frequency during this learning. Furthermore, an ACC-limited deletion of Ca(v)1.2 Ca(2+) channels in mice impaired observational fear learning and reduced behavioral pain responses. These results demonstrate the functional involvement of the affective pain system and Ca(v)1.2 channels of the ACC in observational social fear.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Medo/fisiologia , Giro do Cíngulo/fisiologia , Aprendizagem/fisiologia , Dor/fisiopatologia , Comportamento Social , Animais , Canais de Cálcio Tipo L/deficiência , Canais de Cálcio Tipo L/genética , Córtex Cerebral/fisiologia , Condicionamento Clássico/fisiologia , Medo/psicologia , Feminino , Reação de Congelamento Cataléptica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dor/genética , Dor/psicologia , Medição da Dor/métodos , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
In vitro immunoglobulin E (IgE) production was found to be sensitive to increasing cell concentration in culture wells. While class switching to IgE is intact as suggested by surface IgE staining, ELISPOT analysis provided evidence that the differentiation of IgE committed B cells to the plasma cell stage was arrested at high cell doses. In fact, splitting the cells at higher concentrations after culture initiation increased IgE production. Cells plated at higher doses were found to be more prone to apoptosis as assessed by Annexin staining. Interestingly, inhibiting apoptosis by the use of the caspase inhibitor DEVD significantly increased IgE levels implicating apoptosis in the preferential deletion of IgE expressing cells. These data not only highlight the caveat against using a single B-cell dose for IgE production in vitro but also suggest for the first time a possible IgE regulatory mechanism mediated by cell density.