RESUMO
Autoantibodies are a hallmark of numerous neurological disorders, including multiple sclerosis, autoimmune encephalitides and neuromyelitis optica. Whilst well understood in peripheral myeloid cells, the pathophysiological significance of autoantibody-induced Fc receptor signalling in microglia remains unknown, in part due to the lack of a robust in vivo model. Moreover, the application of therapeutic antibodies for neurodegenerative disease also highlights the importance of understanding Fc receptor signalling in microglia. Here, we describe a novel in vivo experimental paradigm that allows for selective engagement of Fc receptors within the CNS by peripherally injecting anti-myelin oligodendrocyte glycoprotein (MOG) monoclonal antibodies into normal wild-type mice. MOG antigen-bound immunoglobulins were detected throughout the CNS and triggered a rapid and tightly regulated proliferative response in both brain and spinal cord microglia. This microglial response was abrogated when anti-MOG antibodies were deprived of Fc receptor effector function or injected into Fcγ receptor knockout mice and was associated with the downregulation of Fc receptors in microglia, but not peripheral myeloid cells, establishing that this response was dependent on central Fc receptor engagement. Downstream of the Fc receptors, BTK was a required signalling node for this response, as microglia proliferation was amplified in BtkE41K knock-in mice expressing a constitutively active form of the enzyme and blunted in mice treated with a CNS-penetrant small molecule inhibitor of BTK. Finally, this response was associated with transient and stringently regulated changes in gene expression predominantly related to cellular proliferation, which markedly differed from transcriptional programs typically associated with Fc receptor engagement in peripheral myeloid cells. Together, these results establish a physiologically-meaningful functional response to Fc receptor and BTK signalling in microglia, while providing a novel in vivo tool to further dissect the roles of microglia-specific Fc receptor and BTK-driven responses to both pathogenic and therapeutic antibodies in CNS homeostasis and disease.
Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Autoanticorpos/imunologia , Encéfalo/patologia , Microglia/patologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Receptores Fc/metabolismo , Medula Espinal/patologia , Animais , Encéfalo/imunologia , Encéfalo/metabolismo , Proliferação de Células/fisiologia , Camundongos , Microglia/imunologia , Microglia/metabolismo , Medula Espinal/imunologia , Medula Espinal/metabolismoRESUMO
V(D)J gene segment recombination is linked to the cell cycle by the periodic phosphorylation and destruction of the RAG-2 protein at the G1-to-S cell cycle transition. To examine the function of this coupling, we constructed mice in which the phosphorylation site at threonine 490 of RAG-2 was mutated to alanine. The RAG-2(T490A) mutation uncoupled DNA cleavage from cell cycle and promoted aberrant recombination. Similar aberrant recombination products were observed in mice deficient in the Skp2 ubiquitin ligase subunit, which is required for periodic destruction of RAG-2. On a p53-deficient background, the RAG-2(T490A) mutation induced lymphoid malignancies characterized by clonal chromosomal translocations involving antigen receptor genes. Taken together, these observations provide a direct link between the periodic destruction of RAG-2 and lymphoid tumorigenesis. We infer that cell cycle control of the V(D)J recombinase limits the potential genomic damage that could otherwise result from RAG-mediated DNA cleavage.
Assuntos
Ciclo Celular , Proteínas de Ligação a DNA/fisiologia , Rearranjo Gênico , Instabilidade Genômica , Linfoma não Hodgkin/genética , Receptores de Antígenos de Linfócitos T/genética , Substituição de Aminoácidos , Animais , Sequência de Bases , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Técnicas de Introdução de Genes , Genes p53 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Quinases Associadas a Fase S/genética , Organismos Livres de Patógenos Específicos , Linfócitos T/metabolismo , Translocação GenéticaRESUMO
Despite being one of the most common rheumatologic diseases, there is still no disease-modifying drug for primary Sjögren's syndrome (pSS). Advancing our knowledge of the target tissue has been limited by the low dimensionality of histology techniques and the small size of human salivary gland biopsies. In this study, we took advantage of a molecularly validated mouse model of pSS to characterize tissue-infiltrating CD4+ T cells and their regulation by the lymphotoxin/LIGHT signaling axis. Novel cell subsets were identified by combining highly dimensional flow and mass cytometry with transcriptomic analyses. Pharmacologic modulation of the LTßR signaling pathway was achieved by treating mice with LTßR-Ig, a therapeutic intervention currently being tested in pSS patients (Baminercept trial NCT01552681). Using these approaches, we identified two novel CD4+ T cell subsets characterized by high levels of PD1: Prdm1+ effector regulatory T cells expressing immunoregulatory factors, such as Il10, Areg, Fgl2, and Itgb8, and Il21+ effector conventional T cells expressing a pathogenic transcriptional signature. Mirroring these observations in mice, large numbers of CD4+PD1+ T cells were detected in salivary glands from Sjögren's patients but not in normal salivary glands or kidney biopsies from lupus nephritis patients. Unexpectedly, LTßR-Ig selectively halted the recruitment of PD1- naive, but not PD1+, effector T cells to the target tissue, leaving the cells with pathogenic potential unaffected. Altogether, this study revealed new cellular players in pSS pathogenesis, their transcriptional signatures, and differential dependency on the lymphotoxin/LIGHT signaling axis that help to interpret the negative results of the Baminercept trial and will guide future therapeutic interventions.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Receptor beta de Linfotoxina/metabolismo , Linfotoxina-alfa/metabolismo , Glândulas Salivares/imunologia , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/fisiopatologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Anfirregulina/genética , Animais , Biópsia , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Interleucina-10/genética , Interleucinas/genética , Rim/patologia , Nefrite Lúpica/imunologia , Linfotoxina-alfa/genética , Camundongos , Glândulas Salivares/patologia , Transdução de Sinais , Síndrome de Sjogren/terapia , Subpopulações de Linfócitos T/imunologia , Linfócitos T ReguladoresRESUMO
Ischemia-reperfusion injury (IRI) is a leading cause of AKI. This common clinical complication lacks effective therapies and can lead to the development of CKD. The αvß5 integrin may have an important role in acute injury, including septic shock and acute lung injury. To examine its function in AKI, we utilized a specific function-blocking antibody to inhibit αvß5 in a rat model of renal IRI. Pretreatment with this anti-αvß5 antibody significantly reduced serum creatinine levels, diminished renal damage detected by histopathologic evaluation, and decreased levels of injury biomarkers. Notably, therapeutic treatment with the αvß5 antibody 8 hours after IRI also provided protection from injury. Global gene expression profiling of post-ischemic kidneys showed that αvß5 inhibition affected established injury markers and induced pathway alterations previously shown to be protective. Intravital imaging of post-ischemic kidneys revealed reduced vascular leak with αvß5 antibody treatment. Immunostaining for αvß5 in the kidney detected evident expression in perivascular cells, with negligible expression in the endothelium. Studies in a three-dimensional microfluidics system identified a pericyte-dependent role for αvß5 in modulating vascular leak. Additional studies showed αvß5 functions in the adhesion and migration of kidney pericytes in vitro Initial studies monitoring renal blood flow after IRI did not find significant effects with αvß5 inhibition; however, future studies should explore the contribution of vasomotor effects. These studies identify a role for αvß5 in modulating injury-induced renal vascular leak, possibly through effects on pericyte adhesion and migration, and reveal αvß5 inhibition as a promising therapeutic strategy for AKI.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Rim/irrigação sanguínea , Receptores de Vitronectina/antagonistas & inibidores , Traumatismo por Reperfusão/prevenção & controle , Animais , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Mass cytometry has recently emerged as a promising tool for clinical research. However, few studies have demonstrated its benefit for patient stratification and biomarker identification. Primary Sjögren's syndrome (pSS) is a prototype of chronic autoimmune disease, the pathogenesis of which remains unclear and for which treatment does not exist. OBJECTIVE: This observational case-control study was designed to discover new cellular biomarkers and therapeutic targets in patients with pSS. METHODS: Forty-nine patients with pSS and 45 control subjects were enrolled for clinical evaluation and mass cytometry quantification of 34 protein markers in blood. For a third of these subjects, matched labial salivary gland biopsy specimens were also analyzed by mass cytometry and immunohistochemistry. RESULTS: In salivary gland biopsy specimens from patients with pSS, we identified a high number of activated CD8(+) T cells, terminally differentiated plasma cells, and activated epithelial cells, pointing to new pathogenic mechanisms for future clinical intervention. In blood, we identified a 6-cell disease signature defined by decreased numbers of CD4 and memory B lymphocytes, decreased plasmacytoid dendritic cell numbers, and increased representation of activated CD4 and CD8 T cells and plasmablasts. These blood cellular components correlated with clinical parameters and, when taken together, clustered patients into subsets with distinct disease activity and glandular inflammation. CONCLUSION: This first application of mass cytometry to a well-stratified clinical cohort and small biopsy tissues establishes the benefits of such an approach for the discovery of new biomarkers and therapeutic targets. Similar high-dimensional immunophenotyping strategies could be implemented in longitudinal and interventional clinical settings in this and other disease areas.
Assuntos
Biomarcadores , Imunofenotipagem , Glândulas Salivares/metabolismo , Glândulas Salivares/patologia , Síndrome de Sjogren/diagnóstico , Adulto , Biópsia , Linfócitos T CD8-Positivos , Análise por Conglomerados , Biologia Computacional/métodos , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Imunofenotipagem/métodos , Masculino , Pessoa de Meia-Idade , Plasmócitos , Reprodutibilidade dos Testes , Glândulas Salivares Menores/metabolismo , Glândulas Salivares Menores/patologiaRESUMO
B-cell activating factor (BAFF) levels are increased in rheumatoid arthritis, lupus and primary Sjögren's syndrome (pSS). However, BAFF contribution to pathogenesis is not completely understood. In pSS, immune infiltration of the salivary and lacrimal glands leads to xerostomia and xerophtalmia. Glandular B cell hyperactivation, differentiation into germinal center (GC)-like structures and plasma cell accumulation are histopathological hallmarks that were attributed to increased BAFF. Here, we experimentally tested this hypothesis by overexpressing BAFF in a mouse model of pSS. BAFF overexpression enhanced lymphocytic infiltration and MHCII expression on B cells. Increased BAFF also induced B cell differentiation into GC B cells within the autoimmune target tissue. However, even in these conditions, GC B cells only accounted for <1% of glandular B cells, demonstrating that BAFF is not efficiently promoting ectopic GC formation in pSS and warranting further investigation of therapeutics targeting both BAFF and the related TNF-family member APRIL.
Assuntos
Fator Ativador de Células B/imunologia , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Síndrome de Sjogren/imunologia , Animais , Autoimunidade/genética , Autoimunidade/imunologia , Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Linfócitos B/metabolismo , Linfócitos B/patologia , Diferenciação Celular/genética , Células Cultivadas , Citometria de Fluxo , Perfilação da Expressão Gênica/métodos , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Imuno-Histoquímica , Aparelho Lacrimal/imunologia , Aparelho Lacrimal/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Análise de Sequência com Séries de Oligonucleotídeos , Síndrome de Sjogren/genética , Síndrome de Sjogren/metabolismo , Xeroftalmia/genética , Xeroftalmia/imunologia , Xeroftalmia/metabolismo , Xerostomia/genética , Xerostomia/imunologia , Xerostomia/metabolismoRESUMO
Sjögren's syndrome (SS) is a complex autoimmune disease associated with lymphocytic infiltration and secretory dysfunction of salivary and lacrimal glands. Although the etiology of SS remains unclear, evidence suggests that epithelial damage of the glands elicits immune and fibrotic responses in SS. To define molecular changes underlying epithelial tissue damage in SS, we laser capture microdissected (LCM) labial salivary gland epithelia from 8 SS and 8 non-SS controls for analysis by RNA sequencing (RNAseq). Computational interrogation of gene expression signatures revealed that, in addition to a division of SS and non-SS samples, there was a potential intermediate state overlapping clustering of SS and non-SS samples. Differential expression analysis uncovered signaling events likely associated with distinct SS pathogenesis. Notable signals included the enrichment of IFN-γ and JAK/STAT-regulated genes, and the induction of genes encoding secreted factors, such as LTF, BMP3, and MMP7, implicated in immune responses, matrix remodeling and tissue destruction. Identification of gene expression signatures of salivary epithelia associated with mixed clinical and histopathological characteristics suggests that SS pathology may be defined by distinct molecular subtypes. We conclude that gene expression changes arising in the damaged salivary epithelia may offer novel insights into the signals contributing to SS development and progression.
Assuntos
Regulação da Expressão Gênica , Expressão Gênica , Glândulas Salivares/metabolismo , Síndrome de Sjogren/genética , Adulto , Idoso , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Epitélio/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Glândulas Salivares/patologia , Transdução de Sinais/fisiologia , Síndrome de Sjogren/metabolismo , Síndrome de Sjogren/patologiaRESUMO
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
OBJECTIVE: The Outcomes and Assessment Committee at the Virginia Commonwealth University School of Pharmacy was tasked with refining the school's key performance indicators (KPIs) to improve programmatic assessment by focusing on the most important measures. METHODS: Initially, 56 KPIs were tracked, nine of which were university mandated, divided into 10 modules: admissions, community outreach, continuing education, diversity, faculty experience and success, fundraising, graduate program, research and scholarship, staff experience and success, and student experience and success. Using a three-round Delphi consensus technique, KPIs were reviewed by faculty and staff. Each participant responded whether they considered each KPI to be essential or not essential for school quality assessment and improvement. Consensus for the first, second, and third rounds was defined as ≥90%, ≥80%, and ≥75% agreement, respectively. RESULTS: Of 109 faculty and staff invited, 49 participated in the first round, 51 in the second, and 42 in the third. At the end of the third round, accumulated consensus was achieved for 35 out of 88 (39.8%) KPIs that were considered essential and 3 out of 88 (3.4%) that were considered non-essential. Consensus percentage per module was: 15.4% (2/13) admissions, 28.6% (2/7) community outreach, 33.3% (3/9) continuing education, 27.3% (3/11) diversity, 62.5% (5/8) faculty experience and success, 55.6% (5/9) fundraising, 40% (4/10) graduate program, 33.3% (3/9) research and scholarship, 57.1% (4/7) staff experience and success, and 66.7% (4/6) student experience and success. CONCLUSIONS: Ultimately, 35 KPIs achieved consensus as essential to measure achievement of benchmarks for the school, which totals 44 KPIs, including nine university mandated KPIs. The process facilitated faculty and staff involvement in KPI selection and achieved improved focus for programmatic assessment.
RESUMO
Advances in immunosuppression have decreased the incidence of acute rejection, but the development of vasculopathy in the coronary arteries of transplants continues to limit the survival of cardiac allografts. Transplant vasculopathy has also been referred to as accelerated graft arteriosclerosis because it has features of arteriosclerosis, but it is limited to the graft and develops over a period of months to years. Although the pathological features of transplant vasculopathy are well defined, the causative mechanisms are not completely understood. This review focuses on the mechanisms by which antibody and complement can cause or contribute to coronary vasculopathy in cardiac transplants. Antibodies and complement can have independent effects, but the combination of antibodies and complement with inflammatory cells has greater pathogenic potential for the endothelial and smooth muscle cells of the coronary arteries. For example, stimulation through receptors for IgG or complement split products can activate macrophages, but stimulation through combinations of these receptors generates synergistic results. Together, antibodies and complement efficiently integrate the activation of endothelial cells, platelets, and macrophages, which are 3 of the primary components in the pathogenesis of transplant vasculopathy. Recent findings indicate that antibodies and complement produced within the transplant may contribute to vascular pathology in some transplants. Acute rejection caused by antibodies and complement has been treated by combinations of plasmapheresis, intravenous gamma-globulin and monoclonal antibodies to CD20 on B lymphocytes. The effect of these treatment modalities on the development of coronary vasculopathy is unknown.
Assuntos
Anticorpos/fisiologia , Proteínas do Sistema Complemento/fisiologia , Vasos Coronários/imunologia , Vasos Coronários/transplante , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Transplante de Coração/imunologia , Animais , Vasos Coronários/patologia , HumanosRESUMO
Naked mole-rats (Heterocephalus glaber) are fossorial, eusocial rodents that exhibit the unusual capability of moving their lower incisors independently in lateral and rostroventral directions. The evolution of this trait would presumably also involve concurrent alterations in neck musculature to support and control movements of the lower incisors. In order to assess morphological adaptations that might facilitate these movements, we performed detailed dissections of the neck musculature of adult naked mole-rats. In addition to characterizing attachment sites of superficial, suprahyoid, and infrahyoid musculature, we also quantified muscle mass and mandibular features thought to be associated with gape (condyle height, condyle length, and jaw length). Based on muscle attachment sites, the platysma myoides may contribute to lateral movement of the lower incisor and hemi-mandible in naked mole-rats. The large digastric muscle is likely to be a main contributor to rostroventral movement of each lower incisor. The geniohyoid and mylohyoid muscles also likely contribute to rostroventral movements of the lower incisors, and the mylohyoid may also produce lateral spreading of the hemi-mandibles. The transverse mandibular (intermandibularis) muscle likely serves to reposition the lower incisors back to a midline orientation following a movement.
Assuntos
Osso Hioide/anatomia & histologia , Incisivo/fisiologia , Ratos-Toupeira/anatomia & histologia , Movimento , Músculos do Pescoço/anatomia & histologia , Animais , Peso Corporal , Tamanho do ÓrgãoRESUMO
BIN1 is the most important risk locus for Late Onset Alzheimer's Disease (LOAD), after ApoE. BIN1 AD-associated SNPs correlate with Tau deposition as well as with brain atrophy. Furthermore, the level of neuronal-specific BIN1 isoform 1 protein is decreased in sporadic AD cases in parallel with neuronal loss, despite an overall increase in BIN1 total mRNA. To address the relationship between reduction of BIN1 and neuronal cell loss in the context of Tau pathology, we knocked-down endogenous murine Bin1 via stereotaxic injection of AAV-Bin1 shRNA in the hippocampus of mice expressing Tau P301S (PS19). We observed a statistically significant reduction in the number of neurons in the hippocampus of mice injected with AAV-Bin1 shRNA in comparison with mice injected with AAV control. To investigate whether neuronal loss is due to deletion of Bin1 selectively in neurons in presence Tau P301S, we bred Bin1flox/flox with Thy1-Cre and subsequently with PS19 mice. Mice lacking neuronal Bin1 and expressing Tau P301S showed increased mortality, without increased neuropathology, when compared to neuronal Bin1 and Tau P301S-expressing mice. The loss of Bin1 isoform 1 resulted in reduced excitability in primary neurons in vitro, reduced neuronal c-fos expression as well as in altered microglia transcriptome in vivo. Taken together, our data suggest that the contribution of genetic variation in BIN1 locus to AD risk could result from a cell-autonomous reduction of neuronal excitability due to Bin1 decrease, exacerbated by the presence of aggregated Tau, coupled with a non-cell autonomous microglia activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Doença de Alzheimer/patologia , Biomarcadores/metabolismo , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Hipocampo/fisiopatologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/patologia , Proteínas Supressoras de Tumor/fisiologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Comportamento Animal , Encéfalo/metabolismo , Feminino , Perfilação da Expressão Gênica , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Ratos , Proteínas tau/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
Despite Bridging INtegrator 1 (BIN1) being the second most statistically-significant locus associated to Late Onset Alzheimer's Disease, its role in disease pathogenesis remains to be clarified. As reports suggest a link between BIN1, Tau and extracellular vesicles, we investigated whether BIN1 could affect Tau spreading via exosomes secretion. We observed that BIN1-associated Tau-containing extracellular vesicles purified from cerebrospinal fluid of AD-affected individuals are seeding-competent. We showed that BIN1 over-expression promotes the release of Tau via extracellular vesicles in vitro as well as exacerbation of Tau pathology in vivo in PS19 mice. Genetic deletion of Bin1 from microglia resulted in reduction of Tau secretion via extracellular vesicles in vitro, and in decrease of Tau spreading in vivo in male, but not female, mice, in the context of PS19 background. Interestingly, ablation of Bin1 in microglia of male mice resulted in significant reduction in the expression of heat-shock proteins, previously implicated in Tau proteostasis. These observations suggest that BIN1 could contribute to the progression of AD-related Tau pathology by altering Tau clearance and promoting release of Tau-enriched extracellular vesicles by microglia.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Doença de Alzheimer/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas Nucleares/biossíntese , Proteínas Supressoras de Tumor/biossíntese , Proteínas tau/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Vesículas Extracelulares/genética , Vesículas Extracelulares/patologia , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Proteostase , Caracteres Sexuais , Proteínas Supressoras de Tumor/genética , Proteínas tau/genéticaRESUMO
CX3CR1, one of the highest expressed genes in microglia in mice and humans, is implicated in numerous microglial functions. However, the molecular mechanisms underlying Cx3cr1 signaling are not well understood. Here, we analyzed transcriptomes of Cx3cr1-deficient microglia under varying conditions by RNA-sequencing (RNA-seq). In 2-mo-old mice, Cx3cr1 deletion resulted in the down-regulation of a subset of immune-related genes, without substantial epigenetic changes in markers of active chromatin. Surprisingly, Cx3cr1-deficient microglia from young mice exhibited a transcriptome consistent with that of aged Cx3cr1-sufficient animals, suggesting a premature aging transcriptomic signature. Immunohistochemical analysis of microglia in young and aged mice revealed that loss of Cx3cr1 modulates microglial morphology in a comparable fashion. Our results suggest that CX3CR1 may regulate microglial function in part by modulating the expression levels of a subset of inflammatory genes during chronological aging, making Cx3cr1-deficient mice useful for studying aged microglia.
Assuntos
Senilidade Prematura/genética , Receptor 1 de Quimiocina CX3C/deficiência , Microglia/metabolismo , Animais , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Modelos Animais de Doenças , Feminino , Deleção de Genes , Perfil Genético , Inflamação/genética , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neurônios/metabolismo , Receptores de Quimiocinas/deficiência , Transdução de Sinais , TranscriptomaRESUMO
Salivary glands, such as submandibular glands (SMGs), are composed of branched epithelial ductal networks that terminate in acini that together produce, transport and secrete saliva. Here, we show that the transcriptional regulator Yap, a key effector of the Hippo pathway, is required for the proper patterning and morphogenesis of SMG epithelium. Epithelial deletion of Yap in developing SMGs results in the loss of ductal structures, arising from reduced expression of the EGF family member Epiregulin, which we show is required for the expansion of Krt5/Krt14-positive ductal progenitors. We further show that epithelial deletion of the Lats1 and Lats2 genes, which encode kinases that restrict nuclear Yap localization, results in morphogenesis defects accompanied by an expansion of Krt5/Krt14-positive cells. Collectively, our data indicate that Yap-induced Epiregulin signaling promotes the identity of SMG ductal progenitors and that removal of nuclear Yap by Lats1/2-mediated signaling is critical for proper ductal maturation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Epirregulina/metabolismo , Epitélio/embriologia , Morfogênese , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Glândula Submandibular/embriologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Padronização Corporal , Proteínas de Ciclo Celular , Deleção de Genes , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Células-Tronco/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas de Sinalização YAPRESUMO
Fibrotic diseases contribute to 45% of deaths in the industrialized world, and therefore a better understanding of the pathophysiological mechanisms underlying tissue fibrosis is sorely needed. We aimed to identify novel modifiers of tissue fibrosis expressed by myofibroblasts and their progenitors in their disease microenvironment through RNA silencing in vivo. We leveraged novel biology, targeting genes upregulated during liver and kidney fibrosis in this cell lineage, and employed small interfering RNA (siRNA)-formulated lipid nanoparticles technology to silence these genes in carbon-tetrachloride-induced liver fibrosis in mice. We identified five genes, Egr2, Atp1a2, Fkbp10, Fstl1, and Has2, which modified fibrogenesis based on their silencing, resulting in reduced Col1a1 mRNA levels and collagen accumulation in the liver. These genes fell into different groups based on the effects of their silencing on a transcriptional mini-array and histological outcomes. Silencing of Egr2 had the broadest effects in vivo and also reduced fibrogenic gene expression in a human fibroblast cell line. Prior to our study, Egr2, Atp1a2, and Fkbp10 had not been functionally validated in fibrosis in vivo. Thus, our results provide a major advance over the existing knowledge of fibrogenic pathways. Our study is the first example of a targeted siRNA assay to identify novel fibrosis modifiers in vivo.
RESUMO
Systemic sclerosis (SSc) is a chronic autoimmune disorder that can result in extensive tissue damage in the skin and, in advanced cases, internal organs. Vasculopathy, aberrant immune activation, and tissue fibrosis are three hallmarks of the disease that have been identified, with vasculopathy and aberrant immunity being amongst the earliest events. However, a mechanistic link between these processes has not been established. Here, we have identified a novel role of platelet derived growth factor-BB (PDGF-BB)/PDGFRß activation in combination with dermal injury induced by bleomycin as a driver of early, aberrant expression of interferon stimulatory genes (ISGs) and inflammatory monocyte infiltration. Activation of PDGFRß in combination with bleomycin-induced dermal injury resulted in increased dermal thickness, vascular density, monocyte/macrophage infiltration, and exacerbation of tissue injury. Many of these features were dependent on IFNAR-signaling, and an increase in the number of interferon-beta (IFN-ß) producing monocytes cells was found in the skin lesions. Taken together, these results identify a novel link between PDGFRß activation, and Type I IFN-driven vascular maintenance and monocyte/macrophage cell recruitment, and provide a potential explanation linking key features of SSc that were previously thought to be unrelated.
Assuntos
Vasos Sanguíneos/patologia , Modelos Animais de Doenças , Interferon Tipo I/fisiologia , Monócitos/patologia , Proteínas Proto-Oncogênicas c-sis/fisiologia , Escleroderma Sistêmico/patologia , Dermatopatias/patologia , Animais , Becaplermina , Bleomicina/farmacologia , Fibrose , Camundongos , Camundongos Endogâmicos C57BL , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
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
A stranded 5-month-old female Pacific harbor seal (Phoca vitulina richardsi) was presented displaying tachypnea and diminished lung sounds. No neurological abnormalities were noted. The animal was treated for verminous pneumonia, but died 2 wk later. Gross necropsy examination revealed a severe obstructive verminous pneumonia associated with large numbers of Otostrongylus circumlitus. In addition, the majority of the right cerebral hemisphere was absent, with hypoplasia of the left cerebellar hemisphere, absence of the right pyramid, and malformation of the right occipital bone. Histopathologic findings included multifocal thrombosis and inflammation of pulmonary arteries, verminous pneumonia, and mild vacuolation of the subependymal white matter in the third ventricle representing swelling of myelin sheaths and edema. This is the first report of a hemicerebral anomaly in a marine mammal.