RESUMO
B-cell receptor (BCR)-activated B cells contribute to pathogenesis in chronic graft-versus-host disease (cGVHD), a condition manifested by both B-cell autoreactivity and immune deficiency. We hypothesized that constitutive BCR activation precluded functional B-cell maturation in cGVHD. To address this, we examined BCR-NOTCH2 synergy because NOTCH has been shown to increase BCR responsiveness in normal mouse B cells. We conducted ex vivo activation and signaling assays of 30 primary samples from hematopoietic stem cell transplantation patients with and without cGVHD. Consistent with a molecular link between pathways, we found that BCR-NOTCH activation significantly increased the proximal BCR adapter protein BLNK. BCR-NOTCH activation also enabled persistent NOTCH2 surface expression, suggesting a positive feedback loop. Specific NOTCH2 blockade eliminated NOTCH-BCR activation and significantly altered NOTCH downstream targets and B-cell maturation/effector molecules. Examination of the molecular underpinnings of this "NOTCH2-BCR axis" in cGVHD revealed imbalanced expression of the transcription factors IRF4 and IRF8, each critical to B-cell differentiation and fate. All-trans retinoic acid (ATRA) increased IRF4 expression, restored the IRF4-to-IRF8 ratio, abrogated BCR-NOTCH hyperactivation, and reduced NOTCH2 expression in cGVHD B cells without compromising viability. ATRA-treated cGVHD B cells had elevated TLR9 and PAX5, but not BLIMP1 (a gene-expression pattern associated with mature follicular B cells) and also attained increased cytosine guanine dinucleotide responsiveness. Together, we reveal a mechanistic link between NOTCH2 activation and robust BCR responses to otherwise suboptimal amounts of surrogate antigen. Our findings suggest that peripheral B cells in cGVHD patients can be pharmacologically directed from hyperactivation toward maturity.
Assuntos
Linfócitos B/metabolismo , Doença Enxerto-Hospedeiro/metabolismo , Transplante de Células-Tronco Hematopoéticas , Proteínas de Neoplasias/metabolismo , Receptor Notch2/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Aloenxertos , Linfócitos B/patologia , Doença Crônica , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Humanos , Fatores Reguladores de Interferon/biossíntese , Fatores Reguladores de Interferon/genética , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Receptor Notch2/genética , Receptores de Antígenos de Linfócitos B/genética , Tretinoína/farmacologiaRESUMO
Although B cells have emerged as important contributors to chronic graft-versus-host-disease (cGVHD) pathogenesis, the mechanisms responsible for their sustained activation remain unknown. We previously showed that patients with cGVHD have significantly increased B cell-activating factor (BAFF) levels and that their B cells are activated and resistant to apoptosis. Exogenous BAFF confers a state of immediate responsiveness to antigen stimulation in normal murine B cells. To address this in cGVHD, we studied B-cell receptor (BCR) responsiveness in 48 patients who were >1 year out from allogeneic hematopoietic stem cell transplantation (HSCT). We found that B cells from cGVHD patients had significantly increased proliferative responses to BCR stimulation along with elevated basal levels of the proximal BCR signaling components B cell linker protein (BLNK) and Syk. After initiation of BCR signaling, cGVHD B cells exhibited increased BLNK and Syk phosphorylation compared with B cells from patients without cGVHD. Blocking Syk kinase activity prevented relative post-HSCT BCR hyper-responsiveness of cGVHD B cells. These data suggest that a lowered BCR signaling threshold in cGVHD associates with increased B-cell proliferation and activation in response to antigen. We reveal a mechanism underpinning aberrant B-cell activation in cGVHD and suggest that therapeutic inhibition of the involved kinases may benefit these patients.
Assuntos
Linfócitos B/metabolismo , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/metabolismo , Receptores de Antígenos de Linfócitos B/fisiologia , Adulto , Idoso , Fator Ativador de Células B/metabolismo , Linfócitos B/patologia , Proliferação de Células , Células Cultivadas , Doença Crônica , Feminino , Humanos , Ativação Linfocitária/fisiologia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Receptores de Antígenos de Linfócitos B/agonistas , Adulto JovemRESUMO
Chronic myeloid leukemia (CML) is a blood cancer characterized by dysregulated production of maturing myeloid cells driven by the product of the Philadelphia chromosome, the BCR-ABL1 tyrosine kinase. Tyrosine kinase inhibitors (TKIs) have proved effective in treating CML, but there is still a cohort of patients who do not respond to TKI therapy even in the absence of mutations in the BCR-ABL1 kinase domain that mediate drug resistance. To discover novel strategies to improve TKI therapy in CML, we developed a nonlinear mathematical model of CML hematopoiesis that incorporates feedback control and lineage branching. Cell-cell interactions were constrained using an automated model selection method together with previous observations and new in vivo data from a chimeric BCR-ABL1 transgenic mouse model of CML. The resulting quantitative model captures the dynamics of normal and CML cells at various stages of the disease and exhibits variable responses to TKI treatment, consistent with those of CML patients. The model predicts that an increase in the proportion of CML stem cells in the bone marrow would decrease the tendency of the disease to respond to TKI therapy, in concordance with clinical data and confirmed experimentally in mice. The model further suggests that, under our assumed similarities between normal and leukemic cells, a key predictor of refractory response to TKI treatment is an increased maximum probability of self-renewal of normal hematopoietic stem cells. We use these insights to develop a clinical prognostic criterion to predict the efficacy of TKI treatment and design strategies to improve treatment response. The model predicts that stimulating the differentiation of leukemic stem cells while applying TKI therapy can significantly improve treatment outcomes.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Camundongos , Animais , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Mielopoese , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/farmacologia , Camundongos Transgênicos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genéticaRESUMO
Type 2 diabetes is the most prevalent endocrine disease in the world, and recently the gut microbiota have become a potential target for its management. Recent studies have illustrated that this disease may predispose individuals to certain microbiome compositions, and treatments like metformin have been shown to change gut microbiota and their associated metabolic pathways. However, given the limitations and side effects associated with pharmaceuticals currently being used for therapy of diabetes, there is a significant need for alternative treatments. In this study, we investigated the effects of a root extract from Rhodiola rosea in a Leptin receptor knockout (db/db) mouse model of type 2 diabetes. Our previous work showed that Rhodiola rosea had anti-inflammatory and gut microbiome-modulating properties, while extending lifespan in several animal models. In this study, treatment with Rhodiola rosea improved fasting blood glucose levels, altered the response to exogenous insulin, and decreased circulating lipopolysaccharide and hepatic C-reactive protein transcript levels. We hypothesize that these changes may in part reflect the modulation of the microbiota, resulting in improved gut barrier integrity and decreasing the translocation of inflammatory biomolecules into the bloodstream. These findings indicate that Rhodiola rosea is an attractive candidate for further research in the management of type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2 , Microbiota , Rhodiola , Animais , Biomarcadores , Diabetes Mellitus Tipo 2/tratamento farmacológico , Inflamação/tratamento farmacológico , Camundongos , Camundongos Knockout , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Receptores para Leptina/genéticaRESUMO
The haematopoietic system has a highly regulated and complex structure in which cells are organized to successfully create and maintain new blood cells. It is known that feedback regulation is crucial to tightly control this system, but the specific mechanisms by which control is exerted are not completely understood. In this work, we aim to uncover the underlying mechanisms in haematopoiesis by conducting perturbation experiments, where animal subjects are exposed to an external agent in order to observe the system response and evolution. We have developed a novel Bayesian hierarchical framework for optimal design of perturbation experiments and proper analysis of the data collected. We use a deterministic model that accounts for feedback and feedforward regulation on cell division rates and self-renewal probabilities. A significant obstacle is that the experimental data are not longitudinal, rather each data point corresponds to a different animal. We overcome this difficulty by modelling the unobserved cellular levels as latent variables. We then use principles of Bayesian experimental design to optimally distribute time points at which the haematopoietic cells are quantified. We evaluate our approach using synthetic and real experimental data and show that an optimal design can lead to better estimates of model parameters.