Trypanosoma brucei-induced dyshematopoiesis: blocked B lymphopoiesis

描述（由申请人提供）：非洲锥虫是一种抗原高度可变的鞭毛原虫，可引起人和家畜的致命疾病。目前约有30万人患有锥虫病，该疾病威胁着撒哈拉以南非洲另外6 000万人，并严重限制了一个面积相当于美国的地区的农业发展。非洲锥虫在血液和间质液中通过二元裂变进行复制，并受宿主针对其可变外壳抗原的抗体反应控制。随着疾病的进展，受感染的宿主失去了发起有效抗体反应的能力，导致对新出现的锥虫抗原类型控制不力，持续病理并最终死亡。我们的长期目标是了解布鲁氏杆菌如何影响B细胞（抗体形成细胞）系统，并利用这方面的知识来开发战略性干预措施，以保持免疫功能和疾病控制。我们在小鼠模型中进行的分析表明，骨髓和脾脏中早期B细胞祖细胞迅速扩增，但这些器官中新形成的和成熟的B细胞的损失，通过使用锥虫药物Berenil治疗感染小鼠的锥虫清除，可以逆转造血障碍的特征。这些观察结果使我们假设布鲁氏杆菌诱导了B淋巴生成的可逆阻断，阻止了新B细胞和成熟B细胞的补充，从而导致B细胞克隆衰竭和B细胞介导免疫的崩溃。我们将解决这个假设在接下来的具体目标:1)识别B家族发展的阶段是在锥虫属被捕brucei-infected老鼠ii)确定阻止在B细胞发展结果截断或异常B细胞分化,iii)确定阻塞B细胞恢复发展在锥体虫-免费主机,或体外生长,如果是这样,是否t brucei或组件的t . brucei防止逃避阻碍发展。方法包括：(i)利用B细胞发育阶段特异性单克隆抗体进行细胞表面分化抗原谱分析；（ii）利用荧光激活细胞分选（FACS）从正常小鼠和锥虫感染小鼠的脾脏和骨髓中分离出的离散B细胞发育阶段的转录因子和免疫球蛋白基因表达谱分析；（iii）对目标ii中分离的B细胞祖细胞FACS进行发育分析；并在免疫缺陷小鼠（RAG-/-）和体外存在和不存在锥虫及其提取物的条件下生长。公共卫生相关性包括阐明锥虫诱导的B细胞衰竭和宿主产生保护性抗体反应能力丧失的细胞基础。

DESCRIPTION (provided by applicant): African trypanosomes are highly antigenically variable flagellated protozoa that cause fatal disease of people and domestic animals. About 300,000 persons have trypanosomiasis at present and the disease threatens an additional 60 million individuals in sub-Saharan Africa as well as severely limiting agricultural development in an area the size of USA. African trypanosomes replicate by binary fission in the blood and interstitial fluids and are controlled by host antibody responses against their variable coat antigens. Infected hosts lose the ability to mount an effective antibody response as the disease progresses resulting in poor control of newly arising trypanosome antigenic types, sustained pathology and eventually death. Our long term goal is to understand how T. brucei affects the B cell (antibody forming cell) system and to use this knowledge to develop strategic interventions that will preserve immune function and disease control. Our on- going analyses in the mouse model show that there is a rapid expansion of early B cell progenitors in the bone marrow and spleen, but the loss of newly formed and mature B cells from these organs, features of dyshematopoiesis that are reversed by clearance of trypanosomes from infected mice by treatment with the trypanocidal drug Berenil. These observations lead us to hypothesize that T. brucei induces a reversible block in B lymphopoiesis that prevents replenishment of the pools of new and mature B cell, which leads to B- cell clonal exhaustion and the collapse of B cell-mediated immunity. We will address this hypothesis in the following specific aims: i) identify the stage at which B lineage development is arrested in Trypanosoma brucei-infected mice ii) determine whether the block in B cell development results from truncated or aberrant B cell differentiation, iii) determine whether blocked B cells resume development when placed in trypanosome- free hosts, or grown in vitro, and if so, whether T. brucei or components of T. brucei prevent escape from the block in development. Methodologies will include: (i) cell surface differentiation antigen profiling using monoclonal antibodies specific for B cell developmental stages, (ii) transcription factor and immunoglobulin gene expression profiling of discrete B cell developmental stages isolated by fluorescence activate cell sorting (FACS) from the spleen and bone marrow of normal and trypanosome-infected mice, (iii) developmental analyses of B cell progenitors FACS isolated as in aim ii, and grown in immunodeficient mice (RAG-/-) and in vitro in the presence and absence of trypanosomes and extracts. Public heath relevance includes the elucidation of the cellular basis for trypanosome-induced B cell exhaustion and the loss of host ability to make protective antibodies responses. Narrative: Loss of the ability to make protective antibody responses and consequently to control parasitemia is a common feature of African trypanosomiasis in people, domestic and laboratory animals. Using the mouse model we have shown an infection-induced reversible block in B lymphocyte development that prevents the maintenance of pools of newly formed and mature B cell leading to B cell clonal exhaustion and thus, collapse of humoral immune competence. We propose to identify the affected B cell developmental stage and the nature of the developmental block as a step towards elucidating the molecular basis of this newly identified mechanism of disease-associated immuno-depression.