Bone Marrow Interstitial Light-chain Amyloid and its Microenvironment

Amyloid deposition and neighboring tissue responses remain poorly understood. Twenty percent of patients with systemic light-chain amyloidosis (AL) have interstitial marrow amyloid containing clonal Ig light-chain fibrils and apolipoprotein chaperone proteins. We compared CD138-depleted aspirate mononuclear cells (MNC) from marrows of AL patients with (+MA) and without (-MA) interstitial amyloid by gene expression profiling (GEP) and single-cell RNA-sequencing (scRNA seq). GEP showed no differential expression of genes for proteolytic enzymes or apolipoproteins between the groups but +MA cases had significantly up-regulated erythroid genes involved in oxygen transport, including transmembrane and coiled-coil domain family 2 (TMCC2). In +MA marrows at the single-cell level, CD14+ monocytes were increased by 22%, granulocyte-monocyte progenitors decreased by 66%, and erythroid-megakaryocyte progenitors and early and late erythroid progenitors increased up to five-fold. Gene enrichment studies showed that in +MA marrows pathways for TNFa signaling, immune activation, monocyte hypoxia and erythropoiesis were significantly enriched. We also compared peripheral blood and marrow plasma by immunoprecipitation and immunoblot with respect to apolipoproteins and light chains in complex with the erythroid protein TMCC2, and by ELISA for marrow apolipoprotein E and J levels. Apolipoprotein J is strongly associated with light chains in blood and E is not, while in +MA marrow plasma J and E are strongly present in association with light chains. There is also significantly more apolipoprotein E and J in +MA marrow plasma. In summary, marrows with interstitial amyloid provide opportunities to study amyloid's impact on cellular and regenerative activity and chaperone involvement in amyloid formation. Overall design: RNA was obtained from CD138-depleted marrow mononuclear cells (MNC) and used to prepare libraries with the Illumina TruSeq Stranded Total RNA kit for sequencing on the Illumina HiSeq 2500. Following preparation, the libraries were denatured, introduced into the flow cell, and subjected to bridge amplification in order to create clonal clusters of single stranded cDNA molecules. Sequencing and analysis were performed in our core facility as previously described. Bioinformatic analyses were performed on the Tufts University High Performance Cluster (Medford, MA). Tuxedo Tools were used to analyze the RNASeq results. Briefly, reads were mapped to the UCSC hg19 human genome with Tophat 2/Bowtie 2. Normalization and differential expression analyses were performed with Cuffdiff.

淀粉样蛋白沉积及其邻近组织反应的相关机制仍有待深入阐明。20%的系统性轻链淀粉样变性（systemic light-chain amyloidosis, AL）患者会出现间质性骨髓淀粉样变，病灶内含有克隆性免疫球蛋白轻链纤维与载脂蛋白伴侣蛋白。本研究通过基因表达谱（gene expression profiling, GEP）与单细胞RNA测序（single-cell RNA-sequencing, scRNA-seq），对比了伴（+MA组）与不伴（-MA组）间质性骨髓淀粉样变的AL患者骨髓中经CD138去除处理的抽吸标本单个核细胞（mononuclear cells, MNC）的转录组差异。基因表达谱分析显示，两组间蛋白水解酶类基因与载脂蛋白基因的表达无显著差异，但+MA组的氧转运相关红细胞系基因表达显著上调，其中包括跨膜与卷曲螺旋结构域家族2（transmembrane and coiled-coil domain family 2, TMCC2）。单细胞水平分析结果表明，+MA组骨髓中CD14+单核细胞占比升高22%，粒细胞-单核细胞祖细胞占比降低66%，而红系-巨核细胞祖细胞、早晚期红系祖细胞的占比最高可提升至5倍。基因富集分析显示，+MA组骨髓中TNF-α信号通路、免疫激活通路、单核细胞缺氧通路与红细胞生成通路均显著富集。本研究还通过免疫沉淀与免疫印迹实验，对比了外周血与骨髓血浆中与红细胞系蛋白TMCC2结合的载脂蛋白与轻链情况，并通过酶联免疫吸附实验（enzyme-linked immunosorbent assay, ELISA）检测了骨髓血浆载脂蛋白E与J的水平。结果显示，载脂蛋白J在血液中与轻链紧密结合，而载脂蛋白E则无此关联；但在+MA组骨髓血浆中，载脂蛋白J与E均能与轻链形成稳定复合物，且+MA组骨髓血浆中的载脂蛋白E与J含量显著更高。综上，伴间质性淀粉样变的骨髓样本为研究淀粉样蛋白对细胞与再生活性的影响，以及伴侣蛋白在淀粉样蛋白形成过程中的作用提供了优质研究模型。

整体实验设计：本研究从经CD138去除处理的骨髓单个核细胞中提取RNA，使用Illumina TruSeq Stranded Total RNA试剂盒构建测序文库，随后在Illumina HiSeq 2500平台上完成测序。文库制备完成后，经变性处理载入流动池，通过桥式扩增生成单链cDNA分子的克隆簇。测序与数据分析工作在本中心实验室完成，具体方法参照既往文献报道。生物信息学分析在塔夫茨大学高性能计算集群（马萨诸塞州梅德福）上进行。本研究使用Tuxedo Tools工具包分析RNA测序结果，简要流程如下：通过Tophat 2/Bowtie 2将测序reads比对至UCSC hg19人类参考基因组，使用Cuffdiff完成标准化与差异表达分析。