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 TNFα 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. 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.