Impaired Inflammation Resolution Is Alleviated By Resolvin E1 in Murine Immune Bone Marrow Failure
Authors: Grazda Rachel , Rahtes Allison , Maddipati Krishna Rao , Fredman Gabrielle , MacNamara Katherine C. 
Published: December 2024

Introduction: Inflammation resolution is an active process driven by endogenous molecules, such as the specialized pro-resolving lipid mediators (SPMs), that limit cell recruitment, dampen inflammatory signaling, remove dead cells (i.e. efferocytosis), and promote tissue repair. Impaired resolution results in chronic inflammation, which is an essential driver of many diseases from autoimmunity to cardiovascular disease. Severe aplastic anemia (SAA) is a fatal form of bone marrow failure (BMF) characterized by severe pancytopenia and persistent inflammation. It is well-understood that autoreactive T cells mediate the destruction of the hematopoietic stem cells (HSCs) during SAA, however the underlying mechanisms that lead to persistent inflammation are unknown. In this study, we aim to define mechanisms responsible for chronic inflammation in SAA and determine the efficacy of SPM treatment as a novel therapeutic approach.

Methods: To induce BMF, CByB6F1 (H-2b/d) received a sub-lethal dose of radiation (300 RADS) and an intraperitoneal injection of 6.5x107 splenocytes from MHC-mismatched C57BL/6 (H-2b/b) mice. Mice that received only sub-lethal radiation were used as controls. 250 ng Resolvin E1 was administered to mice via intraperitoneal injection every other day, starting day 7. Mice were sacrificed at day 8, 10, 12, and 14 or left for survival studies. Peripheral blood, hind limbs, pelvis, and sternum were harvested at each timepoint. Peripheral blood and bone marrow (BM) cells were used for immunophenotyping via flow cytometry. Lipids from the pelvis were extracted and analyzed via LC/MS. Sort-purified BM monocytes or total BM was analyzed by single cell RNA sequencing (scRNA-seq) using 10X Genomics and the Seurat pipeline. A human scRNAseq BM dataset (GSE181989) was also analyzed.

Results: Despite an expansion of professional phagocytes (i.e. monocytes and macrophages), SAA mice exhibited a 3-fold increase (7.8% to 23.1%) in dead cells in the BM, compared to radiation control. Efferocytosis, as measured by the uptake of fluorescently-labeled dead cells, was significantly reduced during SAA. MerTK is a cell-surface receptor on phagocytes that mediates efferocytosis. Under inflammatory conditions, ADAM17 can cleave MerTK into soluble Mer, impairing efferocytosis. SAA mice exhibit an increase in soluble Mer in the BM. To determine if MerTK cleavage limits efferocytosis, we utilized a mouse model in which MerTK is cleavage resistant (MerTKCR). MerTKCR mice failed to exhibit an increase in efferocytosis or survival. However, SAA mice demonstrated a substantial increase in CD47 (17.6% to 79.8%) on apoptotic BM cells and signal regulatory protein alpha (SIRPα; 0.5% to 40.0%) on monocytes. CD47-SIRPα is a “don't eat me” axis that prevents the activation of efferocytosis by MerTK. Functionally, SIRPα-CD47 blockade was sufficient to recover efferocytosis. scRNAseq and gene ontology enrichment analysis of human SAA BM monocytes demonstrated a significant upregulation in genes that inhibit efferocytosis, including SIRPA, CD300LF, CD300A, CSK, DYSF, and CNN2. Lipidomic analysis of murine BM revealed a reduction in the precursors of SPMs and increased prostaglandins during SAA, indicative of impaired inflammation resolution. Therapeutic administration of the SPM Resolvin E1 (RvE1) improved efferocytosis, BM cellularity, and SPM production. scRNAseq of whole BM indicated that RvE1 enhanced the regulation of hematopoiesis and limited inflammatory pathways, including T cell activation. Importantly, RvE1 therapy increased survival from 0% to 50%, and survivors exhibited a recovery in HSCs and BM cellularity, as well as reduced T cells by 40 days post-induction. ChemR23, the receptor for RvE1, significantly increases on T cells (8.3% to 30.1%) during SAA. To determine if RvE1 directly acts on T cells, ChemR23-/- mice were induced with wild-type donor splenocytes. RvE1 therapy led to a significant reduction in CD8+ T cells, increased HSCs, and 25% survival.

Conclusions: We demonstrate that impaired inflammation resolution contributes to SAA progression and that SPMs, such as RvE1, offer a novel treatment for SAA. These studies begin to fill a substantial knowledge gap concerning the mechanisms of inflammation resolution in the BM and the shortcomings of current immunosuppressant therapies that often fail in treating chronic inflammatory disorders.