70 - Modulation of Bone Marrow Progenitor Niches as a Mechanism Underlying Trained Immunity Induced by In-Vivo CpG-DNA Treatment

Publication Number: 70.5601

Zoe Michael, Boston Children's Hospital, Boston, MA, United States; John F. Pulford, Harvard Medical School, Cambridge, MA, United States; Ekaterina Murzin, Brigham & Women's Hospital, Harvard Medical School, Somerville, MA, United States; Alexandria Byskosh, Brigham and Women’s Hospital, Boston, MA, United States; James Lederer, BWH Harvard Medical School, boston, MA, United States

Background: With the escalating threat of antibiotic resistance, there is an urgent need for innovative therapeutic strategies to enhance immune function. In previous studies, we demonstrated that CpG-DNA preconditioning of mesenchymal stromal cells (MSCs) could amplify their therapeutic activity in a murine model of neutropenic sepsis. However, the biological and molecular effects on MSCs and hematopoietic stem cells (HSCs) in mice given therapeutic doses of CpG-DNA and long-term effects thereof have not yet been explored.

Objective: We sought to evaluate the immunomodulatory effects and transcriptional responses in endogenous MSCs and HSCs after in vivo treatment with CpG-DNA.

Design/Methods: Male C57BL/6 mice were subcutaneously injected with 3 mg/kg Class A CpG-DNA seq2336 (CpG2336) or left untreated as controls. Lineage-negative cells were isolated from bone 24 hours later, by negative selection as previously described, followed by flow sorting for HSC purification and CD45+ depletion to isolate MSCs. Total RNA was extracted using RNeasy Mini kits, and RNA sequencing was conducted to assess transcriptional changes in HSCs. Single-cell RNA sequencing was performed using the 10x Genomics 5’v3 kit. To induce systemic inflammation, a separate group received LPS injections at 1, 4, and 12 weeks post-CpG treatment. Detailed phenotyping of BM stem and progenitor cells via multicolor flow cytometry, peripheral immune cells by CyTOF, and plasma cytokine assays using Luminex were conducted 24 hours later.

Results: Treatment with CpG2336 induced significant increases in BM MSCs and HSCs. Bulk RNA sequencing in HSCs showed that transcriptional responses to CpG2336 modulated inflammatory networks. Gene set enrichment analysis on the differentially expressed genes that were significantly different in HSCs showed that treatment with CpG2336 modified gene networks involved in cell proliferation, interferon alpha and gamma responses, MTOR signaling, IL2/STAT5 signaling, glycolysis, and TNF-a pathways compared to controls. Additionally, scRNA sequencing of endogenous MSCs showed clear delineation of CpG-DNA reactive populations. Animal experiments following LPS injection are ongoing.

Conclusion(s): Our findings underscore the immunomodulatory activities of CpG-DNA in HSCs and support the concept that CpG-DNA therapy has bone marrow regenerative activity in mice. Further investigations are warranted to elucidate the molecular mechanisms underlying the effects of CpG-DNA as potential therapeutics for infections and sepsis in immunocompromised individuals.

Fig 1. In vivo screen for CpG-DNA activity on Bone Marrow progenitor cells.
(A) Mice were given CpG2336 at 3 mg/Kg by subcutaneous injection. At 24hrs bone marrow was prepared for flow cytometry to identify changes in abundance of BM progenitor cells. (B) HSC, Progenitor, and MSC flow cytometry panels used to identify CpG-DNA effects on hematopoiesis. (C) FACS plots showing MSC number differences in equal subsampled stains from mice injected with CpG2336. (D) Statistical analysis sowing significant increase in bone marrow MSCs and HSCs in mice treated with CpG2336.
**p < 0.001, **** p<0.0001 Unpaired t-test

Fig 2. In-vivo CpG treatment induced transcriptional modifications of Bone Marrow progenitor cells.
(A) Mice were given CpG2336 at 3 mg/Kg by subcutaneous injection. At 24hrs, lineage depletion of BM followed by either sorting for RNA extraction or depletion of CD45+ cells for scRNA sequencing was performed. (B) CpG2336 perturbed inflammatory, metabolic and mTORC1 signaling pathways in HSCs. (C) Heatmap of normalized counts per sample of control and CpG2336 groups. Genes present in leading edge of NES pathway from DESeq2 Control vs CpG comparison, filtered p < 0.05, sorted by log2FC, top 5. (D) scRNA sequencing data analysis of non-hematopoietic bone marrow progenitor cells. UMAPs based on (top) cluster and (bottom) relative expression of known MSC markers.

Fig 1. In vivo screen for CpG-DNA activity on Bone Marrow progenitor cells.
(A) Mice were given CpG2336 at 3 mg/Kg by subcutaneous injection. At 24hrs bone marrow was prepared for flow cytometry to identify changes in abundance of BM progenitor cells. (B) HSC, Progenitor, and MSC flow cytometry panels used to identify CpG-DNA effects on hematopoiesis. (C) FACS plots showing MSC number differences in equal subsampled stains from mice injected with CpG2336. (D) Statistical analysis sowing significant increase in bone marrow MSCs and HSCs in mice treated with CpG2336.
**p < 0.001, **** p<0.0001 Unpaired t-test

Fig 2. In-vivo CpG treatment induced transcriptional modifications of Bone Marrow progenitor cells.
(A) Mice were given CpG2336 at 3 mg/Kg by subcutaneous injection. At 24hrs, lineage depletion of BM followed by either sorting for RNA extraction or depletion of CD45+ cells for scRNA sequencing was performed. (B) CpG2336 perturbed inflammatory, metabolic and mTORC1 signaling pathways in HSCs. (C) Heatmap of normalized counts per sample of control and CpG2336 groups. Genes present in leading edge of NES pathway from DESeq2 Control vs CpG comparison, filtered p < 0.05, sorted by log2FC, top 5. (D) scRNA sequencing data analysis of non-hematopoietic bone marrow progenitor cells. UMAPs based on (top) cluster and (bottom) relative expression of known MSC markers.