Oxidized Mitochondrial DNA Engages TLR9 to Activate the NLRP3 Inflammasome in Myelodysplastic Syndromes

Abstract

Myelodysplastic Syndromes (MDS) are bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, Nlrp3 inflammasome (IFM) driven pyroptotic cell death and the induction of interferon-stimulated genes (ISG). Toll-like receptor 9 (TLR9) is an endosomal, DNA sensing pattern recognition receptor that primes and activates the IFM and ISG response through myddosome signaling upon engagement by hypomethylated, CpG-rich DNA. Oxidized newly synthesized mitochondrial DNA (ox-mtDNA) is released into the cytosol upon TLR/IL-1R activation to trigger Nlrp3 IFM activation. Upon lytic pyroptotic cell death, however, ox-mtDNA is released into the extracellular space. We previously reported that concentrations of ox-mtDNA, a native TLR9 ligand, are profoundly increased in MDS patient plasma compared to age-matched controls and other hematologic malignancies (Ward G, et. al. ASH 2018). The aim of this investigation was to determine if ox-mtDNA acts as a danger associated molecular pattern (DAMP) to propagate the inflammatory response and IFM activation in neighboring cells through TLR9. We have shown that MDS hematopoietic stem and progenitor cells (HSPC) redistribute TLR9 to display cell surface TLR9 expression. We hypothesized that this increased surface expression is induced in response to ox-mtDNA in the BM plasma. To test this, SKM1 and U937 cells were incubated for 2 hours with 50ng/mL ox-mtDNA (ND1 gene, unmethylated, amplified with oxidized guanosine) and TLR9 expression was assessed by flow cytometry (FC). Following incubation, both cell lines significantly increased TLR9 surface expression (n=3, p<0.03).We next confirmed that TLR9 interacts with ox-mtDNA by immunofluorescence (IF) microscopy in MDS samples and murine somatic gene mutation (SGM) models (Tet2-/- and Srsf2P95H). We further investigated the relationship between TLR9 and ox-mtDNA during IFM activation. Treatment of SKM1 and U937 cells with the TLR4 ligand LPS and priming agents ATP/nigericin initiates a robust increase in ox-mtDNA that co-localized intracellularly with TLR9 by IF microscopy. Furthermore, upon incubation with ox-mtDNA, ox-mtDNA bound TLR9 is internalized. Additionally, interferon regulatory factor 7 (IRF7), an ISG transcription factor induced by TLR9, translocates to the nucleus following treatment, confirming TLR9 activation in response to ox-mtDNA. We next assessed whether IFM activation by ox-mtDNA is TLR9-dependent. 50ng/mL ox-mtDNA was incubated with 3 leukemic cell lines: SKM1, U937, and THP1 that display varying levels of endogenous TLR9 expression, as well as corresponding TLR9-KO cells generated by CRISPR-Cas9 editing. We found that TLR9 was necessary for IFM initiated caspase-1 cleavage in response to ox-mtDNA, thereby demonstrating the specificity of ox-mtDNA for the TLR9 sensor. Notably, receptor density determined response tempo. SKM1 cells, which have the highest TLR9 receptor density, responded within 1 hour, U937 cells which have an intermediate receptor density respond in 2 hours, while THP1 cells, which do not express endogenous TLR9 never responded to ox-mtDNA treatment. TLR9 knockout abrogated IFM activation in response to ox-mtDNA as demonstrated by caspase-1 glo ® assay (n=5, p<0.03). By western blot, TLR9 knockout cells no longer demonstrate phosphorylated NFk-B, cleaved caspase-1, and cleaved IL-1β in response to ox-mtDNA treatment. We conclude that MDS HSPC display functionally competent TLR9 on the plasma membrane which primes them for response to ox-mtDNA released by neighboring pyroptotic cells. Blocking TLR9 activation may prove to be a novel therapeutic strategy for MDS and suppression of inflammatory BM failure. Disclosures Hou: Celgene: Research Funding; Abbvie, Astellas, BMS, Celgene, Chugai, Daiichi Sankyo, IQVIA, Johnson & Johnson, Kirin, Merck Sharp & Dohme, Novartis, Pfizer, PharmaEssential, Roche, Takeda: Honoraria. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.