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Myelodysplastic Syndromes (MDSs) are bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, including NLRP3 inflammasome driven pyroptotic cell death. We recently reported that the danger-associated molecular pattern (DAMP) oxidized mitochondrial DNA (ox-mtDNA) is diagnostically increased in MDS plasma although the functional consequences remain poorly defined. We hypothesized that ox-mtDNA is released into the cytosol, upon NLRP3 inflammasome pyroptotic lysis, where it propagates and further enhances the inflammatory cell death feed-forward loop onto healthy tissues. This activation can be mediated via ox-mtDNA engagement of Toll-like receptor 9 (TLR9), an endosomal DNA sensing pattern recognition receptor known to prime and activate the inflammasome propagating the IFN-induced inflammatory response in neighboring healthy hematopoietic stem and progenitor cells (HSPCs), which presents a potentially targetable axis for the reduction in inflammasome activation in MDS. We found that extracellular ox-mtDNA activates the TLR9-MyD88-inflammasome pathway, demonstrated by increased lysosome formation, IRF7 translocation, and interferon-stimulated gene (ISG) production. Extracellular ox-mtDNA also induces TLR9 redistribution in MDS HSPCs to the cell surface. The effects on NLRP3 inflammasome activation were validated by blocking TLR9 activation via chemical inhibition and CRISPR knockout, demonstrating that TLR9 was necessary for ox-mtDNA-mediated inflammasome activation. Conversely, lentiviral overexpression of TLR9 sensitized cells to ox-mtDNA. Lastly, inhibiting TLR9 restored hematopoietic colony formation in MDS BM. We conclude that MDS HSPCs are primed for inflammasome activation via ox-mtDNA released by pyroptotic cells. Blocking the TLR9/ox-mtDNA axis may prove to be a novel therapeutic strategy for MDS.

Somatic gene mutations are key determinants of outcome in patients with myelodysplastic syndromes (MDS) and secondary AML (sAML). In particular, patients with TP53 mutations represent a distinct molecular cohort with uniformly poor prognosis. The precise pathogenetic mechanisms underlying these inferior outcomes have not been delineated. In this study, we characterized the immunological features of the malignant clone and alterations in the immune microenvironment in patients with TP53-mutant and wild-type MDS or sAML. Notably, PDL1 expression is significantly increased in hematopoietic stem cells of patients with TP53 mutations, which is associated with MYC upregulation and marked downregulation of MYC’s negative regulator miR-34a, a p53 transcription target. Notably, patients with TP53 mutations display significantly reduced numbers of bone marrow–infiltrating OX40+ cytotoxic T cells and helper T cells, as well as decreased ICOS+ and 4-1BB+ natural killer cells. Further, highly immunosuppressive regulatory T cells (Tregs) (ie, ICOShigh/PD-1−) and myeloid-derived suppressor cells (PD-1low) are expanded in cases with TP53 mutations. Finally, a higher proportion of bone marrow–infiltrating ICOShigh/PD-1− Treg cells is a highly significant independent predictor of overall survival. We conclude that the microenvironment of TP53 mutant MDS and sAML has an immune-privileged, evasive phenotype that may be a primary driver of poor outcomes and submit that immunomodulatory therapeutic strategies may offer a benefit for this molecularly defined subpopulation.

Myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem cell malignancies that can phenotypically resemble other hematologic disorders. Thus, tools that may add to current diagnostic practices could aid in disease discrimination. Constitutive innate immune activation is a pathogenetic driver of ineffective hematopoiesis in MDS through Nod-like receptor protein 3 (NLRP3)–inflammasome-induced pyroptotic cell death. Oxidized mitochondrial DNA (ox-mtDNA) is released upon cytolysis, acts as a danger signal, and triggers inflammasome oligomerization via DNA sensors. By using immortalized bone marrow cells from murine models of common MDS somatic gene mutations and MDS primary samples, we demonstrate that ox-mtDNA is released upon pyroptosis. ox-mtDNA was significantly increased in MDS peripheral blood (PB) plasma compared with the plasma of healthy donors, and it was significantly higher in lower-risk MDS vs higher-risk MDS, consistent with the greater pyroptotic cell fraction in lower-risk patients. Furthermore, ox-mtDNA was significantly higher in MDS PB plasma compared with all other hematologic malignancies studied, with the exception of chronic lymphocytic leukemia (CLL). Receiver operating characteristic/area under the curve (ROC/AUC) analysis demonstrated that ox-mtDNA is a sensitive and specific biomarker for patients with MDS compared with healthy donors (AUC, 0.964), other hematologic malignancies excluding CLL (AUC, 0.893), and reactive conditions (AUC, 0.940). ox-mtDNA positively and significantly correlated with levels of known alarmins S100A9, S100A8, and apoptosis-associated speck-like protein containing caspase recruitment domain (CARD) specks, which provide an index of medullary pyroptosis. Collectively, these data indicate that quantifiable ox-mtDNA released into the extracellular space upon inflammasome activation serves as a biomarker for MDS and the magnitude of pyroptotic cell death.

Neurosurgery for brain tumor resection or epilepsy treatment requires a craniotomy to gain access to the brain. Despite prophylactic measures, infectious complications occur at a frequency of 1–3%, with approximately half caused by Staphylococcus aureus (S. aureus) that forms a biofilm on the bone flap and is recalcitrant to antibiotics. Using scRNA-seq in a mouse model of S. aureus craniotomy infection, this study revealed the complex transcriptional heterogeneity of resident microglia and infiltrating monocytes in the brain in addition to transcriptionally diverse granulocyte subsets in the subcutaneous galea and bone flap. In the brain, trajectory analysis identified the transition of microglia from a homeostatic/anti-inflammatory to pro-inflammatory and proliferative populations, whereas granulocytes in the brain demonstrated a trajectory from a granulocyte myeloid-derived suppressor cell (G-MDSC)-like phenotype to a small population of mature neutrophils. In the galea, trajectory analysis identified the progression from two distinct G-MDSC-like populations to neutrophil clusters enriched for IFN signaling and cell cycle genes. Based on their abundance in the galea and bone flap, PMNs and MDSCs were depleted using anti-Ly6G, which resulted in increased bacterial burden. This revealed a critical role for PMNs in S. aureus containment since MDSCs were found to attenuate PMN antibacterial activity which may explain, in part, why craniotomy infection persists in the presence of PMN infiltrates. These results demonstrate the existence of a transcriptionally diverse leukocyte response that likely influences the chronicity of S. aureus craniotomy infection.

Staphylococcus aureus (S. aureus) is a leading cause of biofilm-associated prosthetic joint infections (PJIs), where leukocyte IL-10 production is critical for biofilm persistence. We screened a S. aureus mutant library that identified the importance of lactate biosynthetic pathways to elicit IL-10 production. A S. aureus Δddh/ldh1/ldh2 mutant, defective in both D- and L-lactate production, led to significantly decreased leukocyte IL-10 expression and re-programmed biofilm-associated monocytes to a pro-inflammatory state, resulting in reduced biofilm burden. Histone acetylation plays a central role in regulating gene expression, and we found that histone deacetylase (HDAC) activity was increased in Δddh/ldh1/ldh2-infected mice compared to WT concomitant with significant decreases in global histone 3 (H3) acetylation and H3 acetylation at the IL-10 promoter, demonstrating that S. aureus-derived lactate is a HDAC inhibitor. Lactate was found to inhibit HDAC11, which resulted in unregulated HDAC6 activity and increased IL-10 production. MDSCs and macrophages produced significantly less IL-10 in the presence of the HDAC6 inhibitor tubastatin A during co-culture with WT but not Δddh/ldh1/ldh2 biofilms, while HDAC11 KO MDSCs and macrophages co-cultured with Δddh/ldh1/ldh2 biofilms produced significantly more IL-10 compared to WT cells due to unchecked HDAC6 action. D-lactate was elevated in synovial fluid of patients with PJI compared to aseptic controls and lactate also promoted IL-10 production by human monocyte-derived macrophages. Collectively, S. aureus lactate-mediated inhibition of HDAC11 plays a critical role during biofilm infection, leading to epigenetic changes that reprogram the host immune response.