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.

Single-cell transcriptomes are established by transcription factors (TFs), which determine a cell's gene-expression complement. Post-transcriptional regulation of single-cell transcriptomes, and the RNA binding proteins (RBPs) responsible, are more technically challenging to determine, and combinatorial TF-RBP coordination of single-cell transcriptomes remains unexplored. We used fluorescent reporters to visualize alternative splicing in single Caenorhabditis elegans neurons, identifying complex splicing patterns in the neuronal kinase sad-1. Most neurons express both isoforms, but the ALM mechanosensory neuron expresses only the exon-included isoform, while its developmental sister cell the BDU neuron expresses only the exon-skipped isoform. A cascade of three cell-specific TFs and two RBPs are combinatorially required for sad-1 exon inclusion. Mechanistically, TFs combinatorially ensure expression of RBPs, which interact with sad-1 pre-mRNA. Thus a combinatorial TF-RBP code controls single-neuron sad-1 splicing. Additionally, we find ‘phenotypic convergence,’ previously observed for TFs, also applies to RBPs: different RBP combinations generate similar splicing outcomes in different neurons.

Dendritic cells (DCs) are professional antigen presenting cells with a great capacity for cross-presentation of exogenous antigens from which robust anti-tumor immune responses ensue. However, this function is not always available and requires DCs to first be primed to induce their maturation. In particular, in the field of DC vaccine design, currently available methodologies have been limited in eliciting a sustained anti-tumor immune response. Mechanistically, part of the maturation response is influenced by the presence of stimulatory receptors relying on ITAM-containing activating adaptor molecules like DAP12, that modulates their function. We hypothesize that activating DAP12 in DC could force their maturation and enhance their potential anti-tumor activity for therapeutic intervention. For this purpose, we developed constitutively active DAP12 mutants that can promote activation of monocyte-derived DC. Here we demonstrate its ability to induce the maturation and activation of monocyte-derived DCs which enhances migration, and T cell stimulation in vitro using primary human cells. Moreover, constitutively active DAP12 stimulates a strong immune response in a murine melanoma model leading to a reduction of tumor burden. This provides proof-of-concept for investigating the pre-activation of antigen presenting cells to enhance the effectiveness of anti-tumor immunotherapies.

This protocol outlines a step-by-step workflow for processing clinical tissues for formalin fixed paraffin embedded (FFPE) samples. Following fixation and storage of the tissues, these processes are used to characterize histological features and identify pathology relevant to osteoarthritis. We have used this protocol to process clinical fixed samples from patients with total knee arthroplasty. Once the surgeon has excised and removed tissues to place the implant, these tissues are transferred in saline to be processed for histology and other processes. Our protocol works for both hard (bone) and soft (synovium, infrapatellar fat pad, and meniscus) tissues. Paraffin histology is commonly performed to identify structural features and pathology of disease, particularly cartilage degeneration in osteoarthritis. Processing of tissues for paraffin histology is an important and impactful step that will influence the results of the sections and images downstream. Therefore, our protocol has been optimized to best preserve all tissue types and structure for histological characterization.

This protocol describes the stepwise process for preparing, sectioning, and immunostaining clinical samples obtained from total knee arthroplasty (TKA) participants. Our protocol works for both hard (bone) and soft (synovium, infrapatellar fat pad, and meniscus) tissues.In our work, we utilize this procedure to visualize innervation, vasculature, and immune cell markers via immunofluorescence imaging. Following fixation of the tissue, these processes are used to characterize histopathological features and identify cellular and tissue pathology.

Vibration nociception threshold in rodents is assessing using a 128 hz tuning fork to stimulate nociception at the knee in rodents. Withdrawal threshold of vibration is determined when the rodent either physically withdrawals their leg and if they vocalize. Vibration nociception threshold is beneficial to measure the variability in vibration nociception across rodents as well as across different pain models or unilateral models such as medial collateral ligament transection and medial meniscus transection (MCLT+MMT).