Streptococcal pyrogenic exotoxin B cleaves GSDMA and triggers pyroptosis.

Gasdermins, a family of five pore-forming proteins (GSDMA-GSDME) in humans expressed predominantly in the skin, mucosa and immune sentinel cells, are key executioners of inflammatory cell death (pyroptosis), which recruits immune cells to infection sites and promotes protective immunity1,2. Pore formation is triggered by gasdermin cleavage1,2. Although the proteases that activate GSDMB, C, D and E have been identified, how GSDMA-the dominant gasdermin in the skin-is activated, remains unknown. Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a major skin pathogen that causes substantial morbidity and mortality worldwide3. Here we show that the GAS cysteine protease SpeB virulence factor triggers keratinocyte pyroptosis by cleaving GSDMA after Gln246, unleashing an active N-terminal fragment that triggers pyroptosis. Gsdma1 genetic deficiency blunts mouse immune responses to GAS, resulting in uncontrolled bacterial dissemination and death. GSDMA acts as both a sensor and substrate of GAS SpeB and as an effector to trigger pyroptosis, adding a simple one-molecule mechanism for host recognition and control of virulence of a dangerous microbial pathogen.

Disulfiram blocks inflammatory TLR4 signaling by targeting MD-2.

Toll-like receptor 4 (TLR4) sensing of lipopolysaccharide (LPS), the most potent pathogen-associated molecular pattern of gram-negative bacteria, activates NF-κB and Irf3, which induces inflammatory cytokines and interferons that trigger an intense inflammatory response, which is critical for host defense but can also cause serious inflammatory pathology, including sepsis. Although TLR4 inhibition is an attractive therapeutic approach for suppressing overexuberant inflammatory signaling, previously identified TLR4 antagonists have not shown any clinical benefit. Here, we identify disulfiram (DSF), an FDA-approved drug for alcoholism, as a specific inhibitor of TLR4-mediated inflammatory signaling. TLR4 cell surface expression, LPS sensing, dimerization and signaling depend on TLR4 binding to MD-2. DSF and other cysteine-reactive drugs, previously shown to block LPS-triggered inflammatory cell death (pyroptosis), inhibit TLR4 signaling by covalently modifying Cys133 of MD-2, a key conserved residue that mediates TLR4 sensing and signaling. DSF blocks LPS-triggered inflammatory cytokine, chemokine, and interferon production by macrophages in vitro. In the aggressive N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease (PD) in which TLR4 plays an important role, DSF markedly suppresses neuroinflammation and dopaminergic neuron loss, and restores motor function. Our findings identify a role for DSF in curbing TLR4-mediated inflammation and suggest that DSF and other drugs that target MD-2 might be useful for treating PD and other diseases in which inflammation contributes importantly to pathogenesis.

Image-seq: spatially resolved single-cell sequencing guided by in situ and in vivo imaging.

Tissue function depends on cellular organization. While the properties of individual cells are increasingly being deciphered using powerful single-cell sequencing technologies, understanding their spatial organization and temporal evolution remains a major challenge. Here, we present Image-seq, a technology that provides single-cell transcriptional data on cells that are isolated from specific spatial locations under image guidance, thus preserving the spatial information of the target cells. It is compatible with in situ and in vivo imaging and can document the temporal and dynamic history of the cells being analyzed. Cell samples are isolated from intact tissue and processed with state-of-the-art library preparation protocols. The technique therefore combines spatial information with highly sensitive RNA sequencing readouts from individual, intact cells. We have used both high-throughput, droplet-based sequencing as well as SMARTseq-v4 library preparation to demonstrate its application to bone marrow and leukemia biology. We discovered that DPP4 is a highly upregulated gene during early progression of acute myeloid leukemia and that it marks a more proliferative subpopulation that is confined to specific bone marrow microenvironments. Furthermore, the ability of Image-seq to isolate viable, intact cells should make it compatible with a range of downstream single-cell analysis tools including multi-omics protocols.

Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress.

Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9-mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism.

Tissue-resident memory T (TRM) cells persist indefinitely in epithelial barrier tissues and protect the host against pathogens. However, the biological pathways that enable the long-term survival of TRM cells are obscure. Here we show that mouse CD8+ TRM cells generated by viral infection of the skin differentially express high levels of several molecules that mediate lipid uptake and intracellular transport, including fatty-acid-binding proteins 4 and 5 (FABP4 and FABP5). We further show that T-cell-specific deficiency of Fabp4 and Fabp5 (Fabp4/Fabp5) impairs exogenous free fatty acid (FFA) uptake by CD8+ TRM cells and greatly reduces their long-term survival in vivo, while having no effect on the survival of central memory T (TCM) cells in lymph nodes. In vitro, CD8+ TRM cells, but not CD8+ TCM cells, demonstrated increased mitochondrial oxidative metabolism in the presence of exogenous FFAs; this increase was not seen in Fabp4/Fabp5 double-knockout CD8+ TRM cells. The persistence of CD8+ TRM cells in the skin was strongly diminished by inhibition of mitochondrial FFA ß-oxidation in vivo. Moreover, skin CD8+ TRM cells that lacked Fabp4/Fabp5 were less effective at protecting mice from cutaneous viral infection, and lung Fabp4/Fabp5 double-knockout CD8+ TRM cells generated by skin vaccinia virus (VACV) infection were less effective at protecting mice from a lethal pulmonary challenge with VACV. Consistent with the mouse data, increased FABP4 and FABP5 expression and enhanced extracellular FFA uptake were also demonstrated in human CD8+ TRM cells in normal and psoriatic skin. These results suggest that FABP4 and FABP5 have a critical role in the maintenance, longevity and function of CD8+ TRM cells, and suggest that CD8+ TRM cells use exogenous FFAs and their oxidative metabolism to persist in tissue and to mediate protective immunity.

Liver stromal cells restrict macrophage maturation and stromal IL-6 limits the differentiation of cirrhosis-linked macrophages.

BACKGROUND & AIMS: Myeloid cells are key regulators of cirrhosis, a major cause of mortality worldwide. Because stromal cells can modulate the functionality of myeloid cells in vitro, targeting stromal-myeloid interactions has become an attractive potential therapeutic strategy. We aimed to investigate how human liver stromal cells impact myeloid cell properties and to understand the utility of a stromal-myeloid coculture system to study these interactions in the context of cirrhosis. METHODS: Single-cell RNA-sequencing analyses of non-cirrhotic (n = 7) and cirrhotic (n = 5) human liver tissue were correlated to the bulk RNA-sequencing results of in vitro cocultured human CD14+ and primary liver stromal cells. Complimentary mechanistic experiments and flow cytometric analysis were performed on human liver stromal-myeloid coculture systems. RESULTS: We found that stromal-myeloid coculture reduces the frequency CD14+ cell subsets transcriptionally similar to liver macrophages, showing that stromal cells inhibit the maturation of monocytes into macrophages. Stromal cells also influenced in vitro macrophage differentiation by skewing away from cirrhosis-linked CD9+ scar-associated macrophage-like cells and towards CD163+ Kupffer cell-like macrophages. We identify IL-6 production as a mechanism by which stromal cells limit CD9+ macrophage differentiation and find that local IL-6 levels are decreased in early-stage human liver disease compared to healthy liver tissue, suggesting a protective role for local IL-6 in the healthy liver. CONCLUSIONS: Our work reveals an unanticipated role for liver stromal cells in impeding the maturation and altering the differentiation of macrophages and should prompt investigations into the role of local IL-6 production in the pathogenesis of liver disease. These studies provide a framework for investigating macrophage-stromal interactions during cirrhosis. LAY SUMMARY: The impact of human liver stromal cells on myeloid cell maturation and differentiation in liver disease is incompletely understood. In this study, we present a mechanistic analysis using a primary in vitro human liver stromal-myeloid coculture system that is translated to liver disease using single-cell RNA sequencing analysis of cirrhotic and non-cirrhotic human liver tissue. Our work supports a role for stromal cell contact in restricting macrophage maturation and for stromal-derived IL-6 in limiting the differentiation of a cirrhotic macrophage subset.

Cistrome Data Browser: expanded datasets and new tools for gene regulatory analysis.

The Cistrome Data Browser (DB) is a resource of human and mouse cis-regulatory information derived from ChIP-seq, DNase-seq and ATAC-seq chromatin profiling assays, which map the genome-wide locations of transcription factor binding sites, histone post-translational modifications and regions of chromatin accessible to endonuclease activity. Currently, the Cistrome DB contains approximately 47,000 human and mouse samples with about 24,000 newly collected datasets compared to the previous release two years ago. Furthermore, the Cistrome DB has a new Toolkit module with several features that allow users to better utilize the large-scale ChIP-seq, DNase-seq, and ATAC-seq data. First, users can query the factors which are likely to regulate a specific gene of interest. Second, the Cistrome DB Toolkit facilitates searches for factor binding, histone modifications, and chromatin accessibility in any given genomic interval shorter than 2Mb. Third, the Toolkit can determine the most similar ChIP-seq, DNase-seq, and ATAC-seq samples in terms of genomic interval overlaps with user-provided genomic interval sets. The Cistrome DB is a user-friendly, up-to-date, and well maintained resource, and the new tools will greatly benefit the biomedical research community. The database is freely available at http://cistrome.org/db, and the Toolkit is at http://dbtoolkit.cistrome.org.

Pharmacological activation of estrogen receptor beta augments innate immunity to suppress cancer metastasis.

Metastases constitute the greatest causes of deaths from cancer. However, no effective therapeutic options currently exist for cancer patients with metastasis. Estrogen receptor ß (ERß), as a member of the nuclear receptor superfamily, shows potent tumor-suppressive activities in many cancers. To investigate whether modulation of ERß could serve as a therapeutic strategy for cancer metastasis, we examined whether the selective ERß agonist LY500307 could suppress lung metastasis of triple-negative breast cancer (TNBC) and melanoma. Mechanistically, while we observed that LY500307 potently induced cell death of cancer cells metastasized to lung in vivo, it does not mediate apoptosis of cancer cells in vitro, indicating that the cell death-inducing effects of LY500307 might be mediated by the tumor microenvironment. Pathological examination combined with flow cytometry assays indicated that LY500307 treatment induced significant infiltration of neutrophils in the metastatic niche. Functional experiments demonstrated that LY500307-treated cancer cells show chemotactic effects for neutrophils and that in vivo neutrophil depletion by Ly6G antibody administration could reverse the effects of LY500307-mediated metastasis suppression. RNA sequencing analysis showed that LY500307 could induce up-regulation of IL-1ß in TNBC and melanoma cells, which further triggered antitumor neutrophil chemotaxis. However, the therapeutic effects of LY500307 treatment for suppression of lung metastasis was attenuated in IL1B-/- murine models, due to failure to induce antitumor neutrophil infiltration in the metastatic niche. Collectively, our study demonstrated that pharmacological activation of ERß could augment innate immunity to suppress cancer metastatic colonization to lung, thus providing alternative therapeutic options for cancer patients with metastasis.

Modeling cis-regulation with a compendium of genome-wide histone H3K27ac profiles.

Model-based analysis of regulation of gene expression (MARGE) is a framework for interpreting the relationship between the H3K27ac chromatin environment and differentially expressed gene sets. The framework has three main functions: MARGE-potential, MARGE-express, and MARGE-cistrome. MARGE-potential defines a regulatory potential (RP) for each gene as the sum of H3K27ac ChIP-seq signals weighted by a function of genomic distance from the transcription start site. The MARGE framework includes a compendium of RPs derived from 365 human and 267 mouse H3K27ac ChIP-seq data sets. Relative RPs, scaled using this compendium, are superior to superenhancers in predicting BET (bromodomain and extraterminal domain) -inhibitor repressed genes. MARGE-express, which uses logistic regression to retrieve relevant H3K27ac profiles from the compendium to accurately model a query set of differentially expressed genes, was tested on 671 diverse gene sets from MSigDB. MARGE-cistrome adopts a novel semisupervised learning approach to identify cis-regulatory elements regulating a gene set. MARGE-cistrome exploits information from H3K27ac signal at DNase I hypersensitive sites identified from published human and mouse DNase-seq data. We tested the framework on newly generated RNA-seq and H3K27ac ChIP-seq profiles upon siRNA silencing of multiple transcriptional and epigenetic regulators in a prostate cancer cell line, LNCaP-abl. MARGE-cistrome can predict the binding sites of silenced transcription factors without matched H3K27ac ChIP-seq data. Even when the matching H3K27ac ChIP-seq profiles are available, MARGE leverages public H3K27ac profiles to enhance these data. This study demonstrates the advantage of integrating a large compendium of historical epigenetic data for genomic studies of transcriptional regulation.

Cistrome Data Browser: a data portal for ChIP-Seq and chromatin accessibility data in human and mouse.

Chromatin immunoprecipitation, DNase I hypersensitivity and transposase-accessibility assays combined with high-throughput sequencing enable the genome-wide study of chromatin dynamics, transcription factor binding and gene regulation. Although rapidly accumulating publicly available ChIP-seq, DNase-seq and ATAC-seq data are a valuable resource for the systematic investigation of gene regulation processes, a lack of standardized curation, quality control and analysis procedures have hindered extensive reuse of these data. To overcome this challenge, we built the Cistrome database, a collection of ChIP-seq and chromatin accessibility data (DNase-seq and ATAC-seq) published before January 1, 2016, including 13 366 human and 9953 mouse samples. All the data have been carefully curated and processed with a streamlined analysis pipeline and evaluated with comprehensive quality control metrics. We have also created a user-friendly web server for data query, exploration and visualization. The resulting Cistrome DB (Cistrome Data Browser), available online at http://cistrome.org/db, is expected to become a valuable resource for transcriptional and epigenetic regulation studies.

CRISPR-DO for genome-wide CRISPR design and optimization.

MOTIVATION: Despite the growing popularity in using CRISPR/Cas9 technology for genome editing and gene knockout, its performance still relies on well-designed single guide RNAs (sgRNA). In this study, we propose a web application for the Design and Optimization (CRISPR-DO) of guide sequences that target both coding and non-coding regions in spCas9 CRISPR system across human, mouse, zebrafish, fly and worm genomes. CRISPR-DO uses a computational sequence model to predict sgRNA efficiency, and employs a specificity scoring function to evaluate the potential of off-target effect. It also provides information on functional conservation of target sequences, as well as the overlaps with exons, putative regulatory sequences and single-nucleotide polymorphisms (SNPs). The web application has a user-friendly genome-browser interface to facilitate the selection of the best target DNA sequences for experimental design. AVAILABILITY AND IMPLEMENTATION: CRISPR-DO is available at http://cistrome.org/crispr/ CONTACT: qiliu@tongji.edu.cn or hanxu@jimmy.harvard.edu or xsliu@jimmy.harvard.eduSupplementary information: Supplementary data are available at Bioinformatics online.

ChiLin: a comprehensive ChIP-seq and DNase-seq quality control and analysis pipeline.

BACKGROUND: Transcription factor binding, histone modification, and chromatin accessibility studies are important approaches to understanding the biology of gene regulation. ChIP-seq and DNase-seq have become the standard techniques for studying protein-DNA interactions and chromatin accessibility respectively, and comprehensive quality control (QC) and analysis tools are critical to extracting the most value from these assay types. Although many analysis and QC tools have been reported, few combine ChIP-seq and DNase-seq data analysis and quality control in a unified framework with a comprehensive and unbiased reference of data quality metrics. RESULTS: ChiLin is a computational pipeline that automates the quality control and data analyses of ChIP-seq and DNase-seq data. It is developed using a flexible and modular software framework that can be easily extended and modified. ChiLin is ideal for batch processing of many datasets and is well suited for large collaborative projects involving ChIP-seq and DNase-seq from different designs. ChiLin generates comprehensive quality control reports that include comparisons with historical data derived from over 23,677 public ChIP-seq and DNase-seq samples (11,265 datasets) from eight literature-based classified categories. To the best of our knowledge, this atlas represents the most comprehensive ChIP-seq and DNase-seq related quality metric resource currently available. These historical metrics provide useful heuristic quality references for experiment across all commonly used assay types. Using representative datasets, we demonstrate the versatility of the pipeline by applying it to different assay types of ChIP-seq data. The pipeline software is available open source at https://github.com/cfce/chilin . CONCLUSION: ChiLin is a scalable and powerful tool to process large batches of ChIP-seq and DNase-seq datasets. The analysis output and quality metrics have been structured into user-friendly directories and reports. We have successfully compiled 23,677 profiles into a comprehensive quality atlas with fine classification for users.

CR Cistrome: a ChIP-Seq database for chromatin regulators and histone modification linkages in human and mouse.

Diversified histone modifications (HMs) are essential epigenetic features. They play important roles in fundamental biological processes including transcription, DNA repair and DNA replication. Chromatin regulators (CRs), which are indispensable in epigenetics, can mediate HMs to adjust chromatin structures and functions. With the development of ChIP-Seq technology, there is an opportunity to study CR and HM profiles at the whole-genome scale. However, no specific resource for the integration of CR ChIP-Seq data or CR-HM ChIP-Seq linkage pairs is currently available. Therefore, we constructed the CR Cistrome database, available online at http://compbio.tongji.edu.cn/cr and http://cistrome.org/cr/, to further elucidate CR functions and CR-HM linkages. Within this database, we collected all publicly available ChIP-Seq data on CRs in human and mouse and categorized the data into four cohorts: the reader, writer, eraser and remodeler cohorts, together with curated introductions and ChIP-Seq data analysis results. For the HM readers, writers and erasers, we provided further ChIP-Seq analysis data for the targeted HMs and schematized the relationships between them. We believe CR Cistrome is a valuable resource for the epigenetics community.

Single-cell analyses of metastatic bone marrow in human neuroblastoma reveals microenvironmental remodeling and metastatic signature.

Neuroblastoma is an aggressive pediatric cancer with a high rate of metastasis to the BM. Despite intensive treatments including high-dose chemotherapy, the overall survival rate for children with metastatic neuroblastoma remains dismal. Understanding the cellular and molecular mechanisms of the metastatic tumor microenvironment is crucial for developing new therapies and improving clinical outcomes. Here, we used single-cell RNA-Seq to characterize immune and tumor cell alterations in neuroblastoma BM metastases by comparative analysis with patients without metastases. Our results reveal remodeling of the immune cell populations and reprogramming of gene expression profiles in the metastatic niche. In particular, within the BM metastatic niche, we observed the enrichment of immune cells, including tumor-associated neutrophils, macrophages, and exhausted T cells, as well as an increased number of Tregs and a decreased number of B cells. Furthermore, we highlighted cell communication between tumor cells and immune cell populations, and we identified prognostic markers in malignant cells that are associated with worse clinical outcomes in 3 independent neuroblastoma cohorts. Our results provide insight into the cellular, compositional, and transcriptional shifts underlying neuroblastoma BM metastases that contribute to the development of new therapeutic strategies.

Single-cell analysis of immune and stroma cell remodeling in clear cell renal cell carcinoma primary tumors and bone metastatic lesions.

BACKGROUND: Despite therapeutic advances, once a cancer has metastasized to the bone, it represents a highly morbid and lethal disease. One third of patients with advanced clear cell renal cell carcinoma (ccRCC) present with bone metastasis at the time of diagnosis. However, the bone metastatic niche in humans, including the immune and stromal microenvironments, has not been well-defined, hindering progress towards identification of therapeutic targets. METHODS: We collected fresh patient samples and performed single-cell transcriptomic profiling of solid metastatic tissue (Bone Met), liquid bone marrow at the vertebral level of spinal cord compression (Involved), and liquid bone marrow from a different vertebral body distant from the tumor site but within the surgical field (Distal), as well as bone marrow from patients undergoing hip replacement surgery (Benign). In addition, we incorporated single-cell data from primary ccRCC tumors (ccRCC Primary) for comparative analysis. RESULTS: The bone marrow of metastatic patients is immune-suppressive, featuring increased, exhausted CD8 + cytotoxic T cells, T regulatory cells, and tumor-associated macrophages (TAM) with distinct transcriptional states in metastatic lesions. Bone marrow stroma from tumor samples demonstrated a tumor-associated mesenchymal stromal cell population (TA-MSC) that appears to be supportive of epithelial-to mesenchymal transition (EMT), bone remodeling, and a cancer-associated fibroblast (CAFs) phenotype. This stromal subset is associated with poor progression-free and overall survival and also markedly upregulates bone remodeling through the dysregulation of RANK/RANKL/OPG signaling activity in bone cells, ultimately leading to bone resorption. CONCLUSIONS: These results provide a comprehensive analysis of the bone marrow niche in the setting of human metastatic cancer and highlight potential therapeutic targets for both cell populations and communication channels.

Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy.

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. Since novel and improved immunotherapies may fill this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies.

Regulation of EZH2 Expression by INPP4B in Normal Prostate and Primary Prostate Cancer.

The phosphatases INPP4B and PTEN are tumor suppressors that are lost in nearly half of advanced metastatic cancers. The loss of PTEN in prostate epithelium initially leads to an upregulation of several tumor suppressors that slow the progression of prostate cancer in mouse models. We tested whether the loss of INPP4B elicits a similar compensatory response in prostate tissue and whether this response is distinct from the one caused by the loss of PTEN. Knockdown of INPP4B but not PTEN in human prostate cancer cell lines caused a decrease in EZH2 expression. In Inpp4b-/- mouse prostate epithelium, EZH2 levels were decreased, as were methylation levels of histone H3. In contrast, Ezh2 levels were increased in the prostates of Pten-/- male mice. Contrary to PTEN, there was a positive correlation between INPP4B and EZH2 expression in normal human prostates and early-stage prostate tumors. Analysis of single-cell transcriptomic data demonstrated that a subset of EZH2-positive cells expresses INPP4B or PTEN, but rarely both, consistent with their opposing correlation with EZH2 expression. Unlike PTEN, INPP4B did not affect the levels of SMAD4 protein expression or Pml mRNA expression. Like PTEN, p53 protein expression and phosphorylation of Akt in Inpp4b-/- murine prostates were elevated. Taken together, the loss of INPP4B in the prostate leads to overlapping and distinct changes in tumor suppressor and oncogenic downstream signaling.

Biocompatible Nanostructured Silver-Incorporated Implant Surfaces Show Effective Antibacterial, Osteogenic, and Anti-Inflammatory Effects in vitro and in Rat Model.

Introduction: Titanium (Ti) and its alloys are widely utilized in endosseous implants. However, their clinical efficacy is marred by complications arising from bacterial infections owing to their inadequate antibacterial properties. Consequently, enhancing the antibacterial attributes of implant surfaces stands as a pivotal objective in the realm of implantable materials research. Methods: In this study, we employed sequential anodization and plasma immersion ion implantation (PIII) technology to fabricate a silver-embedded sparsely titania nanotube array (SNT) on the near-ß titanium alloy Ti-5Zr-3Sn-5Mo-15Nb (TLM) implants. The surface characteristics, antimicrobial properties, biocompatibility, and osteogenic activity of the silver-nanomodified SNT implant (SNT Ag) surface, alongside peri-implant inflammatory responses, were meticulously assessed through a combination of in vitro and in vivo analyses. Results: Compared with polished TLM and SNT, the silver-embedded SNT (SNT Ag) surface retained the basic shape of nanotubes and stably released Ag+ at the ppm level for a long time, which demonstrated an effective inhibition and bactericidal activity against Staphylococcus aureus (SA) while maintaining ideal cytocompatibility. Additionally, the subtle modifications in nanotubular topography induced by silver implantation endowed SNT Ag with enhanced osteogenic activity and mitigated inflammatory capsulation in soft tissue peri-implants in a rat model. Conclusion: Incorporating a silver-embedded SNT array onto the implant surface demonstrated robust antibacterial properties, impeccable cytocompatibility, exceptional osteogenic activity, and the potential to prevent inflammatory encapsulation around the implant site. The Silver-PIII modification strategy emerges as a highly promising approach for surface applications in endosseous implants and trans-gingival implant abutments.

Androgen blockade primes NLRP3 in macrophages to induce tumor phagocytosis.

Immune-based therapies induce durable remissions in subsets of patients across multiple malignancies. However, there is limited efficacy of immunotherapy in metastatic castrate-resistant prostate cancer (mCRPC), manifested by an enrichment of immunosuppressive (M2) tumor- associated macrophages (TAM) in the tumor immune microenvironment (TME). Therefore, therapeutic strategies to overcome TAM-mediated immunosuppression are critically needed in mCRPC. Here we discovered that NLR family pyrin domain containing 3 (NLRP3), an innate immune sensing protein, is highly expressed in TAM from metastatic PC patients treated with standard-of-care androgen deprivation therapy (ADT). Importantly, ex vivo studies revealed that androgen receptor (AR) blockade in TAM upregulates NLRP3 expression, but not inflammasome activity, and concurrent AR blockade/NLRP3 agonist (NLRP3a) treatment promotes cancer cell phagocytosis by immunosuppressive M2 TAM. In contrast, NLRP3a monotherapy was sufficient to enhance phagocytosis of cancer cells in anti-tumor (M1) TAM, which exhibit high de novo NLRP3 expression. Critically, combinatorial treatment with ADT/NLRP3a in a murine model of advanced PC resulted in significant tumor control, with tumor clearance in 55% of mice via TAM phagocytosis. Collectively, our results demonstrate NLRP3 as an AR-regulated "macrophage phagocytic checkpoint", inducibly expressed in TAM by ADT and activated by NLRP3a treatment, the combination resulting in TAM-mediated phagocytosis and tumor control.