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SUMMARY Circular RNAs (circRNAs) are stable RNAs present in cell-free RNA,which may comprise cellular debris and pathogen genomes. Here we investigate the phenomenon and mechanism of cellular uptake and intracellular fate of exogenous circRNAs. Human myeloid cells and B cells selectively internalize extracellular circRNAs. Macrophage uptake of circRNA is rapid,energy-dependent,and saturable. CircRNA uptake can lead to translation of encoded sequences and antigen presentation. The route of internalization influences immune activation after circRNA uptake,with distinct gene expression programs depending on the route of RNA delivery. Genome-scale CRISPR screens and chemical inhibitor studies nominate macrophage scavenger receptor MSR1,toll-like receptors,and mTOR signaling as key regulators of receptor-mediated phagocytosis of circRNAs,a dominant pathway to internalize circRNAs in parallel to macropinocytosis. These results suggest that cell-free circRNA serves as an “eat me” signal and danger-associated molecular pattern,indicating orderly pathways of recognition and disposal. eTOC Blurb: Amaya et. al. explores how cells take up extracellular circular RNAs (CircRNAs) and their impact on immune signaling. Macrophages readily internalize circRNAs,and this study identifies the specific receptors and signaling pathways governing circRNA internalization,highlighting their role as signaling molecules for immune recognition and disposal. Graphical Abstract View Publication

SummaryAerobic exercise training (AET) has emerged as a strategy to reduce cancer mortality,however,the mechanisms explaining AET on tumor development remain unclear. Tumors escape immune detection by generating immunosuppressive microenvironments and impaired T cell function,which is associated with T cell mitochondrial loss. AET improves mitochondrial content and function,thus we tested whether AET would modulate mitochondrial metabolism in tumor-infiltrating lymphocytes (TIL). Balb/c mice were subjected to a treadmill AET protocol prior to CT26 colon carcinoma cells injection and until tumor harvest. Tissue hypoxia,TIL infiltration and effector function,and mitochondrial content,morphology and function were evaluated. AET reduced tumor growth,improved survival,and decreased tumor hypoxia. An increased CD8+ TIL infiltration,IFN-γ and ATP production promoted by AET was correlated with reduced mitochondrial loss in these cells. Collectively,AET decreases tumor growth partially by increasing CD8+ TIL effector function through an improvement in their mitochondrial content and function. Graphical abstract Highlights•Exercise training reduces tumor growth and improves survival in colorectal cancer•Trained mice present tumors with less hypoxia and higher CD8+ T cells infiltration•The production of IFNγ by CD8+ TIL is increased in exercise-trained mice•CD8+ TIL from trained mice show higher mitochondrial density and function Natural sciences; Biological sciences; Biochemistry; Physiology; Immunology; Systems biology; Cancer systems biology View Publication

BackgroundInterleukin-17 (IL-17) family cytokines promote protective inflammation for pathogen resistance,but also facilitate autoimmunity and tumor development. A direct signal of IL-17 to regulatory T cells (Tregs) has not been reported and may help explain these dichotomous responses.MethodsWe generated a conditional knockout of Il17ra in Tregs by crossing Foxp3-YFP-Cre mice to Il17ra-flox mice (Il17ra ΔTreg mice). Subsequently,we adoptively transferred bone marrow cells from Il17ra ΔTreg mice to a mouse model of sporadic colorectal cancer (Cdx2-Cre +/Apc F/+),to selectively ablate IL-17 direct signaling on Tregs in colorectal cancer. Single cell RNA sequencing and bulk RNA sequencing were performed on purified Tregs from mouse colorectal tumors,and compared to those of human tumor infiltrating Treg cells.ResultsIL-17 Receptor A (IL-17RA) is expressed in Tregs that reside in mouse mesenteric lymph nodes and colon tumors. Ablation of IL-17RA,specifically in Tregs,resulted in increased Th17 cells,and exacerbated tumor development. Mechanistically,tumor-infiltrating Tregs exhibit a unique gene signature that is linked to their activation,maturation,and suppression function,and this signature is in part supported by the direct signaling of IL-17 to Tregs. To study pathways of Treg programming,we found that loss of IL-17RA in tumor Tregs resulted in reduced RNA splicing,and downregulation of several RNA binding proteins that are known to regulate alternative splicing and promote Treg function.ConclusionIL-17 directly signals to Tregs and promotes their maturation and function. This signaling pathway constitutes a negative feedback loop that controls cancer-promoting inflammation in CRC. View Publication

AbstractDespite the success of antiretroviral therapy,human immunodeficiency virus (HIV) cannot be cured because of a reservoir of latently infected cells that evades therapy. To understand the mechanisms of HIV latency,we employed an integrated single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) approach to simultaneously profile the transcriptomic and epigenomic characteristics of ∼ 125,000 latently infected primary CD4+ T cells after reactivation using three different latency reversing agents. Differentially expressed genes and differentially accessible motifs were used to examine transcriptional pathways and transcription factor (TF) activities across the cell population. We identified cellular transcripts and TFs whose expression/activity was correlated with viral reactivation and demonstrated that a machine learning model trained on these data was 75%–79% accurate at predicting viral reactivation. Finally,we validated the role of two candidate HIV-regulating factors,FOXP1 and GATA3,in viral transcription. These data demonstrate the power of integrated multimodal single-cell analysis to uncover novel relationships between host cell factors and HIV latency. View Publication

Acute and chronic coronary syndromes (ACS and CCS) are leading causes of mortality. Inflammation is considered a key pathogenic driver of these diseases,but the underlying immune states and their clinical implications remain poorly understood. Multiomic factor analysis (MOFA) allows unsupervised data exploration across multiple data types,identifying major axes of variation and associating these with underlying molecular processes. We hypothesized that applying MOFA to multiomic data obtained from blood might uncover hidden sources of variance and provide pathophysiological insights linked to clinical needs. Here we compile a longitudinal multiomic dataset of the systemic immune landscape in both ACS and CCS (n = 62 patients in total,n = 15 women and n = 47 men) and validate this in an external cohort (n = 55 patients in total,n = 11 women and n = 44 men). MOFA reveals multicellular immune signatures characterized by distinct monocyte,natural killer and T cell substates and immune-communication pathways that explain a large proportion of inter-patient variance. We also identify specific factors that reflect disease state or associate with treatment outcome in ACS as measured using left ventricular ejection fraction. Hence,this study provides proof-of-concept evidence for the ability of MOFA to uncover multicellular immune programs in cardiovascular disease,opening new directions for mechanistic,biomarker and therapeutic studies. Multiomic factor analysis of blood multiomic data,including single-cell transcriptomics,for individuals with either acute or chronic coronary syndrome identifies immune cell signatures that correlate with treatment outcomes. View Publication

Haematopoietic stem cell (HSC) transplantation (HSCT) is the only curative treatment for a broad range of haematological malignancies,but the standard of care relies on untargeted chemotherapies and limited possibilities to treat malignant cells after HSCT without affecting the transplanted healthy cells1. Antigen-specific cell-depleting therapies hold the promise of much more targeted elimination of diseased cells,as witnessed in the past decade by the revolution of clinical practice for B cell malignancies2. However,target selection is complex and limited to antigens expressed on subsets of haematopoietic cells,resulting in a fragmented therapy landscape with high development costs2–5. Here we demonstrate that an antibody–drug conjugate (ADC) targeting the pan-haematopoietic marker CD45 enables the antigen-specific depletion of the entire haematopoietic system,including HSCs. Pairing this ADC with the transplantation of human HSCs engineered to be shielded from the CD45-targeting ADC enables the selective eradication of leukaemic cells with preserved haematopoiesis. The combination of CD45-targeting ADCs and engineered HSCs creates an almost universal strategy to replace a diseased haematopoietic system,irrespective of disease aetiology or originating cell type. We propose that this approach could have broad implications beyond haematological malignancies. An antibody–drug conjugate that targets the pan-haematopoietic marker CD45 combined with transplanted stem cells engineered to be shielded from it can eradicate leukaemic cells while preserving haematopoiesis. View Publication

T lymphocyte activation plays a pivotal role in adaptive immune response and alters the spatial organization of nuclear architecture that subsequently impacts transcription activities. Here,using stochastic optical reconstruction microscopy (STORM),we observe dramatic de-condensation of chromatin and the disruption of nuclear envelope at a nanoscale resolution upon T lymphocyte activation. Super-resolution imaging reveals that such alterations in nuclear architecture are accompanied by the release of nuclear DNA into the cytoplasm,correlating with the degree of chromatin decompaction within the nucleus. The authors show that under the influence of metabolism,T lymphocyte activation de-condenses chromatin,disrupts the nuclear envelope,and releases DNA into the cytoplasm. Taken together,this result provides a direct,molecular-scale insight into the alteration in nuclear architecture. It suggests the release of nuclear DNA into the cytoplasm as a general consequence of chromatin decompaction after lymphocyte activation. The authors show that under the influence of metabolism,T lymphocyte activation de-condenses chromatin,disrupts the nuclear envelope,and releases DNA into the cytoplasm. View Publication

Comparative genomics has revealed the rapid expansion of multiple gene families involved in immunity. Members within each gene family often evolved distinct roles in immunity. However,less is known about the evolution of their epigenome and cis-regulation. Here we systematically profile the epigenome of the recently expanded murine Ly49 gene family that mainly encode either inhibitory or activating surface receptors on natural killer cells. We identify a set of cis-regulatory elements (CREs) for activating Ly49 genes. In addition,we show that in mice,inhibitory and activating Ly49 genes are regulated by two separate sets of proximal CREs,likely resulting from lineage-specific losses of CRE activity. Furthermore,we find that some Ly49 genes are cross-regulated by the CREs of other Ly49 genes,suggesting that the Ly49 family has begun to evolve a concerted cis-regulatory mechanism. Collectively,we demonstrate the different modes of cis-regulatory evolution for a rapidly expanding gene family. The Ly49 gene family mainly encodes inhibitory or activating surface receptors on natural killer cells. Here the authors show that in mice,inhibitory and activating Ly49 genes are regulated by two distinct sets of cis-regulatory elements,and that different Ly49 genes can be cross-regulated. View Publication

Bacteria such as the oral microbiome member Peptostreptococcus anaerobius can exacerbate colorectal cancer (CRC) development. Little is known regarding whether these immunomodulatory bacteria also affect antitumour immune checkpoint blockade therapy. Here we show that administration of P. anaerobius abolished the efficacy of anti-PD1 therapy in mouse models of CRC. P. anaerobius both induced intratumoral myeloid-derived suppressor cells (MDSCs) and stimulated their immunosuppressive activities to impair effective T cell responses. Mechanistically,P. anaerobius administration activated integrin α2β1–NF-κB signalling in CRC cells to induce secretion of CXCL1 and recruit CXCR2+ MDSCs into tumours. The bacterium also directly activated immunosuppressive activity of intratumoral MDSCs by secreting lytC_22,a protein that bound to the Slamf4 receptor on MDSCs and promoted ARG1 and iNOS expression. Finally,therapeutic targeting of either integrin α2β1 or the Slamf4 receptor were revealed as promising strategies to overcome P. anaerobius-mediated resistance to anti-PD1 therapy in CRC. Interactions between Peptostreptococcus anaerobius and host cells promote recruitment and activation of myeloid-derived suppressor cells,leading to anti-PD1 immune checkpoint inhibitor resistance and exacerbated colorectal cancer in mice. View Publication

SummaryThe existing suite of therapies for bone diseases largely act to prevent further bone loss but fail to stimulate healthy bone formation and repair. We describe an endogenous osteopeptide (PEPITEM) with anabolic osteogenic activity,regulating bone remodeling in health and disease. PEPITEM acts directly on osteoblasts through NCAM-1 signaling to promote their maturation and formation of new bone,leading to enhanced trabecular bone growth and strength. Simultaneously,PEPITEM stimulates an inhibitory paracrine loop: promoting osteoblast release of the decoy receptor osteoprotegerin,which sequesters RANKL,thereby limiting osteoclast activity and bone resorption. In disease models,PEPITEM therapy halts osteoporosis-induced bone loss and arthritis-induced bone damage in mice and stimulates new bone formation in osteoblasts derived from patient samples. Thus,PEPITEM offers an alternative therapeutic option in the management of diseases with excessive bone loss,promoting an endogenous anabolic pathway to induce bone remodeling and redress the imbalance in bone turnover. Graphical abstract Highlights•PEPITEM exerts anabolic osteogenic activity to regulate osteoblast-osteoclast coupling•PEPITEM acts directly on osteoblasts to promote formation of new and stronger bone•PEPITEM stimulates an inhibitory paracrine loop via OPG to limit bone resorption•PEPITEM therapy halts disease-induced bone loss in vivo Lewis and Frost et al. identify the anabolic activity of an endogenous osteopeptide (PEPITEM),revealing the cellular and molecular mechanisms by which PEPITEM regulates bone remodeling in vitro and in preclinical disease models,to promote new bone formation. They suggest that PEPITEM offers an alternative therapeutic option for bone loss diseases. View Publication