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Super‐resolution optical imaging tools are crucial in microbiology to understand the complex structures and behavior of microorganisms such as bacteria, fungi, and viruses. However, the capabilities of these tools, particularly when it comes to imaging pathogens and infected tissues, remain limited. MicroMagnify (µMagnify) is developed, a nanoscale multiplexed imaging method for pathogens and infected tissues that are derived from an expansion microscopy technique with a universal biomolecular anchor. The combination of heat denaturation and enzyme cocktails essential is found for robust cell wall digestion and expansion of microbial cells and infected tissues without distortion. µMagnify efficiently retains biomolecules suitable for high‐plex fluorescence imaging with nanoscale precision. It demonstrates up to eightfold expansion with µMagnify on a broad range of pathogen‐containing specimens, including bacterial and fungal biofilms, infected culture cells, fungus‐infected mouse tone, and formalin‐fixed paraffin‐embedded human cornea infected by various pathogens. Additionally, an associated virtual reality tool is developed to facilitate the visualization and navigation of complex 3D images generated by this method in an immersive environment allowing collaborative exploration among researchers worldwide. µMagnify is a valuable imaging platform for studying how microbes interact with their host systems and enables the development of new diagnosis strategies against infectious diseases.

The molecular mechanisms behind recognition of altered self remain unclear. Moore and co-workers show that oxidized low-density lipoprotein (LDL) and β-amyloid trigger inflammatory signaling through a heterodimer of Toll-like receptors 4 and 6. In atherosclerosis and Alzheimer's disease, deposition of the altered self components oxidized low-density lipoprotein (LDL) and amyloid-β triggers a protracted sterile inflammatory response. Although chronic stimulation of the innate immune system is believed to underlie the pathology of these diseases, the molecular mechanisms of activation remain unclear. Here we show that oxidized LDL and amyloid-β trigger inflammatory signaling through a heterodimer of Toll-like receptors 4 and 6. Assembly of this newly identified heterodimer is regulated by signals from the scavenger receptor CD36, a common receptor for these disparate ligands. Our results identify CD36-TLR4-TLR6 activation as a common molecular mechanism by which atherogenic lipids and amyloid-β stimulate sterile inflammation and suggest a new model of TLR heterodimerization triggered by coreceptor signaling events.

Infectious and autoimmune diseases are associated with anemia and thrombocytopenia. A severe form of inflammatory cytopenia called macrophage activation syndrome (MAS) may occur during rheumatological disorders and viral infections. Akilesh et al. show that monocyte recognition of self- or pathogen-derived nucleic acids via Toll-like receptors 7 and 9 (TLR7 and TLR9) drives MAS-like disease in mice. TLR7 or TLR9 signaling in monocytes causes these cells to differentiate into inflammatory hematophagocytes (iHPCs), which are similar to but distinct from red pulp macrophages. Preventing iHPC differentiation by depleting monocytes relieves MAS-like symptoms. When mice were subjected to a model of malarial anemia, MyD88- and endosomal TLR-dependent iHPC differentiation also occurred. Thus, iHPCs may play a role in both MAS-driven and malarial anemia, as well as thrombocytopenia. Science , this issue p. eaao5213 Immune receptor signaling underlies the process whereby inflammatory hemophagocytes drive anemia and thrombocytopenia. Cytopenias are an important clinical problem associated with inflammatory disease and infection. We show that specialized phagocytes that internalize red blood cells develop in Toll-like receptor 7 (TLR7)–driven inflammation. TLR7 signaling caused the development of inflammatory hemophagocytes (iHPCs), which resemble splenic red pulp macrophages but are a distinct population derived from Ly6C hi monocytes. iHPCs were responsible for anemia and thrombocytopenia in TLR7-overexpressing mice, which have a macrophage activation syndrome (MAS)–like disease. Interferon regulatory factor 5 (IRF5), associated with MAS, participated in TLR7-driven iHPC differentiation. We also found iHPCs during experimental malarial anemia, in which they required endosomal TLR and MyD88 signaling for differentiation. Our findings uncover a mechanism by which TLR7 and TLR9 specify monocyte fate and identify a specialized population of phagocytes responsible for anemia and thrombocytopenia associated with inflammation and infection.

In the mammalian gut CD103+ve myeloid DCs are known to suppress inflammation threatened by luminal bacteria, but stimuli driving DC precursor maturation towards this beneficial phenotype are incompletely understood. We isolated CD11+ve DCs from mesenteric lymph nodes (MLNs) of healthy mice; CD103+ve DCs were 8–24 fold more likely than CD103-ve DCs to exhibit extensive of prior phagocytosis of apoptotic intestinal epithelial cells. However, CD103+ve and CD103-ve MLN DCs exhibited similar ex vivo capacity to ingest apoptotic cells, indicating that apoptotic cells might drive immature DC maturation towards the CD103+ve phenotype. When cultured with apoptotic cells, myeloid DC precursors isolated from murine bone marrow and characterised as lineage-ve CD103-ve, displayed enhanced expression of CD103 and β8 integrin and acquired increased capacity to induce T regulatory lymphocytes (Tregs) after 7d in vitro. However, DC precursors isolated from αv-tie2 mice lacking αv integrins in the myeloid line exhibited reduced binding of apoptotic cells and complete deficiency in the capacity of apoptotic cells and/or latent TGF-β1 to enhance CD103 expression in culture, whereas active TGF-β1 increased DC precursor CD103 expression irrespective of αv expression. Fluorescence microscopy revealed clustering of αv integrin chains and latent TGF-β1 at points of contact between DC precursors and apoptotic cells. We conclude that myeloid DC precursors can deploy αv integrin to orchestrate binding of apoptotic cells, activation of latent TGF-β1 and acquisition of the immunoregulatory CD103+ve β8+ve DC phenotype. This implies that a hitherto unrecognised consequence of apoptotic cell interaction with myeloid phagocytes is programming that prevents inflammation.

Integrins play an important role in haematopoietic stem cell (HSC) maintenance in the bone marrow niche. Here, we demonstrate that Periostin (Postn) via interaction with Integrin-αv (Itgav) regulates HSC proliferation. Systemic deletion of Postn results in peripheral blood (PB) anaemia, myelomonocytosis and lymphopenia, while the number of phenotypic HSCs increases in the bone marrow. Postn −/− mice recover faster from radiation injury with concomitant loss of primitive HSCs. HSCs from Postn −/− mice show accumulation of DNA damage generally associated with aged HSCs. Itgav deletion in the haematopoietic system leads to a similar PB phenotype and HSC-intrinsic repopulation defects. Unaffected by Postn, Vav-Itgav −/− HSCs proliferate faster in vitro , illustrating the importance of Postn-Itgav interaction. Finally, the Postn-Itgav interaction inhibits the FAK/PI3K/AKT pathway in HSCs, leading to increase in p27Kip1 expression resulting in improved maintenance of quiescent HSCs. Together, we demonstrate a role for Itgav-mediated outside-in signalling in regulation of HSC proliferation and stemness. Integrins regulate haematopoietic stem cell (HSC) homeostasis and engraftment into the bone marrow (BM) niche upon transplantation. Here, the authors show that HSC quiescence and function in the BM is regulated by the interaction of PERIOSTIN and INTEGRIN αv and subsequent increase in p27Kip1.

The NLRP3 inflammasome is primarily known for producing inflammatory cytokines and inducing pyroptosis. Stuart and colleagues identify an additional role for NLRP3 in driving down the pH of phagosomes. Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates many functions of these organelles that allow phagosomes to participate in processes that are essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3 inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3 inflammasome and caspase-1 in host defense.

The formation of extracellular DNA traps (ETosis) is a first response mechanism by specific immune cells following exposure to microbes. Initially characterized in vertebrate neutrophils, cells capable of ETosis have been discovered recently in diverse non-vertebrate taxa. To assess the conservation of ETosis between evolutionarily distant non-vertebrate phyla, we observed and quantified ETosis using the model ctenophore Mnemiopsis leidyi and the oyster Crassostrea gigas . Here we report that ctenophores – thought to have diverged very early from the metazoan stem lineage – possess immune-like cells capable of phagocytosis and ETosis. We demonstrate that both Mnemiopsis and Crassostrea immune cells undergo ETosis after exposure to diverse microbes and chemical agents that stimulate ion flux. We thus propose that ETosis is an evolutionarily conserved metazoan defense against pathogens. Identifying core mechanisms of immune cells is critical for understanding the evolution of animal immune function. Here, Vandepas et al. report that ctenophore immune-like cells release extracellular DNA traps when exposed to microbes.

An accurate prognostic tool is essential to aid clinical decision-making (e.g., patient triage) and to advance personalized medicine. However, such a prognostic tool is lacking for acute pancreatitis (AP). Increasingly machine learning (ML) techniques are being used to develop high-performing prognostic models in AP. However, methodologic and reporting quality has received little attention. High-quality reporting and study methodology are critical for model validity, reproducibility, and clinical implementation. In collaboration with content experts in ML methodology, we performed a systematic review critically appraising the quality of methodology and reporting of recently published ML AP prognostic models. Using a validated search strategy, we identified ML AP studies from the databases MEDLINE and EMBASE published between January 2021 and December 2023. We also searched pre-print servers medRxiv, bioRxiv, and arXiv for pre-prints registered between January 2021 and December 2023. Eligibility criteria included all retrospective or prospective studies that developed or validated new or existing ML models in patients with AP that predicted an outcome following an episode of AP. Meta-analysis was considered if there was homogeneity in the study design and in the type of outcome predicted. For risk of bias (ROB) assessment, we used the Prediction Model Risk of Bias Assessment Tool. Quality of reporting was assessed using the Transparent Reporting of a Multivariable Prediction Model of Individual Prognosis or Diagnosis-Artificial Intelligence (TRIPOD+AI) statement that defines standards for 27 items that should be reported in publications using ML prognostic models. The search strategy identified 6,480 publications of which 30 met the eligibility criteria. Studies originated from China (22), the United States (4), and other (4). All 30 studies developed a new ML model and none sought to validate an existing ML model, producing a total of 39 new ML models. AP severity (23/39) or mortality (6/39) were the most common outcomes predicted. The mean area under the curve for all models and endpoints was 0.91 (SD 0.08). The ROB was high for at least one domain in all 39 models, particularly for the analysis domain (37/39 models). Steps were not taken to minimize over-optimistic model performance in 27/39 models. Due to heterogeneity in the study design and in how the outcomes were defined and determined, meta-analysis was not performed. Studies reported on only 15/27 items from TRIPOD+AI standards, with only 7/30 justifying sample size and 13/30 assessing data quality. Other reporting deficiencies included omissions regarding human-AI interaction (28/30), handling low-quality or incomplete data in practice (27/30), sharing analytical codes (25/30), study protocols (25/30), and reporting source data (19/30). There are significant deficiencies in the methodology and reporting of recently published ML based prognostic models in AP patients. These undermine the validity, reproducibility, and implementation of these prognostic models despite their promise of superior predictive accuracy. Research Registry (reviewregistry1727).

Integrin signalling triggers cytoskeletal rearrangements, including endocytosis and exocytosis of integrins and other membrane proteins. In addition to recycling integrins, this trafficking can also regulate intracellular signalling pathways. Here we describe a role for αv integrins in regulating Toll-like receptor (TLR) signalling by modulating intracellular trafficking. We show that deletion of αv or β3 causes increased B-cell responses to TLR stimulation in vitro , and αv-conditional knockout mice have elevated antibody responses to TLR-ligand-associated antigens. αv regulates TLR signalling by promoting recruitment of the autophagy component LC3 (microtubule-associated proteins 1 light chain 3) to TLR-containing endosomes, which is essential for progression from NF-κB to IRF signalling, and ultimately for traffic to lysosomes where signalling is terminated. Disruption of LC3 recruitment leads to prolonged NF-κB signalling and increased B-cell proliferation and antibody production. This work identifies a previously unrecognized role for αv and the autophagy components LC3 and atg5 in regulating TLR signalling and B-cell immunity. Integrins can regulate antigen-specific and innate immune receptor signalling, thereby affecting immune cell function. Here the authors show that avß3 integrin controls Toll-like receptor (TLR) signalling by regulating its trafficking to limit TLR-mediated B-cell proliferation and antibody production.

The gastrointestinal tract is constantly challenged by foreign antigens and commensal bacteria but nonetheless is able to maintain a state of immunological quiescence. Recent advances have highlighted the importance of active suppression by regulatory lymphocytes and immunosuppressive cytokines in controlling mucosal immunity. Failures of these mechanisms contribute to the development of inflammatory bowel disease, but how these regulatory networks are established remains unclear. Here, we demonstrate key roles for αv integrins in regulation of mucosal immunity. We report that deletion of αv in the immune system causes severe colitis, autoimmunity, and cancer. Mice lacking immune cell αv have fewer regulatory T (Treg) cells in the colon and corresponding increases in activated T cells and T cell cytokine production, leading to colitis. Using conditional gene targeting, we demonstrate that this is specifically attributable to loss of αv from myeloid cells. Furthermore, we show that gut-associated macrophages and dendritic cells fail both to remove apoptotic cells efficiently and to induce Treg cells. Our results identify a vital role for myeloid αv integrins in generating mucosal Treg cells and emphasize the importance of antigen-presenting cells in establishing immune tolerance.