搜索结果：innate

The clinical translation of cancer vaccines remains limited by the inability to effectively coordinate innate and adaptive immunity. Leveraging the metabolism pattern of the immune system, herein, we report a glucose metabolism-modulatory nanobiohybrid vaccine with superior antitumor immunity via orchestrating cross-presentation-enhanced adaptive immunity and proinflammatory signaling-mediated innate immunity. This nanobiohybrid vaccine is constructed by a nanoscale hydrogen-bonded organic framework (HOF) co-encapsulating glucose oxidase (GOx), and a model antigen, ovalbumin (OVA) (denoted OVA/GOx@HOF). OVA/GOx@HOF enables a GOx-mediated enzyme reaction that consumes glucose and produces H2O2 within dendritic cells (DCs) in controlled kinetics, which disrupts intracellular glucose metabolism and causes redox imbalance but minimally impacts cell viability. These effects induce autophagic machinery in DCs that promotes antigen cross-presentation, thereby boosting cytotoxic T cell response. Meanwhile, through RNA sequencing, we found that OVA/GOx@HOF stimulates innate immunity through an NF-κB signaling-mediated and endogenous hydrogen sulfide (H2S)-enhanced NLRP3 inflammasome pathway, leading to significant DC maturation. In a melanoma tumor model, this nanobiohybrid vaccine effectively inhibits tumor growth and metastasis, and can be applied to encapsulate neoantigens to generate a potent personalized cancer vaccine. Our findings suggested the remarkable potential of enzyme-driven nanobiohybrid materials as a metabolism-modulatory platform for cancer vaccines.

Megakaryocytes, beyond their classical role in platelet formation, are increasingly recognized as immune modulators. This study aimed to investigate the function and activation mechanisms of megakaryocytes during Pseudomonas aeruginosa (PA) infection, focusing on their interaction with bacterial toxins and innate immune pathways. This study evaluated the response of pulmonary megakaryocytes to PA-induced acute pneumonia in mice. Specifically, the activation status of pulmonary megakaryocytes was determined by flow cytometry and sorting, followed by RNA sequencing, combining Gene Ontology and Gene Set Enrichment Analysis. The expression of pro-inflammatory cytokines and chemokines within megakaryocytes after PA infection were detected by qPCR and fluorescent confocal imaging. The effect of megakaryocytes on the chemotaxis and recruitment of neutrophils and monocytes was demonstrated using chemotaxis assays. Additionally, purified PA toxins were injected into the lungs of mice in vivo or added to flow-sorted cells in vitro in order to induce inflammation and evaluate the response of megakaryocytes to a single PA toxin. The activation status of intracellular signaling pathways was verified through inhibitor-mediated blocking experiments. PA infection significantly activates megakaryocytes, which promote the recruitment of phagocytes and enhance the host's defense against PA infection. The innate immune response in the lungs was diminished during the early stages of PA infection among the megakaryocytes depleted mice, resulting in increased bacterial load and higher mortality. Flow cytometry sorting followed by RNA sequencing indicated that megakaryocytes exhibit a typical pro-inflammatory phenotype, along with NF-κB pathways activation. Furthermore, the PA toxin PcrV was identified as a key NF-κB activator on megakaryocytes, as demonstrated by increased expression of inflammatory markers and chemokines in response to PcrV. Inhibition of NF-κB signaling attenuated this pro-inflammatory phenotype of megakaryocytes. This study provides new insights into how megakaryocytes shape the cytokine and chemokine landscape in the lung during PA infection. It highlights the intrinsic interactions between megakaryocytes and phagocytes during pathogen invasion, suggests that managing megakaryocyte activity could be a new strategy to enhance immune defense. Megakaryocytes, traditionally known for their role in platelet production, have recently emerged as key players in immune responses during infections. In this study, we explore the involvement of pulmonary megakaryocytes in the response to PA infection, with a specific focus on the mechanistic activation of megakaryocytes via bacterial toxins. We demonstrate that PA infection triggers significant alterations in chemokine signaling and NF-κB pathways in pulmonary megakaryocytes, leading to the upregulation of pro-inflammatory cytokines and chemotactic factors. Activated pulmonary megakaryocytes effectively promote the recruitment of phagocytes, enhancing the host’s resistance to PA infection. we identify PcrV as a key NF-κB activator in megakaryocytes, evidenced by elevated expression of inflammatory markers and chemokines in response to PcrV. Inhibition of NF-κB signaling dampens this inflammatory response, confirming its critical role in megakaryocyte. Together, these findings provide novel insights into the immune functions of megakaryocytes as active modulators during bacterial infections. This work underscores the intricate crosstalk between megakaryocytes and phagocytes and suggests that targeting megakaryocyte activity may represent a promising strategy for controlling bacterial pneumonia.

Natural killer (NK) cells are innate lymphocytes that play a critical role in protective immunity against diverse intracellular pathogens and cancers. Their primary function is to kill target cells that are infected, malignantly transformed, or coated by antibodies via antibody-dependent cellular cytotoxicity (ADCC). NK cells can also be genetically engineered to express chimeric antigen receptors (CARs) that enable targeted recognition of specific antigens. Quantitative measurement of NK-cell cytotoxicity is essential for assessing baseline functionality and for preclinical evaluation of monoclonal antibodies and CAR-engineering strategies. However, in vitro functional assays remain highly variable across laboratories due to differences in cell preparation, target cells, effector-to-target ratios, co-incubation times, and readout methods, limiting reproducibility and cross-study comparisons. This article presents a standardized protocol for quantitative assessment of NK-cell cytotoxicity using flow cytometry and real-time, live-cell imaging. Primary human NK cells and CAR-expressing NK-92 cells were evaluated for their ability to kill cancer cells and antibody-coated target cells in a 96-well plate format to measure natural cytotoxicity, CAR-mediated killing, and ADCC. Target-cell survival was measured either continuously using live-cell imaging or at a defined time point by flow cytometry, which also enabled phenotypic characterization of NK cells and target cells. These protocols provide a robust framework using routine tissue culture, imaging, and flow cytometry methods to enable reproducible quantification of NK-cell effector functions for studies of innate immunity and NK cell-based immunotherapies.

Neonatal sepsis is a leading cause of morbidity and mortality in neonates. The underdeveloped neonatal immune system, particularly innate immune cells such as monocytes, plays a critical role in susceptibility to infection. Monocyte-mediated regulation of iron metabolism, a key component of "nutritional immunity," is known to influence sepsis outcomes in adults. In this study, we investigated differences in iron sensing, iron-regulated gene expression, and intracellular iron content between neonatal and adult monocytes. Monocytes were isolated from human umbilical cord blood and adult peripheral blood and stimulated in vitro with lipopolysaccharide (LPS), ferric nitrilotriacetate (FeNTA), or the iron chelator deferoxamine (DFO). Transferrin receptor 1 (TfR1) and differentiation markers were analyzed by flow cytometry, intracellular iron content by atomic absorption spectrometry, and metabolic and inflammatory responses via lactate and cytokine measurements. Neonatal monocytes exhibited lower basal TfR1 expression with a trend toward higher intracellular iron. LPS induced TfR1 upregulation exclusively in adult monocytes, while neonatal cells maintained consistently low expression. Although FeNTA increased intracellular iron in both groups, neonatal monocytes accumulated iron less efficiently. These findings indicate fundamental developmental differences in monocyte iron handling and immunometabolic adaptation, which may underlie the distinct immune profile observed in neonatal sepsis. Neonatal sepsis is a leading cause of morbidity and mortality in neonates. Particularly innate immune cells such as monocytes, play a critical role in susceptibility to infection. Monocyte-mediated regulation of iron metabolism, a key component of "nutritional immunity," is known to influence sepsis outcomes in adults. Our findings indicate fundamental developmental differences in monocyte iron handling and immunometabolic adaptation, which may play a role in both the distinct immune profile observed in neonatal sepsis and, consecutively, sepsis outcome. These findings imply that host-directed iron modulation could be a viable strategy to counteract immunoparalysis in neonatal sepsis without compromising cellular activation.

CD24 is a "don't eat me" signal overexpressed across multiple solid tumors and contributes to immune evasion by suppressing macrophage-mediated phagocytosis. Targeting the CD24/Siglec-10 axis represents a novel immuno-oncology strategy to restore innate immune surveillance. We developed PHST001, a humanized IgG4 monoclonal antibody targeting CD24, and evaluated its activity using in vitro phagocytosis assays, xenograft and immune-competent syngeneic mouse models, and ex vivo systems incorporating human immune cells and tumor samples. Nonclinical safety parameters were assessed to evaluate translational feasibility. PHST001 binds CD24 with high affinity and blocks Siglec-10 engagement, resulting in enhanced macrophage-mediated phagocytosis across multiple tumor indications and subtypes, inhibition of primary and metastatic tumor growth, and prolonged survival in preclinical models. PHST001 demonstrated a favorable nonclinical safety profile and exhibited anti-tumor activity as both monotherapy and in combination with standard-of-care treatments, including chemotherapy, radiotherapy, and antibody-drug conjugates (ADCs). Antitumor responses were associated with engagement of tissue-resident macrophages, and in syngeneic models, induction of tumor-reactive T-cell responses, supporting a role for CD24 in coordinating innate and adaptive immune suppression. These findings establish CD24 as a critical regulator of tumor immune evasion and support the clinical development of PHST001 as a CD24-targeted immunotherapy. A Phase I clinical study (NCT06840886) evaluating the safety and tolerability of PHST001 in adult patients with relapsed or refractory solid tumors is ongoing.

The immune system combats viral infections through innate and adaptive responses, with macrophages and dendritic cells playing key roles in pathogen clearance. Neutralizing antibodies aid viral elimination but have variable efficacy against SARS-CoV-2, which uses ACE2 for entry. Human recombinant soluble ACE2 (hrsACE2) acts as a decoy to block viral entry but has limitations in affinity and immune uptake. We produced mannosylated hrsACE2 (Mann-hrsACE2) via a baculovirus expression system to enhance uptake by innate immune cells, aiming to improve viral neutralization and stimulate cell-mediated immunity against SARS-CoV-2. Mann-hrsACE2 protein was produced in Sf9 insect cells using recombinant baculovirus, purified by Ni-NTA chromatography, and verified by SDS-PAGE and Western blot. Its interaction with macrophages was assessed by FITC-labeling and analyzed through fluorescence microscopy and flow cytometry. Expressed Mann-hrsACE2 displayed an upward shift SDS-PAGE and Western blot analysis. Flow cytometry revealed macrophage uptake of Mann-hrsACE2 at 3, 28, and 98% corresponding to concentrations of 1, 10, and 51 nM, respectively. The increased molecular weight of the expressed protein confirmed its glycosylation. Its mannosylation enabled efficient uptake by macrophages, key antigen-presenting cells in the immune system. Furthermore, by binding to SARS-CoV-2, the protein not only inhibits viral entry but also redirects the virus to macrophages for enhanced immune clearance.

Lipid nanoparticles (LNPs) are central to next-generation vaccines, yet candidate selection remains largely empirical, limiting early identification of formulations associated with rare adverse events such as myocarditis. A better understanding of LNP composition-immunogenicity relationships is therefore critical for rational vaccine design. Here, we profiled a panel of clinically relevant LNP formulations across complementary in vitro and in vivo models to define mechanisms underlying innate immune activation and adaptive responses. We identified three distinct cytokine programs: (i) a monocyte chemoattractant protein-1 (MCP-1)-dominated inflammatory response associated with cytotoxic stress; (ii) inflammasome-dependent interleukin-1 beta (IL-1β) secretion requiring pro-inflammatory priming; and (iii) type I and II interferon-dependent responses in which LNPs synergize with interferon gamma (IFNγ) to amplify interferon gamma-induced protein 10 (IP-10) production. Using a design of experiments (DoE) framework with formulation feature analysis, we found that polyethylene glycol-conjugated (PEGylated) lipid content and ionizable lipid identity are key modulators of the IFNγ/IP-10 axis, previously implicated in vaccine-associated myocarditis. In vivo validation showed that innate cytokine responses are strongly influenced by lipid composition, whereas adaptive humoral and cellular responses correlate with transgene expression rather than cytokine magnitude. Collectively, these findings define relationships among LNP composition, cytokine induction, and vaccine efficacy, and provide a framework for rational design and screening of LNP-based vaccines that maximize immunogenicity while minimizing reactogenicity.

Although there are several ongoing clinical trials using neoantigen peptide-based cancer vaccines, challenges still exist to implement in clinical approval such as poor antigen stability, inefficient delivery, and inadequate immune activation. To address these limitations, we developed a polymer-based polyvalent peptide and adjuvant (SPPA) that co-delivers lipid-conjugated tumor-specific peptides with Toll-like receptor 7/8 (3M-052) and a STING (2'3'-cGAMP) agonist. This nanoplatform enables efficient peptide encapsulation, sustained release, and targeted delivery to antigen-presenting cells (APCs), thereby enhancing both innate and adaptive immune responses. We synthesized and characterized a library of lipid-conjugated tumor-associated and neoantigenic peptides. In vitro, SPPA significantly upregulated pro-inflammatory genes and cytokine secretion, confirming robust innate immune activation and demonstrated effective cellular uptake and lymphatic trafficking. In vivo, SPPA alone or in combination with anti-PD-1 antibody (αPD-1) elicited strong cytotoxic T lymphocyte (CTL) responses and inhibited tumor growth in four aggressive syngeneic mouse models: Triple-negative breast cancer (4T1), HER2+ breast cancer (TUBO), lung carcinoma (LLC1), and renal cell carcinoma (RENCA). The combination therapy led to pronounced tumor growth inhibition, survival benefit, and immune cell infiltration, including elevated CD8+IFN-γ+ T cells and M1 macrophages, and reduced regulatory T cells and MDSCs. Spatial transcriptomics revealed localized transcriptional reprogramming, with downregulation of extracellular matrix genes and activation of inflammatory pathways. Collectively, these findings establish SPPA as a potent and versatile nanovaccine platform capable of inducing durable antitumor immunity, especially when combined with immune checkpoint blockade. This approach offers strong translational potential for personalized immunotherapy across diverse solid tumor types.

This experiment was conducted to evaluate the effects of dietary nanocurcumin on growth performance, feed utilization, innate immunity, antioxidant capacity, biochemical indices, and air exposure stress resistance in European seabass (Dicentrarchus labrax) fingerlings. Five isonitrogenous (47% crude protein) and isocaloric (21 MJ/kg dry matter) diets were formulated, supplemented with nanocurcumin at the following levels: 0, 100, 300, 600, and 900 mg/kg. The diets were administered to D. labrax (6.45 ± 0.08 g) for 65 days. Following the feeding trial, the fish were subjected to air exposure stress for 3 min followed by 2 h of recovery before sampling. The results indicated that dietary nanocurcumin significantly enhanced growth performance and feed utilization. Antioxidant status was improved, as evidenced by increased levels of reduced glutathione and catalase, along with decreased concentrations of malondialdehyde and hydrogen peroxide. In addition, nanocurcumin supplementation reduced the activities of hepatic enzymes, including aspartate aminotransferase and alanine aminotransferase, as well as stress indicators such as glucose, cortisol, and lactate dehydrogenase, both before and after air exposure stress. Immune responses were also enhanced, with higher levels of immunoglobulin M, immunoglobulin G, and complement components 3 and 4 observed in fish fed nanocurcumin-supplemented diets. Overall, dietary nanocurcumin improved growth performance, antioxidant capacity, and innate immunity, while mitigating stress responses in D. labrax, with optimum results obtained at a dietary inclusion level of 900 mg/kg.

Mycobacterium tuberculosis (Mtb) remodels host cell functions to support its persistence within macrophages. While infected cells have been extensively studied, the responses of uninfected bystander macrophages in the same microenvironment remain poorly understood. Here, we demonstrate that Mtb infection triggers broad epigenetic and transcriptional reprogramming in bystander macrophages, predominantly via interleukin-1β-dependent nuclear factor-κB signaling from infected cells. These bystander cells acquire active chromatin marks, exhibit distinct gene expression profiles, and display enhanced responsiveness to subsequent immune challenges. Functionally, bystander macrophages restrict intracellular Mtb growth and also show increased responsiveness to heterologous stimuli resembling trained immunity. Our findings uncover a previously underappreciated mechanism of intercellular communication during infection, wherein Mtb-infected macrophages prime neighboring uninfected cells for enhanced defense. This work defines cytokine-mediated reprogramming of both infected and bystander cell subpopulations, and identifies bystander cells as active participants in shaping the population-wide host immune landscape. These insights have implications for understanding innate immune memory and developing strategies to modulate host defense in tuberculosis and other infections. This study reveals an underappreciated role for uninfected bystander macrophages in host defense against Mycobacterium tuberculosis (Mtb). We demonstrate that Mtb-infected macrophages trigger interleukin-1β-mediated epigenetic training in neighboring bystander cells, priming them for enhanced immune responses. These trained macrophages exhibit heightened antimicrobial activity and restrict Mtb growth upon subsequent infection. By uncovering a mechanism through which immune memory-like responses propagate beyond infected cells, our findings redefine the cellular scope of innate immunity during tuberculosis and identify new opportunities to boost host defense through intercellular signaling and epigenetic reprogramming.

While high-dimensional flow cytometry plays critical roles in resolving complex cellular networks, there remains a scarcity of comprehensive panels for the simultaneous profiling of diverse mouse cell types, primarily due to the technical challenges of spectral overlap and the limited availability of mouse-validated reagents. To address this technical gap and resolve diverse cell populations in murine models, we designed a 27-color flow cytometry panel optimized for 3-laser spectral flow cytometers. This optimized panel enables broad and simultaneous detection of 16 distinct cell subsets from both lymphoid and myeloid lineages-including T cells, B cells, plasma cells, NK cells, innate lymphoid cells, dendritic cells, monocytes, macrophages, neutrophils, eosinophils, basophils, mast cells-along with non-immune cells, such as epithelial, endothelial, fibroblast, and neuronal-associated cells. The panel has been successfully applied to various tissues, including spleen, thymus, bone marrow, peripheral blood, mesenteric lymph nodes, peritoneal lavage fluid, gut epithelium, and lamina propria. Applying this panel to a poly(I:C) model, we successfully tracked systemic shifts in monocyte and neutrophil populations and identified a previously unrecognized, Cyp11b1-expressing immune cell subset via reporter expression. This panel will facilitate high-dimensional immune profiling on standard 3-laser cytometers, providing a robust tool for dissecting cellular landscapes across diverse contexts.

Salmonella enterica serovar Reading has been associated with foodborne disease outbreaks from contaminated poultry products. This study investigated the effect of S. Reading inoculation dose on Salmonella prevalence and cecal loads, and the mRNA abundance of immune response genes in turkey poults, with higher doses expected to increase colonization and elicit stronger immune responses. Day-old poults (n = 487) were assigned to four groups based on S. Reading inoculation doses of 2, 4, 6, and 8-log CFU/mL per bird, with 28 birds per pen and 4 replicate pens per treatment group. Additional poults (n = 39) were assigned to a single pen as the non-inoculated control group. On d 7, all birds were inoculated with their respective dose of S. Reading. On d 9 and d 21, the ceca, liver, and spleen were collected from 10 birds per group to assess S. Reading colonization. Cecal tonsils and spleens were collected to measure the mRNA abundance of immune response genes. On d 14, ceca were collected from 19 birds per pen (76 birds/group) to measure S. Reading loads using the SalQuant&#x2122; procedure of the BAX&#xae; PCR System. Higher doses (6 and 8-log) resulted in increased colonization of S. Reading in the ceca on d 9 (p < 0.001) and d 14 (p < 0.001), and increased prevalence of S. Reading-positive cecal samples on d 9 (p = 0.001) and d 14 (p < 0.001) compared to lower doses. The 8-log S. Reading dose resulted in greater mRNA abundance of cytokines IFN-&#x3b3; (p = 0.007) and IL-10 (p = 0.037), and Toll-like receptors (TLR)-4 (p = 0.017) and TLR-15 (p = 0.002) in the cecal tonsils on d 9 compared to all other groups. Inoculation with higher S. Reading doses resulted in greater prevalence among poults and a heightened local innate immune response.

Bacteriophage T7 RNA polymerase (T7 RNAP) is a key enzyme for in vitro transcription (IVT) and plays a central role in the production of synthetic mRNA for research and therapeutic applications. However, IVT frequently generates double-stranded RNA (dsRNA) as an undesired by-product, which can trigger innate immune responses and compromise mRNA quality. Increasing reaction temperatures reduces dsRNA formation, however, the wild-type T7 RNA polymerase exhibits limited stability under such conditions. Therefore, polymerase variants with enhanced thermotolerance enable more robust transcription at elevated temperatures while minimizing dsRNA generation. To address this limitation, we aimed at obtaining T7 RNA polymerase variants with increased thermotolerance using the Protein Repair One Stop Shop (PROSS) web server. Four crystal structures of T7 RNA polymerase, comprising a promoter complex, an initiation complex, and two elongation complexes were used as input for independent PROSS runs. Mutations shared across all four designs for each PROSS index were then combined to generate multi-structure PROSS Combined Designs (PCDs). In the subset evaluated experimentally, PCD9 retained full-length transcription activity at temperatures up to 48 &#xb0;C, whereas wild-type T7 RNA polymerase showed strong loss of activity under the same buffer conditions. At 48 &#xb0;C, PCD9 supported the synthesis of kilobase-length scale transcripts and produced no detectable dsRNA signal in a dot blot assay. In contrast, the wild-type enzyme generated strong dsRNA signals at 37 &#xb0;C and failed to produce detectable RNA at 48 &#xb0;C. Additional PROSS variants derived exclusively from the elongation complex structure were inactive at both 37 &#xb0;C and 48 &#xb0;C. Together, these results show that multi-structure PROSS design can yield a thermotolerant T7 RNA polymerase with improved performance at elevated temperature and reduced dsRNA byproduct formation. The findings also suggest that restricting stability design to a single structural state may not fully capture the requirements of a highly dynamic enzyme.

Cadmium is an environmentally relevant metal toxicant associated with impaired bone health, but the mechanistic architecture linking cadmium exposure to osteoporosis remains insufficiently organized. We developed a cadmium-centered, adverse outcome pathway (AOP)-guided framework by integrating population epidemiology, toxicogenomics, bulk and single-cell transcriptomics, inferred myeloid pseudotime, and spatial transcriptomics. Survey-weighted analyses of NHANES 2013-2014 and 2017-2018 showed that whole-blood cadmium was positively associated with osteoporosis (odds ratio, 1.43; 95% confidence interval, 1.05-1.95), with an increasing exposure-response pattern. In quantile g-computation using total femur bone mineral density as the outcome, cadmium contributed the largest negative-direction weight. Cadmium-related toxicogenomic evidence from the Comparative Toxicogenomics Database was organized into six biologically interpretable key-event modules. Bulk transcriptomic analyses revealed compartment-skewed module representation in femoral tissue and peripheral monocytes, whereas single-cell analysis localized module scores mainly to stromal and myeloid populations. Myeloid pseudotime analysis identified three associated gene programs reflecting innate defense, interferon activation, and remodeling/lipid handling. Spatial transcriptomics further revealed niche-associated module distributions, trabecula-related gradients, and nonrandom spatial clustering, particularly for bone-remodeling, inflammation/cell-fate, and oxidative stress/mitochondrial modules. These findings provide a spatially contextualized, hypothesis-generating AOP framework that organizes cadmium-osteoporosis association evidence with osteoporosis-relevant molecular and spatial patterns. The proposed relationships require validation in longitudinal exposure studies and experimental models.