参考文献

BackgroundThe characteristics of the tumor immunosuppressive microenvironment represent a major challenge that limits the efficacy of immunotherapy. Our previous results suggested that cryo-thermal therapy,a tumor ablation system developed in our laboratory,promotes macrophage M1-type polarization and the complete maturation of DCs to remodel the immunosuppressive environment. However,the cells that respond promptly to CTT have not yet been identified. CTT can cause extensive cell death and the release of danger-associated molecular patterns and antigens. Neutrophils are the first white blood cells recruited to sites of damage and acute inflammation. Therefore,we hypothesized that neutrophils are the initial cells that respond to CTT and are involved in the subsequent establishment of antitumor immunity.MethodsIn this study,we examined the kinetics of neutrophil recruitment after CTT via flow cytometry and immunofluorescence staining and explored the effect of neutrophils on the establishment of systemic antitumor immunity by in vivo neutrophil depletion and in vitro co-culture assays.ResultsWe found that CTT led to a rapid and strong proinflammatory neutrophil response,which was essential for the long-term survival of mice. CTT-induced neutrophils promoted the activation of monocytes via reactive oxygen species and further upregulated the expression of IFN-γ and cytotoxic molecules in T and NK cells. Adoptive neutrophil transfer further enhanced the antitumor efficacy of CTT in tumor models of spontaneous and experimental metastasis.ConclusionThese results reveal the important role of neutrophil‒monocyte interactions in the development of anti-tumor immunity and highlight that CTT could be used as an immunotherapy for targeting neutrophils and monocytes to enhance antitumor immunity. View Publication

BackgroundSepsis survivors exhibit immune dysregulation that contributes to poor long-term outcomes. Phenotypic and functional alterations within the myeloid compartment are believed to be a contributing factor. Here we dissect the cellular and transcriptional heterogeneity of splenic CD11b+Ly6Chigh myeloid cells that are expanded in mice that survive the cecal ligation and puncture (CLP) murine model of polymicrobial sepsis to better understand the basis of immune dysregulation in sepsis survivors.MethodsSham or CLP surgeries were performed on C57BL/6J and BALB/c mice. Four weeks later splenic CD11b+Ly6Chigh cells from both groups were isolated for phenotypic (flow cytometry) and functional (phagocytosis and glycolysis) characterization and RNA was obtained for single-cell RNA-seq (scRNA-seq) and subsequent analysis.ResultsCD11b+Ly6Chigh cells from sham and CLP surviving mice exhibit phenotypic and functional differences that relate to immune function,some of which are observed in both C57BL/6J and BALB/c strains and others that are not. To dissect disease-specific and strain-specific distinctions within the myeloid compartment,scRNA-seq analysis was performed on CD11b+Ly6Chigh cells from C57BL/6J and BALB/c sham and CLP mice. Uniform Manifold Approximation and Projection from both strains identified 13 distinct clusters of sorted CD11b+Ly6Chigh cells demonstrating significant transcriptional heterogeneity and expressing gene signatures corresponding to classical-monocytes,non-classical monocytes,M1- or M2-like macrophages,dendritic-like cells,monocyte-derived dendritic-like cells,and proliferating monocytic myeloid-derived suppressor cells (M-MDSCs). Frequency plots showed that the percentages of proliferating M-MDSCs (clusters 8,11 and 12) were increased in CLP mice compared to sham mice in both strains. Pathway and UCell score analysis in CLP mice revealed that cell cycle and glycolytic pathways were upregulated in proliferating M-MDSCs in both strains. Notably,granule protease genes were upregulated in M-MDSCs from CLP mice. ScRNA-seq analyses also showed that phagocytic pathways were upregulated in multiple clusters including the classical monocyte cluster,confirming the increased phagocytic capacity in CD11b+Ly6Chigh cells from CLP mice observed in ex vivo functional assays in C57BL/6J mice.ConclusionThe splenic CD11b+Ly6Chigh myeloid populations expanded in survivors of CLP sepsis correspond to proliferating cells that have an increased metabolic demand and gene signatures consistent with M-MDSCs,a population known to have immunosuppressive capacity.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-024-00970-0. View Publication

While prostaglandin E2 (PGE2) is produced in human tumor microenvironment (TME),its role therein remains poorly understood. Here,we examine this issue by comparative single-cell RNA sequencing of immune cells infiltrating human cancers and syngeneic tumors in female mice. PGE receptors EP4 and EP2 are expressed in lymphocytes and myeloid cells,and their expression is associated with the downregulation of oxidative phosphorylation (OXPHOS) and MYC targets,glycolysis and ribosomal proteins (RPs). Mechanistically,CD8+ T cells express EP4 and EP2 upon TCR activation,and PGE2 blocks IL-2-STAT5 signaling by downregulating Il2ra,which downregulates c-Myc and PGC-1 to decrease OXPHOS,glycolysis,and RPs,impairing migration,expansion,survival,and antitumor activity. Similarly,EP4 and EP2 are induced upon macrophage activation,and PGE2 downregulates c-Myc and OXPHOS in M1-like macrophages. These results suggest that PGE2-EP4/EP2 signaling impairs both adaptive and innate immunity in TME by hampering bioenergetics and ribosome biogenesis of tumor-infiltrating immune cells. Mechanisms of prostaglandin E2 (PGE2)-mediated immunosuppression in the tumor microenvironment (TME) have been previously reported. Here,the authors profile PGE2 functions in human cancer,suggesting that prostaglandin E2-mediated signaling impairs the activity of human CD8+ T cells and macrophages by altering bioenergetics and ribosome biogenesis. View Publication

Acquired immunodeficiency syndrome (AIDS) occurs when HIV depletes CD4+ helper T cells. Some patients develop AIDS slowly or not at all,and are termed long-term non-progressors (LTNP),and while mutations in the HIV-1 Viral Protein R (vpr) gene such as R77Q are associated with LTNP,mechanisms for this correlation are unclear. This study examines the induction of apoptosis,cell cycle arrest,and pro-inflammatory cytokine release in the HUT78 T cell line following infection with replication-competent wild-type strain NL4-3,the R77Q mutant,or a vpr Null mutant. Our results show a significant enhancement of apoptosis and G2 cell cycle arrest in HUT78 cells infected with R77Q,but not with WT NL4-3 or the vpr Null strain. Conversely,HUT78 cells infected with the WT virus show higher levels of necrosis. We also detected lower TNF and IL-6 release after infection with R77Q vs. WT. The apoptotic phenotype was also seen in the CEM cell line and in primary CD4+ T cells. Protein expression of the R77Q vpr variant was low compared to WT vpr,but expression levels alone cannot explain these phenotypes because the Null virus did not show apoptosis or G2 arrest. These results suggest that R77Q triggers a non-inflammatory apoptotic pathway that attenuates inflammation,possibly contributing to LTNP. View Publication

Natural adjuvants have recently garnered interest in the field of vaccinology as their immunostimulatory effects. In this study,we aimed to investigate the potential use of Peyssonnelia caulifera (PC),a marine alga,as a natural adjuvant for an inactivated split A/Puerto Rico/8/1934 H1N1 influenza vaccine (sPR8) in a murine model. We administered PC-adjuvanted vaccines to a murine model via intramuscular prime and boost vaccinations,and subsequently analyzed the induced immunological responses,particularly the production of antigen-specific IgG1 and IgG2a antibodies,memory T and B cell responses,and the protective efficacy against a lethal viral infection. PC extract significantly bolstered the vaccine efficacy,demonstrating balanced Th1/Th2 responses,increased memory T and B cell activities,and improved protection against viral infection. Notably,within 3 days post-vaccination,the PC adjuvant stimulated activation markers on dendritic cells (DCs) and macrophages at the inguinal lymph nodes (ILN),emphasizing its immunostimulatory capabilities. Furthermore,the safety profile of PC was confirmed,showing minimal local inflammation and no significant adverse effects post-vaccination. These findings contribute to our understanding of the immunomodulatory properties of natural adjuvants and suggest the promising roles of natural adjuvants in the development of more effective vaccines for infectious diseases.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-76736-9. View Publication

Background/Objectives: Asthma is a chronic inflammatory disorder of the airways affecting over 10% of the global population. It is characterized by airway inflammation,mucus hypersecretion,and bronchial hyperresponsiveness,driven predominantly by type 2 helper T cells (Th2) and type 2 innate lymphoid cells (ILC2s) in a subset of patients. However,a significant portion of asthmatic individuals present with “type 2-low” asthma that is often refractory to standard inhaled corticosteroid (ICS) therapy. Therefore,developing innovative therapeutic strategies has become essential. Recent studies have highlighted cannabidiol (CBD) as a promising anti-inflammatory agent capable of modulating immune responses. This study investigates the therapeutic potential of a high-CBD extract (CBD-X) in asthma. Methods: We evaluated the effects of CBD-X on cells involved in asthma pathogenesis using primary human Th2 cells,neutrophils,and asthma mouse model. Results: Our findings indicate that CBD-X extract inhibits Th2 differentiation and reduces the secretion of IL-5 and IL-13,which are crucial cytokines in asthma. Additionally,CBD-X significantly reduces pro-inflammatory cytokines IL-8 and IL-6 in neutrophils and impairs their migration,a critical step in airway inflammation. In a murine asthma model,CBD-X administration led to marked downregulation of IgE and pro-asthmatic cytokines,along with reduced leukocyte,eosinophil,and neutrophil infiltration in lung tissues. Conclusions: These results suggest that CBD-X extract could offer a novel and complementary approach to managing both type 2-high and type 2-low asthma by targeting key inflammatory pathways and modulating immune cell behavior. View Publication

Inflammatory bowel disease (IBD),encompassing Crohn’s disease (CD) and ulcerative colitis (UC),is a chronic inflammatory condition of the gut affecting both adults and children. Neutrophil extracellular traps (NETs) are structures released by activated neutrophils,potentially contributing to tissue damage in various diseases. This study aimed to explore the presence and role of NETs in pediatric IBD. We compared intestinal biopsies and peripheral blood from 20 pediatric IBD patients (UC and CD) to controls. Biopsy staining and techniques for neutrophil activation were used to assess neutrophil infiltration and NET formation. We also measured the enzymatic activity of key NET proteins and evaluated NET formation in UC patients in remission. Both UC and CD biopsies showed significantly higher levels of neutrophils and NETs compared to controls (p < 0.01),with UC exhibiting the strongest association. Peripheral blood neutrophils from UC patients at diagnosis displayed increased NET formation compared to controls and CD patients. Interestingly,NET formation normalized in UC patients following remission-inducing treatment. This pilot study suggests a potential role for NETs in pediatric IBD,particularly UC. These findings warrant further investigation into the mechanisms of NET involvement and the potential for targeting NET formation as a therapeutic strategy. View Publication

In the pathophysiology of osteoarthritis and osteoporosis,articular cartilage and bone represent the target tissues,respectively,but muscle is also involved. Since many changes in energy metabolism occur in muscle with aging,the aim of the present work was to investigate the involvement of carnitine palmitoyl transferase 1b (Cpt1b) in the muscle pathophysiology of the two diseases. Healthy subjects (CTR,n = 5),osteoarthritic (OA,n = 10),and osteoporotic (OP,n = 10) patients were enrolled. Gene expression analysis conducted on muscle and myoblasts showed up-regulation of CPT1B in OA patients; this result was confirmed by immunohistochemical and immunofluorescence analyses and enzyme activity assay,which showed increased Cpt1b activity in OA muscle. In addition,CPT1B expression resulted down-regulated in cultured OP myoblasts. Given the potential involvement of Cpt1b in the modulation of oxidative stress,we investigated ROS levels,which were found to be lower in OA myoblasts,and gene expression of nicotinamide adenine dinucleotide phosphate hydrogen oxidase 4 (Nox4),which resulted up-regulated in OA cells. Finally,the immunofluorescence of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) showed a decreased expression in OP myoblasts,with respect to CTR and OA. Contextually,through an ultrastructural analysis conducted by Transmission Electron Microscopy (TEM),the presence of aberrant mitochondria was observed in OP muscle. This study highlights the potential role of Cpt1b in the regulation of muscle homeostasis in both osteoarthritis and osteoporosis,allowing for the expansion of the current knowledge of what are the molecular biological pathways involved in the regulation of muscle physiology in both diseases. View Publication

Introduction: The maintenance of intestinal homeostasis depends on a complex interaction between the immune system,intestinal epithelial barrier,and microbiota. Alteration in one of these components could lead to the development of inflammatory bowel diseases (IBD). Variants within the autophagy gene ATG16L1 have been implicated in susceptibility and severity of Crohn's disease (CD). Individuals carrying the risk ATG16L1 T300A variant have higher caspase 3-dependent degradation of ATG16L1 resulting in impaired autophagy and increased cellular stress. ATG16L1-deficiency induces enhanced IL-1β secretion in dendritic cells in response to bacterial infection. Infection of ATG16L1-deficient mice with a persistent strain of murine norovirus renders these mice highly susceptible to dextran sulfate sodium colitis. Moreover,persistent norovirus infection leads to intestinal virus specific CD8+ T cells responses. Both Toll-like receptor 7 (TLR7),which recognizes single-stranded RNA viruses,and ATG16L1,which facilitates the delivery of viral nucleic acids to the autolysosome endosome,are required for anti-viral immune responses. Results and discussion: However,the role of the enteric virome in IBD is still poorly understood. Here,we investigate the role of TLR7 and ATG16L1 in intestinal homeostasis and inflammation. At steady state,Tlr7-/- mice have a significant increase in large intestinal lamina propria (LP) granzyme B+ tissue-resident memory CD8+ T (TRM) cells compared to WT mice,reminiscent of persistent norovirus infection. Deletion of Atg16l1 in myeloid (Atg16l1ΔLyz2 ) or dendritic cells (Atg16l1ΔCd11c ) leads to a similar increase of LP TRM. Furthermore,Tlr7-/- and Atg16l1ΔCd11c mice were more susceptible to dextran sulfate sodium colitis with an increase in disease activity index,histoscore,and increased secretion of IFN-γ and TNF-α. Treatment of Atg16l1ΔCd11c mice with the TLR7 agonist Imiquimod attenuated colonic inflammation in these mice. Our data demonstrate that ATG16L1-deficiency in myeloid and dendritic cells leads to an increase in LP TRM and consequently to increased susceptibility to colitis by impairing the recognition of enteric viruses by TLR7. Conclusion: In conclusion,the convergence of ATG16L1 and TLR7 signaling pathways plays an important role in the immune response to intestinal viruses. Our data suggest that activation of the TLR7 signaling pathway could be an attractive therapeutic target for CD patients with ATG16L1 risk variants. View Publication

The development and progression of chronic lymphocytic leukemia (CLL) depend on genetic abnormalities and on the immunosuppressive microenvironment. We have explored the possibility that genetic drivers might be responsible for the immune cell dysregulation that shapes the protumor microenvironment. We performed a transcriptome analysis of coding and non-coding RNAs (ncRNAs) during leukemia progression in the Rag2−/−γc−/− MEC1-based xenotransplantation model. The DLEU2/miR-16 locus was found downmodulated in monocytes/macrophages of leukemic mice. To validate the role of this cluster in the tumor immune microenvironment,we generated a mouse model that simultaneously mimics the overexpression of hTCL1 and the germline deletion of the minimal deleted region (MDR) encoding the DLEU2/miR-15a/miR-16-1 cluster. This model provides an innovative and faster CLL system where monocyte differentiation and macrophage polarization are exacerbated,and T-cells are dysfunctional. MDR deletion inversely correlates with the levels of predicted target proteins including BCL2 and PD1/PD-L1 on murine CLL cells and immune cells. The inverse correlation of miR-15a/miR-16-1 with target proteins has been confirmed on patient-derived immune cells. Forced expression of miR-16-1 interferes with monocyte differentiation into tumor-associated macrophages,indicating that selected ncRNAs drive the protumor phenotype of non-malignant immune cells. View Publication