参考文献

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

IntroductionThe 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 discussionHowever,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.ConclusionIn 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

AbstractMyeloproliferative neoplasms (MPNs) are characterized by an increased production of blood cells due to the acquisition of mutations such as JAK2V617F. TGF‐β,whose secretion is increased in MPN patients,is known to negatively regulate haematopoietic stem cell (HSC) proliferation. Using an isogenic JAK2V617F or JAK2 wild‐type UT‐7 cell line we observed that JAK2V617F cells resist to TGF‐β antiproliferative activity. Although TGF‐β receptors and SMAD2/3 expressions are similar in both cell types,TGF‐β‐induced phosphorylation of SMAD2/3 is reduced in UT‐7 JAK2V617F cells compared with JAK2 WT cells. We confirmed that JAK2V617F mutated cells are resistant to the antiproliferative effect of TGF‐β in a competitive assay as we observed a positive selection of JAK2V617F cells when exposed to TGF‐β. Using cell lines,CD34‐positive cells from MPN patients and bone marrow cells from JAK2V617F knock‐in mice we identified a down regulation of the SHP‐1 phosphatase,which is required for the regulation of HSC quiescence by TGF‐β. The transduction of SHP‐1 cDNA (but not a phosphatase inactive cDNA) restores the antiproliferative effect of TGF‐β in JAK2V617F mutated cells. Finally,SC‐1,a known agonist of SHP‐1,antagonized the selection of JAK2V617F mutated cells in the presence of TGF‐β. In conclusion,we show a JAK2‐dependent down regulation of SHP‐1 in MPN patients' cells which is related to their resistance to the antiproliferative effect of TGF‐β. This may participate in the clonal selection of cancer cells in MPNs. View Publication

While adoptive cell therapies (ACT) have been successful as therapies for blood cancers,they have limited efficacy in treating solid tumours,where the tumour microenvironment excludes and suppresses adoptively transferred tumour-specific immune cells. A major obstacle to improving cell therapies for solid tumours is a lack of accessible and quantitative imaging modalities capable of tracking the migration and immune functional activity of ACT products for an extended duration in vivo.Methods: A high-efficiency magnetophoretic method was developed for facile magnetic labelling of hard-to-label immune cells,which were then injected into tumour-bearing mice and imaged over two weeks with a compact benchtop Magnetic Particle Imager (MPI) design.Results: Labelling efficiency was improved more than 10-fold over prior studies enabling longer-term tracking for at least two weeks in vivo of the labelled immune cells and their biodistribution relative to the tumour. The new imager showed 5-fold improved throughput enabling much larger density of data (up to 20 mice per experiment).Conclusions: Taken together,our innovations enable the convenient and practical use of MPI to visualise the localisation of ACT products in in vivo preclinical models for longitudinal,non-invasive functional evaluation of therapeutic efficacy. View Publication

Sensory neurons sense pathogenic infiltration to drive innate immune responses,but their role in humoral immunity is unclear. Here,using mouse models of Streptococcus pneumoniae infection and Alternaria alternata asthma,we show that sensory neurons are required for B cell recruitment and antibody production. In response to S. pneumoniae,sensory neuron depletion increases bacterial burden and reduces B cell numbers,IgG release,and neutrophil stimulation. Meanwhile,during A. alternata-induced airway inflammation,sensory neuron depletion decreases B cell population sizes,IgE levels,and asthmatic characteristics. Mechanistically,during bacterial infection,sensory neurons preferentially release vasoactive intestinal polypeptide (VIP). In response to asthma,sensory neurons release substance P. Administration of VIP into sensory neuron-depleted mice suppresses bacterial burden,while VIPR1 deficiency increases infection. Similarly,exogenous substance P delivery aggravates asthma in sensory neuron-depleted mice,while substance P deficiency ameliorates asthma. Our data,thus demonstrate that sensory neurons release select neuropeptides which target B cells dependent on the immunogen. Sensory neurons may regulate innate immune cells,but their roles in humoral immunity is still unclear. Here the authors show that bacterial infection and asthma induction induce sensory neuron production of distinct neurotransmitters to dampen B cell responses but differentially target IgG and IgE,respectively,to specifically modulate the symptoms. View Publication

Neutrophil dysfunction is a form of immune suppression in patients with β-thalassemia (Beta-thal),although data on this are limited. In this study,blood from patients and healthy volunteers was analyzed. Flow cytometry analysis demonstrated an increase in immature neutrophils (CD16− CD62L+) and aged (senescent) neutrophils (CD16+ CD62L−) in Beta-thal patients compared to healthy volunteers. The Beta-thal neutrophils demonstrated less prominent chemotaxis and phagocytosis than healthy neutrophils at the baseline. With phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) stimulations,some of the indicators,including the flow cytometry markers (CD11b,CD62L,CD66b,CD63,apoptosis,and reactive oxygen species) and neutrophil extracellular traps (NETs; detected by anti-citrullinated histone 3 immunofluorescence),were lower than the control. Additionally,low-density neutrophils (LDNs),which are found in the peripheral blood mononuclear cell (PBMC) fraction,were observed in Beta-thal patients but not in the control group. The expression of CD11b,CD66b,CD63,arginase I,and ROS in LDNs was higher than the regular normal-density neutrophils (NDNs). The proliferation rate of CD3+ T cells isolated from the PBMC fraction of healthy volunteers was higher than that of the cells from patients with Beta-thal. The incubation of red blood cell (RBC) lysate plus ferric ions with healthy NDNs transformed the NDNs into the aged neutrophils (decreased CD62L) and LDNs. In conclusion,iron overload induces neutrophil diversity along with some dysfunctions. View Publication

BackgroundActivated fibroblast-like synoviocytes (FLS) are drivers of synovitis and structural joint damage in rheumatoid arthritis (RA). Despite the use of disease-modifying drugs,only about 50% of RA patients reach remission in real-world settings. We used an unbiased approach to investigate the effects of standard-of-care methotrexate (MTX) and a Janus kinase inhibitor,tofacitinib (TOFA),on gene expression in RA-FLS,in order to identify untargeted disease mediators.MethodsPrimary RA-FLS were activated by stimulation with interleukin-1β (IL-1β) or platelet-derived growth factor + IL-1β in the presence or absence of MTX or TOFA,with or without additional inhibitors. Co-cultures of synovial cells were performed in direct and indirect systems. Cells were collected for RNA sequencing or qPCR,and supernatants were analyzed for protein concentrations.ResultsSix thousand three hundred fifty genes were differentially expressed,the majority being upregulated,in MTX-treated activated RA-FLS and 970 genes,the majority being downregulated,in TOFA-treated samples. Pathway analysis showed that MTX had largest effects on ‘Molecular mechanisms of cancer’ and TOFA on ‘Interferon signaling’. Targeted analysis of disease-associated genes revealed that MTX increased the expression of cell cycle-regulating genes but also of pro-inflammatory mediators like IL-1α (IL1A) and granulocyte–macrophage colony-stimulating factor,GM-CSF (CSF2). The MTX-promoted expression of CSF2 in activated RA-FLS peaked at 48 h,could be mediated via either NF-κB or AP-1 transcription factors,and was abrogated by IL-1 inhibitors (IRAK4 inhibitor and anakinra). In a co-culture setting,MTX-treatment of activated RA-FLS induced IL1B expression in macrophages.ConclusionsMTX treatment induces secretion of IL-1 from activated RA-FLS which by autocrine signaling augments their release of GM-CSF. This unexpected effect of MTX might contribute to the persistence of synovitis. Supplementary InformationThe online version contains supplementary material available at 10.1186/s13075-024-03406-6. View Publication