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JCI Insight Nov 2023Variation in the preservation of β cell function in clinical trials in type 1 diabetes (T1D) has emphasized the need to define biomarkers to predict treatment response....
Variation in the preservation of β cell function in clinical trials in type 1 diabetes (T1D) has emphasized the need to define biomarkers to predict treatment response. The T1DAL trial targeted T cells with alefacept (LFA-3-Ig) and demonstrated C-peptide preservation in approximately 30% of new-onset T1D individuals. We analyzed islet antigen-reactive (IAR) CD4+ T cells in PBMC samples collected prior to treatment from alefacept- and placebo-treated individuals using flow cytometry and single-cell RNA sequencing. IAR CD4+ T cells at baseline had heterogeneous phenotypes. Transcript profiles formed phenotypic clusters of cells along a trajectory based on increasing maturation and activation, and T cell receptor (TCR) chains showed clonal expansion. Notably, the frequency of IAR CD4+ T cells with a memory phenotype and a unique transcript profile (cluster 3) were inversely correlated with C-peptide preservation in alefacept-treated, but not placebo-treated, individuals. Cluster 3 cells had a proinflammatory phenotype characterized by expression of the transcription factor BHLHE40 and the cytokines GM-CSF and TNF-α, and shared TCR chains with effector memory-like clusters. Our results suggest IAR CD4+ T cells as a potential baseline biomarker of response to therapies targeting the CD2 pathway and warrant investigation for other T cell-related therapies.
Topics: Humans; Diabetes Mellitus, Type 1; CD4-Positive T-Lymphocytes; Alefacept; C-Peptide; Leukocytes, Mononuclear; Biomarkers; Receptors, Antigen, T-Cell
PubMed: 37751304
DOI: 10.1172/jci.insight.167881 -
Molecular Medicine (Cambridge, Mass.) Dec 2023The cGAS-STING signaling pathway is an essential section of the natural immune system. In recent years, an increasing number of studies have shown a strong link between...
BACKGROUND
The cGAS-STING signaling pathway is an essential section of the natural immune system. In recent years, an increasing number of studies have shown a strong link between abnormal activation of the cGAS-STING signaling pathway, a natural immune pathway mediated by the nucleic acid receptor cGAS, and the development and progression of autoimmune diseases. Therefore, it is important to identify an effective compound to specifically downregulate this pathway for disease.
METHODS
The effect of Glabridin (Glab) was investigated in BMDMs and Peripheral blood mononuclear cell (PBMC) by establishing an in vitro model of cGAS-STING signaling pathway activation. An activation model stimulated by DMXAA was also established in mice to study the effect of Glab. On the other hand, we investigated the possible mechanism of action of Glab and the effect of Glab on Trex1-deficient mice.
RESULTS
In this research, we report that Glab, a major component of licorice, specifically inhibits the cGAS-STING signaling pathway by inhibiting the level of type I interferon and inflammatory cytokines (IL-6 and TNF-α). In addition, Glab has a therapeutic effect on innate immune diseases caused by abnormal cytoplasmic DNA in Trex1-deficient mice. Mechanistically, Glab can specifically inhibit the interaction of STING with IRF3.
CONCLUSION
Glab is a specific inhibitor of the cGAS-STING signaling pathway and may be used in the clinical therapy of cGAS-STING pathway-mediated autoimmune diseases.
Topics: Animals; Mice; Autoimmune Diseases; Interferon Type I; Leukocytes, Mononuclear; Nucleotidyltransferases; Isoflavones; Phenols
PubMed: 38066431
DOI: 10.1186/s10020-023-00754-y -
Clinical and Experimental Immunology Dec 2023IL-17A+ CD8+ T-cells, termed Tc17 cells, have been identified at sites of inflammation in several immune-mediated inflammatory diseases. However, the biological function...
IL-17A+ CD8+ T-cells, termed Tc17 cells, have been identified at sites of inflammation in several immune-mediated inflammatory diseases. However, the biological function of human IL-17A+ CD8+ T-cells is not well characterized, likely due in part to the relative scarcity of these cells. Here, we expanded IL-17A+ CD8+ T-cells from healthy donor PBMC or bulk CD8+ T-cell populations using an in vitro polarization protocol. We show that T-cell activation in the presence of IL-1β and IL-23 significantly increased the frequencies of IL-17A+ CD8+ T-cells, which was not further enhanced by IL-6, IL-2, or anti-IFNγ mAb addition. In vitro-generated IL-17A+ CD8+ T-cells displayed a distinct type-17 profile compared with IL-17A- CD8+ T-cells, as defined by transcriptional signature (IL17A, IL17F, RORC, RORA, MAF, IL23R, CCR6), high surface expression of CCR6 and CD161, and polyfunctional production of IL-17A, IL-17F, IL-22, IFNγ, TNFα, and GM-CSF. A significant proportion of in vitro-induced IL-17A+ CD8+ T-cells expressed TCRVα7.2 and bound MR1 tetramers indicative of MAIT cells, indicating that our protocol expanded both conventional and unconventional IL-17A+ CD8+ T-cells. Using an IL-17A secretion assay, we sorted the in vitro-generated IL-17A+ CD8+ T-cells for functional analysis. Both conventional and unconventional IL-17A+ CD8+ T-cells were able to induce pro-inflammatory IL-6 and IL-8 production by synovial fibroblasts from patients with psoriatic arthritis, which was reduced upon addition of anti-TNFα and anti-IL-17A neutralizing antibodies. Collectively, these data demonstrate that human in vitro-generated IL-17A+ CD8+ T-cells are biologically functional and that their pro-inflammatory function can be targeted, at least in vitro, using existing immunotherapy.
Topics: Humans; Interleukin-17; Interleukin-6; Leukocytes, Mononuclear; CD8-Positive T-Lymphocytes; Tumor Necrosis Factor-alpha; Fibroblasts
PubMed: 37367825
DOI: 10.1093/cei/uxad068 -
Frontiers in Immunology 2023Malaria remains a widespread health problem with a huge burden. Severe or complicated malaria is highly lethal and encompasses a variety of pathological processes,...
INTRODUCTION
Malaria remains a widespread health problem with a huge burden. Severe or complicated malaria is highly lethal and encompasses a variety of pathological processes, including immune activation, inflammation, and dysmetabolism. Previously, we showed that adrenal hormones, in particular glucocorticoids (GCs), play critical roles to maintain disease tolerance during infection in mice. Here, GC responses were studied in Cameroon in children with uncomplicated malaria (UM), severe malaria (SM) and asymptomatic controls (AC).
METHODS
To determine the sensitivity of leukocytes to GC signaling on a transcriptional level, we measured the induction of glucocorticoid induced leucine zipper (GILZ) and FK506-binding protein 5 (FKBP5) by GCs in human and murine leukocytes. Targeted tracer metabolomics on peripheral blood mononuclear cells (PBMCs) was performed to detect metabolic changes induced by GCs.
RESULTS
Total cortisol levels increased in patients with clinical malaria compared to AC and were higher in the SM versus UM group, while cortisol binding globulin levels were unchanged and adrenocorticotropic hormone (ACTH) levels were heterogeneous. Induction of both GILZ and FKBP5 by GCs was significantly reduced in patients with clinical malaria compared to AC and in malaria-infected mice compared to uninfected controls. Increased activity in the pentose phosphate pathway was found in the patients, but this was not affected by stimulation with physiological levels of hydrocortisone. Interestingly, hydrocortisone induced increased levels of cAMP in AC, but not in clinical malaria patients.
DISCUSSION
Altogether, this study shows that patients with SM have increased cortisol levels, but also a decreased sensitivity to GCs, which may clearly contribute to the severity of disease.
Topics: Humans; Child; Mice; Animals; Glucocorticoids; Hydrocortisone; Leukocytes, Mononuclear; Receptors, Glucocorticoid; Transcription Factors; Malaria
PubMed: 37492570
DOI: 10.3389/fimmu.2023.1187196 -
Arteriosclerosis, Thrombosis, and... Jan 2024Patients with constitutive activation of DNA-sensing pathway through stimulator of IFN (interferon) genes (STING), such as those with STING-associated vasculopathy with...
BACKGROUND
Patients with constitutive activation of DNA-sensing pathway through stimulator of IFN (interferon) genes (STING), such as those with STING-associated vasculopathy with onset in infancy, develop pulmonary hypertension (PH). However, the role of STING signaling in general PH patients is heretofore undescribed. Here, we seek to investigate the role of STING in PH development.
METHODS
STING expression in patient lung samples was examined. PH was induced in global STING-deficient mice and global type I IFN receptor 1-deficient mice using bleomycin or chronic hypoxia exposure. PH development was evaluated by right ventricular systolic pressure and Fulton index, with additional histological and flow cytometric analysis. VEGF (vascular endothelial growth factor) expression on murine immune cells was quantified and evaluated with multiplex and flow cytometry. Human myeloid-derived cells were differentiated from peripheral blood mononuclear cells and treated with either STING agonist or STING antagonist for evaluation of VEGF secretion.
RESULTS
Global STING deficiency protects mice from PH development, and STING-associated PH seems independent of type I IFN signaling. Furthermore, a role for STING-VEGF signaling pathway in PH development was demonstrated, with altered VEGF secretion in murine pulmonary infiltrated myeloid cells in a STING-dependent manner. In addition, pharmacological manipulation of STING in human myeloid-derived cells supports in vivo findings. Finally, a potential role of STING-VEGF-mediated apoptosis in disease development and progression was illustrated, providing a roadmap toward potential therapeutic applications.
CONCLUSIONS
Overall, these data provide concrete evidence of STING involvement in PH, establishing biological plausibility for STING-related therapies in PH treatment.
Topics: Humans; Animals; Mice; Vascular Endothelial Growth Factor A; Hypertension, Pulmonary; Leukocytes, Mononuclear; Signal Transduction; Interferon Type I
PubMed: 37942608
DOI: 10.1161/ATVBAHA.123.320121 -
Stem Cell Research & Therapy Aug 2023Immunomodulation by mesenchymal stromal cells (MSCs) can occur through trophic factor mechanisms, however, intravenously infused MSCs are rapidly cleared from the body...
BACKGROUND
Immunomodulation by mesenchymal stromal cells (MSCs) can occur through trophic factor mechanisms, however, intravenously infused MSCs are rapidly cleared from the body yet a potent immunotherapeutic response is still observed. Recent work suggests that monocytes contribute to the clearance of MSCs via efferocytosis, the body's natural mechanism for clearing dead and dying cells in a non-inflammatory manner. This begs the questions of how variations in MSC quality affect monocyte phenotype and if viable MSCs are even needed to elicit an immunosuppressive response.
METHODS
Herein, we sought to dissect MSC's trophic mechanism from their efferocytic mechanisms and determine if the viability of MSCs prior to efferocytosis influences the resultant phenotype of monocytes. We cultured viable or heat-inactivated human umbilical cord MSCs with human peripheral blood mononuclear cells for 24 h and observed changes in monocyte surface marker expression and secretion profile. To isolate the effect of efferocytosis from MSC trophic factors, we used cell separation techniques to remove non-efferocytosed MSCs before challenging monocytes to suppress T-cells or respond to inflammatory stimuli. For all experiments, viable and heat-inactivated efferocytic-licensing of monocytes were compared to non-efferocytic-licensing control.
RESULTS
We found that monocytes efferocytose viable and heat-inactivated MSCs equally, but only viable MSC-licensed monocytes suppress activated T-cells and suppression occurred even after depletion of residual MSCs. This provides direct evidence that monocytes that efferocytose viable MSCs are immunosuppressive. Further characterization of monocytes after efferocytosis showed that uptake of viable-but not heat inactivated-MSC resulted in monocytes secreting IL-10 and producing kynurenine. When monocytes were challenged with LPS, IL-2, and IFN-γ to simulate sepsis, monocytes that had efferocytosed viable MSC had higher levels of IDO while monocytes that efferocytosed heat inactivated-MSCs produced the lowest levels of TNF-α.
CONCLUSION
Collectively, these studies show that the quality of MSCs efferocytosed by monocytes polarize monocytes toward distinctive immunosuppressive phenotypes and highlights the need to tailor MSC therapies for specific indications.
Topics: Humans; Monocytes; Leukocytes, Mononuclear; Phagocytosis; Biological Transport; Immunosuppressive Agents; Phenotype
PubMed: 37592321
DOI: 10.1186/s13287-023-03443-z -
Investigative Ophthalmology & Visual... Jul 2023To explore the role of Th2 signaling pathway in allergic conjunctivitis (AC).
PURPOSE
To explore the role of Th2 signaling pathway in allergic conjunctivitis (AC).
METHODS
Serum Th2 cytokines IL-4 or IL-13 of patients with AC were detected using the Meso scale discovery assay to verify the correlation of Th2 immunity and AC pathogenesis. Wistar Han rats were intraperitoneally and subcutaneously injected with ovalbumin (OVA) to establish an experimental AC model and the Th2 signaling pathway was blocked by an investigational neutralizing antibody (CM310). Serum IgE and OVA-specific IgE were detected by ELISA. Conjunctivitis inflammation, infiltration of eosinophils, and mast cell degranulation were detected by histological examination. Immortalized human conjunctival epithelial cells, a conjunctival epithelial cell line, and peripheral blood mononuclear cells of patients with AC were used as the target cells to study the impact of IL-4 or IL-13 on AC progression. Finally, a STAT6 reporter gene system was constructed using immortalized human conjunctival epithelial cells to confirm whether the downstream signaling pathway activated by IL-4 or IL-13.
RESULTS
Serum IL-4 or IL-13 were increased in patients with AC versus healthy individuals. In an OVA-induced rat experimental AC model, blocking the Th2 signaling pathway with CM310, an investigational neutralizing antibody, alleviated the conjunctival symptoms, and decreased serum IgE, suppressed infiltration of eosinophils and mast cell degranulation. Further, an in vitro model showed CM310 suppressed the secretion of inflammatory cytokine from both immune cells and epithelial cells in both patients peripheral blood mononuclear cells and cell line.
CONCLUSIONS
Blocking Th2 signaling pathway alleviates the clinical symptoms and inflammation in AC.
Topics: Humans; Rats; Animals; Mice; Conjunctivitis, Allergic; Interleukin-13; Interleukin-4; Leukocytes, Mononuclear; Rats, Wistar; Inflammation; Immunoglobulin E; Signal Transduction; Cytokines; Ovalbumin; Th2 Cells; Mice, Inbred BALB C; Disease Models, Animal
PubMed: 37486293
DOI: 10.1167/iovs.64.10.30 -
Scientific Reports Aug 2023Recently, TNF receptor type 2 (TNFR2) signaling was found to be involved in the proliferation and activation of regulatory T cells (Tregs), a subpopulation of...
Recently, TNF receptor type 2 (TNFR2) signaling was found to be involved in the proliferation and activation of regulatory T cells (Tregs), a subpopulation of lymphocytes that suppress immune responses. Tregs mediate peripheral immune tolerance, and the disruption of their functions causes autoimmune diseases or allergy. Therefore, cell expanders or regulators of Tregs that control immunosuppressive activity can be used to treat these diseases. We focused on TNFR2, which is preferentially expressed on Tregs, and created tumor necrosis factor-α (TNF-α) muteins that selectively activate TNFR2 signaling in mice and humans, termed R2agoTNF and R2-7, respectively. In this study, we attempted to optimize the structure of muteins to enhance their TNFR2 agonistic activity and stability in vivo by IgG-Fc fusion following single-chain homo-trimerization. The fusion protein, scR2agoTNF-Fc, enhanced the expansion of CD4CD25 Tregs and CD4Foxp3 Tregs and contributed to their immunosuppressive activity ex vivo and in vivo in mice. The prophylactic administration of scR2agoTNF-Fc suppressed inflammation in contact hypersensitivity and arthritis mouse models. Furthermore, scR2-7-Fc preferentially expanded Tregs in human peripheral blood mononuclear cells via TNFR2. These TNFR2 agonist-Fc fusion proteins, which have bivalent structures, are novel Treg expanders.
Topics: Animals; Humans; Mice; Arthritis; Immunosuppressive Agents; Leukocytes, Mononuclear; Receptors, Tumor Necrosis Factor, Type II; T-Lymphocytes, Regulatory; Tumor Necrosis Factor-alpha
PubMed: 37612373
DOI: 10.1038/s41598-023-40925-9 -
Molecular Psychiatry Dec 2023The development of Alzheimer's disease (AD) involves central and peripheral immune deregulation. Gene identification and studies of AD genetic variants of peripheral...
The development of Alzheimer's disease (AD) involves central and peripheral immune deregulation. Gene identification and studies of AD genetic variants of peripheral immune components may aid understanding of peripheral-central immune crosstalk and facilitate new opportunities for therapeutic intervention. In this study, we have identified in a Flanders-Belgian family a novel variant p.E317D in the Toll-like receptor 9 gene (TLR9), co-segregating with EOAD in an autosomal dominant manner. In human, TLR9 is an essential innate and adaptive immune component predominantly expressed in peripheral immune cells. The p.E317D variant caused 50% reduction in TLR9 activation in the NF-κB luciferase assay suggesting that p.E317D is a loss-of-function mutation. Cytokine profiling of human PBMCs upon TLR9 activation revealed a predominantly anti-inflammatory response in contrast to the inflammatory responses from TLR7/8 activation. The cytokines released upon TLR9 activation suppressed inflammation and promoted phagocytosis of Aβ oligomers in human iPSC-derived microglia. Transcriptome analysis identified upregulation of AXL, RUBICON and associated signaling pathways, which may underline the effects of TLR9 signaling-induced cytokines in regulating the inflammatory status and phagocytic property of microglia. Our data suggest a protective role of TLR9 signaling in AD pathogenesis, and we propose that TLR9 loss-of-function may disrupt a peripheral-central immune crosstalk that promotes dampening of inflammation and clearance of toxic protein species, leading to the build-up of neuroinflammation and pathogenic protein aggregates in AD development.
Topics: Humans; Alzheimer Disease; Immunity, Innate; Toll-Like Receptor 9; Male; Female; Cytokines; Microglia; Mutation; Signal Transduction; Aged; Amyloid beta-Peptides; Leukocytes, Mononuclear; Middle Aged; Phagocytosis; Inflammation
PubMed: 37433968
DOI: 10.1038/s41380-023-02166-0 -
Scientific Data Jul 2023Peripheral blood mononuclear cells (PBMCs) are blood cells that are a critical part of the immune system used to fight off infection, defending our bodies from harmful...
Peripheral blood mononuclear cells (PBMCs) are blood cells that are a critical part of the immune system used to fight off infection, defending our bodies from harmful pathogens. In biomedical research, PBMCs are commonly used to study global immune response to disease outbreak and progression, pathogen infections, for vaccine development and a multitude of other clinical applications. Over the past few years, the revolution in single-cell RNA sequencing (scRNA-seq) has enabled an unbiased quantification of gene expression in thousands of individual cells, which provides a more efficient tool to decipher the immune system in human diseases. In this work, we generate scRNA-seq data from human PBMCs at high sequencing depth (>100,000 reads/cell) for more than 30,000 cells, in resting, stimulated, fresh and frozen conditions. The data generated can be used for benchmarking batch correction and data integration methods, and to study the effect of freezing-thawing cycles on the quality of immune cell populations and their transcriptomic profiles.
Topics: Humans; Freezing; Gene Expression Profiling; Immunity; Leukocytes, Mononuclear; Sequence Analysis, RNA; Single-Cell Gene Expression Analysis; Transcriptome
PubMed: 37414801
DOI: 10.1038/s41597-023-02348-z