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Cancer Immunology, Immunotherapy : CII Jul 2022Monocyte-derived Dendritic cells (DCs) have successfully been employed to induce immune responses against tumor-associated antigens in patients with various cancer...
Monocyte-derived Dendritic cells (DCs) have successfully been employed to induce immune responses against tumor-associated antigens in patients with various cancer entities. However, objective clinical responses have only been achieved in a minority of patients. Additionally, generation of GMP-compliant DCs requires time- and labor-intensive cell differentiation. In contrast, Blood DCs (BDCs) require only minimal ex vivo handling, as differentiation occurs in vivo resulting in potentially better functional capacities and survival. We aimed to identify a protocol for optimal in vitro activation of BDCs including the three subsets pDCs, cDC1s, and cDC2s. We evaluated several TLR ligand combinations and demonstrated that polyinosinic:polycytidylic acid [poly(I:C)] and R848, ligands for TLR3 and TLR7/8, respectively, constituted the optimal combination for inducing a positive co-stimulatory profile in all BDC subsets. In addition, TLR3 and TLR7/8 activation led to high secretion of IFN-α and IL-12p70. Simultaneous as opposed to separate tailored activation of pDCs and cDCs increased immunostimulatory capacities, suggesting that BDC subsets engage in synergistic cross-talk during activation. Stimulation of BDCs with this protocol resulted in enhanced migration, high NK-cell activation, and potent antigen-specific T-cell induction.We conclude that simultaneous activation of all BDC subsets with a combination of R848 + poly(I:C) generates highly immunostimulatory DCs. These results support further investigation and clinical testing, as standalone or in conjunction with other immunotherapeutic strategies including adoptive T-cell transfer and checkpoint inhibition.
Topics: Antigens, Neoplasm; Antigens, Viral; Dendritic Cells; Humans; Lymphocyte Activation; Poly I-C; Toll-Like Receptor 3; Toll-Like Receptor 7; Toll-Like Receptor 8
PubMed: 34821951
DOI: 10.1007/s00262-021-03109-w -
Respiratory Research Apr 2021Asthma has been associated with impaired interferon response. Multiple cell types have been implicated in such response impairment and may be responsible for asthma...
BACKGROUND
Asthma has been associated with impaired interferon response. Multiple cell types have been implicated in such response impairment and may be responsible for asthma immunopathology. However, existing models to study the immune response in asthma are limited by bulk profiling of cells. Our objective was to Characterize a model of peripheral blood mononuclear cells (PBMCs) of patients with severe asthma (SA) and its response to the TLR3 agonist Poly I:C using two single-cell methods.
METHODS
Two complementary single-cell methods, DropSeq for single-cell RNA sequencing (scRNA-Seq) and mass cytometry (CyTOF), were used to profile PBMCs of SA patients and healthy controls (HC). Poly I:C-stimulated and unstimulated cells were analyzed in this study.
RESULTS
PBMCs (n = 9414) from five SA (n = 6099) and three HC (n = 3315) were profiled using scRNA-Seq. Six main cell subsets, namely CD4 + T cells, CD8 + T cells, natural killer (NK) cells, B cells, dendritic cells (DCs), and monocytes, were identified. CD4 + T cells were the main cell type in SA and demonstrated a pro-inflammatory profile characterized by increased JAK1 expression. Following Poly I:C stimulation, PBMCs from SA had a robust induction of interferon pathways compared with HC. CyTOF profiling of Poly I:C stimulated and unstimulated PBMCs (n = 160,000) from the same individuals (SA = 5; HC = 3) demonstrated higher CD8 + and CD8 + effector T cells in SA at baseline, followed by a decrease of CD8 + effector T cells after poly I:C stimulation.
CONCLUSIONS
Single-cell profiling of an in vitro model using PBMCs in patients with SA identified activation of pro-inflammatory pathways at baseline and strong response to Poly I:C, as well as quantitative changes in CD8 + effector cells. Thus, transcriptomic and cell quantitative changes are associated with immune cell heterogeneity in this model to evaluate interferon responses in severe asthma.
Topics: Adult; Asthma; Case-Control Studies; Cells, Cultured; Female; Flow Cytometry; Gene Expression Profiling; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Phenotype; Poly I-C; RNA-Seq; Severity of Illness Index; Single-Cell Analysis; Time Factors; Transcriptome; Young Adult
PubMed: 33902571
DOI: 10.1186/s12931-021-01709-9 -
Journal of Immunology Research 2020Mast cells (MCs) are found mainly at the anatomical sites exposed to the external environment; thus, they are localized close to blood vessels, lymphatic vessels, and a...
Mast cells (MCs) are found mainly at the anatomical sites exposed to the external environment; thus, they are localized close to blood vessels, lymphatic vessels, and a multitude of immune cells. Moreover, those cells can recognize invading pathogens through a range of surface molecules known as pathogen recognition receptors (PRRs), mainly Toll-like receptors (TLRs). MCs are extensively engaged in the control and clearance of bacterial infections, but much less is known about their contribution to antiviral host response as well as pathomechanisms of virus-induced diseases. In the study, we employed differentiated mature tissue mast cells freshly isolated from rat peritoneal cavity. Here, we demonstrated that rat peritoneal mast cells (rPMCs) express viral dsRNA-specific TLR3 molecule (intracellularly and on the cell surface) as well as other proteins associated with cellular antiviral response: IRF3, type I and II IFN receptors, and MHC I. We found that exposure of rPMCs to viral dsRNA mimic, i.e., poly(I:C), induced transient upregulation of surface TLR3 (while temporarily decreased TLR3 intracellular expression), type II IFN receptor, and MHC I. TLR3 ligand-stimulated rPMCs did not degranulate but generated and/or released type I IFNs (IFN- and IFN) as well as proinflammatory lipid mediators (cysLTs), cytokines (TNF, IL-1), and chemokines (CCL3, CXCL8). We documented that rPMC priming with poly(I:C) did not affect FcRI-dependent degranulation. However, their costimulation with TLR3 agonist and anti-IgE led to a significant increase in cysLT and TNF secretion. Our findings confirm that MCs may serve as active participants in the antiviral immune response. Presented data on modulated FcRI-mediated MC secretion of mediators upon poly(I:C) treatment suggests that dsRNA-type virus infection could influence the severity of allergic reactions.
Topics: Animals; Cell Degranulation; Cells, Cultured; Cytokines; Female; Hypersensitivity; Mast Cells; Poly I-C; RNA Virus Infections; RNA Viruses; RNA, Double-Stranded; Rats; Rats, Wistar; Receptors, IgE; Toll-Like Receptor 3
PubMed: 32185237
DOI: 10.1155/2020/2140694 -
International Journal of Molecular... Aug 2022Poly (I:C) can work as an immunostimulant and a viral vaccine; however, its functional mechanism in aquatic animals needs to be further investigated. In this study,...
Poly (I:C) can work as an immunostimulant and a viral vaccine; however, its functional mechanism in aquatic animals needs to be further investigated. In this study, comparative transcriptomic analyses were performed to investigate the effects of poly (I:C) on at 12 h and 48 h postinjection. A total of 194 and 294 differentially expressed genes were obtained in the liver and spleen, respectively. At 12 h, poly (I:C) injection could significantly influence the function of the metabolism-related pathways and immune-related pathways in the liver through the upregulation of the genes , , , , and , and the downregulation of the genes , , , and . At 48 h, poly (I:C) could enhance the liver energy metabolism by upregulating the genes and , while it also induced some injury in the cells with the downregulation of the genes and . In the spleen, poly (I:C) could regulate the fish immunity and inflammatory response by upregulating the genes , , , and , and by downregulating the genes , , , , , and at 12 h, and at 48 h, the poly (I:C) had a similar influence as that in the liver. Intersection analyses demonstrated that and were the main functional genes that contributed to the health of the liver. Ten and four genes participated in maintaining the health of the two tissues after 12 h and 48 h, respectively. In summary, our results provided a new insight into ploy (I:C) application in , and it also helped us to better understand the fish response mechanism to the viral vaccine injection.
Topics: Animals; Liver; Perciformes; Poly I-C; Spleen; Transcriptome; Viral Vaccines; Vitamin D3 24-Hydroxylase
PubMed: 36077207
DOI: 10.3390/ijms23179801 -
Viruses Jan 2022SARS-CoV-2, a member of the coronavirus family, is the causative agent of the COVID-19 pandemic. Currently, there is still an urgent need in developing an efficient...
SARS-CoV-2, a member of the coronavirus family, is the causative agent of the COVID-19 pandemic. Currently, there is still an urgent need in developing an efficient therapeutic intervention. In this study, we aimed at evaluating the therapeutic effect of a single intranasal treatment of the TLR3/MDA5 synthetic agonist Poly(I:C) against a lethal dose of SARS-CoV-2 in K18-hACE2 transgenic mice. We demonstrate here that early Poly(I:C) treatment acts synergistically with SARS-CoV-2 to induce an intense, immediate and transient upregulation of innate immunity-related genes in lungs. This effect is accompanied by viral load reduction, lung and brain cytokine storms prevention and increased levels of macrophages and NK cells, resulting in 83% mice survival, concomitantly with long-term immunization. Thus, priming the lung innate immunity by Poly(I:C) or alike may provide an immediate, efficient and safe protective measure against SARS-CoV-2 infection.
Topics: Angiotensin-Converting Enzyme 2; Animals; COVID-19; Cytokine Release Syndrome; Disease Models, Animal; Female; Humans; Immunity, Innate; Lung; Mice; Mice, Transgenic; Poly I-C; SARS-CoV-2; Toll-Like Receptor 3; Viral Load; COVID-19 Drug Treatment
PubMed: 35215785
DOI: 10.3390/v14020189 -
Advanced Healthcare Materials Nov 2022Here, antigen and adjuvant encapsulated dendritic cell-targeted nanoparticles for immune activation in the small intestinal lymphatic system to inhibit melanoma...
Here, antigen and adjuvant encapsulated dendritic cell-targeted nanoparticles for immune activation in the small intestinal lymphatic system to inhibit melanoma development are described. This strategy is demonstrated using chondroitin sulfate-coated nanoparticles (OPGMN) grafted with glycocholic acid and mannose for cationic liposomes encapsulated with ovalbumin as an antigen and polyinosine-polycytidylic acid as a cancer-specific adjuvant. OPGMN is absorbed in the gastrointestinal tract and delivered to the lymph nodes when orally administered. Oral delivery of OPGMN induces increased dendritic cell maturation compared to the intradermal route in the lymph node and induces T helper type 1 and type 2 responses, such as immunoglobulin G1 and G2c, interferon-gamma, and interleukin-2, in the blood. Repeated oral administration of OPGMN increases the population of CD3 CD8 T cells, CD44 CD62L memory T cells, and CD11b CD27 natural killer cells in the blood. OPGMN completely prevents melanoma development in the B16F10-bearing C57BL/6 mouse model by reducing the population of CD4 CD25 Foxp3 regulatory T cells in the blood. This strategy is expected to prevent the recurrence of tumors after various cancer treatments.
Topics: Mice; Animals; Ovalbumin; Poly I-C; CD8-Positive T-Lymphocytes; Mice, Inbred C57BL; Dendritic Cells; Nanoparticles; Antigens; Adjuvants, Immunologic; Lymph Nodes; Melanoma
PubMed: 35835068
DOI: 10.1002/adhm.202200909 -
Journal For Immunotherapy of Cancer Aug 2019Efficient cancer therapy is sought not only for primary tumor treatment but also for the prevention of metastatic cancer growth. Immunotherapy has been shown to prevent...
BACKGROUND
Efficient cancer therapy is sought not only for primary tumor treatment but also for the prevention of metastatic cancer growth. Immunotherapy has been shown to prevent cancer metastasis by inducing antigen-specific immune responses. Indocyanine green (ICG) has a peak spectral absorption at about 800 nm, which makes it a photothermal reagent for direct treatment of solid tumors by photothermal therapy (PTT). Since PTT alone cannot fully induce antigen-specific immune response for prevention of cancer metastasis, the combination of PTT and immunotherapy has been developed as a new strategy of cancer treatment.
METHODS
Thermal responsive liposomes (TRL) were synthesized by incorporating ICG into the lipid bilayer and encapsulating the water-soluble immune stimulatory molecule polyinosinic:polycytidylic acid (poly I:C) in the hydrophilic core. The poly I:C- and ICG-containing TRLs (piTRLs) were analyzed according to size, and their photothermal effect was evaluated following laser irradiation at 808 nm. Moreover, the temperature-dependent release of poly I:C was also measured. For cancer therapy, CT-26 (carcinoma) and B16 (melanoma) cells were subcutaneously inoculated to build the 1st transplanted tumor in BALB/c and C57BL/6 mice, respectively. These mice received a 2nd transplantation with the same cancer cells by intravenous inoculation, for evaluation of the anti-metastatic effects of the liposomes after PTT.
RESULTS
Near-infrared (NIR) laser irradiation increased the temperature of piTRLs and effectively released poly I:C from the liposomes. The increased temperature induced a photothermal effect, which promoted cancer cell apoptosis and dissolution of the 1st transplanted tumor. Moreover, the released poly I:C from the piTRL induced activation of dendritic cells (DCs) in tumor draining lymph node (tdLN). Cancer cell apoptosis and DC-activation-mediated cancer antigen-specific immune responses further prevented growth of lung metastatic cancer developed following intravenous transplantation of cancer cells.
CONCLUSION
These results demonstrated the potential usage of a piTRL with laser irradiation for immuno-photothermal therapy against various types of cancer and their metastases.
Topics: Animals; Humans; Immunotherapy; Indocyanine Green; Liposomes; Mice; Neoplasm Metastasis; Neoplasms; Poly I-C; Spectroscopy, Near-Infrared
PubMed: 31412934
DOI: 10.1186/s40425-019-0702-1 -
PloS One 2016Viral infections are often accompanied by pulmonary microvascular leakage and vascular endothelial dysfunction via mechanisms that are not completely defined. Here, we...
Viral infections are often accompanied by pulmonary microvascular leakage and vascular endothelial dysfunction via mechanisms that are not completely defined. Here, we investigated the effect of the Toll-like receptor 3 (TLR3) ligand polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic analog of viral double-stranded RNA (dsRNA) commonly used to simulate viral infections, on the barrier function and tight junction integrity of primary human lung microvascular endothelial cells. Poly(I:C) stimulated IL-6, IL-8, TNFα, and IFNβ production in conjunction with the activation of NF-κB and IRF3 confirming the Poly(I:C)-responsiveness of these cells. Poly(I:C) increased endothelial monolayer permeability with a corresponding dose- and time-dependent decrease in the expression of claudin-5, a transmembrane tight junction protein and reduction of CLDN5 mRNA levels. Immunofluorescence experiments revealed disappearance of membrane-associated claudin-5 and co-localization of cytoplasmic claudin-5 with lysosomal-associated membrane protein 1. Chloroquine and Bay11-7082, inhibitors of TLR3 and NF-κB signaling, respectively, protected against the loss of claudin-5. Together, these findings provide new insight on how dsRNA-activated signaling pathways may disrupt vascular endothelial function and contribute to vascular leakage pathologies.
Topics: Adaptor Proteins, Vesicular Transport; Chemokines; Claudin-5; Down-Regulation; Endothelial Cells; Humans; Interferon Regulatory Factor-3; Lung; NF-kappa B; Permeability; Poly I-C; RNA, Messenger; Signal Transduction; Tight Junctions; Toll-Like Receptor 3
PubMed: 27504984
DOI: 10.1371/journal.pone.0160875 -
Molecular Neurobiology Apr 2022Microglia are resident macrophages in the central nervous system that are involved in immune responses driven by Toll-like receptors (TLRs). Microglia-mediated...
Microglia are resident macrophages in the central nervous system that are involved in immune responses driven by Toll-like receptors (TLRs). Microglia-mediated inflammation can lead to central nervous system disorders, and more than one TLR might be involved in these pathological processes. The cysteine peptidase cathepsin X has been recognized as a pathogenic factor for inflammation-induced neurodegeneration. Here, we hypothesized that simultaneous TLR3 and TLR4 activation induces synergized microglia responses and that these phenotype changes affect cathepsin X expression and activity. Murine microglia BV2 cells and primary murine microglia were exposed to the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)) and the TLR4 ligand lipopolysaccharide (LPS), individually and simultaneously. TLR3 and TLR4 co-activation resulted in increased inflammatory responses compared to individual TLR activation, where poly(I:C) and LPS induced distinct patterns of proinflammatory factors together with different patterns of cathepsin X expression and activity. TLR co-activation decreased intracellular cathepsin X activity and increased cathepsin X localization at the plasma membrane with concomitant increased extracellular cathepsin X protein levels and activity. Inhibition of cathepsin X in BV2 cells by AMS36, cathepsin X inhibitor, significantly reduced the poly(I:C)- and LPS-induced production of proinflammatory cytokines as well as apoptosis. Additionally, inhibiting the TLR3 and TLR4 common signaling pathway, PI3K, with LY294002 reduced the inflammatory responses of the poly(I:C)- and LPS-activated microglia and recovered cathepsin X activity. We here provide evidence that microglial cathepsin X strengthens microglia activation and leads to subsequent inflammation-induced neurodegeneration. As such, cathepsin X represents a therapeutic target for treating neurodegenerative diseases related to excess inflammation.
Topics: Animals; Cysteine; Inflammation; Ligands; Lipopolysaccharides; Mice; Microglia; Poly I-C; Toll-Like Receptor 3; Toll-Like Receptor 4
PubMed: 35066760
DOI: 10.1007/s12035-021-02694-2 -
Frontiers in Immunology 2022Recent studies have shown that corn-derived cationic α-D-glucan nanoparticles, known as Nano-11, significantly increase the immune response when used as a vaccine...
Recent studies have shown that corn-derived cationic α-D-glucan nanoparticles, known as Nano-11, significantly increase the immune response when used as a vaccine adjuvant in mice and in pigs. Furthermore, the nanoparticles can be formulated with other immunostimulators such as poly(I:C), which further enhances the immune response. The current experiments were aimed at elucidating the mechanism of action of Nano-11 alone and in combination with poly(I:C). The effect of these adjuvants on porcine monocyte-derived dendritic cells (Mo-DCs) was determined by RNA-sequencing, supplemented with flow cytometry, cytokine analysis, and Western blots. Adsorption of poly(I:C) to Nano-11 reduced its cytotoxicity for Mo-DCs. Exposure of Mo-DCs to Nano-11 and Nano-11/poly(I:C) induced differential expression of 979 and 2016 genes, respectively. Gene Ontology enrichment and KEGG pathway analysis revealed many changes in gene expression related to inflammation, innate immunity, immune response to infections, and metabolism. Nano-11 and Nano-11/poly(I:C) induced maturation of the Mo-DCs as indicated by increased expression of costimulatory molecules and MHC II. Increased expression of genes downstream of p38 MAPK activation revealed a role for this signaling pathway in the activation of Mo-DCs by the adjuvants. This was confirmed by Western blot and inhibition of TNF-secretion upon incubation with the p38 inhibitor SB203580. These experiments provide insights into the mechanism of action of the novel adjuvants Nano-11 and Nano-11/poly(I:C).
Topics: Adjuvants, Immunologic; Adjuvants, Pharmaceutic; Animals; Cytokines; Dendritic Cells; Glucans; Mice; Nanoparticles; Poly I-C; RNA; Swine; p38 Mitogen-Activated Protein Kinases
PubMed: 36131928
DOI: 10.3389/fimmu.2022.990900