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Journal For Immunotherapy of Cancer Sep 2021Tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer and hinder the antitumoral efficacy of...
BACKGROUND
Tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer and hinder the antitumoral efficacy of most treatments currently applied in the clinic. Previous studies have evaluated the antitumoral immune response triggered by (TLR) agonists, such as poly(I:C), imiquimod (R837) or resiquimod (R848) as monotherapies; however, their combination for the treatment of cancer has not been explored. This study investigates the antitumoral efficacy and the macrophage reprogramming triggered by poly(I:C) combined with R848 or with R837, versus single treatments.
METHODS
TLR agonist treatments were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry using primary human and murine M-CSF-differentiated macrophages. Cytotoxic activity of TLR-treated macrophages toward cancer cells was evaluated with an in vitro functional assay by flow cytometry. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry, RT-qPCR, multispectral immunophenotyping, quantitative proteomic experiments, and protein-protein interaction analysis.
RESULTS
Results demonstrated the higher efficacy of poly(I:C) combined with R848 versus single treatments or combined with R837 to polarize macrophages toward M1-like antitumor effectors in vitro. In vivo, the intratumoral synergistic combination of poly(I:C)+R848 significantly prevented tumor growth and metastasis in lung cancer and fibrosarcoma immunocompetent murine models. Regressing tumors showed increased infiltration of macrophages with a higher M1:M2 ratio, recruitment of CD4 and CD8 T cells, accompanied by a reduction of immunosuppressive CD206 TAMs and FOXP3/CD4 T cells. The depletion of both CD4 and CD8 T cells resulted in complete loss of treatment efficacy. Treated mice acquired systemic antitumoral response and resistance to tumor rechallenge mediated by boosted macrophage cytotoxic activity and T-cell proliferation. Proteomic experiments validate the superior activation of innate immunity by poly(I:C)+R848 combination versus single treatments or poly(I:C)+R837, and protein-protein-interaction network analysis reveal the key activation of the STAT1 pathway.
DISCUSSION
These findings demonstrate the antitumor immune responses mediated by macrophage activation on local administration of poly(I:C)+R848 combination and support the intratumoral application of this therapy to patients with solid tumors in the clinic.
Topics: Animals; Antiviral Agents; Cell Line, Tumor; Combined Modality Therapy; Drug Synergism; Humans; Imidazoles; Immunotherapy; Mice; Neoplasms; Poly I-C; Tumor-Associated Macrophages
PubMed: 34531246
DOI: 10.1136/jitc-2021-002408 -
Cancer Biology & Therapy Oct 2017TLR3 belong to the Toll-like receptors family, it is mainly expressed on immune cells where it senses pathogen-associated molecular patterns and initiates innate immune... (Review)
Review
TLR3 belong to the Toll-like receptors family, it is mainly expressed on immune cells where it senses pathogen-associated molecular patterns and initiates innate immune response. TLR3 agonist poly(I:C) was developed to mimic pathogens infection and boost immune system activation to promote anti-cancer therapy. Accordingly, TLR agonists were included in the National Cancer Institute list of immunotherapeutic agents with the highest potential to cure cancer. Besides well known effects on immune cells, poly(I:C) was also shown, in experimental models, to directly induce apoptosis in cancer cells expressing TLR3. This review presents the current knowledge on the mechanism of poly(I:C)-induced apoptosis in cancer cells. Experimental evidences on positive or negative regulators of TLR3-mediated apoptosis induced by poly(I:C) are reported and strategies are proposed to successfully promote this event in cancer cells. Cancer cells apoptosis is an additional arm offered by poly(I:C), besides activation of immune system, for the treatment of various type of cancer. A further dissection of TLR3 signaling would contribute to greater resolution of the critical steps that impede full exploitation of the poly(I:C)-induced apoptosis. Experimental evidences about negative regulator of poly(I:C)-induced apoptotic program should be considered in combinations with TLR3 agonists in clinical trials.
Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Humans; Immunity, Innate; Neoplasms; Poly I-C; Prognosis; Signal Transduction; Toll-Like Receptor 3
PubMed: 28881163
DOI: 10.1080/15384047.2017.1373220 -
Nature Communications Sep 2023Inhibition of glycolysis in immune cells and cancer cells diminishes their activity, and thus combining immunotherapies with glycolytic inhibitors is challenging....
Inhibition of glycolysis in immune cells and cancer cells diminishes their activity, and thus combining immunotherapies with glycolytic inhibitors is challenging. Herein, a strategy is presented where glycolysis is inhibited in cancer cells using PFK15 (inhibitor of PFKFB3, rate-limiting step in glycolysis), while simultaneously glycolysis and function is rescued in DCs by delivery of fructose-1,6-biphosphate (F16BP, one-step downstream of PFKFB3). To demonstrate the feasibility of this strategy, vaccine formulations are generated using calcium-phosphate chemistry, that incorporate F16BP, poly(IC) as adjuvant, and phosphorylated-TRP2 peptide antigen and tested in challenging and established YUMM1.1 tumours in immunocompetent female mice. Furthermore, to test the versatility of this strategy, adoptive DC therapy is developed with formulations that incorporate F16BP, poly(IC) as adjuvant and mRNA derived from B16F10 cells as antigens in established B16F10 tumours in immunocompetent female mice. F16BP vaccine formulations rescue DCs in vitro and in vivo, significantly improve the survival of mice, and generate cytotoxic T cell (Tc) responses by elevating Tc1 and Tc17 cells within the tumour. Overall, these results demonstrate that rescuing glycolysis of DCs using metabolite-based formulations can be utilized to generate immunotherapy even in the presence of glycolytic inhibitor.
Topics: Female; Animals; Mice; Immunotherapy; Glycolysis; Adjuvants, Immunologic; Fructose; Poly I-C; Dendritic Cells; Neoplasms
PubMed: 37660049
DOI: 10.1038/s41467-023-41016-z -
Frontiers in Immunology 2022Major Histocompatibility Complex (MHC)-I and -II genes are upregulated in intestinal epithelial cells (IECs) during active inflammatory bowel diseases (IBD), but little...
Major Histocompatibility Complex (MHC)-I and -II genes are upregulated in intestinal epithelial cells (IECs) during active inflammatory bowel diseases (IBD), but little is known about how IBD-relevant pro-inflammatory signals and IBD drugs can regulate their expression. We have previously shown that the synthetic analog of double-stranded RNA (dsRNA) Polyinosinic:polycytidylic acid (Poly(I:C)), induces interferon stimulated genes (ISGs) in colon organoids (colonoids). These ISGs may be involved in the induction of antigen presentation. In the present study, we applied colonoids derived from non-IBD controls and ulcerative colitis patients to identify induction and effects of IBD-drugs on antigen presentation in IECs in the context of Tumor Necrosis Factor (TNF)-driven inflammation. By RNA sequencing, we show that a combination of TNF and Poly(I:C) strongly induced antigen-presentation gene signatures in colonoids, including expression of MHC-II genes. MHC-I and -II protein expression was confirmed by immunoblotting and immunofluorescence. TNF+Poly(I:C)-dependent upregulation of MHC-II expression was associated with increased expression of Janus Kinases as well as increased activation of transcription factor Signal transducer and activator of transcription 1 (). Accordingly, pre-treatment of colonoids with IBD-approved pan-Janus Kinase (JAK) inhibitor Tofacitinib led to the downregulation of TNF+Poly(I:C)-dependent MHC-II expression associated with the abrogation of STAT1 activation. Pre-treatment with corticosteroid Budesonide, commonly used in IBD, did not alter MHC-II expression. Collectively, our results identify a regulatory role for IBD-relevant pro-inflammatory signals on MHC-II expression that is influenced by Tofacitinib.
Topics: Colon; Epithelium; Humans; Inflammatory Bowel Diseases; Janus Kinase Inhibitors; Major Histocompatibility Complex; Piperidines; Poly I-C; Pyrimidines; Tumor Necrosis Factor-alpha
PubMed: 35655783
DOI: 10.3389/fimmu.2022.882277 -
Oncoimmunology 2023Toll-like receptor 3 (TLR3) agonists such as polyinosinic:polycytidylic acid (poly(I:C)) have immunostimulatory effects that can be taken advantage of to induce...
Toll-like receptor 3 (TLR3) agonists such as polyinosinic:polycytidylic acid (poly(I:C)) have immunostimulatory effects that can be taken advantage of to induce anticancer immune responses in preclinical models. In addition, poly(I:C) has been introduced into clinical trials to demonstrate its efficacy as an adjuvant and to enhance the immunogenicity of locally injected tumors, thus reverting resistance to PD-L1 blockade in melanoma patients. Here, we report the pharmacokinetic, pharmacodynamic, mechanistic and toxicological profile of a novel TLR3 agonist, TL-532, a chemically synthesized double-stranded RNA that is composed by blocks of poly(I:C) and poly(A:U) (polyadenylic - polyuridylic acid). In preclinical models, we show that TL-532 is bioavailable after parenteral injection, has an acceptable toxicological profile, and stimulates the production of multiple chemokines and interleukins that constitute pharmacodynamic markers of its immunostimulatory action. When given at a high dose, TL-532 monotherapy reduced the growth of bladder cancers growing on mice. In addition, in immunodeficient mice lacking formylpeptide receptor-1 (FPR1), TL-532 was able to restore the response of orthotopic subcutaneous fibrosarcoma to immunogenic chemotherapy. Altogether, these findings may encourage further development of TL-532 as an immunotherapeutic anticancer agent.
Topics: Animals; Mice; Toll-Like Receptor 3; Adjuvants, Immunologic; Melanoma; Poly I-C
PubMed: 37389102
DOI: 10.1080/2162402X.2023.2227510 -
Journal of Experimental & Clinical... Jun 2021Immunotherapy is currently under intensive investigation as a potential breakthrough treatment option for glioblastoma. Given the anatomical and immunological... (Review)
Review
Immunotherapy is currently under intensive investigation as a potential breakthrough treatment option for glioblastoma. Given the anatomical and immunological complexities surrounding glioblastoma, lymphocytes that infiltrate the brain to develop durable immunity with memory will be key. Polyinosinic:polycytidylic acid, or poly(I:C), and its derivative poly-ICLC could serve as a priming or boosting therapy to unleash lymphocytes and other factors in the (immuno)therapeutic armory against glioblastoma. Here, we present a systematic review on the effects and efficacy of poly(I:C)/poly-ICLC for glioblastoma treatment, ranging from preclinical work on cellular and murine glioblastoma models to reported and ongoing clinical studies. MEDLINE was searched until 15 May 2021 to identify preclinical (glioblastoma cells, murine models) and clinical studies that investigated poly(I:C) or poly-ICLC in glioblastoma. A systematic review approach was conducted according to PRISMA guidelines. ClinicalTrials.gov was queried for ongoing clinical studies. Direct pro-tumorigenic effects of poly(I:C) on glioblastoma cells have not been described. On the contrary, poly(I:C) changes the immunological profile of glioblastoma cells and can also kill them directly. In murine glioblastoma models, poly(I:C) has shown therapeutic relevance as an adjuvant therapy to several treatment modalities, including vaccination and immune checkpoint blockade. Clinically, mostly as an adjuvant to dendritic cell or peptide vaccines, poly-ICLC has been demonstrated to be safe and capable of eliciting immunological activity to boost therapeutic responses. Poly-ICLC could be a valuable tool to enhance immunotherapeutic approaches for glioblastoma. We conclude by proposing several promising combination strategies that might advance glioblastoma immunotherapy and discuss key pre-clinical aspects to improve clinical translation.
Topics: Animals; Brain Neoplasms; Cancer Vaccines; Carboxymethylcellulose Sodium; Clinical Trials as Topic; Glioblastoma; Humans; Immune Checkpoint Inhibitors; Immunotherapy; Mice; Poly I-C; Polylysine
PubMed: 34172082
DOI: 10.1186/s13046-021-02017-2 -
Pharmacology & Therapeutics May 2015Increasing epidemiological and experimental evidence implicates gestational infections as one important factor involved in the pathogenesis of several neuropsychiatric... (Review)
Review
Increasing epidemiological and experimental evidence implicates gestational infections as one important factor involved in the pathogenesis of several neuropsychiatric disorders. Corresponding preclinical model systems based upon maternal immune activation (MIA) by treatment of the pregnant female have been developed. These MIA animal model systems have been successfully used in basic and translational research approaches, contributing to the investigation of the underlying pathophysiological mechanisms at the molecular, cellular and behavioral levels. The present article focuses on the application of a specific MIA rodent paradigm, based upon treatment of the gestating dam with the viral mimic polyinosinic-polycytidilic acid (Poly(I:C)), a synthetic analog of double-stranded RNA (dsRNA) which activates the Toll-like receptor 3 (TLR3) pathway. Important advantages and constraints of this animal model will be discussed, specifically in light of gestational infection as one vulnerability factor contributing to the complex etiology of mood and psychotic disorders, which are likely the result of intricate multi-level gene×environment interactions. Improving our currently incomplete understanding of the molecular pathomechanistic principles underlying these disorders is a prerequisite for the development of alternative therapeutic approaches which are critically needed in light of the important drawbacks and limitations of currently available pharmacological treatment options regarding efficacy and side effects. The particular relevance of the Poly(I:C) MIA model for the discovery of novel drug targets for symptomatic and preventive therapeutic strategies in mood and psychotic disorders is highlighted in this review article.
Topics: Animals; Disease Models, Animal; Drug Discovery; Humans; Mental Disorders; Models, Immunological; Poly I-C
PubMed: 25562580
DOI: 10.1016/j.pharmthera.2015.01.001 -
Viruses May 2023Immunopathology in hepatitis B virus (HBV) infection is driven by innate and adaptive immunity. Whether the hepatitis B surface antigen (HBsAg) affects hepatic antiviral...
Immunopathology in hepatitis B virus (HBV) infection is driven by innate and adaptive immunity. Whether the hepatitis B surface antigen (HBsAg) affects hepatic antiviral signalling was investigated in HBV-transgenic mouse models that either accumulate (Alb/HBs, Tg[Alb1HBV]Bri44), lack (Tg1.4HBV-s-mut3) or secrete (Tg1.4HBV-s-rec (F1, Tg1.4HBV-s-mut × Alb/HBs) the HBsAg. Herein, the responsiveness of TLR3 and RIG-I in primary parenchymal and non-parenchymal liver cells was determined in vitro and in vivo. Cell type-specific and mouse strain-dependent interferon, cytokine and chemokine expression were observed by LEGENDplex™ and validated by quantitative PCR. In vitro, the hepatocytes, liver sinusoidal endothelial cells and Kupffer cells of Tg1.4HBV-s-rec mice showed poly(I:C) susceptibilities similar to the wild-type controls, while in the remaining leucocyte fraction the interferon, cytokine and chemokine induction was reduced. On the contrary, poly(I:C)-injected 1.4TgHBV-s-rec mice showed suppressed interferon, cytokine and chemokine levels in hepatocytes but increased levels in the leucocyte fraction. Thus, we concluded that liver cells of Tg1.4HBV-s-rec mice, which produce HBV particles and release the HBsAg, responded to exogenous TLR3/RIG-I stimuli in vitro but exhibited a tolerogenic environment in vivo.
Topics: Mice; Animals; Hepatitis B virus; Mice, Transgenic; Hepatitis B Surface Antigens; Toll-Like Receptor 3; Endothelial Cells; Hepatocytes; Liver; Interferons; Cytokines; Hepatitis B; Poly I-C
PubMed: 37243287
DOI: 10.3390/v15051203 -
RNA Biology Jan 2022Germline-encoded pattern recognition receptors [PRRs] in mammalian cells function in the detection of molecular patterns associated with pathogen invasion or cellular...
Germline-encoded pattern recognition receptors [PRRs] in mammalian cells function in the detection of molecular patterns associated with pathogen invasion or cellular damage. A PRR subset is activated by the atypical presence and location of double-stranded RNA [dsRNA] or its synthetic analogue polyinosinic-polycytidylic acid [poly(I:C)], triggering pro-inflammatory signalling and death in many cell types. Poly(I:C) has been tested as a sole or combination cancer therapy in preclinical studies and clinical trials. The purpose of this study was to evaluate the effects of poly(I:C) transfection via electroporation on cell lines from a cancer of epithelial origin, 4T1 mammary carcinoma, and a cancer of mesenchymal origin, WEHI 164 fibrosarcoma. The effects of the poly(I:C) delivery on cell metabolism implicate the induction of cell death. A pro-inflammatory response was demonstrated by mRNA upregulation and the secretion of Type I interferon and several cytokines and chemokines. The mRNAs of dsRNA sensor DExD/H-box helicase 58/retinoic acid-inducible gene I protein [Ddx58/RIG-I] and sensor/co-sensor DEAH-box helicase 9 [Dhx9] were not regulated, but the mRNAs of RNA sensors toll-like receptor 3 [TLR3], interferon-induced with helicase C domain 1/melanoma differentiation-associated protein 5 [Ifih1/MDA5] and Z-DNA binding protein 1 [Zbp1] and co-sensors DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 [Ddx60] and interferon-inducible protein 204 [Ifi204] were upregulated in both cell lines. The mRNAs encoding signalling pathways components were present or upregulated in both cell types. These data demonstrate that RNA sensing effects can be amplified by electroporation delivery, potentially expanding the practicality of this immunotherapeutic approach.
Topics: Animals; Carcinoma; DEAD Box Protein 58; DEAD-box RNA Helicases; Fibrosarcoma; Interferon Type I; Mammals; Mice; Poly I-C; RNA, Double-Stranded; RNA, Messenger; RNA-Binding Proteins; Transfection
PubMed: 35737804
DOI: 10.1080/15476286.2022.2084861 -
Poultry Science Aug 2021Exosomes are small membrane vesicles that contain proteins and nucleic acids derived from secretory cells and mediate intracellular communication. Immune cell-derived...
Exosomes are small membrane vesicles that contain proteins and nucleic acids derived from secretory cells and mediate intracellular communication. Immune cell-derived exosomes regulate immune responses and gene expression of recipient cells. Macrophages recognize viral dsRNA via Toll-like receptor 3, thereby inducing the activation of transcription factors such as interferon regulatory factor 3 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In this study, we aimed to identify the immunomodulatory functions of exosomes derived from chicken macrophages (HD11) stimulated with polyinosinic-polycytidylic acid (poly[I:C]); exosomes were then delivered into HD11 cells and CU91 chicken T cells. Exosomes purified from poly(I:C)-activated macrophages stimulated the expression of type I interferons, proinflammatory cytokines, anti-inflammatory cytokines, and chemokines in HD11 and CU91 cells. Moreover, poly(I:C)-stimulated exosomes induced the NF-κB signaling pathway by phosphorylating TAK1 and NF-κB1. Therefore, we suggest that after the activation of Toll-like receptor 3 ligands following infection with dsRNA virus, chicken macrophages regulate the immune response of naive macrophages and T cells through the NF-κB signaling pathway. Furthermore, poly(I:C)-activated exosomes can be potentially utilized as immunostimulators.
Topics: Animals; Chickens; Exosomes; Immunity; Macrophages; NF-kappa B; Poly I-C
PubMed: 34174563
DOI: 10.1016/j.psj.2021.101247