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Neurobiology of Disease Nov 2023Peripheral contact to pathogen-associated molecular patterns (PAMPs) evokes a systemic innate immune response which is rapidly relayed to the central nervous system...
Peripheral contact to pathogen-associated molecular patterns (PAMPs) evokes a systemic innate immune response which is rapidly relayed to the central nervous system (CNS). The remarkable cellular heterogeneity of the CNS poses a significant challenge to the study of cell type and stimulus dependent responses of neural cells during acute inflammation. Here we utilized single nuclei RNA sequencing (snRNAseq), serum proteome profiling and primary cell culture methods to systematically compare the acute response of the mammalian brain to the bacterial PAMP lipopolysaccharide (LPS) and the viral PAMP polyinosinic:polycytidylic acid (Poly(I:C)), at single cell resolution. Our study unveiled convergent transcriptional cytokine and cellular stress responses in brain vascular and ependymal cells and a downregulation of several key mediators of directed blood brain barrier (BBB) transport. In contrast the neuronal response to PAMPs was limited in acute neuroinflammation. Moreover, our study highlighted the dominant role of IFN signalling upon Poly(I:C) challenge, particularly in cells of the oligodendrocyte lineage. Collectively our study unveils heterogeneous, shared and distinct cell type and stimulus dependent acute responses of the CNS to bacterial and viral PAMP challenges. Our findings highlight inflammation induced dysregulations of BBB-transporter gene expression, suggesting potential translational implications on drug pharmacokinetics variability during acute neuroinflammation. The pronounced dependency of oligodendrocytes on IFN stimulation during viral PAMP challenges, emphasizes their limited molecular viral response repertoire.
Topics: Animals; Lipopolysaccharides; Neuroinflammatory Diseases; Pathogen-Associated Molecular Pattern Molecules; Central Nervous System; Inflammation; Mammals
PubMed: 37913832
DOI: 10.1016/j.nbd.2023.106339 -
Frontiers in Endocrinology 2023Preeclampsia (PE), a hypertensive pregnancy disorder, can originate from varied etiology. Placenta malperfusion has long been considered the primary cause of PE....
Preeclampsia (PE), a hypertensive pregnancy disorder, can originate from varied etiology. Placenta malperfusion has long been considered the primary cause of PE. However, we and others have showed that this disorder can also result from heightened inflammation at the maternal-fetal interface. To advance our understanding of this understudied PE subtype, it is important to establish validated rodent models to study the pathophysiology and test therapies. We evaluated three previously described approaches to induce inflammation-mediated PE-like features in pregnant rats: 1) Tumor necrosis factor-α (TNF-α) infusion via osmotic pump from gestational day (GD) 14-19 at 50ng/day/animal; 2) Polyinosinic:polycytidylic acid (Poly I:C) intraperitoneal (IP) injections from GD 10-18 (alternate days) at 10mg/kg/day/animal; and, 3) Lipopolysaccharide (LPS) IP injections from GD 13-18 at 20ug-70ug/kg/day per animal. Maternal blood pressure was measured by tail-cuff. Upon sacrifice, fetal and placenta weights were recorded. Placenta histomorphology was assessed using H&E sections. Placenta inflammation was determined by quantifying TNF-α levels and inflammatory gene expression. Placenta metabolic and mitochondrial health were determined by measuring mitochondrial respiration rates and placenta NAD/NADH content. Of the three rodent models tested, we found that Poly I:C and LPS decreased both fetal weight and survival; and correlated with a reduction in region specific placenta growth. As the least effective model characterized, TNF-α treatment resulted in a subtle decrease in fetal/placenta weight and placenta mitochondrial respiration. Only the LPS model was able to induce maternal hypertension and exhibited pronounced placenta metabolic and mitochondrial dysfunction, common features of PE. Thus, the rat LPS model was most effective for recapitulating features observed in cases of human inflammatory PE. Future mechanistic and/or therapeutic intervention studies focuses on this distinct PE patient population may benefit from the employment of this rodent model of PE.
Topics: Pregnancy; Female; Humans; Rats; Animals; Pre-Eclampsia; Tumor Necrosis Factor-alpha; Lipopolysaccharides; Hypertension; Inflammation; Poly I
PubMed: 37842294
DOI: 10.3389/fendo.2023.1219205 -
Stem Cell Research & Therapy Nov 2023Mesenchymal stromal cells (MSCs) have regenerative and immunomodulatory properties, making them suitable for cell therapy. Toll-like receptors (TLRs) in MSCs respond to...
BACKGROUND
Mesenchymal stromal cells (MSCs) have regenerative and immunomodulatory properties, making them suitable for cell therapy. Toll-like receptors (TLRs) in MSCs respond to viral load by secreting immunosuppressive or proinflammatory molecules. The expression of anti-inflammatory molecules in MSCs can be altered by the concentration and duration of exposure to the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)). This study aimed to optimize the preconditioning of MSCs with poly(I:C) to increase immunosuppressive effects and to identify MSCs with activated TLR3 (prMSCs).
METHODS
Flow cytometry and histochemical staining were used to analyze MSCs for immunophenotype and differentiation potential. MSCs were exposed to poly(I:C) at 1 and 10 μg/mL for 1, 3, and 24 h, followed by determination of the expression of IDO1, WARS1, PD-L1, TSG-6, and PTGES2 and PGE2 secretion. MSCs and prMSCs were cocultured with intact (J) and activated (J) Jurkat T cells. The proportion of proliferating and apoptotic J and J cells, IL-10 secretion, and IL-2 production after cocultivation with MSCs and prMSCs were measured. Liquid chromatography-mass spectrometry and bioinformatics analysis identified proteins linked to TLR3 activation in MSCs.
RESULTS
Poly(I:C) at 10 μg/mL during a 3-h incubation caused the highest expression of immunosuppression markers in MSCs. Activation of prMSCs caused a 18% decrease in proliferation and a one-third increase in apoptotic J cells compared to intact MSCs. Cocultures of prMSCs and Jurkat cells had increased IL-10 and decreased IL-2 in the conditioned medium. A proteomic study of MSCs and prMSCs identified 53 proteins with altered expression. Filtering the dataset with Gene Ontology and Reactome Pathway revealed that poly(I:C)-induced proteins activate the antiviral response. Protein‒protein interactions by String in prMSCs revealed that the antiviral response and IFN I signaling circuits were more active than in native MSCs. prMSCs expressed more cell adhesion proteins (ICAM-I and Galectin-3), PARP14, PSMB8, USP18, and GBP4, which may explain their anti-inflammatory effects on Jurkat cells.
CONCLUSIONS
TLR3 activation in MSCs is dependent on exposure time and poly(I:C) concentration. The maximum expression of immunosuppressive molecules was observed with 10 µg/mL poly(I:C) for 3-h preconditioning. This priming protocol for MSCs enhances the immunosuppressive effects of prMSCs on T cells.
Topics: Humans; Toll-Like Receptor 3; Interleukin-10; Interleukin-2; Proteomics; Immunosuppressive Agents; Anti-Inflammatory Agents; Antiviral Agents; Ubiquitin Thiolesterase
PubMed: 38031182
DOI: 10.1186/s13287-023-03579-y -
The Journal of Investigative Dermatology Jul 2023Actinic keratoses and cutaneous squamous cell carcinomas are associated with infections with human papillomavirus of genus beta (betaHPV) in immunosuppressed patients....
Actinic keratoses and cutaneous squamous cell carcinomas are associated with infections with human papillomavirus of genus beta (betaHPV) in immunosuppressed patients. To date, targeted therapy against betaHPV-associated skin cancer does not exist because of the large number of betaHPV without defined high-risk types. In this study, we hypothesized that the activation of innate antiviral immunity in the skin, asymptomatically infected with betaHPV, induces an antitumor response by in situ autovaccination and prevents the formation of betaHPV-associated skin cancer. To test this, we used the preclinical keratin-14-HPV8 transgenic mouse model, which develops skin tumors after mechanical wounding. Remarkably, treatment with the antiviral immune response activating polyinosinic-polycytidylic acid (poly[I:C]) completely prevented cutaneous tumor growth. The induction of the IFN-induced genes Cxcl10 and Ifit1 by poly(I:C) depended on MDA5 activation. Increased numbers of total and activated CD4 and CD8 T cells were detected in poly(I:C)-treated skin. T cells were found in the skin of poly(I:C)-treated mice but not in the skin tumors of untreated mice. T-cell depletion showed a predominant role of CD4 T cells in poly(I:C)-mediated tumor prevention. Our findings identify the MDA5 ligand poly(I:C) as a promising candidate for in situ autovaccination approaches, which might serve as a treatment strategy against betaHPV-related skin diseases.
Topics: Humans; Mice; Animals; Mice, Transgenic; Poly I-C; Skin Neoplasms; Skin; Antiviral Agents
PubMed: 36584911
DOI: 10.1016/j.jid.2022.12.007 -
Frontiers in Immunology 2023In the tumor microenvironment (TME), tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer....
BACKGROUND
In the tumor microenvironment (TME), tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer. Toll-like receptors (TLRs) ligands, such as poly(I:C) or resiquimod (R848) are able to reprogram TAMs towards M1-like antitumor effector cells. The objective of our work has been to develop and evaluate polymeric nanocapsules (NCs) loaded with poly(I:C)+R848, to improve drug stability and systemic toxicity, and evaluate their targeting and therapeutic activity towards TAMs in the TME of solid tumors.
METHODS
NCs were developed by the solvent displacement and layer-by-layer methodologies and characterized by dynamic light scattering and nanoparticle tracking analysis. Hyaluronic acid (HA) was chemically functionalized with mannose for the coating of the NCs to target TAMs. NCs loaded with TLR ligands were evaluated for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry, using primary human monocyte-derived macrophages. For 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 and multispectral immunophenotyping.
RESULTS
We have developed polymeric NCs loaded with poly(I:C)+R848. Among a series of 5 lead prototypes, protamine-NCs were selected based on their physicochemical properties (size, charge, stability) and characterization, showing good biocompatibility on primary macrophages and ability to stimulate their production of T-cell attracting chemokines (CXCL10, CCL5) and to induce M1-like macrophages cytotoxicity towards tumor cells. In mouse tumor models, the intratumoral injection of poly(I:C)+R848-protamine-NCs significantly prevented tumor growth and lung metastasis. In an orthotopic murine lung cancer model, the intravenous administration of poly(I:C)+R848-prot-NCs, coated with an additional layer of HA-mannose to improve TAM-targeting, resulted in good antitumoral efficacy with no apparent systemic toxicity. While no significant alterations were observed in T cell numbers (CD8, CD4 or Treg), TAM-reprogramming in treated mice was confirmed by the relative decrease of interstitial alveolar macrophages, having higher CD86 expression but lower CD206 and Arg1 expression in the same cells, in treated mice.
CONCLUSION
Mannose-HA-protamine-NCs loaded with poly(I:C)+R848 successfully reprogram TAMs , and reduce tumor progression and metastasis spread in mouse tumors.
Topics: Humans; Animals; Mice; Tumor-Associated Macrophages; Nanocapsules; Mannose; Lung Neoplasms; Disease Models, Animal; Protamines; Tumor Microenvironment; Imidazoles
PubMed: 38259462
DOI: 10.3389/fimmu.2023.1334800 -
Cancer Medicine Jul 2023Improving the potency of immune response is paramount among issues concerning immunotherapy of muscle-invasive bladder cancer (MIBC).
BACKGROUND
Improving the potency of immune response is paramount among issues concerning immunotherapy of muscle-invasive bladder cancer (MIBC).
METHODS
On the basis of immune subtypes, we investigated possible molecular mechanisms involved in tumor immune escape in MIBC. According to the 312 immune-related genes, three MIBC immune subtypes were clustered.
RESULTS
Cluster 2 subtype is characterized by FGFR3 mutations and has a better clinical prognosis. However, the expression levels of MHC-I and immune checkpoints genes were the lowest, indicating that this subtype is subject to immune escape and has a low response rate to immunotherapy. Bioinformatics analysis and immunofluorescence staining of clinical samples revealed that the FGFR3 is involved in the immune escape in MIBC. Besides, after FGFR3 knockout with siRNA in RT112 and UMUC14 cells, the TLR3/NF-kB pathway was significantly activated and was accompanied by upregulation of MHC-I and PD-L1 gene expression. Furthermore, the use of TLR3 agonists poly(I:C) can further improve the effect.
CONCLUSION
Together, our results suggest that FGFR3 might involve in immunosuppression by inhibition of NF-kB pathway in BC. Considering that TLR3 agonists are currently approved for clinical treatment as immunoadjuvants, our study might provide more insights for improving the efficacy of immunotherapy in MIBC.
Topics: Humans; NF-kappa B; Toll-Like Receptor 3; B7-H1 Antigen; Urinary Bladder Neoplasms; Prognosis; Muscles; Receptor, Fibroblast Growth Factor, Type 3
PubMed: 37283287
DOI: 10.1002/cam4.6172 -
Journal of Neuroimmune Pharmacology :... Sep 2023Impairment of specific cognitive domains in schizophrenia has been associated with prefrontal cortex (PFC) catecholaminergic deficits. Among other factors, prenatal...
BACKGROUND
Impairment of specific cognitive domains in schizophrenia has been associated with prefrontal cortex (PFC) catecholaminergic deficits. Among other factors, prenatal exposure to infections represents an environmental risk factor for schizophrenia development in adulthood. However, it remains largely unknown whether the prenatal infection-induced changes in the brain may be associated with concrete switches in a particular neurochemical circuit, and therefore, if they could alter behavioral functions.
METHODS
In vitro and in vivo neurochemical evaluation of the PFC catecholaminergic systems was performed in offspring from mice undergoing maternal immune activation (MIA). The cognitive status was also evaluated. Prenatal viral infection was mimicked by polyriboinosinic-polyribocytidylic acid (poly(I:C)) administration to pregnant dams (7.5 mg/kg i.p., gestational day 9.5) and consequences were evaluated in adult offspring.
RESULTS
MIA-treated offspring showed disrupted recognition memory in the novel object recognition task (t = 2.30, p = 0.031). This poly(I:C)-based group displayed decreased extracellular dopamine (DA) concentrations compared to controls (t = 3.17, p = 0.0068). Potassium-evoked release of DA and noradrenaline (NA) were impaired in the poly(I:C) group (DA: F[10,90] = 43.33, p < 0.0001; F[1,90] = 1.224, p = 0.2972; F[10,90] = 5.916, p < 0.0001; n = 11); (NA: F[10,90] = 36.27, p < 0.0001; F[1,90] = 1.841, p = 0.208; F[10,90] = 8.686, p < 0.0001; n = 11). In the same way, amphetamine-evoked release of DA and NA were also impaired in the poly(I:C) group (DA: F[8,328] = 22.01, p < 0.0001; F[1,328] = 4.507, p = 0.040; F[8,328] = 2.319, p = 0.020; n = 43); (NA: F[8,328] = 52.07; p < 0.0001; F[1,328] = 4.322; p = 0.044; F[8,398] = 5.727; p < 0.0001; n = 43). This catecholamine imbalance was accompanied by increased dopamine D and D receptor expression (t = 2.64, p = 0.011 and t = 3.55, p = 0.0009; respectively), whereas tyrosine hydroxylase, DA and NA tissue content, DA and NA transporter (DAT/NET) expression and function were unaltered.
CONCLUSIONS
MIA induces in offspring a presynaptic catecholaminergic hypofunction in PFC with cognitive impairment. This poly(I:C)-based model reproduces catecholamine phenotypes reported in schizophrenia and represents an opportunity for the study of cognitive impairment associated to this disorder.
Topics: Pregnancy; Female; Mice; Animals; Humans; Dopamine; Brain; Prefrontal Cortex; Cognitive Dysfunction; Amphetamine; Norepinephrine; Prenatal Exposure Delayed Effects; Poly I-C; Disease Models, Animal
PubMed: 37208550
DOI: 10.1007/s11481-023-10070-1 -
Cell Reports Aug 2023Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe clinical disorders that mainly develop from viral respiratory infections, sepsis, and...
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe clinical disorders that mainly develop from viral respiratory infections, sepsis, and chest injury. Antigen-presenting cells play a pivotal role in propagating uncontrolled inflammation and injury through the excess secretion of pro-inflammatory cytokines and recruitment of immune cells. Autophagy, a homeostatic process that involves the degradation of cellular components, is involved in many processes including lung inflammation. Here, we use a polyinosinic-polycytidylic acid (poly(I:C))-induced lung injury mouse model to mimic viral-induced ALI/ARDS and show that disruption of autophagy in macrophages exacerbates lung inflammation and injury, whereas autophagy induction attenuates this process. Therefore, induction of autophagy in macrophages can be a promising therapeutic strategy in ALI/ARDS.
Topics: Animals; Mice; Antigen-Presenting Cells; Macrophages; Autophagy; Acute Lung Injury; Poly I-C; Respiratory Distress Syndrome
PubMed: 37590140
DOI: 10.1016/j.celrep.2023.112990 -
IScience Jul 2023Interferon-induced transmembrane proteins (IFITMs) are restriction factors that block many viruses from entering cells. High levels of type I interferon (IFN) are...
Interferon-induced transmembrane proteins (IFITMs) are restriction factors that block many viruses from entering cells. High levels of type I interferon (IFN) are associated with adverse pregnancy outcomes, and IFITMs have been shown to impair the formation of syncytiotrophoblast. Here, we examine whether IFITMs affect another critical step of placental development, extravillous cytotrophoblast (EVCT) invasion. We conducted experiments using / models of EVCT, mice treated with the IFN-inducer poly (I:C), and human pathological placental sections. Cells treated with IFN-β demonstrated upregulation of IFITMs and reduced invasive abilities. Transduction experiments confirmed that IFITM1 contributed to the decreased cell invasion. Similarly, migration of trophoblast giant cells, the mouse equivalent of human EVCTs, was significantly reduced in poly (I:C)-treated mice. Finally, analysis of CMV- and bacterial-infected human placentas revealed upregulated IFITM1 expression. These data demonstrate that high levels of IFITM1 impair trophoblast invasion and could explain the placental dysfunctions associated with IFN-mediated disorders.
PubMed: 37434700
DOI: 10.1016/j.isci.2023.107147 -
Scientific Reports Nov 2023Proinflammatory cytokine interleukin (IL)-6 was associated with disease severity in patients with COVID-19. The mechanism underlying the excessive IL-6 production by...
Proinflammatory cytokine interleukin (IL)-6 was associated with disease severity in patients with COVID-19. The mechanism underlying the excessive IL-6 production by SARS-Cov-2 infection remains unclear. Respiratory viruses initially infect nasal or bronchial epithelial cells that produce various inflammatory mediators. Here, we show that pretreatment of human bronchial epithelial cells (NCl-H292) with interferon (IFN)-γ (10 ng/mL) markedly increased IL-6 production induced by the toll-like receptor (TLR) 3 agonist poly(I:C) (1 µg/mL) from 0.4 ± 0.1 to 4.1 ± 0.4 ng/mL (n = 3, P < 0.01). A similar effect was observed in human alveolar A549 and primary bronchial epithelial cells. TLR3 knockdown using siRNA in NCl-H292 cells diminished the priming effects of IFN-γ on poly(I:C)-induced IL-6 production. Furthermore, the Janus kinase (JAK) inhibitor tofacitinib (1 µM) inhibited IFN-γ-induced upregulation of TLR3, and suppressed poly(I:C)-induced IL-6 production. Quantitative chromatin immunoprecipitation revealed that IFN-γ stimulated histone modifications at the IL-6 gene locus. Finally, IFN-γ priming significantly increased lung IL-6 mRNA and protein levels in poly(I:C)-administrated mice. Thus, priming bronchial epithelial cells with IFN-γ increases poly(I:C)-induced IL-6 production via JAK-dependent TLR3 upregulation and chromatin remodeling at the IL-6 gene locus. These mechanisms may be involved in severe respiratory inflammation following infection with RNA viruses.
Topics: Animals; Humans; Mice; Epithelial Cells; Interferon-gamma; Interleukin-6; Interleukin-8; Poly I-C; Toll-Like Receptor 3
PubMed: 38030681
DOI: 10.1038/s41598-023-48422-9