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Cell Biochemistry and Biophysics Jun 2024Toll-like receptors 3 (TLR3) are innate immune receptors expressed on a wide range of cell types, including glial cells. Inflammatory responses altered by hyperglycemia...
Toll-like receptors 3 (TLR3) are innate immune receptors expressed on a wide range of cell types, including glial cells. Inflammatory responses altered by hyperglycemia highlight the need to explore the molecular underpinnings of these changes in cellular models. Therefore, here we estimated TLR3-mediated response of astrocytes cultured at normal (NG, 5 mM) and high (HG, 22.5 mM) glucose concentrations for 48 h before stimulation with polyinosinic:polycytidylic acid Poly(I:C) (PIC) for 6 h. Seahorse Extracellular Flux Analyzer (Seahorse XFp) was used to estimate the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Although adaptation to HG affected ECAR and OCR, the stimulation of cells with PIC had no effect on ECAR. PIC reduced maximal OCR, but this effect disappeared upon adaptation to HG. PIC-stimulated release of cytokines IL-1β, IL-10 was reduced, and that of IL-6 and iNOS was increased in the HG model. Adaptation to HG reduced PIC-stimulated synthesis of COX-derived oxylipins measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Adaptation to HG did not alter PIC-stimulated p38 activity, ERK mitogen-activated protein kinase, STAT3 and ROS production. Metformin exhibited anti-inflammatory activity, reducing PIC-stimulated synthesis of cytokines and oxylipins. Cell adaptation to high glucose concentration altered the sensitivity of astrocytes to TLR3 receptor activation, and the hypoglycemic drug metformin may exert anti-inflammatory effects under these conditions.
PubMed: 38918312
DOI: 10.1007/s12013-024-01380-z -
Journal of Controlled Release :... Jun 2024The development of high-purity antigens promotes the urgent need of novel adjuvant with the capability to trigger high levels of immune response....
The development of high-purity antigens promotes the urgent need of novel adjuvant with the capability to trigger high levels of immune response. Polyinosinic-polycytidylic (Poly(I:C)) is a synthetic double-stranded RNA (dsRNA) that can engage Toll-like receptor 3 (TLR3) to initiate immune responses. However, the Poly(I:C)-induced toxicity and inefficient delivery prevent its applications. In our study, combination adjuvants are formulated by aluminum oxyhydroxide nanorods (AlOOH NRs) and Poly(I:C), named Al-Poly(I:C), and the covalent interaction between the two components is further demonstrated. Al-Poly(I:C) mediates enhanced humoral and cellular immune responses in three antigen models, i.e., HBsAg virus-like particles (VLPs), human papilloma virus (HPV) VLPs and varicella-zoster virus (VZV) glycoprotein E (gE). Further mechanistic studies demonstrate that the dose and molecular weight (MW) of Poly(I:C) determine the physicochemical properties and adjuvanticity of the Al-Poly(I:C) combination adjuvants. Al-Poly(I:C) with higher Poly(I:C) dose promotes antigen-bearing dendritic cells (DCs) recruitment and B cells proliferation in lymph nodes. Al-Poly(I:C) formulated with higher MW Poly(I:C) induces higher activation of helper T cells, B cells, and CTLs. This study demonstrates that Al-Poly(I:C) potentiates the humoral and cellular responses in vaccine formulations. It offers insights for adjuvant design to meet the formulation requirements in both prophylactic and therapeutic vaccines.
PubMed: 38914205
DOI: 10.1016/j.jconrel.2024.06.054 -
Developmental and Comparative Immunology Jun 2024Increasing evidence has been shown that OTUB1, a member of OTU deubiquitinases, is of importance in regulating the immune system. However, its molecular identification...
Increasing evidence has been shown that OTUB1, a member of OTU deubiquitinases, is of importance in regulating the immune system. However, its molecular identification and functional characterization in teleosts are still rarely known. In this work, we cloned the otub1 of miiuy croaker (Miichthys miiuy), analyzed its sequence, structure, and evolution at genetic and protein levels, and determined its function in the antiviral immune response. The complete open reading frame (ORF) of miiuy croaker otub1 is 843 bp in length, encoding 280 amino acids. Miiuy croaker Otub1 has an OTU domain at the carboxyl terminus, which is a common functional domain that exists in OTU deubiquitinases. Molecular characteristics and evolution analysis results indicated that miiuy croaker Otub1, especially its functional domain, is highly conserved during evolution. The luciferase reporter assays showed that miiuy croaker Otub1 could significantly inhibit the poly(I:C) and IRF3-induced IFN1 and IFN-stimulated response element (ISRE) activation. Further experiments showed that miiuy croaker Otub1 decreases Irf3 protein abundance by promoting its proteasomal degradation. These data suggest that the evolutionarily conserved Otub1 acts as a suppressor in controlling antiviral immune response by promoting Irf3 proteasomal degradation in miiuy croaker.
PubMed: 38914152
DOI: 10.1016/j.dci.2024.105218 -
ACS Omega Jun 2024The suppression of the host's innate antiviral immune response by SARS-CoV-2, a contributing factor to the severity of disease, has been considerably studied in recent...
The suppression of the host's innate antiviral immune response by SARS-CoV-2, a contributing factor to the severity of disease, has been considerably studied in recent years. Many of these studies have focused on the actions of the structural proteins of the virus because of their accessibility to host immunological components. However, less is known about SARS-CoV-2 nonstructural and accessory proteins in relation to viral evasion. Herein, we study SARS-CoV-2 nonstructural proteins Orf3a, Orf6, and Nsp9 in a mimicked virus-infected state using poly(I:C), a synthetic analog of viral dsRNA, that elicits the antiviral immune response. Through genome-wide expression profiling, we determined that Orf3a, Orf6, and Nsp9 all modulate the host antiviral signaling transcriptome to varying extents, uniquely suppressing aspects of innate immune signaling. Our data suggest that SARS-CoV-2 Nsp9 hinders viral detection through suppression of RIG-I expression and antagonizes the interferon antiviral cascade by downregulating NF-kB and TBK1. Our data point to unique molecular mechanisms through which the different SARS-CoV-2 proteins suppress immune signaling and promote viral evasion. Nsp9 in particular acts on major elements of the host antiviral pathways to impair the antiviral immune response.
PubMed: 38911767
DOI: 10.1021/acsomega.4c02631 -
Fish & Shellfish Immunology Jun 2024Receptors of type I interferon (IFNR) play a vital role in the antiviral immune response. However, little is known about the negative regulatory role of the IFNR....
Two cytokine receptor family B (CRFB) members in orange-spotted grouper Epinephelus coioides, EcCRFB3 and EcCRFB4, negatively regulate interferon immune responses to assist nervous necrosis virus replication.
Receptors of type I interferon (IFNR) play a vital role in the antiviral immune response. However, little is known about the negative regulatory role of the IFNR. Nervous necrosis virus (NNV) is one of the most significant viruses in cultured fish, resulting in great economic losses for the aquaculture industry. In this study, two orange-spotted grouper (Epinephelus coioides) cytokine receptor family B (CRFB) members, EcCRFB3 and EcCRFB4 were cloned and characterized from NNV infected grouper brain (GB) cells. The open reading frame (ORF) of EcCRFB3 consists of 852 bp encoding 283 amino acids, while EcCRFB4 has an ORF of 1004 bp encoding 333 amino acids. The mRNA levels of EcCRFB3 or EcCRFB4 were significantly upregulated after NNV infection and the stimulation of poly (I:C) or NNV-encoded Protein A. In addition, EcCRFB3 or EcCRFB4 overexpression facilitated NNV replication, whereas EcCRFB3 or EcCRFB4 silencing resisted NNV replication. Overexpressed EcCRFB3 or EcCRFB4 inhibited the expression of IFN-I --induced ISGs. Taken together, our research provides the first evidence in fish demonstrating the role of IFN receptors to regulate the IFN signaling pathway negatively. Our findings enrich the understanding of the functions of IFNRs and reveal a novel escape mechanism of NNV.
PubMed: 38909635
DOI: 10.1016/j.fsi.2024.109718 -
The Journal of Allergy and Clinical... Jun 2024Endolysosomal compartments are acidic and contain low pH-dependent proteases, and these conditions are exploited by respiratory viruses, such as SARS-CoV-2 and influenza...
BACKGROUND
Endolysosomal compartments are acidic and contain low pH-dependent proteases, and these conditions are exploited by respiratory viruses, such as SARS-CoV-2 and influenza virus, for escaping into the cytosol. Moreover, endolysosomes contain various pattern recognition receptors (PRRs), which respond to virus-derived pathogen-associated molecular patterns (PAMPs) by production of pro-inflammatory cytokines/chemokines. However, excessive pro-inflammatory responses can lead to a potentially lethal cytokine storm.
OBJECTIVES
Here we investigated the endosomal PRR expression profile in primary human small airway epithelial cells (HSAECs), and whether blockade of endolysosomal acidification affects their cytokine/chemokine production after challenge with virus-derived stimulants.
METHODS
HSAECs were exposed to stimulants mimicking virus-derived PAMPs, either in the absence or presence of compounds causing blockade of endolysosomal acidification, followed by measurement of cytokine expression and release.
RESULTS
We show that toll-like receptor 3 (TLR3) is the major endosomal PRR expressed by HSAECs, and that TLR3 expression is strongly induced by TLR3 agonists, but not by a range of other PRR agonists. We also demonstrate that TLR3 engagement with its agonists elicits a robust pro-inflammatory cytokine/chemokine response, which is profoundly suppressed through blockade of endolysosomal acidification, by bafilomycin A1, monensin, or niclosamide. Using TLR3 reporter cells, it was confirmed that TLR3 signaling is strongly induced by Poly(I:C) and that blockade of endolysosomal acidification efficiently blocked TLR3 signaling. Finally, we show that blockade of endolysosomal acidification causes a reduction in the levels of TLR3 mRNA and protein.
CONCLUSION
These findings show that blockade of endolysosomal acidification suppresses TLR3-dependent cytokine and chemokine production in HSAECs.
CLINICAL IMPLICATION
These findings may be exploited for therapeutic strategies aiming to ameliorate the cytokine storm in response to respiratory virus infection.
PubMed: 38906273
DOI: 10.1016/j.jaci.2024.05.031 -
European Journal of Microbiology &... Jun 2024Cell cultures are models in biological and medical research to understand physiological and pathological processes. Cell lines are not always available depending on cell...
Cell cultures are models in biological and medical research to understand physiological and pathological processes. Cell lines are not always available depending on cell type and required species. In addition, the immortalization process often affects cell biology. Primary cells generally maintain a greater degree of similarity in short-term culture to the cells in tissue. Goal of this study was to verify the suitability of chicken primary epithelial caecal cells (PECCs) for in vitro investigations of host‒pathogen interactions. Epithelial nature of PECCs was confirmed by detection of tight and adherens junctions and cobblestone-like cell morphology. Sialic acids distribution was similar to that in caecal cyrosections. To understand the capacity of PECCs to respond to microbial challenges, the Toll-like receptors (TLRs) repertoire was determined. Exposure of PECCs to polyinosinic-polycytidylic acid (poly(I:C)) or lipopolysaccharide (LPS) led to upregulation of type I and III interferon (IFN) as well as interleukin (IL-) 1β, IL-6 and IL-8 mRNA expression. Overall, the PECCs showed properties of polarized epithelial cells. The presence of TLRs, their differential expression, as well as pattern recognition receptor dependent immune responses enable PECCs to act as suitable in vitro model for host‒pathogen interaction studies, which are difficult to conduct under in vivo conditions.
PubMed: 38905002
DOI: 10.1556/1886.2024.00053 -
Brain, Behavior, and Immunity Jun 2024Rodent models of maternal immune activation (MIA) are increasingly used as experimental tools in preclinical research of immune-mediated neurodevelopmental disorders and...
Rodent models of maternal immune activation (MIA) are increasingly used as experimental tools in preclinical research of immune-mediated neurodevelopmental disorders and mental illnesses. Using a viral-like MIA model that is based on prenatal poly(I:C) exposure in mice, we have recently identified the existence of subgroups of MIA-exposed offspring that show dissociable behavioral, transcriptional, brain network and inflammatory profiles even under conditions of genetic homogeneity and identical MIA. Here, we tested the hypothesis that the intrauterine positions of fetuses, which are known to shape individual variability in litter-bearing mammals through variations in fetal hormone exposure, may contribute to the variable outcomes of MIA in mice. MIA was induced by maternal administration of poly(I:C) on gestation day 12 in C57BL/6N mice. Determining intrauterine positions using delivery by Cesarean section (C-section), we found that MIA-exposed offspring developing between female fetuses only (0M-MIA offspring) displayed significant deficits in sociability and sensorimotor gating at adult age, whereas MIA-exposed offspring developing between one or two males in utero (1/2M-MIA offspring) did not show the same deficits. These intrauterine position effects similarly emerged in male and female offspring. Furthermore, while MIA elevated fetal brain levels of pro- and anti-inflammatory cytokines independently of the precise intrauterine position and sex of adjacent fetuses during the acute phase, fetal brain levels of TNF-α remained elevated in 0M-MIA but not 1/2M-MIA offspring until the post-acute phase in late gestation. As expected, 1/2M offspring generally showed higher testosterone levels in the fetal brain during late gestation as compared to 0M offspring, confirming the transfer of testosterone from male fetuses to adjacent male or female fetuses. Taken together, our findings identify a novel source of within-litter variability contributing to heterogeneous outcomes of short- and long-term effects in a mouse model of MIA. In broader context, our findings highlight that individual differences in fetal exposure to hormonal and inflammatory signals may be a perinatal factor that shapes risk and resilience to MIA.
PubMed: 38897330
DOI: 10.1016/j.bbi.2024.06.015 -
Advanced Healthcare Materials Jun 2024Immunotherapy has emerged as a powerful weapon against lung cancer, yet only a fraction of patients respond positively to the treatment. Poly(I:C) effectively triggers...
Immunotherapy has emerged as a powerful weapon against lung cancer, yet only a fraction of patients respond positively to the treatment. Poly(I:C) effectively triggers both innate and adaptive immunity. It is also capable of inducing immunogenic cell death (ICD) in tumor cells. However, its efficacy is hindered by its instability in vivo and limited cellular uptake. To address this, we encapsulated poly(I:C) in cRGD-targeted polymersomes (t-PPIC), which significantly increased its stability and uptake, resulting in the vital activation of dendritic cells (DCs) and apoptosis of lung tumor cells in vitro. In a murine LLC lung tumor model, systemic administration of t-PPIC effectively suppressed tumor growth and led to striking survival benefits, with 40% of the mice becoming tumor-free. Notably, t-PPIC provoked stronger apoptosis and ICD in tumor tissue and elicited a more potent stimulation of DCs, recruitment of NK cells, and activation of CD8 T cells, compared to free poly(I:C) and nontargeted PPIC controls. Furthermore, when combined with immune checkpoint inhibitors or radiotherapy, t-PPIC amplified the antitumor immune response, resulting in complete regression in 60% of the mice. These compelling findings underscore the potential of integrin-targeted polymersomal poly(I:C) to enhance antitumor immunity by simultaneously inducing ICD and systemic immune activation. This article is protected by copyright. All rights reserved.
PubMed: 38896790
DOI: 10.1002/adhm.202400784 -
BioRxiv : the Preprint Server For... Jun 2024Alterations induced by maternal immune activation (MIA) during gestation impact the subsequent neurodevelopment of progeny, a process that in humans, has been linked to...
Alterations induced by maternal immune activation (MIA) during gestation impact the subsequent neurodevelopment of progeny, a process that in humans, has been linked to the development of several neuropsychiatric conditions. To undertake a comprehensive examination of the molecular mechanisms governing MIA, we have devised an in vitro model based on neural stem cells (NSCs) sourced from fetuses carried by animals subjected to Poly I:C treatment. These neural progenitors demonstrate proliferative capacity and can be effectively differentiated into both neurons and glial cells. Transcriptomic, proteomic, and phosphoproteomic analyses conducted on these cellular models, in conjunction with counterparts from control treatments, revealed discernible shifts in the expression levels of a specific subset of proteins implicated in neuronal function. Noteworthy, we found an absence of congruence between these alterations at the transcriptomic level, suggesting that differences in protein translation contribute to the observed dysregulation. Furthermore, the phosphoproteomic data highlighted a discernible discrepancy in the basal phosphorylation of proteins between differentiated cells from both experimental groups, particularly within proteins associated with cytoskeletal architecture and synaptic functionality, notably those belonging to the MAP family. Observed alterations in MAP phosphorylation were found to potentially have functional consequences as they correlate with changes in neuronal plasticity and the establishment of neuronal synapses. Our data agrees with previous published observations and further underscore the importance of MAP2 phosphorylation state on its function and the impact that this protein has in neuronal structure and function.
PubMed: 38895311
DOI: 10.1101/2024.06.07.597886