Polyinosinic-polycytidylic Acid Research Articles

Toll-like receptor 21 in Labeo rohita recognizes double-stranded RNA and lipopolysaccharides by engaging the critical motifs in the LRR domain and gets activated against bacterial assaults

TLR (Toll-like receptor)-21 is a non-mammalian TLR and exhibits a unique function within the innate immune systems of fishes, birds, and amphibians. Despite its important role as PRR (pattern recognition receptor), research on TLR21 in many fish species, as well as in rohu (Labeo rohita), remains relatively limited. This article describes the molecular cloning of LrTLR21 (TLR21 in L. rohita), 3D (3-dimensional) modelling of its LRR (leucine-rich repeat)-regions, prediction of LRR18 to LRR20 as the LPS (lipopolysaccharide)-binding site, and LRR1 to LRR5 and LRR21 to LRR23 as the poly I:C (polyinosinic-polycytidylic acid) binding sites. It also describes the response of LrTLR21 in response to Aeromonas hydrophila and Edwardsiella tarda infections and PAMPs (pathogen-associated molecular patterns) (LPS and poly I:C)-stimulations. The ORF (open reading frame) of the LrTLR21 comprises 2955 nucleotides, encoding 984 aa (amino acid) residues with molecular weight and isoelectric point of 113.791 kDa and ∼8.79, respectively. Domain analysis of the deduced LrTLR21 displayed the existence of a signal peptide (residues 1–24), 26 LRR regions (residues 61–685), a TM (transmembrane) domain (residues 736–758), and a TIR (Toll/interleukin-1 receptor) domain (residues 790–937). The 3D model of LrTLR21-LRR regions has parallel β-sheets and few α-helices. Phylogenetically, LrTLR21 is closely related to the Onychostoma macrolepis and Carassius gibelio TLR21, and during ontogenesis, it is expressed in most of the developmental stages. In rohu fingerlings, it is consistently expressed in all examined tissues viz., skin, liver, heart, blood, eye, muscle, kidney, intestine, brain, gill, and spleen. Upon exposure to E. tarda and A. hydrophila infections, as well as LPS and poly I:C stimulations, the expression of LrTLR21 is significantly (p < 0.05) increased in the blood, kidney, liver, and gill. In the RBCs (red blood cells), PBLs (peripheral blood leukocytes), and kidney macrophages, LrTLR21 is also significantly induced following in-vivo and in-vitro LPS and poly I: C-stimulations. These findings on LrTLR21 are the first ones showing its structural insights and PAMPs binding motifs and its key role in recognizing pathogens and their PAMPs in RBCs, PBLs, and macrophages.

In vitro analysis of antiviral immune response against avian influenza virus in chicken tracheal epithelial cells.

Avian influenza virus (AIV) infections first affect the respiratory tract of chickens. The epithelial cells activate the host immune system, which leads to the induction of immune-related genes and the production of antiviral molecules against external environmental pathogens. In this study, we used chicken tracheal epithelial cells (TECs) in vitro model to investigate the immune response of the chicken respiratory tract against avian respiratory virus infections. Eighteen-day-old embryonic chicken eggs were used to culture the primary chicken TECs. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC) analysis of epithelial cell-specific gene makers were performed to confirm the characteristics, morphology, and growth pattern of primary cultured chicken TECs. Moreover, to investigate the cellular immune response to AIV infection or polyinosinic-polycytidylic acid (poly (I:C)) treatment, the TECs were infected with the H5N1 virus or poly (I:C). Then, immune responses were validated by RT-qPCR and western blotting. The TECs exhibited polygonal morphology and formed colony-type cell clusters. The RT-qPCR results showed that H5N1 infection induced a significant expression of antiviral genes in TECs. We found that TECs treated with poly (I:C) and exposed to AIV infection-mediated activation of signaling pathways, leading to the production of antiviral molecules (e.g., pro-inflammatory cytokines and chemokines), were damaged due to the loss of junction proteins. We observed the activation of the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, which are involved in inflammatory response by modulating the release of pro-inflammatory cytokines and chemokines in TECs treated with poly (I:C) and pathway inhibitors. Furthermore, our findings indicated that poly (I:C) treatment compromises the epithelial cell barrier by affecting junction proteins in the cell membrane. Our study highlights the utility of in vitro TEC models for unraveling the mechanisms of viral infection and understanding host immune responses in the chicken respiratory tract.

Extracellular RNA Induces Neutrophil Recruitment Via Toll-Like Receptor 3 During Venous Thrombosis After Vascular Injury.

Venous thromboembolism is associated with endothelial cell activation that contributes to the inflammation-dependent activation of the coagulation system. Cellular damage is associated with the release of different species of extracellular RNA (eRNA) involved in inflammation and coagulation. TLR3 (toll-like receptor 3), which recognizes (viral) single-stranded or double-stranded RNAs and self-RNA fragments, might be the receptor of these species of eRNA during venous thromboembolism. Here, we investigate how the TLR3/eRNA axis contributes to venous thromboembolism. Thrombus formation and size in wild-type and TLR3 deficient (-/-) mice were monitored by ultrasonography after venous thrombosis induction using the ferric chloride and stasis models. Mice were treated with RNase I, with polyinosinic-polycytidylic acid, a TLR3 agonist, or with RNA extracted from murine endothelial cells. Gene expression and signaling pathway activation were analyzed in HEK293T cells overexpressing TLR3 in response to eRNA or in human umbilical vein endothelial cells transfected with a small interference RNA against TLR3. Plasma clot formation on treated human umbilical vein endothelial cells was analyzed. Thrombosis exacerbated eRNA release invivo and increased eRNA content within the thrombus. RNase I treatment reduced thrombus size compared with vehicle-treated mice (P<0.05). Polyinosinic-polycytidylic acid and eRNA treatments increased thrombus size in wild-type mice (P<0.01 and P<0.05), but not in TLR3-/- mice, by reinforcing neutrophil recruitment (P<0.05). Mechanistically, TLR3 activation in endothelial cells promotes CXCL5 (C-X-C motif chemokine 5) secretion (P<0.001) and NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation (P<0.05). Finally, eRNA triggered plasma clot formation invitro (P<0.01). We show that eRNA and TLR3 activation enhance venous thromboembolism through neutrophil recruitment possibly through secretion of CXCL5, a potent neutrophil chemoattractant.

Poly I: C Alleviated Duck Intestinal Injury Infected with Duck Viral Enteritis by Inhibiting Apoptosis.

Duck enteritis virus (DEV) may lead to vascular injury, gastrointestinal mucosal erosion, lymphoid organ injury, and Polyinosinic-polycytidylic acid (Poly I:C) has an antiviral effectby inducing low levels of interferon. The purpose of this study was to explore the pathogenesis of DEV-induced intestinal injury in ducks and to verify the therapeutic effects of different concentrations of Poly I:C. In this study, duck enteritis model was established by infecting healthy Pekin ducks with DEV. Duck intestinal tissues were extracted from normal control group, model group, and treatment group with different doses of Poly I:C. In vivo, HE and TUNEL staining were used to observe the morphological changes and apoptosis. In vitro, the proliferation and apoptosis of duck intestinal epithelial cells were evaluated by MTT assay, TUNEL staining, and flow cytometry. The results showed that Poly I:C protected ducks from DEV toxicity by improving intestinal morphology and inhibiting apoptosis. In addition, the antiviral effect of Poly I:C on DEV was found in a dose-dependent manner, with a more relatively obvious effect at a high dose of Poly I:C. All in all, these results demonstrated that Poly I:C played a vital role in the apoptosis induced by DEV in ducks and modest dose of Poly I:C treatment worked well and may provide important reference for the development of new antiviral drugs in the future.

Vortioxetine improved schizophrenia-like behavioral deficits in a Poly I:C-induced maternal immune activation model of schizophrenia in rats.

Several studies provide clear evidence that exposure to various infections during pregnancy are linked with an increased risk for schizophrenia. In preclinical studies, administration of polyinosinic-polycytidylic acid (Poly I:C) in pregnant rodents can induce maternal immune activation leading to impairments in brain function in the offspring. The aim of this study was to investigate the effect of vortioxetine, a multimodal selective serotonin reuptake inhibitor (SSRI), in the pathophysiology of Poly I:C-induced schizophrenia-like model in rats. For this purpose, Poly I:C (8 mg/kg, ip) was injected into pregnant animals 14 days after mating, and tail blood was taken for determination of IL-6 levels after 2 h. At postnatal days 83-86, behavioral tests were performed. Our results revealed that Poly I:C caused impairments in prepulse inhibition, novel object recognition, social interaction, and open-field tests. Chronic administration of vortioxetine (2.5, 5, and 10 mg/kg, ip, postnatal days 69-83) caused significant improvements in these deficits. Overall, our findings indicate that vortioxetine may provide new therapeutic approaches for the treatment of schizophrenia. We think that increased serotonergic activity in frontal brain regions may provide the ameliorative effect of vortioxetine, especially on negative and cognitive symptoms. Therefore, it will be useful to determine the efficacy of vortioxetine with combined drugs with further studies.

Molecular characterization, expression profiling, and functional analysis of prohibitin 1 in red seabream, Pagrus major

Prohibitin 1 (PHB1) is ubiquitously expressed in multiple compartments within cells and is involved in the cell cycle, cell signaling, apoptosis, transcriptional regulation, and mitochondrial biogenesis at the cellular level and in the inflammation-associated and immunological functions of B and T lymphocytes. PHB1 is an important protein that performs antioxidant regulation and immune functions inside and outside cells but has not been sufficiently studied in teleost fish. Our study aimed to elucidate the functional properties and gain new insights into the biological processes and immune system of red seabream (Pagrus major), a commercially important fish cultured in South Korea and East Asia. PHB1 mRNA was most abundantly expressed in the head kidney of healthy red seabream, and significant changes in its expression were observed after artificial infection with bacteria and viruses. On analysis, reporter gene was also significantly upregulated by polyinosinic-polycytidylic acid, lipopolysaccharides, and hydrogen peroxide. Consequent to the functional characterization of PHB1 in cells via recombinant protein preparation, the activity of leukocytes was enhanced and the reactive oxygen species-induced stress in red blood cells was reduced. The results reveal the functional characteristics of PHB1 and provide new insights into the biological processes and immune system of P. major, with beneficial implications in the study of stress responses.

Interferon-stimulated gene 56 positively regulates Toll-like receptor 3-mediated CXCL10 expression in human renal proximal tubular epithelial cells.

Viral infections in tubular epithelial cells lead to the production of inflammatory cytokines by innate immunity, causing tubulointerstitial nephritis. TLR3 recognizes viral infections and acts via the activation of interferon (IFN)/IFN-stimulated genes (ISGs). This study investigates the role of ISG56, a representative ISG, in TLR3 signaling in cultured human renal proximal tubular epithelial cells (hRPTECs). To this end, hRPTECs were stimulated by a synthetic TLR3 ligand, polyinosinic-polycytidylic acid (poly IC), recombinant human interferon-β [r(h)IFN-β] or Japanese encephalitis virus (JEV) infection and assayed for inflammatory cytokine mRNA expression by RT-qPCR, and protein expression via western blotting or ELISA. ISG56 was expressed by poly IC or r(h)IFN-β and IFN-β knockdown reduced poly IC-induced expression of ISG56 and CXCL10. Moreover, ISG56 knockdown reduced poly IC- or r(h)IFN-β-induced expression of CXCL10 at the same time as increasing JEV growth and reducing CXCL10 expression induced by JEV infection. Overall, TLR3 signaling induced IFN-β-dependent expression of ISG56 and CXCL10. We show that ISG56 possibly plays a critical role in antiviral immunity of hRPTECs by positive regulation of IFN-β-mediated CXCL10 expression downstream of TLR3.

Morphological and immunological characterization of primary cultured chicken caecal epithelial cells.

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.

Selenium nanoparticles enhance mucosal immunity against Mycobacterium bovis infection

Selenium nanoparticles (SeNPs) enhance the immune response as adjuvants, increasing the efficacy of viral vaccines, including those for COVID-19. However, the efficiency of mucosal SeNPs in boosting vaccine-induced protective immunity against tuberculosis remains unclear. Therefore, this study aims to investigate whether the combination of SeNPs with the AH antigen (Ag85A-HspX) can boost respiratory mucosal immunity and thereby enhance the protective effects against tuberculosis. We synthesized SeNPs and assessed their impact on the immune response and protection against Mycobacterium bovis (M. bovis) as a mucosal adjuvant in mice, administered intranasally at a dose of 20 µg. SeNPs outperformed polyinosinic-polycytidylic acid (Poly IC) in stimulating the maturation of bone marrow-derived dendritic cells (BMDCs), which enhanced antigen presentation. SeNPs significantly activated and proliferated tissue-resident memory T cells (TRMs) and effector CD4+ T cells in the lungs. The vaccines elicited specific antibody responses in the respiratory tract and stimulated systemic Th1 and Th17 immune responses. Immunization with AH and SeNPs led to higher levels of mucosal secretory IgA in bronchoalveolar lavage fluid (BALF) and secretory IL-17 in splenocytes. Moreover, SeNPs immunized mice showed reduced M. bovis infection loads and inflammatory lesions in the lungs post-challenge. Notably, immunization with AH and SeNPs significantly reduced bacterial load in the lungs, achieving the lowest levels compared to all other tested groups. This study calls for pre-clinical investigation of AHB-SeNPs as an anti-bovine tuberculosis vaccine and for exploring its human vaccine potential, which is anticipated to aid in the development of innovative vaccines or adjuvants.

Protective Effects of Long Double-Stranded RNA with Different CpG Motifs against Miamiensis avidus and Viral Hemorrhagic Septicemia Virus (VHSV) Infections in Olive Flounder (Paralichthys olivaceus)

The South Korean aquaculture industry has incurred considerable production losses due to various infectious diseases. Artificially synthesized polyinosinic–polycytidylic acid (poly I:C), structurally similar to double-stranded RNA (dsRNA) and cytidine–phosphate–guanosine oligodeoxynucleotides, can enhance immune responses and protect against diseases. Here, we investigated dsRNA molecules with different cytidine–phosphate–guanosine (CpG) motifs (dsRNA-CpGMix) as fused agents to treat Miamiensis avidus and viral hemorrhagic septicemia virus (VHSV) infection in olive flounders. We further investigated the efficacy of specific sequence motifs in dsRNA in modulating immunostimulatory effects. Fish treated with poly I:C or dsRNA-CpGMix exhibited higher survival rates than the control group. Olive flounder leukocytes stimulated with poly I:C or dsRNA-CpGMix showed increased scuticocidal activity in the presence of inactivated immune sera. dsRNA with CpG motif sequences induced higher resistance against M. avidus and VHSV infections than dsRNA without CpG motif sequences, and the dsRNA-CpGMix group showed upregulated ISG15 or Mx compared to the dsRNA-GFP group. Thus, dsRNA containing CpG motifs can be used as effective immunostimulants to enhance resistance against viral and parasitic diseases in olive flounder. The specific sequences of the CpG motifs in dsRNA may be important for enhancing immune responses and resistance against M. avidus and VHSV infections in olive flounders.

Abstract B025: A genome-wide CRISPR screen identifies synthetic lethality of double-stranded RNA with BRCA1 loss

Abstract Double-stranded RNAs (dsRNAs) are potent immunostimulatory nucleic acids of viral origin but also physiologically produced by mammalian cells. Recent studies demonstrated that elevating dsRNA levels, either exogenously through dsRNA mimetics like Polyinosinic-polycytidylic acid (pI:C) or endogenously through epigenetic modulation of retroviral elements, can elicit anticancer effects. In response to dsRNA, cells elicit a dual response consisting in Interferon (IFN) production and translational shutdown associated, in some cells, with cell death. These two responses are linked in a feed-forward loop in which induced cytokines themselves enhance dsRNA sensing by the death-inducing pathway, complicating our understanding of the cell-intrinsic mechanisms at play. The 2',5'-oligoadenylate synthetase (OAS)-RNASEL system is widely thought to be the main responsible for dsRNA-induced cell death, but it remains unclear how its activity leads to cell death. To elucidate these mechanisms, we decided to minimize the impact of interferon modulation by studying dsRNA-induced death in interferon-saturated conditions. We performed a genome-wide CRISPR screen for factors involved in dsRNA-induced death in our interferon-saturated conditions in HT29 cells. Pathway enrichment analysis of screen hits revealed that RNA surveillance, RNA Polymerase II Transcription Initiation, Mitochondrial translation and respiratory chain and DNA Repair genes have a protective role against dsRNA increase. In particular, we identifiedBRCA1 but not BRCA2 as a candidate hit. The differential sensitivity of BRCA1-KO cells was subsequently validated in cloncal populations. Ongoing research is aimed at validating this interaction in vivo models. Our work shed light on potential new mechanisms involved in dsRNA dependent cell death, revealing a connection with DNA damage and BRCA1. If confirmed, these findings may pave the way for the use of dsRNA mimetics, possibly in conjunction with immune checkpoint inhibitors, in the treatment of BRCA1-mutated tumors. Citation Format: Luca Mazzarella, Deborah Trastulli, Sara Galavotti, Gianluca Vozza, Veronica Pinamonti, Nunzia Zagaria. A genome-wide CRISPR screen identifies synthetic lethality of double-stranded RNA with BRCA1 loss [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B025.

The Effect of Exosomes Isolated from Poly (I:C) Treated Human Wharton's Jelly Mesenchymal Stem Cells on CD4+CD25+Foxp3+ Regulatory T Cells

Mesenchymal stem cells (MSCs) are a potential cell therapy candidate for autoimmune and inflammatory diseases due to their multilineage capacity and immune modulating function. MSCs exert immunomodulatory effects on target cells through the secretion of exosomes. Inflammatory conditions such as Toll-like receptors (TLRs) engagement can change the biological functions and immunomodulatory activities of MSCs and the contents of exosomes derived from MSCs are changed. Regulatory T-cells (Treg) are crucial for maintaining immune cell homeostasis and self-tolerance. Our study aimed to investigate the impact of isolated exosomes from hWJ-MSCs that were treated with Poly (I:C) on regulatory CD4 CD25 Foxp3 T-cells. MSCs were harvested from human umbilical cord Wharton’s Jelly by explant method. Stem cells were treated by Polyinosinic-polycytidylic acid sodium salt (Poly (I:C)) for 48 hours. Exosomes were extracted from supernatant of cells and Scanning electron microscopy (SEM) and Dynamic light scattering (DLS) were performed for them. Peripheral blood mononuclear cells (PBMCs) isolated from the healthy donors were stimulated with PHA (Phytohemagglutinin) and co-cultured with Poly (I:C) treated hWJ-MSCs derived exosome and untreated hWJ-MSCs derived exosome or without hWJ-MSCs-derived exosome for 6 days. Then, frequency of CD4+CD25+ Foxp3+ regulatory T cells was measured by flow cytometry. Our results showed that exosomes isolated from Poly (I:C) treated hWJ-MSCs significantly increased frequency of CD4+CD25+ Foxp3+ regulatory T cells compared to the untreated hWJ-MSCs derived exosome group and control group. Stimulation by TLR3 improved the anti-inflammatory features of exosomes that were derived from hWJ-MSCs by increasing the frequency of Treg cells.

TLR21 is involved in the NF-κB and IFN-β pathways in largemouth bass (Micropterus salmoides) and interacts with TRIF but not with the Myd88 adaptor

Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-β (IFN-β) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-β (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.

A newly identified scallop MyD88 interacts with TLR and functions in innate immunity

Myeloid differentiation factor-88 (MyD88) is a key adaptor of the toll-like receptor (TLR) signaling pathway and plays a crucial role in innate immune signal transduction in animals. However, the MyD88-mediated signal transduction mechanism in shellfish has not been well studied. In this study, a new MyD88 gene, CfMyD88-2, was identified in the Zhikong scallop, Chlamys farreri. The 1779 bp long open reading frame encodes 592 amino acids. The N-terminus of CfMyD88-2 contains a conserved death domain (DD), followed by a TIR (TLR/Interleukin-1 Receptor) domain. The results of the multi-sequence comparison showed that the TIR domain sequences were highly conserved. Phylogenetic analysis revealed that CfMyD88-2 was first associated with Mizuhopecten yessoensis MyD88-4 and Argopecten irradians MyD88-4. CfMyD88-2 mRNA was expressed in all scallop tissues, as detected by qRT-PCR, and the expression level was the highest in the mantle and hepatopancreas. In addition, CfMyD88-2 mRNA expression significantly increased after pathogen-associated molecular patterns (PAMPs, such as lipopolysaccharide, peptidoglycan, or polyinosinic-polycytidylic acid) stimulation. The results of the co-immunoprecipitation experiments in HEK293T cells showed that both CfMyD88-1 and CfMyD88-2 interacted with the TLR protein of scallops, suggesting the existence of more than one functional TLR-MyD88 signaling axis in scallops. Dual luciferase reporter gene assays indicated that the overexpressed CfMyD88-2 in HEK293T cells activated interferon (IFN) α, IFN-β, IFN-γ, and NF-κB reporter genes, indicating that the protein has multiple functions. The results of the subcellular localization experiment uncovered that CfMyD88-2 was mainly localized in the cytoplasm of human cells. In summary, the novel identified CfMyD88-2 can respond to the challenge of PAMPs, participate in TLR immune signaling, and may activate downstream effector genes such as NF-κB gene. These research results will be useful in advancing the theory of innate immunity in invertebrates and provide a reference for the selection of disease-resistant scallops in the future.

NETs contribute to psoriasiform skin inflammation: A novel therapeutic approach targeting IL-36 cytokines by a small molecule tetrahydroxystilbene glucoside

BackgroundPsoriasis, a chronic immune-mediated skin disease with pathological features such as aberrant differentiation of keratinocytes, dermal-epidermal inflammation, and angiogenesis. 2,3,5,4′-Tetrahydroxy stilbene 2-Ο-β-d-glucoside (2354Glu) is a natural small molecule polyhydrostilbenes isolated from Polygonum multiglorum Thunb. The regulation of IL-36 subfamily has led to new pharmacologic strategies to reverse psoriasiform dermatitis. PurposeHere we investigated the therapeutic potential of 2354Glu and elucidated the underlying mechanism in psoriasis. MethodsThe effects of 2354Glu on IL-36 signaling were assessed by psoriasiform in vivo, in vitro and ex vivo model. The in vivo mice model of psoriasis-like skin inflammation was established by applying imiquimod (IMQ), and the in vitro and ex vitro models were established by stimulating mouse primary keratinocyte, human keratinocytes cells (HaCaT) and ex vivo skin tissue isolated from the mice back with Polyinosine-polycytidylic acid (Poly(I:C)), IMQ, IL-36γ and Lipopolysaccharide (LPS) respectively. Moreover, NETs formation was inhibited by Cl-amidine to evaluate the effect of NETs in psoriatic mouse model. The effects of 2354Glu on skin inflammation were assessed by western blot, H&E, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay and real-time quantitative PCR. ResultsIn Poly(I:C)-stimulated keratinocytes, the secretion of IL-36 was inhibited after treatment with 2354Glu, similar to the effects of TLR3, P2X7R and caspase-1 inhibitors. In aldara (imiquimod)-induced mice, 2354Glu (100 and 25 mg/kg) improved immune cell infiltration and hyperkeratosis in psoriasis by directly targeting IL-36 in keratinocytes through P2X7R-caspase-1. When treatment with 2354Glu (25 mg/kg) was insufficient to inhibit IL-36γ, NETs reduced pathological features and IL-36 signaling by interacting with keratinocytes to combat psoriasis like inflammation. Conclusion: These results indicated that NETs had a beneficial effect on psoriasiform dermatitis. 2354Glu alleviates psoriasis by directly targeting IL-36/P2X7R axis and NET formation, providing a potential candidate for the treatment of psoriasis.

Poly (I:C)-Induced microRNA-30b-5p Negatively Regulates the JAK/STAT Signaling Pathway to Mediate the Antiviral Immune Response in Silver Carp (Hypophthalmichthys molitrix) via Targeting CRFB5.

In aquaculture, viral diseases pose a significant threat and can lead to substantial economic losses. The primary defense against viral invasion is the innate immune system, with interferons (IFNs) playing a crucial role in mediating the immune response. With advancements in molecular biology, the role of non-coding RNA (ncRNA), particularly microRNAs (miRNAs), in gene expression has gained increasing attention. While the function of miRNAs in regulating the host immune response has been extensively studied, research on their immunomodulatory effects in teleost fish, including silver carp (Hyphthalmichthys molitrix), is limited. Therefore, this research aimed to investigate the immunomodulatory role of microRNA-30b-5p (miR-30b-5p) in the antiviral immune response of silver carp (Hypophthalmichthys molitrix) by targeting cytokine receptor family B5 (CRFB5) via the JAK/STAT signaling pathway. In this study, silver carp were stimulated with polyinosinic-polycytidylic acid (poly (I:C)), resulting in the identification of an up-regulated miRNA (miR-30b-5p). Through a dual luciferase assay, it was demonstrated that CRFB5, a receptor shared by fish type I interferon, is a novel target of miR-30b-5p. Furthermore, it was found that miR-30b-5p can suppress post-transcriptional CRFB5 expression. Importantly, this study revealed for the first time that miR-30b-5p negatively regulates the JAK/STAT signaling pathway, thereby mediating the antiviral immune response in silver carp by targeting CRFB5 and maintaining immune system stability. These findings not only contribute to the understanding of how miRNAs act as negative feedback regulators in teleost fish antiviral immunity but also suggest their potential therapeutic measures to prevent an excessive immune response.

Molecular characterization, expression analysis and function identification of Pf_IL-12p35, Pf_IL-23p19 and Pf_IL-12p40 genes in yellow catfish (Pelteobagrus fulvidraco)

The interleukin-12 (IL-12) family is a class of heterodimeric cytokines that play crucial roles in pro-inflammatory and pro-stimulatory responses. Although some IL-12 and IL-23 paralogues have been found in fish, their functional activity in fish remains poorly understood. In this study, Pf_IL-12p35a/b, Pf_IL-23p19 and Pf_IL-12p40a/b/c genes were cloned from yellow catfish (Pelteobagrus fulvidraco), four α-helices were found in Pf_IL-12p35a/b and Pf_IL-23p19. The transcripts of these six genes were relatively high in mucus and immune tissues of healthy individuals, and in gill leukocytes. Following Edwardsiella ictaluri infection, Pf_IL-12p35a/b and Pf_IL-23p19 mRNAs were induced in brain and kidney (or head kidney), Pf_IL-12p40a mRNA was induced in gill, and Pf_IL-12p40b/c mRNAs were induced in brain and liver (or skin). The mRNA expression of these genes in PBLs was induced by phytohaemagglutinin (PHA) and polyinosinic-polycytidylic acid (poly I:C), while lipopolysaccharides (LPS) induced the mRNA expression of Pf_IL-12p35a and Pf_IL-12p40b/c in PBLs. After stimulation with recombinant (r) Pf_IL-12 and rPf_IL-23 subunit proteins, either alone or in combination, mRNA expression patterns of genes related to T helper cell development exhibited distinct differences. The results suggest that Pf_IL-12 and Pf_IL-23 subunits may play important roles in regulating immune responses to pathogens and T helper cell development.

Interaction between maternal immune activation and postpartum immune stress in neuropsychiatric phenotypes

Epidemiological evidence has shown that maternal infection is a notable risk factor for developmental psychiatric disorders. Animal models have corroborated this link and demonstrated that maternal immune activation (MIA) induces long-term behavioural deficits and neuroimmunological responses to subsequent immune stress in offspring. However, it is unclear whether MIA offspring are more sensitive or more tolerant to immunological challenges from postnatal infections. Pregnant mice were weighed and injected with a single dose of polyinosinic-polycytidylic acid (poly I:C) or saline at gestational day 9.5, and their male offspring were exposed to poly I:C or saline again during adolescence, adulthood, and middle life. After a two-week recovery from the last exposure to poly I:C, the mice underwent behavioural and neuroendophenotypic evaluations. Finally, the mice were sacrificed, and the expression levels of inflammatory factors and the activation levels of glial cells in the cerebral cortex and hippocampus were evaluated. We found MIA mice have lifelong behavioural deficits and glial activation abnormalities. Postpartum infection exposure at different ages has different consequences. Adolescent and middle life exposure prevents sensorimotor gating deficiency, but adult exposure leads to increased sensitivity to MK-801. Moreover, MIA imposed a lasting impact on the neuroimmune profile, resulting in an enhanced cytokine-associated response and diminished microglial reactivity to postnatal infection. Our results reveal an intricate interplay between prenatal and postpartum infection in neuropsychiatric phenotypes, which identify potential windows where preventive or mitigating measures could be applied.

Structure-Activity Relationship Analysis of Fluoxetine for Suppression of Inflammatory Cytokine Production.

There is accumulating evidence that selective serotonin reuptake inhibitors (SSRIs), clinically used as antidepressants, have a beneficial effect on inflammatory diseases such as coronavirus disease 2019 (COVID-19). We previously compared the inhibitory effects of five U.S. Food and Drug Administration (FDA)-approved SSRIs on the production of an inflammatory cytokine, interleukin-6 (IL-6), and concluded that fluoxetine (FLX) showed the most potent anti-inflammatory activity. Here, we investigated the structure-activity relationship of FLX for anti-inflammatory activity towards J774.1 murine macrophages. FLX suppressed IL-6 production induced by the TLR3 agonist polyinosinic-polycytidylic acid (poly(I : C)) with an IC50 of 4.76 µM. A derivative of FLX containing chlorine instead of the methylamino group lacked activity, suggesting that the methylamino group is important for the anti-inflammatory activity. FLX derivatives bearing an N-propyl or N-(pyridin-3-yl)methyl group in place of the N-methyl group exhibited almost the same activity as FLX. Other derivatives showed weaker activity, and the N-phenyl and N-(4-trifluoromethyl)benzyl derivatives were inactive. The chlorine-containing derivative also lacked inhibitory activity against TLR9- or TLR4-mediated IL-6 production. These derivatives showed similar structure-activity relationships for TLR3- and TLR9-mediated inflammatory responses. However, the activities of all amino group-containing derivatives against the TLR4-mediated inflammatory response were equal to or higher than the activity of FLX. These results indicate that the substituent at the nitrogen atom in FLX strongly influences the anti-inflammatory effect.

LINC01002 functions as a ceRNA to regulate FRMD8 by sponging miR-4324 for the development of COVID-19

BackgroundSyndrome coronavirus-2 (SARS-CoV-2) has developed various strategies to evade the antiviral impact of type I IFN. Non-structural proteins and auxiliary proteins have been extensively researched on their role in immune escape. Nevertheless, the detailed mechanisms of structural protein-induced immune evasion have not been well elucidated.MethodsHuman alveolar basal epithelial carcinoma cell line (A549) was stimulated with polyinosinic-polycytidylic acid (PIC) and independently transfected with four structural proteins expression plasmids, including nucleocapsid (N), spike (S), membrane (M) and envelope (E) proteins. By RT-qPCR and ELISA, the structural protein with the most pronounced inhibitory effects on IFN-β induction was screened. RNA-sequencing (RNA-Seq) and two differential analysis strategies were used to obtain differentially expressed genes associated with N protein inhibition of IFN-β induction. Based on DIANA-LncBase and StarBase databases, the interactive competitive endogenous RNA (ceRNA) network for N protein-associated genes was constructed. By combining single-cell sequencing data (GSE158055), lncRNA-miRNA-mRNA axis was further determined. Finally, RT-qPCR was utilized to illustrate the regulatory functions among components of the ceRNA axis.ResultsSARS-CoV-2 N protein inhibited IFN-β induction in human alveolar epithelial cells most significantly compared with other structural proteins. RNA-Seq data analysis revealed genes related to N protein inhibiting IFNs induction. The obtained 858 differentially expressed genes formed the reliable ceRNA network. The function of LINC01002-miR-4324-FRMD8 axis in the IFN-dominated immune evasion was further demonstrated through integrating single-cell sequencing data. Moreover, we validated that N protein could reverse the effect of PIC on LINC01002, FRMD8 and miR-4324 expression, and subsequently on IFN-β expression level. And LINC01002 could regulate the production of FRMD8 by inhibiting miR-4324.ConclusionSARS-CoV-2 N protein suppressed the induction of IFN-β by regulating LINC01002 which was as a ceRNA, sponging miR-4324 and participating in the regulation of FRMD8 mRNA. Our discovery provides new insights into early intervention therapy and drug development on SARS-CoV-2 infection.