Fish cell cultures as in vitro models of inflammatory responses elicited by immunostimulants. Expression of regulatory genes of the innate immune response

Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single virus/gene basis. Here, we present a medium-throughput high-content cell-based assay to reveal the immunosuppressive effects of viral proteins. To test the predictive power of our approach, we developed a library of 800 genes encoding known, predicted, and uncharacterized human viral genes. We find that previously known immune suppressors from numerous viral families such as Picornaviridae and Flaviviridae recorded positive responses. These include a number of viral proteases for which we further confirmed that innate immune suppression depends on protease activity. A class of predicted inhibitors encoded by Rhabdoviridae viruses was demonstrated to block nuclear transport, and several previously uncharacterized proteins from uncultivated viruses were shown to inhibit nuclear transport of the transcription factors NF-kB and IRF3. We propose that this pathway-based assay, together with early sequencing, gene synthesis, and viral infection studies, could partly serve as the basis for rapid in vitro characterization of novel viral proteins.

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The Hepatitis C Virus-Induced Membranous Web in Liver Tissue

Cells ◽

10.3390/cells7110191 ◽

2018 ◽

Vol 7 (11) ◽

pp. 191

Author(s):

Emmanuelle Blanchard ◽

Philippe Roingeard

Keyword(s):

Immune Response ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Innate Immune Response ◽

Liver Tissue ◽

Hepatoma Cell ◽

Membrane Vesicles ◽

Innate Immune ◽

Host Cell Membrane

Host cell membrane rearrangements induced by the hepatitis C virus (HCV) have been exclusively studied in vitro. These studies have shown that HCV induces double-membrane vesicles (DMVs), which probably serve to separate replication sites from the cytoplasmic sensors of the innate immune response. We report for the first time the observation of HCV-induced membrane rearrangements in liver biopsy specimens from patients chronically infected with HCV. Unlike observations performed in vitro, the membranous web detected in liver tissue seems essentially made of clusters of single-membrane vesicles derived from the endoplasmic reticulum and close to lipid droplets. This suggests that the DMVs could be a hallmark of laboratory-adapted HCV strains, possibly due to their ability to achieve a high level of replication. Alternatively, the concealment of viral RNA in DMVs may be part of innate immune response mechanisms particularly developed in hepatoma cell lines cultured in vitro. In any case, this constitutes the first report showing the differences in the membranous web established by HCV in vitro and in vivo.

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microRNA-210 and microRNA-3570 Negatively Regulate NF-κB-Mediated Inflammatory Responses by Targeting RIPK2 in Teleost Fish

Frontiers in Immunology ◽

10.3389/fimmu.2021.617753 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hui Su ◽

Renjie Chang ◽

Weiwei Zheng ◽

Yuena Sun ◽

Tianjun Xu

Keyword(s):

Immune Response ◽

Signaling Pathway ◽

Innate Immune Response ◽

Inflammatory Responses ◽

Innate Immune ◽

Regulatory Role ◽

Inflammatory Cytokine Production ◽

Immune Genes ◽

Lower Vertebrates ◽

Antimicrobial Immunity

Pathogen infection can cause the production of inflammatory cytokines, which are key mediators that cause the host’s innate immune response. Therefore, proper regulation of immune genes associated with inflammation is essential for immune response. Among them, microRNAs (miRNAs) as gene regulator have been widely reported to be involved in the innate immune response of mammals. However, the regulatory network in which miRNAs are involved in the development of inflammation is largely unknown in lower vertebrates. Here, we identified two miRNAs from miiuy croaker (Miichthys miiuy), miR-210 and miR-3570, which play a negative regulatory role in host antibacterial immunity. We found that the expressions of miR-210 and miR-3570 were significantly upregulated under the stimulation of Gram-negative bacterium vibrio harveyi and LPS (lipopolysaccharide). Induced miR-210 and miR-3570 inhibit inflammatory cytokine production by targeting RIPK2, thereby avoiding excessive inflammation. In particular, we found that miR-210 and miR-3570 negatively regulate antimicrobial immunity by regulating the RIPK2-mediated NF-κB signaling pathway. The collective results indicated that both miRNAs are used as negative feedback regulators to regulate RIPK2-mediated NF-κB signaling pathway and thus play a regulatory role in bacteria-induced inflammatory response.

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An In Vitro Assessment of Immunostimulatory Responses to Ten Model Innate Immune Response Modulating Impurities (IIRMIs) and Peptide Drug Product, Teriparatide

Molecules ◽

10.3390/molecules26247461 ◽

2021 ◽

Vol 26 (24) ◽

pp. 7461

Author(s):

Claire K. Holley ◽

Edward Cedrone ◽

Duncan Donohue ◽

Barry W. Neun ◽

Daniela Verthelyi ◽

...

Keyword(s):

Immune Response ◽

Immune Responses ◽

Innate Immune Response ◽

Innate Immune ◽

Cytokine Secretion ◽

Poly I:C ◽

In Vitro Assays ◽

Innate Immune Responses ◽

Vitro Assay

Understanding, predicting, and minimizing the immunogenicity of peptide-based therapeutics are of paramount importance for ensuring the safety and efficacy of these products. The so-called anti-drug antibodies (ADA) may have various clinical consequences, including but not limited to the alteration in the product’s distribution, biological activity, and clearance profiles. The immunogenicity of biotherapeutics can be influenced by immunostimulation triggered by the presence of innate immune response modulating impurities (IIRMIs) inadvertently introduced during the manufacturing process. Herein, we evaluate the applicability of several in vitro assays (i.e., complement activation, leukocyte proliferation, and cytokine secretion) for the screening of innate immune responses induced by ten common IIRMIs (Bacillus subtilis flagellin, FSL-1, zymosan, ODN2006, poly(I:C) HMW, poly(I:C) LMW, CLO75, MDP, ODN2216, and Escherichia coli O111:B4 LPS), and a model biotherapeutic Forteo™ (teriparatide). Our study identifies cytokine secretion from healthy human donor peripheral blood mononuclear cells (PBMC) as a sensitive method for the in vitro monitoring of innate immune responses to individual IIRMIs and teriparatide (TP). We identify signature cytokines, evaluate both broad and narrow multiplex cytokine panels, and discuss how the assay logistics influence the performance of this in vitro assay.

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Ursodeoxycholic Acid (UDCA) Mitigates the Host Inflammatory Response during Clostridioides difficile Infection by Altering Gut Bile Acids

Infection and Immunity ◽

10.1128/iai.00045-20 ◽

2020 ◽

Vol 88 (6) ◽

Author(s):

Jenessa A. Winston ◽

Alissa J. Rivera ◽

Jingwei Cai ◽

Rajani Thanissery ◽

Stephanie A. Montgomery ◽

...

Keyword(s):

Immune Response ◽

Bile Acid ◽

Inflammatory Response ◽

Innate Immune Response ◽

Innate Immune ◽

Colonization Resistance ◽

Content Type ◽

Clostridioides Difficile

ABSTRACT Clostridioides difficile infection (CDI) is associated with increasing morbidity and mortality posing an urgent threat to public health. Recurrence of CDI after successful treatment with antibiotics is high, thus necessitating discovery of novel therapeutics against this enteric pathogen. Administration of the secondary bile acid ursodeoxycholic acid (UDCA; ursodiol) inhibits the life cycles of various strains of C. difficile in vitro, suggesting that the FDA-approved formulation of UDCA, known as ursodiol, may be able to restore colonization resistance against C. difficile in vivo. However, the mechanism(s) by which ursodiol is able to restore colonization resistance against C. difficile remains unknown. Here, we confirmed that ursodiol inhibits C. difficile R20291 spore germination and outgrowth, growth, and toxin activity in a dose-dependent manner in vitro. In a murine model of CDI, exogenous administration of ursodiol resulted in significant alterations in the bile acid metabolome with little to no changes in gut microbial community structure. Ursodiol pretreatment resulted in attenuation of CDI pathogenesis early in the course of disease, which coincided with alterations in the cecal and colonic inflammatory transcriptome, bile acid-activated receptors nuclear farnesoid X receptor (FXR) and transmembrane G-protein-coupled membrane receptor 5 (TGR5), which are able to modulate the innate immune response through signaling pathways such as NF-κB. Although ursodiol pretreatment did not result in a consistent decrease in the C. difficile life cycle in vivo, it was able to attenuate an overly robust inflammatory response that is detrimental to the host during CDI. Ursodiol remains a viable nonantibiotic treatment and/or prevention strategy against CDI. Likewise, modulation of the host innate immune response via bile acid-activated receptors FXR and TGR5 represents a new potential treatment strategy for patients with CDI.

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Human PSC-Derived Hepatocytes Express Low Levels of Viral Pathogen Recognition Receptors, but Are Capable of Mounting an Effective Innate Immune Response

International Journal of Molecular Sciences ◽

10.3390/ijms21113831 ◽

2020 ◽

Vol 21 (11) ◽

pp. 3831

Author(s):

Lena Fischer ◽

Baltasar Lucendo-Villarin ◽

David C. Hay ◽

Cliona O’Farrelly

Keyword(s):

Immune Response ◽

Innate Immune Response ◽

Innate Immune ◽

Receptor Expression ◽

Cytochrome P450 3A ◽

Metabolic Switch ◽

Viral Pathogen ◽

Pathogen Recognition Receptors ◽

Viral Responses

Hepatocytes are key players in the innate immune response to liver pathogens but are challenging to study because of inaccessibility and a short half-life. Recent advances in in vitro differentiation of hepatocyte-like cells (HLCs) facilitated studies of hepatocyte–pathogen interactions. Here, we aimed to define the anti-viral innate immune potential of human HLCs with a focus on pattern recognition receptor (PRR)-expression and the presence of a metabolic switch. We analysed cytoplasmic PRR and endosomal toll-like receptor (TLR)-expression, as well as activity and adaptation of HLCs to an inflammatory environment. We found that transcript levels of retinoic acid inducible gene I (RIG-I), melanoma differentiation antigen 5 (MDA5), and TLR3 became downregulated during differentiation, indicating the acquisition of a more tolerogenic phenotype, as expected in healthy hepatocytes. HLCs responded to activation of RIG-I by producing interferons (IFNs) and IFN-stimulated genes. Despite low-level levels of TLR3, receptor expression was upregulated in an inflammatory environment. TLR3 signalling induced expression of proinflammatory cytokines at the gene level, indicating that several PRRs need to interact for successful innate immune activation. The inflammatory responsiveness of HLCs was accompanied by the downregulation of cytochrome P450 3A and 1A2 activity and decreased serum protein production, showing that the metabolic switch seen in primary hepatocytes during anti-viral responses is also present in HLCs.

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Differential modulation of innate immune response by epinephrine and estradiol

Hormone Molecular Biology and Clinical Investigation ◽

10.1515/hmbci-2016-0046 ◽

2017 ◽

Vol 30 (3) ◽

Cited By ~ 3

Author(s):

Sona Margaryan ◽

Armenuhi Hyusyan ◽

Anush Martirosyan ◽

Shushan Sargsian ◽

Gayane Manukyan

Keyword(s):

Immune Response ◽

Innate Immune Response ◽

Inflammatory Responses ◽

Innate Immune ◽

Dependent Manner ◽

Induced Expression ◽

Short Term Exposure ◽

Tnf Α ◽

Vitro Effect ◽

Dose Dependent

AbstractBackgroundAlthough it is widely accepted that catecholamines and estrogens influence immunity and have consequences for health, their effect on innate immunity (e.g. monocytes and neutrophils) is still not fully investigated.Materials and methodsOur study aimed to analyze the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, monocyte chemoattractant protein (MCP)-1 and IL-8 by whole blood cells following short-term exposure to epinephrine (Epi) and 17β-estradiol (E2) in the presence or absence of lipopolysaccharide (LPS). We also evaluated the in vitro effect of these hormones on expression of β2 integrin (CD11b/CD18) and L-selectin (CD62L) by circulating neutrophils and monocytes in the blood of healthy subjects.ResultsEpi has shown a potential to modulate the production of pro-inflammatory mediators. Its exposure resulted in significantly increased production of IL-8 in a dose-dependent manner. On the contrary, a dose-dependent suppression of LPS-induced production of IL-1β, IL-8, and MCP-1 by Epi was observed. In neutrophils, a modest rise in CD11b expression was observed after Epi exposure. Simultaneously, Epi suppressed LPS-induced expression of CD11b and CD18. In monocytes, Epi suppressed LPS-induced expression of C11b. E2 inhibited LPS-induced TNF-α production and caused a significant decrease in CD62L expression in both cell populations. No significant changes were observed after double exposure of cells with Epi and E2.ConclusionsThus, our results show that Epi and E2 differentially modulate the innate immune response and have a dual effect on cytokine modulation. The findings suggest that the observed immunoregulatory role of Epi and E2 may influence the outcome in endotoxin responses and can be critical in the regulation of inflammatory responses.

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Differential immunomodulatory effect of vitamin D (1,25 (OH)2 D3) on the innate immune response in different types of cells infected in vitro with infectious bursal disease virus

Poultry Science ◽

10.1016/j.psj.2020.06.006 ◽

2020 ◽

Vol 99 (9) ◽

pp. 4265-4277

Author(s):

J. Jaime ◽

D.S. Vargas-Bermúdez ◽

A. Yitbarek ◽

J. Reyes ◽

J.C. Rodríguez-Lecompte

Keyword(s):

Vitamin D ◽

Immune Response ◽

Disease Virus ◽

Innate Immune Response ◽

Infectious Bursal Disease Virus ◽

Innate Immune ◽

Infectious Bursal Disease ◽

Different Types ◽

Bursal Disease

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The transcription factor NRF2 enhances melanoma malignancy by blocking differentiation and inducing COX2 expression

Oncogene ◽

10.1038/s41388-020-01477-8 ◽

2020 ◽

Vol 39 (44) ◽

pp. 6841-6855 ◽

Cited By ~ 3

Author(s):

Christina Jessen ◽

Julia K. C. Kreß ◽

Apoorva Baluapuri ◽

Anita Hufnagel ◽

Werner Schmitz ◽

...

Keyword(s):

Oxidative Stress ◽

Immune Response ◽

Transcription Factor ◽

Prostaglandin E2 ◽

Innate Immune Response ◽

Stress Responses ◽

Melanoma Cells ◽

Innate Immune ◽

Dependent Manner

AbstractThe transcription factor NRF2 is the major mediator of oxidative stress responses and is closely connected to therapy resistance in tumors harboring activating mutations in the NRF2 pathway. In melanoma, such mutations are rare, and it is unclear to what extent melanomas rely on NRF2. Here we show that NRF2 suppresses the activity of the melanocyte lineage marker MITF in melanoma, thereby reducing the expression of pigmentation markers. Intriguingly, we furthermore identified NRF2 as key regulator of immune-modulating genes, linking oxidative stress with the induction of cyclooxygenase 2 (COX2) in an ATF4-dependent manner. COX2 is critical for the secretion of prostaglandin E2 and was strongly induced by H2O2 or TNFα only in presence of NRF2. Induction of MITF and depletion of COX2 and PGE2 were also observed in NRF2-deleted melanoma cells in vivo. Furthermore, genes corresponding to the innate immune response such as RSAD2 and IFIH1 were strongly elevated in absence of NRF2 and coincided with immune evasion parameters in human melanoma datasets. Even in vitro, NRF2 activation or prostaglandin E2 supplementation blunted the induction of the innate immune response in melanoma cells. Transcriptome analyses from lung adenocarcinomas indicate that the observed link between NRF2 and the innate immune response is not restricted to melanoma.

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