Development of an in vitro immunobiotic evaluation system against rotavirus infection in bovine intestinal epitheliocytes

2017 ◽  
Vol 8 (2) ◽  
pp. 309-321 ◽  
Author(s):  
H. Kobayashi ◽  
P. Kanmani ◽  
T. Ishizuka ◽  
A. Miyazaki ◽  
J. Soma ◽  
...  
Keyword(s):  
Immune Response ◽  
Innate Immune Response ◽  
Evaluation System ◽  
Innate Immune ◽  
Poly I:C ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial ◽  
Antiviral Immune Response ◽  
Comprehensive Information

The bovine intestinal epithelial cell line (BIE cells) expresses the Toll-like receptor (TLR)3 and is able to mount an antiviral immune response after the stimulation with poly(I:C). In the present study, we aimed to further characterise the antiviral defence mechanisms in BIE cells by evaluating the innate immune response triggered by rotavirus (RV) infection. In addition, we attempted to determine whether immunobiotic bifidobacteria are able to confer protection of BIE cells against RV infection by beneficially modulating the antiviral immune response. RV OSU (porcine) and UK (bovine) effectively infected BIE cells, while a significant lower capacity to infect BIE cells was observed for human (Wa) and murine (EW) RV. We observed that viral infection in BIE cells triggered TLR3/RIG-I-mediated immune responses with activation of IRF3 and TRAF3, induction of interferon beta (IFN-β) and up-regulation of inflammatory cytokines. Our results also demonstrated that preventive treatments with Bifidobacterium infantis MCC12 or Bifidobacterium breve MCC1274 significantly reduced RV titres in infected BIE cells and differentially modulated the innate immune response. Of note, both strains significantly improved the production of the antiviral factor IFN-β in RV-infected BIE cells. In conclusion, this work provides comprehensive information on the antiviral immune response of BIE cells against RV, that can be further studied for the development of strategies aimed to improve antiviral defences in bovine intestinal epithelial cells. Our results also demonstrate that BIE cells could be used as a newly immunobiotic evaluation system against RV infection for application in the bovine host.

scholarly journals Lactobacillus delbrueckii CRL 581 Differentially Modulates TLR3-Triggered Antiviral Innate Immune Response in Intestinal Epithelial Cells and Macrophages

2021 ◽  
Vol 9 (12) ◽  
pp. 2449
Author(s):  
Mariano Elean ◽  
Leonardo Albarracin ◽  
Kohtaro Fukuyama ◽  
Binghui Zhou ◽  
Mikado Tomokiyo ◽  
...  
Keyword(s):  
Immune Response ◽  
Epithelial Cells ◽  
Innate Immune Response ◽  
Intestinal Epithelial Cells ◽  
Starter Culture ◽  
Innate Immune ◽  
Lactobacillus Delbrueckii ◽  
Poly I:C ◽  
Intestinal Epithelial ◽  
Antiviral Immune Response

Lactobacillus delbrueckii subsp. lactis CRL 581 beneficially modulates the intestinal antiviral innate immune response triggered by the Toll-like receptor 3 (TLR3) agonist poly(I:C) in vivo. This study aimed to characterize further the immunomodulatory properties of the technologically relevant starter culture L. delbrueckii subsp. lactis CRL 581 by evaluating its interaction with intestinal epithelial cells and macrophages in the context of innate immune responses triggered by TLR3. Our results showed that the CRL 581 strain was able to adhere to porcine intestinal epithelial (PIE) cells and mucins. The CRL 581 strain also augmented the expression of antiviral factors (IFN-α, IFN-β, Mx1, OAS1, and OAS2) and reduced inflammatory cytokines in PIE cells triggered by TLR3 stimulation. In addition, the influence of L. delbrueckii subsp. lactis CRL 581 on the response of murine RAW macrophages to the activation of TLR3 was evaluated. The CRL 581 strain was capable of enhancing the expression of IFN-α, IFN-β, IFN-γ, Mx1, OAS1, TNF-α, and IL-1β. Of note, the CRL 581 strain also augmented the expression of IL-10 in macrophages. The results of this study show that the high proteolytic strain L. delbrueckii spp. lactis CRL 581 was able to beneficially modulate the intestinal innate antiviral immune response by regulating the response of both epithelial cells and macrophages relative to TLR3 activation.

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

Molecules ◽  
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.

Lactobacilli stimulate the innate immune response and modulate the TLR expression of HT29 intestinal epithelial cells in vitro

2009 ◽  
Vol 133 (1-2) ◽  
pp. 86-93 ◽  
Author(s):  
María G. Vizoso Pinto ◽  
Manuel Rodriguez Gómez ◽  
Stephanie Seifert ◽  
Bernhard Watzl ◽  
Wilhelm H. Holzapfel ◽  
...  
Keyword(s):  
Immune Response ◽  
Epithelial Cells ◽  
Innate Immune Response ◽  
Intestinal Epithelial Cells ◽  
Innate Immune ◽  
Intestinal Epithelial

scholarly journals TLR3-mediated NF-κB signaling in human esophageal epithelial cells

2009 ◽  
Vol 297 (6) ◽  
pp. G1172-G1180 ◽  
Author(s):  
Diana M. Lim ◽  
Sneha Narasimhan ◽  
Carmen Z. Michaylira ◽  
Mei-Lun Wang
Keyword(s):  
Immune Response ◽  
Epithelial Cells ◽  
Inflammatory Response ◽  
Innate Immune Response ◽  
Innate Immune ◽  
Poly I:C ◽  
Esophageal Epithelium ◽  
Esophageal Epithelial Cells ◽  
Stimulation Of

Despite its position at the front line against ingested pathogens, very little is presently known about the role of the esophageal epithelium in host innate immune defense. As a key player in the innate immune response, Toll-like receptor (TLR) signaling has not been well characterized in human esophageal epithelial cells. In the present study, we investigated the inflammatory response and signaling pathways activated by TLR stimulation of human esophageal cells in vitro. Using quantitative RT-PCR, we profiled the expression pattern of human TLRs 1–10 in primary esophageal keratinocytes (EPC2), immortalized nontransformed esophageal keratinocytes (EPC2-hTERT), and normal human esophageal mucosal biopsies and found that TLRs 1, 2, 3, and 5 were expressed both in vivo and in vitro. Using the cytokine IL-8 as a physiological read out of the inflammatory response, we found that TLR3 is the most functional of the expressed TLRs in both primary and immortalized esophageal epithelial cell lines in response to its synthetic ligand polyinosinic polycytidylic acid [poly(I:C)]. Through reporter gene studies, we show that poly(I:C)-induced NF-κB activation is critical for the transactivation of the IL-8 promoter in vitro and that nuclear translocation of NF-κB occurs at an early time point following poly(I:C) stimulation of esophageal epithelial cells. Importantly, we also show that poly(I:C) stimulation induces the NF-κB-dependent esophageal epithelial expression of TLR2, leading to enhanced epithelial responsiveness of EPC2-hTERT cells to TLR2 ligand stimulation, suggesting an important regulatory role for TLR3-mediated NF-κB signaling in the innate immune response of esophageal epithelial cells. Our findings demonstrate for the first time that TLR3 is highly functional in the human esophageal epithelium and that TLR3-mediated NF-κB signaling may play an important regulatory role in esophageal epithelial homeostasis.

scholarly journals Cyprinus carpio TRIF Participates in the Innate Immune Response by Inducing NF-κB and IFN Activation and Promoting Apoptosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Rongrong Liu ◽  
Xiaoye Liu ◽  
Meijiao Song ◽  
Yue Qi ◽  
Hua Li ◽  
...  
Keyword(s):  
Immune Response ◽  
Common Carp ◽  
Innate Immune Response ◽  
Cyprinus Carpio ◽  
Critical Role ◽  
Innate Immune ◽  
Poly I:C ◽  
Luciferase Reporter

TRIF, an important adaptor downstream of Toll-like receptor signaling, plays a critical role in the innate immune response. In this study, the full-length coding sequence of TRIF from common carp (Cyprinus carpio L.) was cloned and characterized. Bioinformatics analysis showed that common carp TRIF exhibited a conserved TIR domain and had the closest relationship with grass carp TRIF. Expression analysis revealed that TRIF was constitutively expressed in the examined tissues of common carp, with the highest expression in the spleen and the lowest expression in the head kidney, and could be upregulated under Aeromonas hydrophila and poly(I:C) stimulation in vivo and under poly(I:C), LPS, PGN, flagellin, and Pam3CSK4 stimulation in vitro. Laser confocal microscopy showed that common carp TRIF colocalized with the Golgi apparatus. A luciferase reporter assay showed that carp TRIF elicited the activity of ifn-1 and nf-κb through the C-terminal domain. Additionally, crystal violet staining and qPCR assays revealed that carp TRIF inhibited the replication of SVCV in epithelioma papulosum cyprini (EPC) cells. Then, the signaling downstream of carp TRIF was investigated. Coimmunoprecipitation and Western blotting analysis demonstrated that carp TRIF interacted with TBK1 and augmented the expression of TRAF6 and phosphorylation of TBK1. Overexpression of carp TRIF significantly enhanced the expression of interferon-stimulated genes and inflammatory cytokines. Furthermore, flow cytometric (FCM) analysis suggested that carp TRIF induced apoptosis through the activation of caspase-8. In summary, our study indicated that TRIF plays an essential role in the innate immune responses of common carp against bacterial and viral infection.

scholarly journals Protective Effects of Lactoferrin against SARS-CoV-2 Infection In Vitro

Nutrients ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 328 ◽  
Author(s):  
Claudio Salaris ◽  
Melania Scarpa ◽  
Marina Elli ◽  
Alice Bertolini ◽  
Simone Guglielmetti ◽  
...  
Keyword(s):  
Immune Response ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Plaque Assay ◽  
Immune Response Gene ◽  
Intestinal Epithelial ◽  
Antiviral Immune Response ◽  
Qrt Pcr ◽  
Anti Inflammatory

SARS-CoV-2 is a newly emerging virus that currently lacks curative treatments. Lactoferrin (LF) is a naturally occurring non-toxic glycoprotein with broad-spectrum antiviral, immunomodulatory and anti-inflammatory effects. In this study, we assessed the potential of LF in the prevention of SARS-CoV-2 infection in vitro. Antiviral immune response gene expression was analyzed by qRT-PCR in uninfected Caco-2 intestinal epithelial cells treated with LF. An infection assay for SARS-CoV-2 was performed in Caco-2 cells treated or not with LF. SARS-CoV-2 titer was determined by qRT-PCR, plaque assay and immunostaining. Inflammatory and anti-inflammatory cytokine production was determined by qRT-PCR. LF significantly induced the expression of IFNA1, IFNB1, TLR3, TLR7, IRF3, IRF7 and MAVS genes. Furthermore, LF partially inhibited SARS-CoV-2 infection and replication in Caco-2 intestinal epithelial cells. Our in vitro data support LF as an immune modulator of the antiviral immune response with moderate effects against SARS-CoV-2 infection.

High content screening and computational prediction reveal viral genes that suppress innate immune response

2021 ◽  
Author(s):  
Tai L Ng ◽  
Erika J Olson ◽  
Tae Yeon Yoo ◽  
H. Sloane Weiss ◽  
Yukiye Koide ◽  
...  
Keyword(s):  
Immune Response ◽  
Innate Immune Response ◽  
Nuclear Transport ◽  
Computational Prediction ◽  
Viral Proteins ◽  
Innate Immune ◽  
Type I ◽  
Viral Genes ◽  
Genes Encoding

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.

scholarly journals The Hepatitis C Virus-Induced Membranous Web in Liver Tissue

Cells ◽  
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.

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

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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