Innate immune response of human epidermal keratinocytes and dermal fibroblasts
            to in vitro
            incubation of
            Trichophyton benhamiae
            DSM
            6916

The LabEx Milieu Interieur (MI) project is a clinical study centered on the detailed characterization of the baseline and induced immune responses in blood samples from 1,000 healthy donors. Analyses of these samples has lay ground for seminal studies on the genetic and environmental determinants of immunologic variance in a healthy cohort population. In the current study we developed in vitro methods enabling standardized quantification of MI-cohort-derived primary fibroblasts responses. Our results show that in vitro human donor cohort fibroblast responses to stimulation by different MAMPs analogs allows to characterize individual donor immune-phenotype variability. The results provide proof-of-concept foundation to a new experimental framework for such studies. A bio-bank of primary fibroblast lines was generated from 323 out of 1,000 healthy individuals selected from the MI-study cohort. To study inter-donor variability of innate immune response in primary human dermal fibroblasts we chose to measure the TLR3 and TLR4 response pathways, both receptors being expressed and previously studied in fibroblasts. We established high-throughput automation compatible methods for standardized primary fibroblast cell activation, using purified MAMPS analogs, poly I:C and LPS that stimulate TLR3 and TLR4 pathways respectively. These results were in turn compared with a stimulation method using infection by HSV-1 virus. Our “Add-only” protocol minimizes high-throughput automation system variability facilitating whole process automation from cell plating through stimulation to recovery of cell supernatants, and fluorescent labeling. Images were acquired automatically by high-throughput acquisition on an automated high-content imaging microscope. Under these methodological conditions standardized image acquisition provided for quantification of cellular responses allowing biological variability to be measured with low system noise and high biological signal fidelity. Optimal for automated analysis of immuno-phenotype of primary human cell responses our method and experimental framework as reported here is highly compatible to high-throughput screening protocols like those necessary for chemo-genomic screening. In context of primary fibroblasts derived from donors enrolled to the MI-clinical-study our results open the way to assert the utility of studying immune-phenotype characteristics relevant to a human clinical cohort.

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Unveiling inter individual variability of human fibroblast innate immune response using robust cell-based protocols

10.1101/2020.06.04.133264 ◽

2020 ◽

Author(s):

A. Chansard ◽

N. Dubrulle ◽

M. Poujol de Mollens ◽

P. B. Falanga ◽

T. Stephen ◽

...

Keyword(s):

Immune Response ◽

Clinical Study ◽

Innate Immune Response ◽

High Throughput ◽

Dermal Fibroblasts ◽

Innate Immune ◽

Primary Fibroblast ◽

Immune Phenotype ◽

Primary Fibroblasts

ABSTRACTThe LabEx Milieu Interieur (MI) project is a clinical study centered on the detailed characterization of the baseline and induced immune responses in blood samples from 1000 healthy donors. Analyses of these samples has lay ground for seminal studies on the genetic and environmental determinants of immunologic variance in a healthy cohort population. In the current study we developed in vitro methods enabling standardized quantification of MI-cohort-derived primary fibroblasts responses. Our results show that in vitro human donor cohort fibroblast responses to stimulation by different MAMPs analogs allows to characterize individual donor immune-phenotype variability. The results provide proof-of-concept foundation to a new experimental framework for such studies.A bio-bank of primary fibroblast lines was generated from 323 out of 1,000 healthy individuals selected from the MI-study cohort. To study inter-donor variability of innate immune response in primary human dermal fibroblasts we chose to measure the TLR3 and TLR4 response pathways, both receptors being expressed and previously studied in fibroblasts. We established high-throughput automation compatible methods for standardized primary fibroblast cell activation, using purified MAMPS analogs, poly I:C and LPS that stimulate TLR3 and TLR4 pathways respectively. These results were in turn compared with a stimulation method using infection by HSV-1 virus. Our “Add-only” protocol minimizes high-throughput automation system variability facilitating whole process automation from cell plating through stimulation to recovery of cell supernatants, and fluorescent labeling. Images were acquired automatically by high-throughput acquisition on an automated high-content imaging microscope. Under these methodological conditions standardized image acquisition provided for quantification of cellular responses allowing biological variability to be measured with low system noise and high biological signal fidelity. Optimal for automated analysis of immuno-phenotype of primary human cell responses our method and experimental framework as reported here is highly compatible to high-throughput screening protocols like those necessary for chemo-genomic screening. In context of primary fibroblasts derived from donors enrolled to the MI-clinical-study our results open the way to assert the utility of studying immune-phenotype characteristics relevant to a human clinical cohort.

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

10.1101/2021.12.14.472572 ◽

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.

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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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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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Persea americanaMill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/391247 ◽

2013 ◽

Vol 2013 ◽

pp. 1-12 ◽

Cited By ~ 8

Author(s):

Maria del R. Ramos-Jerz ◽

Socorro Villanueva ◽

Gerold Jerz ◽

Peter Winterhalter ◽

Alexandra M. Deters

Keyword(s):

Chlorogenic Acid ◽

Dermal Fibroblasts ◽

Persea Americana ◽

Epidermal Keratinocytes ◽

Hacat Keratinocytes ◽

Human Epidermal Keratinocytes ◽

Residual Solvent ◽

Keratinocyte Proliferation ◽

Normal Human

Methanolic avocado (Persea americanaMill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Theirin vitroinfluence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fractionM.2composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore,M.2increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fractionM.6increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fractionM.7contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes.

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