Differential induction of Toll-like receptor gene expression in equine monocytes activated by Toll-like receptor ligands or TNF-α

Kwon, Soyoung; Vandenplas, Michel L.; Figueiredo, Monica D.; Salter, Caroline E.; Andrietti, A.L.P.; Robertson, Tom; Moore, James N.; Hurley, David J.

doi:10.1016/j.vetimm.2010.07.015

Cited by 14 publications

(13 citation statements)

References 23 publications

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“…Neutrophils and monocytes were isolated by density-gradient centrifugation over Histopaque 1077 as previously described (29,32). Neutrophils were suspended at a final concentration of 3 3 10 7 cells/ml in RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine, 2 mM sodium pyruvate, and 50 mg/ml gentamicin.…”

Section: Cell Isolation and Culturementioning

confidence: 99%

“…This primer set was also used for end-point RT-PCR assays. A second set of internal primers (forward: 59-TCCATGGAGGGTT-GTGATGA-39 and reverse: 59-CCCCGGAACTTTGTGACAAT-39) for equine TLR5 was designed using PrimerExpress software and validated for SYBR Green quantitative RT-PCR (qRT-PCR) gene expression assays as previously described for equine gene expression studies (32). Primers for measuring equine TLR4 expression by qRT-PCR have previously been reported (32).…”

Section: Identification Of a Partial Equine Tlr5 Mrna Sequence Requirmentioning

confidence: 99%

“…A second set of internal primers (forward: 59-TCCATGGAGGGTT-GTGATGA-39 and reverse: 59-CCCCGGAACTTTGTGACAAT-39) for equine TLR5 was designed using PrimerExpress software and validated for SYBR Green quantitative RT-PCR (qRT-PCR) gene expression assays as previously described for equine gene expression studies (32). Primers for measuring equine TLR4 expression by qRT-PCR have previously been reported (32). Differences in TLR gene expression were calculated by relative quantification against 18S rRNA using the D threshold cycle (C T ) method, in which DC T = gene of interest C T 2 18S rRNA C T .…”

Section: Identification Of a Partial Equine Tlr5 Mrna Sequence Requirmentioning

confidence: 99%

“…The equine target genes of interest in this study were TNF-a, IL-1b, and IL-10, with gene expression analysis done by qRT-PCR assay as previously described (32). Approximately 1 3 10 7 isolated neutrophils in the aforementioned medium were added to sterile polystyrene tubes and incubated for 1 or 4 h at 37˚C in a 5% CO 2 atmosphere in the presence or absence of either 100 ng/ml LPS or 200 ng/ml flagellin.…”

Section: Rna Isolation Cdna Synthesis and Real-time Qrt-pcr Assays mentioning

confidence: 99%

“…Only samples having 260/280 nm absorbance ratios between 2.0 and 2.2 as measured on a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) were processed for cDNA synthesis using the High Capacity cDNA Archive kit (Applied Biosystems) with 100 ng RNA as template. Real-time quantitative PCR assays using SYBR Green as a detector were performed in an Applied Biosystems 7900HT (Applied Biosystems) sequence detection system, with eukaryotic 18S rRNA assays serving as endogenous control as previously described (32). The validated primer sets for equine TLR4, TNF-a, cyclooxygenase-2 (COX-2), and IL-10 qRT-PCR assays have been previously reported (36).…”

Section: Rna Isolation Cdna Synthesis and Real-time Qrt-pcr Assays mentioning

confidence: 99%

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Disparities in TLR5 Expression and Responsiveness to Flagellin in Equine Neutrophils and Mononuclear Phagocytes

Kwon

Gewirtz

Hurley

et al. 2011

The Journal of Immunology

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As sentinel cells of the innate immune system, neutrophils and mononuclear phagocytes use specific TLRs to recognize the conserved molecular patterns that characterize microbes. This study was performed to compare the responses of equine neutrophils and mononuclear phagocytes to LPS and flagellin, components of bacteria that are recognized by TLR4 and TLR5, respectively. Neutrophils and mononuclear phagocytes isolated from healthy horses were incubated in vitro with LPS, flagellin, or pronase-inactivated flagellin in the presence or absence of polymyxin B. Production of reactive oxygen species and expression of mRNA for proinflammatory cytokines were used as readouts for activation of neutrophils; production of TNF-α was used for the mononuclear cells. Western blot analysis and flow cytometry were used to detect TLR5 protein in both cell types. Although the neutrophils responded to both LPS and flagellin by producing reactive oxygen species and expressing mRNA for proinflammatory cytokines, flagellin had no stimulatory effect on monocytes or macrophages. Although both neutrophils and monocytes expressed mRNA for TLR5, it appeared to be translated into protein only by the neutrophils. Incubation with neither LPS nor IFN-γ altered TLR5 expression by the monocytes. These findings indicate that flagellin has disparate effects on neutrophils and mononuclear phagocytes isolated from horses, a species that is exquisitely sensitive to the TLR4 ligand, LPS, and that equine mononuclear phagocytes, unlike corresponding cells of other mammalian species, lack surface expression of TLR5 and do not respond to flagellin.

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Section: Cell Isolation and Culturementioning

confidence: 99%

Section: Identification Of a Partial Equine Tlr5 Mrna Sequence Requirmentioning

confidence: 99%

Section: Identification Of a Partial Equine Tlr5 Mrna Sequence Requirmentioning

confidence: 99%

Section: Rna Isolation Cdna Synthesis and Real-time Qrt-pcr Assays mentioning

confidence: 99%

Section: Rna Isolation Cdna Synthesis and Real-time Qrt-pcr Assays mentioning

confidence: 99%

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Disparities in TLR5 Expression and Responsiveness to Flagellin in Equine Neutrophils and Mononuclear Phagocytes

Kwon

Gewirtz

Hurley

et al. 2011

The Journal of Immunology

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show abstract

Types of Vaccines

Bordin

Cohen

2015

Equine Clinical Immunology

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Diseases of the Alimentary Tract

Jones¹,

Fecteau²,

Hullinger³

et al. 2020

Large Animal Internal Medicine

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Differential induction of Toll-like receptor gene expression in equine monocytes activated by Toll-like receptor ligands or TNF-α

Cited by 14 publications

References 23 publications

Disparities in TLR5 Expression and Responsiveness to Flagellin in Equine Neutrophils and Mononuclear Phagocytes

Disparities in TLR5 Expression and Responsiveness to Flagellin in Equine Neutrophils and Mononuclear Phagocytes

Types of Vaccines

Diseases of the Alimentary Tract

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