Establishment of a novel probe-based RT-qPCR approach for detection and quantification of tight junctions reveals age-related changes in the gut barriers of broiler chickens.

Michael Hess,Jörg R Aschenbach,Wageha A Awad,J Sophia Von Buchholz,Tomohito Higashi,Ivana Bilic,Taniya Mitra

doi:10.1371/journal.pone.0248165

Michael Hess, Jörg R Aschenbach + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0248165

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Abstract

Tight junctions (TJs) play a dominant role in gut barrier formation, therefore, resolving the structures of TJs in any animal species is crucial but of major importance in fast growing broilers. They are regulated in molecular composition, ultrastructure and function by intracellular proteins and the cytoskeleton. TJ proteins are classified according to their function into barrier-forming, scaffolding and pore-forming types with deductible consequences for permeability. In spite of their importance for gut health and its integrity limited studies have investigated the TJs in chickens, including the comprehensive evaluation of TJs molecular composition and function in the chicken gut. In the actual study sequence-specific probes to target different TJ genes (claudin 1, 3, 5, 7, 10, 19, zonula occludens 1 (ZO1), occludin (OCLN) and tricellulin (MD2)) were designed and probe-based RT-qPCRs were newly developed. Claudin (CLDN) 1, 5, ZO1 and CLDN 3, 7, MD2 were engulfed in multiplex RT-qPCRs, minimizing the number of separate reactions and enabling robust testing of many samples. All RT-qPCRs were standardized for chicken jejunum and caecum samples, which enabled specific detection and quantification of the gene expression. Furthermore, the newly established protocols were used to investigate the age developmental changes in the TJs of broiler chickens from 1–35 days of age in the same organ samples. Results revealed a significant increase in mRNA expression between 14 and 21days of age of all tested TJs in jejunum. However, in caecum, mRNA expression of some TJs decreased after 1 day of age whereas some TJs mRNA remained constant till 35 days of age. Taken together, determining the segment-specific changes in the expression of TJ- proteins by RT-qPCR provides a deeper understanding of the molecular mechanisms underpinning pathophysiological changes in the gut of broiler chickens with various etiologies.

Highlights

Intestinal epithelial cells are tightly connected by intercellular junctional complexes that regulate the passage of ions and molecules through the paracellular pathway and are crucial for the integrity of the epithelial barrier
To confirm that the optimized Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) protocol is applicable among different animals, three biological replicates and two technical replicates were used for each standardization protocol (Figs 1–4)
Sequence-specific probes for different Tight junctions (TJs) genes were designed and probe-based RTqPCRs were established in chickens to possibly monitor gut health and integrity on an mRNA level

Summary

Introduction

Intestinal epithelial cells are tightly connected by intercellular junctional complexes that regulate the passage of ions and molecules through the paracellular pathway and are crucial for the integrity of the epithelial barrier. Tight junctions play a crucial part in the physiological function of epithelial cells. The degree of sealing of tight junctions varies according to external stimuli along with physiological and pathological conditions [4]. Depending on their localization, two different types of TJ can be distinguished: i) the bi-cellular tight junction at cell-cell contacts of two neighbouring cells and ii) the tri-cellular tight junction at the meeting point of three cells, which leads to the formation of a paracellular tube. A loss of TJs’ integrity enables an easy paracellular migration of pathogens as well as the uncontrolled diffusion of toxins from the gut mucosa into the whole body [5,6,7,8]

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