Expressions of TLR4, MyD88 and IRAK4 in the Skin of Chinese Brown Frog (Rana Dybowskii) During the Breeding and Pre-Hibernation Periods

Toll-Like Receptors (TLRs) are membrane-bound proteins that
recognize invading organisms with Pathogen-Associated Molecular
Patterns (PAMPs) and Damage-Related Molecular Patterns (DAMPs)...


Introduction
Toll-Like Receptors (TLRs) are membrane-bound proteins that recognize invading organisms with Pathogen-Associated Molecular Patterns (PAMPs) and Damage-Related Molecular Patterns (DAMPs) [1]. TLRs activated by PAMP or DAMP could upregulate chemokines and inflammatory cytokines and participate in various intracellular signaling pathways to regulate the inflammatory responses (Brown et al., 2011). TLR4, the first TLR to be identified by Medzhitov in 1997, is the only receptor that mediates the immune response through the MyD88-dependent signaling pathway and the MyD88independent signaling pathway and is a key component of the innate immune system [2]. TLR4 is expressed in epithelial cells and activated TLR4 causes activation of Nuclear factor κB (NF-κB) through multiple downstream intracellular signals, and then synthesizes pro-allergic cytokines [3]. TLR4 recruit's adaptor proteins such as MyD88 to IRAK4 and TNF receptor associated factor 6 (TRAF6), finally induces the activation of COX2, which plays a key regulatory role in skin inflammation Sherwani (2018).
MyD88 is an adaptor protein for most TLR signaling pathways in regulation of the innate immunity [4]. Upon recruitment by TLRs, MyD88 activates NF-κB, which leads to the subsequent transcription and expression of inflammatory genes such as TNF-α, IL-1 and IL-6 Palti [5]. Studies have shown that MyD88-dependent signaling could exacerbate allergic inflammation, and the lack of MyD88 could protect mice with skin barrier function from spontaneous skin damage [6,7]. IRAK4 is a serine-threonine kinase that is critical in the signaling cascade and is central to MyD88-dependent signaling. IRAK4-deficiency animals completely lost their immune response to bacterial and viral challenge, which indicate that IRAK4 plays an important role in innate immunity [6]. The skin of the amphibian is directly exposed to the complex environment. As an organ that is in direct contact with the external environment, it is vulnerable to external factors such as predators, microorganisms, parasites and physical damage [9]. To resist the harmful effects of environmental factors, the skin of amphibians has formed a stable defense mechanism in the long-term evolution process. The main function of the skin immune system is to promote wound healing and immune tolerance [10].
The Chinese brown frog (Rana dybowskii) is distributed in highhumidity environments in China, Japan, Korea and eastern Siberia [11]. The breeding period of the Rana dybowskii is from early April to early May, and from early September to early October is the prehibernation period, it migrates to the river or pond, waiting for winter. Their skin therefore acts as the first line of defense against invading pathogens. Granular glands in the skin tissue of Rana dybowskii synthesize and secrete biologically active molecules such as antibacterial peptides and antioxidants associated with innate immunity, which contribute to epithelial defense [12]. Previous studies have shown that the expression of NF-κB, a downstream factor of the TLR signaling pathway, is present in Rana dybowskii skin, suggesting that NF-κB may play a significant role in the skin immune system of Rana dybowskii [11]. In addition, MyD88 is expressed in various tissues of Rana dybowskii and has a conserved structure, which might be an important component of the innate immunity in Rana dybowskii [13]. In this study, we investigated the expressions of TLR4, MyD88 and IRAK4 in Rana dybowskii skin during the breeding and pre-hibernation periods to clarify the roles of TLR4, MyD88 and IRAK4 in the skin immune system of Rana dybowskii.

Histology
The skin samples were dehydrated by a certain concentration of ethanol and xylene, and immersed and embedded in paraffin [10]. Sagittal serial sections (5 μm) were adhered on slides coat-

Real-time PCR Quantification
The mRNA expressions of TLR4, MyD88 and IRAK4 during the breeding and pre-hibernation periods were analyzed by real-time PCR using one-step SYBR PrimeScript RT-PCR kit (TaKaRa Company, Dalian, China). Tissues dissected from 3 to 10 individuals were pooled from Rana dybowskii to analyze expression in the skin. The primers for real-time PCR analysis were designed using the Primer 3 program ( Transcript levels of the target genes were normalized to the β-actin after correcting for differences in amplification efficiency. The expression level of each target mRNA relative to β-actin mRNA was determined using the 2-ΔΔCt method.

Statistical Analysis
Statistical comparisons were made with Student's t-test using GraphPad Prism 6. A value of P < 0.05 was considered statistically significant.

Histological Structure of Rana dybowskii Skin
The

Immunolocalizations of TLR4, MyD88 and IRAK4 in Rana dybowskii Skin
The

The Expressions of TLR4, MyD88 and IRAK4 in Rana dybowskii Skin
The results of Western blot analysis of TLR4, MyD88 and

Discussion
The present results demonstrated that TLR4, MyD88 and IRAK4 were localized in epithelial and gland cells of the skin of Rana dybowskii in the breeding and pre-hibernation periods. The mRNA expression levels of TLR4 and IRAK4 were not significantly different in two periods, while the mRNA expression level of MyD88 in pre-hibernation was lower than that of the breeding period. In addition, the protein expression levels of TLR4, MyD88 and IRAK4 did not differ between the breeding and pre-hibernation periods.
These findings suggested that TLR4, MyD88 and IRAK4 might be involved in the regulation of Rana dybowskii skin immune function during the breeding and pre-hibernation periods.
Amphibian skin glands are basically composed of two types: mucus glands and granular glands. The mucous glands secrete mucus to keep it moist and prevent mechanical damage to delicate skin and delay evaporation of water [14]. Granular glands could synthesize a variety of chemicals such as peptides and alkaloids, to provide protection against fungal and bacterial infections and to resist predators [15]. In addition, glandular secretions are closely related to various biological effects such as cytotoxic, bactericidal, fungicidal, lytic, anesthetic, and phenomenal [16]. One previous report has shown that proteins in the skin mucus of Chinese giant salamander participated in the physiological activities of the skin through various ways, including defense, immune response, wound healing and breathing [16]. Previous studies have shown that inflammatory factors such as IL-1β, IL-1R, IL-6 and TNF-α were expressed in the skin of Rana dybowskii, indicating that these cytokines might be involved in skin immune and inflammatory responses against external aggressors [17,11]. The present study with the corresponding ligands [19]. These results indicated that IRAK4 is a downstream adapter molecule of MyD88 that is critically required for the induction of many TLR/IL-1R-dependent immune responses [24]. Endogenous IRAK4 interacts with IRAK1 and TRAF6 in an IL-1-dependent manner, and its overexpression induces activation of NF-κB and mitogen-activated protein (MAP) kinase pathways, which in turn causes inflammation and immune responses [25]. Moreover, defects in IRAK4 would cause severe TLR signaling and NF-κB signaling, suggesting that IRAK4 plays an important role in TLR-mediated pathways [26,27]. Our results In summary, our previous studies have shown that IL-1β, IL-1R, IL-6, TNF-α and NF-κB were found in the skin of Rana dybowskii, and these cytokines may be involved in immune and inflammatory responses [17,11]. The present study demonstrated that TLR4, MyD88 and IRAK4 were expressed in Rana dybowskii skin during the breeding and pre-hibernation periods. These results suggested that TLR4 might regulate the secretion of these cytokines via the MyD88 signaling pathway, which plays a regulatory role in the immune response of Rana dybowskii skin. The data presented here will greatly aid the dissection of TLR4 signaling pathway in the skin of amphibians.