Toll-like Receptors of the Ascidian Ciona intestinalis

Abstract

Key transmembrane proteins in the innate immune system, Toll-like receptors (TLRs), have been suggested to occur in the genome of non-mammalian organisms including invertebrates. However, authentic invertebrate TLRs have been neither structurally nor functionally investigated. In this paper, we originally present the structures, localization, ligand recognition, activities, and inflammatory cytokine production of all TLRs of the ascidian Ciona intestinalis, designated as Ci-TLR1 and Ci-TLR2. The amino acid sequence of Ci-TLR1 and Ci-TLR2 were found to possess unique structural organization with moderate sequence similarity to functionally characterized vertebrate TLRs. ci-tlr1 and ci-tlr2 genes were expressed predominantly in the stomach and intestine as well as in hemocytes. Ci-TLR1 and Ci-TLR2 expressed in HEK293 cells, unlike vertebrate TLRs, were localized to both the plasma membrane and endosomes. Intriguingly, both Ci-TLR1 and Ci-TLR2 stimulate NF-B induction in response to multiple pathogenic ligands such as double-stranded RNA, and bacterial cell wall components that are differentially recognized by respective vertebrate TLRs, revealing that Ci-TLRs recognize broader pathogen-associated molecular patterns than vertebrate TLRs. The Ci-TLR-stimulating pathogenic ligands also induced the expression of Ci-TNF␣ in the intestine and stomach where Ci-TLRs are expressed. These results provide evidence that the TLR-triggered innate immune systems are essentially conserved in ascidians, and that Ci-TLRs possess "hybrid" biological and immunological functions, compared with vertebrate TLRs. Moreover, it is presumed that chordate TLR ancestors also acquired the Ci-TLR-like multiple cellular localization and pathogen-associated molecular pattern recognition.

Characteristic and functional analysis of Toll-like receptors (TLRs) in the lophotrocozoan, Crassostrea gigas, reveals ancient origin of TLR-mediated innate immunity

“…The lack of PAMPs recognition was consistent with previous reports for amphioxus and Drosophila [6,29]. In contrast, Sasaki et al demonstrated that ascidian Cionaintestinalis TLRs show low level responses to high concentrations of PAMPs [30], however, such a response is too weak to distinguish from C. intestinalis TLR's authentic ability and the background response of cell line. However, these evidences suggest that specific PAMP recognition of TLR might appear during evolution of vertebrates.…”

Immune-directed support of rich microbial communities in the gut has ancient roots

“…At a minimum, Ciona could represent the basic functional requirements for homeostasis in the gut of chordates. Ciona possesses a competent innate immune system (Azumi et al, 2003; Shida et al, 2003) and includes AMPs (Di Bella et al, 2011; Fedders and Leippe, 2008), two TLRs with multi-ligand binding potential (Sasaki et al, 2009), GNBPs, lipopolysaccharide binding protein (LBP), a variety of C-type lectins and other PRRs, tumor necrosis factor (TNF) (Azumi et al, 2003), mannose binding lectin (MBL) (Skjoedt et al, 2010), complement protein C3 (Marino et al, 2002), as well as the variable region-containing chitin-binding proteins (VCBPs), a relatively small family of immunoglobulin domain-containing proteins (Dishaw et al, 2011). …”

“…Like most other animals, Ciona likely uses AMPs as a first line of defense (Di Bella et al, 2011; Fedders and Leippe, 2008; Lu et al, 2014); however, response to specific bacterial products also can be viewed as being consistent with the patterns of expression of TLRs and other PRRs in gut tissues, resulting in the induction of pro-inflammatory molecules, e.g., TNF (Sasaki et al, 2009). Although the Ciona genome encodes only two TLRs, both are expressed, along with VCBPs, in distinct locations along the gut.…”

“…Ciona TLRs (Sasaki et al, 2009), as well as additional PRRs [e.g., MBL (Skjoedt et al, 2010)], are differentially expressed in unique sections of the gut. The innate immune repertoire of Ciona likely is augmented by VCBPs.…”

“…Ciona intestinalis has been shown previously to be a relevant model system for studies of development (Baghdiguian et al, 2007; Cañestro et al, 2003; Davidson, 2007; Katz, 1983; Meinertzhagen and Okamura, 2001; Sasakura et al, 2009; Shi et al, 2005) and immune defense (Fujita et al, 2004; Parrinello et al, 2008; Sasaki et al, 2009; Zucchetti et al, 2009) and presently is being developed for studies of gut host-bacterial interactions (Dishaw et al, 2012a; Dishaw et al, 2011). By permitting the experimental dissection of the conversation between host and microbiota at the onset of colonization, the Ciona model promises to reveal chordate rules that govern the biology of colonization.…”