Evolution of Lipopolysaccharide (LPS) Recognition and Signaling: Fish TLR4 Does Not Recognize LPS and Negatively Regulates NF-κB Activation

Abstract

It has long been established that lower vertebrates, most notably fish and amphibians, are resistant to the toxic effect of LPS. Furthermore, the lack of a TLR4 ortholog in some fish species and the lack of the essential costimulatory molecules for LPS activation via TLR4 (i.e., myeloid differentiation protein 2 (MD-2) and CD14) in all the fish genomes and expressed sequence tag databases available led us to hypothesize that the mechanism of LPS recognition in fish may be different from that of mammals. To shed light on the role of fish TLRs in LPS recognition, a dual-luciferase reporter assay to study NF-κB activation in whole zebrafish embryos was developed and three different bony fish models were studied: 1) the gilthead seabream (Sparus aurata, Perciformes), an immunological-tractable teleost model in which the presence of a TLR4 ortholog is unknown; 2) the spotted green pufferfish (Tetraodon nigroviridis, Tetraodontiformes), which lacks a TLR4 ortholog; and 3) the zebrafish (Danio rerio, Cypriniformes), which possesses two TLR4 orthologs. Our results show that LPS signaled via a TLR4- and MyD88-independent manner in fish, and, surprisingly, that the zebrafish TLR4 orthologs negatively regulated the MyD88-dependent signaling pathway. We think that the identification of TLR4 as a negative regulator of TLR signaling in the zebrafish, together with the absence of this receptor in most fish species, explains the resistance of fish to endotoxic shock and supports the idea that the TLR4 receptor complex for LPS recognition arose after the divergence of fish and tetrapods.

Isolation and analysis of a novel grass carp toll-like receptor 4 (tlr4) gene cluster involved in the response to grass carp reovirus

“…Both identify LPS; however, the response of bird TLR4 to LPS is much weaker than mammalian TLR4 (Keestra and van Putten, 2008). As paralogs of mammalian TLR4, fish TLR4 does not have the ability to identify LPS (Sepulcre et al, 2009); however, we have demonstrated that that grass carp TLR4s are associated with GCRV infection. This implies that the function of TLR4 varies significantly in different species.…”

“…Comprehensive sequence and phylogenetic analyses have revealed that the zebrafish tlr4 genes are paralogous, rather than orthologous, to human tlr4 (Sullivan et al, 2009). A fusion protein containing the zebrafish TLR4 extracellular region and mouse TLR4 transmembrane and intracellular regions cannot identify LPS (Sullivan et al, 2009) and it has been suggested that zebrafish TLR4 recognizes unknown PAMPs (Sullivan et al, 2009;Sepulcre et al, 2009). Although most fish lost tlr4 during evolution (Oshiumi et al, 2003;Baoprasertkul et al, 2007), the structure and function study of the small number of fish which retained tlr4 may provide evolutionary clues.…”

Expression regulation and functional characterization of a novel interferon inducible gene Gig2 and its promoter

“…Besides TLR3 fish has TLR22, a fish-specific TLR member, which can also recognize dsRNA to induce IFN through TICAM1 pathway (Matsuo et al, 2008). Study on the mechanism of LPS recognition found that fish TLR4 does not sense LPS but negatively regulated NF-kB activation (Sepulcre et al, 2009). Unlike mammalian IRF3 that is constitutively expressed in most tissues and is not modulated at the transcriptional level, trout IRF3 is markedly up-regulated by Poly I:C and rIFN (Holland et al, 2008).…”

Characterization of bbtTICAM from amphioxus suggests the emergence of a MyD88-independent pathway in basal chordates

“…Unlike MyD88, no homolog of TICAM1 or TICAM2 has been identified in non-chordates such as Drosophila, Cnidaria [8], or sea urchin [10,11]. Although TICAM1 orthologs could be found in the early vertebrates lamprey [14] and zebrafish [15][16][17][18][19], they do not function in the same manner as those found in mammals. For example, human TICAM1 can induce the secretion of type I interferon by activating transcriptional factors interferon-regulatory factor (IRF) 3 and IRF7 via interaction with tumor necrosis factor receptor-associated factor 6 (TRAF6) [20], while zebrafish TICAM1, localized on the Golgi apparatus [18], induces production of zebrafish IFN in a IRF3/7-independent manner and activates NF-κB via interaction with RIP, but not with TRAF6 [17].…”

“…TICAM2 specifically acts as a bridge for TICAM1 to be successfully recruited when TLR4 recognizes LPS by coupling with CD14 and MD-2 [5,24,25]. Since endotoxin recognition by TLR4 is absent in lamprey [14], jawfish [19], and Xenopus [22], it was previously believed that the endotoxin recognition complex and the downstream signaling pathway in mammals arose after the divergence of fish and tetrapods [19]. Thus, the absence of TICAM2 in bony fish and the rapid evolution from tetrapods to mammals may have been associated with the development of the functional requirement for the formation of the endotoxin recognition complex.…”