Endotoxin recognition: in fish or not in fish?

Abstract

The interaction between pathogens and their multicellular hosts is initiated by activation of pathogen recognition receptors (PRRs). These receptors, that include most notably members of the toll-like receptor (TLR) family, recognize specific pathogen-associated molecular patterns (PAMPs). TLR4 is a central part of the receptor complex that is involved in the activation of the immune system by lipopolysaccharide (LPS) through the specific recognition of its endotoxic moiety (Lipid A). This is a critical event that is essential for the immune response to Gram-negative bacteria as well as the etiology of endotoxic shock. Interestingly, compared to mammals, fish are resistant to endotoxic shock. This in vivo resistance concurs with in vitro studies demonstrating significantly lowered sensitivity of fish leukocytes to LPS activation. Further, our in vitro analyses demonstrate that in trout mononuclear phagocytes, LPS fails to induce antiviral genes, an event that occurs downstream of TLR4 and is required for the development of endotoxic shock. Finally, an in silico approach that includes mining of different piscine genomic and EST databases, reveals the presence in fish of all of the major TLR signaling elements except for the molecules specifically involved in TLR4-mediated endotoxin recognition and signaling in mammals. Collectively, our analysis questions the existence of TLR4-mediated cellular responses to LPS in fish. We further speculate that other receptors, in particular beta-2 integrins, may play a primary role in the activation of piscine leukocytes by LPS.

Figure 1. Dose-dependent TNF2 induction by different PAMPs

Differentiated rtMOCs, prepared as previously described [91], were stimulated for 12-hours at various PAMP concentrations in the presence of 10% fetal bovine serum (FBS). TNF2 expression in total RNA was analyzed using Northern Blot (upper 4 panels) and RT-PCR (lower 3 panels) as previously described [47]. Equal loading of RNA for Northern blotting was verified by staining ribosomal RNA with ethidium bromide prior to transfer, while the quality of the cDNA used for RT-PCR was confirmed by beta-actin amplification (data not shown). Northern data for the effects of E. coli and P. aeruginosa phenol-extracted LPS, zymosan and MDP from Iliev et al., 2005 [47]. Ultra-pure LPS from Invivogen and phenol-extracted V. anguillarum provided by Dr. J. Bogwald, University of Tromso (Tromso, Norway). The data concerning the activation of mammalian macrophages was derived from the following papers: [–94] for LPS, [95] for zymosan and [96,97] for MDP.

Figure 2. Polymyxin B does not prevent LPS activation of rtMOCs

Differentiated rtMOCs were stimulated with varying concentrations of E. coli LPS in the presence (+) or the absence (−) of polymyxin (Sigma) B. Panel A: Northern blot of TNF2 induction after stimulation for 12 hours in the presence of 10% FBS. Panel B: RT-PCR of TNF2 induction after 3 hours of stimulation in the presence of 5% homologous trout serum. Methods as described or referenced in Fig. 1.

Figure 3. Differential gene induction by LPS from E. coli and poly(I:C)

Differentiated rtMOCs were stimulated for varying times with 50 μg/ml of LPS or poly(I:C). Non-stimulated cells (“C”) were left untreated for 24 hours. Expression of IFN-α, IP-10-like, TNF2, COX-2 and beta-actin were analyzed in total RNA using RT-PCR. Results from two experiments are presented. The first experiment includes treatments with phenol-extracted LPS and poly(I:C). The second includes treatments with ultra-pure LPS and poly(I:C). The TNF2 bands across each row (i.e., under LPS and poly(I:C) stimulation) were obtained from the same experiment and were visualized on the same gel. Therefore, they can be directly compared.

Figure 4. Phylogenetic analyses. Panel A: TRIF and TRAM are closely related; Multidimensional scaling (MDS) of the molecular distances between the TIR domains of the TLR-adapter proteins

TICAM2 is only found in mammals, suggesting a recent divergence from TICAM1. The distance between the gene families compared to the distances within the gene families is so great that it is possible that portraying this information as a molecular tree could be misleading. MDS permits visualization of the proximity of TICAM1 and TICAM2 with respect to MYD88 and TIRAP. Distances are computed with PROTDIST at alignment positions with at least 80% non-gap characters (i.e., only from the TIR domain) from alignments by CLUSTALX. MDS is performed as described in [98]. Panel B: Molecular tree of the Toll-like LRR multigene family; CD180, CD14 and TLRS5 belong to the same superfamily as the vertebrate TLRs. The LRR domains of TLR4 are more closely related to CD180 than they are to any other TLR. The LRR domains of TLR5 are more closely related to TLRS5 than they are to any other TLR. This molecular tree is derived from a CLUSTALX alignment, followed by PROTDIST and FITCH from the PHYLIP package. Distance is calculated only from the 600 alignment positions with at least 80% non-gap characters (out of 855), up to the end of the CD180 alignment (i.e., not including the TIR domain of the TLRs). A single TLR is used to represent TLR clades described in [14], with additional illustrative TLRs from the TLR4 and TLR5 clades. CD180 is monophyletic, but TLRS5 is not. This difference in relationships suggests differences in mechanisms of evolution and selection pressures and thus differences in functional relationships. Mammals do not possess TLR5S. Xt: Xenopus tropicalis - frog; Gg: Gallus gallus - chicken; Hs: Homo sapiens; Dr: Danio rerio - zebrafish; Md: Monodelphis domestica - opossum; Mm: Mus musculus - mouse; Cf: Canis familiaris - dog; Om: Oncorhynchus mykiss - trout; Tr: Takifugu rubripes - Torafugu pufferfish; Tn: Tetraodon nigroviridis - Spotted green pufferfish.

Figure 5. A schematic representation of the model of the CD14/LY96/TLR4-dependent response to LPS

The right panel summarizes on the key observations that question the presence of this model in fish. The TLR4 homodimerization is not depicted for schematic clarity. See text for details.