Differential effects of lipopolysaccharide on mouse sensory TRP channels
Graphical abstract
Introduction
The detection of pathogen-associated molecules relies on pattern recognition receptors (PRR) that identify specific molecular motifs, highly conserved across species [1]. Among these PRRs, Toll-like receptors (TLR) have been described as sentinels for distinct viral and bacterial components [2]. For instance, lipopolysaccharides (LPS), a major component of the wall of gram-negative bacteria, are recognized by TLR4 in antigen presenting cells and innate immune cells [3]. Upon LPS ligation, TLR4 activation triggers the production of pro-inflammatory cytokines and chemokines (TNFα, interleukin (IL)-6, IL-1) that induce immune-mediated inflammation through the recruitment of leukocytes [3,4].
Pain associated to infections was first described as sensitization and activation of nociceptors by inflammatory mediators found in the cytokine cocktail secreted by immune cells [5,6]. However, recent evidence indicates that bacterial components can directly sensitize and activate sensory afferent neurons. For instance, it has been proposed that bacterial-derived N-formyl peptides, pore-forming toxins and LPS produce pain by inducing depolarization and firing in nociceptive neurons [[7], [8], [9]]. Furthermore, we recently reported that the cation channel TRPA1 can be activated by LPS, leading to pain and neurogenic inflammation in mice [10] and to aversive responses in Drosophila melanogaster [11]. In the former study, we found that E. coli LPS induces a concentration dependent activation of mouse sensory neurons isolated from nodose and trigeminal ganglia. Up to the concentrations of 3 and 10 μg/ml the responses to LPS occurred exclusively in neurons responsive to the TRPA1 agonist cinnamaldehyde (CA). However, at higher concentrations (> 20 μg/ml), LPS induced responses in neurons that did not express TRPA1 (cinnamaldehyde-insensitive). Furthermore, the genetic ablation of Trpa1 significantly reduced, but did not completely abolish, the responses to LPS. These data demonstrate that TRPA1 is not the only excitatory Ca2+permeable channel mediating LPS effects.
Ligand promiscuity has risen as one of the key features of the TRP channel superfamily [[12], [13], [14], [15], [16]]. For instance, allyl isothiocyanate (AITC), initially described as a TRPA1 specific agonist, can also activate TRPV1 at higher concentrations, leading to aversive and pain responses and visceral irritation [17]. Furthermore, we have recently shown that epithelial TRPV4 is activated by LPS, inducing nitric oxide production and increased mucociliary beat frequency [18].
In this study, we tested the hypothesis that other sensory TRP channels may be involved in the TRPA1-independent effects of LPS. For this, we first characterized the effects of LPS on recombinant TRPV1, TRPV2, TRPM3 and TRPM8 channels heterologously expressed in HEK293T cells. We found that TRPV1 and TRPM3 are also sensitive to LPS, although at higher concentrations than for TRPA1. On the other hand, TRPM8 responded to LPS only at temperatures lower than physiological ones, and TRPV2 appeared to be LPS-insensitive. Using freshly isolated dorsal root ganglion neurons, we determined the relative roles of these channels ex vivo, by comparing the LPS-induced responses in sensory neurons isolated from wild type, Trpa1 KO, Trpv1 KO and double Trpa1/Trpv1 KO mice. Together with our previous results [10], we conclude that TRPA1 and TRPV1 are the main contributors to the acute responses to LPS in sensory neurons at physiological temperatures.
Section snippets
Reagents
Reagents were purchased from Sigma-Aldrich (Bornem, Belgium), unless stated otherwise.
Animals
Trpv1 knock-out (KO) mice were obtained from The Jackson Laboratory (http://jaxmice.jax.org/strain/003770.html) and Trpa1 KO and double Trpv1/Trpa1 KO mice have been described earlier [17,19]. All knockout strains were backcrossed at least nine times into the C57BL/6 J background, and C57BL/6 J mice were used as wild type (WT) controls. Mice of all genotypes were housed under identical conditions, with a
TRPV1 is activated by LPS
First, we sought to determine whether LPS activates TRPV1 using intracellular Ca2+ imaging and whole-cell patch-clamp recordings in HEK293T cells transfected with human TRPV1. In accordance with our previous report [10], LPS was ineffective at the low concentration of 6 μg/ml. However, at higher concentrations it induced intracellular Ca2+ increase in TRPV1-expressing cells (capsaicin-sensitive; Fig. 1A and B). Notably, the amplitude of the responses did not follow a simple dependence with the
Discussion
The initial hypothesis for pain associated to bacterial infection featured the sensitization and activation processes in nociceptors by pro-inflammatory mediators secreted by leukocytes in response to bacterial endotoxins [5,6]. The spectrum of mechanisms of activation of nociceptors has been recently expanded to several bacterial-derived compounds. Chiu et al. have recently reported that N-formylated peptides (N-FP) and the pore-forming toxin (PFT) αHL from gram-positive bacteria contribute to
Author contribution
B.B. conducted the electrophysiological recordings. Y.A.A. and A.S. conducted Ca2+ imaging experiments. B.B., Y.A.A. and K.T. wrote the manuscript. A.L.-R. and T.V. contributed to the interpretation of data. K.T. conceived, designed and supervised the project. All authors edited and accepted the final version of the manuscript.
Conflict of interest
No conflict of interest is declared by the authors.
Acknowledgements
We thank all the members of the laboratories of Ion Channel Research (KU Leuven) for helpful discussions and Melissa Benoit for maintaining the cell culture. Y.A.A. held a Postdoctoral Mandate of the KU Leuven and is currently a Postdoctoral Fellow of the Fund for Scientific Research Flanders (FWO). B.B. was funded by a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT). This work was supported by grants from the Belgian Federal Government (IUAP P7/13), the FWO (G.0702.12)
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These authors contributed equally to this work.