TLR4 Signaling Selectively and Directly Promotes CGRP Release from Vagal Afferents in the Mouse
Lin Jia, Syann Lee, Jessica A. Tierney, Joel K. Elmquist, Michael D. Burton, Laurent Gautron
Abstract
There has been a long-standing debate regarding the role of peripheral afferents in mediating rapid-onset anorexia among other responses elicited by peripheral inflammatory insults. Thus, the current study assessed the sufficiency of peripheral afferents expressing toll-like receptor 4 (TLR4) to the initiation of the anorexia caused by peripheral bacterial lipopolysaccharide (LPS). We generated a Tlr4 null (Tlr4<sup>LoxTB</sup>) mouse in which Tlr4 expression is globally disrupted by a loxP-flanked transcription blocking (TB) cassette. This novel mouse model allowed us to restore the endogenous TLR4 expression in specific cell types. Using Zp3-Cre and Na<sub>v</sub>1.8-Cre mice, we produced mice that express TLR4 in all cells (Tlr4<sup>LoxTB</sup> X Zp3-Cre) and in peripheral afferents (Tlr4<sup>LoxTB</sup> X Na<sub>v</sub>1.8-Cre), respectively. We validated the Tlr4<sup>LoxTB</sup> mice, which were phenotypically identical to previously reported global TLR4 knock-out mice. Contrary to our expectations, the administration of LPS did not cause rapid-onset anorexia in mice with Na<sub>v</sub>1.8-restricted TLR4. The later result prompted us to identify Tlr4-expressing vagal afferents using <i>in situ</i> hybridization (ISH). <i>In vivo</i>, we found that Tlr4 mRNA was primarily enriched in vagal Na<sub>v</sub>1.8 afferents located in the jugular ganglion that co-expressed calcitonin gene-related peptide (CGRP). <i>In vitro</i>, the application of LPS to cultured Na<sub>v</sub>1.8-restricted TLR4 afferents was sufficient to stimulate the release of CGRP. In summary, we demonstrated using a new mouse model that vagally-expressed TLR4 is selectively involved in stimulating the release of CGRP but not in causing anorexia.