TLR4 Signaling Selectively and Directly Promotes CGRP Release from Vagal Afferents in the Mouse

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(LoxTB)) 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(v)1.8-Cre mice, we produced mice that express TLR4 in all cells (Tlr4(LoxTB) X Zp3-Cre) and in peripheral afferents (Tlr4(LoxTB) X Na(v)1.8-Cre), respectively. We validated the Tlr4(LoxTB) 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(v)1.8-restricted TLR4. The later result prompted us to identify Tlr4-expressing vagal afferents using in situ hybridization (ISH). In vivo, we found that Tlr4 mRNA was primarily enriched in vagal Na(v)1.8 afferents located in the jugular ganglion that co-expressed calcitonin gene-related peptide (CGRP). In vitro, the application of LPS to cultured Na(v)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.