TLR4 signaling selectively and directly promotes CGRP release from vagal afferents in the mouse
Visual 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 LoxTB ) mouse in which Tlr4 expression is globally disrupted by a
... ly 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 January/February 2021, 8(1) ENEURO.0254-20.2020 1-16 Research Article: New Research 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.