Background and aims:
Visceral hypersensitivity is considered the core pathophysiological mechanism that causes abdominal pain in patients with irritable bowel syndrome (IBS). Fungal dysbiosis has been proved to contribute to visceral hypersensitivity in IBS patients. However, the underlying mechanisms for Dectin-1, a major fungal recognition receptor, in visceral hypersensitivity are poorly understood. This study aims to explore the role of Dectin-1 in visceral hypersensitivity and elucidate the impact of Dectin-1 activity on the channel function of transient receptor potential vanilloid type 1 (TRPV1), a key molecule involved in visceral hypersensitivity.
Methods:
Visceral hypersensitivity model was established by the intrarectal administration of 0.1 ml TNBS (130 μg/ml dissolved in 30% ethanol) into the colon of 8-10 weeks old C57BL/6 mice. Laminarin, a Dectin-1 antagonist, administration and gene knockout (Clec7a^-/-) approaches were used to interrupt the function of Dectin-1 (Fig. 1A). Colorectal distension-electromyogram (CRD-EMG) recording was performed to assess visceral sensitivity. Western blot was used to detected Dectin-1 and TRPV1 expression in dorsal root ganglion (DRG) neurons and distal colon. Immunostaining experiment was performed to determine the colocalization of Dectin-1 and TRPV1 in DRG neurons. Calcium imaging study was performed to assay TRPV1-mediated calcium influx in acutely dissociated DRG neurons.
Results:
Application of laminarin or genetic deletion of Clec7a alleviated TNBS-induced visceral hypersensitivity in mice (Fig. 1B-G). The expression of Dectin-1 was upregulated in the DRG and colon of IBS mice (Fig. 1H). Colocalization of Dectin-1 and TRPV1 was observed in DRG neurons (Fig. 1I). However, the expression of TRPV1 in the DRG has no significantly difference between vehicle and TNBS group (Fig. 2A). TRPV1-mediated calcium influx was increased in TNBS group (Fig. 2B). Importantly, administration of curdlan, a Dectin-1 agonist, increased TRPV1-mediated calcium influx, while laminarin reduced that (Fig. 2D-G).
Conclusions:
Dectin-1 receptors contribute to the development of visceral hypersensitivity in IBS or IBD in remission and activation of Dectin-1 induces TRPV1 sensitization.

<b>Figure 1 Application of laminarin or genetic deletion of <i>Clec7a</i> alleviated TNBS-induced visceral hypersensitivity in mice</b><br /> A) Schematic diagram showing the timelines to establish the TNBS-induced visceral hypersensitivity and administer laminarin. B) The rate of body weight change. C) Images of H&E staining of colonic sections, D) CRD-EMG results on the 28th day after vehicle or TNBS injection without or with concurrent administration of laminarin. E) The rate of body weight change of in <i>Clec7a<sup>-/-</sup></i> mice. F) Images of H&E staining of colonic sections of <i>Clec7a<sup>-/-</sup></i> mice. G) CRD-EMG results on the 28th day after intrarectal injection of vehicle or TNBS in in wild-type and <i>Clec7a<sup>-/-</sup></i> mice. H) The expression of the Dectin-1 protein in the colon and T12-L1 and L6-S1 DRGs. I) Immunofluorescence (IF) staining of Dectin-1 and TRPV1 in L6 DRG. H&E Scale bar = 100 μm. IF Scale bar = 100 or 25 μm. Data are show as the means ± SEM.

Figure 1 Application of laminarin or genetic deletion of Clec7a alleviated TNBS-induced visceral hypersensitivity in mice
A) Schematic diagram showing the timelines to establish the TNBS-induced visceral hypersensitivity and administer laminarin. B) The rate of body weight change. C) Images of H&E staining of colonic sections, D) CRD-EMG results on the 28th day after vehicle or TNBS injection without or with concurrent administration of laminarin. E) The rate of body weight change of in Clec7a^-/- mice. F) Images of H&E staining of colonic sections of Clec7a^-/- mice. G) CRD-EMG results on the 28th day after intrarectal injection of vehicle or TNBS in in wild-type and Clec7a^-/- mice. H) The expression of the Dectin-1 protein in the colon and T12-L1 and L6-S1 DRGs. I) Immunofluorescence (IF) staining of Dectin-1 and TRPV1 in L6 DRG. H&E Scale bar = 100 μm. IF Scale bar = 100 or 25 μm. Data are show as the means ± SEM.

<b>Figure 2 Dectin-1 Induces TRPV1 Sensitization.</b><br /> A) The expression of the TRPV1 protein in the T12-L1 and L6-S1 DRGs. B-C) Calcium imaging of TNBS-injected group compared with the vehicle group. B) Traces of calcium imaging upon stimulation with capsaicin (1 μM) followed by KCl (3 μM). C) Change in the ratio of F340 to F380 in the vehicle- or TNBS-injected group. D-E) Calcium imaging of pretreatment with curdlan. D) Traces of calcium imaging after stimulation with capsaicin (1 μM) followed by KCl (3 μM) in DRG neurons treated with the vehicle or different doses of curdlan. E) Change in the ratio of F340 to F380. F-G) Calcium imaging of pretreatment with laminarin. F) Traces of calcium imaging after stimulation with capsaicin (1 μM) followed by KCl (3 μM) in DRG neurons treated with the vehicle or different doses of laminarin. G) Change in the ratio of F340 to F380. Data are show as the means ± SEM. Cap, capsaicin; CPZ, Capsazepine.

Figure 2 Dectin-1 Induces TRPV1 Sensitization.
A) The expression of the TRPV1 protein in the T12-L1 and L6-S1 DRGs. B-C) Calcium imaging of TNBS-injected group compared with the vehicle group. B) Traces of calcium imaging upon stimulation with capsaicin (1 μM) followed by KCl (3 μM). C) Change in the ratio of F340 to F380 in the vehicle- or TNBS-injected group. D-E) Calcium imaging of pretreatment with curdlan. D) Traces of calcium imaging after stimulation with capsaicin (1 μM) followed by KCl (3 μM) in DRG neurons treated with the vehicle or different doses of curdlan. E) Change in the ratio of F340 to F380. F-G) Calcium imaging of pretreatment with laminarin. F) Traces of calcium imaging after stimulation with capsaicin (1 μM) followed by KCl (3 μM) in DRG neurons treated with the vehicle or different doses of laminarin. G) Change in the ratio of F340 to F380. Data are show as the means ± SEM. Cap, capsaicin; CPZ, Capsazepine.

Ying Zhang

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