Fig. 4

TRPV1-initiated signaling in one eye disrupts mucosal tolerance in the opposite ocular surface by inducing contralateral release of substance P. a Experimental design for dissecting the inter-eye neurogenic inflammatory response affecting the ocular mucosal response to ovalbumin (OVA). From days 1 to 4, capsaicin was repeatedly applied to the right eye (OD) while at the same time saline or a substance P blocker was instilled on the left eye (OS). Antigen was instilled on the left eye from days 2 to 5, and then s.c. immunization was performed with complete Freund’s adjuvant (CFA). The antigen-specific immune response was measured by footpad swelling in a delayed-type hypersensitivity (DTH) challenge. b Antigen-specific swelling in control immunized and tolerized mice that only received OVA and in mice that received capsaicin (Cap) and/or substance P blocker (aNK1R) on either eye before immunization (Imm). Some mice were injected subconjunctivally with CFA. Footpad swelling was measured 48 h after s.c. OVA injection and expressed as the difference with the saline-injected contralateral footpad. Pooled data (mean ± SD) from 4 independent experiments with at least 3 mice/group (two-way ANOVA with Bonferroni’s correction). c Dot plot (left) and representative confocal micrographs (right) of nuclear localization of NF-κB p65 protein (green) relative to individual cell expression (blue: TO-PRO-3, red: phalloidin) in the conjunctival epithelium of mice that received saline (Ct) or capsaicin (Cap) on the right eye. Both eyes from each mouse were harvested 48 h after daily instillation and analyzed separately. Some animals also received aNK1R (PDTC) on the opposite (O) eye. Mean ± SD of at least 100 cells analyzed from samples from 3 mice/group (representative example of 3 independent experiments), analyzed by one-way ANOVA with Bonferroni’s correction. d Dot plot (left) and representative confocal micrographs (right) of nuclear localization of NF-κB p65 protein stained as for c and obtained from cultures of murine conjunctival epithelial cells exposed for 15 min to fresh medium (Ct), NF-κB activation inhibitor (PDTC), substance P (SP), a combination of both, and benzalkonium chloride (BAK). Mean ± SD of at least 100 cells from 3 replicates (representative example of 3 independent experiments), analyzed by one-way ANOVA with Bonferroni’s correction. For all panels, asterisk (*) indicates a statistically significant difference by the corresponding test