Fig 2: Effects of TSLPR neutralization mediated downregulation on the severity of P. aeruginosa keratitis. Mouse corneas were subconjunctivally injected 5 μL control IgG or anti-TSLP at −4 hours. At 0 hours, the corneas were inoculated with 1.0 × 104 CFU of P. aeruginosa. (A) Micrographs and clinical scores of the control (IgG) and TSLPR neutralizing antibody treated corneas at 1, 2, and 3 dpi. Note the increased opacification in anti-TSLPR–treated corneas (right panels). The infected corneas were excised, homogenized, and subjected to (B) bacteria counting, (C) MPO determination (units/cornea), and (D) ELISA determination for IL1β. The results are representative for two independent experiments. **P < 0.01, (t-test).

Fig 3: The expression of TSLP and IL-23/IL-17 in DC depleted, P. aeruginosa–infected B6 mouse corneas. CD11c-DTR and WT mice were injected with 100 ng of DT in 100 µL of PBS IP 24 hours before 1.0 × 104 CFU of P. aeruginosa inoculation of the cornea. (A) Micrographs of the WT or CD11c-DTR mice treated with DT and inoculated P. aeruginosa at 1 dpi. Note that the DC depleted corneas had much high average clinical score (n = 5). (B) The corneas were excised from the enucleated eyes, homogenized, and subjected to real-time PCR analysis of TSLP, IL-23, IL-17A, and IL-17C. The results are presented as fold increase with the levels of naïve corneas (NL) set as 1. The figure is representative of two independent experiments. *P < 0.05. **P < 0.01 (1-way ANOVA, with Bonferroni correction).

Fig 4: TSLP expression and secretion in primary human corneal epithelial cells in response to P. aeruginosa challenge. HCECs (p4) grown overnight in KBM were treated with heat-killed P. aeruginosa (MOI = 50) for different times. Cell lysates as well as culture media were collected and subjected to PCR and Western blotting analyses. (A) TSLP expression in cultured HCECs challenged with heat-killed P. aeruginosa for 1, 2, 4, and 8 hours was analyzed by real-time PCR. The results are representative of two independent experiments, each with three corneas. *P < 0.05. **P < 0.01 (1-way ANOVA, with Bonferroni Correction). (B) The steady-state expression of TSLP in HCEC cell lysates at indicated times were analyzed by Western blot. (C) TSLP secretion was analyzed by Western blotting of the concentrated culture media of HCECs challenged with heat-killed P. aeruginosa. The data shown are representative of two independent experiments.

Fig 5: TSLP and TSLPR expression in P. aeruginosa–infected mouse corneal epithelial cells. The centers of B6 mouse corneas were gently scratched with 26-gauge needles (1 mm long; 3 lines) and incubated with 1.0 × 104 CFU of P. aeruginosa. At the indicated times, mouse CECs were collected and subjected to (A) RT-PCR, (B, C) Real-time PCR for TSLP and TSLPR expression in naïve and P. aeruginosa infected mouse CECs, respectively. (D) TSLP expression in mouse CECs was assessed using ELISA. Error bars show standard deviations. The results are representative of two independent experiments, each with 3 corneas. *P < 0.05. **P < 0.01 (1-way ANOVA, with Bonferroni correction).