Interferon regulatory factor-1 in flagellin-induced reprogramming: potential protective role of CXCL10 in cornea innate defense against Pseudomonas aeruginosa infection

Abstract

Purpose: We previously showed that pre-exposure of the cornea to Toll-like receptor (TLR)5 ligand flagellin induces strong protective innate defense against microbial pathogens and hypothesized that flagellin modulates gene expression at the transcriptional levels. Thus, we sought to determine the role of one transcription factor, interferon regulatory factor (IRF1), and its target gene CXCL10 therein.

Methods: Superarray was used to identify transcription factors differentially expressed in Pseudomonas aeruginosa-challenged human corneal epithelial cells (CECs) with or without flagellin pretreatment. The expression of CXCL10, IRF1, LI-8(CXCL2), and IFNγ was determined by PCR, immunohistochemistry, Western/dot blotting, and/or ELISA. IRF1 knockout mice, CXCL10 and IFNγ neutralization, and NK cell depletion were used to define in vivo regulation and function of CXCL10. The severity of P. aeruginosa was assessed using clinical scoring, slit-lamp microscopy, bacterial counting, polymorphonuclear leukocytes (PMN) infiltration, and macrophage inflammatory protein 2/Chemokine (C-X-C motif) ligand 2 (MIP-2/CXCL2) expression.

Results: Flagellin pretreatment drastically affected P. aeruginosa-induced IRF1 expression in human CECs. However, flagellin pretreatment augmented the P. aeruginosa-induced expression of Irf1 and its target gene Cxcl10 in B6 mouse corneas. Irf1 deficiency reduced infection-triggered CXCL10 expression, increased keratitis severity, and attenuated flagellin-elicited protection compared to values in wild-type (WT) controls. CXCL10 neutralization in the cornea of WT mice displayed pathogenesis similar to that of IRF1⁻/⁻ mice. IFNγ receptor neutralization and NK cell depletion prevented flagellin-augmented IRF1 and CXCL10 expression and increased the susceptibility to P. aeruginosa infection in mouse corneas.

Conclusions: IRF1 plays a role in the corneal innate immune response by regulating CXCL10 expression. IFNγ-producing NK cells augment the epithelial expression of IRF1 and CXCL10 and thus contribute to the innate defense of the cornea against P. aeruginosa infection.

Keywords: CXCL10; IRF1; cell reprogramming; inflammation; toll-like receptors.

Figure 1

Reprogramming of transcription factor expression caused by flagellin pretreatment in primary HCECs. Normal or flagellin-pretreated (50 ng/mL, 24 hours) cells were challenged with live PAO1 (1:50 MOI) for 4 hours, and transcription factor RNA expression alterations were quantified by comparing RNA fold changes of PAO1-infected normal cells versus PAO1-infected flagellin-pretreated cells using the real-time RT² Profiler PCR Array (A). For confirmation of PCR array results, cells were cultured and challenged under the same conditions and lysed and subjected to Western blotting analyses with IRF1 and β-actin antibodies (D). Cell culture media were collected 4 hours after bacterial challenge for the analyses of IL-8 by ELISA (B) and hBD2 using slot blot (C). P values were generated using unpaired Student's t-test (**P < 0.01). The figure is a representative of four independent experiments.

Figure 2

Detection of Cxcl10 expression in corneal epithelia of B6 WT and Irf1^−/− mice. Wild-type and Irf1^−/− B6 mice were topically pretreated with 500 ng flagellin in 5 μL PBS, a dosage we showed to induce protection in B6 mice, or 5 μL PBS for 24 hours, and then infected with PAO1 at 10⁵ CFU for 6 hours. The epithelial cells were collected and RNA was isolated for semiquantitative RT-PCR (A) and quantitative real-time PCR (B) analysis of Cxcl10, Irf1, and mBd3. Gapdh was used as the internal control. P values were generated using unpaired Student's t-test (*P < 0.05). The figure is a representative of three independent experiments.

Figure 3

Distribution of CXCL10 expression in corneal epithelia of WT and Irf1^−/− mice. WT and Irf1^−/− B6 mice were topically pretreated with 500 ng flagellin in 5 μL PBS, a dosage we showed to induce protection in B6 mice, or 5 μL PBS for 24 hours, and then infected with PAO1 at 10⁵ CFU for 6 hours. Mouse eyes were enucleated in OCT, sectioned, stained with mouse anti-CXCL10 antibody, and examined with fluorescence microscopy middle panels with DAPI used to stain the nuclei (A, B, D, G, J, K, M, N, P, Q) and ×4 lens to view the entire mouse corneas (insert [i] in [E, H) or confocal microscopy (C, E, F, H, I, L, O, R). Scale bars are given in the figures.

Figure 4

IRF1 deficiency increased the severity of Pseudomonas keratitis at 3 dpi and attenuated flagellin-induced protection in B6 mice. WT or IRF1^−/− B6 mice were topically pretreated with 500 ng flagellin or PBS for 24 hours, and then infected with ATCC 19660 at 10⁴ CFU for 3 days. Keratitis progression was photographed (A), and clinical scores were assigned at 1, 2, and 3 dpi (B). The whole cornea was then collected for viable bacterial count (C), MPO activity assay (D), and CXCL2 ELISA (E). P values were generated using unpaired Student's t-test (*P < 0.05; **P < 0.01). The figure is a representative of three independent experiments.

Figure 5

Neutralization of CXCL10 activity in the cornea attenuated flagellin-induced protection in B6 mice at 3 dpi. WT mice were injected with neutralizing anti-CXCL10 antibody (3 μg in 5 μL) or isotype rabbit IgG 24 hours prior to topical PBS or flagellin pretreatment, and then infected with ATCC 19660 at 10⁴ CFU. Keratitis progression was photographed (A), and clinical scores were assigned at 1, 2, and 3 dpi (B). The corneas were then excised and homogenized for viable bacterial count (C), MPO activity assay (D), and CXCL2 ELISA (E). P values were generated using unpaired Student's t-test (**P < 0.01). The figure is a representative of three independent experiments.

Figure 6

Neutralization of IFNγR2 activity in the cornea attenuated epithelial CXCL10 production and flagellin-induced protection in B6 mice. WT mice were injected with IFNγ receptor neutralizing anti-IFNγR2 antibody (3 μg in 5 μL) or isotype rabbit IgG 24 hours prior to topical PBS or flagellin pretreatment, and then infected with ATCC 19660 at 10⁴ CFU. At 20 hours postinfection the corneas were photographed (A) and then harvested for immunohistochemistry (B), RT-PCR (C) analysis of CXCL10 expression, bacterial count (D), MPO activity assay (E), and CXCL2 ELISA (F). P values were generated using unpaired Student's t-test (*P < 0.05; **P < 0.01). The figure is a representative of six corneas (n = 3 mice) per condition.

Figure 7

Neutralization of NK cells in the cornea blocked IFNγ, IRF1, and CXCL10 expression and compromised flagellin-induced corneal protection in B6 mice. WT mice were injected with NK cell neutralizing anti-NK1.1 antibody (3 μg in 5 μL) or isotype rabbit IgG 24 hours prior to topical PBS or flagellin pretreatment, and then infected with ATCC 19660 at 10⁴ CFU. At 20 hours postinfection the corneas were photographed (A) and then harvested for bacterial count (B); MPO activity assay (C); CXCL2 (D) and CXCL10 (F) ELISA; and real-time PCR analysis of IFNγ, IRF1, and CXCL10 expression (E). P values were generated using unpaired Student's t-test (**P < 0.01). The figure is a representative of six corneas (n = 3 mice) per condition.