Hyperglycemia-Suppressed Acod1 Expression Contributes to Innate Immune Deficiency in Pseudomonas aeruginosa Keratitis

Abstract

Purpose: Diabetes mellitus (DM) patients are at higher risk for infections, which are often more severe. This study investigated the role of aconitate decarboxylase 1 (Acod1) and its product, itaconate, in innate defense against bacterial keratitis and its impairment in type 1 DM mice.

Methods: Wild-type or normal (NL), streptozotocin-induced DM, and Acod1-/- mice were inoculated with Pseudomonas aeruginosa (Pa) with or without 4-octyl itaconate (4-OI), a cell-permeable derivative of itaconate. Keratitis severity was determined by photography, clinical scores, Pa burden (cfu), and myeloperoxidase (MPO) activity. Gene expression was determined by quantitative PCR. Immune and Acod1-positive cells were determined by immunohistochemistry.

Results: DM mice expressed lower levels of Acod1 in B6 mouse corneas, and Pa infection triggered its upregulation, mostly in infiltrated cells. Acod1 deficiency increased the severity of Pa keratitis and significantly augmented the expression of Il-1β, Il-1ra, and Ccl3, but not Ccl2, at 1 day post-infection (dpi). Acod1-/- increased neutrophil but decreased macrophage infiltration. 4-OI prevented Pa infection in NL corneas (P = 6.7E-05) and alleviated Pa keratitis in DM corneas (P = 0.000204) at 3 dpi. Hyperglycemia augmented Pa infection-induced Il-1β, Il-1ra, and particularly Ccl3, but not Ccl2. In DM corneas, 4-OI greatly dampened the expression of CCL3 but not CCL2, compared to DM corneas without the treatment. The presence of 4-OI significantly reduced the severity of Pa keratitis in Acod1-deficient mice.

Conclusions: Acod1/itaconate is crucial for mediating protective immune responses against Pa infection in both NL and DM corneas. Acod1 activation and/or itaconate-based therapies may offer promising adjunctive treatments for microbial keratitis in patients with diabetes.

Figure 1.

Immunohistochemistry determination of Acod1 expression and distribution in NL and type 1 diabetes mellitus (T1DM) corneas at 1 dpi. STZ-T1DM (duration 10 weeks) and age-matched NL mice were infected with 2000 and 10,000 cfu Pa, respectively. At 1 dpi, the infected NL (A, C) and DM (B, D) corneas were excised, processed for cryostat section, and stained with antibody against Acod1 (green), followed by Alexa Fluor 594–conjugated goat anti-rabbit IgG. The Acod1 images were merged with DAPI staining for nuclei.

Figure 2.

Acod1 knockout mice developed severe Pa keratitis in B6 mice. The corneas of Acod1^–/– (B6 background, 10 weeks old) and age-matched WT mice were scarified and inoculated with indicated numbers of Pa (10,000 cfu) at 0 hour. (A–D) The infected corneas were photographed (A) and assigned clinical scores (B); bacterial burden is presented as colony-forming units of Pa per cornea (C) and MPO determination (units/cornea) (D). The results represent two independent experiments (n = 5 each), and P values were generated using the nonparametric Mann–Whitney U test for clinical scores and one-way ANOVA for colony-forming units and MPO. *P < 0.05, **P < 0.01.

Figure 3.

Effects of Acod1 deficiency on the expression of cytokines/chemokines, Ripk3, and Tnfaip3 in Pa-infected B6 mouse corneas at 1 dpi. Eight-week-old WT and Acod1 knockout mice were inoculated with 10,000 cfu of Pa. Infected corneas were collected at 1 dpi with noninfected corneas as the controls (0 hour, WT, and AC^–/–) and were subjected to real-time PCR analysis. The results are presented as the increase (fold) over the value for noninfected WT corneas (set at a value of 1) after normalization to the level of β-actin, used as the internal control. P values were generated by two-tailed Student's t-test to compare NL with DM corneas at the indicated time. *P < 0.05, **P < 0.01. The data are representative of two independent experiments with three mice per group.

Figure 4.

Immunohistochemical detection of neutrophils and macrophages in WT and Acod1^–/– mice corneas at 1 dpi. The cryostat sections of Pa-infected WT and Acod1^–/– mouse corneas at 1 dpi were stained with NIMP-R14 for neutrophils and F4/80 for macrophages. Arrows indicate representatives of DAPI-stained cells with multiple spots of F4/80 staining. E, epithelial layer; S, stroma.

Figure 5.

4-OI prevents infection in NL corneas and protects DM corneas from severe Pa keratitis in B6 mice. STZ-T1DM (duration 10 weeks) and age-matched NL mice were treated with or without 4-OI and inoculated with 2000 and 10,000 cfu Pa, respectively. (A, B) At 1 and 3 dpi, the corneas were photographed (A) and clinically scored (B). (C, D) The corneas were then excised at 3 dpi and subjected to colony-forming unit counting (CFU/corneas) (C) and MPO determination (units/cornea) (D). The results represent two independent experiments (n = 5 each), and P values were generated using the nonparametric Mann–Whitney U test for clinical scores and one-way ANOVA for colony-forming units and MPO. **P < 0.01. The lowercase letters in panels B to D refer to the corneas presented in A.

Figure 6.

Effects of 4-OI on the expressions of cytokines/chemokines, Ripk3, and Tnfaip3 in Pa-infected NL and DM corneas at 1 dpi. STZ-T1DM (duration 10 weeks) and age-matched NL mice were inoculated with 2000 and 10,000 cfu Pa, respectively. Infected corneas treated with 4-OI (NL+I or DM+I) or without 4-OI (NL or DM) were collected at 1 dpi with noninfected corneas as the controls (0 hour, NL, and DM) and were subjected to real-time PCR analysis. The results are presented as the increase (fold) over the value for noninfected NL and DM corneas (set at a value of 1) after normalization to the level of β-actin as the internal control. P values were generated by one-way ANOVA and followed by two-tailed Student's t-test to compare NL with DM corneas. *P < 0.05, **P < 0.01. The data are representative of two independent experiments with three mice per group.

Figure 7.

4-OI improves the outcome of Pa keratitis in Acod1 KO mice. Acod1^–/– mice corneas were treated with or without 4-OI at −4 hours and inoculated with the indicated 10,000 cfu Pa at 0 hour. (A–D) The infected corneas were photographed (A) and assigned clinical scores (B); bacterial burden is presented as colony-forming units of Pa per cornea (C), and MPO determination (units/cornea) (D). The results represent two independent experiments (n = 5 each). P values were generated using the nonparametric Mann–Whitney U test for clinical scores and one-way ANOVA for colony-forming units and MPO. *P < 0.05, **P < 0.01.