Diabetes Exacerbates Pseudomonas aeruginosa Keratitis in Streptozotocin-Induced and db/db Mice via Altering Programmed Cell Death Pathways

Abstract

Purpose: Patients with diabetes have a higher incidence of infections, which are often more severe. This study aimed to investigate the impact of hyperglycemia on bacterial keratitis caused by Pseudomonas aeruginosa (Pa) in two mouse models of diabetes, streptozotocin-induced type 1 diabetes mellitus (T1DM) and db/db type 2 diabetes mellitus.

Methods: The susceptibility of corneas to Pa was assessed by determining the inocula required to cause infectious keratitis. Dead or dying cells were identified using TUNEL staining or immunohistochemistry. Specific inhibitors were used to evaluate the role of cell death modulators in Pa keratitis. Cytokines and Treml4 expressions were analyzed using quantitative PCR, and the role of Treml4 in keratitis was determined using small interfering RNA technology.

Results: DM corneas required significantly fewer inocula to develop Pa keratitis, with T1DM corneas requiring 750 inocula and type 2 diabetes mellitus corneas requiring 2000 inocula, compared with 10,000 inocula required for normal (NL) mice. T1DM corneas had more TUNEL-positive and fewer F4/80-positive cells than NL corneas. Phospho-caspase 8 (apoptosis) and -RIPK3 (necroptosis) staining was more intense in the epithelial and stromal layers of NL and T1DM corneas, respectively. Pa keratitis was augmented by targeting caspase-8 and prevented by RIPK3 inhibition in both NL and T1DM mice. Hyperglycemia suppressed IL-17A/F and augmented IL-17C, IL-1β, IL-1Ra, and TREML4, the downregulation of which protected T1DM corneas from Pa infection by suppressing necroptosis. RIPK3 inhibition blocked Pa infection in db/+ mice and significantly decreased the severity of keratitis in db/db mice.

Conclusions: Hyperglycemia exacerbates bacterial keratitis in B6 mice by skewing apoptosis toward necroptosis. Preventing or reversing this transition may serve as an adjunct therapy for treating microbial keratitis in patients with diabetes.

Figure 1.

Susceptibility and severity of Pa keratitis in NL and T1DM B6 mice. The corneas of STZ-T1DM B6 (duration 10 weeks) and age-matched NL mice were scarified and inoculated with indicated numbers of Pa (CFU) at 0 h. The infected corneas were photographed (A) and assigned clinical scores (A’) at 1 and 3 dpi. At 3 dpi, the corneas were excised and subjected to bacterial counting (B) presented as CFU of Pa per cornea and to MPO determination (units/cornea) (C). The results are representative of three independent experiments (n = 5 each), and P values were generated using the nonparametric Mann–Whitney U test for clinical scores and two-way ANOVA for CFU and MPO. *P < 0.05; ** P < 0.01. The lowercase letters in (A’–C) referred to the corneas presented in (A).

Figure 2.

TUNEL and macrophage staining of NL and T1DM corneas at 1 dpi at a lesion site. 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 immunostained with TUNEL staining for dead cells (green) and F4/80 for macrophages (red). The TUNEL and F4/80 images were merged and superimposed with DAPI staining for nuclei. Arrows, macrophages costained with TUNEL; arrowheads, TUNEL-positive cells or cell debris that are not near macrophages. (B and D) High magnification of (A and B), respectively.

Figure 3.

Immunohistochemical detection of c-Casp8–positive innate immune cells in NL and DM corneas at 1 dpi. The cryostat sections of Pa-infected NL and DM corneas at 1 dpi used in Figure 2 were stained with c-Casp8 antibody and costained with F4/80 (macrophages) or NIMP-R14 (polymorphonuclear leucocytes). Arrows, representative of neutrophils or macrophages that were also c-Casp8 positive, indicative of apoptosis. E, epithelial layer; S, stroma. Scale bar, 100 µm.

Figure 4.

Immunohistochemical detection of p-RIPK3–positive innate immune cells in NL and DM corneas at 1 dpi. The cryostat sections of Pa-infected NL and DM corneas at 1 dpi used in Figure 2 were stained with antibodies against phospho-RIPK3 and co-stained with F4/80 (macrophages) or NIMP-R14 (polymorphonuclear leucocytes). Arrows, representative of neutrophils or macrophages that were also p-RIPK3 positive, indicative of necroptosis. E, epithelial layer; S, stroma. Scale bar, 100 µm.

Figure 5.

Effects of Casp-8 or RIPK3 inhibition on Pa keratitis in NL and T1DM corneas. STZ-T1DM (duration 10 weeks) and age-matched NL mice were inoculated with 2000 and 10,000 CFU Pa, respectively. At 1 dpi, the corneas were photographed (A) and clinically scored (B). The corneas were then excised and subjected to CFU 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 CFU and MPO. **P < 0.01. The lowercase letters in (B–D) refer to the corneas presented in (A).

Figure 6.

Effects of hyperglycemia on the expressions of the IL-1, IL-17 families of cytokines and TremL4 in B6 mouse corneas during Pa infection. STZ-T1DM (duration 10 weeks) mice were inoculated with 10,000 (DM 6pi), 2000 (DM 1dpi), or 750 (DM750 3dpi) CFU of Pa while age-matched NL mice inoculated with 10,000 CFU. Infected corneas were collected at the indicated time with noninfected cornea as the controls (0 h, 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 value 1) after normalization to the level of β-actin as the internal control. P values were generated by a two-tailed Student's t-test to compare NL with DM corneas at the indicated time; * P < 0.05 and ** P < 0.01. Data are representative of three independent experiments with three mice per group.

Figure 7.

TREML4 affecting the outcome of Pa keratitis by promoting necroptosis in DM corneas. STZ-T1DM (duration 10 weeks) were treated with the control (Aa) and Treml4 siRNA (Ab and Ac) via subconjunctival injection of 5 µL of siRNA –24 and –6 hours and inoculated with 2000 (Ab) or 1000 (Aa and Ac) CFU of Pa at 0 h. At 1 dpi, all corneas were photographed (A) and a clinical score was assigned (B). One group (n = 3) was subjected to CFU (C) and MPO (D) determination. Another group of corneas was subjected to IHC analysis of neutrophils (NIMP-R14, green) and p-MLKL (necroptotic cells, red) (E). Arrows, NIMP-R14 and p-MLKL positive; arrowheads, p-MLKL positive, NIMP-R14 negative. E, epithelium; S, stroma. Scale bar, 10 µm.

Figure 8.

Susceptibility and severity of Pa keratitis in db/db mice. (A) Ten-week-old db/db and db/+ (n = 6, 3 male and 3 female) were scarified and inoculated with indicated numbers of Pa (CFU) at 0 h. The infected corneas were photographed (A) and assigned clinical scores at 1 (B) and 3 dpi (C). At 3 dpi, the corneas were excised and subjected to bacterial counting (white number in A) presented as CFU of Pa per cornea and to MPO determination (units/cornea) (D). The letters in (B–D) correspond with the letters in (A). The results represent three independent experiments (n = 5 each). P values were generated using the nonparametric Mann–Whitney U test for clinical scores and two-way ANOVA for CFU and MPO. * P < 0.05; ** P < 0.01.

Figure 9.

Suppression of necroptosis improves the outcome of Pa keratitis in db/+ and db/db mice. RIPK3 inhibitor GSK-872 was administrated through subconjunctival injection 4 hours before Pa inoculation. The infected corneas were photographed (A) and assigned clinical scores at 1 dpi (numbers in corneal micrograph). At 1 dpi, the corneas were excised and subjected to bacterial counting presented as CFU of Pa per cornea (B), to MPO determination (units/cornea, C), and to qPCR analysis of IL-1β and IL-1Ra (fold increase) (D). The results are representative of two independent experiments (n = 6), and P values in (B–D) were generated using one-way ANOVA. ** P < 0.01.