Aging, But Not Sex and Genetic Diversity, Impacts the Pathobiology of Bacterial Endophthalmitis

Abstract

Purpose: Age, sex, and genetics are important biological variables in determining an individual's susceptibility or response to infectious agents; however, their role has not been evaluated in intraocular infections. In this study, we comprehensively examined the impact of these host biological factors in the pathogenesis of experimental bacterial endophthalmitis.

Methods: Endophthalmitis was induced by intravitreal injection of bacteria (Staphylococcus aureus) in the eyes of male and female C57BL/6 mice of different ages: group I (young, 6-8 weeks), group II (mid-age, 18-20 weeks), and group III (old, 1 year). Highly heterogeneous outbred J:DO mice were used for genetic diversity analysis. Eyes were subjected to clinical examination, retinal function testing using electroretinography (ERG), histopathological analysis (hematoxylin and eosin staining), and bacterial burden estimation. The levels of inflammatory mediators were measured using qPCR and ELISA, and the infiltration of neutrophils was determined by flow cytometry.

Results: Both inbred C57BL/6 and diversity outbred (J:DO) mice were equally susceptible to S. aureus endophthalmitis, as evidenced by a time-dependent increase in clinical scores, bacterial burden, intraocular inflammation, and retinal tissue damage, in addition to decreased retinal function. However, no significant differences were observed in disease severity and innate responses in male versus female mice. Older mice (group III) exhibited higher clinical scores coinciding with increased bacterial proliferation and intraocular inflammation, resulting in enhanced disease severity. Moreover, bone-marrow-derived macrophages from old mice exhibited reduced phagocytic activity but increased inflammatory response toward S. aureus challenge.

Conclusions: Age, but not sex, is an important biological variable in bacterial endophthalmitis. Identification of pathways underlying altered innate immunity and impaired bacterial clearance in aging eyes could provide new insights into the pathobiology of intraocular infections in elderly patients.

Figure 1.

Both male and female C57BL/6 mice are equally susceptible to S. aureus endophthalmitis. Eyes of C57BL/6 male and female mice (6–8 weeks of age, n = 10–12 each sex) were infected with S. aureus (SA), strain RN6390 (5000 CFU/eye), by intravitreal injection. Eyes with PBS injection were used as control (C). (A) Representative micrograph shows that SA induced corneal haze/opacity in the infected eyes. (B) Disease progression was graded on a five-point clinical scoring system (score of 0 indicating a healthy eye and scores of 1–4 indicating increasing severity). (C) At indicated time points, the whole-eye lysates were used to estimate the intraocular bacterial burden by serial dilution and the plate count method (represented as CFU/eye). (D) Enucleated eyes were fixed and stained with H&E at the indicated time points. (E) Scotopic ERG was performed at the indicated time points. The bar graph represents the a- and b-wave amplitudes retained at the indicated time points. Statistical analysis was performed using Student's t-test (male vs. female; B, C) or two-way ANOVA with Tukey's multiple comparison test (control vs. SA and male vs. female; E). ^**P < 0.005; ^***P < 0.0005. C, cornea; AC, anterior chamber; L, lens; VC, vitreous chamber; R, retina; ONH, optic nerve head; ns, not significant.

Figure 2.

The inflammatory response in C57BL/6 mice is not influenced by sex bias. C57BL/6 male and female mice (6–8 weeks of age, n = 10 each sex) were infected with S. aureus (SA), strain RN6390 (5000 CFU/eye), by intravitreal injection. Eyes with PBS injection were used as control. (A) At the designated time points, the retinas were harvested and subjected to qPCR for the indicated cytokine or chemokine. (B) The whole-eye lysates were subjected to ELISA to measure the protein levels of the indicated cytokines or chemokines. (C) At the indicated time points, neural retinas were harvested, and single-cell suspensions were stained with anti-CD45–PECy5 and anti-Ly6G–FITC antibodies to estimate PMN infiltration. The bar graph represents the percent retinal PMN infiltration. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test (control vs. SA and male vs. female). ^*P < 0.05; ^**P < 0.005; ^***P < 0.0005.

Figure 3.

Genetic diversity does not influence a sex bias to S. aureus endophthalmitis. Eyes of J:DO male and female mice (6–8 weeks of age, n = 8–10 each sex) were infected with S. aureus (SA), strain RN6390 (5000 CFU/eye), by intravitreal injection. Eyes with PBS injection were used as control. (A) Representative micrograph shows that SA induced corneal haze/opacity in the infected eyes. (B) Disease progression was graded on a five-point clinical scoring system. (C) At the indicated time points, the whole-eye lysates were used to estimate the intraocular bacterial burden by serial dilution and the plate count method (represented as CFU/eye). (D) The whole-eye lysates were subjected to ELISA to measure the protein levels of the indicated cytokines or chemokines. (E) At the designated time points, scotopic ERG was performed, and the data are presented as amplitudes of the a- and b-waves. Statistical analysis was performed using Student's t-test (male vs. female; B, C) or two-way ANOVA with Tukey's multiple comparison test (control vs. SA and male vs. female; D, E). ^*P < 0.05; ^**P < 0.005; ^***P < 0.0005.

Figure 4.

Aging does not change the sex bias in S. aureus endophthalmitis. Mid-age (18–20 weeks of age) C57BL/6 male and female mice eyes (n = 8–10 each sex) were infected with S. aureus (SA), strain RN6390 (5000 CFU/eye), by intravitreal injection. Eyes with PBS injection were used as control. (A) Representative micrograph shows that SA induced corneal haze/opacity in the infected eyes. (B) Disease progression was graded on a five-point clinical scoring system and represented as a mean clinical score. (C) At the indicated time points, the whole-eye lysates were used to estimate the intraocular bacterial burden by serial dilution and the plate count method (represented as CFU/eye). (D) At the indicated time points, eyes were enucleated, fixed, and stained with H&E. (E) Scotopic ERG was performed at the indicated time points, and the data are presented as amplitudes of the a- and b-waves. Statistical analysis was performed using Student's t-test (male vs. female; B, C) or two-way ANOVA with Tukey's multiple comparison test (control vs. SA and male vs. female; E). ^***P < 0.0005.

Figure 5.

Aging increases disease severity in S. aureus endophthalmitis. Old (1 year of age, n = 10) and young (6–8 weeks of age, n = 10) C57BL/6 female mice eyes were infected with S. aureus (SA), strain RN6390 (5000 CFU/eye), by intravitreal injection. Eyes with PBS injection were used as control. (A) Representative micrograph shows that SA induced corneal haze/opacity in the infected eyes. (B) Disease progression was graded on a five-point clinical scoring system and represented as a mean clinical score. (C) At the indicated time points, the whole-eye lysates were serially diluted and plated for intraocular bacterial burden estimation (represented as CFU/eye). (D) At the designated time points, the retinas were harvested and subjected to qPCR to measure the mRNA transcripts of the indicated cytokines or chemokines. (E) The whole-eye lysates were subjected to ELISA to measure the protein levels of the indicated cytokines or chemokines. Statistical analysis was performed using Student's t-test (male vs. female; B, C) or two-way ANOVA with Tukey's multiple comparison test (control vs. SA and male vs. female; D, E). ^#,*P < 0.05; ^##,**P < 0.005; ^###,***P < 0.0005.

Figure 6.

Aging modulates retinal function in bacterial endophthalmitis. Old (1 year of age) and young (6–8 weeks of age) C57BL/6 female mice eyes (n = 10) were infected with S. aureus (SA), strain RN6390 (5000 CFU/eye), by intravitreal injection. Eyes with PBS injection were used as control. Scotopic ERG was performed at the indicated time points. The bar graph represents the a- and b-wave amplitudes retained at the respective time points. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test (control vs. SA and young vs. old). ^#P < 0.05; ^***P < 0.0005.

Figure 7.

BMDMs from aged mice exhibit increased inflammatory response and impaired antibacterial activity. (A) BMDMs from young (6–8 weeks of age) and old (1 year of age) mice were seeded in six-well plates and infected with S. aureus (SA) (MOI 10:1) for 6 hours. Uninfected cells were used as control. Following infection, both control and SA-infected cells were subjected to qPCR for the indicated inflammatory mediators. (B) Conditioned media were subjected to ELISA to quantify protein levels of the indicated cytokines and chemokines. (C) To assess the phagocytic and intracellular killing activity, BMDMs from young and old mice were challenged with SA for 2 hours. After 2 hours of infection, cells were rinsed to remove extracellular bacteria and incubated with fresh medium containing gentamicin (200 µg/mL) up to the indicated time points. At the desired time point, cells were lysed, and the viable bacterial counts were quantitated via serial dilution and plate count. At 1 hour, more CFU were observed in BMDMs from young mice, indicating that the number of internalized bacteria was greater as compared to the old mice, whereas at 24 hours there were more CFU in BMDMs from the old mice compared to their younger counterparts, indicating increased intracellular killing by BMDMs from the younger mice. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test (control vs. SA and young vs. old). Data represent mean ± SD from three independent experiments. ^#,*P < 0.05; ^##,**P < 0.005; ^###,***P < 0.0005.