Butyrate Ameliorates Intraocular Bacterial Infection by Promoting Autophagy and Attenuating the Inflammatory Response

Abstract

Despite an important link between the gut and ocular health, the role of the gut-eye axis remains elusive in ocular infections. In this study, we investigated the role of butyrate, a gut microbial metabolite, in the pathobiology of intraocular bacterial (Staphylococcus aureus) infection, endophthalmitis. We found that intravitreal administration of butyrate derivatives, sodium butyrate (NaB), or phenylbutyrate (PBA) reduced intraocular bacterial growth and retinal inflammatory response. The ocular tissue architecture and retinal function were preserved in butyrate-treated eyes. In cultured mouse bone marrow-derived macrophages (BMDMs) and human retinal Müller glia, NaB or PBA treatment reduced S. aureus-induced inflammatory response by inhibiting NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome. However, in vivo data showed NLRP3-independent effects of butyrate. The butyrate-treated mouse retina and cells exhibited induced expression of antimicrobial molecules CRAMP (LL37) and S100A7/A8, resulting in increased bacterial phagocytosis and killing. Moreover, butyrate treatment enhanced AMP-activated protein kinase (AMPK)-dependent autophagy and promoted the co-localization of CRAMP in autophagosomes, indicating autophagy-mediated bacterial killing. Furthermore, pharmacological inhibition of autophagy in mice revealed its role in butyrate-mediated protection. Finally, butyrate exhibited synergy with antibiotic in promoting endophthalmitis resolution. Collectively, our study demonstrated the protective mechanisms of butyrate in ameliorating bacterial endophthalmitis. Therefore, butyrate derivatives could be explored as immunomodulatory and anti-bacterial therapeutics to improve visual outcomes in ocular bacterial infections.

Keywords: AMPK; Staphylococcus aureus; autophagy; butyrate; endophthalmitis; eye; inflammation; innate immunity; retina.

FIG 1

Butyrate ameliorates bacterial endophthalmitis in mice. Endophthalmitis was induced in C57BL/6 mice eyes (n = 4) by intravitreal injection of Staphylococcus aureus. Eyes were treated with various doses (5, 10, 20 μg/eye) of sodium butyrate (NaB) or phenylbutyrate (PBA) via intravitreal injection at 6 h postinfection. (A) Timeline of induction of endophthalmitis, butyrate treatment, and assays used to monitor disease progression. (B) Quantitation of intraocular bacterial burden by serial dilution and plate count, represented as CFU/eye. (C) Upper panel: representative slit-lamp images showing corneal haze/opacity. Lower panel: hematoxylin and eosin (H&E) staining of eyes enucleated at 30 h postinfection. (D) Corneal opacity was measured using ImageJ and represented as integrated pixel intensity. (E) H&E was quantified by pathology score, with 4 being the worst and 0 being the best. (E) Scotopic electroretinography (ERG) response as measured by the percentage of a- and b-wave amplitudes retained in comparison to uninfected control eyes, with values kept at 100%. Data represent the culmination of two independent experiments and are shown as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons (panels B and D). **, P < 0.001; ***, P < 0.0001; ns, not significant.

FIG 2

Butyrate treatment reduces the inflammatory response and neutrophil infiltration in the eyes. Endophthalmitis was induced in C57BL/6 mouse eyes by intravitreal inoculation of 5,000 CFU of S. aureus strain RN6390. After 6 h, eyes were treated with butyrate derivatives (10 μg/eye) NaB or PBA via intravitreal injection. (A) Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of indicated inflammatory cytokines or chemokines in whole eye lysates (n = 6). (B) Flow cytometry was performed using retinal single-cell suspensions to determine neutrophil infiltration (CD45⁺Ly6G⁺ cells). Bar graph represents the percentage of neutrophil infiltration with or without butyrate derivative treatment. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparison test (panels A and B). **, P < 0.001; ***, P < 0.0001; ns = not significant.

FIG 3

Butyrate attenuates S. aureus-induced inflammatory response in cultured cells. (A and B) Mouse bone marrow-derived macrophages (BMDMs) from C57BL/6 mice (n = 4) and (C and D) human retinal Müller glia (MIO-M1 cell line) were pretreated with NaB or PBA (3 mM) for 2 h followed by infection with S. aureus (MOI = 10:1) for 6h. The transcript levels of inflammatory cytokines were measured by quantitative PCR (qPCR) (A and C), and ELISA was used to estimate protein levels (B and D). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparison test by comparing S. aureus-infected samples with or without butyrate treatment. **, P < 0.001, ***, P < 0.0001.

FIG 4

Butyrate inhibits S. aureus-induced NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome. BMDMs from C57BL/6 mice (n = 4/condition) were either left untreated or pretreated with NaB or PBA at 3 mM for 2 h, followed by S. aureus (MOI = 10:1) challenge for 6 h. (A and B) qPCR was performed to determine Nlrp3 and Caspase1 expression. Data are expressed as relative fold change by normalizing gene expression to the endogenous β-actin gene. (C) Western blot detection of NLRP3 and (D) densitometry analysis was performed using ImageJ; data are expressed as relative fold changes normalized to the loading control HSP90. Data represent the culmination of two independent experiments and are shown as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparison test by comparing S. aureus-infected samples with or without NaB or PBA treatment. ***, P < 0.0001; ns = not significant.

FIG 5

NLRP3 inhibition exacerbates the disease pathology and diminishes the anti-inflammatory properties of butyrate. C57BL/6 mice eyes (n = 4) were injected with the selective NLRP3 inhibitor MCC950 (50 μM/eye) 12 h before induction of endophthalmitis. After 6 h postinfection, eyes were treated with butyrate derivatives (10 μg/eye) NaB or PBA. (A) Representative slit-lamp images showing corneal haze/opacity, with increased corneal opacity (B) represented as integrated pixel intensity by ImageJ quantification. (C) Quantitation of intraocular bacterial burden by serial dilution and plate count represented as CFU/eye (n = 4). (D) ELISA was performed to measure levels of indicated inflammatory cytokines or chemokines in whole-eye lysates (n = 4). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparison test. *, P < 0.05; **, P < 0.001; ***, P < 0.0001; ns = not significant.

FIG 6

Butyrate treatment promotes bacterial phagocytosis and killing. (A) BMDMs from C57BL/6 mice and (B) human retinal Müller glia (MIO-M1 cell line) were pretreated with NaB (3 mM) for 2 h followed by challenge with S. aureus (MOI = 10:1). After 2 h of infection, cells were rinsed to remove extracellular bacteria and incubated with fresh medium containing gentamicin (200 μg/mL) for the indicated time points. At the desired time points, cells were lysed, and viable bacterial counts were quantitated via serial dilution and plate count. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparison test (A and B). Data represent mean ± SD from three independent experiments. *, P < 0.05; **, P < 0.001; ns = not significant.

FIG 7

Butyrate treatment induced the expression of antimicrobial molecules both in vitro and in vivo. (A) BMDMs from C57BL/6 mice (n = 6/condition) and (B and C) human retinal Müller glia (MIO-M1 cell line) were either left untreated or pretreated with NaB or PBA (3 mM) for 2 h followed by infection with green fluorescent protein (GFP)-expressing S. aureus (green) at 6 h for qPCR or 3 h for immunostaining. The expression of genes (e.g., S100a7, S100a8, Cramp, LL37) with antimicrobial functions was assessed by qPCR (A and B) or immunostaining for S100A7 (red) or LL37 (red) expression, and images were captured at ×60 original magnification (C). To assess the expression of these genes in vivo, mouse retinal tissue was used for qPCR (D). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparison test (panels A, B, and D). Data represent mean ± SD from three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.0001; ns = not significant.

FIG 8

Butyrate potentiates autophagy and autophagosome-mediated bacterial killing. (A to C) BMDMs from C57BL/6 mice (n = 3) were pretreated with 3-methyladenine (3-MA) (1 mM), NaB, or PBA (3 mM) for 2 h, followed by infection with S. aureus (MOI = 10:1) for 3 or 6 h. (A and C) Western blot analysis was performed to detect p62, LC3BII, pAMPK, total AMP-activated protein kinase (AMPK), pACC, total ACC, β-actin, and HSP90. (B and D) Densitometric analysis of immunoblots were performed using ImageJ. Data are expressed as relative fold change by normalizing the expression of proteins with respect to the housekeeping controls β-actin or HSP-90. (E) Protein expression of autophagy markers p62 and LC3BII was determined in human retinal Müller glia (MIO-M1 cell line) by immunostaining. Images were captured at ×60 magnification. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparison test (B and D). *, P < 0.01; **, P < 0.001; ***, P < 0.0001; ns = not significant.

FIG 9

Butyrate treatment potentiates the co-localization of LL37 and LC3BII. Human retinal Müller glia (MIO-M1 cell line) were pretreated with NaB or PBA (3 mM) for 2 h followed by challenge with S. aureus (MOI = 10:1) for 3 h. (A) Representative images showing immunostaining for LC3BII (green), LL37 (red), and DAPI (4′,6-diamidino-2-phenylindole; blue, a cell nuclear stain). All images were captured at ×60 magnification. (B) Bar graph showing the fraction of LL37 and LC3BII co-localization cells/field. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparison test. Data represent mean ± SD from two independent experiments. **, P < 0.001.

FIG 10

Autophagy inhibition exacerbates endophthalmitis and diminishes the protective effects of butyrate. C57BL/6 mice eyes (n = 4) were injected with selective autophagy inhibitor 3-MA (500 μM/eye) 12 h before induction of endophthalmitis. At 6 h postinfection, eyes were treated with NaB or PBA (10 μg/eye) via intravitreal injections. (A) Representative slit-lamp images showing corneal haze/opacity and (B) increased corneal opacity represented as integrated pixel intensity by ImageJ quantification. (C) Quantitation of intraocular bacterial burden by serial dilution and plate count represented as CFU/eye (n = 4). (D) ELISA was performed to measure the levels of indicated inflammatory cytokines or chemokines in whole-eye lysates (n = 4). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparison test. *, P < 0.01; **, P < 0.001; ***, P < 0.0001; ns = not significant.

FIG 11

Mechanisms underlying butyrate treatment mediated protection in the eye. Butyrate derivatives (NaB or PBA) exert both antibacterial and anti-inflammatory properties in mouse eyes and cultured cells (macrophages and Müller glia). Butyrate treatment activates AMPK, promotes selective autophagy, induces antimicrobial peptide expression, and inhibits NLRP3 expression. This ameliorates bacterial endophthalmitis by increasing bacterial clearance and reducing intraocular inflammation.