Modulation of immune signaling, bacterial clearance, and corneal integrity by toll-like receptors during streptococcus pneumoniae keratitis

Abstract

Purpose: Bacterial keratitis, without effective antimicrobial treatment, leads to poor patient prognosis. Even after bacterial clearance, the host inflammatory response can contribute to corneal damage. Though Streptococcus pneumoniae (pneumococcus) is a common cause of bacterial keratitis, the role of host innate immunity during pneumococcal keratitis is not well characterized. This study investigated the role of Toll-like receptors (TLRs) during pneumococcal keratitis.

Materials and methods: C57BL/6, as well as TLR2(-/-) and TLR4(-/-) mice, were infected with S. pneumoniae, and infected corneas were examined for 21 days. Quantitative real-time reverse-transcriptase polymerase chain reaction was performed using primers for genes involved in the inflammatory response and TLR signaling. Bacterial survival and leukocyte invasion were examined over a 72-h period.

Results: The corneal expression of TLR2, TLR4, and other inflammatory genes was increased at 72 h post-infection (p.i.) compared to uninfected C57BL/6 scratch controls. TLR2(-/-) mice showed a significant increase in bacterial survival at 24 h p.i. likely due to decreased neutrophil infiltration; however, after Day 5 p.i. observed clinical scores of TLR2(-/-) and C57BL/6 mice were not significantly different. In contrast, permanent corneal damage was observed for TLR4(-/-) mice over 21 days. Initially, both TLR(-/-) mouse strains exhibited lower expression levels in many immune genes, but returned to similar or elevated levels compared to C57BL/6 mice by 72 h p.i.

Conclusions: TLR2 and TLR4 are involved in the response to pneumococcal keratitis and TLR2 may aid in bacterial clearance by recruitment of neutrophils to the cornea, whereas TLR4 may be necessary to modulate the immune response to limit cellular damage.

FIGURE 1

TLR expression in infected C57BL/6 corneas. Infected and scratched C57BL/6 corneas were harvested at 24 h p.i. (A and B) and 72 h p.i. (C and D). Harvested corneas were stained with anti-TLR2 (A and C) or anti-TLR4 (B and D) antibodies. Representative corneal sections (left panel) and individual epithelial cells (right panel) for infected and scratched corneas stained with either anti-TLR2 or anti-TLR4 are provided. Expression of both TLRs was detected at both 24 and 72 h p.i. The anti-TLR antibodies stained more strongly in infected corneas than in scratched corneas at both time points (compare top panel pair to bottom panel pair in each group). The antibody isotype control corneal section and individual epithelial cells shows diffuse, non-specific staining (E). Scale bar for corneal sections represents 10 μm and for individual epithelial cells represents 5 μm.

FIGURE 2

Mean clinical scores. C57BL/6, TLR2^−/− and TLR4^−/− mice were inoculated with 10⁸ total CFU of pneumococci and gross corneal damage was measured using the clinical score rubric from Day 1 to Day 21 p.i. TLR2-deficient mutants showed decreased damage at 24 h and increased damage at 48, 72 and 96 h p.i. compared to C57BL/6 mice. Damage observed in TLR2-deficient mice was similar to C57BL/6 mice over the 21-day period. TLR4-deficient mutants showed increased damage compared to C57BL/6 mice over the 21-day period. Error bars represent SEM at each time point. Stars indicate significance (p < 0.05).

FIGURE 3

Mean neutrophil counts of corneal histology sections. Limbus-to-limbus counts were obtained from three sections each for each mouse strain and time point. “Scratch” represents C57BL/6 corneal scratch controls. Asterisks denote statistical significance compared to C57BL/6 corneas (p < 0.05). Error bars represent SEM at each time point.

FIGURE 4

Histology of C57BL/6, TLR2^−/− and TLR4^−/− mouse corneas. Corneas were stained with anti-neutrophil elastase antibody. Representative histology slides showing neutrophils stained with anti-neutrophil elastase as well as isotype controls and whole cornea sections at 24 h p.i. H/E-stained corneas for each group are also shown. Panel A: C57BL/6 corneas at 24 h p.i. Panel B: TLR2^−/− corneas at 24 h p.i. Panel C: TLR4^−/− corneas at 24 h p.i. Neg: isotype control antibody for each group. Pos: anti-elastase antibody stained corneas for each group. Corneal cross-section scale bar represents 10 μm. Individual neutrophil panel scale bar represents 5 μm.

FIGURE 5

Immune gene expression in infected C57BL/6 corneas. C57BL/6 corneas were harvested at 24 and 72 h p.i. and qRT-PCR performed on 84 immune genes with error bars representing SEM for each gene. Granulocyte maturation genes and chemokines (CSF2, CSF3 and CXCL10) were upregulated as well as cytokines (IL-10, IL-2 and IL-6) and TLR signaling genes (MyD88, TRIF and TRAM). Upregulation decreased but was still >2-fold at 72 h.

FIGURE 6

Immune gene expression in TLR2^−/− corneas. TLR2^−/− corneas were harvested at 24 and 72 h p.i. and qRT-PCR performed using primers for 84 immune genes with error bars representing SEM for each gene. Granulocyte maturation genes and chemokines (CSF2, CSF3 and CXCL10) were all downregulated as well as cytokines (IL-10, IL-2 and IL-6) and TLR signaling genes (MyD88, TRIF, TRAM and TIRAP). Upregulated expression levels were observed at 72 h p.i.

FIGURE 7

Immune gene expression in TLR4^−/− corneas. TLR4^−/− corneas were harvested at 24 and 72 h p.i. and qRT-PCR performed using primers for 84 immune genes with error bars representing SEM for each gene. Granulocyte maturation genes and chemokines (CSF2, CSF3 and CXCL10) were all downregulated as well as cytokines (IL-10, IL-2 and IL-6) and TLR signaling genes (MyD88, TRIF and TRAM) except for TIRAP. Slightly downregulated expression of these genes was observed at 72 h p.i. with the exception of CSF3 and TIRAP.

FIGURE 8

Detection of Th1 and Th2-associated cytokines. C57BL/6 mice were infected with 10⁸ total CFU and their corneas were harvested at 24 and 72 h p.i. Cytokine protein concentrations were determined using a CBA. Low concentrations (<5 pg/mL) of the Th1-associated IFN-y and IL-2 were detected for infected, scratched and uninfected C57BL/6 corneas at 24 (A) and 72 h p.i. (B). Higher concentrations of the Th2-associated IL-6 and IL-10 were detected at both time points though not significantly higher than scratch or uninfected controls. Error bars represent SEM for each cytokine.