TLR4 contributes to the host response to Klebsiella intraocular infection

Abstract

Purpose/aim: Klebsiella pneumoniae causes a blinding infection called endogenous endophthalmitis. The role of innate immune recognition of K. pneumoniae in the eye during infection is not known. We hypothesized that intraocular recognition of K. pneumoniae was mediated by Toll-like receptor (TLR)-4 and may be dependent on MagA-regulated hypermucoviscosity.

Materials and methods: Experimental endophthalmitis was induced in C57BL/6J or TLR4(-/-) mice by intravitreal injection of 100 CFU of wild type or ΔmagA K. pneumoniae. Infection and inflammation were quantified by determining viable K. pneumoniae per eye, retinal responses via electroretinography, myeloperoxidase activity of infiltrating neutrophils and the proinflammatory cytokine and chemokine response.

Results: C57BL/6J and TLR4(-/-) mice could not control intraocular wild-type K. pneumoniae growth. TLR4(-/-) mice were less able than C57BL/6J to control the intraocular growth of ΔmagA K. pneumoniae. Retinal function testing suggested that infection with ΔmagA K. pneumoniae resulted in less retinal function loss. There was a TLR4-dependent delay in initial neutrophil recruitment, regardless of the infecting organism. The proinflammatory cytokine/chemokine data supported these results. These findings were not due to an inability of TLR4(-/-) neutrophils to recognize or kill K. pneumoniae.

Conclusions: These studies suggest that TLR4 is important in the early intraocular recognition and host response to K. pneumoniae. However, the role of MagA in TLR4-mediated intraocular recognition and subsequent inflammation is less clear.

Keywords: Endophthalmitis; Klebsiella pneumoniae; Toll-like receptor 4 (TLR4); eye; hypermucoviscosity.

Figure 1. Comparison of Intraocular Bacterial Growth in C57BL/6J and TLR4-Deficient Mice

C57BL/6J or TLR4^−/− mice were infected with either wild type or ΔmagA K. pneumoniae. At the indicated time points, eyes were harvested for bacterial counts. n≥5 eyes per time point. *;p<0.01 for C57BL/6J ΔmagA vs. TLR4^−/− ΔmagA.

Figure 2. Evaluation of Retinal Function

After infection with K. pneumoniae, mice were dark adapted for at least 6 hours. At the indicated time points, mice were anesthetized as described above and subjected to electroretinography. The average of n>6 eyes ± SEM is shown for each time point. *;p≤0.01 for C57BL/6J –wild type K. pneumoniae. vs. TLR4^−/−–wild type K. pneumoniae

Figure 3. Ocular Histology of C57BL/6J and TLR4^−/− Mice during Experimental K. pneumoniae Endophthalmitis

At the indicated time points postinfection, mice were euthanized and whole globes were harvested, fixed, sectioned and stained with hematoxylin and eosin as described above. Each eye is representative of an n≥3, except the 24 hour time point for wild type K. pneumoniae in C57BL/6J mice. At this time point only one eye had not undergone pthisis and could be removed intact.

Figure 4. Comparison of Retinal Histology During Experimental K. pneumoniae Endophthalmitis

Sections of C57BL/6J and TLR4^−/− eyes infected with wild type or ΔmagA K. pneumoniae were stained with hematoxylin and eosin. There was a marked delay in the infiltration of immune cells into the vitreous of C57BL/6J mice infected with the ΔmagA strain as well as in TLR4^−/− mice infected with either strain. Retinal folding and thickening of the inner plexiform layer can be seen in wild type-infected retinas. RPE; retinal pigment epithelium, PR; photoreceptors, ONL; outer nuclear layer, OPL; outer plexiform layer, INL; inner nuclear layer, IPL; inner plexiform layer, GCL; ganglion cell layer, ILM; inner limiting membrane. All sections are 20x magnification.

Figure 5. Neutrophil Recruitment to the Eye is Delayed in TLR4^−/− mice

At the indicated time points postinfection, mice were euthanized and whole globes were harvested, homogenized in PBS and used to determine the amount MPO present in the sample by ELISA. *;p≤0.05.

Figure 6. Delay in Proinflammatory Cytokine Production in TLR4^−/− Mice

C57BL/6J or TLR4^−/− mice were infected with wild type or ΔmagA K. pneumoniae and eyes were assayed for KC, TNFα, or MIP-1α by ELISA. n≥5 eyes except for KC in C57BL/6J mice at 24 hours where n=3. Error bars represent standard deviation, *; p≤0.01, †; p<0.01 when values below the LOD were estimated by. $\frac{\mathit{LOD}}{\sqrt{2}}$.

Figure 7. Killing of K. pneumoniae by Neutrophils

Casein-elicited peritoneal neutrophils were purified as described in the Methods. Cells and bacteria were mixed in a 1:1 ratio and incubated at 37°C for the indicated times when cell pellets were washed and supernatants pooled. Data in A is displayed as percent of input bacteria recovered ± standard deviation and represents an n= 3 repeated on separate days (total of n=6). A, wild type; B, TLR4^−/− mice. Pel; pellet, supe; supernatant. *, p<0.01.