TLR4 modulates inflammatory gene targets in the retina during Bacillus cereus endophthalmitis

Abstract

BackgroundEndophthalmitis is a serious intraocular infection that frequently results in significant inflammation and vision loss. Because current therapeutics are often unsuccessful in mitigating damaging inflammation during endophthalmitis, more rational targets are needed. Toll-like receptors (TLRs) recognize specific motifs on invading pathogens and initiate the innate inflammatory response. We reported that TLR4 contributes to the robust inflammation which is a hallmark of Bacillus cereus endophthalmitis. To identify novel, targetable host inflammatory factors in this disease, we performed microarray analysis to detect TLR4-dependent changes to the retinal transcriptome during B. cereus endophthalmitis.ResultsC57BL/6 J and TLR4−/− mouse eyes were infected with B. cereus and retinas were harvested at 4 h postinfection, a time representing the earliest onset of neutrophil infiltration. Genes related to acute inflammation and inflammatory cell recruitment including CXCL1 (KC), CXCL2 (MIP2-α), CXCL10 (IP-10), CCL2 (MCP1), and CCL3 (MIP1-α)) were significantly upregulated 5-fold or greater in C57BL/6 J retinas. The immune modulator IL-6, intercellular adhesion molecule ICAM1, and the inhibitor of cytokine signal transduction SOCS3 were upregulated 25-, 11-, and 10-fold, respectively, in these retinas. LIF, which is crucial for photoreceptor cell survival, was increased 6-fold. PTGS2/COX-2, which converts arachidonic acid to prostaglandin endoperoxide H2, was upregulated 9-fold. PTX3, typically produced in response to TLR engagement, was induced 15-fold. None of the aforementioned genes were upregulated in TLR4−/− retinas following B. cereus infection.ConclusionsOur results have identified a cohort of mediators driven by TLR4 that may be important in regulating pro-inflammatory and protective pathways in the retina in response to B. cereus intraocular infection. This supports the prospect that blocking the activation of TLR-based pathways might serve as alternative targets for Gram-positive and Gram-negative endophthalmitis therapies in general.Electronic supplementary materialThe online version of this article (10.1186/s12886-018-0764-8) contains supplementary material, which is available to authorized users.

Expression of Bacillus cereus Virulence-Related Genes in an Ocular Infection-Related Environment

“…However, the role of cereolyin O in the pathogenesis of B. cereus endophthalmitis remains unknown. We reported that Toll-like receptor 4 (TLR4) contributes to the robust inflammatory response to B. cereus during endophthalmitis and identified a cohort of TLR4-dependent inflammatory mediators that are upregulated in the retina 4 h following B. cereus infection [22]. This suggests that B. cereus is capable of directly or indirectly activating TLR4.…”

“…Therefore, ribosomal RNA was depleted using the Ribo-Zero rRNA Removal kit for bacterial rRNA (Illumina, San Diego, CA, USA) according to the manufacturer's instructions, and depletion was assessed via quantitative PCR using primers specific to the B. cereus 16s ribosomal RNA. 100 ng aliquots of RNA before and after depletion were subjected to qPCR using the iTaq™ Universal SYBR ® Green One-Step kit (Bio-Rad, Hercules, CA, USA) [22]. The forward and reverse primers were used at a final concentration of 300 nM.…”

“…The forward and reverse primers were used at a final concentration of 300 nM. The samples were run on a Bio-Rad ® CFX96 Touch™ Real-Time PCR System (Bio-Rad) [22]. Dissociation curves were used to assess the successful amplification of the desired product, and the threshold cycle (C T ) was used to determine relative amounts of transcripts between RNA samples before and after depletion.…”

“…B. cereus ATCC 14,579 was studied as a prototype B. cereus species with a sequenced and annotated genome. This particular strain has also been well characterized in terms of its virulence and pathogenesis in endophthalmitis [9,16,19,22,23]. It has 13 rRNA loci, and rRNA constitutes the bulk of the total RNA.…”

Toll‐like receptor 2 (TLR2) engages endoplasmic reticulum stress sensor IRE1α to regulate retinal innate responses in Staphylococcus aureus endophthalmitis

“…[8][9][10] Studies from both our laboratory and others have sought to elucidate the role of Toll-like receptors (TLRs) in the initiation of innate defense mechanisms in bacterial endophthalmitis. 4,8,[11][12][13][14][15] Of particular interest is TLR2, which has been found to orchestrate protective retinal innate immune responses in Staphylococcal endophthalmitis via modulating intraocular inflammation, and induction of antimicrobial peptides. 4,[8][9][10][11]15 Endoplasmic reticulum (ER) is an intricate cellular organelle present in eukaryotic cells.…”