Innate Immune System Activation, Inflammation and Corneal Wound Healing

Abstract

Corneal wounds resulting from injury, surgeries, or other intrusions not only cause pain, but also can predispose an individual to infection. While some inflammation may be beneficial to protect against microbial infection of wounds, the inflammatory process, if excessive, may delay corneal wound healing. An examination of the literature on the effect of inflammation on corneal wound healing suggests that manipulations that result in reductions in severe or chronic inflammation lead to better outcomes in terms of corneal clarity, thickness, and healing. However, some acute inflammation is necessary to allow efficient bacterial and fungal clearance and prevent corneal infection. This inflammation can be triggered by microbial components that activate the innate immune system through toll-like receptor (TLR) pathways. In particular, TLR2 and TLR4 activation leads to pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) activation. Similarly, endogenous molecules released from disrupted cells, known as damage-associated molecular patterns (DAMPs), can also activate TLR2, TLR4 and NFκB, with the resultant inflammation worsening the outcome of corneal wound healing. In sterile keratitis without infection, inflammation can occur though TLRs to impact corneal wound healing and reduce corneal transparency. This review demonstrates the need for acute inflammation to prevent pathogenic infiltration, while supporting the idea that a reduction in chronic and/or excessive inflammation will allow for improved wound healing.

Keywords: cornea; healing; inflammation; innate immune system; phosphatidylglycerol; toll-like receptors; wound.

Figure 1

Monitoring Corneal Wound Healing. Corneal epithelial wounds are created with an Alger brush, which is used to scrape off the corneal epithelium. Exposure of the underlying stroma allows visualization of the wound with the dye fluorescein, which binds to the stroma but not the intact epithelium. The rate of wound healing can be monitored over time by measuring the percentage of the wound area remaining as visualized with fluorescein. Agents of interest can be topically applied to the corneal surface (once or repeatedly) to determine their impact on corneal wound healing.

Figure 2

Toll-like Receptor Signaling Pathway. Toll-like receptors are located in the plasma membrane (TLR-2/-1, TLR-2/-6, TLR-4 and TLR-5) or the endosome (TLR-3, TLR-7, TLR-8 and TLR-9) where they are engaged by the indicated microbial components to initiate signaling through myeloid differentiation primary response 88 (MyD88) and in some cases the adaptor TIR domain-containing adaptor protein (TIRAP). MyD88 interaction with interleukin-1 receptor-associated kinase (IRAK) protein kinases activates TNF receptor-associated factor-6 (TRAF6) and through the adaptors TAB2 and 3, the kinase (TGFβ)-activated kinase 1 (TAK1). TAK1 phosphorylates and activates inhibitory kappaB (IκB) kinases (IKK)-α and -β, which are in complex with a regulatory subunit, NFκB essential modulator (NEMO), also known as IKKγ. Phosphorylation of IκB targets it for degradation thereby releasing the transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). NFκB translocates into the nucleus where it promotes the expression of pro-inflammatory mediators. TLR-4 signaling also involves lipopolysaccharide (LPS)-binding protein (LBP), which helps to present LPS to the TLR-4 receptor complex, which also includes myeloid differentiation protein-2 (MD-2) and the co-receptor cluster of differentiation-14 (CD-14). LPS binding to TLR-4 can also activate a MyD88-independent pathway through TIR domain-containing adaptor inducing interferon-β (TRIF) and translocation-associated membrane protein (TRAM) to activate TRAF3 to induce type I interferon expression; dsRNA = double-stranded RNA, ssRNA = single-stranded RNA and CpG DNA = CpG-rich DNA. Created with Biorender.com.

Figure 3

Innate Immune System Activation upon Corneal Wounding. As shown, corneal wounding results in the release of cytokines like interleukin-1 (IL-1), which can lead to keratocyte apoptosis and the release of endogenous molecules, called danger- or damage-associated molecular patterns (DAMPs); DAMPs can also be released from the injured corneal epithelial cells. DAMPs are able to activate pattern recognition receptors of the innate immune system such as toll-like receptors-2 and -4 (TLR2/4), which can function through the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway to induce the production and release of additional cytokines and chemokines that induce infiltration of innate and adaptive immune cells into the cornea. This immune cell recruitment can then lead to chronic sterile inflammation, which can in turn, impair vision. The phospholipid phosphatidylglycerol has been found to inhibit TLR2/4 activation by DAMPs and microbial components known as pathogen-associated molecular patterns. This effect, as well as its ability to enhance corneal wound healing, suggest the possibility that it might be useful as a therapy to reduce inflammation and improve corneal wound healing.