Nuclear Factor-κB: Central Regulator in Ocular Surface Inflammation and Diseases

Laboratory Science
JAMES V. JESTER, PHD, EDITOR
Nuclear Factor-kB: Central Regulator in OcularSurface Inflammation and Diseases

WANWEN LAN, BSC, 1 ANDREA PETZNICK, PHD, 1 SUZI HERYATI, MD, 2 MAULA RIFADA, MD, 2

AND LOUIS TONG, FRCS, PHD 1,3,4

ABSTRACT The nuclear factor-kB (NF-kB) is a key tran-scription factor pathway that is responsible for many keybiological processes, such as inflammation, apoptosis, stressresponse, corneal wound healing, angiogenesis, and lymph-angiogenesis. Numerous recent studies have investigatedNF-kB in the context of ocular surface disorders, includingchemical injury, ultraviolet radiation-induced injury, micro-bial infections, allergic eye diseases, dry eye, pterygium, andcorneal graft rejection. The purpose this article is tosummarize key findings with regard to the pathways regu-lating NF-kB and processes governed by the NF-kB pathway.In the innate defense system, NF-kB is involved in signalingfrom the toll-like receptors 2, 3, 4, 5 and 7, which areexpressed in conjunctival, limbal, and corneal epithelial cells.These determine the ocular responses to infections, such asthose caused by Pseudomonas aeruginosa, Staphylococcusaureus, adenovirus, and herpes simplex-1 virus. Naturalangiogenic inhibitors enhance NF-kB, and this may occurthrough the mitogen-activated protein kinases andperoxisome proliferator-activated receptor g. In alkali injury,inhibition of NF-kB can reduce corneal angiogenesis,suggesting a possible therapeutic strategy. The evaluation ofNF-kB inhibitors in diseases is also discussed, including emodin,besifloxacin, BOL-303242-X (mapracorat), thymosin-b4,

Accepted for publication April 2012.

From the 1Singapore Eye Research Institute, Singapore, 2Cicendo EyeHospital, Bandung, Indonesia, 3Corneal and External Eye Disease Service,Singapore National Eye Center, Singapore, and 4Duke-NUS GraduateMedical School, Singapore.

Grant Support: National Medical Research Council grants NMRC/TCR/002-SERI/2008, NMRC/1206/2009, NMRC/CSA/013/2009, SinghealthFoundation Grant SHF/FG325S/2007.

The authors have no commercial or proprietary interest in any concept orproduct discussed in this article.

Single-copy reprint requests to Louis Tong, MD (address below).

Corresponding author: Louis Tong, MD, Singapore National Eye Center, 11Third Hospital Avenue, Singapore 168751. Tel: 65-62277255. Fax: 65-6322-4599. E-mail address: [email protected]

© 2012 Elsevier Inc. All rights reserved. The Ocular Surface ISSN: 1542-0124. Lan W, Petznick A, Heryati S, Rifada M, Tong L. Nuclear factor-kB:Central regulator in ocular surface inflammation and diseases.2012;10(3):137-148.

THE OCULAR SURFACE / JULY 2012, VOL.

epigallocatechin gallate, Perilla frutescens leaf extract andIKKb-targeting short interfering RNA.

KEY WORDS angiogenesis, apoptosis, inflammation, NF-kB,signal transduction, transcription factors, wound healing

I. INTRODUCTION TO NUCLEAR FACTOR-kB (NF-kB)PATHWAY IN THE OCULAR SURFACE

N F-kB is a ubiquitous transcription factor that,through target genes, regulates key processes suchas inflammation, apoptosis, stress response, wound

healing, angiogenesis, and lymphangiogenesis.1 NF-kB/Relproteins consist of p50 (originating from p105 or NF-kB1), p52 (derived from p100 or NF-kB2), c-Rel, RelB,and p65 (RelA). The p65, the most commonly studiedmember, interacts with NF-kB inhibitor protein (IkB) inthe cell cytoplasm. Upon activation by various types ofpathological or physiological stimulation, IkB is phosphory-lated and degraded. The p65 then translocates to the cellularnucleus, regulating transcription of NF-kB target genes.1

It is well known that NF-kB regulation is cell- andtissue-specific.2 Because of the unique anatomical and phys-iological requirements of the cornea and the special immu-nological microenvironment of the ocular surface,inflammatory processes in the ocular surface can havesequelae and outcomes very different from those in otherparts of the body.3 NF-kB plays an important role in thedevelopment of the cornea and conjunctiva.4,5 IkB kinase(IKK)a, upstream of IkB, is essential for differentiation ofcorneal and conjunctival epithelium.4 More importantly,immunological and inflammatory pathways regulating NF-kB and its downstream processes (Figure 1, Table 1) canpotentially be exploited in the treatment of human ocularsurface diseases (Figure 2).1

There has been an increase in the number of studies onNF-kB in the context of the ocular surface in recent years,including its involvement in chemical injury, ultraviolet(UV) radiation-induced injury, microbial infections, allergiceye diseases, dry eye, pterygium, and corneal graft rejection.The purpose of this article is to summarize key findings ofNF-kB research in the ocular surface and its related celltypes. This includes three main categories: regulation ofthe ocular surface innate immunity, regulation of ocular

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OUTLINE

I. Introduction to Nuclear Factor-kB (NF-kB) Pathway inthe Ocular Surface

II. Regulation of Ocular Surface Innate Immunity

A. Bacterial Infections

1. Gram-negative Bacterial Keratitis

2. Toll-like Receptors 4 and 5 in Gram-negativeBacterial Keratitis

3. Toll-like Receptor 2 in Gram-positive BacterialKeratitis

B. Viral Infections

C. Fungal and Protozoan Infections

III. Regulation of Ocular Surface Angiogenesis andWound Healing Response

A. Regulation of Vascular Endothelial Growth Factorand Lymphangiogenesis

B. Regulation of Matrix Metalloproteinases

C. Diseases of Wound Healing Regulated by NF-kB

1. Regulation of Alkali- and Mechanically InducedWounds

2. Regulation of Pterygium

IV. Regulation of Inflammatory Response against Stress

A. Types of Stress That Trigger NF-kB Signaling

B. Stimulation by Specific Cytokines

1. IFNg

2. TNFa

3. IL-1 and IL-17

C. Regulation of Ocular Surface Diseases withInflammatory Features

V. Novel Drugs in Ocular Surface Intervention

VI. Conclusion and Summary

VII. Method of Literature Search

Abbreviations

AGE Advanced glycation end productsCCL Chemokine (C-C motif) ligandCOX Cyclo-oxygenaseds Double-strandedEGCG Epigallocatechin gallateERK Extracellular signal-regulated kinaseFn-14 Fibroblast growth factor inducible 14hBD Human beta defensinHCEC Human conjunctival epithelial cellHSV Herpes simplex virusICAM Intercellular adhesions moleculeIFN InterferonIKK NF-kB inhibitor protein (IkB)-kinaseIL InterleukiniNOS Inducible nitric oxide synthaseIP-10 IFN-induced protein of 10 kDaI-TAC IFN-inducible T-cell alpha-chemoattractantIkB NF-kB inhibitor proteinJNK c-Jun N-terminal kinaseLPS LiposaccharideMCP Monocyte chemoattractant proteinMIG Monokine induced by IFN- gMMP Matrix metalloproteinaseMnSOD Manganese superoxide dismutaseNF-kB Nuclear factor-kBPAMP Pathogen-associated molecular patternPI3K Phosphoinositide 3-kinasepoly(I:C) Polyinosinic-polycytidylic acidRANKL Receptor activator of NF-kB ligandRANTES Regulated on Activation, Normal T

Expressed and SecretedTGF Transforming growth factorTLR Toll-like receptorTNF Tumor necrosis factorTRAF TNF receptor-associated factorTWEAK TNF-like weak inducer of apoptosisUV UltravioleVCAM Vascular cell adhesion protein

NUCLEAR FACTOR-kB IN OCULAR SURFACE DISEASE / Lan, et al

surface angiogenesis and wound healing response, and regu-lation of inflammatory response against stress. We alsodescribe recent drugs used in ocular surface interventionvia NF-kB targeting.

VEGF Vascular endothelial growth factor

II. REGULATION OF OCULAR SURFACE INNATEIMMUNITY

Microbial infections, such as bacterial, viral, and fungalkeratitis, are common and devastating sight-threateningconditions in the eye.6 The innate immunity is the firstline of defense against microbes, and the toll-like receptors(TLR) form a family of pattern recognition receptors thatplay an important role in innate host defense in the ocularsurface.7-9 These receptors initiate a rapid host innateimmune response to microbial components, known aspathogen-associated molecular patterns (PAMPs), whichdetermine the outcome of many infections.7-9

A. Bacterial Infections1. Gram-negative Bacterial Keratitis

One of the most commonly encountered clinical condi-tions is contact lens wear-associated Gram-negative bacterial

138 THE OCULAR SURFACE / JULY 2012, V

cornea ulcers, such as those caused by Pseudomonasaeruginosa.6 Hypoxic conditions created by contact lenswear facilitate internalization of P. aeruginosa via its cellularreceptor, the cystic fibrosis transmembrane conductancechannel.10 This internalization, as well as hypoxia alone,results in elevated NF-kB activation via nuclear translocationand a worsening of inflammation consequently leading tocorneal destruction. The use of an NF-kB inhibitor, kameba-kaurin, can block the P. aeruginosa-induced rise in cytokines,namely interleukin (IL)-6, -8 and tumor necrosis factor(TNF)-a, reducing the severity of inflammation.11

In the cornea, the neuropeptide substance P is a potentimmunoregulator, and C57BL/6 mice injected intraperitone-ally with substance P showed worsening of P. aeruginosa-induced corneal perforation. Both transcript and proteinlevels of NF-kB were elevated, along with increased neutro-phils and bacteria counts in the cornea. The authors

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Figure 1. Schematic illustrating pathwaysinvolving NF-kB in the ocular surface. Moleculesupstream and downstream of corneal epithelialcells and corneal-derived fibroblasts are shown.Processes that may accumulate free radicals orreactive oxygen species are shown on the right.The NF-kB targets may have anti-oxidant orpro-oxidant effects, as shown. TNFa ¼ tumornecrosis factor; IL ¼ interleukin; IFNg ¼ inter-feron g; TLR ¼ toll-like receptor; Pl3K ¼phospholinositol-3 kinase; MCP-1 ¼ monocytechemoattractant protein-1; RANTES ¼ Regu-lated on Activation, Normal T Expressed andSecreted; hBD2 ¼ human beta-defensins 2; LL-37 ¼ cathelicidin; HLA-DR ¼ human leukocyteantigen-D related; MMP ¼ matrix metal-loproteinase; iNOS ¼ nitric oxide synthase;ICAM1 ¼ intercellular adhesion molecule 1;VCAM1 ¼ vascular cell adhesion molecule 1;MnSOD ¼ manganese superoxide dismutase.


postulated that substance P may augment infection suscep-tibility by upregulating proinflammatory molecules via NF-kB stimulation.12 It is not known if changes in tear levels ofsubstance P can result in differences in the activation of NF-kB in ocular surface epithelial cells.

In contrast, NF-kB is involved in the upregulation ofhuman beta defensin (hBD)-2 after P. aeruginosa

Table 1. Biological processes regulated by the NFkBpathway and the known NF kB signalingcomponents involved in these processes. Themediators of the biological processes knownto be involved are also shown.

BiologicalProcesses

NFkBmodulator

Mediators/Triggers

Angiogenesis p65/p50 VEGF-A, VEGF-B,VEGFR-1,VEGFR-3

Lymphangiogenesis p65/p50 VEGF-C, VEGF-D,VEGFR-3

Immune recognition p65/p50 TLR2,3,4, 7 andhBD2

Cytokine regulation RelB, p100,p52, IkBx

RANTES, IL-8, TNF-a, IFN-g, IL-1b

Redox regulation p65/p50 MnSOD, iNOS

Cell migration/proliferation

IKK-b TNF-a, MCP1, p38,JNK

Matrix remodeling p65/p50 MMPs eg MMP7and MMP14


infection.13 Defensins are natural broad-spectrum antimi-crobial molecules found in tears and other ocular tissues.The expression of hBD-2 in human corneal epithelial cellsis mediated by IL-1b and TNFa, and expression of hBD-2by IL-1b can be blocked by NF-kB inhibitors.14 The longdistal portion of the promoter of hBD-2 is responsive toIL-1b.15 These studies therefore show that cytokines mayhave a protective effect against infections by upregulatinghBD-2 expression, but it is not known if this upregulationby itself can arrest infection.

2. Toll-like Receptors 4 and 5 in Gram-negative BacterialKeratitisLiposaccharide (LPS), a component of Gram-negative

bacteria, is recognized by TLR4.16 In cultured corneal fibro-blasts, LPS induced an increase in NF-kB activity,17 whichstimulated production of cytokines IL-6 and -8.18 The addi-tion of soluble CD14 or LPS-binding protein (LBP) to LPSpotentiated the expression of intercellular adhesions mole-cule (ICAM)-1, IL-8 and monocyte chemoattractant protein(MCP)-1 in corneal fibroblasts.19

NF-kB activation in human conjunctival epithelial cells(HCEC) incubated with LPS was mediated by TLR4 andTNF receptor-associated factor (TRAF)-6.20 HCEC incu-bated with LPS demonstrated a dose-dependent inductionof NF-kB activity.20 In a study more directly relevant toclinical scenarios, LPS-induced keratitis in rats was associ-ated with NF-kB activation.21 The severity of the keratitiscan be reduced by subconjunctival injection of inhibitorsof NF-kB, ie, pyrrolidine dithiocarbamate22 or emodin.23

A family of calcium-binding proinflammatory proteins,the S100 proteins, has been found to be increasingly



Figure 2. Schematic showing ocular surfacediseases involving NF-kB, their triggers, and thebiological processes that are disturbed. Theextreme right boxes show the inhibitors thathave been used to inhibit NF-kB in these ocularsurface diseases. UV ¼ ultraviolet; UVB ¼ultraviolet-B; TLR ¼ toll-like receptor; ROS ¼reactive oxygen species; NO ¼ nitric oxide;COS ¼ cyclo-oxygenase; IKKb ¼ inhibitor ofnuclear factor-kB kinase, EGCG ¼ epi-gallocatechin gallate; TNF Ig ¼ tumor necrosisfactor immunoglobulin; DHMEQ ¼ dehy-dromethylepoxyquinomicin; siRNA ¼ shortinterfering RNA.


important in inflammation. The expression of S100A9,a member of this family, may be dependent on LPS-stimulated NF-kB activity and may play a role in keratitis,as demonstrated in Wilstar rats.24 The implications ofS100A9 on severity of keratitis are currently unclear.

Flagellin is the protein that forms the whip-like processor flagellum of bacteria. TLR5 recognizes the flagellincomponent of P. aeruginosa, and downstream signalingresults in production of proinflammatory cytokines.However, when corneal epithelial cells have been primedby low-dose flagellin, this immune signaling was reduced,suggesting a protective mechanism against inflammation.25

Yet, not all beneficial effects of flagellin are NF-kB-dependent. Flagellin may also have non-NF-kB-dependentmechanisms of aiding corneal epithelial wound closure.26

3. Toll-like Receptor 2 in Gram-positive BacterialKeratitisThe ocular surface is dominated by Gram-postive

commensals, such as coagulase-negative Staphylococcusspp and Streptococcus spp. Such bacteria also form a signifi-cant proportion of microbes isolated from cases of severeinfectious corneal keratitis. TLR2 is able to detect bacterialproducts such as Staphylococcus aureus protein A, so thisTLR is the main type involved in Gram-postive bacterialkeratitis.

The addition of S. aureus protein A stimulated cornealepithelial cells to activate NF-kB and produce TNF-a andIL-8,25,27 as well as a variety of molecules, such as ICAM-1,antimicrobial molecules hBD-2, cathelicidin (LL-37),cytokine-inducible nitric oxide synthase (iNOS) and thehomeostasis molecule manganese superoxide dismutase


(MnSOD).28 Not all the effects of Gram-postive bacterialinfections are mediated by NF-kB. For example, afterS. aureus infection, the expression of IL-8 by conjunctivalepithelial cells was not dependent on NF-kB.29

B. Viral InfectionsViral keratitis can present clinically in a variety of ways,

including severe cases with sight-threatening cornealstromal keratitis, opacity, and vascularization.30 The maintypes of viruses that cause sight-threatening disease are theherpes simplex viruses (HSVs) and the adenoviruses.

Infection of the cornea by HSV-1 is associated withupregulation of various cytokines via the NF-kB pathway.31

HSV-1 has the ability to block apoptosis of infected cornealepithelial cells, thus evading one mechanism of host defenseagainst this virus.32 It was found that the inhibition ofapoptosis was partly due to the degradation of IkBa, andtranslocated NF- kB in the cellular nucleus then stimulatedthe transcription of anti-apoptotic genes.32

Clinical improvement of herpetic stromal keratitis maybe due to a combination of immunostimulatory and immu-nosuppressive effects.33 As mentioned above, effects of NF-kB differ with cell types. The compound brassinosteroid-2can block HSV-1-induced NF-kB activation in humancorneal epithelial and conjunctival cells and reduce IL-6production by LPS-stimulated murine macrophage cells,but it enhances IL-6 production in the corneal and conjunc-tival epithelial cells.33 This suggests that potential benefits ofin vitro drug effects must be studied in the context ofdiseases in vivo.

The TLRs involved in antiviral defenses are TLR3 and -7.HSV-1 infection of human corneal epithelial cells results in




expression of TLR7 and suppression of TLR3,34 whichrecognizes double-stranded (ds) RNA. While most PAMPreceptors recognize dsRNA, only TLR3 recognizes dsRNAproduced by HSV during infection.

Because polyinosinic-polycytidylic acid [poly(I:C)], ananalog of viral dsRNA, can be recognized by TLR3 asa ligand in the same way as viral dsRNA, it is a useful modelfor evaluating innate defenses mediated by TLR3. It wasshown in human corneal fibroblasts that through TLR3,poly(I:C) activated NF-kB and induced adhesion moleculesimportant for inflammation, the vascular cell adhesionprotein (VCAM)-1 and ICAM-1,35 as well as moleculesinvolved in wound healing, the matrix metalloproteinases(MMPs)1 and 336 (see section III). The expression ofpoly(I:C)-induced MMP-1 and MMP-3 can be inhibitedby triptolide, an herbal extract from Tripterygium wilfordiiused in traditional Chinese medicine.37 Poly(I:C) alsoinduced TLR3 upregulation and expression of cytokinessuch as IL-6 and -8, propagating the proinflammatoryresponse.38 Similarly, in human corneal epithelial cells,poly(I:C) stimulation resulted in NF-kB activation andIL-6 and -8 production.39

Poly(I:C)-induced NF-kB activation can be suppressedby glucocorticoids such as dexamethasone and cyclosporine,leading to a modified immune response of the cornea toHSV and other viruses.40 This may explain the “double-edged sword” that steroid treatment represents in herpetickeratitis: while it is crucial for arresting cell destructioncaused by inflammation in viral infections, it also impairshost defense against the virus itself.

Other viruses that have been linked to NF-kB activation inthe ocular surface include adenovirus and the respiratorysyncytial virus. The respiratory syncytial virus, a commoncauseof respiratory tract infections, is commonly found in cornealswabs. Infection of corneal epithelial cells by this virus resultsin the activation of NF-kB and production of proinflammatorychemokines, such as monokine induced by interferon (IFN)-g(MIG), IFN-inducible T-cell alpha-chemoattractant (I-TAC)and IFN-induced protein of 10 kDa (IP-10).41

In adenoviral keratitis, NF-kB activation may be trig-gered by upstream p38, c-Jun N-terminal kinase (JNK)and extracellular signal-regulated kinase (ERK) pathways,42

leading to IL-8 transcription.43 The NF-kB activationfollowing adenovirus type 19 infection is also downstreamof phosphoinositide 3-kinase (PI3K) pathway.44 Therefore,in viral infections of the ocular surface, multiple stress-and cell proliferation-related signaling pathways canconverge on NF-kB.

C. Fungal and Protozoan InfectionsMycotic keratitis is a severe fungal infection of the eye,

commonly caused by Fusarium spp or Aspergillus spp, andis often associated with severe visual morbidity as well asa high rate of therapeutic keratoplasty surgeries.45,46 Thishighlights the importance of understanding and modulatinghost defenses against fungi, which are known to be mediatedby TLR2 and TLR4.


TLR2 and TLR4 can be stimulated by Aspergillus fungalinfections,47 leading to NF-kB activation and secretion ofTNF-a, IL-6, IL-8,48,49 IL-1b49 and IL-10.50 After additionof zymosan, a component of fungus, to human cornealepithelial cells, the TLR2-NF-kB pathway was stimulated,leading to production of proinflammatory cytokines TNF-a and IL-1b, chemokines IL-8 and Regulated on Activation,Normal T Expressed and Secreted (RANTES), and MMP1,3 and 9.51 The addition of zymosan to human corneal fibro-blasts similarly resulted in release of IL-6 and IL-8.52

Acanthamoeba are free-living protozoans, which, in rarecircumstances, invade the cornea without an evident epithe-lial defect,53 causing a devastating keratitis that is oftenresistant to most forms of therapy.54 A study has shownthat the role of TLR4 was not limited to Gram-negativebacterial or fungal infections, but also extended to parasiticinfection. The addition of Acanthamoeba protozoans to ratcorneas induced the expression of cytokines via TLR4 andNF-kB.55

III. REGULATION OF OCULAR SURFACE ANGIO-GENESIS AND WOUND HEALING RESPONSE

Wound healing is an important component of ocularsurface diseases such as mechanical abrasions andchemical-induced keratitis. Wound healing in the corneais exemplified by a finely orchestrated series of eventsinvolving cell types such as corneal epithelial cells, fibro-blasts and myofibroblasts, and regulated by molecules suchas vascular endothelial growth factor (VEGF), MMPs andvarious inflammatory cytokines.56 Epithelial migration isa key process in wound healing involving one of theupstream regulators of NF-kB, IKKb,57 which is known toplay an important role in the homeostasis and inflammatoryprocesses of the ocular surface epithelium. In the cornea,unlike skin, avoidance of angiogenesis during wound heal-ing is critical in preventing loss of transparency.

A. Regulation of Vascular Endothelial Growth Factorand LymphangiogenesisThere is a very high incidence rate of corneal neovascu-

larization in the USA.58 In the cornea, transparency dependson a balance between proangiogenic factors, such as VEGFand transforming growth factor (TGF)-b, and antiangio-genic factors. Naturally occurring antiangiogenic factorshave been characterized in the corneal epithelium. Theseinclude factors that inhibit corneal new vessels directly,such as soluble VEGFR-1 and VEGFR-3 and neostatins,and those that inhibit corneal neovascularization throughproteolysis, such as MMP-7 and MMP-14.58 In a diseasecontext or in severe inflammation, there is an abnormalincrease in proangiogenic factors or reduction in antiangio-genic factors, causing a tilt toward angiogenesis.

The normal purpose of the lymphatic system is forreturning extracellular fluid to the blood circulation andfor antigen presentation (the afferent limb of the immuneresponse).58 The cornea is normally devoid of blood andlymph vessels, accounting for its immune privilege.58 Both




angiogenesis and lymphangiogenesis in the cornea can occuras a result of severe inflammation, resulting in poor vision.58

Using distinct markers for lymphatics (such as VEGFR-3), itis now possible to visualize lymphangiogenesis duringcorneal inflammation.58

Members of the VEGF family bind to different receptorsto exert their effects.58 VEGF-A is the main member respon-sible for normal hemangiogenesis. Shorter isoforms ofVEGF-A can be secreted freely and cause an increase invascular permeability and inflammation. VEGF-A isinvolved in several steps of angiogenesis, such as proteolyticactivities (dissolution of the membrane of the originalvessel), proliferation and migration of vascular endothelialcells, and the formation of capillary tubes. VEGF-C additionresults in VEGFR-3 forming homodimers or heterodimerswith VEGFR-2, whereas both VEGF-C and VEGF-D canbind to VEGFR-3, resulting in its phosphorylation anddownstream signaling that induces differentiation of venousendothelial cells into lymphangioblasts. Formation oflymphatics allows antigen presentation directly from thecorneal stroma and hence can propagate inflammationthrough the adaptive immune response.

VEGF was observed to be upregulated near silver nitrateapplications on rat corneas, increasing vascular permeabilityof limbal blood vessels and inducing vascular endothelial cellmigration and proliferation, resulting in corneal angiogen-esis.59 The activation of NF-kB in the mouse cornea leadsto upregulation of VEGF-A, VEGF-C and VEGF-D, andthus plays a role not only in angiogenesis, but also in corneallymphangiogenesis.60 VEGF-C is one of the upregulatedmolecules after alkali injury,61 pointing to a role for lym-phangiogenesis in chemical injury.

B. Regulation of Matrix MetalloproteinasesThe MMPs are a family of zinc-binding proteases

important for remodeling of extracellular matrix in healthand disease.58 MMP-2 and -7 have been known to playa role in promoting angiogenesis by allowing tissue invasionby vascular endothelial cells, or inhibiting angiogenesis bygenerating antiangiogenic fragments from inactive precur-sors.58 MMP-9 or gelatinase B located in the basementmembrane of the corneal epithelium and superficial stromamay cause epithelial barrier disruption and has been shownto play a role in dry eye disease both in vivo in mice62 orhumans,63 and in vitro.64

NF-kB activating molecules have been implicated inwound healing involving MMP. Platelet activating factorincreases NF-kB binding to MMP-9 promoters in humancorneal epithelial cells, resulting in upregulated expressionof MMP-9.65 In corneal myofibroblasts, fibroblast growthfactor inducible 14 (Fn-14) and its ligand, TNF-like weakinducer of apoptosis (TWEAK), interact to cause anincrease in MMP-1 and MMP-3 expression, which couldbe abrogated by TGF-b1 expression. TWEAK also inducesphosphorylation of NF-kB in the stimulated corneal myofi-broblasts. It was postulated that competition between NF-kBproteins and Smad proteins activated by TGF-b1 control the


expression of MMPs.66 Receptor activator of NF-kB ligand(RANKL) levels in murine corneal epithelium increasedafter epithelial scrape, suggesting the involvement of NF-kB signaling in corneal wound healing. RANKL was alsodetected in monocytes that appeared in the wound1-2 days after wounding.67 However, further studies areneeded to characterize the functions of RANKL in thepreliminary study.

C. Diseases of Wound Healing Regulated by NF-kB1. Regulation of Alkali- and Mechanically Induced

WoundsChemical injuries in the eye, especially in the cornea, can

have a very poor prognosis in severe cases, ie, when there issignificant loss of limbal progenitor cells, resulting in condi-tions like an opaque and vascularized cornea with poor vision.Alkali injury models of angiogenesis have been investi-gated.68-70 In all these studies, NF-kB was found to mediateangiogenesis. Reactive oxygen species play a role in angiogen-esis in alkali burn animal models, and the use of the NF-kBinhibitor DHMEQ was shown to reduce corneal angiogen-esis.71 Blocking of NF-kB inhibitor SN-50 accelerated thecorneal epithelial proliferation and promoted wound heal-ing.68 Over-expression of Smad-7 molecule blocks Smadsignal and attenuates the NF- kB signal, reducing invasionof monocytes and expression of MCP-1 and MMP-9, andabolishing myofibroblast formation in mice with alkalinecorneal burns.72 Other models of angiogenesis induced byTNFa,73 IL-1b74 and cautery75 also implicated NF-kB activa-tion. In a mechanically induced cornea wounding model inC57/B6 mice, NF-kB activation was observed in the conjunc-tival epithelium after scraping the cornea.76

2. Regulation of PterygiumPterygium is a UV radiation-associated ocular surface

condition characterized by excessive fibroblast proliferation,matrix degradation and abnormal vascularity.77 This lesiontherefore involves abnormal wound healing and angiogen-esis. The pathogenesis of pterygium is unknown, althoughsun exposure and free radical production have been impli-cated in its formation. As NF-kB is involved in oxidativestress signaling,1 it is not surprising that it may play a rolein pterygium pathology.

Phosphorylation of IkBa has been found to be increasedin pterygium tissues relative to control conjunctivaltissues,78 and this may set up a chain of events leading toformation or progression of inflammation. Both inflamma-tion and angiogenesis can be a consequence of cytokines,chemokines, growth factors and proteolytic enzymesproduced in the pathology of pterygium. One such cytokineis the chemokine (C-C motif) ligand (CCL)5 or RANTES,belonging to the IL-8 superfamily. RANTES serves asa selective chemoattractant for memory T lymphocytesand monocytes involved in immune response. Upregulationof RANTES was more pronounced after treatment ofpterygium-derived fibroblasts with TNFa compared toTenon fibroblasts, suggesting a greater susceptibility of




pterygium fibroblasts to NF-kB activation via TNFastimulation.78

Interestingly, treatment of pterygium-derived fibroblastswith TNFa resulted in nuclear localization of RelB, p100and p52.78 These are components of the alternate NF-kBpathway, as opposed to the canonical NF-kB pathwaydescribed in all other sections of this review. Unlike thecanonical pathway, the alternate pathway is characterizedby a slower response and a different type of triggering ligands.

VEGF is upregulated in pterygium.79 Recently, therehave been a few studies investigating antiangiogenesis strat-egies for treatment of pterygium, which were based on tar-geting of VEGF, but varying degrees of clinical success werereported.80 However, there is no direct evidence that inpterygium the upregulation of VEGF is caused by activationof NF-kB.

IV. REGULATION OF INFLAMMATORY RESPONSEAGAINST STRESS

Regulation of NF-kB is pivotal to processes like theproduction of inflammatory cytokines. This is well illus-trated by studying the IkBx, a regulator of NF-kB. TheIkBx (-/-) mice present with chronic ocular surfaceinflammation and loss of goblet cells. There was also aninflammatory infiltration of CD45R/B220þ and CD4þ cellsin the subconjunctival matrix.81

A. Types of Stress That Trigger NF-kB SignalingThe two main types of stress that trigger NF-kB

signaling are hyperosmolar stress and UV radiation-induced stress.

Hyperosmolar stress activates NF-kB pathway incorneal82-85 and limbal epithelial cells.86 Hyperosmolarstress is believed to be a common factor in the pathogenesisof dry eye syndrome in humans. In dry eye syndrome,affected glands that are involved in the secretion of tearsmay be the lacrimal glands, meibomian glands, and themucus-secreting goblet and conjunctival epithelial cells.Dry eye associated lacrimal gland and ocular surface dysre-gulation is often observed in diabetes. In lacrimal glands ofa diabetic murine model, NF-kB expression and advancedglycation end products (AGE) were found to be greaterthan in controls.87 AGE binding to AGE receptor triggersNF-kB activation. In aging rats, NF-kB activity and AGEwere also increased alongside insulin resistance, suggestingan involvement of NF-kB in metabolic events and tearfilm dysfunction related to age.88

NF-kB is activated following UVB exposure.89,90 Photo-keratitis induced by UVB can be reduced by zerumbone,a potent NF-kB inhibitor.91 Human corneal epithelialstress-induced apoptotic response is in part due to down-regulation of CCCTC binding factor, a nuclear transcriptionfactor. Inhibition of NF-kB can reduce apoptosis bypreventing this downregulation.92 Similarly in mice, lornox-icam suppressed the UVB-induced activation of NF-kB andexerted a protective effect on the cornea after UVBexposure.93


Apart from hyperosmolar stress and UVB-inducedchanges, there are other forms of stimuli which can activateNF-kB. Exposure to coal dust, for example, is another formof irritation that can upregulate NF-kB in the ocular surfaceof humans and rabbits.94 Reactive oxygen species have beenknown to have stimulatory or inhibitory effects on NF-kBsignaling, and certain NFkB target genes are important forregulating the amount of reactive oxygen species in thecell (Figure 1).95,96

B. Stimulation by Specific CytokinesRegardless of the initial trigger, ocular surface inflamma-

tion may be propagated by the effect of specific cytokinessecreted into the tear bathing the ocular surface epithelialcells.

1. IFNgIFNg and anti-Fas antibody treatments activate NF-kB

in Chang conjunctival epithelial cells.97 IFNg-induced up-regulation of NF-kB is responsible for HLA-DR expressionin conjunctival epithelial cells in patients with Sjögrensyndrome, an autoimmune disorder characterized by drymouth and dry eyes.69 HLA-DR expression is consideredto be a histological marker of inflammation.

2. TNFaTNFa has multiple cellular effects mediated by NF-kB

signaling, ranging from cell death to production of chemo-kines. TNFa induced apoptosis in corneal fibroblaststhrough NF-kB,98 whereas exposure to hypochlorite sup-pressed the TNFa-induced increase in NF-kB activity.99 Incontrast, TNFa promoted cell survival in corneal epithelialcells through Kþ channel activity.100 The TNFa-inducedtranscription of IL-8 and MCP1 was driven by differentkinds of NF-kB dimers, the p65p65 homodimers andp50p65 heterodimers, respectively.101 TNFa receptorexpression pattern might be dependent on the biologicalcontext and the time course of the stimulation, etc., affectingthe signaling.102

3. IL-1 and IL-17IL-1 and IL-17 are also involved in the complex regula-

tion of ocular surface inflammation. IL-1b activated NF-kBin corneal fibroblasts,103 which could be inhibited bydexamethasone and triptolide.103 NF-kB plays a role in theIL-1-induced collagen degradation by corneal fibroblasts,a process that is believed to be important in infective cornealulcers.104 In corneal fibroblasts, TNFa and IL-1 inducedIL-11 secretion via an NF-kB mechanism.105 IL-11 is ananti-apoptotic and anti-inflammatory cytokine. IL-1aexpression and autocrine loop is dependent on NF-kB. Incorneal stromal cells, absence of NF-kB activation reducedIL-1a expression,106 whereas TGF-b, retinoic acid and dexa-methasone, which can affect NF-kB DNA-binding affinity,inhibited IL-1a expression.107

IL-17 induced NF-kB activation and produced inflam-matory mediators in primary limbal epithelial cells.108




Work on a mouse model of dry eye indicated that autoim-munity in dry eye is regulated by resistance of Th17 T cellsto suppression by T regulatory cells.109

C. Regulation of Ocular Surface Diseases withInflammatory FeaturesThe two main conditions described here are allergic eye

disease and ocular surface surgical intervention. Althoughstudies cited above also implicate the role of NF-kB inanother inflammatory disease, dry eye syndrome, the directevidence that NF-kB is activated in an in vivo model of dryeye disease does not exist.

Ocular allergic keratoconjunctivitis is a common condi-tion with a significant health burden.110 The conjunctivalepithelial cells in humans with epidermic keratoconjunctivitisdemonstrated activation of NF-kB.76 Degranulation of mastcells is an important component in allergic eye disease. In acti-vated human mast cells, NF-kB activation is increased. Thisphenomenon has been studied in ovalbumin-treated mice,which demonstrated increased mast cell and eosinophilicinfiltration, typical hallmarks of allergic eye disease. However,on treatment with extract powder of P. frutescens var. acutaKudo and rosmarinic acid, NF-kB activation was suppressed,with reduction in serum histamine, IL-1b, IL-6 and TNFaproteins in the nasal mucosa.111

The two scenarios after ocular surface surgeries thatinvolve NF-kB are corneal graft rejection and wound heal-ing after corneal refractive surgeries, where signaling occursin the corneal endothelial and epithelial cells, respectively.

The exiting of leukocytes into extravascular compart-ments, such as the anterior chamber, and subsequent inter-action with donor corneal tissue are critical inflammatoryprocesses that may be involved in corneal graft rejection.Leukocyte adhesion to corneal endothelium is enhancedby TNFa in an NF-kB-dependent way, and this can be sup-pressed by treatment with dexamethasone steroid.112 Cyto-kines such as IL-1, IFNg and TNFa in corneal grafts areable to trigger NF-kB activation in corneal endothelial cells,leading to generation of nitric oxide and playing a role ingraft rejection.113 In photorefractive keratectomy for refrac-tive corrections, a part of the corneal epithelium is removedto enable direct application of the excimer laser to theunderlying stromal bed. During wound healing after photo-refractive keratectomy in rabbits, NF-kB activation playeda role in the migrating process of the corneal epitheliumvia expression of cyclo-oxygenase (COX).114

V. NOVEL DRUGS IN OCULAR SURFACEINTERVENTION

Apart from some of the inhibitors mentioned in theabove sections, there are other NF-kB interventions thatmay be exploited for therapeutic purposes (Figure 2). Ananti-TNFa strategy has been used clinically in peripheralulcerative keratitis,115,116 although the effects on NF-kBhave not been evaluated in those reports. Since there area huge number of papers on the inhibition of ligands thatpotentially can activate NF-kB, the rest of this section refers


only to the reports that specifically investigated NF-kBinhibition.

The use of a tetranortriterpenoid compound from leavesof Melia azedarach was beneficial for herpetic stromal kera-titis in mice.117 It is intriguing that a fluoroquinolone anti-microbial besifloxacin is able to inhibit IkB degradationand showed an anti-inflammatory response in IL-1b-treatedhuman corneal epithelial cells.84 Further evidence of efficacyand safety should now be sought in vivo. BOL-303242-X,a selective glucocorticoid receptor agonist is able to actas an anti-inflammatory agent that selectively targets theNF-kB, p38 and c-Jun kinases.118,119

The use of emodin120 from a Chinese herb and epigallo-catechin gallate (EGCG)121 from green tea have beendescribed above. In particular, the use of EGCG in cornealneovascularization has been reported.122

TNFa-induced NF-kB and injury-induced cytokineproduction can be suppressed by thymosin b4.123,124

Cationic nanopolymers with a IKKb silencing siRNA havebeen used successfully in human Tenon fibroblasts to down-regulate NF-kB and reduce proliferation of cells.125 If theseexperiments can be repeated in vivo, it will represent animportant anti-scarring strategy to improve success ratesfor glaucoma filtration surgery.

Innovative applications in biotechnology and industryhave also exploited research in the NF-kB pathway. Oneexample is the use of NF-kB activation in toxicity screensusing an organ culture system, avoiding the more expensiveand ethically challenging use of animals in testing ofcosmetics and drugs.126

VI. CONCLUSION AND SUMMARYSince NF-kB is an ubiquitous transcription factor with

tissue-specific effects, it is important for ocular surfacescientists and practitioners to focus on research relevant tothe ocular surface. Although most of the treatmentsdescribed have not been used in mainstream treatment ofocular surface diseases, the use of anti-inflammatory NF-kB inhibitors has been applied to other ocular inflammatorydiseases, such as uveitis; certain therapies that have beenused clinically, such as anti-TNFa in autoimmune periph-eral ulcerative keratitis, are likely to involve NF-kB.127,128

More therapies in specific clinical contexts in ocular surfacedisease may be available in the future.

VII. METHOD OF LITERATURE SEARCHThe keywords “NF-kB” and “eye” were searched for in

the Entrez Pubmed database up to September 2011 andthe article abstracts found were examined for research inocular surface tissues or cell types or in animal models ofocular surface diseases. This yielded 100 articles. These arereferenced in this paper, together with other more generalreferences for introductory purposes in each section.

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Nuclear Factor-κB: Central Regulator in Ocular Surface Inflammation and Diseases

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