Localization and Gene Expression of Human β-Defensin 9 at...

Localization and Gene Expression of Human �-Defensin9 at the Human Ocular Surface Epithelium

Imran Mohammed,1 Hanif Suleman,1 Ahmad M. Otri,1 Bina B. Kulkarni,1 Peng Chen,1

Andrew Hopkinson,1,2 and Harminder S. Dua1,2

PURPOSE. Antimicrobial peptides (AMPs) are multifunctionalhost defense molecules. Human �-defensin 9 (HBD9) has pre-viously been shown to be downregulated during ocular surface(OS) infection or inflammation. Here, the authors aimed tostudy localization of HBD9 protein in different OS regions andto determine the role of Toll-like receptors (TLRs), nucleotideoligomerization domain (NOD)-like receptors, and proinflam-matory cytokines in HBD9 expression.

METHODS. Immunolocalization of HBD9 protein was carried outon the normal human OS regions (cornea, limbus, and conjunc-tiva). Quantitative PCR analysis of HBD9 mRNA was performed inSV40-transformed human corneal epithelial cells (hCECs) treatedfor different durations with synthetic pathogen-associated molec-ular patterns (PAMPs) and recombinant cytokines.

RESULTS. HBD9 protein was constitutively expressed on OSepithelia. Corneal and limbal epithelia and corneal stromademonstrated modest levels of HBD9, whereas conjunctivalepithelium demonstrated high levels of HBD9 protein. TLR02,TLR03, TLR04, and TLR05 were shown to modulate HBD9mRNA in hCECs. Similarly, NOD2 and IL-1� were also shownto alter HBD9 in a time-dependent manner. In response toinfection-related PAMPs and inflammatory cytokines, an initialincrease in HBD9 mRNA levels was observed, followed by asignificant downregulation.

CONCLUSIONS. This is the first demonstration of HBD9 proteinexpression at different OS regions. The authors also deter-mined the role of various innate immune receptors in HBD9mRNA modulation. Further understanding of the signalingmechanisms involved in the initial response of HBD9 to infec-tion or inflammation is likely to indicate future therapeuticdirections with this AMP. (Invest Ophthalmol Vis Sci. 2010;51:4677–4682) DOI:10.1167/iovs.10-5334

The interaction of pathogens and epithelial cells is recog-nized to be an active process in which epithelial cells

participate in innate host defense by expressing a variety of

proinflammatory cytokines, chemokines, and antimicrobialpeptides (AMPs).1 Several studies have demonstrated the local-ization of different families of AMPs, including defensins andcathelicidins in ocular surface (OS) epithelium.2 Localization ofhuman �-defensin (HBD)-1 to HBD-3 was reported in superfi-cial layers of normal and inflamed corneal and conjunctivalepithelium,3–5 whereas LL-37 distribution was shown only innormal human corneal tissue.6 A newer member of the �-de-fensin family, DEFB109, also known as HBD9, has been shownto be constitutively present in gingival keratinocytes and to bedownregulated in response to Candida albicans.7 In our pre-vious report, we demonstrated a reduced expression of HBD9in bacterial-, viral-, acanthamoeba-, and dry eye-related OS dis-ease compared with controls.8 To further analyze the expres-sion of HBD9, we characterized the distribution of this AMP innormal cornea, conjunctiva, and limbal tissue using an immu-nofluorescence technique.

The ocular surface epithelium has been shown to provideimmune surveillance against invading pathogens by means of awide gamut of pathogen recognition receptors such as Toll-likereceptors (TLRs) and nucleotide oligomerization domain(NOD)-like receptors (NLRs). TLRs are highly conserved andhomologous receptors found in different species of plant andanimal kingdoms.1 To date, 11 human and 13 mouse TLRs havebeen identified, and each is capable of recognizing pathogen-associated molecular patterns (PAMPs) derived from variousinfection-causing microbes.9 TLR02, TLR04, TLR05, and TLR11reside on the cell membrane and recognize lipopeptides ofGram-positive bacteria, lipopolysaccharide (LPS) of Gram-neg-ative bacteria, flagellin and propellin, respectively.9 TLR03,TLR07/08, and TLR09 are sequestered in endosomes and re-spond to double-stranded RNA from Herpes-simplex virus andsingle-stranded RNA from influenza virus and West Nile virus,respectively.9 As yet the PAMP for TLR10 is unknown. Of allNOD-like receptors, NOD-1 and NOD-2 are well-characterizedproteins, present endosomally, and recognize �-D-glutamyl-meso-diaminopimelic acid (iE-DAP) and muramyl dipeptide(MDP), the synthetic components of peptidoglycan, respec-tively.9,10

Here, we investigated the role of known human TLRs and ofNOD-1 and NOD-2 at the corneal epithelium in relation to theexpression of HBD9. In addition, we studied the effects ofIL-1� and TNF� on HBD9 expression.

METHODS

Reagents and Antibody

Synthetic PAMPs—namely Pam3CSK4, LPS, Poly I-C, Flagellin, R848,CpG ODN, MDP, and iE-DAP—were purchased from Invivogen (SanDiego, CA). Recombinant human IL-1� and human TNF� were pro-cured from Calbiochem (Nottingham, UK). Purified polyclonal rabbitanti–human HBD9 was custom synthesized (Eurogentec, Southamp-ton, UK) for immunofluorescence analysis.

From the Larry A. Donoso Laboratory for Eye Research, Division ofOphthalmology and Visual Sciences, The University of Nottingham,Nottingham, United Kingdom.

2These authors should be regarded as equivalent senior authors.Supported by a British Eye Research Foundation–Fight for Sight

PhD Studentship (IM) and by the Royal Blind Asylum and School/Scottish National Institution for the War Blinded and Royal College ofSurgeons of Edinburgh.

Submitted for publication February 6, 2010; revised March 11,2010; accepted March 19, 2010.

Disclosure: I. Mohammed, None; H. Suleman, None; A.M. Otri,None; B.B. Kulkarni, None; P. Chen, None; A. Hopkinson, None;H.S. Dua, None

Corresponding author: Harminder S. Dua, Division of Ophthalmol-ogy and Visual Sciences, Queens Medical Centre, Eye & ENT Building,University Hospital, Nottingham NG7 2UH, UK;[email protected].

Immunology and Microbiology

Investigative Ophthalmology & Visual Science, September 2010, Vol. 51, No. 9Copyright © Association for Research in Vision and Ophthalmology 4677

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Tissue Processing

Human cadaver eyes were collected after obtaining local ethics com-mittee approval and prior consent from the donors or their relatives inadherence with the tenets of the Declaration of Helsinki. Cadaverocular tissue was processed within 48 hours of death under asepticconditions. A 15-mm trephine was used to dissect the corneoscleralbutton, maintaining a 3-mm frill of conjunctiva around the limbus. Thebutton was divided into eight segments and then snap-frozen in pre-chilled isopentane. Frozen tissue blocks were stored at �80°C untilfurther use.

Immunofluorescence

Six-micrometer sections of normal ocular surface tissue on 2% 3-amin-opropyltriethoxysilane–coated glass slides were acetone fixed andblocked with normal goat serum (Invitrogen, Paisley, UK). Washingstep was performed with 1% BSA � 0.1% Triton-X premixed solution.The sections were then incubated with primary antibody against HBD9(1:50; polyclonal rabbit anti–human antibody; Eurogentec, Liege, Bel-gium). The sections were washed and incubated with secondary anti-body Alexa Fluor 555 (anti–rabbit; Invitrogen) for 30 minutes, followedby counterstaining with DAPI (4�, 6-diamidino-2-phenylindole) for 4minutes. The slides were mounted with fluorescent mounting medium(Dako, Ely, UK) and examined under a fluorescent microscope (B51X;Olympus, Tokyo, Japan) using imaging software (CELL-F; Olympus,Southend-on-Sea, UK). The images were edited in image editing soft-ware (Photoshop CS4; Adobe Systems, San Jose, CA).

SV40-Transformed Human Corneal EpithelialCell Line

SV40-transformed human corneal epithelial cells (hCECs) were a kindgift from Felicity Rose (School of Pharmacy, The University of Notting-ham, Nottingham, UK). Cells were maintained in medium (Epilife;Cascade Biologics, Paisley, UK) containing human keratinocyte growthsupplement (HKGS; Cascade Biologics), antibiotic mixture (gentamicin

and amphotericin B; Cascade Biologics), and anti–mycoplasma agent(Plasmocin; Invivogen). The hCECs (8 � 104) were plated onto 12-wellculture plates and grown until they reached 70% confluence in humid-ified conditions (5% CO2, 37°C). Before treatment, hCECs were starvedovernight in HKGS-free media.

Total RNA Extraction

Cells were collected in lysis buffer (Buffer RLT; Qiagen, Crawley, UK),and total RNA was extracted according to the manufacturer’s instruc-tions (RNeasy mini kit; Qiagen). Briefly, the cell lysate was homoge-nized with a spin column (QIAshredder; Qiagen) and mixed with 70%ethanol in equal volume (1:1). The mixture was then applied onto spincolumns and centrifuged at 10,000 rpm for 15 seconds. The filtrate wasdiscarded, and the spin column was washed with buffer RW1 and thenwith buffer RPE by centrifugation. Total RNA was eluted in RNase-freewater and quantified (Nanodrop Spectrophotometer; Thermo FisherScientific, Loughborough, UK).

Complementary DNA Synthesis

Total RNA (2000 ng) was reverse transcribed into complementary DNA(cDNA) with a reverse transcription kit (Quantitect; Qiagen). Briefly,total RNA was mixed with genomic DNA wipeout buffer (Qiagen) onice, and the volume was adjusted with water. The mixture was incu-bated at 42°C for 3 minutes and then placed on ice. The reversetranscription enzyme mix (Quantitect reverse transcriptase, QuantitectRT buffer, and RT primer mix; Qiagen) was prepared on ice and mixedwith total RNA mixture. The final mixture was then incubated at 42°Cfor 30 minutes, followed by reverse transcriptase deactivation at 95°Cfor 3 minutes. Samples were stored at �20°C until further analysis.

Quantitative Polymerase Chain Reaction

Quantitative polymerase chain reaction (qPCR) analysis of HBD9mRNA was performed using pre-optimized assay (TaqMan,Hs02760065_g1; Applied Biosystems, Warrington, UK) on the

FIGURE 1. Localization of HBD9 protein. Six-micrometer frozen section of full-thickness normal OS tissue and human tonsil tissue was stained withpolyclonal antibody against HBD9 (rabbit anti–human; 1:50 dilution). Secondary antibody (goat anti–rabbit IgG; 1:300 dilution) was used to detectprimary antibody. Top: merged images of both HBD9 (yellow) and nuclei (DAPI; blue) staining at 200� magnification. Bottom: HBD9 and DAPImerged images at 400� magnification. Negative control staining with secondary antibody alone showed no immunoreactive staining.

4678 Mohammed et al. IOVS, September 2010, Vol. 51, No. 9


Mx3005p real-time PCR instrument (Stratagene, La Jolla, CA). Forrelative quantification, HBD9 levels were normalized against 18S rRNAendogenous control (4319413E; Applied Biosystems) levels. The qPCRexperimental setup was carried out as detailed in the manufacturer’sprotocol (Applied Biosystems). Initially, template cDNA was diluted to1:2 using nuclease-free water to perform the qPCR in triplicate. Eachreaction was prepared to 20 �L final reaction volume with 10 �L of 2�gene expression master mix, 1 �L of 20� assay (primers and probemix; TaqMan; Applied Biosystems), 5 �L diluted cDNA, and 4 �Lnuclease-free water. Appropriate negative (nontemplate control andreverse transcriptase control) and positive (human reference RNA;Stratagene) controls were also run in each experiment. Raw data wereacquired (MxPro software, version 4.01; Stratagene) on the computerlinked to the qPCR machine. The data were then analyzed on aspreadsheet (Excel; Microsoft, Redmond, WA) using the ��-Ctmethod.

Statistical Analysis

qPCR data were statistically analyzed on statistical software (SPSS, 16.0version; IBM, Chicago, IL) with significance set at P � 0.05. UsingStudent’s t-test, we statistically compared HBD9 mRNA expression intreated samples with those obtained in untreated control. All data wererepresented as mean � SE of three independent experiments per-formed in triplicate.

RESULTS

Immunolocalization of DEFB109/HBD9 in NormalOS Tissue Sections

As shown in Figure 1, DEFB109/HBD9 was constitutively ex-pressed in all regions of normal OS tissue sections. It waspredominantly present in the uppermost layers of the cornealepithelium, with a decline in intensity in basal layers. A fewkeratocytes in corneal stroma also stained for HBD9. HBD9 wasalso localized in all layers of limbal epithelium, with no stainingdemonstrated in limbal keratocytes. Notably, the conjunctivalepithelium demonstrated strong expression of HBD9 protein,with decreased staining in basal layers. Human tonsil tissuesection, which served as a control, was strongly positive forHBD9.

TLRs Play an Essential Role in DEFB109/HBD9Expression in hCECs

To determine whether HBD9 expression is modulated in re-sponse to the activation of TLRs, we assessed mRNA expres-sion of HBD9 using the qPCR method. As shown in Figure 2,treatment of hCECs for different durations (1–24 hours) withPAMPs of TLRs resulted in the immediate upregulation ofHBD9 mRNA. Stimulation of hCECs with Pam3CSK4 resulted in

FIGURE 2. TLRs are the key modula-tors of HBD9 mRNA in hCECs. Cellswere incubated with (A) Pam3CSK4,(B) poly I-C, (C) LPS, (D) flagellin, (E)R848, and (F) CpG ODN for the indi-cated time points. HBD9 mRNA levelswere measured by qPCR. All data rep-resent mean � SEM of three samplesrepeated three times. *P � 0.05; **P �0.001. Expression levels are normal-ized against the control untreated sam-ple, which has been maintained at 1 ineach of the graphs (note differentscales in y-axes).

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significant upregulation of HBD9 at 1 hour (5.4-fold increase;P � 0.0077), with a sudden decline at the end of 3 hours thatremained low until 9 hours. As indicated, after 24 hours oftreatment, HBD9 expression reached 3.9-fold (P � 0.0237; Fig.2A). In response to poly I-C, a time-dependent increase inHBD9 level (up to 3 hours; P � 0.0435) was observed. How-ever, after a surge, HBD9 transcript levels reached baselineexpression at 6 hours and remained at the same level until 24hours (Fig. 2B). Similar to the TLR02-mediated response, treat-ment of hCECs with LPS, a TLR04 PAMP, resulted in increasedexpression of HBD9 mRNA at 1 hour (1.83-fold), with a rapiddecline at 3 hours. However, after 6 hours, HBD9 transcriptlevels increased again (reaching the level seen at 1 hour) andremained unchanged until 24 hours (Fig. 2C). HBD9 expres-sion pattern in response to Flagellin, a TLR05 stimulant,matched the response mediated by the Pam3CSK4/TLR02 axisexcept for a rise at 24 hours. A surge of HBD9 mRNA at 1 hour(1.9-fold; P � 0.0191) was followed by a plunge in levels belowthe baseline expression until 24 hours (Fig. 2D). Stimulation ofTLR07/08 and TLR09 with R-848 (Fig. 2E) and CpG ODN (Fig.2F), respectively, did not affect HBD9 mRNA expression inhCECs.

NOD2 Modulates DEFB109/HBD9 Expressionin hCECs

To investigate whether NOD-like receptors regulate HBD9mRNA expression, we treated hCECs with synthetic PAMPs ofNOD1 and NOD2 in a time-dependent manner. As shown inFigure 3A, treatment of cells with iE-DAP demonstrated aninsignificant upregulation of HBD-9 at all time points. How-ever, a 70% decrease in baseline expression of HBD9 was notedat 24 hours. In contrast to iE-DAP, we observed an immediateresponse of HBD9 mRNA to NOD2 stimulant MDP at 1 hour(1.58-fold; P � 0.0228) that was followed by a time-dependentdecrease in transcript levels, reaching 50% below the baselineexpression at 6 hours (P � 0.0019). This was followed by a

gradual increase in mRNA, with maximal levels noted at 24hours (Fig. 3B).

IL-1� Is an Important Regulator ofDEFB109/HBD9 Expression in hCECs

To determine the effect of proinflammatory cytokines onHBD9 expression, we treated the cells without or with humanrecombinant IL-1� and TNF�, alone or in combination for 24hours. As shown in Figure 4A, a 4.4-fold increase in HBD9mRNA expression was noted in response to IL-1� (P �0.0139), and a modest and insignificant upregulation of HBD9transcript was observed after incubation with TNF� in combi-nation with IL-1� or by itself. To further understand the role ofIL-1�, we conducted a time-course treatment study. As indi-cated in Figure 4B, a significant downregulation of HBD9 wasevident after 3 hours (0.04-fold; P � 0.0004) of stimulation,which remained at the same level until the end of 9 hours(0.09-fold; P � 0.0119). This was followed by a sharp increasein HBD9 mRNA levels at 24 hours (4.43-fold; P � 0.0419).

DISCUSSION

In this study, we have demonstrated the constitutive presenceof the HBD9 protein in the cornea, conjunctiva, and limbus.HBD9 was shown to be highly concentrated in superficiallayers of healthy corneal epithelium, suggesting an immediaterole of these AMPs in OS defense against invading pathogens.Notably, we also observed the presence of HBD9 in the cornealstroma, the layer beneath the epithelium. In the normal state,the corneal stroma provides structural support to the corneaand a second barrier against microbial intrusion. The latterresponse is provided by the presence of Langerhans cells(dendritic cells), immunoglobulins (IgG and IgA), andAMPs.11–15 On infection or breach in the corneal epithelium,the local stromal immune response is activated, recruiting

FIGURE 3. NOD1 and NOD2 modu-late HBD9 mRNA expression inhCECs. Cells were incubated with (A)iE-DAP or (B) MDP for the indicatedtime points. HBD9 mRNA levels weremeasured by qPCR. All data representmean � SEM of three samples re-peated three times. *P � 0.05; **P �0.001.

FIGURE 4. IL-1� is an important regu-lator of HBD9 mRNA expression inhCECs. (A) Effect of proinflammatorycytokines on HBD9. Cells were incu-bated with IL-1�, TNF� or combina-tion of both for 24 hours and HBD9mRNA expression was evaluated. (B)Effect of IL-1� on HBD9. Cells treatedwith IL-1� for different durations wereprocessed for measurement of HBD9mRNA levels by qPCR. All data repre-sents means � SEM of three samplesrepeated three times. *P � 0.05; **P �0.001; ***P � 0.0001.



polymorphonuclear cells (PMNs) and lymphocytes (B- and T-cells) to the site.14,16,17 Thus, it could be proposed that HBD9,similar to its family members HBD218 and HBD3,19 might alsoplay a role in adaptive immunity. We noted an increasedexpression of HBD9 in the conjunctiva compared with thecornea. The normal conjunctiva contains resident lymphocytesand neutrophils and has a well-defined vasculature. Inflamma-tory mediators and growth factors produced locally or arrivingthrough the blood vessels could influence the differential ex-pression of HBD9 in the conjunctiva compared with the avas-cular cornea.20,21

�-Defensins are highly expressed at the OS. Ikeda et al.22

have shown the absence of mRNA expression of the human�-defensin-2 ortholog, mBD-2, in the limbal region of the ex-perimentally developed allergic conjunctivitis (EAC) mousemodel compared with control. Notably, conjunctival and cor-neal epithelia showed positive expression of mBD2 in the EACmodel. Terai et al.5 have demonstrated stronger expression ofHBD1 in the normal limbus and the conjunctiva than in thecornea. The expression of HBD9 is high at the limbus and thecornea, and, like its family members, it could play a significantrole in allergic and infectious conjunctivitis.

Downregulation of HBD9 mRNA in response to infectiousstimuli has previously been demonstrated.7,8 Innate immunereceptors such as TLRs and NLRs are widely studied because oftheir high affinity for pathogen recognition and modulation ofcytokine and AMP expression.1 In this study we have demon-strated the role of TLRs, NLRs, and IL-1� in HBD9 mRNAexpression. Activation of TLR02 and TLR04 has been impli-cated in HBD2 expression in various epithelia, such as lung,23

tracheobronchus,24 intestine,25 and cornea.26 Consistent withprevious studies on other defensins, we have demonstrated animportant role of TLR02 in the modulation of HBD9 expressionin hCECs. Kumar et al.26 demonstrated an increased expressionof HBD2 mRNA in hCECs after 6 hours of treatment with 10�g/mL Pam3CSK4. However, using a similar cell line modeland a much lower concentration of 1 �g/mL Pam3CSK4, wedemonstrated TLR02-induced HBD9 mRNA expression at anearlier time point, 1 hour. Notably, after 6 hours, we foundHBD9 mRNA rapidly declined below baseline expression, sup-porting our ex vivo study results.8 Using intestinal cell lines,Vora et al.25 showed TLR04 induced HBD2 expression in re-sponse to treatment with LPS after 5 hours. In our study, wefound that the LPS induced HBD9 after 1 hour and remained atthe same level until 24 hours, suggesting that the family mem-bers of �-defensins work in tandem against infectious stimuli.

Poly (I-C), the TLR03 agonist, has been shown to augmentHBD2 expression in gingival,27 uterine,28 and fallopian tube29

epithelial cells. In hCECs, we demonstrated a threefold in-crease in HBD9 mRNA expression in response to poly I-C in 3hours, suggesting an immediate role of HBD9 against viralinfections similar to HBD2.

Flagellin, a bacterial flagella filament protein, has been re-ported to induce HBD2 expression by TLR05 in human carci-noma (Caco-2) and murine enteroendocrine (STC-1) cell linemodels.30,31 Here, using synthetic flagellin protein, we demon-strated a rapid induction of HBD9 mRNA expression in hCECs.After a surge, we noted a sudden decrease in mRNA levels,with the lowest concentration attained at the end of 9 hours.Thus, further investigation is needed to fully understand themechanism behind the downregulation of HBD9 mRNA inresponse to bacterial infection.

Uehara et al.32 have demonstrated the involvement ofTLR07/08 in the induction of HBD2 expression in severalhuman epithelial cell line models. Similarly, bacterial DNA orsynthetic oligonucleotides containing CpG motif (CpG ODN)–activated TLR09-dependent HBD2 expression has been re-ported in sinonasal33 and respiratory epithelial cells.34 In con-

trast to these studies, we noted an insignificant effect ofTLR07/08 and TLR09 activation on HBD9 gene expression inhCECs. The role of these TLRs in the expression of other OSAMPs remains to be investigated.

NOD-like receptors such as NOD1 and NOD2 are involvedin the recognition of bacterial stimuli.10 Similar to TLRs, NOD1and NOD2 have been shown to trigger HBD2 expression.35,36

Mutation in NOD2 has been implicated in Crohn’s disease andin the subsequent reduced expression of �-defensins in thePaneth cells of affected persons.37,38 In this study, we noted asignificant role of NOD2 in HBD9 induction in hCECs. Recentstudies39,40 have demonstrated that the TLRs and NLRs syner-gistically enhance the immune response of innate and adaptiveimmune cell types. Therefore, further studies are needed tofully understand the synergistic effect of NOD1 and NOD2agonists on TLR-induced HBD9 expression in hCECs.

Proinflammatory cytokines such as IL-1� and TNF� havebeen widely studied because of their pronounced effects ondefensin and cathelicidin expression in a variety of surfaces. Inaddition to TLR02,26 IL-1� has been shown to induce AMPs atthe human OS.41–43 In this study, we demonstrated a signifi-cant effect of IL-1�, but not of TNF�, in HBD9 expression inhCECs. Unlike TLRs, which induced expression at earlier timepoints, IL-1� induced HBD9 mRNA expression at 24 hours oftreatment. Notably, IL-1� downregulated the expression ofHBD9 from 3 to 9 hours.

HBD9 is unique in that its expression is both downregulatedand upregulated in a time-dependent manner in response tothe activation of different TLRs and NLRs with specific PAMPsand IL-1�. The variable regulation of HBD9 expression maysuggest that it may have other functions, such as an interme-diary between innate and adaptive immune system and immu-nomodulation, in addition to the attributed antimicrobial ef-fect. Furthermore, the finding of HBD9 in various layers of theocular surface expands the body of knowledge related to thisAMP and highlights the role that AMPs may play in ocularsurface defense.

References

1. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innateimmunity. Cell. 2006;124:783–801.

2. McIntosh RS, Cade JE, Al-Abed M, et al. The spectrum of antimi-crobial peptide expression at the ocular surface. Invest Ophthal-mol Vis Sci. 2005;46:1379–1385.

3. Haynes RJ, Tighe PJ, Dua HS. Innate defence of the eye by antimi-crobial defensin peptides. Lancet. 1998;352:451–452.

4. Gottsch JD, Li Q, Ashraf MF, O’Brien TP, Stark WJ, Liu SH. Defensingene expression in the cornea. Curr Eye Res. 1998;17:1082–1086.

5. Terai K, Sano Y, Kawasaki S, et al. Effects of dexamethasone andcyclosporin A on human beta-defensin in corneal epithelial cells.Exp Eye Res. 2004;79:175–180.

6. Huang LC, Petkova TD, Reins RY, Proske RJ, McDermott AM.Multifunctional roles of human cathelicidin (LL-37) at the ocularsurface. Invest Ophthalmol Vis Sci. 2006;47:2369–2380.

7. Premratanachai P, Joly S, Johnson GK, McCray PB Jr, Jia HP,Guthmiller JM. Expression and regulation of novel human beta-defensins in gingival keratinocytes. Oral Microbiol Immunol.2004;19:111–117.

8. Abedin A, Mohammed I, Hopkinson A, Dua HS. A novel antimicro-bial peptide on the ocular surface shows decreased expression ininflammation and infection. Invest Ophthalmol Vis Sci. 2008;49:28–33.

9. Kumar H, Kawai T, Akira S. Pathogen recognition in the innateimmune response. Biochem J. 2009;420:1–16.

10. Franchi L, McDonald C, Kanneganti TD, Amer A, Nunez G. Nucle-otide-binding oligomerization domain-like receptors: intracellularpattern recognition molecules for pathogen detection and hostdefense. J Immunol. 2006;177:3507–3513.

IOVS, September 2010, Vol. 51, No. 9 Localization and Gene Expression of HBD9 4681


11. Gillette TE, Chandler JW, Greiner JV. Langerhans cells of theocular surface. Ophthalmology. 1982;89:700–711.

12. Knop E, Knop N. Anatomy and immunology of the ocular surface.Chem Immunol Allergy. 2007;92:36–49.

13. McClellan KA. Mucosal defense of the outer eye. Surv Ophthal-mol. 1997;42:233–246.

14. Sack RA, Nunes I, Beaton A, Morris C. Host-defense mechanism ofthe ocular surfaces. Biosci Rep. 2001;21:463–480.

15. Narayanan S, Miller WL, McDermott AM. Expression of humanbeta-defensins in conjunctival epithelium: relevance to dry eyedisease. Invest Ophthalmol Vis Sci. 2003;44:3795–3801.

16. Bron AJ, Seal DV. The defences of the ocular surface. TransOphthalmol Soc U K. 1986;105(pt 1):18–25.

17. Chandler JW. Immunology of the ocular surface. Int OphthalmolClin. 1985;25:13–23.

18. Yang D, Chertov O, Bykovskaia SN, et al. Beta-defensins: linkinginnate and adaptive immunity through dendritic and T cell CCR6.Science. 1999;286:525–528.

19. Funderburg N, Lederman MM, Feng Z, et al. Human -defensin-3activates professional antigen-presenting cells via Toll-like recep-tors 1 and 2. Proc Natl Acad Sci U S A. 2007;104:18631–18635.

20. Allansmith MR, Greiner JV, Baird RS. Number of inflammatory cellsin the normal conjunctiva. Am J Ophthalmol. 1978;86:250–259.

21. Detorakis ET, Zaravinos A, Spandidos DA. Growth factor expres-sion in ophthalmic pterygia and normal conjunctiva. Int J MolMed. 25:513–516.

22. Ikeda A, Nakanishi Y, Sakimoto T, Shoji J, Sawa M, Nemoto N.Expression of beta defensins in ocular surface tissue of experimen-tally developed allergic conjunctivitis mouse model. Jpn J Oph-thalmol. 2006;50:1–6.

23. Birchler T, Seibl R, Buchner K, et al. Human Toll-like receptor 2mediates induction of the antimicrobial peptide human beta-de-fensin 2 in response to bacterial lipoprotein. Eur J Immunol.2001;31:3131–3137.

24. Hertz CJ, Wu Q, Porter EM, et al. Activation of Toll-like receptor 2on human tracheobronchial epithelial cells induces the antimicro-bial peptide human beta defensin-2. J Immunol. 2003;171:6820–6826.

25. Vora P, Youdim A, Thomas LS, et al. Beta-defensin-2 expression isregulated by TLR signaling in intestinal epithelial cells. J Immunol.2004;173:5398–5405.

26. Kumar A, Zhang J, Yu FS. Toll-like receptor 2-mediated expressionof beta-defensin-2 in human corneal epithelial cells. Microbes In-fect. 2006;8:380–389.

27. Mahanonda R, Sa-Ard-Iam N, Eksomtramate M, et al. Cigarettesmoke extract modulates human beta-defensin-2 and interleukin-8expression in human gingival epithelial cells. J Periodontal Res.2009;44:557–564.

28. Schaefer TM, Fahey JV, Wright JA, Wira CR. Innate immunity in thehuman female reproductive tract: antiviral response of uterine

epithelial cells to the TLR3 agonist poly(I:C). J Immunol. 2005;174:992–1002.

29. Ghosh M, Schaefer TM, Fahey JV, Wright JA, Wira CR. Antiviralresponses of human fallopian tube epithelial cells to toll-like re-ceptor 3 agonist poly(I:C). Fertil Steril. 2008;89:1497–1506.

30. Palazzo M, Balsari A, Rossini A, et al. Activation of enteroendocrinecells via TLRs induces hormone, chemokine, and defensin secre-tion. J Immunol. 2007;178:4296–4303.

31. Ogushi K, Wada A, Niidome T, et al. Salmonella enteritidis FliC(flagella filament protein) induces human beta-defensin-2 mRNAproduction by Caco-2 cells. J Biol Chem. 2001;276:30521–30526.

32. Uehara A, Fujimoto Y, Fukase K, Takada H. Various human epithe-lial cells express functional Toll-like receptors, NOD1 and NOD2to produce anti-microbial peptides, but not proinflammatory cyto-kines. Mol Immunol. 2007;44:3100–3111.

33. Ramanathan M Jr, Lee WK, Dubin MG, Lin S, Spannhake EW, LaneAP. Sinonasal epithelial cell expression of toll-like receptor 9 isdecreased in chronic rhinosinusitis with polyps. Am J Rhinol.2007;21:110–116.

34. Platz J, Beisswenger C, Dalpke A, et al. Microbial DNA induces ahost defense reaction of human respiratory epithelial cells. J Im-munol. 2004;173:1219–1223.

35. Boughan PK, Argent RH, Body-Malapel M, et al. Nucleotide-bindingoligomerization domain-1 and epidermal growth factor receptor:critical regulators of beta-defensins during Helicobacter pyloriinfection. J Biol Chem. 2006;281:11637–11648.

36. Voss E, Wehkamp J, Wehkamp K, Stange EF, Schroder JM, HarderJ. NOD2/CARD15 mediates induction of the antimicrobial peptidehuman beta-defensin-2. J Biol Chem. 2006;281:2005–2011.

37. Elphick D, Liddell S, Mahida YR. Impaired luminal processing ofhuman defensin-5 in Crohn’s disease: persistence in a complexwith chymotrypsinogen and trypsin. Am J Pathol. 2008;172:702–713.

38. Peyrin-Biroulet L, Chamaillard M. NOD2 and defensins: translatinginnate to adaptive immunity in Crohn’s disease. J Endotoxin Res.2007;13:135–139.

39. Uehara A, Takada H. Functional TLRs and NODs in human gingivalfibroblasts. J Dent Res. 2007;86:249–254.

40. Takada H, Uehara A. Enhancement of TLR-mediated innate im-mune responses by peptidoglycans through NOD signaling. CurrPharm Des. 2006;12:4163–4172.

41. Shin JS, Kim CW, Kwon YS, Kim JC. Human beta-defensin 2 isinduced by interleukin-1beta in the corneal epithelial cells. ExpMol Med. 2004;36:204–210.

42. McDermott AM, Redfern RL, Zhang B, Pei Y, Huang L, Proske RJ.Defensin expression by the cornea: multiple signalling pathwaysmediate IL-1beta stimulation of hBD-2 expression by human cor-neal epithelial cells. Invest Ophthalmol Vis Sci. 2003;44:1859–1865.

43. Ruan X, Chodosh J, Callegan MC, et al. Corneal expression of theinflammatory mediator CAP37. Invest Ophthalmol Vis Sci. 2002;43:1414–1421.



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