Reductions in claudin-1 may enhance susceptibility to herpes simplex virus 1 infections in atopic dermatitis
2011; Elsevier BV; Volume: 128; Issue: 1 Linguagem: Inglês
10.1016/j.jaci.2011.02.014
ISSN1097-6825
AutoresAnna De Benedetto, Mark K. Slifka, Nicholas Rafaels, I‐Hsin Kuo, Steve N. Georas, Mark Boguniewicz, Tissa Hata, Lynda C. Schneider, Jon M. Hanifin, Richard L. Gallo, David C. Johnson, Kathleen C. Barnes, Donald Y.M. Leung, Lisa A. Beck,
Tópico(s)Allergic Rhinitis and Sensitization
ResumoTo the Editor: Atopic dermatitis (AD) is the most common inflammatory skin disease, affecting up to 20% of children in the United States, and is characterized by an increased susceptibility to cutaneous infections.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 2Boguniewicz M. Leung D.Y. Recent insights into atopic dermatitis and implications for management of infectious complications.J Allergy Clin Immunol. 2010; 125: 4-13Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar One in 10 subjects with AD has difficulty clearing cutaneous infections with a host of viruses including herpes simplex, vaccinia, human papilloma, and/or molluscum contagiosum.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar This typically manifests as more extensive cutaneous and sometimes systemic disease and/or resistance to standard therapies. One of the rarer but more severe viral complications is eczema herpeticum (EH), caused by herpes simplex virus (HSV)–1 that results in a widespread skin infection.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar EH can be complicated by viremia and multiorgan involvement including keratoconjunctivitis, meningitis, and encephalitis.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Although we have shown that subjects with AD with more severe disease, greater allergen sensitization, and enhanced TH2 polarity are at greatest risk for this viral complication, the molecular mechanisms responsible for this remain unclear.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Disruption of the epidermal barrier is now recognized as a cardinal feature of AD pathogenesis. The skin has 2 barrier structures: the stratum corneum (SC) and tight junctions (TJs). It is widely accepted that the SC in subjects with AD is dysfunctional. The loss-of-function mutations in filaggrin, a structural protein important for its hygroscopic properties, have been reproducibly and robustly associated with AD3O'Regan G.M. Sandilands A. McLean W.H. Irvine A.D. Filaggrin in atopic dermatitis.J Allergy Clin Immunol. 2009; 124: R2-R6Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar and more recently with EH.4Gao P.S. Rafaels N.M. Hand T. Murray T. Boguniewicz M. Hata T. et al.Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 507-513Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar TJs are found on opposing membranes of stratum granulosum keratinocytes, directly below the SC. TJs consist of a complex of adhesive and scaffolding proteins that control the passage of water, ions, and solutes through the paracellular pathway.5Niessen C.M. Tight junctions/adherens junctions: basic structure and function.J Invest Dermatol. 2007; 127: 2525-2532Crossref PubMed Scopus (508) Google Scholar We have recently demonstrated that the nonlesional epithelium of subjects with AD has remarkable bioelectric abnormalities indicative of a TJ defect that may be the consequence of reduced levels of claudin-1 (CLDN1), a key TJ adhesive protein.6De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar This is in line with the landmark work of Furuse and Tsukita,7Furuse M. Hata M. Furuse K. Yoshida Y. Haratake A. Sugitani Y. et al.Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice.J Cell Biol. 2002; 156: 1099-1111Crossref PubMed Scopus (1230) Google Scholar whose CLDN1 knockout mouse established the importance of epidermal TJ and CLDN1. These mice died within 24 hours of birth with wrinkled skin, severe dehydration, and increased epidermal permeability as measured by dye studies and transepidermal water loss.7Furuse M. Hata M. Furuse K. Yoshida Y. Haratake A. Sugitani Y. et al.Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice.J Cell Biol. 2002; 156: 1099-1111Crossref PubMed Scopus (1230) Google Scholar In our AD studies, we also observed an inverse correlation between CLDN1 expression and markers of TH2 polarity (total eosinophil counts and serum total IgE).6De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar This has led us to speculate that TJ defects may promote greater TH2 polarity, which is a characteristic of subjects with EH.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar We hypothesized that TJ defects such as those we observed in subjects with AD may promote HSV-1 infections on that basis. In addition, viruses often commandeer intercellular junction proteins as a means of viral entry.5Niessen C.M. Tight junctions/adherens junctions: basic structure and function.J Invest Dermatol. 2007; 127: 2525-2532Crossref PubMed Scopus (508) Google Scholar For example, wild-type HSV-1 infects keratinocytes through a complex interaction involving the viral envelope glycoprotein gD with either nectin-1 (PVRL1) or HSV entry mediator.8Gonzalez-Mariscal L. Garay E. Lechuga S. Virus interaction with the apical junctional complex.Front Biosci. 2009; 14: 731-768Crossref Scopus (28) Google Scholar, 9Geraghty R.J. Krummenacher C. Cohen G.H. Eisenberg R.J. Spear P.G. Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor.Science. 1998; 280: 1618-1620Crossref PubMed Scopus (784) Google Scholar Nectin-1, a Ca2Boguniewicz M. Leung D.Y. Recent insights into atopic dermatitis and implications for management of infectious complications.J Allergy Clin Immunol. 2010; 125: 4-13Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar-independent cell adhesion protein of the immunoglobulin superfamily, colocalizes with E-cadherin and β-catenin to form another intercellular junctional complex called the adherens junction. Previous studies have shown that the susceptibility of human keratinocytes to HSV-1 infection is inversely related to the degree of cell-cell contact and confluency.9Geraghty R.J. Krummenacher C. Cohen G.H. Eisenberg R.J. Spear P.G. Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor.Science. 1998; 280: 1618-1620Crossref PubMed Scopus (784) Google Scholar, 10Schelhaas M. Jansen M. Haase I. Knebel-Morsdorf D. Herpes simplex virus type 1 exhibits a tropism for basal entry in polarized epithelial cells.J Gen Virol. 2003; 84: 2473-2484Crossref PubMed Scopus (51) Google Scholar This suggests that healthy junctional complexes may be a key deterrent to the spread of HSV-1 from one keratinocyte to another. In these studies, we tested the hypothesis that the silencing of epidermal CLDN1 would enhance HSV-1 infectivity of human keratinocytes. We had previously demonstrated that reductions in CLDN1 adversely affected measures of TJ integrity.6De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Primary human keratinocytes (PHKs) were isolated from foreskin as previously described6De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar and transiently transfected with scrambled (control) or CLDN1 (100 nmol/L) small interfering RNA (siRNA; Santa Cruz Biotechnology, Santa Cruz, Calif) by using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, Calif; see this article's complete Methods in the Online Repository at www.jacionline.org). We have previously demonstrated that this knockdown approach led to a >50% reduction in claudin-1 transcripts and adversely affected TJ function as demonstrated by a 50% reduction in transepithelial electrical resistance and doubling of the 50% permeability.6De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Importantly, this level of CLDN1 reduction mirrored what we had observed in AD samples. Twenty-four to 48 hours posttransfection, PHKs were differentiated in Dulbecco modified Eagle medium (DMEM) with 10% heat-inactivated FBS (HI-FBS; Invitrogen/Gibco) for 24 hours and subsequently infected with the highly virulent HSV-1 strain F (provided by Dr D. C. Johnson, Oregon Health and Science University) at a multiplicity of infection of 0.1 (see this article's complete Methods in the Online Repository). The multiplicity of infection was chosen based on findings from viral titration (0.01-10 multiplicity of infection) studies on differentiated and undifferentiated PHKs, respectively, used as negative and positive controls (data not shown). After 2 hours, the viral inoculum was removed, and PHKs were cultured for 24 hours in DMEM containing 5% HI-FBS and 0.4% human-γ-globulin (0.5 mg/mL; Sigma, St Louis, Mo) to neutralize extracellular virus. HSV-infected PHKs were fixed in 4% formaldehyde and stained with a polyclonal rabbit anti–HSV-1 (1:500; Dako, Carpinteria, Calif) antibody followed by Alexa Fluor 488 donkey-antirabbit IgG H+L (1:1000; Invitrogen) and 4',6-diamidino-2-phenyl-indole, dihydrochloride (DAPI; 1:10,000; Invitrogen). For each sample, 6 random fields were captured at identical acquisition settings and analyzed computationally to quantify differences objectively in focal forming units (FFUs; see complete Methods in the Online Repository). A FFU was defined as a cluster of 3 or more adjacent HSV-1–positive cells. CLDN1 depletion significantly increased the number and size of HSV-1 FFUs (Fig 1, A) compared with scrambled siRNA–transfected PHK (Fig 1, B). An average of 4.8 ± 0.7 FFUs/field were counted in CLDN1 knockdown PHKs, whereas only 2.5 ± 0.5 FFUs/field were detected in the control cells (P = .05; n = 6; Fig 1, C). The diameters of FFUs were significantly greater in keratinocytes whose CLDN1 expression was reduced (207 ± 22 μm) compared with controls (152 ± 40 μm; P = .03; n = 6; Fig 1, D). Similar findings were observed when evaluating FFU area (P = .04; n = 6; Fig 1, E) in PHKs after CLDN1 knockdown (15,649 ± 4367 pixels) compared with control transfection (9902 ± 4943 pixels). The increased frequency of HSV-1–infected cells in CLDN1 knockdown PHKs was confirmed by an infectious center assay (see this article's complete Methods in the Online Repository). More CLDN1 siRNA–transfected PHKs were infected with HSV-1 (38 ± 6%; n = 3) than control PHKs (28 ± 7%; n = 3; P = .003; see this article's Fig E1 in the Online Repository at www.jacionline.org). Importantly, the observed increase in HSV-1 infectivity in CLDN1 knockdown PHKs could not be explained by reduced expression of nectin-1, the HSV-1 receptor. Indeed, nectin-1 mRNA expression was unaffected by CLDN1 silencing (n = 5; see this article's Fig E2 in the Online Repository at www.jacionline.org). However, we cannot rule out the possibility that reductions in CLDN1 and the effects on TJ function might reduce nectin-1 membrane localization. It is also important to highlight that our in vitro model is sufficient to induce functional TJ as demonstrated by enhanced transepithelial resistance and reduced permeability.6De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar However, it is not enough to induce terminal differentiation (eg, filaggrin expression and cornified envelope). Although this might be a limitation of the methodology, we believe this condition well reflects the in vivo situation. In AD subjects with history of EH (ADEH+), often the virus spreading is a result of reactivation of a silent infection (inside-out), suggesting that the first barrier structure the virus will encounter is indeed the TJ (located in the stratum granulosum, just below the SC). As part of the National Institute of Allergy and Infectious Diseases–funded Atopic Dermatitis and Vaccinia Network (ADVN; reviewed in a previous publication by Beck et al1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), we performed a preliminary study to evaluate whether CLDN1 polymorphisms were associated with EH in AD subjects. To do this, we used a haplotype-tagging SNP approach with genetic markers available in the public arena (n = 132 in single nucleotide polymorphism database) in 414 European Americans and 328 African Americans (see this article's complete Methods and Table E1 in the Online Repository at www.jacionline.org). We previously reported modest associations (P = .003-.05) between SNP variants throughout the CLDN1 gene and AD that were more significant among African Americans.6De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar In this study, we addressed whether genetic variations in CLDN1 contribute to risk of EH in both North American AD populations. The EH subphenotype was defined as subjects with AD who had at least 1 EH episode documented by an ADVN investigator (or a physician affiliated with the same academic center) or diagnosed clinically by an outside physician where the HSV infection was confirmed by PCR, tissue immunofluorescence, Tzanck smear, and/or culture. The study was approved by the institutional review boards at National Jewish Health, Johns Hopkins University, Oregon Health and Science University, the University of California San Diego, Children's Hospital of Boston, and the University of Rochester Medical Center. All subjects gave written informed consent before participation. To determine whether SNPs in CLDN1 were associated with EH, the Cochran-Armitage trend test using the PLINK software (http://pngu.mgh.harford.edu/∼purcell/plink/) was performed within each AD population and confirmed with the adaptive permutation test. We observed only modest associations between CLDN1 SNPs and EH in our North American AD populations. We wondered whether the strong association we observed between both filaggrin (FLG) null mutations R501X and 2282del4 and EH (odds ratio [OR], 10.1 [value range: 4.7-22.1]; P = 1.99 × 1011Shimazaki J. Higa K. Kato N. Satake Y. Barrier function of cultivated limbal and oral mucosal epithelial cell sheets.Invest Ophthalmol Vis Sci. 2009; 50: 5672-5680Crossref PubMed Scopus (27) Google Scholar) was masking a lesser effect from CLDN1 variants.4Gao P.S. Rafaels N.M. Hand T. Murray T. Boguniewicz M. Hata T. et al.Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 507-513Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar To address this, we excluded subjects who had 1 or both of these FLG mutations and reanalyzed the associations with all CLDN1 SNPs. In the European American group, an intronic SNP (rs3774032; OR, 0.59 [0.35-0.98]; P = .037) was marginally associated with EH (Fig 2) but not significantly after adaptive permutations adjustment (P = .10). When excluding subjects with a FLG mutation (n = 29 [36.3%]; Table I), the association became slightly more significant (OR, 0.44 [0.22-0.85]; P = .0225) and still significant after adaptive permutations adjustment (P = .035). Interestingly, 1 additional intronic SNP emerged (rs3732923; OR, 1.93 [1.21-3.07]; P = .0010; adaptive permutation, P = .0005; Fig 2). Although the EH sample size in the African American population was too small (n = 21) for meaningful analyses, a SNP in the promoter region was associated with EH (rs3954259; OR, 2.16 [1.02-4.59]; P = .040; adaptive permutation, P = .033). Limitations of these studies are the small sample size and the superficial coverage of CLDN1 loci. Nevertheless, these findings implicate CLDN1 mutations in susceptibility to widespread HSV skin infections in subjects with AD, especially those who do not have a FLG mutation.Table IADVN registry participant characteristicsCharacteristicEuropean AmericanAfrican AmericanADEH+ADEH–ADEH+ADEH–Sample size9316521155Males, n (%)49 (52.7)47 (28.5)8 (38.1)35 (22.6)Age (y), mean (SD)22.9 (21.5)38.0 (14.5)22.8 (19.0)36.4 (11.0)AD onset <5 y, n (%)77 (97.5)101 (78.3)14 (93.3)77 (68.1)IgE level, median (interquartile range)*Total serum IgE levels were significantly higher in patients with ADEH+ compared with both patients with ADEH– and nonatopic, healthy controls even after adjusting for age (P < .001).2225 (611-9034)252 (85-1275)3505 (571-9429)425 (129-1194)EASI,†EASI was determined by the percentage of eczema area on a 7-point ordinal scale: 0, no eruption; 1, <10%; 2, 10% to 29%; 3, 30% to 49%; 4, 50% to 69%; 5, 70% to 89%; and 6, 90% to 100%. median (interquartile range)‡EASI was significantly higher among patients with ADEH+ compared with patients with ADEH– in both racial groups even after adjusting for age (P < .001).10.0 (4.4-15.8)3.2 (1.3-8.4)10.9 (6.8-15.8)3.0 (1.5-7.4)FLG null allele carrier, n (%)29 (36.3)36 (22.1)2 (12.5)9 (6.0)ADEH+, AD with a history of eczema herpeticum; ADEH–, AD without a history of EH; EASI, Eczema Area and Severity Index.∗ Total serum IgE levels were significantly higher in patients with ADEH+ compared with both patients with ADEH– and nonatopic, healthy controls even after adjusting for age (P < .001).† EASI was determined by the percentage of eczema area on a 7-point ordinal scale: 0, no eruption; 1, <10%; 2, 10% to 29%; 3, 30% to 49%; 4, 50% to 69%; 5, 70% to 89%; and 6, 90% to 100%.‡ EASI was significantly higher among patients with ADEH+ compared with patients with ADEH– in both racial groups even after adjusting for age (P < .001). Open table in a new tab ADEH+, AD with a history of eczema herpeticum; ADEH–, AD without a history of EH; EASI, Eczema Area and Severity Index. In conclusion, this study and previously reported work implicate both SC and TJ epidermal barrier defects as a mechanism to explain the susceptibility of subjects with AD to widespread cutaneous infections with HSV and possibly other viral pathogens.4Gao P.S. Rafaels N.M. Hand T. Murray T. Boguniewicz M. Hata T. et al.Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 507-513Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar In support of this notion, our patients with EH more commonly experienced infections with molluscum contagiosum than subjects with AD with no history of EH (29% vs 2%, respectively).1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar More of our subjects with EH (16%) had recurrent HSV keratitis compared with only 1% of subjects with AD with no history of EH.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar It is possible that the same mechanism may responsible for their ocular disease because corneal epithelium possesses TJs and these structures express claudin-1.11Shimazaki J. Higa K. Kato N. Satake Y. Barrier function of cultivated limbal and oral mucosal epithelial cell sheets.Invest Ophthalmol Vis Sci. 2009; 50: 5672-5680Crossref PubMed Scopus (27) Google Scholar, 12Yoshida Y. Ban Y. Kinoshita S. Tight junction transmembrane protein claudin subtype expression and distribution in human corneal and conjunctival epithelium.Invest Ophthalmol Vis Sci. 2009; 50: 2103-2108Crossref PubMed Scopus (64) Google Scholar In this study, we show both mechanistic and genetic results that implicate TJs as a critical barrier structure important in containing the spread of epidermal HSV-1 infections. Defects in TJ compared with the SC may be particularly relevant in viral infections in which the virus enters from a basolateral direction, as is the case with HSV reactivation. Collectively, this work highlights a previously underappreciated role for TJ proteins in cutaneous host defense and may lead to new therapeutic strategies. We acknowledge Melissa Dubois, PhD, and Todd W. Wisner at Oregon Health and Science University (Beaverton, Ore) for helpful suggestions on HSV-1 experiments. Jonathan Rebhahn, MS, at the University of Rochester Medical Center (Rochester, NY) developed the software to quantify FFUs. Mary Bolognino, BS, at the University of Rochester Medical Center (Rochester, NY) helped with PHK studies. We also acknowledge several groups whose efforts made the clinical enrollment possible: ADVN coordinators (Patricia Taylor, NP; Trista Berry, BS; Susan Tofte, FNP; Shahana Baig-Lewis, MPH; Peter Brown, BS; Lisa Heughan, BA, CCRC; Meggie Nguyen, BS; Doru Alexandrescu, MD; Lorianne Stubbs, RC; Reena Vaid, MD; Diana Lee, MD), ADVN regulatory advisors (Judy Lairsmith, RN), biological sample tracking (Johns Hopkins University, Tracey Hand, MSc, Jessica Scarpola; University of Rochester Medical Center, Mary Bolognino, MS), National Institute of Allergy and Infectious Diseases–Division of Allergy, Immunology and Transplantation oversight (Marshall Plaut, MD, and Joy Laurienzo Panza, RN, BSN), Rho, Inc, statistical support (Daniel Zaccaro, MS; Brian Armstrong, MPH) Rho, Inc, general study support and oversight (Jamie Reese, BS; Susi Lieff, PhD; Gloria David, PhD, MHSc), and all the patients who participated in this study. Human keratinocytes (PHKs) were isolated from neonatal foreskin.E1De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar PHKs were cultured in keratinocyte-SFM (Invitrogen/Gibco, Carlsbad, Calif) with 1% Pen/Strep, 0.2% amphotericin B (Invitrogen/Gibco). To differentiate PHK, cells were grown in DMEM (Invitrogen/Gibco) with 10% HI-FBS (Invitrogen/Gibco) and 1% Pen/Strep, 0.2% amphotericin B (Invitrogen/Gibco). Primary human keratinocytes were plated on glass coverslips at 2 to 3 × 105 cells/well in a 6-well plate or at 2 to 3 × 104 cells/filter in Transwell inserts (Costar; Corning Inc, Corning, New York; Polyester Membrane, 0.4 μm pore size, 6.5 mm insert) in keratinocyte-SFM without antibiotics. The day after plating, cells were transfected with claudin-1 specific or control (scrambled) siRNAs (Santa Cruz Biotechnology, Santa Cruz, Calif) by using Lipofectamine 2000 Transfection Reagent (Invitrogen). Infection studies were performed by using the highly virulent HSV-1 strain F (provided by Dr D. C. Johnson). Twenty-four to 48 hours posttransfection with 100 nmol/L claudin-1 or control siRNA, the cells were differentiated in DMEM with 10% heat-inactivated FBS for 24 hours. Cells were washed twice with HBSS and infected with HSV-1 strain F at a multiplicity of infection of 0.1 in DMEM containing 1% heat-inactivated FBS at 37°C, with rocking every 15 minutes. After 2 hours, the viral inoculum was removed, and the cells were washed 2 times with HBSS and incubated in DMEM containing 5% HI-FBS and 0.4% human-γ-globulin (Sigma; final concentration, 0.5 mg/mL) for 24 hours to neutralize any extracellular virus. It is important to note that our PHKs are grown under differentiating conditions that induce functional TJs as demonstrated by enhanced transepithelial resistance and reduced permeability,E1De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar but they do not fully differentiate, as evidenced by the lack of filaggrin expression. Herpes simplex virus–infected PHKs were washed 2 times with PBS and fixed in 4% formaldehyde/PBS for 20 minutes at room temperature. A polyclonal rabbit anti–HSV-1 (Dako) antibody diluted 1:500 in PBS/1% BSA was placed on the PHK for 1 hour at 37°C followed by Alexa Fluor 488 donkey-antirabbit IgG H+L (1:1000; Invitrogen) and DAPI (1:10,000; Invitrogen). Coverslips were mounted onto slides with SlowFade (Invitrogen). For each sample, 6 random fields were captured at identical acquisition settings. Images were stored in Portable Network Graphics format and analyzed computationally to quantify differences objectively in FFUs. HSV-1 infected cells were assigned to the green channel. Cell density was calculated by counting the number of DAPI-labeled nuclei, assigned to the blue channel. Images were analyzed by using MATLAB (MathWorks, Inc, Natick, MA) to enumerate FFUs, and total cell number. The following FFU measurements were also taken: major axis length (μm), area (μm2), and pixel area (px). The infected cell channel (green) was converted to a binary image using the Otsu method for threshold determination.E2Otsu N. A threshold selection method from gray-level histograms.IEEE Transactions on Systems, Man, and Cybernetics. 1979; 9: 62-66Crossref Google Scholar A morphologic closing (dilation followed by erosion) was performed to reduce noise and fuse individual infected cells into colonies. A distance transform was computed from the binary image, followed by a watershed transform. The colony binary image was then multiplied by its watershed image, the result was dilated slightly, and its perimeter was drawn. Only cells with sizes greater than a predefined threshold were included for further analysis. The infected cell channel (green) was filtered to reduce effects of noise. The resulting image was inverted, followed by a watershed transform. The infected cell watershed image was then multiplied by the colony watershed image to identify cells within previously identified colonies. Finally, a perimeter was drawn around cell borders. Twenty-four hours postinfection, PHKs were treated with 0.05% trypsin-EDTA (Invitrogen/Gibco) for 5 minutes at 37°C followed by gently scraping to lift the cells, washed 3 times in DMEM containing 1% HI-FBS, and vortexed for 30 seconds to disrupt cell clumps. Live cells were counted by using Trypan blue exclusion, and scalar PHK dilutions (1000 to 1 cells in 1 mL media) were plated onto pregrown monolayers of Vero cells in 6-well plates. After 2 hours, 1 mL DMEM containing 5% HI-FBS and 0.4% human-γ-globulin (Sigma; final concentration, 0.5 mg/mL) was added to each well. After 3 days of incubation at 37°C, cells were fixed (75% methanol/25% acetic acid) and stained with 0.1% crystal violet. Plaques were counted by 2 independent investigators, and results were expressed as a percentage of infected PHKs. Reverse transcription was performed from total RNA by using the iScript cDNA Synthesis kit (Bio-Rad) according to the manufacturer's protocol. Quantitative PCR (eg, real-time PCR) was performed by using the iQ SYBER Green Supermix assay system from Bio-Rad Laboratories. All PCR amplifications were carried out in triplicate on an iQ5 Multicolor real-time PCR detection system (Bio-Rad). Primers were designed and synthesized by Integrated DNA Technologies. Primers used in the study were as follows: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (forward: GAA GGT GAA GGT CGG AGT C, and reverse: GAA GAT GGT GAT GGG ATT TC), CLDN1 (forward: CGA TGA GGT GCA GAA GAT GA, and reverse: CCA GTG AAG AGA GCC TGA CC), and PVRL1 (nectin-1; forward: AGC CAT TAA GGA GAA ACG A, and reverse: TTC CCA ATT TCT CTG CTC T). Relativ
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