SUPPLEMENTARY MATERIAL

Supplementary material is linked to the onlineversion of the paper at www.jidonline.org, and athttp://dx.doi.org/10.1016/j.jid.2015.12.005.

CM Sweeney et al.b-Defensins-3 Activate LCs and Exacerbate Psoriasis

Our data show that one-third ofpatients with PD have autoantibodiesto Col XVII that also bind to THþ neu-rons. The lack of reactivity of PD auto-antibodies with skin suggests theautoantibodies develop from neuronalCol XVII. The subset of patients withneurologic disease that develop BPlikely result from epitope spreading toregions of Col XVII that are pathogenicin skin. A key question that remains iswhether Col XVII autoantibodies have adetrimental effect on PD or are other-wise predictive of disease onset/out-comes. Finally, these data suggest lossof tolerance to neuronal Col XVII maycontribute to the risk for pemphigoid.

CONFLICT OF INTERESTThe authors state no conflicts of interest.

ACKNOWLEDGMENTSThis material is based on work supported in part byVA Merit Review grant 1l01CX000317-01 (JAF)from the Department of Veterans Affairs, VeteransHealth Administration, Office of Research andDevelopment, Biomedical Laboratory Researchand Development 1BX001680-01 and a KO8award from the National Institutes of Health (NIH),NS078100 (NSN), an award from the Aging Mindand Brain Institute (NSN, JAF) and the NIH CTSA:2UL1 TR000442-06.We gratefully acknowledge thetechnical assistance of Rupasree Srikantha, ColleenFullenkamp, and Dennis Porto, MD. Martin Cas-sell, PhD, kindly provided sections of human sub-stantia nigra. David Moser, PhD, providedassistance in arranging the recruitment of patients.

Abbreviations: DC, dendritic cell; HBD3, human b-db-defensin 14; moLC, monocyte-derived Langerhans

Accepted manuscript published online 18 December2016

Kelly A.N. Messingham1,Samantha Aust1, Joseph Helfenberger1,Krystal L. Parker2, Susan Schultz3,Julie McKillip1,Nandakumar S. Narayanan2 andJanet A. Fairley1,4,*

1Department of Dermatology, University ofIowa, Iowa City, IA; 2Department of Neurologyand Aging Mind and Brain Initiative, Universityof Iowa, Iowa City, IA; 3Department ofPsychiatry, Carver College of Medicine,University of Iowa, Iowa City, IA; and4Veterans Administration Medical Center, IowaCity, IA*Corresponding author e-mail: janet-fairley@uiowa.edu

REFERENCES

Bastuji-Garin S, Joly P, Lemordant P, Sparsa A,Bedane C, Delaporte E, et al. Risk factors forbullous pemphigoid in the elderly: a prospec-tive case-control study. J Invest Dermatol2011;131:637e43.

Chen J, Li L, Chen J, Zeng Y, Xu H, Song Y,Wang B. Sera of elderly bullous pemphigoidpatients with associated neurological diseasesrecognize bullous pemphigoid antigens in thehuman brain. Gerontology 2011;57:211e6.

Diaz LA, Ratrie III H, Saunders HS, Futamura S,Squiquera HL, Anhalt GJ, et al. Isolation of ahuman epidermal cDNA corresponding to the180-kD autoantigen recognized by bullous

efensin 3; LC, Langerhans cell; MBD14, murinecell

2015; corrected proof published online 27 January

pemphigoid and herpes gestationis sera. J ClinInvest 1990;86:1088e94.

Jordon RE, Sams WM, Beutner EH. Comple-ment immunofluorescent staining in bullouspemphigoid. J Lab Clin Med 1969;74:548e56.

Langan S, Groves RW, West J. The relationshipbetween neurological disease and bullouspemphigoid: a population-based case-controlstudy. J Invest Dermatol 2011;131:631e6.

Liu Z, Giudice GJ, Swartz SJ, Fairley JA, Till GO,Troy JL, et al. The role of complement inexperimental bullous pemphigoid. J Clin Invest1995;95:1539e44.

Seppanen A. Collagen XVII: a shared antigenin neurodermatological interactions? [e-pubahead of print] Clin Dev Immunol 2013; http://dx.doi.org/10.1155/2013/240570.

Seppanen A, Miettinen R, Alafuzoff I. Neuronalcollagen XVII is localized to lipofuscin gran-ules. Neuroreport 2010;21:1090e4.

Stanley JR, Tanaka T, Mueller S, Klaus-Kovtun V,Roop D. Isolation of a complementary DNA forbullous pemphigoid antigen by use of patients’autoantibodies. J Clin Invest 1988;82:1864e70.

Stinco G, Codutti R, Scarbolo M, Valent F,Patrone P. A retrospective epidemiologicalstudy on the association of bullous pemphigoidand neurological diseases. Acta Derm Venereol2005;85:136e9.

Taghipour K, Chi C-C, Vincent A, Groves RW,Venning V, Wojnarowska F. The associationof bullous pemphigoid with cerebrovasculardisease and dementia: a case-control study.Arch Dermatol 2010;146:1251e4.

Yang YW, Chen YH, Xirasagar S, Lin HC.Increased risk of stroke in patients with bullouspemphigoid: a population-based follow-upstudy. Stroke 2011;42:319e23.

Human b-Defensin 3 and Its Mouse OrthologMurine b-Defensin 14 Activate LangerhansCells and Exacerbate Psoriasis-Like SkinInflammation in Mice

Journal of Investigative Dermatology (2016) 136, 723e727; doi:10.1016/j.jid.2015.12.011

TO THE EDITORGenetic variations in the proin-flammatory cytokine IL-23 are associ-ated with psoriasis, a common,inflammatory skin disorder (Nair et al.,2009). IL-23 is primarily produced bydendritic cells (DCs) and plays a path-ogenic role in psoriasis by directing the

development of T helper type 17 cells(Di Cesare et al., 2009). Langerhanscells (LCs), the main DC subtype in theepidermis, can participate in both im-munity and the induction of tolerance(Igyarto et al., 2011; Seneschal et al.,2012), yet their role in psoriasis re-mains unclear. Mouse models of

disease have yielded contradictory re-sults (Glitzner et al., 2014; Wohn et al.,2013; Yoshiki et al., 2014). However,the migration of LCs is impaired inhuman disease, and it was suggestedthat the inflammatory environmentof psoriasis may affect their function(Cumberbatch et al., 2006). This studysought to clarify the role of LC role indisease with specific focus on the pro-duction of IL-23.

Punch biopsies (6 mm) were obtainedfrom psoriasis patients and healthy

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Figure 1. IL-23 production by Langerhans cells (LCs) is enhanced in psoriasis. (a) Epidermal suspensions from healthy controls and psoriasis patients were

treated with monensin for 6 hours and IL-23 expression by LCs (live, CD45þCD1aþCD207 cells) was analyzed by flow cytometry. Results are given as

mean � standard error of the mean for 6e8 donors. (b) Monocyte-derived LCs from psoriasis patients (PSOR) and controls (HC) were stimulated with zymosan

(10 mg/ml). After 24 hours, the concentration of IL-23 was determined by ELISA. (c, d) Epidermal LCs from psoriasis patients (PSOR) and healthy controls (HC)

were treated with (c) zymosan or (d) human b-defensin 3 (HBD3) for 24 hours. Monensin was added for the final 12 hours, and the expression of IL-23 by

LCs was determined by flow cytometry. Results are given as mean � standard error of the mean for 4e8 donors. Representative plots are pregated on live

CD45þCD207þCD1aþ LCs. (e) IL-23 expression by epidermal LCs from psoriasis patients (PSOR) and healthy controls (HC) (as in d). Results are given as

mean � standard error of the mean for 6e8 donors, *P < 0.05, **P < 0.01, ***P < 0.001.

CM Sweeney et al.b-Defensins-3 Activate LCs and Exacerbate Psoriasis

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controls after written informed patientconsent and ethical approval were ob-tained from St. Vincent’s Ethics andMedical Research Committee. LCs inepidermal suspensions were identifiedas live single CD45þCD1aþCD207þ

cells (Figure 1a; see SupplementaryMaterials online). A significantly higherpercentage of epidermal LCs expressedIL-23p19 or coexpressed p40 and p19in psoriatic lesional and perilesionalbiopsies compared with LCs fromhealthy epidermis (P < 0.01, P < 0.05;Figure 1a). There was no significant

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difference in the expression of IL-12p40only (Figure 1a). These results suggestthat under inflammatory conditions, LCsare capable of producing proin-flammatory cytokines that promote Thelper type 17 cell development.

Since monocyte-derived LCs (moLCs)play an important role in repopulatingthe epidermis during inflammation-induced migration (Ginhoux et al.,2006), we next examined IL-23 pro-duction by moLCs. LC differentiationwas confirmed by examining theexpression of CD1a, E-cadherin, and

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CD207 (see Supplementary Figure S1online). moLCs from psoriasis patientswere found to express significantlyhigher levels of IL-23 upon zymosanstimulation compared with moLCs fromhealthy controls (P < 0.01; Figure 1b).Furthermore, although zymosan in-duced a small but significant expressionof IL-23 by epidermal LCs from healthycontrols (P< 0.05; Figure 1c), it inducedsignificantly more IL-23 by epidermalLCs from psoriasis patients, suggestingthat factors associated with the inflam-matory microenvironment of psoriasis

Figure 2. Human b-defensin 3 (HBD3) is enhanced in psoriasis and its ortholog mouse b-defensin 14 (MBD14) exacerbates psoriasis-like inflammation

in mice, which is associated with an increase in IL-23p19 expression by Langerhans cells (LCs). (a) HBD3 mRNA and (b) protein expression was determined

in the (a) skin (n ¼ 8) and (b) serum (n ¼ 25) of psoriasis patients before and after (n ¼ 6e7) treatment and from control skin (HC; n ¼ 3) and serum (HC; n ¼ 13)

by reverse transcriptase-PCR and ELISA, respectively. (c) Psoriasis-like skin disease was induced in C57BL/6 mice with topical application of Aldara cream;

intradermal administration of MBD14 (1 mg in phosphate buffered saline) and topical application of Aldara; or intradermal administration of MBD14 (1 mgin phosphate buffered saline) only for 7 days. Matched control ears received vehicle treatment. (c) Ear thickness was measured using a thickness gauge (Hitec)

before initiation of the experiment on day 0 and every day for 7 days. Clinical thickness of control (vehicle) and treated ears are shown as mean score � standard

error of the mean (n ¼ 5e6). (d, e) Ears from mice were removed and frozen in optimal cutting temperature compound. (d) Psoriasis severity and (e) expression

of IL-23p19 (red) by LCs (green) counterstained with 40,6-diamidino-2-phenylindole (DAPI; blue) were determined using hematoxylin and eosin staining and

three-color immunofluorescence, respectively. Bar ¼ 100 mm. (d) Representative control image displayed. (e) High magnification inset (magnification �60) from

displayed images are depicted. Magnification �40 also depicted for MBD14&Aldara group (bottom inset). (f) Percentage of IL-23þ LCs shown as mean score �standard error of the mean. n ¼ 5e6, *P < 0.05, **P < 0.01, ***P < 0.001.

CM Sweeney et al.b-Defensins-3 Activate LCs and Exacerbate Psoriasis

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Supplementary material is linked to the onlineversion of the paper at www.jidonline.org, and athttp://dx.doi.org/10.1016/j.jid.2015.12.011.

CM Sweeney et al.b-Defensins-3 Activate LCs and Exacerbate Psoriasis

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cooperate with zymosan to promote IL-23 expression. We next sought to deter-mine the mechanism driving IL-23expression by LCs in psoriasis. Antimi-crobial peptides in complex with self-nucleotides have been implicated inthe pathogenesis of psoriasis throughthe production of proinflammatory cy-tokines by plasmacytoid (Lande et al.,2007) and myeloid DC (Ganguly et al.,2009). Human b-defensin 3 (HBD3), asmall antimicrobial peptide, is chemo-tactic for immune cells and is enhancedin psoriasis (Lande et al., 2015). HBD3in complexwith self-DNA (Tewary et al.,2013) was shown to induce IFN-a pro-duction by plasmacytoid DC (Tewaryet al., 2013) and in cooperation withother antimicrobial peptides can breaktolerance to self-DNA (Lande et al.,2015). Other studies have shown thatHBD3 alone enhances the expression ofcostimulatory molecules on LCs (Ferriset al., 2013), suggesting that HBD3may modulate antigen-presenting cellsin psoriasis. Given its effects on LCs, weexamined whether HBD3 plays a role inthe dysregulation of LCs in psoriasis.HBD3 induced IL-23 and enhancedzymosan-induced IL-23 production byhealthy moLCs (see SupplementaryFigure S2 online). Moreover, HBD3alone induced the production of IL-23by epidermal LCs from psoriasis pa-tients and healthy controls (P < 0.01;Figure 1d). However, HBD3-inducedIL-23 production was significantlyincreased in psoriasis patients comparedwith healthy controls (P < 0.01;Figure 1e), suggesting that increased IL-23 in psoriasis is a result of HBD3 andthe psoriatic environment (possiblyendogenous HBD3). These results indi-cate that HBD3 may play a pathogenicrole in psoriasis.

We next examined the expression ofHBD3 in psoriasis. The mRNA expres-sion of HBD3 was increased in psoria-sis skin during active disease anddecreased upon clearance of psoriasiswith UVB treatment to levels compa-rable with that of healthy controls(P < 0.05; Figure 2a). Similar resultswere obtained in the serum (P < 0.01;P < 0.05, Figure 2b). To confirm thepathogenic role of HBD3 in psoriasis,we next examined the effect of HBD3in a murine model of disease. Psoriasis-like skin inflammation was inducedin C57BL/6 mice with imiquimod

Journal of Investigative Dermatology (2016), Volum

formulated in a commercially availablecream (Aldara, MEDA Pharmaceuticals,Dublin, Ireland); Aldara and murine b-defensin 14 (MBD14), a murine ortho-log of HBD3 (Hinrichsen et al., 2008);or MBD14 only for 7 days. As expected,application of Aldara cream resultedin psoriasis-like skin inflammationas measured by increased epidermalthickness (Figure 2c), acanthosis,desquamation, parakeratosis, and infil-tration (Figure 2d). However, MBD14exacerbated epidermal ear thicknessand psoriasis-like skin inflammation(P < 0.01, P < 0.001; Figure 2c and d),and MBD14 alone induced a mild dis-ease (Figure 2c and d). To investigatethe role of LCs in this model,we examined IL-23p19 expression byLCs by immunofluorescence (seeSupplementary Materials). IL-23p19was strongly expressed in ears thatreceived Aldara or Aldara plus MBD14(Figure 2e) but was absent in controlskin (Figure 2e). From 25% to 35% ofLCs expressed IL-23p19 in epidermisfrom inflamed skin (Figure 2f) inducedwith Aldara (mean 25.64%) or Aldaraplus MBD14 (mean 35.4%). In accor-dance with a milder clinical pheno-type, 15% of LCs expressed IL-23p19 inepidermis from mice treated withMBD14 only (Figure 2f). These resultssuggest that MBD14 exacerbatespsoriasis-like skin inflammation andalone induces a mild disease in mice,which is associated with an increasedexpression of IL-23p19 by LCs. Thisstrongly suggests that HBD3 plays apathogenic role in psoriasis by pro-moting the expression of IL-23 by LCsand that LCs likely act as sources of IL-23 in disease. Recent studieshave demonstrated that conventionallangerin-negative DC produce IL-23 toinduce psoriasis-like inflammation inmice and suggest that LCs are dispens-able for the induction of disease (Wohnet al., 2013). In contrast, it was shownthat LCs are a major DC source of IL-23during psoriasis-like inflammation andare essential for disease induction(Yoshiki et al., 2014). It is worth notingthat each of these studies differed intheir precise methods of disease in-duction or LC ablation but establishedIL-23 as a key player in drivingpsoriasis-like inflammation. Although itis unclear whether LCs play a role in theinduction of psoriasis-like inflammation

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in mice, our results demonstrate thatLCs are capable of producing IL-23 indisease. Thus, it may be that althoughLCs are not necessary for the initial in-duction of disease, activation of LCs byHBD3 sustains and amplifies an estab-lished inflammatory response. Regard-less, our study clearly demonstrates thatHBD3 drives the production of IL-23 byLCs in psoriasis, which contributes tothe pathogenesis of disease.

CONFLICT OF INTERESTThe authors state no conflict of interest.

ACKNOWLEDGMENTSCS is funded by the Health Research Award fromthe Health Research Board of Ireland.

Cheryl M. Sweeney1,*, Shane E. Russell2,Anna Malara1, Genevieve Kelly1,Rosalind Hughes1, Anne-Marie Tobin1,Karoline Adamzik1, Patrick T. Walsh2

and Brian Kirby1

1School of Medicine and Medical Sciences,University College Dublin, Education andResearch Centre, St. Vincent’s UniversityHospital, Dublin, Ireland; and 2School ofMedicine, Trinity College Dublin, NationalChildren’s Research Centre, Our Lady’sChildren’s Hospital, Crumlin, Dublin, Ireland*Corresponding author e-mail: Cheryl.sweeney@ucd.ie

REFERENCES

Cumberbatch M, Singh M, Dearman RJ,Young HS, Kimber I, Griffiths CE. ImpairedLangerhans cell migration in psoriasis. J ExpMed 2006;203:953e60.

Di Cesare A, Di Meglio P, Nestle FO. The IL-23/Th17 axis in the immunopathogenesis of pso-riasis. J Invest Dermatol 2009;129:1339e50.

Ferris LK, Mburu YK, Mathers AR, Fluharty ER,Larregina AT, Ferris RL, et al. Human beta-defensin 3 induces maturation of human Lang-erhans cell-like dendritic cells: an antimicrobialpeptide that functions as an endogenous adju-vant. J Invest Dermatol 2013;133:460e8.

Ganguly D, Chamilos G, Lande R, Gregorio J,Meller S, Facchinetti V, et al. Self-RNA-antimi-crobial peptide complexes activate humandendritic cells through TLR7 and TLR8. J ExpMed 2009;206:1983e94.

Ginhoux F, Tacke F, Angeli V, Bogunovic M,Loubeau M, Dai XM, et al. Langerhans cellsarise from monocytes in vivo. Nat Immunol2006;7:265e73.

Glitzner E, Korosec A, Brunner PM, Drobits B,Amberg N, Schonthaler HB, et al. Specific rolesfor dendritic cell subsets during initiation andprogression of psoriasis. EMBO Mol Med2014;6:1312e27.

H-j Kim et al.Effects of Visible Light on Innate Immunity

Hinrichsen K, Podschun R, Schubert S,Schroder JM, Harder J, Proksch E. Mouse beta-defensin-14, an antimicrobial ortholog ofhuman beta-defensin-3. Antimicrob AgentsChemother 2008;52:1876e9.

Igyarto BZ, Haley K, Ortner D, Bobr A, Gerami-Nejad M, Edelson BT, et al. Skin-resident mu-rine dendritic cell subsets promote distinct andopposing antigen-specific T helper cell re-sponses. Immunity 2011;35:260e72.

Lande R, Chamilos G, Ganguly D, Demaria O,Frasca L, Durr S, et al. Cationic antimicrobialpeptides in psoriatic skin cooperate to breakinnate tolerance to self-DNA. Eur J Immunol2015;45:203e13.

Lande R, Gregorio J, Facchinetti V, Chatterjee B,Wang YH, Homey B, et al. Plasmacytoiddendritic cells sense self-DNA coupled withantimicrobial peptide. Nature 2007;449:564e9.

Abbreviations: AMPs, antimicrobial peptides; HBD,epithelial keratinocytes; poly I:C, polyinosinic-polycyreceptors; UVA, ultraviolet A

Accepted manuscript published online 11 December2016

Nair RP, Duffin KC, Helms C, Ding J, Stuart PE,Goldgar D, et al. Genome-wide scan revealsassociation of psoriasis with IL-23 and NF-kappaB pathways. Nat Genet 2009;41:199e204.

Seneschal J, Clark RA, Gehad A, Baecher-Allan CM, Kupper TS. Human epidermalLangerhans cells maintain immune homeostasisin skin by activating skin resident regulatoryT cells. Immunity 2012;36:873e84.

Tewary P, de la Rosa G, Sharma N,Rodriguez LG, Tarasov SG, Howard OM,et al. beta-Defensin 2 and 3 promote theuptake of self or CpG DNA, enhance IFN-alpha production by human plasmacytoiddendritic cells, and promote inflammation.J Immunol 2013;191:865e74.

Wohn C, Ober-Blobaum JL, Haak S,Pantelyushin S, Cheong C, Zahner SP, et al.Langerin(neg) conventional dendritic cells

human beta-defensins; NHEKs, normal humantidylic acid; SNO, S-nitrosylated; TLRs, Toll-like

2015; corrected proof published online 13 January

produce IL-23 to drive psoriatic plaque forma-tion in mice. Proc Natl Acad Sci U S A2013;110:10723e8.

Yoshiki R, Kabashima K, Honda T, Nakamizo S,Sawada Y, Sugita K, et al. IL-23 from Lang-erhans cells is required for the developmentof imiquimod-induced psoriasis-like derma-titis by induction of IL-17A-producing gam-madelta T cells. J Invest Dermatol 2014;134:1912e21.

This work is licensedunder a Creative Commons

Attribution-NonCommercial-NoDer-ivatives 4.0 International License.To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/4.0/

Short Wavelength Visible Light SuppressesInnate Immunity-Related Responsesby Modulating Protein S-Nitrosylationin Keratinocytes

Journal of Investigative Dermatology (2016) 136, 727e731; doi:10.1016/j.jid.2015.12.004

TO THE EDITORThe solar radiation spectrum reachingthe earth’s surface consists mostlyof ultraviolet A (UVA), visible, andinfrared light. UVA irradiation has clin-ical applications for patients requiringlocal immune-suppression therapy(Weatherhead et al., 2012). However,UV radiation is carcinogenic and is notrecommended for long-term treatment(Kunisada et al., 2007). Thus, visiblelight-based therapies with less harmfuleffects on human skin are desirable. Forexample, blue light (400e450 nm) irra-diation is used therapeutically to treatsevere atopic dermatitis (Becker et al.,2011), 632.8-nm light enhances cellproliferation, and red light (550e670nm) accelerates epidermal permeabilitybarrier recovery after disruption (Dendaand Fuziwara, 2008; Hu et al., 2007).However, the mechanisms underlyingvarious effects of visible light are notclear.

Human skin exhibits innate immuneresponses, such as epithelial defensevia antimicrobial peptides (AMPs), andthe release of proinflammatory cyto-kines involved in the recognition ofmicrobes via toll-like receptors (TLRs)(Gallo and Nakatsuji, 2011; Meyeret al., 2007). Thus, we investigated theeffects of visible light on innate immu-nity. The survival rate of normal humanepithelial keratinocytes (NHEKs) wasnot affected by visible light irradiation(Supplementary Figure S1 online). Vio-let or blue light downregulated themRNA expression levels of AMPs afterone or three exposures (Figure 1a, band Supplementary Figure S2a online).By contrast, UV irradiation upregulatedAMPs, except for LL-37 (SupplementaryFigure S2b). The expression levelsof AMPs (human beta-defensins (HBD-1, -3)) and proinflammatory cytokines(RANTES, MCP-1, and IL-8) decreasedafter violet light irradiation in 3D skin

(Supplementary Figure S2c). This violetlight-induced downregulation of AMPsaffected bacterial survival. Bacteriagrew better in a violet light-irradiatedNHEK-conditioned medium thanin a control NHEK-conditioned me-dium, irrespective of polyinosinic-polycytidylic acid (poly I:C), whichamplifies innate immune responses(Supplementary Figure S3 online).

We examined whether violet light in-fluences TLR ligand-induced responses.Poly I:C, but not Pam3, lipopolysaccha-ride, or CpG, increased HBD-1, -2, and-3 simultaneously, which was signifi-cantly decreased by violet light.Flagellin-induced increases in HBD-2and S100A7were reduced by violet light(Figure 1c). Poly I:C-induced increasesin proinflammatory cytokines weredecreased by violet light in NHEKs and3D models (Supplementary Figure S4aonline). Unlike violet light, red lightdid not affect the poly I:C-inducedaugmentation of AMPs and proin-flammatory cytokines (SupplementaryFigure S4b).

When TLRs are activated via ligands,the NF-kB signaling cascade is activatedand proinflammatory cytokines are

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