Accepted Article Preview: Published ahead of advance online publication Toll-Like Receptor 3 Increases Allergic and Irritant Contact Dermatitis Naomi Nakamura, Risa Tamagawa-Mineoka, Mayumi Ueta, Shigeru Kinoshita, Norito Katoh

Cite this article as: Naomi Nakamura, Risa Tamagawa-Mineoka, Mayumi Ueta, Shigeru Kinoshita, Norito Katoh, Toll-Like Receptor 3 Increases Allergic and Irritant Contact Dermatitis, Journal of Investigative Dermatology accepted article preview 17 September 2014; doi: 10.1038/jid.2014.402. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

Received 10 August 2013; revised 30 July 2014; accepted 18 August 2014; Accepted article preview online 17 September 2014

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Toll-like Receptor 3 Increases Allergic and Irritant Contact Dermatitis

Naomi Nakamura,1* Risa Tamagawa-Mineoka,1* Mayumi Ueta,2 Shigeru Kinoshita2 and Norito Katoh1

Departments of 1Dermatology and 2Ophthalmology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan *These authors contributed equally to this work.

Correspondence: Risa Tamagawa-Mineoka, Department of Dermatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan. Tel. & Fax: +81-75-251-5586 E-mail: [email protected]

Short title: The role of TLR3 in cutaneous inflammation

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Abbreviations used ACD: Allergic contact dermatitis CFSE: Carboxyfluorescein succinimidyl ester CHS: Contact hypersensitivity FITC: Fluorescein isothiocyanate ICD: Irritant contact dermatitis IL: Interleukin IP-10: Interferon- -inducible protein 10 KC: Keratinocyte chemoattractant PolyI:C: Polyinosinic:polycytidylic acid RANTES: Regulated on activation, normally T-cell expressed and secreted RT-PCR: Real-time polymerase chain reaction Tg: Transgenic TLR: Toll-like receptor Tlr3 KO: Toll-like receptor 3 knockout TNCB: 2,4,6-Trinitro-1-chlorobenzene TNF: Tumor necrosis factor

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ABSTRACT There is increasing recognition of the role of Toll-like receptor (TLR)-3 in non-infectious inflammatory diseases, but the function of TLR3 in inflammatory skin diseases is unclear. We investigated the functions of TLR3 in allergic and irritant contact dermatitis. The contact hypersensitivity (CHS) response was lower in Toll-like receptor 3 knockout (Tlr3 KO) mice, and was greater in TLR3 transgenic mice, than in wild-type mice after challenge with 2,4,6-trinitro-1-chlorobenzene (TNCB). Adoptive transfer of immunized lymph node cells from Tlr3 KO mice induced CHS in wild-type recipients. In contrast, adoptive transfer of those from wild-type mice did not fully induce CHS in Tlr3 KO recipients. The irritant contact dermatitis reaction following croton oil application was lower in Tlr3 KO mice, and was greater in TLR3 transgenic mice, than in wild-type mice. Maturation, migration and antigen presentation of dendritic cells and proliferation of lymphocytes between wild-type mice and Tlr3 KO mice were comparable. These results show that TLR3 enhances antigen-independent skin inflammation in the elicitation phase of allergic contact dermatitis and in irritant contact dermatitis.

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INTRODUCTION The innate immune system is the first line of defense against pathogens. This system recognizes pathogens based on pathogen-associated molecular patterns (Ishii and Akira, 2008; Kawai and Akira, 2011). The mammalian pattern recognition receptors responsible for this process are known as Toll-like receptors (TLRs). The TLR family consists of at least 11 members, and is expressed on immune cells such as macrophages, dendritic cells, granulocytes, natural killer cells, and T cells, and nonimmune cells such as fibroblasts and epithelial cells (Ishii and Akira, 2008; Kawai and Akira, 2011). Signaling through TLRs results in activation of nuclear factor- B, interferon-regulatory factor, and their target genes, and the coordinated activation of several transcription factors regulate expression of antimicrobial genes, cytokines, and costimulatory molecules (Ishii and Akira, 2008; Kawai and Akira, 2011). These innate immune responses then help to prime subsequent adaptive immune responses to the pathogens.

TLR3 recognizes viral double-stranded RNA and polyinosinic:polycytidylic acid (Poly(I:C)), a mimic of double-stranded RNA, leading to immune responses that are characterized by the production of type I interferons and proinflammatory cytokines (Ishii and Akira, 2008; Kawai and Akira, 2011). It has been shown that TLR3 plays

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roles not only in viral infection but also in non-infectionus inflammatory responses in allergic disorders (Fransson et al., 2005; Prescott et al., 2008; Ueta et al., 2009; Nuolivirta et al. 2012) and skin injury (Lai Y et al., 2009). We have found that TLR3 enhances eosinophil infiltration in experimental allergic conjunctivitis (Ueta et al., 2009). Nuolivirta et al. (2012) have shown that virus-induced asthma exacerbations are associated with TLR expression, especially that of TLR3. Furthermore, infants with allergic diseases have increased perinatal TLR3 responses (Prescott et al., 2008). Upregulation of TLRs 2, 3 and 4 has also been found in allergic rhinitis (Fransson et al., 2005). These findings imply that TLR3 plays important roles in enhancement of allergic inflammation.

Allergic contact dermatitis (ACD) is a common allergic inflammatory skin disease characterized by pruritic eczematous lesions. Recent studies have shown that TLRs are involved in the pathomechanism of ACD. TLR2 promotes a TH1 response by inducing interferon- production from dendritic cells in contact hypersensitivity (Jin et al., 2009). TLR4 plays a crucial role in the development of contact hypersensitivity (CHS) via immune cells including dendritic cells and natural killer T cells (Askenase et al., 2005; Martin et al., 2008; Yokoi et al., 2009). Epicutaneous immunization with protein

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antigen induces suppression of subsequent CHS, but epicutaneous application of protein antigen with TLR2, TLR3, TLR4, or TLR9 ligands reverses inhibition of CHS (Ptak et al., 2006; Majewska et al., 2009). Interestingly, recent reports suggest that hapten sensitization can produce endogenous TLR ligands and activate innate immune responses, leading to skin inflammation. For example, Ni2+ induces CHS by directly activating human TLR4 (Schmidt et al., 2010; Martin et al., 2011), while 2,4,6-trinitro-1-chlorobenzene (TNCB) and other organic contact allergens activate TLR2 and TLR4 via degradation of hyaluronic acid (Termeer et al., 2002; Scheibner et al., 2006; Martin et al., 2008; Martin et al., 2008; Freudenberg et al., 2009; Martin et al., 2011). These findings suggest that TLRs are deeply involved in the pathogenesis of ACD. However, the function of TLR3 in skin inflammation including ACD remains to be defined.

In the current study, we investigated the function of TLR3 in inflammatory skin diseases. We first examined whether TLR3 signaling affects CHS using Toll-like receptor 3 knockout (Tlr3 KO) and transgenic (Tg) mice. We then investigated whether TLR3 is involved in the sensitization and elicitation periods, assessed the role of TLR3 in non-antigen-specific responses in a mouse model of irritant contact dermatitis (ICD),

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and evaluated possible roles of TLR3 signaling in cells that are important in inflammatory skin diseases.

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RESULTS Inhibition of the CHS response in Tlr3 KO mice We investigated involvement of TLR3 in the CHS reaction by measuring ear thickness after hapten challenge in wild-type (WT), Tlr3 KO, and TLR3 Tg mice. TNCB was applied to the abdominal skin a week prior to ear application. We previously confirmed that TLR3 mRNA expression was greater in TLR3 Tg mice than in WT mice and was undetectable in Tlr3 KO mice (Ueta et al., 2009). Interestingly, the degree of ear-swelling in Tlr3 KO mice was significantly lower than that in WT mice at 24 hours (Figure 1a) and 48 hours (Supplementary Figure S1a online) after TNCB challenge, while the ear-swelling response in TLR3 Tg mice was greater than that in WT mice (Figure 1a and Supplementary Figure S1a online). Histology of the ear skin at 24 hours (Figure 1b) and 48 hours (Supplementary Figure S1b online) after TNCB challenge showed extensive leukocyte infiltration and edema in the dermis of WT mice. Such reactions were substantially lower in the ear skin of Tlr3 KO mice than in WT mice. In contrast, greater leukocyte infiltration and edema were observed in TLR3 Tg mice after TNCB challenge than in WT mice (Figure 1b and Supplementary Figure S1b online). Fewer inflammatory cells were observed in Tlr3 KO mice after TNCB challenge than in control mice (Figure 1c and Supplementary Figure S1c online), whereas more

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inflammatory cells were observed in TLR3 Tg mice than in WT mice (Figure 1c and Supplementary Figure S1c online). To examine the irritant effects of TNCB, TNCB was topically applied on the ear skin of non-sensitized mice. The results showed almost no inflammation in the skin (Supplementary Figure S2 online), indicating that TNCB has no strong effect on the skin as an irritant. These findings suggest that TLR3 signaling is involved in the CHS response.

No requirement for TLR3 in the sensitization phase of the CHS response The CHS response consists of two phases: sensitization and elicitation. To identify the action phase of TLR3, we first examined the role of TLR3 in the sensitization period using an adoptive transfer-induced CHS model. Lymph node cells were isolated from regional lymph nodes of TNCB-sensitized Tlr3 KO or WT mice, and adoptively transferred into naïve WT mice or immunodeficient NOD/ShiJic-scid JCl mice. Immediately after transfer, recipient mice were challenged with TNCB on the ears. The degrees of ear-swelling (Figure 2a and Supplementary Figure S3a online) and the numbers of inflammatory cells (Figure 2b and Supplementary Figure S3b online) did not differ significantly between WT mice and Tlr3 KO mice as the donors of lymph node cells. These results show that the sensitization phase was not the action phase of

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TLR3.

Requirement for TLR3 in the elicitation phase of the CHS response We next assessed the possible role of TLR3 in the elicitation period of CHS. When lymph node cells from sensitized WT mice were transferred to naïve Tlr3 KO or WT mice, the Tlr3 KO recipient mice showed significantly lower ear-swelling than the WT recipients (Figure 2a). The number of inflammatory cells in Tlr3 KO mice was also lower than that in WT mice (Figure 2b). These results show that TLR3 exerts its function during the elicitation phase of the CHS reaction.

Inhibition of irritation response in Tlr3 KO mice To investigate whether involvement of TLR3 depends on an antigen-specific response, we examined the cutaneous response after application of the irritant croton oil in WT, Tlr3 KO, and TLR3 Tg mice. In contrast to ACD, ICD evolves as a consequence of direct toxic effects of chemical agents including croton oil and shows non-antigen-specific responses (Corsini and Galli, 1998). Ear thickness was determined at 6 hours after topical application of croton oil. The degree of ear-swelling in Tlr3 KO mice was lower than that in WT mice, while the ear-swelling response was greater in

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TLR3 Tg mice than in WT mice (Figure 3a). Histology of the ear skin at 6 hours after croton oil application showed extensive leukocyte infiltration and edema in the dermis of WT mice. The degree of infiltration in the ear skin of Tlr3 KO mice was significantly lower than that in Tlr3 KO mice. In contrast, greater leukocyte infiltration and edema were observed in TLR3 Tg mice than in Tlr3 KO mice (Figure 3b). The number of inflammatory cells in Tlr3 KO mice was also lower than that in WT mice (Figure 3c), whereas more inflammatory cells were observed in TLR3 Tg mice than in WT mice (Figure 3c). These results suggest that TLR3 is involved in ICD as well as in ACD.

Unimpaired function of dendritic cells and lymphocytes Dendritic cells and lymphocytes play a protagonistic role in the pathogenesis of ACD (Sebastiani et al., 2002). To examine the possible role of TLR3 in cutaneous dendritic cells in inflammation, we examined the migration and maturation of these cells after application of fluorescein isothiocyanate (FITC) on the shaved abdominal skin of Tlr3 KO and WT mice. The numbers of FITC+ CD11c+, FITC+ CD86+ and FITC+ MHC class II+ cutaneous dendritic cells accumulating in the lymph nodes at 24 hours after FITC application in Tlr3 KO mice did not differ significantly from those in WT mice (Figure 4a).

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The lymphocyte stimulatory capacity of dendritic cells was evaluated in a carboxyfluorescein succinimidyl ester (CFSE)-staining assay. Lymph node cells of C57BL/6 mice were incubated with allogeneic BALB/c WT or Tlr3 KO splenic CD11c+ dendritic cells, and the fluorescence intensity of CFSE-labeled lymph node cells was analyzed by flow cytometry. The results showed no difference in the lymph node cell proliferation rate between WT and Tlr3 KO mice (Figure 4b). These results indicate that TLR3 did not affect migration, maturation and antigen presentation of dendritic cells.

Proliferation of lymphocytes in Tlr3 KO and WT mice was also evaluated in a CFSE-staining assay. Lymph node cells of BALB/c WT or Tlr3 KO mice were incubated with allogeneic C57BL/6 splenic CD11c+ dendritic cells and the fluorescence intensity of CFSE-labeled lymph node cells were analyzed by flow cytometry. There was no difference in the lymph node cell proliferation rate between WT and Tlr3 KO mice (Figure 4c). These results indicate that TLR3 expression did not affect the lymphocyte proliferation function.

Cytokine and chemokine expression in the elicitation phase of the CHS response

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Cytokines and chemokines play important roles in the elicitation of ACD and ICD. Therefore, we examined the expressions of the cytokines and chemokines, that are important for the development of ACD and ICD, in cutaneous tissue of TNCB-induced CHS. Our results show that the expressions of interleukin (IL)-1α, IL-1 , Tumor necrosis factor (TNF)-α, interferon- -inducible protein 10 (IP-10), and regulated on activation, normally T-cell expressed and secreted (RANTES) tended to be elevated in the whole tissue of TNCB-treated ears in WT mice compared with that in Tlr3 KO mice, although the differences of IL-1

TNF-α, and RANTES were not statistically

significant (Figure 5).

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DISCUSSION In this study, the CHS response was inhibited in Tlr3 KO mice and enhanced in TLR3 Tg mice. The elicitation phase was the major action phase of TLR3 in the CHS based on the results of adoptive transfer experiments of lymph node cells from TNCB-sensitized animals. The ICD response was also suppressed in Tlr3 KO mice and increased in TLR3 Tg mice. Our results suggest that TLR3 plays an important role in enhancement of skin inflammation in the elicitation phase of ACD and in ICD.

In the sensitization phase of ACD, cutaneous dendritic cells ingest hapten that has penetrated into the skin, migrate to regional lymph nodes, and then present the antigens to naïve T cells. This process leads to the clonal expansion of antigen-specific T cells that can be recruited to the skin. Recent studies have shown that the activation of TLRs through the recognition of pathogens or their products can induce maturation and migration of dendritic cells and antigen-specific T cells expansion (Akira et al., 2001). TLR2 and TLR4 signaling is important for dendritic cell function and generation of effector T cells in the sensitization phase of the CHS response (Martin et al., 2008; Yokoi et al., 2009). Because dendritic cells and lymphocytes express TLR3 (Applequist et al., 2002), we expected possible involvement of these cells in TLR3-dependent

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mechanisms of the CHS response. Therefore, we investigated whether TLR3 signaling affects the sensitization phase of the CHS response and the functions of dendritic cells and lymphocytes. Our results suggest that the sensitization phase is not strongly related with TLR3 signaling based on the results of adoptive transfer experiments of lymph node cells from TNCB-sensitized mice. Furthermore, we found that the migration of cutaneous dendritic cells to draining lymph nodes did not decrease in Tlr3 KO mice compared with that in WT mice. Lack of TLR3 expression also did not affect maturation and antigen presentation of dendritic cells, and lymphocyte proliferation induced by stimulation of antigen was not changed in Tlr3 KO mice. These findings suggest that TLR3 in dendritic cells and lymphocytes may not be closely associated with the development of ACD and ICD.

The inflammatory responses in the elicitation phase of the CHS depend on both antigen-dependent and -independent mechanisms (Grabbe and Schwarz, 1998; Honda et al., 2013). Upon reexposure to haptens, antigen-specific T cells are activated by cutaneous antigen-presenting cells, produce numerous mediators, and induce activation of several cell types in the skin. Haptens also directly activate keratinocytes, in an antigen-nonspecific manner (Sebastiani et al., 2002; Honda et al., 2013). These

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reactions also affect each other, leading to the exacerbation of inflammation. In this study, we indicate that TLR3 is associated with development of the elicitation phase of ACD and ICD. In addition, our results show no significant difference of the function of dendritic cells and lymphocytes between Tlr3 KO and WT mice. Taken together, these findings suggest that TLR3 may be involved in antigen-independent cutaneous responses in the elicitation phase of ACD and ICD.

Cytokines and chemokines are important mediators in the elicitation phase of ACD and ICD. TNF-α, IL-1α and IL-1β play a crucial role in eliciting CHS and ICD (Corsini et al., 1998; Watanabe et al., 2007). With regard to chemokines, deficiency of IP-10 exhibits a reduced CHS response (Dufour et al., 2002). Blocking the receptor against RANTES decreases inflammatory responses in ACD and ICD by inhibiting leukocyte recruitment (Canavese et al., 2010). Administration of antibodies against keratinocyte chemoattractant (KC) suppresses the CHS response via inhibition of neutrophil recruitment (Dilulio et al., 1999). Our results show that the expressions of IL-1α, IL-1 , TNF-α, IP-10, and RANTES tended to be elevated in the whole tissue of TNCB-treated ears in WT mice compared with that in Tlr3 KO mice, although the differences of IL-1

TNF-α, and RANTES were not statistically significant.

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Resident skin cells, such as keratinocytes, fibroblasts, and mast cells, play important roles in recruitment of leukocytes to inflamed skin via release of cytokines and chemokines. Previous studies have shown that cytokines and chemokines that are important for inflammatory responses are secreted from epithelial cells and fibroblasts by stimulation with poly(I:C) (Ueta et al., 2005; Kato et al., 2007; Ueta et al., 2009; Farina et al., 2010; Ueta et al., 2010).

Therefore, the production of IL-1α, IL-1 ,

TNF-α, IP-10 and RANTES, that tended to be elevated in the whole tissue of TNCB-induced dermatitis in WT mice compared with that in Tlr3 KO mice, was examined in poly(I:C)-stimulated primary cultures of keratinocytes and dermal fibroblasts from WT mice and Tlr3 KO mice. Quantitative real-time polymerase chain reaction (RT-PCR) detected TLR3 in primary keratinocytes from WT mice, but little TLR3 in those from Tlr3 KO mice (data not shown). The production of IP-10 and RANTES was significantly increased in primary keratinocytes and fibroblasts from WT mice after poly(I:C) stimulation, but not in those from Tlr3 KO mice (Supplementary Figure S4 and S5 online). In contrast, the expressions of IL-1α, IL-1 , and TNF-α mRNA were not increased in primary keratinocytes and fibroblasts from WT mice after poly(I:C) stimulation (data not shown). These findings imply that keratinocytes and

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fibroblasts might release mediators such as IP-10 and RANTES via TLR3 signaling, leading to an increased CHS response.

Mast cells and macrophages also express TLR3 (Applequist et al., 2002; Orinska et al., 2005) and contribute to the pathogenesis of inflammatory skin diseases including CHS via production of various inflammatory mediators (Sebastiani et al., 2002; Tuckermann et al., 2007). However, our results showed no difference in degranulation of bone marrow-derived mast cells between WT and Tlr3 KO mice (data not shown). In addition, the defect of TLR3 did not affect the phagocytic function and lymphocyte stimulatory capacity of macrophages (data not shown). Therefore, TLR3 in mast cells and macrophages may not mainly be related to inflammatory responses.

Our results showed that TLR3 affected skin inflammation in the absence of an exogenous viral pathogen. TLRs recognize endogenous danger signals associated with tissue damage (Seong and Matzinge, 2004; Tsan and Gao, 2004), and recent studies have suggested that RNA released from necrotic cells such as damaged keratinocytes (Lai et al., 2009; Zhang et al., 2011; Bernard et al., 2012) and neutrophils (Cavassani et al., 2008) might serve as a ligand for TLR3 in various cell types, including

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keratinocytes (Karikó et al., 2004; Cavassani et al., 2008; Lai et al., 2009; Zhang et al., 2011; Bernard et al., 2012). Bernard et al. (2012) demonstrated that UV-damaged self RNAs or UV-damaged keratinocytes are detected by TLR3. Furthermore, UVB irradiation increases cytokine production in the skin tissue and affects the CHS response in a TLR3-dependent manner. Previous electron microscopic studies demonstrated that necrosis of epidermal keratinocytes and Langerhans cells is seen in ACD and ICD (Willis et al., 1986; Willis et al., 1989). Therefore, RNA released from cells damaged by an inflammatory stimulus may activate TLR3 in keratinocytes and dermal fibroblast in ACD and ICD. However, the endogenous TLR3 ligands are unknown in the mouse models of ACD and ICD. We are currently studying the mechanisms of cutaneous inflammatory responses regulated by endogenous TLR3 ligands.

In conclusion, our data show that TLR3 enhances antigen-independent skin inflammation and imply the possible involvement of mediators released from resident skin cells in development of skin inflammation via TLR3 signaling.

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MATERIALS AND METHODS Mice Male BALB/c and C57BL/6 mice and NOD/ShiJic-scid JCl mice were obtained from Shimizu Laboratory Supplies (Kyoto, Japan) and CLEA (Tokyo, Japan), respectively, and used at age 6 to 8 weeks. Male Tlr3 KO and TLR3 Tg mice were generated as previously described (Yamamoto et al., 2003; Negishi et al., 2008), back-crossed more than 7 generations to BALB/c mice (Ueta et al., 2009) and used at age 6 to 8 weeks. All mice were housed in a specific pathogen-free barrier facility and screened regularly for pathogens. This experimental procedure was approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine.

Induction of ACD with TNCB Mice were sensitized by application on the shaved abdominal skin of 25 μl of 1% (w/v) TNCB in acetone/olive oil (4:1) or 25 μl of vehicle alone. At 7 days after sensitization, 20 μl (10 μl on the dorsal side and 10 μl on the ventral side) of 1% TNCB was applied to the right ear. The same amount of acetone/olive oil (4:1) was administered to the left ear as a control. Ear thickness was measured at a predetermined site with a dial thickness gauge (Shinwa Rules, Niigata, Japan). The ear-swelling response was

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measured before elicitation and at 24 and 48 hours after elicitation and defined as the difference in ear thickness before and after elicitation.

Induction of ICD with croton oil Mice received topical application of 20 μl of 2% (v/v) croton oil (Sigma-Aldrich) in acetone/olive oil (4:1) to the dorsal and ventral sides of the right ear. The same amount of acetone/olive oil (4:1) was administered to the left ear as a control. The ear-swelling response was measured before and 6 hours after elicitation and defined as the difference in ear thickness before and after application.

Statistical Analysis All data are expressed as means ± SD. Statistical significance was assessed using one-way analysis of variance followed by a Tukey multiple comparison test. A Student t-test was used for comparison of mRNA expression in primary keratinocytes and fibroblasts. A P value of less than 0.05 was considered significant in all analyses.

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CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We thank Prof. Tadatsugu Taniguchi and Hideo Negishi for TLR3 Tg mice, Prof. Shizuo Akira for Tlr3 KO mice, and Tsunao Kishida and Osam Mazda for NOD/ShiJic-scid JCl mice. This work was supported in part by a grant from the Japanese Ministry of Education, Science, Sports, and Culture to R.T.M., M.U., and N.K. This work also was conducted as a part of the BioBank Japan Project supported by the Ministry of Education, Culture, Sports, Science and Technology of the Japanese government.

SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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Figure Legends Figure 1. Impaired CHS response in Tlr3 KO mice. (a) Change in ear-swelling responses of WT (n=12), Tlr3 KO (n=6) and TLR3 Tg (n=11) mice. (b) Histologic examination of ear skin. Hematoxylin and eosin. Original magnification ×100. Scale bar = 50 μm. (c) Numbers of cells infiltrated in the dermis. Data are expressed as the means ± SD. **P

Toll-like receptor 3 increases allergic and irritant contact dermatitis.

There is increasing recognition of the role of Toll-like receptor 3 (TLR3) in noninfectious inflammatory diseases, but the function of TLR3 in inflamm...
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