Abstract

TLR2 recognizes cell wall components of Staphylococcus aureus, which colonizes >90% of atopic eczematous skin lesions. The regulatory mechanisms of TLR2 signaling in the skin remain unclear. Allergin-1, an inhibitory immunoglobulin-like receptor containing an ITIM, is expressed on mast cells (MCs) and inhibits IgE-mediated anaphylaxis in mice. Here, we show that Allergin-1 inhibits TLR2-mediated activation of, and inflammatory cytokine production by, MCs in vitro. Compared with wild-type mice, Allergin-1-deficient mice showed enhanced ear swelling with enhanced collagen deposition and greater Ly6G+ neutrophil recruitment after intra-dermal injection of Pam2CSK4 into pinnae. Using Mas–TRECK mice, which is an MC deletion system based on il4 enhancer elements, we also demonstrated that Allergin-1 on MCs is responsible for the Pam2CSK4-induced ear swelling. These results suggest that Allergin-1 on skin MCs suppresses TLR2-induced dermatitis.

Introduction

Mast cells (MCs) play pivotal roles in allergic reactions in barrier tissues, including skin (1). MCs are activated through both IgE-dependent and IgE-independent pathways to express various TLRs, which recognize diverse components of pathogens and initiate host defense mechanisms (2). Among TLRs, TLR2 is the predominant receptor for Staphylococcus aureus, which colonizes >90% of atopic eczematous skin lesions (3). Although TLR2 ligand is known to activate MCs (4), the regulation of TLR2 signaling in MCs remains incompletely understood.

Allergin-1, an immunoglobulin-like receptor bearing an ITIM in its cytoplasmic region, is highly expressed on MCs. We previously reported that Allergin-1 recruits SHP-1 and SHP-2, which inhibits high-affinity receptors involved in IgE (FcεRI)-mediated signaling and subsequent MC activation, and thus suppresses IgE-mediated systemic and cutaneous anaphylaxis in mice (5). Because SHP-1 and SHP-2 down-regulate TLR-mediated production of pro-inflammatory cytokines (6, 7), we hypothesized that Allergin-1 might influence TLR2-mediated signaling. Here, we examined whether Allergin-1 suppresses Pam2CSK4-induced TLR2 signaling in the MCs of mice.

Methods

Mice

Wild-type (WT) C57BL/6N mice were purchased from Clea Japan (Tokyo, Japan). Allergin-1-deficient (Allergin-1−/−) mice were generated in our laboratory, as previously described (5). Mas–TRECK mice on a C57BL/6 background have been described previously (8). All experiments used female mice (age: 8–13 weeks) that were bred in the specific pathogen-free facilities at the University of Tsukuba, and all procedures followed the guidelines of the animal ethics committee of the University of Tsukuba.

Cells and qualitative PCR analysis

Bone marrow-derived cultured MCs (BMMCs) were prepared from mice as described previously (5) and stimulated with 1 µg ml−1 Pam2CSK4 (InvivoGen, San Diego, CA, USA). Total RNA was isolated from cell pellets (TRIzol; Thermo Fisher Scientific, Waltham, MA, USA) and used as a template for reverse transcription reactions (High-Capacity cDNA RT Kit; Applied Biosystems, Foster City, CA, USA). Real-time PCR analysis of Il6, Tnfsf2, Il1b and Gapdh (measured as an internal control for normalization of data) was performed by using an ABI7500 sequence detector (Applied Biosystems) and Power SYBR Green PCR Master Mix (Applied Biosystems). The primers were: Il6 (forward, 5′-gaggataccactcccaacagacc-3′; reverse 5′-aagtgcatcatcgttgttcataca-3′), Tnfsf2 (5′-gggccaccacgctcttc-3′; 5’-ggtctgggccatagaactgatg-3′), Il1b (5′-actcaactgtgaaatgccacc-3′; 5′-tgatactgcctgcctgaagc-3′) and Gapdh (5′-tggtgaaggtcggtgtgaac-3′; 5′-atgaaggggtcgttgatggc-3′). mRNA transcript levels were determined relative to that in the unstimulated WT BMMCs. All experiments were performed in triplicate.

Antibodies

Anti-mouse Allergin-1 (TX83; mouse IgG1) was described previously (5). TX83 was biotinylated with Biotinylation Kit (Sulfo-OSu; DOJIN, Tokyo, Japan). mAbs to mouse c-Kit (ACK45), Ly6C (AL-21), CD11c (HL3), Siglec-F (E50-2440) and CD16/32 (2.4G2) were purchased from BD Biosciences (San Jose, CA, USA). Anti-mouse CD45.2 (104), FcεRIα (MAR1) and CD11b (M1/70) were purchased from eBioscience (San Diego, CA, USA). Anti-mouse Ly6G (1A8) was purchased from TONBO Biosciences (San Diego, CA, USA). Anti-Gr-1 (RB6-8C5) hybridoma provided by Dr Kazumasa Ogasawara (Shiga University of Medical Science) was used for neutrophil depletion.

Cytometric bead array analysis

The concentrations of various inflammatory cytokines and chemokines were measured by using cytometric bead array analysis (BD Biosciences) according to the manufacturer’s instructions.

Pam2CSK4-induced dermatitis model

The TLR2 ligand-induced dermatitis model was reported previously (9). The left pinnae of WT, Allergin-1−/−, diphtheria toxin (DT; Sigma-Aldrich, St Louis, MO, USA) treated Mas-TRECK and DT-treated Mas-TRECK mice intra-dermally injected with 1 × 106 WT or Allergin-1−/− BMMCs 5 weeks previously were sensitized by intra-dermal injection of Pam2CSK4 (4 µg). Neutrophil-depleted mice were generated by intra-peritoneal injection of anti-Gr-1 (RB6-8C5, 50 µg) mAb on days −2, −1, +1, +3 and +5 and were injected intra-dermally with Pam2CSK4 (4 µg) on day 0. Ear thickness at three sites was measured daily by using a micrometer; these three measures were averaged and reported as the average ear thickness after treatment.

Flow cytometry

Ears were treated with enzyme mixture containing collagenase IV and DNase I (300 U ml−1 and 50 U ml−1, respectively; Worthington Biochemical, Lakewood, NJ, USA) for 1 h at 37°C with shaking. Cells were treated with anti-CD16/32 mAb to avoid binding to FcγR on ice for 10 min prior to incubation with the indicated combination of antibodies. Stained cells were acquired on a BD LSR Fortessa (BD Biosciences) and analyzed using Flowjo software (Treestar, Inc., San Carlos, CA, USA).

Histology

Ears were fixed in 10% formalin and were embedded in paraffin. Ear sections were stained with HE or with Masson’s trichrome. For analysis of epidermal and collagen-deposited dermal thickness, randomly selected regions in the specimen were measured by using a fluorescent microscope (BZ-X710; Keyence) and its associated software. Epidermal and collagen-deposited dermal thicknesses were expressed as mean ± SEM µm of ear skin.

Statistics

Statistical analyses were performed with the unpaired Student’s t-test. P values <0.05 were considered statistically significant.

Results and discussion

To investigate the role of Allergin-1 in the TLR2-triggered production of inflammatory cytokines, we used the TLR2/TLR6-binding diacylated lipopeptide Pam2CSK4 to stimulate BMMCs derived from WT or Allergin-1-deficient mice. Quantitative RT–PCR analysis showed that the mRNA expression of Il1b, Tnfsf2 and Il6 was significantly higher in Allergin-1-deficient BMMCs than in WT BMMCs at 2 h after stimulation with Pam2CSK4 (Fig. 1A). Notably, Il1b expression in Allergin-1-deficient BMMCs was ~20 times that in WT BMMCs (Fig. 1A). Consistent with these results, Allergin-1-deficient BMMCs produced more TNF-α and IL-6 than did WT BMMCs (Fig. 1B).

Fig. 1.

Allergin-1 inhibits TLR2 signaling in BMMCs from mice. (A) Quantitative RT–PCR analyses of pro-inflammatory cytokine expression by BMMCs at 2 h after stimulation with Pam2CSK4 (1 µg ml−1). (B) Cytometric bead array analyses of pro-inflammatory cytokine production by BMMCs at 24 h after stimulation with Pam2CSK4 (10 µg ml−1). WT, C57BL/6N mice; KO, Allergin-1-deficient mice; GADPH, glyceraldehyde 3-phophate dehydrogenase (used as an internal control for normalization). *P < 0.05; **P < 0.01; ***P < 0.005.

Fig. 1.

Allergin-1 inhibits TLR2 signaling in BMMCs from mice. (A) Quantitative RT–PCR analyses of pro-inflammatory cytokine expression by BMMCs at 2 h after stimulation with Pam2CSK4 (1 µg ml−1). (B) Cytometric bead array analyses of pro-inflammatory cytokine production by BMMCs at 24 h after stimulation with Pam2CSK4 (10 µg ml−1). WT, C57BL/6N mice; KO, Allergin-1-deficient mice; GADPH, glyceraldehyde 3-phophate dehydrogenase (used as an internal control for normalization). *P < 0.05; **P < 0.01; ***P < 0.005.

Because Allergin-1 suppressed the Pam2CSK4-induced production of neutrophil chemoattractants, including IL-6, TNF-α and IL-1β, which promote the immune defense against S. aureus infections (10, 11), we next tested whether Allergin-1 inhibits TLR2 signaling-mediated dermatitis. Pam2CSK4 was injected intra-dermally into the left pinnae of WT or Allergin-1-deficient mice, and ear swelling was measured daily thereafter. Ear swelling in WT mice peaked at day 4 and subsequently declined; in contrast, swelling on day 1 was greater in Allergin-1-deficient mice than in WT mice and remained elevated throughout the study (Fig. 2A). In addition, the Ly6G+ neutrophil population in ear tissue 1 day after Pam2CSK4 injection was greater in Allergin-1-deficient mice than in WT mice (Fig. 2B). Histological analysis demonstrated that epidermal thickness was comparable between WT and Allergin-1-deficient mice at 8 days after the intra-dermal injection of Pam2CSK4 (Fig. 2C). However, we observed that Allergin-1-deficient mice exhibited a significantly greater amount of collagen deposition, as analyzed by staining with Masson’s trichrome, than did WT mice (Fig. 2D). Although TNF-α, IL-6 and IL-1β are reported to promote fibrosis (12), increased production of these cytokines in response to Pam2CSK4 injection might be involved in augmented ear swelling in Allergin-deficient mice. Further analyses are required to clarify whether this is also the case.

Fig. 2.

Loss of Allergin-1 exacerbates TLR2 ligand-induced dermatitis. (A) The left pinnae of C57BL/6N WT (n = 15) and Allergin-1 knockout (KO, n = 14) mice were immunized i.d. with Pam2CSK4 (4 µg), after which ear swelling was measured. Data are reported as the means ± SEM of four independent experiments. (B) Frequency of CD45.2+ Ly6G+ CD11b+ neutrophils in ear skin at 1 day after i.d. injection of Pam2CSK4. HE staining (C) and Masson’s trichrome staining (MT) (D) of the ear skin at 8 days after i.d. injection of Pam2CSK4. Epidermal thickness (C) and collagen deposition (D) were evaluated as mean + SEM per millimeter of ear pinna. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.

Fig. 2.

Loss of Allergin-1 exacerbates TLR2 ligand-induced dermatitis. (A) The left pinnae of C57BL/6N WT (n = 15) and Allergin-1 knockout (KO, n = 14) mice were immunized i.d. with Pam2CSK4 (4 µg), after which ear swelling was measured. Data are reported as the means ± SEM of four independent experiments. (B) Frequency of CD45.2+ Ly6G+ CD11b+ neutrophils in ear skin at 1 day after i.d. injection of Pam2CSK4. HE staining (C) and Masson’s trichrome staining (MT) (D) of the ear skin at 8 days after i.d. injection of Pam2CSK4. Epidermal thickness (C) and collagen deposition (D) were evaluated as mean + SEM per millimeter of ear pinna. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.

After we had confirmed that Allergin-1 is expressed on skin-resident myeloid cells, including FcεRI+ c-Kit+ MCs, CD11b+ cells, Langerin+ cells and Ly6G+ neutrophils (Fig. 3A), we investigated whether Allergin-1 on MCs contributes to Pam2CSK4-induced ear swelling. For this, we used Mas–TRECK mice, which carry a DT-inducible MC deletion system that incorporates Il4 enhancer elements (8). Flow cytometry analysis confirmed the lack of MCs in ear tissue after the injection of DT in Mas–TRECK mice (Fig. 3B and C). Skin MCs in DT-treated Mas–TRECK mice were reconstituted through intra-dermal injection of either WT or Allergin-1-deficient BMMCs (Fig. 3B). Pam2CSK4-induced ear swelling in DT-treated Mas–TRECK mice reconstituted with WT BMMCs was similar to that of their non-reconstituted counterparts (Fig. 3D). However, ear swelling was more severe in DT-treated mice reconstituted with Allergin-1-deficient BMMCs than in those reconstituted with WT BMMCs (Fig. 3D). These results indicate that Pam2CSK4-induced MC activation was specifically inhibited by Allergin-1 in skin.

Fig. 3.

Reconstitution of skin MCs in DT-treated Mas–TRECK mice. (A) FACS analysis demonstrated Allergin-1 expression on skin-resident MCs (CD45.2+ PI FcεRI+ c-Kit+), eosinophils (CD45.2+ PI Siglec-F+), neutrophils (CD45.2+ PI CD11b+ Ly6G+), CD11b+ cells (CD45.2+ PI CD11b+ Ly6G) and Langerin+ cells (CD45.2+ PI CD11b+ I-Ab+ Langerin+). Black lines indicate WT C57BL/6N mice; gray lines represent Allergin-1 knockout (KO) mice. (B) The experimental design for reconstitution of skin MCs in DT-treated Mas–TRECK mice. (C) FACS detection of reconstituted MCs in skin at 5 weeks after i.d. injection of BMMCs in DT-treated Mas–TRECK mice. (D) Left ear pinnae of WT or Allergin-1-deficient BMMC-reconstituted DT-treated Mas–TRECK mice were injected with Pam2CSK4, after which ear swelling was measured. (E) Neutrophil-depleted mice were generated by i.p. injection of anti-Gr-1 mAb on days −2, −1, +1, +3 and +5 and were injected i.d. with Pam2CSK4 on day 0, after which ear swelling was measured. (F) DT-treated or DT-untreated Mas–TRECK mice were i.d. injected with Pam2CSK4. Frequency of CD45.2+ Ly6G+ CD11b+ neutrophils in ear skin at 1 day after injection was determined by flow cytometry analysis. Data are reported as the means ± SEM of two independent experiments. *P < 0.05; **P < 0.01; ****P < 0.001.

Fig. 3.

Reconstitution of skin MCs in DT-treated Mas–TRECK mice. (A) FACS analysis demonstrated Allergin-1 expression on skin-resident MCs (CD45.2+ PI FcεRI+ c-Kit+), eosinophils (CD45.2+ PI Siglec-F+), neutrophils (CD45.2+ PI CD11b+ Ly6G+), CD11b+ cells (CD45.2+ PI CD11b+ Ly6G) and Langerin+ cells (CD45.2+ PI CD11b+ I-Ab+ Langerin+). Black lines indicate WT C57BL/6N mice; gray lines represent Allergin-1 knockout (KO) mice. (B) The experimental design for reconstitution of skin MCs in DT-treated Mas–TRECK mice. (C) FACS detection of reconstituted MCs in skin at 5 weeks after i.d. injection of BMMCs in DT-treated Mas–TRECK mice. (D) Left ear pinnae of WT or Allergin-1-deficient BMMC-reconstituted DT-treated Mas–TRECK mice were injected with Pam2CSK4, after which ear swelling was measured. (E) Neutrophil-depleted mice were generated by i.p. injection of anti-Gr-1 mAb on days −2, −1, +1, +3 and +5 and were injected i.d. with Pam2CSK4 on day 0, after which ear swelling was measured. (F) DT-treated or DT-untreated Mas–TRECK mice were i.d. injected with Pam2CSK4. Frequency of CD45.2+ Ly6G+ CD11b+ neutrophils in ear skin at 1 day after injection was determined by flow cytometry analysis. Data are reported as the means ± SEM of two independent experiments. *P < 0.05; **P < 0.01; ****P < 0.001.

To examine whether neutrophils were involved in the increased Pam2CSK4-induced ear swelling in Allergin-1-deficient mice, WT and Allergin-1-deficient mice were injected with anti-Gr-1 mAb to deplete neutrophils. Allergin-1-deficient mice still showed significantly more severe ear swelling than did WT mice after injection with Pam2CSK4 even after neutrophil depletion (Fig. 3E), indicating that neutrophils were not involved in the increased Pam2CSK4-induced ear swelling in Allergin-1-deficient mice. Moreover, both DT-treated and DT-untreated Mas–TRECK mice showed significantly increased neutrophil recruitment after injection with Pam2CSK4 (Fig. 3F), suggesting that Allergin-1-expressing cells such as CD11b+ cells, rather than MCs, might be responsible for Pam2CSK4-induced neutrophil recruitment in ear tissue. Together, these results suggest that although Allergin-1 on MCs plays an important role in suppression of Pam2CSK4-induced ear swelling, neutrophils are dispensable for the increased Pam2CSK4-induced ear swelling in Allergin-1-deficient mice.

The importance of ITIM domains in shaping TLR2-mediated immune responses is demonstrated by the finding that the ITIM-bearing receptors PIR-B and CEACAM-1 directly interact with the pathogens S. aureus and Moraxella catarrhalis, respectively (13, 14). Although pathogens frequently utilize ITIM-containing receptors to evade immune detection, the ITIM-bearing receptors that down-regulate TLR2 signaling to maintain host homeostasis are largely unknown. We found here that Allergin-1 on MCs specifically inhibited TLR2 ligand-induced dermatitis in mice.

Allergin-1 is also expressed at high level on myeloid cells, including CD11b+ cells and neutrophils (Fig. 3A). CD11b+ cells consist of dendritic cell and monocyte/macrophage lineages that strongly express TLR2 (15). The recruitment of neutrophils in response to Pam2CSK4 might be induced by CD11b+ cells in DT-treated Mas–TRECK mice. Therefore, Allergin-1 may also inhibit TLR2 signaling in CD11b+ cells as well, and Allergin-1 on MCs and CD11b+ cells may cooperate to promote TLR2-mediated inflammation in vivo.

The ligand for Allergin-1 remains undetermined. However, since a fusion protein consisting of the extracellular domain of mouse Allergin-1 and the Fc portion of human IgG1 bound to BMMCs (unpublished observation), it is possible that the Allergin-1 ligand might be expressed on MCs and cis- or trans-interaction of Allergin-1 to the ligand on MCs may transmit an inhibitory signal via Allergin-1 in MCs.

Funding

This work was supported in part by grants provided by Japan Society for the Promotion of Science (KAKENHI) (grant numbers 15H01365 and 16H06387 to A.S. and 15H04862 to S.T.-H.) and was funded by Ono Pharmaceutical Co., Ltd. The sponsor had no control over the interpretation, writing or publication of this work.

Acknowledgements

We thank S. Tochihara and Y. Nomura for secretarial assistance. S.T. performed most of the experiments and analyzed the data; S.M. and S. Shibagaki did experiments; M.I., S. Shibayama and M.K. contributed to experimental design; S.T.-H. designed and did experiments, analyzed data and wrote the paper; and A.S. supervised the overall project and wrote the paper.

Conflict of interest statement: M.I. and S. Shibayama are employees of Ono Pharmaceutical Company. This does not alter the authors’ adherence to all the International Immunology policies on sharing data and materials. S.T., S.T.-H., S. Shibagaki, S.M., M.K. and A.S. declare no conflicts of interest.

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