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. 2014 Mar;124(3):1309-19.
doi: 10.1172/JCI67947.

Hyaluronan digestion controls DC migration from the skin

Jun Muto, Yasuhide Morioka, Kenshi Yamasaki, Margaret Kim, Andrea Garcia, Aaron F Carlin, Ajit Varki, Richard L Gallo

Hyaluronan digestion controls DC migration from the skin

Jun Muto et al. J Clin Invest. 2014 Mar.

Abstract

The breakdown and release of hyaluronan (HA) from the extracellular matrix has been hypothesized to act as an endogenous signal of injury. To test this hypothesis, we generated mice that conditionally overexpressed human hyaluronidase 1 (HYAL1). Mice expressing HYAL1 in skin either during early development or by inducible transient expression exhibited extensive HA degradation, yet displayed no evidence of spontaneous inflammation. Further, HYAL1 expression activated migration and promoted loss of DCs from the skin. We subsequently determined that induction of HYAL1 expression prior to topical antigen application resulted in a lack of an antigenic response due to the depletion of DCs from the skin. In contrast, induction of HYAL1 expression concurrent with antigen exposure accelerated allergic sensitization. Administration of HA tetrasaccharides, before or simultaneously with antigen application, recapitulated phenotypes observed in HYAL1-expressing animals, suggesting that the generation of small HA fragments, rather than the loss of large HA molecules, promotes DC migration and subsequent modification of allergic responses. Furthermore, mice lacking TLR4 did not exhibit HA-associated phenotypes, indicating that TLR4 mediates these responses. This study provides direct evidence that HA breakdown controls the capacity of the skin to present antigen. These events may influence DC function in injury or disease and have potential to be exploited therapeutically for modification of allergic responses.

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Figures

Figure 1
Figure 1. Generation of HYAL1-overexpressing mice.
(A) Schematic for conditional overexpression of HYAL1. Filled triangles represent loxP sites. (B) Fluorescence images of transgenic mouse pups (approximately 2 days old) expressing GFP (CAG-GFPfloxed-HYAL1 Tg mice: CAG-GFP and EIIa/HYAL1 mice) and EIIa-Cre mice. (C) Quantitative PCR demonstrating HYAL1 overexpression in normal skin from EIIa/HYAL1 mice and control (CAG-GFP) mice (*P < 0.05; n = 6). (D) Frozen sections of skin from HYAL1-overexpressing (K14/HYAL1) and control (CAG-GFP) mice were stained with an antibody recognizing HYAL1 (red). Scale bar: 100 μm; n = 5 per group. (E) HA immunostaining. Frozen sections of skin from EIIa/HYAL1 mice and control EIIa-Cre mice were stained with HABP and FITC-streptoavidin (green) and DAPI for nuclei (blue). Scale bar: 100 μm; n = 5 per group. (F) The size distribution of HA extracted from skin, as analyzed by agarose gel electrophoresis. A similar total amount of HA was loaded in each lane, as determined by carbazole assay. Gel was stained with Stains-All. Lane 1, molecular weight markers; lane 2, HA extracted from the skin of control EIIa-Cre mice; lane 3, HA extracted from skin of HYAL1-expressing (EIIa/HYAL1) mice shows decrease in average detectable size to <27 kDa; lane 4, human umbilical cord HA as standard. (G) Skin of control (CAG-GFP) mice and HYAL1-overexpressing (EIIa/HYAL1) mice stained with H&E. Scale bar: 100 μm. Data are representative of 3 independent experiments with similar results.
Figure 2
Figure 2. Decreased DC number in the epidermis and dermis of HYAL1-overexpressing mice.
(A) Representative dot plots of MHC class II and CD11c double-positive viable DCs from epidermal sheets of transgenic mice (control, CAG-GFP mice). Numbers within plots denote the percentage of cells within the respective gates. IA/IE, MHC class II. (B) Percentage of MHC class II+CD11c+ DCs in epidermal sheets of mice by flow cytometry (***P < 0.001; n = 3). (C) Immunohistochemistry of MHC class II in epidermal sheets. Scale bar: 50 μm; n = 6. (D) Dermal single cell suspensions of K14/HYAL1 mice and control (K14-Cre) mice were subjected to flow cytometry analysis, and MHC class II– and CD11c-positive cell numbers were assessed (*P < 0.05; n = 3). Mean ± SEM. (E) Number of langerin-positive cells among MHC class II– and CD11c-positive cells (*P < 0.05; n = 3). Mean ± SEM. (F) Flow cytometry analysis of dermal single cell suspensions showing expression of CD11b and CD103 from K14/HYAL1 and control (K14-Cre) mice. Cells were gated on langerin+, MHC class II+, and CD11c+ cells. (G) Cells gated on langerin-negative, MHC class II+, and CD11c+ cells showing expression of CD11b and CD103. Numbers represent the percentage of the cells in the indicated gate. Data are representative of 3 independent experiments.
Figure 3
Figure 3. Decreased DC number in the epidermis of K14CreERT/HYAL1 mice after tamoxifen application.
(A) DCs per mm2 were determined by counting MHC class II immunostained cells in 8 different microscopic fields of the epidermal sheets 0, 24, 48, and 72 hours after topical tamoxifen application. Mean and SEM are shown on the graph (n = 4). (B) 0, 24, 48, 72 hours after tamoxifen treatment, epidermal sheets were harvested and stained with MHC class II antibody. Scale bar: 50 μm. (C) 48 hours after tamoxifen treatment, epidermal sheets were stained with CD80 antibody. Scale bar: 50 μm. (D) Increased trafficking of skin DCs in tamoxifen-dependent HYAL1-overexpressing transgenic mice. Representative plots of eFluor670 fluorescence plotted against CD11c from DLNs of transgenic mice 24 hours after painting eFluor670 dissolved in acetone on shaved and tamoxifen- or vehicle-treated abdominal skin. (E) Number of CD11c+eFluor670+ DCs in DLNs of tamoxifen-dependent HYAL1-overexpressing mice (K14CreERT/HYAL1, black bar) compared with vehicle-treated control mice (K14CreERT/HYAL1, gray bar) (n = 4). (F) Representative dot plots of CD11c against side scatter from DLNs of transgenic mice 72 hours after tamoxifen or vehicle treatment on abdominal skin. (G) Percentage of CD11c+ DCs in DLNs of tamoxifen-dependent HYAL1-overexpressing mice (K14CreERT/HYAL1, black bar) compared with vehicle-treated control mice (K14CreERT/HYAL1, gray bar) (n = 4). Mean and SEM are shown on the graph. Data are representative of 2 separate experiments with similar results. *P < 0.05, ***P < 0.001.
Figure 4
Figure 4. OligoHA induces DC emigration and maturation.
(A) DCs per mm2 were determined by counting MHC class II immunostained cells in 8 different microscopic fields of the epidermal sheets 0, 2, 4, 8, and 24 hours after oligoHA (400 μg) or PBS injection of C57BL/6 WT mice. (B) Frequency of CD80+ cells in MHC class II+ cells in the epidermal sheets after injection. (C) Two hours after injection, epidermal sheets were harvested and stained with MHC class II or CD80 monoclonal antibody. Scale bar: 50 μm. (D) Increased DCs in DLNs after oligoHA injection (400 μg). Representative dot plots of FITC fluorescence plotted against CD11c from DLNs of C57BL/6 WT mice 24 hours after painting FITC dissolved in acetone on shaved abdominal skin. Data are representative of 2 or 3 separate experiments with similar results. Mean and SEM are shown on the graphs (n = 4). ***P < 0.001.
Figure 5
Figure 5. Constitutive overexpression of HYAL1 in the skin suppresses CHS.
(A) Restoration of the CHS response in HYAL1-overexpressing (K14/HYAL1, black bar) mice compared with control (CAG-GFP, gray bar) mice. The data represent the increase in ear thickness for groups of 6 mice. (B) Representative histopathology of the ears of K14/HYAL1 mice and control mice elicited with vehicle or DNFB. Scale bar: 200 μm. (C) Defective trafficking of skin DCs in HYAL1-overexpressing transgenic mice. Representative dot plots of eFluor670 fluorescence plotted against CD11c in viable cells from DLNs of transgenic mice 24 hours after painting eFluor670 dissolved in 1:1 acetone/dibutylphthalate on shaved abdominal skin. (D) Number of CD11c+eFluor670+ DCs in DLNs of transgenic mice (n = 3). (E) LN cells from sensitized CAG-GFP mice were adoptively transferred by i.v. injection into K14/HYAL1 and CAG-GFP mice. Ear elicitation with DNFB and measurement of ear thickness were performed for groups of 4 mice. (F) LN cells from sensitized K14/HYAL1 and CAG-GFP mice were adoptively transferred by i.v. injection into CAG-GFP mice. Ear elicitation with DNFB and measurement of ear thickness were performed for groups of 4 mice. (G) CHS response in C57BL/6 WT mice injected subcutaneously with oligoHA (black bar) or PBS (gray bar) in the dorsal skin 72 hours before sensitization with DNFB (n = 5). Mean ± SEM. *P < 0.05, ***P < 0.001. Data are representative of 3 independent experiments.
Figure 6
Figure 6. Tamoxifen-dependent overexpression of HYAL1 or injection of HA tetrasaccharides accelerates sensitization of CHS.
(A and B) CHS response after early elicitation (1.5 days after sensitization) in tamoxifen-dependent HYAL1-overexpressing mice (K14CreERT/HYAL1, black bar) compared with acetone/DMSO-treated control mice (K14CreERT/HYAL1, gray bar). The data represent the increase in ear thickness for groups of 4 mice. (C and D) CHS response after early elicitation (1.5 days after sensitization) in mice injected with oligoHA (400 μg, black bar) or PBS (gray bar) subcutaneously at the same time and site of sensitization. The results represent the increase in the ear thickness of groups of 5 mice. Mean ± SEM. ***P < 0.001. Data are representative of 2 independent experiments.
Figure 7
Figure 7. Modification of DC function by HA catabolism is TLR4 dependent.
(A) DCs per mm2 were determined by counting MHC class II immunostained cells in 8 different microscopic fields of the epidermal sheets of K14/HYAL1 (HYAL1), K14-Cre (K14), TLR4-deficient K14/HYAL1, and TLR4-deficient K14-Cre mice (n = 4). (B) Increased DCs in DLNs after injection with oligoHA subcutaneously at the same time as and site of FITC application. Number of CD11c+FITC+ DCs in DLNs of oligoHA-injected C57BL/6 WT mice and TLR4-deficient mice compared with those in PBS-injected control mice 24 hours after painting FITC on shaved abdominal skin was analyzed by flow cytometry (n = 4). (C) Evaluation of CHS in TLR4-deficient HYAL1-overexpressing mice. The black bar and the dark gray bar represent TLR4-deficient K14/HYAL1 and control mice (K14-Cre), respectively. The data represent the increase in ear thickness for groups of 5 mice. (D) CHS response in C57BL/6 WT mice and TLR4-deficient mice injected subcutaneously with oligoHA or PBS in the dorsal skin 72 hours before sensitization with DNFB (n = 5). (E) Increase in ear thickness after early elicitation with DNFB (1.5 days after sensitization). C57BL/6 WT mice and TLR4-deficient mice were injected subcutaneously with oligoHA or PBS at the same time and site of sensitization (n = 5). Mean and SEM are shown on the graph. *P < 0.05, ***P < 0.001. Data are representative of 2 independent experiments.
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