MR1 deficiency enhances IL-17-mediated allergic contact dermatitis

Abstract

Major histocompatibility complex (MHC) class Ib molecules present antigens to subsets of T cells primarily involved in host defense against pathogenic microbes and influence the development of immune-mediated diseases. The MHC class Ib molecule MHC-related protein 1 (MR1) functions as a platform to select MR1-restricted T cells, including mucosal-associated invariant T (MAIT) cells in the thymus, and presents ligands to them in the periphery. MAIT cells constitute an innate-like T-cell subset that recognizes microbial vitamin B₂ metabolites and plays a defensive role against microbes. In this study, we investigated the function of MR1 in allergic contact dermatitis (ACD) by examining wild-type (WT) and MR1-deficient (MR1^-/-) mice in which ACD was induced with 2,4-dinitrofluorobenzene (DNFB). MR1^-/- mice exhibited exaggerated ACD lesions compared with WT mice. More neutrophils were recruited in the lesions in MR1^-/- mice than in WT mice. WT mice contained fewer MAIT cells in their skin lesions following elicitation with DNFB, and MR1^-/- mice lacking MAIT cells exhibited a significant increase in IL-17-producing αβ and γδ T cells in the skin. Collectively, MR1^-/- mice displayed exacerbated ACD from an early phase with an enhanced type 3 immune response, although the precise mechanism of this enhancement remains elusive.

Keywords: allergy; delayed-type hypersensitivity; gamma/delta T cells; innate T cells; neutrophils.

Figure 1

MR1^-/- mice develop augmented ACD compared with WT mice. (A) Wild-type (WT, C57BL/6; B6, closed circle) mice and B6.MR1^-/- (MR1^-/-, open circle) mice were sensitized with 0.5% DNFB and challenged after five days on the left pinna with vehicle only or on the right pinna with 0.2% DNFB. The thickness of the pinnae was then measured with a digital micrometer on day 0 (day of challenge), day 1, and day 2. The increment in thickness of the sensitized pinna was presented as ΔEar swelling (mm), as described in the Materials and Methods. (B) Histology of vehicle-painted (control) and DNFB-painted (experimental) pinnae obtained from WT and MR1^-/- mice. (C) The net thickness of the pinnae including that of vehicle control deduced from the measurements of morphometric analyses of histological specimens is presented as Ear thickness (mm). Representative data of at least three experiments of four to five mice/experiment. Mann–Whitney U test. **p < 0.01.

Figure 2

More neutrophils are recruited into the ACD-induced pinnae in MR1^-/- mice than WT mice. (A) Representative flow cytometric profiles of the cells infiltrated into the pinnae prepared two days after challenge with enzymatic degradation, as described in the Materials and Methods and analyzed according to the gating described for Supplementary Figure 1A . The square gate (CD11b⁺Ly6G^hi cells) indicates neutrophils. (B) Frequency of the CD45⁺ fraction and cell number in MR1^{- /-} mice compared with the fraction and cell number of WT mice represented by panel (A). (C) The expression of Cxcl1, Cxcl2, Csf3, and Il17 related with neutrophil recruitment was examined with mRNA obtained from the left pinnae (vehicle control) and the right pinnae (DNFB) two days after challenge. Representative data of at least three experiments of four to eight mice/experiment. Mann–Whitney U test. *p < 0.05, **p < 0.01.

Figure 3

T-cell subsets in both vehicle control and DNFB-painted pinnae two days after challenge in WT and MR1^-/- mice. (A) Representative flow cytometric profiles of T-cell subsets in vehicle- and DNFB-painted pinnae. The cells were prepared as for Figure 2 and analyzed according to the gating of Supplementary Figure 1B for γδ T and αβ T cells. The γδ^hi fraction is designated epidermal γδ T cells (Epi γδ) and the γδ^lo fraction as dermal γδ T cells (Der γδ). (B) Graphs of the frequencies and cell numbers for the αβ T, Epi γδ T, and Der γδ T cells represented in panel (A). (C) Representative flow cytometric profiles of MAIT cells, analyzed with the gated fraction of lymphocyte CD45⁺ cells stained with 5-OP-RU/MR1-tetramer (kindly provided by NTCF, Atlanta, GA, USA) and anti-TCRβ mAb in vehicle control and DNFB-painted pinnae of WT mice two days after challenge. (D) Graphs of the frequencies and cell numbers for MAIT cells for the WT mice represented in panel (C). MR1 ^-/- mice lack MR1-restricted cells, including MAIT cells, graphs were not demonstrated (n.d.), with no difference between control and DNFB groups in trace amounts. Representative data of at least three experiments of three to four mice/experiment. Mann–Whitney U test. *p < 0.05, **p < 0.01.

Figure 4

Cytokine production by antigen-specific T cells in draining lymph nodes. Lymph node T cells harvested from inguinal lymph nodes at day 5 in vehicle- and DNFB-painted WT and MR1^-/- mice were cultured for three days in the presence and absence of DNBS (100 μg/mL). Cytokines (IL-10, IL-17A, TNF-α, IFN-γ, IL-6) in the supernatant were quantified as described in the Materials and Methods. Representative data of two experiments of three to four mice/experiment. Mann–Whitney U test. **p < 0.01.

Figure 5

T-helper (Th) cell subsets in draining lymph nodes from WT and MR1^-/- mice. Cells in inguinal lymph nodes after five days of sensitization were obtained and stained for analyses described in the Materials and Methods. (A) Frequency of the CD3⁺ population and number of CD3⁺CD4⁺ cells (Th cells) in WT and MR1^-/- mice. (B) Frequency and number of T-bet⁺ (Th1) cells (upper right panels) in the Th gate shown in (A). (C) Representative flow cytometric profiles of CD3⁺CD4⁺ Foxp3⁺ and RORγt⁺ cell populations in WT or MR1^-/- mice. (D) Frequencies and numbers of RORγt⁺Foxp3^- (Th17; left panels), RORγt^-Foxp3⁺ (Treg; middle panels), and RORγt⁺Foxp3⁺ (stable Treg effector; right panels) cells in WT and MR1^-/- mice represented by panel (C). (E) Representative flow cytometric profiles of IL-17A or IFN-γ intracellular staining in CD3⁺CD4⁺ cells. Intracellular staining of IL-17A and IFN-γ in Th cells following stimulation with PMA and ionomycin for 4 h. T cells were obtained from draining lymph nodes of WT or MR1^-/- mice five days after sensitization at shaved abdominal skin sites. (F) Frequencies and cell numbers of IL-17A⁺ (left panels) or IFN-γ⁺ cell populations (right panels) represented by panel (E) Representative data of at least three experiments of three mice/experiment. Mann–Whitney U test. *p < 0.05.

Figure 6

T-cell subsets in pinnae of unsensitized or sensitized mice and gene expression in sensitized pinnae. (A) Flow cytometric profiles of Vγ2⁺ γδ T cells (gated as the polygon; upper panels) in γδ T cells and intracellular IL-17A in gated Vγ2⁺ T cells (lower panels) in WT and MR1^-/- mice. T cells obtained from unsensitized pinnae were stimulated with PMA and ionomycin in vitro for 4 h. The expression of intracellular IL-17A was then analyzed in the Vγ2⁺ population in the total γδ T cells by flow cytometry. (B) Frequency of IL-17A⁺ population in the total γδ T cells (left panel) or in the Vγ2⁺ cells (right panel) in WT and MR1^-/- mice represented by panel (A). (C) Representative flow cytometric profiles of dermal Vγ2⁺ γδ T cells (gated as the polygon) in vehicle control and DNFB-painted pinnae in WT and MR1^-/- mice two days after challenge. (D) Frequencies and numbers of Vγ2⁺ γδ T cells in vehicle-painted (veh) and DNFB-painted (DNFB) pinnae. (E) Frequencies and numbers of CD4⁺CD3⁺ cells (Th) in DNFB-painted pinnae of WT and MR1^-/- mice. (F) Flow cytometric profiles of RORγt and Foxp3 staining for the Th cells exhibited in E for WT (left panel) and MR1^-/- (right panel) mice. (G) Frequencies and numbers of RORγt⁺Foxp3^- (Th17; left panels) cells and RORγt^-Foxp3⁺ (Treg; middle panels) cells represented in panel (F). (H) Relative expression of Il17 (left panel) and Il1b (right panel) mRNA in pinnae 6 h after DNFB challenge in WT and MR1^-/- mice. Representative data of at least two experiments of three to five mice/experiment. Mann–Whitney U test. *p < 0.05, **p < 0.01.