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. 2018 Sep 25;30(10):457-470.
doi: 10.1093/intimm/dxy045.

Goblet cell-produced retinoic acid suppresses CD86 expression and IL-12 production in bone marrow-derived cells

Yangyan Xiao  1   2 , Cintia S de Paiva  1 , Zhiyuan Yu  1 , Rodrigo G de Souza  1 , De-Quan Li  1 , Stephen C Pflugfelder  1
Affiliations

Goblet cell-produced retinoic acid suppresses CD86 expression and IL-12 production in bone marrow-derived cells

Yangyan Xiao et al. Int Immunol. .

Abstract

Conjunctival goblet cell loss in ocular surface diseases is accompanied by increased number of interleukin-12 (IL-12)-producing antigen-presenting cells (APCs) and increased interferon-γ (IFN-γ) expression. This study tested the hypothesis that mouse conjunctival goblet cells produce biologically active retinoic acid (RA) that suppresses CD86 expression and IL-12 production by myeloid cells. We found that conditioned media from cultured conjunctival goblet cells (CjCM) suppressed stimulated CD86 expression, NF-κB p65 activation and IL-12 and IFN-γ production in unstimulated and lipopolysaccharide-stimulated cultured bone marrow-derived cells (BMDCs) containing a mixed population of APCs. Goblet cell-conditioned, ovalbumin-loaded APCs suppressed IFN-γ production and increased IL-13 production in co-cultured OTII cells. The goblet cell suppressive activity is due in part to their ability to synthesize RA from retinol. Conjunctival goblet cells had greater expression of aldehyde dehydrogenases Aldh1a1 and a3 and ALDEFLUOR activity than cornea epithelium lacking goblet cells. The conditioning activity was lost in goblet cells treated with an ALDH inhibitor, and a retinoid receptor alpha antagonist blocked the suppressive effects of CjCM on IL-12 production. Similar to RA, CjCM increased expression of suppressor of cytokine signaling 3 (SOCS3) in BMDCs. SOCS3 silencing reversed the IL-12-suppressive effects of CjCM. Our findings indicate that conjunctival goblet cells are capable of synthesizing RA from retinol secreted by the lacrimal gland into tears that can condition APCs. Evidence suggests goblet cell RA may function in maintaining conjunctival immune tolerance and loss of conjunctival goblet cells may contribute to increased Th1 priming in dry eye.

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Figures

Fig. 1.
Fig. 1.
BMDC culture phenotype and suppressive effects of CjCM. (A) Representative laser confocal images of conjunctival section co-stained with anti-MHCII (red), WGA lectin (green) that binds to goblet cell glycoproteins and DAPI for nuclear DNA (white). Arrows point to MHCII+ dendritic-shaped APCs below the epithelium and in the superficial stroma. Inset in left image of MHCII+ DC adjacent to WGA+ goblet cell in basal conjunctival epithelium. NC = negative control with second antibody only. (B) Experimental design for preparation of conjunctival (goblet cell) epithelial conditioned media (Epi culture, upper) and BMDC culture (lower) treatment groups. (C) Representative density plots of BMDCs cultured in control IMDM media (top) or in conjunctival (goblet cell) epithelial conditioned media (CjCM, bottom) from days 6 to 9. The plots on the right were gated from the CD11c−CD11b+ cells. Day 9 = control BMDCs; Day 9 CjCM = BMDCs treated with conjunctival (goblet cell) epithelial conditioned media; Ly6C = monocyte marker; F4/80 = macrophage marker.
Fig. 2.
Fig. 2.
MHCII and CD86 expression in defined populations of cultured bone marrow-derived (myeloid) cells. (A) MHC (top) and CD86 (bottom) MFI histograms in the entire population of cultured bone marrow-derived (myeloid) cells (all cells) and in defined gated populations (CD11c+CD11b−, CD11c+CD11b+, CD11c−CD11b+F4/80−Ly6c+ monocytes, CD11c−CD11b+F4/80+Ly6c+ macrophages) in LPS or CjCM plus LPS-treated day 9 cultured BMDCs (no bead selection). MFI values and the percentage of positive cells are provided below each histogram. (B) MHCII (top) and CD86 (bottom) MFI histograms in LPS or CjCM plus LPS-treated CD11c+ cells bead sorted from day 9 cultured BMDCs. MFI values and the percentage of positive cells are provided below each histogram.
Fig. 3.
Fig. 3.
Expression of inflammatory mediators by BMDCs. (A) Effects of goblet cell conditioned media (CjCM) or RA compared to control day 9 untreated group on relative fold expression of Th1 cytokines IL-12 and IFN-γ in cultured BMDCs with or without LPS treatment, measured by RT–PCR. Results expressed as mean ± SEM, n = 4, ***P < 0.005. (B) IL-12 intra-cellular staining of BMDCs detected by flow cytometry (mean ± SEM, n = 3). Untreated (UT; i.e. no LPS) versus LPS treated within group, *P < 0.05; †P < 0.05 comparison between day 9 UT versus CjCM UT. (C) Level of NF-kB p65 phosphorylation in DCs with/without CjCM or RA treatment. Day 9 BMDCs were seeded in 96-well plates treated without or with LPS for 45 min and phospho-p65 (ser536) and total p65 were measured by cell-based enzyme-linked immunosorbent assay. Results expressed as %phospho-p65/total p65 are mean ± SEM of three independent experiments. (D) Effects of CjCM or RA compared to control group on relative fold expression of chemokine receptor CCR7 and ICAM-1 in BMDCs (mean ± SEM, n = 4), ****P < 0.001. UT versus LPS within groups: *P < 0.05, **P < 0.01; between-group comparisons: †P < 0.05, ††P < 0.01, †††P < 0.005.
Fig. 4.
Fig. 4.
Antigen presentation assay. LPS-treated bone marrow-derived myeloid APCs conditioned with CjCM or RA as described in Fig. 1(B) were harvested on day 9, loaded with OVA peptide for 2 h, then co-cultured with OTII CD4+T cells (1:5 ratio). After 3 days of co-culture, cells were harvested for IFN-γ and IL-13 intra-cellular staining. This experiment was performed three times with n = 3 each time. D9 = control APCs; CjCM = APCs treated with conjunctival conditioned media; RA = APCs treated with 10 nM RA; FMO = fluorescence minus one negative control. (A) Representative contour plots of IFN-γ+CD4+ T cells in different treatment groups shown on the left, and fold change in the percentage of IFN-γ+CD4+ cells relative to day 9 control cells in the bar graph on the right. (B) Representative contour plots of IL-13+CD4+ cells in different treatment groups shown on the left, and fold change in the percentage of IL-13+CD4+ cells relative to day 9 control cells in the bar graph on the right.
Fig. 5.
Fig. 5.
Expression of RA-metabolizing enzymes by conjunctival goblet cells. (A) RT–PCR to measure the expression of ALDH1 isotypes was performed on RNA harvested from cornea and conjunctival epithelial cultures on day 11 and BMDCs on day 9. K = corneal cells, Cj = conjunctival cells, DC = bone marrow derived cells. (B) ALDH activity (ALDHact) was measured in cultured corneal and conjunctival epithelium in vitro. Single cell suspensions were prepared from cornea and conjunctival epithelial cultures on day 11. DEAB, an ALDH inhibitor, was used as a negative control for ALDH staining (control tube, gray dots). In the absence of DEAB, ALDH+ cells (black dots), which are characterized by high ALDH activity and low SSC, could be detected and distinguished from the ALDH− subset. (C) ALDHact was evaluated in ex vivo suspensions of mouse conjunctival cells that were prepared immediately after euthanasia. ALDHact was over 4-fold higher in the CD45− population consisting primarily of epithelial cells (right), compared to the CD45+ BMDC population (left). (D) Immunofluorescent staining for ADH4, ALDH1a3 and RBP1 in mouse conjunctival tissue sections. WGA lectin stains glycoproteins, including goblet cells mucins. The negative control is secondary antibody only. (E) Immunofluorescent staining for ADH4, ALDH1a3 and RBP1 in mouse cornea tissue sections. (F) RT–PCR to measure expression of ADH4 and RBP1 genes in RNA harvested from cornea and conjunctival epithelial cultures on day 11. **P < 0.01, ****P < 0.001.
Fig. 6.
Fig. 6.
Conjunctival goblet cells condition ALDH activity in BMDCs and produce RA. (A) ALDH activity assayed in BMDC treatment groups on day 9, with DEAB-treated cells (gray dots) serving as a negative control for ALDH staining, revealed that CjCM increased the percentage of ALDH+ cells (ALDHbri), n = 3 and experiment performed three times. (B) Statistical comparison of Aldefluor activity using two-way analysis of variance, *P < 0.05, **P < 0.01, ***P < 0.005 means UT versus LPS within groups. (C–F) The supernatant from the conjunctival culture treated with DEAB from day 5 to day 7 was harvested on day 11, then used to condition BMDCs on day 6 (CjCM + DEAB), with CjCM as positive control (CjCM) and media only as negative control (D9). LPS was added to each of the three groups of cultured BMDCs on day 8. After 24 h, the cell suspension was stained with the Aldefluor reagent and CD86. These studies were performed three times with n = 3 per group. (C, D) Representative dot plots (C) and statistical comparisons (D) of ALDH activity assayed in these three groups. (E, F) CD86+ cells displayed as histogram – UT in gray and LPS-treated in black line (E) and the statistical comparisons (F). (G) The F9-RARE-lacZ reporter cell cells were seeded into wells of a 96-well plate, then media (KSFM + 3% FBS), media + vitamin A palmitate (1 nM), conjunctival conditioned media (CjCM) from C57BL/6J or Spdef KO or cornea (KCM) epithelial cultures with or without vitamin A (retinol) palmitate (1 µM), or RA in different concentrations were added. After 16 h, the supernatants were removed and a β-galactosidase assay was performed. Each group was repeated three times with n = 4 per run. The absorbance (OD) values from different concentrations of RA were used to generate the calibration curve (left), and the OD values obtained with different treatment groups are shown on the right. (H) The RARα antagonist, Ro41-5253 (RO), was added to cultured BMDCs 2 h before CjCM or RA on day 6, and RNA harvested for PCR for IL-12 (left) or SOCS3 (right) after 4-h LPS stimulation on day 8. D9 = control BMDCs; D9 RO = BMDCs with 1 µM RO added; CjCM = BMDCs treated with conjunctival conditioned media; RO CjCM= BMDCs with both RO and CjCM added; RA = BMDCs cells treated with 10 nM RA; RO RA = BMDCs with both 1 µM RO and 10 nM RA added; UT = untreated (without LPS stimulation); LPS = BMDCs treated with LPS. UT versus LPS within groups: *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001; between-group comparisons: †P < 0.05, ††P < 0.01, †††P < 0.005.
Fig. 7.
Fig. 7.
SOCS3 neutralization. (A) SOCS3 and β-actin western blot of day 9 (D9) cultured BMDCs and D9 BMDCs treated with conjunctival conditioned media (CjCM) and RA. (B) Compared to a control scrambled sequence, SOCS3 siRNA inhibited expression of SOCS3 transcripts at 24 h in BMDCs with or without LPS stimulation measured by RT–PCR. (C) SOCS3 western blot of LPS-stimulated BMDCs after 24-h treatment with control scrambled sequence or SOCS3 siRNA LPS (left) and the SOCS3/β-actin ratio measured from the western blot (right). (D) SOCS3 gene silencing reversed the suppressive effects of CjCM and RA on IL-12 expression in BMDCs. Untreated (UT) versus LPS within groups: *P < 0.05, **P < 0.01, ***P < 0.005; between-group (D9) comparisons: ††P < 0.01, †††P < 0.005. (E) CjCM suppressed expression of IL-12 in BMDCs stimulated with LPS or hyperosmolar (450mOsm) culture media.
Fig. 8.
Fig. 8.
Conjunctival goblet cells are capable of metabolizing vitamin A secreted by the lacrimal gland as retinol into tears into RA that can suppress maturation and Th1 cytokine production by APCs that are stimulated by danger signals [desiccating/osmotic stress or pattern associated microbial products (PAMPs)]. IFN-γ produced by Th1 cells in the dry eye conjunctiva can cause goblet cell loss/dysfunction resulting in reduced APC conditioning.
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