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. 2020 Sep 14;161(3):230–244. doi: 10.1111/imm.13246

p38 MAPK signaling mediates retinoic acid‐induced CD103 expression in human dendritic cells

Mandi M Roe 1, Marziah Hashimi 1, Steve Swain 1, Krista M Woo 1, Diane Bimczok 1,✉
PMCID: PMC7576877  PMID: 32737889

Summary

Retinoic acid (RA) is an active derivative of vitamin A and a key regulator of immune cell function. In dendritic cells (DCs), RA drives the expression of CD103 (integrin α E), a functionally relevant DC subset marker. In this study, we analyzed the cell type specificity and the molecular mechanisms involved in RA‐induced CD103 expression. We show that RA treatment caused a significant up‐regulation of CD103 in differentiated monocyte‐derived DCs and blood DCs, but not in differentiated monocyte‐derived macrophages or T cells. DC treatment with an RA receptor α (RARα) agonist led to an increase in CD103 expression similar to that in RA treatment, whereas RARA gene silencing with small interfering RNA blocked RA‐induced up‐regulation of CD103, pointing to a major role of RARα in the regulation of CD103 expression. To elucidate RA‐induced signaling downstream of RARα, we used Western blot analysis of RA‐treated DCs and showed a significant increase of p38 mitogen‐activated protein kinase (MAPK) phosphorylation. In addition, DCs cultured with RA and a p38 MAPK inhibitor had a significantly reduced expression of CD103 compared with DCs cultured with RA only, indicating that p38 MAPK is involved in CD103 regulation. In summary, these findings suggest that the RA‐induced expression of CD103 is specific to DCs, is mediated primarily through RARα and involves p38 MAPK signaling.

Keywords: CD103, dendritic cells, p38 mitogen‐activated protein kinase, retinoic acid

Retinoic acid induces expression of CD103 in human monocyte‐derived and blood CD1c+ and CD141+ dendritic cells, but not in macrophages or T cells. CD103 expression in human dendritic cells is mediated through RARα and p38 MAPK signaling.

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Abbreviations

cDC

conventional dendritic cell

DC

dendritic cell

integrin αE

CD103

ITGAE

CD103 gene name

ITGB7

β 7 gene name

MAPK

mitogen‐activated protein kinase

MDM

monocyte‐derived macrophage

MoDC

monocyte‐derived dendritic cell

RA

retinoic acid

RAR

retinoic acid receptor

siRNA

small interfering RNA

TGF‐β

transforming growth factor‐β

TGFβRII

TGF‐β receptor II

Introduction

Retinoic acid (RA) is the major active derivative of vitamin A, an essential dietary micronutrient in humans and other mammals. RA has long been recognized as a key factor in embryonic tissue development and the maintenance of ocular function and plays important roles in mucosal immunity. 1 , 2 , 3 Specifically, RA is involved in regulating lymphocyte homing to mucosal sites, 4 , 5 , 6 induction of regulatory T cells 7 and mucosal dendritic cell (DC) function and development. 4 , 8 The relevance of vitamin A and its active mediator RA for gastrointestinal immune function is illustrated by the fact that vitamin A deficiency leads to an increased risk for diarrheal infections and inflammatory bowel disease. 9 , 10 , 11

Importantly, a significant number of animal studies have revealed an association between DC production of RA, the DC response to RA and DC expression of CD103, the α‐chain of integrin αEβ7. 7 , 8 , 12 , 13 , 14 CD103 is a functionally important subset marker for gastrointestinal DCs in mice and is also expressed on mucosal DC subsets in humans. 7 , 15 , 16 Analyses from our laboratory and others have demonstrated that RA drives the expression of CD103 (integrin αE) as well as the corresponding β‐chain integrin β 7 on human DCs. 13 , 17 , 18 Several studies in mice suggest that signaling through retinoic acid receptor α (RARα), one of the three retinoic acid receptors (RAR), is the major pathway for RA‐mediated regulation of DC development and function; 4 , 19 however, whether the same is true for human DCs is unclear.

Retinoic acid exhibits pleiotropic effects through both genomic and non‐genomic signaling pathways. 20 , 21 , 22 , 23 Canonical genomic RA signaling involves direct modulation of gene expression through binding of RARs that function as transcription factors to RA response elements on the DNA. 24 , 25 , 26 Alternatively, RA can also elicit non‐genomic effects through interactions with cytoplasmic signaling cascades such as p38 mitogen‐activated protein kinase (MAPK) and extracellular signal‐regulated kinase 1 signaling. 21 , 23 , 27 Our previous analyses showed that RA signaling leading to CD103 expression in human DCs intersects with transforming growth factor‐β (TGF‐β) signaling, as a small molecule inhibitor of the TGF‐β receptor prevented RA‐induced CD103 up‐regulation. 13 In activated CD8 T cells, TGF‐β leads to strong induction of CD103 expression. 28 , 29 However, TGF‐β alone does not drive CD103 expression in DCs, as we and others have shown, 13 , 17 pointing to complex interactions between RA signaling and TGF‐β signaling pathways. Overall, these observations led us to investigate the non‐canonical signaling pathways downstream of RA that may be responsible for the regulation of CD103 expression.

We here investigated the cell type specificity of RA‐induced CD103 expression and the molecular mechanisms that drive RA‐dependent expression of CD103 in human DCs. Our experiments showed that RA increased expression of CD103 in monocyte‐derived dendritic cells (MoDCs) and blood DCs and to a lesser extent in monocytes, but not in differentiated macrophages or T cells. Moreover, induction of CD103 was dependent on signaling through RARα and p38 MAPK, but not SMAD2/3. We also showed that inhibition of nuclear factor of activated T cells (NFAT) diminished the expression of CD103 in DCs in the presence of RA, suggesting that NFAT may be a downstream target of p38 MAPK in RA‐dependent signaling.

Materials and methods

 

Human donors

Whole blood was obtained from a pool of approximately 20 healthy male and female volunteers aged 21–60 years with informed consent. For each experiment, three to five donors were randomly selected from this pool. Collection of human blood samples was approved by Montana State University’s Institutional Review Board, protocol #DB082817.

Cell isolation and culture

CD14+ monocytes were isolated from heparinized whole blood by centrifugation and magnetic antibody cell sorting (MACS) sorting, as previously shown. 13 Monocytes were cultured in X‐vivo medium (Lonza, Basel, Switzerland) supplemented with 100 U/l penicillin, 100 µg/l streptomycin, 50 µg/ml gentamycin, 5 mm HEPES and 2 mm l‐glutamine (all Hyclone, Logan, UT). To induce differentiation of a monocyte‐derived macrophage phenotype, 25 ng/ml recombinant human macrophage colony‐stimulating factor (PeproTech, Rocky Hill, NJ) was added to the culture medium. To induce differentiation of MoDCs, 25 ng/ml recombinant human granulocyte–macrophage colony‐stimulating factor and 7 ng/ml recombinant human interleukin‐4 (R&D Systems, Minneapolis, MN) were added to the culture medium. Aliquots of all monocyte samples were tested for tumor necrosis factor‐α secretion after 24 hr using an enzyme‐linked immunosorbent assay (R&D Systems), and monocytes with spontaneous tumor necrosis factor‐α secretion >100 pg/ml were considered pre‐activated and were excluded from the analyses as described previously. 13 , 14 Unretained, monocyte‐depleted cells from CD14 MACS sorting were used for lymphocyte experiments and were also cultured in X‐vivo medium. To induce T‐cell proliferation, Human T‐Activator CD3/CD28 Dynabeads® (Thermo Fisher Scientific, Waltham, MA) were added to the lymphocyte cultures at 0·5 µl/ml, and proliferation was determined by dye dilution assay using CellTraceViolet (Thermo Fisher Scientific). Human blood DCs were isolated from peripheral blood mononuclear cells by MACS using the Blood Dendritic Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany).

Reagents and antibodies

Retinoic acid was obtained from Sigma‐Aldrich (St. Louis, MO) and was used at a physiological concentration of 100 nm. All RA‐treated cells were handled under red light to prevent RA degradation. The following RA agonists and antagonists were applied to MoDC cultures during the first 3 days of culture: RARα agonist AM80 (1–10 µm), RARβ agonist CD2314 (1–10 µm), RARγ agonist CD437 (1–10 µm), RARα antagonist BMS195614 1–50 nm (all from Tocris Bioscience, Bristol, UK) and RA antagonist Ro41‐5253 (60 nm) (from Sigma‐Aldrich). Intracellular signaling pathways were targeted by applying the following reagents to mature MoDC cultures for 1 hr at 37° before the addition of RA: the p38 MAPK inhibitor SB202190 (10 µm; Sigma‐Aldrich) and an NFAT peptide inhibitor (10–50 µm, Tocris Bioscience). The following anti‐human monoclonal antibodies were used for flow cytometry: CD103 (integrin α E), integrin β 7 (both eBioscience Inc., San Diego, CA), CD3, phopho‐SMAD2/3, CD13, CD14, CD80 (all BD Biosciences, San Jose, CA), CD4 (Tonbo Biosciences, San Diego, CA), and CD1c and CD141 (both BioLegend, San Diego, CA). A fixable live/dead stain was applied to all flow cytometry samples (Thermo Fisher Scientific). The following antibodies were used for Western blot analysis: p38 MAPK, pThr180/182 p38 MAPK and GAPDH (all Cell Signaling, Danvers, MA).

Flow cytometry

Cells were stained with the antibodies listed above. Isotype controls for each antibody were used to control for non‐specific binding. We used a live/dead yellow fixable stain to exclude non‐viable cells from our analyses. Surface antibody staining was performed as described previously. 13 Following antibody staining, MoDCs were fixed with Cytofix (BD Biosciences) and analyzed on an LSR II or an LSR Fortessa Flow Cytometer (BD Biosciences). FACS data were analyzed using flowjo x software (Tree Star, Ashland, OR). MoDCs were gated based upon size, single cells and live cells. For intracellular detection of phosphorylated SMAD2/3, MoDCs were cultured for 3 days, harvested and allowed to rest for 2 hr before stimulation with RA, 5 ng TGF‐β 1 (R&D Systems), or TGF‐β 1 and 50 µm TGF‐βRII inhibitor, SB431542 (Tocris Bioscience) for 30 min. Cells then were incubated in pre‐warmed Cytofix (BD Biosciences) at 37° for 10 min. Cells were washed and resuspended in Perm Buffer III (Thermo Fisher Scientific) for 30 min at 4° and then were processed for staining with antibodies.

RARα siRNA knock down

Small interfering RNA (siRNA) specific to RARα was used to inhibit RARα expression in MoDCs. MoDCs were differentiated for 3 days before treatment, then resuspended in BTX electroporation solution (BTX, Holliston, MA) and 5 µg RARα siRNA or control scramble siRNA (Santa Cruz Biotechnology, Dallas, TX) and were electroporated using program U002 for eukaryotic cells on a Nucleofector™ 2b device (Lonza). MoDCs were immediately added to pre‐warmed media and incubated for 24 hr before the addition of RA. Following 24 hr of culture with RA, MoDCs were harvested for quantitative reverse transcription–polymerase chain reaction (qRT‐PCR) analysis of RARα, CD103 and integrin β 7 expression.

Western blot

Monocyte‐derived DCs cultured in the presence of RA for 3 days were harvested for Western blot into RIPA lysis buffer for 30 min at 4°, then centrifuged at 10 000 g for 15 min. Supernatant was recovered and mixed with Laemmli sample buffer (Bio‐Rad Laboratories, Hercules, CA). Protein was quantified with a BCA assay following the manufacturer’s protocol (Thermo Fisher Scientific). Equal quantities of protein, 1–3 µg per lane, were run on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel for 1 hr at 140 V. Protein was transferred to a polyvinylidene fluoride membrane (Bio‐Rad Laboratories) for 40 min at 100 V. Membranes were blocked with Tris–HCl‐buffered saline–Tween‐20, 5% bovine serum albumin, sodium fluoride, proteinase inhibitors and phosphatase inhibitors (all Thermo Fisher Scientific). Membranes were incubated overnight with the primary antibody, washed and then incubated with the secondary antibody conjugated with horseradish peroxidase for 1 hr at room temperature. Membranes were developed with SuperSignal™ West Pico PLUS (Thermo Fisher Scientific) per the manufacturer’s specifications and then were imaged on a FluorChemR system (Protein Simple, San Jose, CA). Band intensity was quantified using imagej 1.52p software. 30

Gene expression analysis by qRT‐PCR

RNA extraction from MoDCs was performed with a Direct‐zol kit (Zymo Research, Irvine, CA) per the manufacturer’s specifications. RNA was quantified using a Nanodrop1000 (Thermo Fisher Scientific). An iScript™ cDNA synthesis kit (Bio‐Rad Laboratories) was used to generate cDNA. TaqMan Mastermix and primer/probes (Thermo Fisher Scientific) for each gene of interest and the GAPDH housekeeping gene were used for qRT‐PCR, as previously described. 13 Samples were amplified on a Lightcycler®96 (Roche Holding AG, Basel, Switzerland), using the following protocol: 1 cycle for uracil N‐glycosylase activation at 50° for 2 min; 1 cycle for hot start at 95° for 10 min; 40 cycles of amplification as follows: 95° for 15 seconds, 60° for 1 min. Fluorescence was measured after each of these cycles. Data analysis was performed using the Pfaffl method. 31

Statistics

Data were analyzed using graphpad prism 8·3.1 (GraphPad Software, San Diego, CA). Results are presented as individual data points and/or mean ± standard deviation (SD). Differences between values were analyzed for statistical significance by Student’s t‐test, the non‐parametric Kruskal–Wallis test, one‐way or two‐way analysis of variance with appropriate multiple comparisons tests. Differences were considered significant at P < 0·05.

Results

RA induces CD103 expression in monocytes and MoDCs, but not in differentiated monocyte‐derived macrophages

In a recent study, we demonstrated that human MoDCs that were differentiated in the presence of RA for 3 days significantly up‐regulated CD103 and integrin β 7 expression. 13 To determine whether the ability to up‐regulate CD103 was specific to DCs and required early, long‐term RA exposure, we here performed parallel experiments with monocytes, monocyte‐derived macrophages (MDMs) and MoDCs with varying RA exposure protocols. MACS‐purified CD14+ monocytes were treated with RA alone or in combination with either granulocyte–macrophage colony‐stimulating factor and interleukin‐4 to induce differentiation to MoDCs or in combination with macrophage colony‐stimulating factor to induce differentiation to MDMs. Interestingly, a one‐day exposure of freshly isolated blood monocytes to RA was sufficient to increase the percentage of CD103‐expressing cells in monocytes and to a greater extent in monocytes cultured in the presence of DC or macrophage‐polarizing cytokines (P < 0·01; Fig. 1a–c). We next differentiated monocytes into either MoDCs or MDMs for 3 days before adding RA for another 24 hr (Fig. 1d). Differentiation was confirmed based on significantly decreased CD14 expression in the MoDCs compared with monocytes and MDMs (P = 0·008, see Supplementary material, Fig. S1a,b), and significant up‐regulation of CD13 in both MoDCs and MDMs (P = 0·009, see Supplementary material, Fig. S1c). 32 Interestingly, RA treatment of all three cell types led to a significantly increased expression of CD14 (P = 0·004, see Supplementary material, Fig. S1b). However, in contrast to our previous study, where MoDCs that were differentiated in the presence of RA showed decreased expression of HLA‐DR, CD83 and CD86, 13 a 1‐day exposure of differentiated MoDCs to RA failed to down‐regulate HLA‐DR (see Supplementary material, Fig. S1d) or co‐stimulatory molecules (not shown). Importantly, as shown in Fig. 1e,f, a 24‐hr exposure to RA induced a significant increase in CD103 expression in differentiated MoDCs, but not in MDMs. These observations indicate that RA‐dependent induction of CD103 is cell‐type specific, with DCs being particularly responsive to RA treatment. Importantly, the data shown here together with the data from our previous study 13 indicate that the ability of RA to induce CD103 expression is highly robust regarding timing and length of RA exposure.

Figure 1.

Figure 1

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Retinoic acid (RA) induces increased expression of CD103 in monocytes and monocyte‐derived dendritic cells (MoDCs), but not in differentiated monocyte‐derived macrophages (MDMs). (a) Schematic of experimental design for (b) and (c). Isolated CD14+ monocytes were cultured for 1 day with or without RA. Interleukin‐4 (IL‐4) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) were added to differentiate monocytes into MoDCs, and macrophage colony‐stimulating factor (M‐CSF) was added to differentiate monocytes into MDMs. (b) FACS analysis of CD103 expression on monocytes cultured under different conditions; pooled data from three independent experiments; individual data points, mean ± SD are shown. (c) Representative histograms of RA‐treated and untreated cells from (b). (d) Schematic of experimental design for panels (e) and (f). CD14+ monocytes were cultured for 3 days with IL‐4/GM‐CSF or with M‐CSF. On day 3, RA was added to the culture for 1 day and cells were harvested for FACS analysis. (e) Pooled FACS data from three or four independent experiments showing CD103 expression on different cell types. (f) Representative histograms of data in (e). Isotype control – gray histogram, CD103 antibody – black solid line. One‐way analysis of variance with Dunnett’s multiple comparisons test; *P ≤ 0·05. **P ≤ 0·01.

RA does not induce CD103 expression in CD4+ or CD8+ T cells

Previous reports have shown that TGF‐β can induce CD103 expression on T cells, 28 , 33 , 34 , 35 but neither TGF‐β 1 nor TGF‐β 2 induced CD103 expression by human DCs in our previous report, 13 again pointing to cell‐type‐specific regulatory mechanisms for CD103 induction. To determine whether RA would induce the expression of CD103 expression in T cells, we treated lymphocytes with RA. Following 24 hr of culture, the cells were harvested and stained for T‐cell markers and CD103 (Fig. 2a). Gating strategies used to identify CD4+ and CD8+ T ‐cell populations are shown in Fig. 2(b). Interestingly, RA did not significantly increase the expression of CD103 on CD4+ or CD8+ T cells (Fig. 2c,d). A small increase of CD103 expression was seen in CD8+ T cells treated with TGF‐β 1 or TGF‐β 2 (Fig. 2d), consistent with previous reports, 28 , 33 , 34 but TGF‐β had no apparent effect on CD103 expression by CD4+ T cells (Fig. 2c). Similarly, no CD103 expression was detected with any of the treatments in CD14‐depleted CD3− cells, which contain a large proportion of B cells (data not shown).

Figure 2.

Figure 2

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Retinoic acid (RA) does not induce significant CD103 expression in blood T cells. (a) Schematic of experimental design for panels (b) to (d). CD14‐depleted peripheral blood mononuclear cells (PBMCs) were cultured for 1 day with or without RA, transforming growth factor β 1 (TGF‐β 1) or TGF‐β 2 and harvested for FACS analysis. (b) Gating schematic for analysis of CD8+ and CD4+ T cells. (c, d) Pooled data from four independent experiments show (c) no CD103 expression on CD4+ T cells and (d) low CD103 expression on CD8+ T cells with or without addition of RA, TGF‐β 1 or TGF‐β 2. (e) Schematic of experimental design for panels (f) to (i). PBMCs remaining after CD14+ monocyte depletion were cultured for 5 days with or without RA and anti‐CD3/CD28 beads and were harvested for FACS analysis. (f) Pooled data from three experiments and (g) representative FACS histograms showing the percentage of proliferating T cells after each treatment as measured by CellTrace Violet for CD4+ T cells (top graphs) and CD8+ T cells (bottom graphs). Two‐way analysis of variance with Dunnet’s multiple comparisons test; *indicates statistical significance (P < 0·01). (h) Pooled FACS data from three experiments (with mean ± SD) and (i) representative dotplots showing CD103 expression on proliferating CD4+ and CD8+ T cells after 5 days in culture with or without RA treatment.

We next analyzed whether T cells respond to RA after polyclonal stimulation with anti‐CD3/CD28 beads (Fig. 2e). The presence of RA in addition to the anti‐CD3/CD28 significantly increased proliferation of CD8+ T cells (P ≤ 0·01) and slightly increased proliferation of CD4+ T cells (Fig. 2f,g). However, CD103 expression in both CD4+ and CD8+ T cells remained unchanged, regardless of experimental condition (Fig. 2h,i), although CD103 expression overall was higher in cells that had divided. In summary, these data suggest that the ability of RA to induce CD103 expression is specific to the myeloid cell compartment, particularly DCs.

RA‐induced expression of CD103 in human MoDCs is mediated primarily by RARα

Retinoic acid signaling is mediated through three different RAR subtypes with different expression patterns and target genes, RARα, RARβ, or RARγ. 36 In order to elucidate the RA signaling pathway involved in RA‐induced CD103 expression, we used agonists specific for the different RARs, which we added to MoDC cultures for 3 days during MoDC differentiation (Fig. 3a). Treatment of MoDCs with 1 and 10 µm AM80, an RARα‐specific agonist, induced an increase in the percentage of CD103+ cells similar to that induced by treatment with 100 nm of RA (Fig. 3b,d). In contrast, the RARβ agonist CD2314 did not increase the expression of CD103 in MoDCs, whereas treatment with the RARγ agonist CD437 caused a significant increase at 10 µm, but not at 1 µm (Fig. 3b,d). Similar patterns were observed when geometric mean fluorescence intensity was analyzed (see Supplementary material, Table S1). CD103 (integrin α E) generally forms heterodimers with integrin β 7, and we have previously shown that CD103 and integrin β 7 had similar expression patterns when MoDCs were cultured with RA. 13 However, none of the RAR agonists caused a significant increase in integrin β 7 expression, although the overall β 7 expression pattern in response to the agonist treatment was similar to that of CD103 (Fig. 3c, and see Supplementary material, Table S2).

Figure 3.

Figure 3

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Retinoic acid receptor α (RARα) mediates RA‐induced expression of CD103 on monocyte‐derived dendritic cells (MoDCs). (a) Schematic of experimental design for (b) to (d). MoDCs were cultured with RA, AM80 (RARα agonist), CD2314 (RARβ agonist), or CD437 (RARγ agonist) before harvest for FACS analysis after 3 days. RAR agonists were added at concentrations of 1 µm or 10 µm. Expression of (b) CD103 and (c) integrin β 7 on MoDCs was analyzed by FACS. Pooled data from three independent experiments; mean ± SD; one‐way analysis of variance with Tukey’s multiple comparisons test. Symbols indicate significant difference compared with medium control; *P ≤ 0·05; **P ≤ 0·01; ***P ≤ 0·001. (d) Representative data showing CD103 expression on MoDCs with RA and RAR agonists. Isotype control – gray histogram, CD103 antibody – black solid line. (e) Schematic of experimental design for panels (f) to (h). MoDCs were differentiated for 3 days before addition of scramble siRNA or RARα siRNA. On day 4, RA was added to the culture medium for 1 day before harvest and analysis by qRT‐PCR. (f) Relative gene expression of RARA, the gene encoding for RARα, after treatment with RARα siRNA; representative experiment with n = 2 technical replicates. Relative gene expression of (g) ITGAE, gene encoding CD103, and (h) ITGB7, gene encoding integrin β 7, of MoDCs after treatment with RA and RARα siRNA. RT‐qPCR analyzed using the Pfaffl method 31 and normalized to GAPDH. Pooled data from three independent experiments; mean ± SD. *Reveals statistically significant difference from media control (P ≤ 0·05). One‐way analysis of varaince with Tukey’s multiple comparisons test.

Having demonstrated that signaling through RARα is most efficient at inducing CD103 expression in DCs, we next used siRNA knockdown of RARA to inhibit RARα signaling. For knockdown experiments, MoDCs were differentiated for 3 days and then treated with RARα siRNA or a non‐specific scramble siRNA to block DC expression of RARα. RA was added to the MoDCs 24 hr after siRNA treatment, and cells were harvested 24 hr after RA treatment (Fig. 3e). The shorter RA exposure was used, because siRNA decreased DC viability. As expected, RARα siRNA reduced gene expression of the RARA mRNA (Fig. 3f). Gene expression analysis revealed that the RA treatment protocol in this experiment caused a significant up‐regulation of ITGAE (CD103) gene expression in cells treated with an unspecific siRNA. Importantly, MoDCs treated with RARα siRNA and RA showed a significant decrease in ITGAE expression compared with MoDCs that were treated with RA and scramble siRNA (Fig. 3g). A similar gene expression pattern was observed with ITGB7 (gene name of integrin β 7) mRNA, although observed changes were not significant (Fig. 3h). Interestingly, experiments with two different RARα inhibitors led to a paradoxical increase in CD103 expression (see Supplementary material, Fig. S2), pointing to the involvement of additional feedback mechanisms. Overall, our data suggest that the RA‐induced expression of CD103 in human DCs is predominantly mediated through RARα signaling.

TGF‐β 1 but not RA drives phosphorylation of SMAD2/3 in human DCs

We have previously shown that inhibition of the TGF‐β signaling pathway disrupts the RA‐induced increase in CD103 expression in DCs. 13 However, the addition of TGF‐β 1 or TGF‐β 2 during MoDC differentiation did not influence CD103 expression. 13 Similarly, concurrent exposure of MoDCs to both RA and TGF‐β did not lead to greater CD103 expression than exposure to RA alone (data not shown). Several previous studies in other cell types indicate that there may be functional interactions between RA and TGF‐β signaling pathways. 13 , 37 Therefore, we here evaluated whether RA could induce the phosphorylation of SMAD2/3 in DCs, as SMAD2/3 activation is the major signaling pathway downstream of the TGF‐β receptor. MoDCs were differentiated for 3 days in the presence of RA, following our standard protocol. Alternatively, TGF‐β 1, or TGF‐β 1 and the TGF‐β signaling inhibitor SB431542, were added to cultures of differentiated MoDCs for 30 min to induce SMAD2/3 activation (Fig. 4a). As expected, there was a significant increase in the phosphorylation of SMAD2/3 when MoDCs were cultured with TGF‐β 1, which was blocked upon the addition of SB431542 (Fig. 4b,c). Interestingly, we found no increase in the phosphorylation of SMAD2/3 when MoDCs were cultured in the presence of RA (Fig. 4b,c). This suggests that, consistent with our previous observations, RA signaling in human DCs likely interacts with TGF‐β signaling 13 but is independent of the SMAD pathway.

Figure 4.

Figure 4

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Retinoic acid (RA) does not significantly increase the phosphorylation of SMAD2/3 in monocyte‐derived dendritic cells (MoDCs). (a) Schematic of experimental design. MoDCs were cultured with or without RA for 3 days. Transforming growth factor‐β 1 (TGF‐β 1) or TGF‐β 1 plus SB431542, a TGF‐β 1 inhibitor, were added to the cell culture, and cells were harvested for FACS analysis of SMAD2/3 phosphorylation 2 hr later. (b) FACS analysis of SMAD2/3 phosphorylation in MoDCs, pooled data from three independent experiments; mean ± SD. (c) Representative FACS histograms of SMA2/3 phosphorylation. Isotype control – gray‐shaded, SMAD2/3 antibody – solid black line. **Indicates statistical significance from the medium only control, P ≤ 0·01. Kruskal–Wallis one‐way analysis of variance with Dunn’s multiple comparisons test.

RA increases the phosphorylation of p38 MAPK in human MoDCs

A review of the literature revealed that p38 MAPK is involved in both TGF‐β and RA signaling pathways. Importantly, Yu et al. demonstrated that TGF‐β‐induced p38 MAPK signaling is independent of SMAD2/3 activation. 38 In addition, several recent studies have established that RA can increase the phosphorylation of p38 MAPK in multiple cell types. 20 , 21 , 23 , 27 , 39 Based upon these observations and our current and previous data that demonstrated involvement of TGF‐β pathways, 13 but not SMAD signaling, we investigated whether p38 MAPK plays a role in the RA‐induced expression of CD103 on DCs. MoDCs cultured with RA for 3 days were harvested for Western blot analysis to investigate p38 MAPK expression and phosphorylation (Fig. 5a). Interestingly, we found that MoDCs cultured with RA demonstrated significantly increased phosphorylation of p38 MAPK compared with MoDCs cultured with medium alone (Fig. 5b,c). Baseline p38 MAPK expression was not affected by RA treatment of the DCs (Fig. 5b). These data demonstrate that p38 MAPK is activated by RA signaling in human DCs.

Figure 5.

Figure 5

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Retinoic acid (RA)‐induced CD103 expression in MoDCs is dependent on p38 mitogen‐activated protein kinase (MAPK) signaling. (a) Schematic of experimental design. Monocyte‐derived dendritic cells (MoDCs) were cultured for 3 days with RA or SB202190, a p38 MAPK inhibitor, before harvest for Western blot, PCR or FACS analysis. (b) Representative Western blot of MoDCs cultured with RA. (c) Band intensity of phosphorylated p38 protein normalized to GAPDH and total p38 MAPK protein. Pooled data from three independent experiments; mean ± SD. (d) Relative ITGAE gene expression of MoDCs cultured with RA and SB202190. Gene expression was analyzed by TaqMan qRT‐PCR, data were analyzed using the Pfaffl method. Pooled data from three independent experiments; mean ± SD. FACS analysis of CD103 (e, f) expression and (g) integrin β 7 expression in MoDCs treated with RA and/or SB202190 (e) Pooled data (n = 3) and (f) representative data for CD103 expression. Isotype control – gray histogram, CD103 antibody – black solid line. (g) Pooled data for β 7 expression; n = 4. *Represents statistically significant difference from medium control at P ≤ 0·05. One‐way analysis of variance with Tukey’s multiple comparisons test.

RA‐induced expression of CD103 in human MoDCs is dependent on p38 MAPK signaling

We next determined whether p38 MAPK activation is involved in the RA‐induced expression of CD103. To that end, MoDCs were differentiated for 3 days in the presence of RA and the p38 MAPK inhibitor SB202190 (Fig. 5a). When MoDCs were cultured with RA, we found the expected significant increase in ITGAE mRNA (Fig. 5d). However, MoDCs cultured with both RA and the p38 MAPK inhibitor showed ITGAE mRNA levels similar to baseline (Fig. 5d). In contrast, addition of the p38 inhibitor did not prevent the RA‐induced up‐regulation of IL10, IL23A and IL12A and down‐regulation of pro‐inflammatory cytokines IL6 and TNFA (see Supplementary material, Fig. S3), suggesting that the regulatory mechanisms for ITGAE gene expression are unique to this integrin. Notably, in contrast to monocytes treated with RA for 18 hr, 14 ALDH1A2 and RDH10 were not significantly up‐regulated in MoDCs differentiated in the presence of RA for 3 days (see Supplementary material, Fig. S3), consistent with our previous study 13 and pointing to a transient regulation of RA biosynthesis pathways by RA.

We next used flow cytometry to confirm that inhibition of p38 signaling inhibits the RA‐induced increase in CD103 protein expression. Indeed, when MoDCs were cultured with RA and the p38 MAPK inhibitor, the RA‐induced CD103 protein expression was completely abrogated (Fig. 5e,f). Integrin β 7 followed the same pattern of expression, although no statistically significant difference was established (Fig. 5g). Similar trends were observed when geometric mean fluorescence intensity was analyzed (see Supplementary material, Tables S1 and S2). These data indicate that RA‐induced expression of CD103 in human DCs is dependent on p38 MAPK signaling.

RA induces CD103 in human blood DCs through a p38 MAPK‐dependent pathway

In our previous study, we were unable to demonstrate induction of CD103 expression in primary DCs isolated from human stomach tissue. 13 We hypothesized that the inability of gastric DCs to respond to RA was due to tissue‐specific mechanisms. However, an alternative interpretation of these observations could be that MoDCs are not representative of primary human DC populations in their responsiveness to RA. To determine whether human DCs other than MoDCs up‐regulate CD103 in response to RA, we here used primary DCs from human blood to determine whether RA would induce increased CD103 expression through a p38‐dependent pathway. We used magnetic beads to enrich CD141+ conventional DC1s (cDC1s), CD1c+ cDC2s, and CD304+ plasmacytoid DCs (pDCs) from peripheral human blood and treated the cells with RA in the presence or absence of the p38 inhibitor for 24 hr (Fig. 6a,b). This exposure protocol was used, because blood DCs are already differentiated, but have a short lifespan. Importantly, both cDC1s and cDC2s showed significantly increased expression of CD103 following RA treatment (Fig. 6c–f). This increase in CD103 expression was completely prevented by adding the p38 inhibitor SB202190 to the RA‐treated cultures (Fig. 6c–f). A similar trend was observed for CD304+ pDCs; however, the percentage of CD304+ pDCs in our blood DCs was generally too low to provide reliable data (data not shown). These data demonstrate that primary human DCs respond to RA treatment by increasing CD103 expression through a p38‐dependent pathway, as also shown for MoDCs.

Figure 6.

Figure 6

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Retinoic acid (RA)‐induced expression of CD103 on blood dendritic cell (DC) subsets is dependent on p38 mitogen‐activated protein kinase (MAPK) signaling. (a) Schematic of experimental design. Blood DCs were isolated for healthy adult donors and cultured for 1 day with the addition of RA and SB202190, a p38 MAPK inhibitor, before harvest and FACS analysis. (b) FACS gating strategy of human blood DC subsets; CD141 – cDC1s, CD1c – cDC2s. Representative FACS histograms of CD103 expression on (c) cDC1s and (d) cDC2s. Isotype control – gray histogram, CD103 antibody – black solid line. (e, f) Pooled FACS data showing CD103 expression by CD141+ cDC1s and CD1c+ cDC2s. Individual data, mean ± SD, n = 5. One‐way analysis of variance with Tukey’s multiple comparisons test; *P ≤ 0·05, **P ≤ 0·01, **P ≤ 0·001.

Inhibition of NFAT signaling prevents RA‐induced up‐regulation of CD103 in MoDCs

Previous studies have described that p38 MAPK can activate the transcription factor NFAT. 40 , 41 Moreover, NFAT has been shown to interact with the enhancer region of CD103 in T cells. 28 Based on these studies, we next asked whether NFAT activation might contribute to the induction of CD103 expression downstream of RARα and p38 MAPK in human DCs. MoDCs were differentiated for 3 days and then were exposed to RA and various concentrations of an NFAT peptide inhibitor for 24 hr (Fig. 7a). Importantly, as seen for the p38 inhibitor, the NFAT peptide inhibitor caused a dose‐dependent decrease in the expression of CD103 in MoDCs cultured with RA that was significant at 50 µm (Fig. 7b,c and see Supplementary material, Table S1). These data suggest that NFAT may be involved in the RA‐induced expression of CD103 in human DCs downstream of RARα and p38. However, further experimental verification is needed to link RARα, p38 MAPK and NFAT signaling to CD103 expression following DC exposure to RA.

Figure 7.

Figure 7

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Inhibition of nuclear factor of activated T cells (NFAT1c) abrogates retinoic acid (RA) ‐induced expression of CD103 on monocyte‐derived dendritic cells (MoDCs). (a) Schematic of experimental design. MoDCs were differentiated for 3 days before the addition of RA and 10–50 µm NFAT inhibitor. MoDCs were harvested 1 day after addition of RA and NFAT inhibitor and CD103 expression was analyzed by FACS. (b) Expression of CD103 on MoDCs treated with RA and different concentrations of NFAT inhibitor by FACS analysis; n = 4 independent experiments; mean ± SD. (c) Representative FACS histograms of CD103 expression. Isotype control – gray‐shaded histogram, CD103 antibody (black solid line). Indicates statistical significance. One‐way analysis of variance with Tukey’s multiple comparisons test, *P ≤ 0·05.

Discussion

Vitamin A is a key dietary micronutrient that is involved in the regulation of growth and development, vision and mucosal integrity and that promotes anti‐inflammatory immune responses. 1 , 2 , 3 In the mammalian immune system, most effects of vitamin A are mediated by its major metabolite, RA. 42 In a previous study that investigated the effect of RA on human DCs, we showed that RA drives the expression of CD103 (integrin α E) and integrin β 7. 13 However, the molecular signaling pathway that regulates CD103 expression remains unknown.

Retinoic acid is involved in the establishment and maintenance of DC populations within the intestinal mucosa. 4 , 5 , 8 Specifically, RA has an essential role in the development of pre‐mucosal DCs that give rise to intestinal DCs, 5 and studies in mice showed that the development of the cDC2 population in the small intestinal and mesenteric lymph nodes is dependent on RA signaling through RARα. 4 , 8 It has been well established in human and mouse studies that RA increases RA biosynthesis in DCs through a positive feedback loop. 12 , 14 Moreover, murine CD103+ mesenteric lymph node DCs have higher expression of Aldh1a2 than CD103− DCs, 2 and are potent inducers of T regulatory cells and gut homing molecule expression. 16 , 43 Taken together, these data point to multiple functionally relevant interactions between RA production, CD103 expression and tolerogenic DC function in the gut. Therefore, we here sought to define the regulatory pathways that control these interactions in more detail.

Our experiments revealed that RA‐driven expression of CD103 is specific to DCs and monocytic cells. Hence, CD103 up‐regulation was consistently observed in MoDCs, regardless of differentiation stage, and also in different subsets of human blood DCs. Interestingly, both cDC1s and cDC2s isolated from human blood were more responsive to the RA than MoDCs, regardless of the RA treatment protocol used. Monocytes cultured in media alone or with cytokines to differentiate into either a macrophage or DC phenotype showed a moderate increase in the expression of CD103 upon culture with RA. In contrast, differentiated monocyte‐derived macrophages treated with RA showed no significant induction of CD103 expression. Similarly, we saw no change in CD103 expression in RA‐treated resting or proliferating CD4+ and CD8+ T cells.

To elucidate the signaling pathway involved in RA‐induced expression of CD103, we investigated the RAR involved. RA can interact with three different nuclear receptors, RARα, RARβ and RARγ, all of which form heterodimers with the retinoid X receptor (RXR) to induce gene expression upon RA stimulation. 36 We here established that RA‐induced CD103 expression is predominantly RARα‐dependent. MoDCs cultured with RA or the RARα agonist, AM80, had a fivefold increase in CD103 expression, and to a lesser extent integrin β 7. In contrast, the RARβ agonist caused only a minor increase in CD103 expression. With the RARγ agonist, high‐dose treatment was required to induce CD103 up‐regulation, which could be due to the cross‐reactivity with RARα that the RARγ agonist exhibits at higher concentrations. 44 The importance of RARα in RA‐mediated CD103 expression was further confirmed through siRNA knockdown experiments, which led to the abrogation of RA‐induced CD103 expression.

Retinoic acid can modify gene expression through two distinct signaling mechanisms. Classical genomic RA effects involve the binding of RAR to target DNA as a transcription factor, which can either repress or induce transcription of target genes. 20 , 21 , 22 , 23 , 45 Non‐genomic RA effects involve the activation of kinase cascades in the cytoplasm, which can indirectly impact gene expression through alternate pathways. 20 , 21 , 23 , 27 , 39 In our investigations to understand the RA‐induced signaling that leads to CD103 expression in DCs, we looked to identify non‐genomic RA involvement. We previously showed that in the presence of RA, TGF‐β signaling plays a role in the expression of CD103. Inhibition of the TGF‐β receptor II (TGF‐βRII) blocked RA‐induced up‐regulation of CD103 in human DCs. 13 Similarly, knockout mice with a DC‐specific deletion of TGF‐βR had fewer CD103+ DCs and in turn a decrease in Foxp3+ T regulatory cells. 46 In human adenocarcinoma cells, RA‐induced expression of VE‐cadherin was abrogated when TGF‐βR was inhibited. 37 Interestingly, our experiments showed that RA‐induced CD103 expression was independent of SMAD2/3 activation, the major signaling pathway downstream of the TGF‐βR. p38 MAPK is a known component of the TGF‐β signaling cascade, and p38 MAPK activation by TGF‐β is independent of SMAD signaling. 38 Therefore, we hypothesized that p38 MAPK signaling could also be involved in RA‐dependent CD103 expression. The results from Western blot analysis of p38 MAPK support this hypothesis, as RA treatment led to increased activation of p38 MAPK, as shown by increased levels of phosphorylated p38 MAPK. Notably, RA treatment of MoDCs did not alter overall p38 MAPK expression, pointing to altered signaling pathways consistent with non‐genomic activation pathways rather than altered expression due to genomic regulation. Our experiments using an inhibitor of p38 MAPK strongly suggest that p38 MAPK signaling is involved in the induction of CD103 expression in both MoDCs and blood DC subsets, because p38 MAPK inhibition in RA‐treated MoDCs resulted in a more than twofold decrease of CD103 expression compared with DCs cultured with RA only. The role of p38 MAPK in RA signaling has previously been demonstrated in human osteoblasts and mouse embryonic fibroblasts. 23 , 39 Specifically, the additive effect of RA and TGF‐β on VEGF expression in osteoblasts was highly dependent on the RA‐induced p38 MAPK signaling. 39 Additionally, in murine embryonic fibroblasts, RA‐driven expression of Cyp26A1 was abrogated when p38 MAPK signaling was blocked. 23 Hence, the results from our investigations with human DCs corroborate previous reports that have demonstrated the importance of p38 MAPK in RA signaling.

We next sought to evaluate the downstream mediators of p38 MAPK signaling. Multiple studies have shown that p38 MAPK can have direct interactions with the immune cell transcription factor NFAT. 40 , 41 Furthermore, investigations by Mokrani et al. revealed that NFAT interacts with the enhancer region of CD103 in T cells. 28 Our results point to a role for NFAT in CD103 regulation downstream of RARα and p38 MAPK, as increasing concentrations of NFAT inhibitor prevented RA‐induced up‐regulation of DC CD103 expression in a dose‐dependent manner. Future experiments will evaluate whether the RARα‐p38‐NFAT axis is the major pathway for the induction of CD103 in human blood DCs, monocytes and MoDCs. Whether RA induction of CD103 also occurs in human tissues is still unclear. Hence, we were unable to show significant up‐regulation of CD103 in RA‐treated human gastric DCs. 13 In mice, vitamin A deficiency leads to decreased numbers of CD103+ CD11b+ DCs, but not of CD103+ CD11b− DCs in the intestine, 8 suggesting that additional mechanisms contribute to the regulation of CD103 downstream of RA.

In summary, we have elucidated a putative signaling pathway involved in the RA‐induced expression of CD103 in human DCs. Our data show that expression of CD103 in human DCs was dependent on RARα and p38 MAPK signaling. Importantly, this effect was cell specific for MNPs, because T cells did not respond in the same way. A lack of sufficient nutritional RA has global health implications directly related to DC function, and CD103 expression has key functional correlates in mucosal immunity. Therefore, unraveling the DC‐specific signaling pathways downstream of RA leading to CD103 expression contributes to our overall understanding of these important molecular players.

Disclosures

The authors declare no conflict of interest.

Author contributions

MMR developed the project, designed and performed experiments, analyzed data, critically interpreted the data, and wrote and revised the manuscript; MH performed experiments, analyzed data and revised the manuscript; SS planned experiments, analyzed data and revised the manuscript; KMW performed experiments; DB developed the project, designed the experiments, analyzed the data, critically interpreted the data, wrote and revised the manuscript, and provided funding for the project.

Supporting information

Figure S1. One‐day RA treatment of monocytes, MoDCs and MDMs increases expression of CD14, but does not alter HLA‐DR or CD13 expression.

Click here for additional data file. (381.1KB, png)

Figure S2. Inhibitors of RARα increase the expression of CD103 in MoDCs.

Click here for additional data file. (699.8KB, png)

Figure S3. MoDC gene expression after treatment with RA and p38 inhibitor.

Click here for additional data file. (380.7KB, png)

Table S1. CD103 Mean Fluorescence Intensity.

Table S2 β7 Integrin Mean Fluorescence Intensity.

Click here for additional data file. (19.6KB, docx)

Acknowledgements

Funding for this study was provided by the National Institutes of Health (NIH) Grants P20GM103474 (pilot funding to DB), R03 DK107960 and U01 EB029242 (to DB) and the USDA National Institute of Food and Agriculture Hatch Project 1015768 (DB); the American Association of Immunologists (AAI) Careers in Immunology Fellowship (MMR and DB). We appreciate the generous support of our core facilities through the M.J. Murdock Charitable Trust and the NIH National Institute of General Medical Sciences IDeA Program P30GM110732. The authors greatly appreciate the support from the Montana State University Agricultural Experimental Station for the Flow Cytometry Core Facility at Montana State University; Drs Mark Jutila, Jovanka Voyich, Edward Schmidt and Douglas Kominsky for providing useful discussions; Andy Sebrell and Melissa Donovan for laboratory support. The authors’ sincerest thanks go to all the volunteers who donated blood samples for this study.

Data availability statement

The data that support the findings of this study are available from the corresponding author, D.B., upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. One‐day RA treatment of monocytes, MoDCs and MDMs increases expression of CD14, but does not alter HLA‐DR or CD13 expression.

Click here for additional data file. (381.1KB, png)

Figure S2. Inhibitors of RARα increase the expression of CD103 in MoDCs.

Click here for additional data file. (699.8KB, png)

Figure S3. MoDC gene expression after treatment with RA and p38 inhibitor.

Click here for additional data file. (380.7KB, png)

Table S1. CD103 Mean Fluorescence Intensity.

Table S2 β7 Integrin Mean Fluorescence Intensity.

Click here for additional data file. (19.6KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, D.B., upon reasonable request.

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