Beta 1-integrin ligation and TLR ligation enhance GM-CSF–induced ALDH1A2 expression in dendritic cells, but differentially regulate their anti-inflammatory properties

Retinoic acid (RA)–producing CD103+ mature dendritic cells (DCs) in mesenteric lymph nodes (MLNs) play crucial roles in gut immunity. GM-CSF and RA contribute to the expression of the RA-producing enzyme ALDH1A2. However, additional signals appeared to be required for inducing ALDH1A2high mature DCs from immature DCs. We found here that TLR ligands (Ls) and immobilized E-cadherin could provide such signals in FLT3-L–generated bone marrow (BM)–derived DCs after treatment with GM-CSF and the RA receptor agonist Am80. The TLR-L-treated DCs produced proinflammatory cytokines unlike normal ALDH1A2high MLN-DCs, whereas the E-cadherin-treated DCs did not. Immobilized VCAM-1 and semaphorin 7 A exerted effects similar to those of E-cadherin. Soluble anti-integrin β1 antibodies or inhibitors of integrin signaling molecules suppressed the effects of these immobilized proteins, whereas immobilized anti-integrin β1 antibodies enhanced the GM-CSF/Am80-induced ALDH1A2 expression without inducing proinflammatory cytokines. Sequential stimulation of splenic pre-DCs with GM-CSF/Am80 and immobilized E-cadherin or anti-integrin β1 antibody also induced differentiation to mature DCs with high ALDH activity. The E-cadherin-treated BM-DCs induced gut-tropic Foxp3+ T cells and alleviated DSS–induced colitis, whereas the TLR-L-treated DCs aggravated DSS–induced colitis. The results suggest that integrin β1-mediated signals contribute to the differentiation and maturation of RA-producing anti-inflammatory DCs.

Scientific RepoRts | 6:37914 | DOI: 10.1038/srep37914 Flow cytometry. ALDH activity in individual cells was estimated using ALDEFLUOR staining kits (Stemcell Technologies) as previously described 14 . Cells were stained with indicated mAbs conjugated to various fluorochromes in the presence of anti-CD16/CD32 mAb. Intracellular staining of Foxp3 was performed using mouse/ rat Foxp3 staining sets (eBioscience). For intracellular cytokine staining, CD4 + T cells obtained from cultures were restimulated for 5 h with PMA (50 ng/ml) and ionomycin (750 ng/ml) (both from Calbiochem). Monensin (3 μ M; Sigma-Aldrich) was added to the cultures for the last 2 h. After surface staining, the cells were fixed with Fixation Buffer (BioLegend), and intracellular cytokine staining was performed according to the manufacturer's protocol. Analysis was performed on a FACSAria or FACSCalibur with CellQuest Pro software (BD Biosciences).
Dextran sulfate sodium (DSS)-induced acute colitis. Colitis was induced by administration of DSS as described previously 24 with slight modifications. Briefly, male C57BL/6 mice aged 8-9 wk were administered 2% DSS (m.w. 36,000-50,000; MP Biochemicals, Solon, OH) in their drinking water ad libitum for 4 days followed by feeding with regular drinking water. The mice were injected intraperitoneally with BM-DCs (1 × 10 6 in 0.1 ml of PBS/mouse) or PBS alone on days 0 and 2, and their body weights were recorded daily. The mice were sacrificed on day 10, and their colon lengths were assessed. Statistical analysis. Statistical comparisons were performed using the one-way ANOVA with Tukey-Kramer multiple comparisons test and the two-tailed unpaired Student's t test. Values less than 0.05 were considered statistically significant.

Two-day stimulation with the combination of GM-CSF and RAR ligands induces both CD103
and ALDH1A2 expression in FLT3-L-generated BM-DCs, and subsequent stimulation with E-cadherin significantly enhances the ALDH1A2 expression without inducing proinflammatory cytokines. In FLT3-L-generated BM-DCs, GM-CSF and the RAR agonist Am80 synergistically enhanced Aldh1a2 expression and ALDH activity in BM-DCs after 1 day of culture (Fig. 1a). Simultaneous stimulation of BM-DCs with GM-CSF and the TLR2/1-L Pam3CSK4 in the presence or absence of Am80 induced high levels of ALDH activity, as we previously reported 14 , but reduced CD103 expression (Fig. 1a). On the contrary, stimulation of BM-DCs with only the combination of GM-CSF and Am80 for 2 days followed by replating (transferring the cells to new culture plates) and 1-day culture enhanced ALDH activity (Fig. 1b). Replating by itself moderately enhanced the effect of GM-CSF/Am80 on ALDH activity and the expression of maturation markers (data not shown). The addition of Pam3CSK4 to the replated culture markedly enhanced ALDH activity and the expression of Aldh1a2 and surface maturation markers including CD40, CD86, and MHC class II without reducing GM-CSF/Am80-induced CD103 expression (Fig. 1b,c). Similar results were obtained using pattern recognition receptor (PRR)-Ls including LPS, CpG ODN, and curdlan instead of Pam3CSK4 (see Supplementary Fig. S1). The PRR-L-treated DCs produced proinflammatory cytokines including IL-6, IL-12p40, IL-23p19, and TNF-α (Fig. 1d, also see Supplementary Fig. S1). However, it is known that CD103 + MLN-DCs from steady-state SPF mice weakly produce proinflammatory cytokines upon stimulation through CD40 or with LPS 9 . Therefore, alternative stimuli appeared to contribute to the development of CD103 + MLN-DCs with high ALDH activity (ALDH high ) in naïve SPF mice.
To identify signals that enhance ALDH1A2 expression but induce low levels of proinflammatory cytokine production, we first hypothesized that CD103 might be involved in the induction of ALDH1A2 expression in the intestinal DCs. Most ALDH1A2 + MLN-DCs and LP-DCs express CD103, whereas intestinal epithelial cells (IECs) express E-cadherin, which can bind to CD103 25 . Furthermore, IECs are known to promote ALDH1A expression in DCs 26,27 . Simultaneous stimulation of BM-DCs with GM-CSF/Am80 and immobilized E-cadherin/ Fc chimera protein induced ALDH activity, but this strategy could not enhance the CD103 expression in ALDH + cells (Fig. 1a). Conversely, stimulation of BM-DC with GM-CSF/Am80 for 2 days followed by 1-day culture with E-cadherin markedly enhanced ALDH activity and the expression of CD40, CD86, MHC class II, and Aldh1a2 without suppressing the GM-CSF/Am80-induced CD103 expression (Fig. 1b,c). Similar results were obtained using RA instead of Am80 (data not shown). This protocol also enhanced CCR7 expression, which is required for the migration of LP-DCs to MLNs (Fig. 1b) 28 . Both Aldh1a2 expression and ALDH activity in MLN-DCs were much higher than those in LP-DCs in naïve SPF mice, and were diminished in MLN-DCs and LP-DCs in GM-CSF-deficient mice (see Supplementary Fig. S2), suggesting that the present protocol may mimic the GM-CSF-dependent maturation process of migratory RA-producing DCs in the intestine. Unlike PRR-L, E-cadherin did not trigger GM-CSF/Am80-treated BM-DCs to produce proinflammatory cytokines (Fig. 1d). E-cadherin partly suppressed the Pam3CSK4-induced production of IL-12p40 and IL-23p19 but not that of IL-6. However, after treatment with E-cadherin, Pam3CSK4 induced much lower levels of these three cytokines, suggesting that the E-cadherin-treated ALDH1A2 high CD103 + DCs do not become proinflammatory as easily as immature DCs.
As the small intestine is rich in TGF-β 29 , we also examined the effect of TGF-β on BM-DCs. The addition of TGF-β together with GM-CSF and Am80 to BM-DC cultures enhanced their expression of CD103 but rather suppressed ALDH activity and the expression of CD86 and Aldh1a2 after subsequent culture with or without E-cadherin or Pam3CSK4 (see Supplementary Fig. S3).
Conventional DCs from the LP, PPs, and MLNs and pre-DCs from spleens of normal mice express integrin β1, but only weakly express integrin α1. Unlike FLT3-L-generated BM-DCs, conventional DCs in the LP, PPs, and MLNs of naïve mice only weakly expressed α 1 (Fig. 3a). They expressed Relative expression levels are presented as the mean + SD of triplicate samples relative to that of the cells cultured in medium alone. (d) Effects of simultaneous or sequential stimulation of GM-CSF/Am80-treated BM-DCs with E-cadherin/Fc and Pam3CSK4 on the production of IL-6, IL-12p40, and IL-23p19. GM-CSF/ Am80-treated BM-DCs were stimulated for 1 day with Pam3CSK4 and immobilized E-cadherin/Fc or each one alone. Aliquots of the E-cadherin/Fc-treated BM-DCs were further stimulated for 1 day with Pam3CSK4 (E-cad → Pam3) or E-cadherin/Fc (E-cad → E-cad). Cytokine concentrations in the culture supernatants were assessed by ELISA. Results are presented as the mean + SD of triplicate samples. Statistical significance was determined by the one-way ANOVA with Tukey-Kramer multiple comparisons test. ***p < 0.001. Data are representative of three independent experiments. significant levels of integrins β 1 and α 4, and low levels of integrins α 5, α 6, and α V (Fig. 3a). However, no significant expression of E-cadherin and Klrg1 was detected (Fig. 3a, and data not shown). Furthermore, integrin α 1 expression on pre-DCs was also extremely weak (Fig. 3b,c). Therefore, integrin α 1 appears to contribute little to the E-cadherin-dependent stimulation of intestinal DCs.
The analysis was performed using pre-DCs from the SPLs of normal mice and those of mice injected with B16-FLT3L cells. The B16-FLT3L injection did not affect integrin α 1 expression on pre-DCs and DCs in the SPL, although it enhanced the expression of some integrins, including CD103. Stimulation of CD11c + MHC class II − CD103 − pre-DCs from the SPL of B16-FLT3L-injected mice (Fig. 3d) with GM-CSF/Am80 followed by 1-day culture in E-cadherin or anti-β 1 mAb (KMI6)-coated plates enhanced the ALDH activity of the cells (Fig. 3e). The results suggest that integrin α 1 expression is not essential for the effect of E-cadherin and the anti-β 1 mAb.

FAK and its downstream signaling molecules are involved in the E-cadherin-and integrin
β1-mediated generation of mature ALDH1A2 high DCs. Integrin-dependent signaling often involves the activation of FAK, also known as protein tyrosine kinase 2, and the subsequent activation of PI3K and Akt, also known as protein kinase B 39 . The FAK inhibitor PF 573228, the PI3K inhibitor LY294002, and the Akt inhibitor triciribine significantly prevented immobilized E-cadherin or anti-integrin β 1 mAb (KMI6) from enhancing ALDH activity in GM-CSF/Am80-treated BM-DCs (Fig. 5). The MEK inhibitor PD98059 and the mTOR inhibitor rapamycin moderately suppressed the enhancement. The β -catenin inhibitor ICG-001 weakly suppressed this enhancement, and an NF-κ B inhibitor failed to suppress it. These results support that the E-cadherin-dependent effect on DCs involves integrin-dependent signaling.

ALDH1A2 high DCs generated by GM-CSF/Am80 and E-cadherin induce Foxp3 + T cells, but not Th1 or Th17 cells, and alleviate colitis.
When GM-CSF/Am80-treated BM-DCs were subsequently stimulated with the TLR2/1-L Pam3CSK4, they acquired the capacity to induce the differentiation of naïve CD4 + T cells to IFN-γ -producing T cells and IL-17A-producing T cells but not to Foxp3 + T cells even in the presence of TGF-β (Fig. 6). Significant populations of these IFN-γ + T cells and IL-17A + T cells expressed integrin α 4β 7 (Fig. 6b). Similar results were obtained by using other PRR-Ls including LPS, CpG ODN, and curdlan (data not shown). On the contrary, when GM-CSF/Am80-treated BM-DCs were subsequently stimulated with E-cadherin or the anti-integrin β 1 mAb (KMI6), these DCs could efficiently induce Foxp3 + T cells in the presence of TGF-β , but they induced few to no IFN-γ − or IL-17A-producing T cells (Fig. 6). Therefore, E-cadherin-or integrin β 1-dependent regulation of proinflammatory cytokine production appears to be critical for inducing regulatory T cells and suppressing proinflammatory T cell differentiation. A significant population of the induced Foxp3 + T cells expressed integrin α 4β 7 and CCR9 (Fig. 6a), suggesting that gut-tropic iTregs were induced.
To examine whether the E-cadherin-induced ALDH1A2 high CD103 + DCs ameliorate inflammatory bowel diseases, we adoptively transferred them into DSS-induced colitis model mice. These DCs significantly suppressed DSS-induced body weight loss, without completely preventing it (Fig. 7a). Contrarily, DCs treated with Pam3CSK4 enhanced body weight loss, mouse mortality, and colon shortening (Fig. 7), suggesting that the maturation signal for GM-CSF-and RA-induced semi-mature DCs is critical for determining their proinflammatory or anti-inflammatory nature. The results collectively indicate that the sequential stimulation of FLT3-L-generated BM-DCs with GM-CSF/Am80 and E-cadherin efficiently induce anti-inflammatory DCs that can induce gut-tropic regulatory T cells.

Discussion
The combination of GM-CSF and RA or Am80 cooperatively enhanced ALDH1A2 expression in pre-DCs as well as BM-DCs, and induced CD103 expression after 2 days of culture. Both GM-CSF and RA appear to be constantly available in the small intestine and MLNs. However, the expression levels of both ALDH1A2 and CD86 in GM-CSF/RA-or GM-CSF/Am80-treated BM-DCs were much lower than those in ALDH1A2 high MLN-DCs. Therefore, to mimic the generation of ALDH1A2 high MLN-DCs, additional signals are likely to be required for enhancing their ALDH1A2 expression and maturation. We previously employed TLR-Ls with GM-CSF for promoting the maturation and ALDH1A2 expression in BM-DCs 14 . Indeed, TLR-L or dectin-L enhanced their maturation and ALDH1A2 expression after 2 days of stimulation with GM-CSF and Am80 as well. However, they induced proinflammatory cytokine production, and the treated DCs could not induce Foxp3 + T cells, but induced IFN-γ + T cells and a small number of IL-17A + T cells bearing the gut-homing receptor integrin α 4β 7. Although RA can suppress the differentiation of both Th1 and Th17 cells 40 , certain levels of RA appear to be essential for Th1 cell lineage stability 8 , for Th17 differentiation 41 , and for the induction of gut-homing Th17 42 .
Contact between DCs and IECs that express E-cadherin is known to enhance ALDH1A2 expression in DCs 26,27 . Accordingly, our preliminary results indicated that co-culture of GM-CSF/Am80-treated BM-DCs with the IEC line CMT93 enhanced the induction of ALDH high DCs, and that a blocking Ab to integrin β 1 inhibited the effect. Furthermore, we found that stimulation of GM-CSF/Am80-treated BM-DCs with immobilized E-cadherin significantly enhanced their maturation and ALDH1A2 expression as well as their ability to induce Foxp3 + T cell differentiation in the presence of TGF-β . The results led us to speculate that semi-mature LP-DCs may become anti-inflammatory mature DCs by interacting with E-cadherin on IECs in steady state but become proinflammatory mature DCs by interacting with pathogen-derived PRR-L.
No evidence was obtained for the role of CD103 in the effect of E-cadherin. On the contrary, a blocking Ab to integrin β 1 inhibited the effect, and immobilized anti-integrin β 1 Ab could mimic the effect, suggesting that E-cadherin stimulated DCs partly through cross-linking of integrin β 1. Accordingly, immobilized VCAM-1/Fc and semaphorin 7 A/Fc, which binds to integrin β 1, could induce similar effects, and a blocking Ab to integrin β 1 suppressed the effects. Semaphorin 7 A is expressed on IECs and activated T cells 37,38 , whereas VCAM-1 is mainly expressed on endothelial cells in the intestine 43 . Therefore, semaphorin 7 A but not VCAM-1 may contribute to the DC maturation step. Heterodimers of integrin β 1 and some α integrins can bind to extracellular matrix glycoproteins including collagens, fibronectins, and laminins 36 . Extracellular matrix molecules are abundant in the intestinal LP and thus may partly contribute to steady-state DC maturation.
In addition to E-cadherin, TLR-Ls, and dectin-Ls, other stimuli may also contribute to the maturation or maintenance of ALDH1A2 + DCs. It has been revealed that 4-1BB (CD137) expressed on ALDH high CD103 + MLN-DCs plays a role in the maintenance of their ALDH1A2 expression 44 . In our culture system, preliminary results indicated that 4-1BB expression was induced in a subset of BM-DCs after culturing with GM-CSF for 2 days, but that its expression was significantly suppressed by adding Am80. It remains to be clarified whether 4-1BB-mediated signaling and integrin β 1-mediated signaling interact with each other in the regulation of ALDH1A2 expression and DC maturation. It was recently reported that culturing BM cells with the combination of FLT3-L and GM-CSF for 16 days efficiently induced CD11c high B220 − CD103 high conventional DCs 45 . Although these DCs did not express maximal levels of ALDH1A2, they appeared to induce T cell-mediated protective immunity in vivo. Resident DCs may also intrinsically reduce the severity of DSS colitis 46 . However, it has been reported that DSS induces production of proinflammatory cytokines by BM-DCs generated with GM-CSF and IL-4 47 . Furthermore, intestinal inflammation may abrogate the tolerogenic properties of CD103 + MLN-DCs, including Aldh1a2 expression 48 . However, we found that inoculation of mature ALDH1A2 high DCs stimulated by E-cadherin in vitro could significantly suppress DSS-induced weight loss but that mature ALDH1A2 high DCs stimulated with Pam3CSK4 significantly enhanced body weight loss, colon shortening, and mortality. Although mature DCs have diminished capacity for macropinocytosis, they can continue to capture and present Ag in vivo 49,50 . The maturation levels of DCs for inoculation might be important for the  Male C57BL/6 mice aged 8-9 wk were administered 2% DSS in their drinking water ad libitum for 4 days, followed by feeding with regular drinking water. On days 0 and 2, the mice were injected intraperitoneally with PBS alone or BM-DCs cultured for 2 days in the presence of GM-CSF and Am80 and subsequently stimulated for 1 day with immobilized E-cadherin/Fc or Pam3CSK4. Body weight changes and the survival ratios of individual groups of mice (n = 6-9) after DSS treatment are shown in (a) and (b), respectively. (c) Colon length was determined at day 10 after DSS treatment. Results are presented as the mean + SEM. Statistical significance was determined using the one-way ANOVA with Tukey-Kramer multiple comparisons test. *p < 0.05 (versus PBS in (a)).
Scientific RepoRts | 6:37914 | DOI: 10.1038/srep37914 maintenance of their anti-inflammatory capacity in vivo. GM-CSF/Am80-treated semi-mature BM-DCs could easily become proinflammatory or anti-inflammatory depending on the subsequent stimulation. After stimulation with E-cadherin, these DCs appeared to become more resistant to TLR stimulation to trigger proinflammatory cytokine production. In addition, these DCs appeared to be capable of inducing iTregs that expressed the gut-homing receptors integrin α 4β 7 and CCR9. DSS treatment is known to enhance the expression of the α 4β 7 ligand MAdCAM-1 in the colon 51 . Although the CCR9 ligand CCL25 is not significantly expressed in the healthy colon unlike MAdCAM-1, DSS treatment induces CCL25 expression in the inflamed colon, and CCL25/ CCR9 interactions regulate inflammatory immune responses in the large intestinal mucosa 52 . As monocytes/ macrophages and neutrophils are major colitogenic effector cells in DSS-treated mice 53 , the effect of the in vitrogenerated anti-inflammatory DCs might be indirect through induction of gut-homing Tregs. In addition, RA produced by the inoculated DCs might contribute to the attenuation of intestinal inflammation by promoting IL-22 synthesis 54 .
Collectively, the present study suggests that FLT3-L-generated immature BM-DC acquire CD103 expression and moderate levels of RA-producing capacity upon stimulation with GM-CSF/RA or GM-CSF/Am80, and that subsequent stimulation with immobilized E-cadherin efficiently generates mature RA-producing anti-inflammatory DCs. It is likely that integrin β 1-mediated signaling contributes to the E-cadherin effect and similar effects of other integrin β 1-binding molecules. However, further studies are required to clarify the role of integrin β 1-mediated signaling in the in vivo generation of intestinal ALDH high CD103 + DC.