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.2016 Nov 29:6:37914.
doi: 10.1038/srep37914.

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

Affiliations

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

Aya Yokota-Nakatsuma et al. Sci Rep..

Abstract

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.

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Figures

Figure 1
Figure 1. Delayed stimulation with TLR-L or E-cadherin of GM-CSF/Am80-treated BM-DCs enhances their ALDH1A2 expression without reducing CD103 expression.
(a) Representative flow cytometric profiles of the ALDH activity and surface CD103 expression of BM-DCs cultured for 1 day with Pam3CSK4 or immobilized E-cadherin/Fc in the presence or absence of GM-CSF and Am80. (b–c) BM-DCs were cultured for 2 days with GM-CSF and Am80 and subsequently stimulated for 1 day with Pam3CSK4 or immobilized E-cadherin/Fc. (b) Representative flow cytometric profiles of ALDH activity and expression of the indicated surface molecules. Solid lines represent isotype controls. (c)Aldh1a2 expression was assessed by real-time PCR. 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.
Figure 2
Figure 2. Integrin β1 contributes to the E-cadherin effect on GM-CSF/Am80-induced ALDH1A2 expression in DCs without inducing proinflammatory cytokines.
(a) Representative flow cytometric profiles of integrin and E-cadherin expression on BM-DCs cultured for 2 days in the presence or absence of GM-CSF and Am80. Solid lines represent staining with isotype control mAbs. (b) Effects of blocking mAbs on the induction of ALDHhigh DCs. GM-CSF/Am80-treated BM-DCs were pretreated for 15 min with blocking mAbs to integrins and stimulated for 1 day with immobilized E-cadherin/Fc. Percentages of ALDHhigh cells were assessed by flow cytometry. Results are presented as the mean + SD (triplicate samples) relative to the control culture with the isotype control mAb. Statistical significance was determined using Student’st-test. ***p < 0.001 versus control mAb. (c–e) GM-CSF/Am80-treated BM-DCs were stimulated for 1 day with immobilized anti-integrin β1 mAb (KMI6), or rat IgG2a isotype control mAb, or Pam3CSK4. (c) Representative flow cytometric profiles of ALDH activity and expression of indicated surface molecules. Solid lines represent staining with isotype control mAbs. (d)Aldh1a2 expression assessed by real-time PCR. Relative expression levels are presented as the mean + SD of triplicate samples relative to that of the cells cultured in medium alone with rat IgG2a isotype control mAb. (e) Cytokine concentrations assessed by ELISA. Results are presented as the mean + SD of triplicate samples. Statistical significance was determined via the one-way ANOVA with Tukey–Kramer multiple comparisons test. ***p < 0.001. Data are representative of three independent experiments.
Figure 3
Figure 3. Integrin β1-mediated stimulation enhances ALDH activity in GM-CSF/Am80-treated pre-DCs in an integrin α1-independent fashion.
(a) Representative flow cytometric profiles of integrin and E-cadherin expression on DCs from the small intestinal LP, PPs, and MLNs of C57BL/6 mice. (b–c) Representative flow cytometric profiles of integrin and E-cadherin expression on DCs (CD11c+MHC class II+) and pre-DCs (CD11c+ MHC class II−) from SPLs of naïve (b) or B16-FLT3L-injected (c) C57BL/6 mice. (d) Scheme illustrating the sorting of CD11c+MHC class II−CD103− pre-DCs from SPLs of B16-FLT3L-injected C57BL/6 mice. (e) CD11c+MHC class II−CD103− pre-DCs isolated from the SPL of B16-FLT3L-injected mice were cultured for 2 days with GM-CSF and Am80 and subsequently stimulated for 1 day with immobilized anti-integrin β1 mAb (KMI6), rat IgG2a isotype control mAb, or E-cadherin/Fc. Percentages of ALDHhigh cells were assessed by flow cytometry. Results are presented as the mean + SD of triplicate samples. Statistical significance was determined via the one-way ANOVA with Tukey–Kramer multiple comparisons test. *p < 0.05, ***p < 0.001. Data are representative of three independent experiments.
Figure 4
Figure 4. VCAM-1 and semaphorin 7 A enhance GM-CSF/Am80-induced ALDH1A2 expression in BM-DCs via integrin β1 without inducing proinflammatory cytokines.
BM-DCs were cultured for 2 days with GM-CSF and Am80 and subsequently stimulated for 1 day with Pam3CSK4 or immobilized MAdCAM-1/Fc, VCAM-1/Fc, semaphorin 7 A/Fc, or E-cadherin/Fc. (a–b) Representative flow cytometric profiles of ALDH activity and CD86 expression are shown. (c) Cytokine concentrations in the culture supernatants were assessed by ELISA. (d) The blocking anti-integrin β1 (9EG7) mAb or control rat IgG2a mAb was added in the last 1-day culture. The expression ofAldh1a2 was assessed by real-time PCR, and its level was normalized withRplp0 and quantified with the 2−ΔCt value. 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. NS: not significant, ***p < 0.001.
Figure 5
Figure 5. FAK and its downstream signaling molecules contribute to the E-cadherin– and integrin β1–mediated generation of ALDH1A2high DCs.
GM-CSF/Am80-treated BM-DCs were pretreated for 15 min with inhibitors of FAK, PI3K, Akt, β-catenin, mTOR, MEK/ERK, and NF-κB and stimulated for 1 day with immobilized E-cadherin/Fc or anti-integrin β1 (KMI6). Percentages of ALDHhigh cells were assessed by flow cytometry. Results are presented as the mean + SD (triplicate samples) relative to the control culture treated with the vehicle and are representative of three independent experiments. Statistical significance was determined using the Student’st-test. *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle.
Figure 6
Figure 6. ALDH1A2high DCs generated with GM-CSF/Am80 and E-cadherin induce gut-tropic Foxp3+ T cells but not Th1 or Th17 cells.
GM-CSF/Am80-treated BM-DCs were stimulated for 1 day with immobilized E-cadherin/Fc, immobilized anti-integrin β1 mAb (KMI6), or Pam3CSK4, treated for 2 h with the OVA peptide P323-339, washed, and then cocultured for 5 days with naïve CD4+ T cells from OT-II/Rag2−/− mice in the presence (a) or absence (b–c) of TGF-β. (a) Representative flow cytometric profiles of intracellular Foxp3 expression and surface expression of integrin α4β7 or CCR9 of gated CD4+ T cells. (b) Representative flow cytometric profiles of intracellular expression of IFN-γ or IL-17A and surface expression of integrin α4β7 of CD4+ T cells restimulated for 5 h with PMA and ionomycin. (c) Cytokine concentrations in the supernatants of DC-T cell cocultures were assessed by ELISA. Results are presented as the mean + SD of triplicate samples. Statistical significance was determined using the one-way ANOVA with Tukey–Kramer multiple comparisons test. ***p < 0.001. Data are representative of three independent experiments.
Figure 7
Figure 7. ALDH1A2high DCs generated by treatment with GM-CSF/Am80 and E-cadherin alleviate DSS-induced acute colitis.
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)).
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