TLR2-dependent activation of β-catenin pathway in dendritic cells induces regulatory responses and attenuates autoimmune inflammation

Abstract

Dendritic cells (DCs) sense microbes via multiple innate receptors. Signals from different innate receptors are coordinated and integrated by DCs to generate specific innate and adaptive immune responses against pathogens. Previously, we have shown that two pathogen recognition receptors, TLR2 and dectin-1, which recognize the same microbial stimulus (zymosan) on DCs, induce mutually antagonistic regulatory or inflammatory responses, respectively. How diametric signals from these two receptors are coordinated in DCs to regulate or incite immunity is not known. In this study, we show that TLR2 signaling via AKT activates the β-catenin/T cell factor 4 pathway in DCs and programs them to drive T regulatory cell differentiation. Activation of β-catenin/T cell factor 4 was critical to induce regulatory molecules IL-10 (Il-10) and vitamin A metabolizing enzyme retinaldehyde dehydrogenase 2 (Aldh1a2) and to suppress proinflammatory cytokines. Deletion of β-catenin in DCs programmed them to drive Th17/Th1 cell differentiation in response to zymosan. Consistent with these findings, activation of the β-catenin pathway in DCs suppressed chronic inflammation and protected mice from Th17/Th1-mediated autoimmune neuroinflammation. Thus, activation of β-catenin in DCs via the TLR2 receptor is a novel mechanism in DCs that regulates autoimmune inflammation.

Figure: 1. Zymosan activates β-catenin/TCF pathway via a mechanism involving TLR2

(A, B) Expression of active β-catenin by CD11c⁺ splenic DCs from WT or TLR2^−/− stimulated with zymosan (25 μg/ml) or curdlan (25 μg/ml) or depleted zymosan (dep zymo; 25 μg/ml) at indicated time points as assessed by intracellular staining and flow cytometry. (C) Expression of active β-catenin by CD11c⁺ splenic DCs from WT stimulated with Pam-2-cys (2 μg/ml) or Pam-3-cys (2μg/ml) or LPS (5 μg/ml) or CpG (5 μg/ml) for 3 h and assessed by intracellular staining and flow cytometry. (D) Active β-catenin expression in CD11c⁺ CD11b^– and CD11c⁺ CD11b^– splenic DC subsets was assessed by intracellular staining and flow cytometry. (E) β-galactosidase expression in CD11c⁺ splenic DCs isolated from TCF-reporter mice stimulated with zymosan (25 μg/ml ) or curdlan (25 μg/ml) or depleted zymosan (dep zymo; 25 μg/ml) for 18 h, and assessed by intracellular staining and flow cytometry. (F, G) Axin2 mRNA expression by splenic DCs from WT or TLR2^−/− stimulated with zymosan (25 μg/ml ) or curdlan (25 μg/ml) or depleted zymosan (dep zymo; 25 μg/ml). (H) β-galactosidase expression in CD11c⁺ splenic DCs isolated from TCF-reporter mice stimulated with stimulated with Pam-2-cys (2 μg/ml) or Pam-3-cys (2μg/ml) or LPS (5 μg/ml) or CpG for 18 h, and assessed by intracellular staining and flow cytometry. (I) Intracellular expression of bcatenin (green) and nuclei (blue) in splenic DCs isolated from WT mice cultured in medium alone or with zymosan for 3 hr. Purified CD11c+ splenic DCs were stimulated with or without zymosan (25 mg/ml) for 3 hours and permeabilized with BD Fix and Perm buffer. Cells were incubated with FITC-labeled b-catenin (1:100 dilution; Cell Signaling) for 1 hr and nuclie were stained with DAPI. Three images were acquired for each field using a Zeiss Axiovert LSM-410 confocal microscope (showing FITC and DAPI simultaneously on the cells). (J) β-galactosidase expression in CD11c⁺ splenic DCs isolated from Axin2-reporter mice stimulated with zymosan (25 μg/ml) or curdlan (25 μg/ml) or depleted zymosan, and assessed by intracellular staining and flow cytometry. Data are from one experiment representative of three independent experiments. *P<0.01

Figure 2. Zymosan-mediated activation of β-catenin in DCs promotes T regulatory cell differentiation and suppresses T_H1/T_H17 cell differentiation

(A, B) CD11c⁺ splenic DCs from WT (β-cat^fl/fl) and β-cat^ΔDC were stimulated with zymosan (25 μg/ml) or curdlan (25 μg/ml) and after 10 h DCs (2 × 10⁴) were washed and co-cultured with naïve CD4⁺CD62L⁺ OT-II T cells (1 × 10⁵/ well) with OVA peptide (2 μg/ml) and TGF-β (1 ng/ml). After 4 d, OT-II cells were restimulated for 6 h with plated bound anti-CD3 and anti-CD28. Foxp3 expression and, intracellular production of IL-17, IFN-γ and IL-10 by CD4 T cell were assessed by intracellular staining and flow cytometry. Data are from one experiment representative of three. (C) WT (β-cat^fl/fl) and β-cat^ΔDC mice reconstituted with naïve CD4⁺CD62L⁺ OT-II T cells and were injected i.v. with class II– restricted OVA_323–339 peptide (50 μg) plus PBS, or OVA_323–339 (50 μg) plus zymosan (50 μg). Four days after challenge, unfractioned spleen cells were restimulated in vitro for 5 h with anti-CD3 and -CD28 antibodies in the presence of brefeldin A. Percent CD4⁺ OT-II⁺ cells positive for Foxp3, IL-17, IFN-γ and IL-10 as assessed by intracellular staining and flow cytometry. Data are representative of two independent experiments. (D) unfractioned spleen cells from immunized mice as described in C were restimulated with OVA peptide (1 μg/ml) in culture for 48 h and cytokines in the supernatants were quantified by ELISA (n= 4, samples). *P<0.01; **P<0.001

Figure 3. β-catenin/TCF-4 signaling pathway induces RA synthesizing enzyme (Aldh1a2) and IL-10, and suppresses inflammatory cytokines in splenic DCs

(A) Purified CD11c⁺ splenic DCs from WT (β-cat^fl/fl) and β-cat^ΔDC mice were cultured in media alone or with zymosan (25 μg/ml). (A) After 24 h, mRNA expression of Aldh1a2, IL-10, IL-6 and IL-23 relative to the expression GAPDH was analyzed by RT-PCR (n= 3 samples). (B) ALDH activity on purified CD11c⁺ splenic DCs from WT (β-cat^fl/fl) and β- cat^ΔDC without (gray) or with (black) zymosan treatment. Data are from one experiment representative of two independent experiments. (C) Purified CD11c⁺ splenic DCs from WT (β-cat^fl/fl) and β-cat^ΔDC mice were cultured in media alone or with zymosan as described in A. After 24h, cytokines in the cell culture supernatants were quantified by ELISA. Data are representative of three experiments. (D) β-cat^fl/fl and β-cat^ΔDC mice were injected with PBS or zymosan (25 mg/ml) by i.v. route. Mice were sacrificed at indicated time points, and blood samples and spleens were collected. Induction of Aldh1a2 mRNA expression in purified CD11c⁺ splenic DCs from treated mice was analyzed by RT-PCR and serum cytokine levels were analyzed by ELISA. (E) Purified CD11c⁺ splenic DCs from TCF4^fl/fl and TCF4^ΔDC mice were cultured in media alone or with zymosan (25 μg/ml). After 24 h, expression of Aldh1a2 and IL-10 mRNAs relative to the expression of GAPDH was analyzed by RT-PCR (n= 3 samples). (F) ALDH activity on purified CD11c⁺ splenic DCs from WT (TCF4^fl/fl) and TCF4^ΔDC without (gray) or with (black) zymosan treatment. Data are from one experiment representative of two independent experiments. *P<0.01; **P<0.001; ***P<0.0001

Figure 4. PI3K/AKT mediated signals activate β-catenin in DCs

(A) Representative histograms of pThr³⁰⁸ AKT in CD11c⁺ SPDCs from WT or TLR2^−/− stimulated with zymosan (25 μg/ml) after 3 h as assessed by intracellular staining and flow cytometry. (B) Representative histograms of pSer⁵⁵² β-cat or pSer⁹ GSK-3β or pERK1/2 in CD11c⁺ splenic DCs from WT or AKT1^−/− stimulated with zymosan (25 μg/ml) in the presence or absence of AKT inhibitor VII (5 μM) for 3 h. (C) Purified CD11c⁺ splenic DCs from WT mice stimulated with zymosan (25 μg/ml) in presence or absence of ERK inhibitor or AKT inhibitor. After 24 h expression of RALDH2 mRNA relative to the expression of mRNA encoding GAPDH was analyzed by RT-PCR (n= 3 samples). IL-10 cytokine levels in the culture supernatants were quantified by ELISA. (D) Representative histograms of pSer⁵⁵² β-cat or pSer⁹ or pAKT in CD11c⁺ splenic DCs from WT mice stimulated with zymosan (25 μg/ml) in the presence or absence of Erk inhibitor (U0126; 1μM) for 3 h. (E) Representative histograms of pERK1/2 in CD11c⁺ SPDCs from WT or β-cat^ΔDC stimulated with zymosan (25 μg/ml) after 3 h as assessed by intracellular staining and flow cytometry. (F) CD11c⁺ SPDCs from WT were stimulated with zymosan (25 μg/ml) or curdlan (25 μg/ml) and after 10 h DCs (2 × 10⁴) in the presence or absence of AKT inhibitor VII (5 μM) or Erk inhibitor (U0126; 1 μM). After 10 h DCs were washed and co-cultured with naïve CD4⁺CD62L⁺ OT-II T cells (1 × 10⁵/ well ) with OVA peptide (2 μg/ml) and TGF-β (1 ng/ml). After 4 d, OT-II cells were restimulated for 6 h with plated bound anti-CD3 and anti-CD28. Foxp3 expression and, intracellular production of IFN-γ by CD4 T cell were assessed by intracellular staining and flow cytometry. Data are from one experiment representative of three. *P<0.01; **P<0.001; ***P<0.0001

Figure 5. Activation of β-catenin suppresses the development of clinical EAE and chronic inflammation

(A, B, C) β-cat^fl/fl and β-cat^ΔDC were immunized with 100 μg of MOG_35-55 + CFA or MOG_35-55 + CFA + zymosan or MOG +IFA + zymosan on days 0. Mice also received 250 ng of pertussis toxin on days 0 and 2. The progression of EAE disease severity in different group of mice was monitored on various days post immunization. (D, E) Mononuclear cells were isolated from CNS tissue on day 18 after immunization and restimulated in vitro for 5 h with anti-CD3 and -CD28 antibodies in the presence of brefeldin A. Induction of IFN-γ, IL-17 and IL-10 was assessed by intracellular staining and flow cytometry by gating on CD4⁺ T cells. Numbers in FACS plots represent percentage of cells positive for the indicated protein. Data are from one experiment representative of two. (F, G) Percentage of Foxp3⁺ Tregs, Tr1⁺, IFN-γ⁺(T_H1) and IL-17⁺(T_H17) cells assessed in the CNS of β-cat^fl/fl and β-cat^ΔDC immunized mice at day 18. Data represent mean (± s.d.) of 6 mice per group. (H) Total RNA was isolated from purified CD11c⁺ SPDCs of β-cat^fl/fl and β-cat^ΔDC immunized mice at day 18 as described above, and expression of IL23p19 and IL-6 mRNA was analyzed RT-PCR (n= 3 samples). Data are representative of two independent experiments. *P<0.01; **P<0.001; ***P<0.0001.

Figure 6. Mechanism of regulation of IL-10 and Aldh1a2 by β-catenin/TCF4 pathway in DCs

Innate sensing of zymosan via TLR2 efficiently induces Akt and Erk activation. Akt then activates β-catenin by directly phosphorylating it at Ser⁵⁵² and indirectly by inactivating GSK-3β activity. Activated β-Catenin interacts with TCF4 in the nucleus, and transcriptionally induces IL-10 and Aldh1a2 gene expression in DCs, which are critical for inducing T regulatory responses and limiting T_H1/T_H17 cell differentiation. Activation of Erk and Akt/β-catenin pathway work synergistically to induce IL-10 and Aldh1a2. Dectin-1 signaling does not play a major role in β-catenin activation but promotes induction of pro-inflammatory cytokines that are critical of T_H1/T_H17 cell differentiation.