Dendritic cells (DCs) are professional APCs that play a crucial role in initiating robust immune responses against invading pathogens while inducing regulatory responses to the body's tissues and commensal microorganisms. A breakdown of DC-mediated immunological tolerance leads to chronic inflammation and autoimmune disorders. However, cell-intrinsic molecular regulators that are critical for programming DCs to a regulatory state rather than to an inflammatory state are not known. In this study, we show that the activation of the TCF4 transcription factor in DCs is critical for controlling the magnitude of inflammatory responses and limiting neuroinflammation. DC-specific deletion of TCF4 in mice increased Th1/Th17 responses and exacerbated experimental autoimmune encephalomyelitis pathology. Mechanistically, loss of TCF4 in DCs led to heightened activation of p38 MAPK and increased levels of proinflammatory cytokines IL-6, IL-23, IL-1β, TNF-α, and IL-12p40. Consistent with these findings, pharmacological blocking of p38 MAPK activation delayed experimental autoimmune encephalomyelitis onset and diminished CNS pathology in TCF4ΔDC mice. Thus, manipulation of the TCF4 pathway in DCs could provide novel opportunities for regulating chronic inflammation and represents a potential therapeutic approach to control autoimmune neuroinflammation.
This article is featured in Top Reads, p.1223
Multiple sclerosis (MS) is a chronic autoimmune inflammatory condition that leads to multifocal demyelination in the white matter of the human CNS. Using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS, studies have shown that dendritic cells (DCs) play a critical role in the initiation and development of CNS pathology (1–3). DCs are a specialized subset of APCs that form a critical link between innate and adaptive immune cells (4, 5). Accumulating evidence suggests that DCs play a pivotal role in instigating inflammation as well as suppressing inflammation and restoring immune homeostasis (4, 5). Recent studies have shown that ablation of DCs in mice breaks self-tolerance of CD4+ T cells and results in spontaneous fatal autoimmunity (6, 7). Likewise, depletion of DCs in mice resulted in stronger inflammatory responses with exacerbated EAE (7, 8). Furthermore, depletion of specific DC subsets in lymphoid or nonlymphoid tissues during the acute or relapse phase of EAE results in stronger inflammatory responses and exacerbates the disease (9–12). Besides, other studies have shown that these cells lose their regulatory properties, resulting in uncontrolled chronic inflammation (13–15). DCs contribute to CNS pathology through differentiation and activation of naive CD4+ T cells to myelin-specific Th1 and Th17 cells (16, 17). Conversely, emerging evidence suggests that DCs are also critical in resolving inflammation and limiting immune-mediated pathology in EAE by producing immune regulatory factors and driving regulatory T cell (Treg) differentiation and activation (3, 4, 5). Thus, DCs play a key role in bridging innate and adaptive immunity. Although DCs are present in low numbers in the CNS under homeostatic conditions, their numbers increase drastically during autoimmune inflammation, infection, or trauma (18). However, cell-intrinsic molecular regulators that are critical for programming DCs to a regulatory state rather than to an inflammatory state are not known. Thus, understanding these events may present promising new targets for therapeutic intervention of various autoimmune and chronic inflammatory conditions.
Aberrant activation of the wingless/integrated (Wnt) signaling pathway occurs in several inflammatory diseases, including neurodegenerative and neuroinflammatory diseases (19–21). Many studies have documented that Wnt ligands are highly expressed in several chronic inflammatory diseases and autoimmune diseases (22–24). The T cell factor/lymphoid enhancer–binding factor (TCF/LEF) family of transcription factors are known to be critical for embryogenesis and in the development of hematopoietic stem cells and immune cells (25). The TCF/LEF family comprises four NFs, namely TCF1, LEF1, TCF3, and TCF4 (also designated as TCF7, LEF1, TCF7L1, and TCF7L2) (25). They act as one of the main downstream mediators of the Wnt/β-catenin signaling pathway (25, 26), and DCs highly express the TCF4 isoform (27, 28). However, the functional and biological significance of TCF4 in DCs in regulating ongoing inflammation and establishing immune homeostasis is poorly understood.
In the current study, we report that during the induction and effector phase of EAE, TCF4 activation in DCs plays a key role in regulating the magnitude of inflammatory responses and limiting collateral damage to the host. Accordingly, our data demonstrate that the DC-specific deletion of TCF4 in mice results in severe EAE pathology. This was because of heightened activation of p38 MAPK and increased expression of proinflammatory cytokines by DCs lacking TCF4, resulting in increased Th1 and Th17 cell polarization. In contrast, pharmacological blocking of p38 MAPK activation in TCF4-deficient DCs markedly reduced the expression of proinflammatory cytokines and diminished EAE severity in TCF4ΔDC mice. These results reveal a novel mechanism by which the TCF4 in DCs controls chronic inflammation and limits immune-mediated pathology in EAE. Thus, manipulating TCF4 activation in DCs may represent a convenient therapeutic approach to improve the outcome of MS and other inflammatory diseases.
Materials and Methods
C57BL/6, CD11c-cre (29), TCF/LEF-reporter mice (30), and 2D2 myelin oligodendrocyte glycoprotein (MOG)–specific TCR transgenic mice (31) were originally obtained from The Jackson Laboratory and bred on site. TCF4 floxed mice (32) were kindly provided by Dr. Melinda L Angus-Hill (University of Utah) and were crossbred to CD11c-cre mice to generate mice lacking TCF4 in DCs (TCF4ΔDC) (27). The successful cre-mediated deletion was confirmed by PCR and protein expression analyses. All mice were housed under specific pathogen-free conditions in the Laboratory Animal Services of Augusta University. Animal care protocols were approved by the Institutional Animal Care and Use Committee of Augusta University.
Reagents and Abs
MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) was purchased from AnaSpec. Abs against mouse CD4 (GK1.5), CD8a (53-6.7), CD45 (30-F11), Foxp3 (FJK-16s), IL-10 (JES5-16E3), TNF-α (MP6-XT22), CD11c (N418), CD90.1 (HIS51), IL-17 (TC11-18H10), and IFN-γ (XMG1.2) were purchased from eBioscience and BioLegend. GFP, phospho-p38 MAPK, p38 MAPK, and TCF4 Abs were obtained from Cell Signaling Technology. p38 MAPK inhibitor (SB203580) was purchased from Calbiochem.
EAE induction experiments were performed as described in our previous studies (33, 34). EAE was induced by s.c. immunization in the hind flanks on day 0 using 100 µg of the MOG35–55 peptide emulsified in CFA containing 2.5 mg/ml heat-inactivated Mycobacterium tuberculosis (Difco Laboratories). Mice also received 250 ng of pertussis toxin (List Biological Laboratories) i.p. on days 0 and 2 postimmunization (pi). Disease severity was assessed on different days pi, according to the following scale: 0, no disease; 1, flaccid tail; 2, hind limb weakness; 3, hind limb paralysis; 4, forelimb weakness; and 5, moribund. In some experiments, EAE-induced TCF4FL/FL and TCF4ΔDC were treated with either p38 MAPK inhibitor (5 mg/kg) or vehicle by i.p. injection every 2 d starting on day 3 pi.
CNS leukocyte isolation
Mice were euthanized with CO2 and perfused through the left ventricle with PBS. The brain and spinal cord were removed from each animal and dissected into small fragments followed by digestion with collagenase type 4 (1 mg/ml) in complete DMEM plus 2% FBS for 30 min at 37°C. Leukocytes were isolated using 40% Percoll (Sigma-Aldrich) and then were stained for CD4+ T cells expressing Foxp3 and different intracellular cytokines.
CD11c+ cell isolation
CD11c+ DCs were purified from the CNS or draining lymph nodes (DLNs) and spleen (pooled together) as previously described (33). In brief, the spleen or brain was cut into small fragments and then digested with collagenase type 4 (1 mg ml−1) in complete DMEM plus 2% FBS for 30 min at 37°C. Cells were washed twice and enriched for CD11c+ DCs with the CD11c-specific microbeads from Miltenyi Biotec. The resulting purity of CD11c+ DCs was ∼95%. Enriched CD11c+ DCs (106 cells/ml) from immunized mice were cultured ex vivo for 24 h. The supernatants were collected for cytokine analysis by ELISA, whereas cells were collected for gene expression analysis by RT-PCR (34, 35).
Single-cell suspensions from CNS leukocytes, DLNs, and spleen were suspended in PBS containing 5% FBS. After incubation for 15 min at 4°C with the blocking Ab 2.4G2 (anti-FcγRIII/I), the cells were stained at 4°C for 30 min with the appropriately labeled Abs. Samples were then washed two times in PBS containing 5% FBS. The samples were either immediately analyzed at this point or fixed in PBS containing 2% paraformaldehyde and stored at 4°C. Intracellular staining for phospho-p38 MAPK-PE and GFP-FITC was performed using rabbit mAb or with appropriate isotype control in TBS containing 1% BSA. For intracellular cytokine staining (ICS), single-cell suspensions from the DLNs or CNS were ex vivo stimulated with PMA/ionomycin and brefeldin A/monensin for 6 h at 37°C. The cells were then stained for CD4 and CD8 followed by intracellular staining of IFN-γ, IL-17A, TNF-α, and IL-10. To measure Foxp3+ Tregs or phospho-p38 MAPK or GFP expression, the corresponding Abs were added after permeabilization and fixation of cells. Flow cytometric analyses were performed using a FACS LSRII system (BD Biosciences), and the data were analyzed using FlowJo software (Ashland, OR).
MOG38–49-IAb tetramers were kindly provided by the National Institutes of Health Tetramer Core Facility at Emory University. CNS-infiltrating leukocytes were resuspended in PBS containing 5% FBS followed by tetramer staining for 1 h at 37°C as described in a previous study (34, 36). After 1 h, cells were stained with anti-CD4 (APC) and anti-CD45 (Alexa Fluor 700) for 30 min on ice. Cells were washed three times, followed by data acquisition on the flow cytometer. A nonspecific peptide tetramer was used as a negative control. The percentage of tetramer-PE–positive cells was determined in CD4-positive populations based on negative control staining.
2D2 CD4+ T cell adoptive transfer
TCF4FL/FL and TCF4ΔDC recipient mice were reconstituted with 2.5 × 106 2D2 TCR transgenic CD4+ T cells followed by EAE immunization, as described in our previous study (34). Five days later, DLNs were removed, and after RBC lysis, in vitro recall responses were assayed by restimulating DLN cells (2 × 106/ml) for 6 h with PMA/ionomycin in the presence of brefeldin A/monensin for intracellular cytokine detection.
In vitro culture of murine DCs and T cells
In vitro 2D2 CD4+ T cell differentiation was performed as previously described (33, 37). In brief, purified DLNs and splenic CD11c+ DCs (pooled together) (106 cells/ml) from immunized mice on day 5 were cultured together with naive 2D2 CD4+CD62L+ T cells (105) in 200 μl RPMI-1640 complete medium in 96-well round-bottom plates in the presence of the MOG35–55 peptide (1 μg/ml). In some experiments, DCs purified from immunized mice treated with p38 inhibitor or vehicle were cultured with naive 2D2 T cells. After 96 h, cells were restimulated with PMA/ionomycin for 6 h in the presence of brefeldin A/monensin for intracellular cytokine detection.
Quantitative real-time PCR
Total RNA was isolated from purified DLNs and spleen (pooled together) or CNS-infiltrating CD11c+ DCs using the QIAGEN RNeasy Mini Kit, according to the manufacturer's protocol (QIAGEN). cDNA was generated using the superscript First-Strand Synthesis System for RT-PCR and random hexamer primers (Invitrogen), according to the manufacturer's protocol. cDNA was used as a template for quantitative real-time PCR using SYBR Green Master Mix (Bio-Rad Laboratories) and gene-specific primers, as described in our previous studies (33, 34). Gene expression across samples was normalized relative to the housekeeping gene GAPDH.
Spinal cords from EAE-induced mice were removed after perfusion and fixed using 10% formalin in PBS. Fixed spinal cords were embedded in paraffin and sectioned, after which the sections were stained with H&E to study leukocyte infiltration and pathology as well as Luxol fast blue stain to demonstrate CNS demyelination (34, 37).
Statistical analyses were conducted using Prism (GraphPad). Mean clinical scores were analyzed using the Mann–Whitney nonparametric t test. The statistical significance of differences in the means ± SD of cytokines released by cells of various groups was calculated with the Student t test (one-tailed).
Deletion of TCF4 in DCs exacerbates EAE pathology
To investigate the selective role of TCF4 in DC function during autoimmune neuroinflammation, we first assessed whether TCF4 is activated in DCs during autoimmune neuroinflammation using the TCF/LEF-GFP reporter mice (30). In this reporter mice, GFP reporter gene is under the control of six copies of a TCF/Lef response element (30). We immunized TCF-reporter mice with the MOG35–55 peptide plus CFA and assessed GFP expression in DCs isolated from the spleen during the preclinical stage of EAE phase (day 5 pi) and disease phase (day 14 pi). We observed a marked increase in GFP expression in splenic DC of TCF/LEF-GFP reporter mice pi during the induction and disease phase of EAE (Fig. 1A). Likewise, CNS-infiltrating DCs from the reporter mice showed increased GFP expression during the clinical stage of EAE (Fig. 1A). Next, we analyzed GFP expression in DC subsets, macrophages (MΦs), and T cells. We observed increased GFP expression in CD8+ DCs (CD45+MHC class II [MHC II]+CD11c+CD11b−CD8α+), CD11b+ DCs (CD45+MHC II+CD11c+CD11b+CD8α−), plasmacytoid DCs (pDCs) (CD45+CD11c+B220+PDCA-1+), and MΦs (CD45+MHC II+CD11c−CD11b+CD64+F4/80+) (Supplemental Fig. 1A). Of note, we do not detect any reporter gene expression in CD4+ and CD8+ T cells during the preclinical stage of EAE phase (day 5 pi) (Supplemental Fig. 1A). We asked whether increased GFP expression in DCs is due to changes in the TCF4 expression during EAE. Enriched splenic and CNS DCs from the EAE mice showed similar TCF4 mRNA expression levels under steady-state conditions as well as during the induction and effector phase of the disease (Supplemental Fig. 1B). These results suggest that TCF4 is activated in DCs in EAE.
To determine the selective role of TCF4 in DC function in EAE, we deleted TCF4 specifically in DCs (TCF4ΔDC) by crossing TCF4 floxed (WTFL/FL) mice to CD11c-cre mice. Subsequently, we confirmed the efficient deletion of TCF4 by quantitating the mRNA and protein expression levels of TCF4 in the splenic DCs isolated from the WTFL/FL and TCF4ΔDC mice. As expected, we noted a marked decrease in the TCF4 mRNA and protein levels in TCF4-deficient DCs compared with the wild-type (WT) DCs (Supplemental Fig. 1C, 1D). Moreover, TCF4 mRNA levels were markedly decreased in DC subsets in TCF4ΔDC mice (Supplemental Fig. 1E). However, TCF4 mRNA levels were comparable in splenic MΦs isolated from WTFL/FL and TCF4ΔDC mice, demonstrating efficient target gene deletion in DCs (Supplemental Fig. 1E). Next, we investigated whether TCF4 deficiency affects DC numbers during the steady state. We did not observe any significant differences in the frequency and total number of splenic CD11c+MHC II+ DCs between WTFL/FL and TCF4ΔDC mice (Supplemental Fig. 1F). Similarly, the frequencies of DC subsets in the spleen were comparable between WTFL/FL and TCF4ΔDC mice (Supplemental Fig. 1G), suggesting that TCF4 deletion does not affect the development and differentiation of these DC subsets.
Next, we analyzed EAE disease progression and pathology in the WTFL/FL and TCF4ΔDC mice. WTFL/FL mice immunized with the MOG35–55 peptide plus CFA showed onset of neurologic impairment occurring around day 14. In contrast, TCF4ΔDC mice showed an earlier onset of neurologic impairment and developed a more severe form of EAE (Fig. 1B). The histopathological analysis of CNS showed a marked increase in leukocyte infiltration and intense demyelination in TCF4ΔDC mice compared with WTFL/FL mice (Fig. 1C, 1D). In line with these observations, flow cytometric analysis confirmed a significant increase in the total number of CD45+ cells in the CNS of TCF4ΔDC mice. Further characterization of CNS-infiltrating leukocytes in TCF4ΔDC mice showed more DCs (CD45hiMHC IIhiCD11c+), MΦs (CD45hiMHC IIhiCD11c−CD11b+CD64+), monocytes (CD45hiMHC II−CD11c−CD11b+Ly6ChiLy6Glow), neutrophils (CD45hiMHC II−CD11c−CD11b+Ly6ClowLy6Ghi), and CD4+ T cells (Fig. 1E). Collectively, these data suggest that DC-specific expression of TCF4 is critical for limiting autoimmune CNS pathology.
TCF4ΔDC mice display increased infiltration of Th1 and Th17 cells in the CNS during EAE
CNS-infiltrating CD4+ effector T cells contribute to the pathogenesis of EAE (16, 17, 38), whereas the CD4+ Tregs play a key role in limiting the disease severity and associated tissue injury. Thus, we next investigated if increased EAE severity observed in TCF4ΔDC mice was due to effector CD4+ T cell subsets. There was a significant increase in the frequency of the MOG38–49 tetramer–specific CD4+ T cells in the CNS of TCF4ΔDC mice when compared with WT control mice (Fig. 2A, 2B). ICS of CD4+ T cells showed a significant increase in the frequencies of IFN-γ+, IL-17+, and TNF-α+ CD4+ T cells in the CNS of TCF4ΔDC mice (Fig. 2C, 2D). In contrast, we observed a marked decrease in the frequency of IL-10+CD4+ regulatory (type 1 Treg [Tr1]) cells in the CNS of TCF4ΔDC mice compared with WT control mice, whereas the frequency of Foxp3+CD4+ T cells was comparable in both groups (Fig. 2C, 2D). Consistent with these observations, following ex vivo MOG stimulation, leukocytes isolated from the CNS of TCF4ΔDC mice produced markedly higher levels of IFN-γ, IL-17A, and TNF-α and lower levels of IL-10 compared with the leukocytes isolated from the CNS of WTFL/FL mice (Fig. 2E). Collectively, these data suggest that DC-specific expression of TCF4 is critical for limiting autoimmune CNS pathology, indicating a possible regulatory role for TCF4 in DCs during ongoing neuroinflammation.
TCF4 programs DCs to induce IL-10 and limit proinflammatory cytokine expression
These results prompted us to examine whether TCF4 regulates DC function through the expression of inflammatory and anti-inflammatory cytokines during the preclinical stage and clinical stage of EAE. We observed significantly increased mRNA levels for IL-6, TNF-α, IL-1β, IL-23p19, and IL-12p40 and a decreased level of IL-10 in DCs isolated from the DLNs and spleen of TCF4ΔDC mice compared with WT control mice (Fig. 3A). In line with this observation, TCF4-deficient DCs produced markedly higher levels of IL-6, IL-1β, TNF-α, IL-23, and IL-12p40 and reduced levels of IL-10 compared with WT DCs (Fig. 3B). In addition to the effector T cells, DCs infiltrating the CNS contributes to the pathogenesis by reactivating the primed T cells and functions as effector cells to cause CNS lesions. Thus, we examined the expression of inflammatory and anti-inflammatory cytokines in CNS-infiltrating DCs. As shown in (Fig. 3C, there was a significant increase in mRNA levels of IL-6, IL-1β, TNF-α, IL-23p19, and IL-12p40 but decreased levels of IL-10 in TCF4-deficient DCs compared with WT control DCs infiltrating CNS. Thus, our data demonstrates that in DCs, TCF4 is critical for limiting inflammatory cytokine expression while inducing IL-10 during the induction and effector phase of EAE.
TCF4 activation in DCs limits Th1 and Th17 differentiation
As DCs dictate the fate of naive CD4+ T cells through differential production of pro- and anti-inflammatory cytokines (39), we further considered the functional relevance of DC-specific TCF4-mediated signaling in naive CD4+ T cell differentiation. First, we performed an ex vivo CD4+ T cell differentiation assay by coculturing naive MOG-specific 2D2 T cells with DCs isolated from the DLNs and spleen of WTFL/FL and TCFΔDC mice challenged with the MOG35–55 peptide plus CFA. T cells cultured with TCF4-deficient DCs contained significantly higher frequencies of Th1 and Th17 cells compared with T cells cultured with WT DCs (Fig. 4A, 4B). Further, analysis of the culture supernatant showed that T cells cultured with TCF4-deficient DCs produced markedly higher levels of IFN-γ and IL-17A than that of T cells cultured with WT DCs (Fig. 4C). Therefore, deletion of TCF4 in DCs augments Th1 and Th17 cell differentiation.
To extend these observations in vivo, we adoptively transferred naive CD4+CD25− T cells from 2D2 mice into WTFL/FL and TCFΔDC mice and then challenged these mice with the MOG35–55 peptide plus CFA. There was a significant increase in the number of CD4+2D2+ T cells in the DLNs of TCF4ΔDC mice when compared with WT mice on day 5 postchallenge (Fig. 4D). In line with these observations, intracellular cytokine analysis showed a significant increase in donor T cell differentiation toward Th1 and Th17 cells in TCF4ΔDC mice compared with the WT mice (Fig. 4E). Consistent with these observations, donor T cells isolated from the DLNs and spleen of TCF4ΔDC mice produced markedly higher levels of IFN-γ and IL-17A compared with the donor T cells isolated from the WT mice in response to ex vivo MOG33–55 peptide stimulation (Fig. 4F). Therefore, TCF4 signaling in DCs limits Ag-specific Th1 and Th17 cell differentiation and expansion in vivo.
Next, we asked whether increased inflammatory Th1 and Th17 cell differentiation observed in TCF4ΔDC mice is due to altered DC maturation and activation. Characterization of DLN and splenic DCs from TCF4ΔDC mice showed no significant difference in the expression of MHC II, CD40, CD80, and CD86 as compared with control WT mice on day 5 pi (Fig. 4G). Collectively, our results indicate that TCF4 activation in DCs regulates the differentiation of naive CD4+ T cells into Th1 and Th17 cells through the regulation of DC-specific cytokine production.
TCF4 limits the expression of inflammatory factors and Th1/Th17 cell differentiation by regulating p38 MAPK activation in DCs
The p38α MAPK signaling pathway is critical for the expression of inflammatory factors and plays a key role in the pathogenesis of EAE and other inflammatory diseases (40, 41). Thus, we assessed the activation status of p38α MAPK in DCs in TCF4ΔDC and WT mice during the induction phase and disease phase of EAE. DCs isolated from the DLNs and spleen of TCF4ΔDC mice showed a marked increase in the phosphorylated (active) form of p38α MAPK compared with DCs isolated from WT control mice both during the induction and effector phase of EAE (Fig. 5A, 5B). Likewise, the activity of p38α MAPK was markedly elevated in CNS-infiltrating DCs isolated from TCF4ΔDC mice as compared with CNS DCs isolated from WT control mice (Fig. 5A).
Based on these observations, we hypothesized that in EAE, TCF4 regulates the expression of proinflammatory cytokines by controlling the p38α MAPK activation in DCs. Thus, we examined the effect of blocking the p38α pathway in TCF4-deficient DCs on the expression of inflammatory and anti-inflammatory cytokines. To test this, we immunized TCF4ΔDC mice with the MOG33–55 peptide plus CFA (MOG) and treated with either SB203580 or vehicle. As shown in (Fig. 5C, DCs isolated from TCF4ΔDC mice with SB203580 produced markedly lower levels of IL-6, TNF-α, IL-23, and IL-12p40 compared with DCs isolated from mice treated with vehicle. However, p38 inhibitor treatment had no effect on IL-1β and IL-10 production by TCF4-deficient DCs (Fig. 5C). These results indicate that TCF4 limits the expression of proinflammatory cytokines by regulating the activity of p38α MAPK.
Next, we asked whether blocking p38α MAPK activation in TCF4-deficient DCs affects Th1/Th17 cell differentiation. For this, we performed ex vivo 2D2 T cell differentiation assay by coculturing naive 2D2 T cells with DCs isolated from TCF4ΔDC mice immunized with the MOG35–55 peptide plus CFA (MOG) and treated with either SB203580 or vehicle. T cells cultured with TCF4-deficient DCs obtained from mice treated with vehicle contained higher frequencies of Th1/Th17 cells compared with T cells cultured with TCF4-deficient DCs obtained from mice treated with SB203580 (Fig. 5D). Further, analysis of the culture supernatant showed that SB203580 treatment of TCF4-deficient DCs markedly reduced IFN-γ and IL-17A produced by T cells (Fig. 5E). Based on these results, we conclude that TCF4 activation in DCs limits Th1/Th17 differentiation by regulating the activity of p38α.
Pharmacological blocking of p38 MAPK activation limits EAE pathology in TCF4ΔDC mice
Our data have thus far indicated that p38 MAPK is downstream of the TCF4 pathway in DCs. So, we explored the possible implication of pharmacologically blocking the p38 MAPK pathway in TCF4ΔDC mice during ongoing neuroinflammation. Thus, we assessed whether blocking this pathway could ameliorate EAE pathology in TCF4ΔDC mice. To study this, we immunized TCF4ΔDC mice with the MOG33–55 peptide plus CFA (MOG) and treated with either SB203580 or vehicle i.p. every 2 d from day 3 pi. The treatment of TCF4ΔDC mice with p38 inhibitor markedly delayed EAE onset and reduced EAE severity compared with vehicle-treated control mice (Fig. 6A). Consistent with disease severity score, histopathological analysis of the CNS showed reduced inflammation as marked by reduced infiltration of leukocytes and diminished demyelination in the p38 inhibitor–treated group compared with mice in the vehicle-treated group (Fig. 6B, 6C). Besides, flow cytometric analysis confirmed a marked decrease in the total number of leukocytes as well as decreased number of DCs, MΦs, monocytes, neutrophils, and CD4+ T cells in the CNS of TCF4ΔDC mice treated with SB203580 compared with the vehicle-treated group (Fig. 6D). Further characterization of CNS-infiltrating CD4+ T cells showed a significant reduction in the frequencies of the MOG38–49 tetramer–specific CD4+ T cells, Th1, and Th17 cells upon SB203580 treatment (Fig. 6E–G). Consistent with these observations, leukocytes isolated from the CNS of TCF4ΔDC mice treated with the p38 inhibitor produced markedly lower levels of IFN-γ and IL-17A compared with the leukocytes isolated from the CNS of vehicle-treated mice in response to ex vivo stimulation with the MOG33–55 peptide (Fig. 6H). Collectively, these results indicate that TCF4 activation in DCs controls CNS inflammation and EAE disease severity by regulating the p38 MAPK pathway.
The current study defines an essential role for the TCF4 transcription factor in DCs in controlling CNS inflammation and EAE disease severity. Accordingly, DC-specific deletion of TCF4 led to an increased expression of IL-6, TNF-α, IL-1β, IL-23, and IL-12p40 with diminished production of IL-10 during the induction and effector phase of EAE. Consequently, the absence of TCF4 signaling in DCs suppressed Treg responses yet promoted Th1 and Th17 cell differentiation. Accordingly, mice lacking TCF4 in DCs exhibited a very severe EAE pathology characterized by intense demyelination in CNS with increased effector CD4+ T cell infiltration in the CNS. Mechanistically, TCF4 controls the expression of proinflammatory cytokines and neuroinflammation by regulating p38 activity in DCs. Finally, pharmacological blocking of p38 MAPK activation in TCF4-deficient DCs markedly reduced the expression of proinflammatory cytokines and delayed EAE onset with diminished neuropathology in TCF4ΔDC mice. These data indicate an important role for TCF4 in DCs in controlling CNS inflammation during EAE and represent a logical target to control chronic inflammatory conditions such as MS.
Aberrant activation of the Wnt signaling pathway occurs in several inflammatory diseases, including neurodegenerative and neuroinflammatory diseases (19, 20, 42). Many studies have documented that Wnt ligands are highly expressed in several chronic inflammatory diseases and autoimmune diseases (22–24). The coreceptors, low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6), and coactivator β-catenin are critical for mediating canonical Wnt signaling in APCs (21, 26, 42). In murine models of intestinal inflammation and colitis-associated colon cancer, recent studies have shown that the canonical Wnt pathway in DCs induce T regulatory response and suppress intestinal inflammation and inflammation-associated colon cancer. Our previous studies have shown that in conditional knockout mice that specifically lack LRP5/6 (LRP5/6ΔDC) or β-catenin (β-catΔDC) in DCs are more susceptible to inflammatory diseases (28, 43). These mice develop more severe EAE (34). β-Catenin interacts with several transcription factors, including TCFs, PPARγ (44, 45), Foxo (46–48), VDR (46, 49), IRF3 (50), and IRF8 (51). Yet, downstream mediators of LRP5/6–β-catenin signaling in DCs are not known. The present study shows that TCF4 is one of the key downstream mediators of the LRP5/6–β-catenin signaling in DCs. As seen in LRP5/6ΔDC and β-catΔDC mice (34), DC-specific deletion of TCF4 in mice increased Th1/Th17 responses and exacerbated EAE pathology. Furthermore, β-catenin target genes such as Axin1, Axin 2, and WISP1 are markedly decreased in DCs isolated from TCF4ΔDC mice during the induction and effector phase of the disease (Supplemental Fig. 1H). Many of the LRP5/6- and β-catenin–controlled cytokines, such as IL-12, IL-6, IL-23, and TNF-α, are elevated in TCF4ΔDC mice. These proinflammatory cytokines are also elevated in MS (52–54) and are essential to induce EAE (55, 56). These observations strongly suggest that TCF4 is a key downstream mediator of the LRP5/6–β-catenin signaling in DCs. In mice and humans, multiple subsets of DCs exist in both lymphoid (CD8α+ DCs, pDCs, and CD11b+ DCs) and nonlymphoid tissues (CD103+ DCs, pDCs, and CD11b+ DCs)2, 3. Depletion of DCs in mice resulted in stronger inflammatory responses with exacerbated EAE7, 8. Furthermore, depletion of specific DC subsets in lymphoid or nonlymphoid tissues during the acute or relapsing phase of EAE results in stronger inflammatory responses and exacerbates the disease9-12. The present study shows that the TCF4 pathway is active in all three major DC subsets (CD8α+ DCs, CD11b+ DCs, and pDCs). Deletion of TCF4 does not dramatically alter the frequencies and number of CD8α+ DCs, CD11b+ DCs, and pDCs. However, further studies are warranted to understand the role of TCF4 in these subsets in regulating CNS inflammation. Although not analyzed in the current study, it is possible that TCF4 might modulate EAE by regulating DC migration.
A fine balance between Tregs versus pathological Th1/Th17 cells underlies disease progression in many inflammatory diseases, including EAE. Past studies have shown that LRP5/6–β-catenin signaling in APCs limits chronic inflammation through the induction of Tregs while limiting the differentiation of pathological Th1/Th17 cells (28, 34, 43). Accumulating evidence suggests that Th1/Th17 cells play an important role in the pathogenesis of EAE and MS (57–59). In contrast, Tregs such as Foxp3+ Tregs and IL-10–producing Tr1 cells play a pivotal role in neuroinflammation and EAE disease severity (59). Our studies show that TCF4-deficient DCs are potent in inducing MOG-specific Th1/Th17 cell differentiation, at least in part because of increased production of IL-6, IL-23, and IL-12. Indeed, conditional deletion of TCF4 in DCs exacerbated EAE severity because of a marked increase in Th1/Th17 cells and a decrease in IL-10–producing Tr1 cells in the CNS. A recent study on intestinal inflammation has shown that the β-catenin directly regulates IL-10 production through TCF4 in intestinal DCs and MΦs (28). Furthermore, β-catenin/TCF4 signaling regulates the expression of inflammatory factors through the autocrine effects of IL-10 on colonic APCs (28). Although not analyzed in the current study, it is possible that TCF4-mediated signals in DCs might modulate EAE by inducing Tr1 cell differentiation and regulating the suppressive function of Foxp3+ Tregs.
Innate immune receptors, including TLR-mediated signaling in DCs, play a critical role in the initiation of EAE. These receptors on DCs sense various danger signals and induce the activation of several signaling networks with the secretion of cytokines that drive the differentiation of naive CD4+ and CD8+ T cells to pathological effector T cells or Tregs (39). Activation of most TLRs on DCs induces secretion of key proinflammatory cytokines that promote Th1 or Th17 cell differentiation (5, 39). M. tuberculosis, an adjuvant used for the induction of EAE, activates several TLRs on APCs. p38 MAPK is one of the key downstream mediators of TLR signaling and is critical for the production of proinflammatory cytokines upon TLR activation (60). The p38α MAPK signaling pathway plays a key role in the pathogenesis of EAE and other inflammatory diseases (40, 61). Moreover, p38 expression and activation in DCs are critical for the expression of the proinflammatory factors that drive Th1/Th17 cell differentiation (62). Indeed, DC-specific deletion of p38α in mice increased the expression of the proinflammatory cytokines and Th1/Th17 responses during EAE (62). More importantly, pharmacological inhibitors of p38 markedly reduced Th1/Th17 cell differentiation and ameliorated EAE in mice (40). Our data indicate that TCF4 in DCs limits the expression of proinflammatory cytokines and Th1/Th17 differentiation by regulating the activity of p38α. Accordingly, loss of TCF4 in DCs led to heightened activation of p38 MAPK and increased levels of proinflammatory cytokines IL-6, IL-23, IL-1β, TNFα, and IL-12. Consistent with these observations, pharmacological blocking of p38 MAPK activation in TCF4-deficient DCs markedly reduced the expression of proinflammatory cytokines and decreased its ability to drive Th1/Th17 cell differentiation. Besides, the treatment of TCF4ΔDC mice with p38 inhibitor markedly delayed EAE onset and reduced EAE severity. Although not analyzed in the current study, further studies are warranted to understand whether SB203580 treatment affects the Ag-presenting capacity or the migratory properties of DCs in TCF4ΔDC mice. Although our data indicate an important role for TCF4 in modulating p38 activity in DCs during EAE, it is not clear whether TCF4 regulates p38 activity directly or indirectly. A recent study has shown that the β-catenin/TCF4/IL-10 signaling axis in intestinal APCs regulates IL-6, IL-12, and IL-23 through the suppression of p38 (28). It is possible that TCF4 could regulate p38 activation indirectly through IL-10. In the current study, we also show that TCF4 is critical for IL-10 production by DCs during EAE. However, p38 inhibition in TCF4-deficient DCs failed to restore the IL-10 production, suggesting that TCF4 regulates IL-10 production independent of p38.
In summary, our study reveals an important role for TCF4 in DCs in exerting a protective effect on CNS inflammation during EAE by regulating the expression of key proinflammatory and anti-inflammatory mediators. Furthermore, our study indicates a regulatory role for the TCF4 during ongoing neuroinflammation, in which activation of this transcription factor in DCs limits the uncontrolled differentiation of naive CD4+ T cells to Th1 and Th17 cells. Thus, manipulation of the TCF4 pathway in DCs could provide novel opportunities for regulating chronic inflammation and represents a potential therapeutic approach to control autoimmune neuroinflammation.
We thank Dr. Melinda L Angus-Hill (University of Utah) for kindly providing TCF4 floxed mice. We also thank Jeanene Pihkala for technical help with FACS sorting and analysis and Janice Randall for expert technical assistance with the mice used in this study.
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Awards (DK097271 and DK123360) and Augusta University Awards (IGPB0003 and ESA00041) (to S.M.).
The online version of this article contains supplemental material.
Abbreviations used in this article
draining lymph node
experimental autoimmune encephalomyelitis
intracellular cytokine staining
low density lipoprotein receptor-related protein 5 and 6
- MHC II
MHC class II
myelin oligodendrocyte glycoprotein
T cell factor/lymphoid enhancer–binding factor
type 1 Treg
regulatory T cell
The authors have no financial conflicts of interest.