Transforming growth factor beta-activated kinase 1 (TAK1)-dependent checkpoint in the survival of dendritic cells promotes immune homeostasis and function

Abstract

Homeostatic control of dendritic cell (DC) survival is crucial for adaptive immunity, but the molecular mechanism is not well defined. Moreover, how DCs influence immune homeostasis under steady state remains unclear. Combining DC-specific and -inducible deletion systems, we report that transforming growth factor beta-activated kinase 1 (TAK1) is an essential regulator of DC survival and immune system homeostasis and function. Deficiency of TAK1 in CD11c⁺ cells induced markedly elevated apoptosis, leading to the depletion of DC populations, especially the CD8⁺ and CD103⁺ DC subsets in lymphoid and nonlymphoid tissues, respectively. TAK1 also contributed to DC development by promoting the generation of DC precursors. Prosurvival signals from Toll-like receptors, CD40 and receptor activator of nuclear factor-κB (RANK) are integrated by TAK1 in DCs, which in turn mediated activation of downstream NF-κB and AKT-Foxo pathways and established a gene-expression program. TAK1 deficiency in DCs caused a myeloid proliferative disorder characterized by expansion of neutrophils and inflammatory monocytes, disrupted T-cell homeostasis, and prevented effective T-cell priming and generation of regulatory T cells. Moreover, TAK1 signaling in DCs was required to prevent myeloid proliferation even in the absence of lymphocytes, indicating a previously unappreciated regulatory mechanism of DC-mediated control of myeloid cell-dependent inflammation. Therefore, TAK1 orchestrates a prosurvival checkpoint in DCs that affects the homeostasis and function of the immune system.

Dendritic cells (DCs) are a heterogeneous population of immune cells specialized to capture, process, and present antigens to T lymphocytes (1). DCs can be divided into three main populations: conventional DCs (cDCs), type I IFN-secreting plasmacytoid DCs (pDCs), and migratory DCs (2–4). cDCs can be further divided into CD8⁺ and CD8^– DC subsets in lymphoid organs, whereas the CD103⁺ DC subset in nonlymphoid organs serves as a functional equivalent of CD8⁺ cDCs in lymphoid organs (2–4). Homeostasis of DCs is maintained through a dynamic balance of three main mechanisms. First, DCs are continuously replenished by blood-borne precursors that are further derived from macrophage and DC precursor (MDP) and common DC precursor (CDP) populations in the bone marrow (BM) (2, 5). Second, ∼5% of DCs are dividing at any given time. Although limited, the self-renewal ability in situ contributes to the maintenance of the DC population (6–8). Finally, apoptotic cell death of DCs is a central mechanism for down-modulating DC numbers to ensure immune homeostasis (9). Recent studies have provided important insight into the regulation of DC survival by Bcl-2 family members, including the antiapoptotic members Bcl-2 and Bcl-xL and the proapoptotic Bim (10–12). Expression of these Bcl-2 family proteins is dynamically regulated by signals transduced from prosurvival cytokines, ligands for Toll-like receptors (TLRs), and other extracellular stimuli, thereby dictating the decision between survival and death (9). However, it is largely unexplored how the expression of Bcl-2–related proteins and apoptosis of DCs are regulated by the intracellular signaling network.

Despite a well-established role for DCs to induce antigen-specific immune activation and tolerance (13, 14), how DC survival affects immune homeostasis in the steady state, in the absence of encounter with foreign antigens, remains incompletely understood. Enhancing DC survival through transgenic expression of the baculoviral caspase inhibitor p35 or deletion of Fas leads to chronic lymphocyte activation and systemic autoimmunity (15, 16), whereas increasing DC numbers by FMS-like tyrosine kinase 3 ligand (FLT3L) administration protects mice from autoimmunity via inducing regulatory T-cell (T_reg) accumulation (17–19). Conversely, constitutive ablation of cDCs through the expression of diphtheria toxin in CD11c⁺ cells has been reported to cause fatal T-cell–mediated autoimmune disease (20) or a myeloid proliferative syndrome without obvious alterations of T-cell homeostasis (21). Mechanistically, peripheral DCs have been implicated in promoting homeostatic proliferation and survival of T cells (22–24) as well as inducing a low-level tonic signaling in naive T cells to program their responsiveness to foreign antigens (25). Moreover, although a direct role for DCs to promote T_reg expansion and accumulation has been established by recent elegant studies (17, 26), how this effect contributes to immune homeostasis is not fully understood (20, 21, 27, 28). Because many of the previous studies have used nonphysiological approaches to modulate DC survival, how endogenous regulators of DC survival influence immune homeostasis and function remains to be established.

Transforming growth factor beta-activated kinase 1 (TAK1) (encoded by Map3k7) is arguably the most widely used MAP kinase kinase kinase in the immune system (29). The essential role of TAK1 in host immune defense was first demonstrated in Drosophila (30, 31) and later confirmed in murine cells following stimulation through TLRs and proinflammatory cytokine receptors (32, 33). TAK1 also mediates the intracellular sensor pathway mediated by nucleotide-binding oligomerization domain 1 (NOD1) and NOD2 (34, 35), but TLR8-induced activation of NF-κB and JNK is independent of TAK1 (36). In lymphocytes, TAK1 is an essential component of antigen receptor signaling and promotes lymphocyte proliferation and survival and adaptive immune functions (33, 37–40). Moreover, TAK1 is critical for the survival of hematopoietic stem cells and progenitors (41). These results indicate a cell context-dependent function for TAK1 in the immune and hematopoietic systems.

Whereas a role for TAK1 in the initiation of innate immune responses upon pathogen recognition is well established, its role in the homeostatic control of innate immune cells such as DCs has not been examined. To investigate the function of TAK1 in DCs, we generated DC-specific TAK1-deficient mice and found that TAK1 was essential for the homeostasis of DCs by promoting their survival. Using an inducible deletion system, we further identified a direct role of TAK1 to actively maintain mature DCs and BM precursors. Moreover, TAK1 deficiency in DCs caused a myeloid proliferative disorder, disrupted T-cell homeostasis under steady state, and prevented effective T-cell priming and T_reg generation. Our studies demonstrate that a TAK1-mediated checkpoint in DC survival has a key role in the homeostasis and function of the innate and adaptive immune systems.

Results

Cell-Autonomous Role of TAK1 in Regulating DC Populations.

To investigate the function of TAK1 in DCs, we generated DC-specific TAK1-deficient mice by crossing mice bearing floxed and null alleles of the Map3k7 gene with transgenic mice expressing Cre under the control of the CD11c promoter to generate Map3k7^flox/null CD11c-Cre mice (called “Map3k7^DC mice” hereafter) (37, 42). TAK1 was efficiently deleted in splenic DCs but not T cells from these mice (SI Appendix, Fig. S1A). Flow cytometry analysis of splenic DC populations in Map3k7^DC mice showed that the percentage and cell number of cDC (CD11c⁺MHC-II⁺) and pDC (CD11c^lomPDCA-1⁺) populations were markedly reduced compared with those of wild-type (WT) mice (Fig. 1A). Within cDCs, the CD8⁺ subset was more profoundly affected than the CD8^– (CD11b⁺) subset (Fig. 1A). Similar defects were observed in other lymphoid organs, including mesenteric lymph nodes (MLNs) and thymus (Fig. 1B and SI Appendix, Fig. S1B). Moreover, nonlymphoid organs, such as the liver, had lower DC frequency with a preferential reduction of the CD103⁺ DC subset (Fig. 1C). This result is consistent with the recent findings that nonlymphoid CD103⁺ DCs and lymphoid organ-resident CD8⁺ cDCs are developmentally and functionally related (8, 43–45). These results reveal a key role for TAK1 to regulate DC populations in both lymphoid and nonlymphoid organs.

Fig. 1.

TAK1 has a key cell-autonomous role in regulating DC populations. (A) Flow cytometry of splenic cDC, pDC, CD8⁺ cDC, and CD11b⁺ cDC populations in WT and Map3k7^DC mice. MHC-II, MHC class II. (Right) The proportion and cell numbers of the splenic DC populations in WT and Map3k7^DC mice. (B and C) Flow cytometry of DC populations in the MLNs (B) and liver (C) of WT and Map3k7^DC mice. (D) Flow cytometry of the contributions of spike BM-derived (CD45.1.2⁺) and WT or Map3k7^DC donor BM-derived cells (CD45.2.2⁺) in the mixed chimeras after 2 mo of reconstitution. Data are representative of three to five independent experiments. Data represent the mean ± SEM.

To address whether the reduction of DCs in Map3k7^DC mice was an intrinsic defect, we generated mixed BM chimeras. Specifically, we transferred WT or Map3k7^DC BM cells (CD45.2.2⁺; donor) and WT BM cells (CD45.1.2⁺; spike) at a ratio of 1:1 into lethally irradiated WT (CD45.1.1⁺) recipients. After reconstitution, we analyzed splenic DC populations in the mixed chimeras. Compared with WT BM-derived donor cells, Map3k7^DC donor cells in the chimeras contained a greatly reduced DC population (Fig. 1D). Therefore, TAK1 has a key cell-autonomous function in maintaining the DC pool.

TAK1 Promotes DC Survival and Development but Not Proliferation.

The overall size of DC populations is dependent upon the rates of apoptosis and proliferation as well as replenishment from DC precursors (2, 5, 9). We first measured caspase activity, a hallmark of apoptotic cell death (46). The percentage of caspase-positive DCs from Map3k7^DC mice was significantly higher compared with that in WT cells (Fig. 2A). Further, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay also revealed elevated apoptosis of Map3k7^DC DCs (Fig. 2B).

Fig. 2.

Deletion of TAK1 results in excessive apoptosis of DCs and loss of MDPs. (A) Caspase activity in freshly isolated WT and Map3k7^DC splenic DCs, assessed by staining with FITC-conjugated VAD-FMK. (Right) The proportion of caspase⁺ cells. (B) Measurement of apoptosis of WT and Map3k7^DC splenic DCs by TUNEL staining. (C) Flow cytometry of BrdU incorporation into splenic DCs of WT and Map3k7^DC mice after an in vivo BrdU pulse for 24 h. (Right) The percentage of proliferating splenic DCs in WT and Map3k7^DC mice. (D) Flow cytometry of MDPs (Lin^–Sca-1^–CSF1R⁺) in the BM Lin^– cells of WT and Map3k7^CreER mice after 3 d of tamoxifen treatment in vivo. (Right) The proportion of MDPs among BM Lin^– cells. (E) BM cells from WT and Map3k7^CreER mice were cultured with FLT3L in the presence of 0.5 μM 4-OHT. The expression of CSF1R and CD11c was analyzed by flow cytometry at day 4 (Left) and the percentage of CSF1R⁺ cells in CD11c^– cells was calculated (Right). Data are representative of three independent experiments. Data represent the mean ± SEM.

We next tested the possibility that the decreased DC population in Map3k7^DC mice was partly ascribed to defective DC proliferation in situ (6–8). Notably, TAK1 is essential for mediating cell cycle progression in lymphocytes (33, 37–40). We therefore measured DC proliferation in vivo using the Bromodeoxyuridine (BrdU) incorporation assay. DCs in WT and Map3k7^DC mice incorporated BrdU to a comparable degree (Fig. 2C), indicating the lack of a role for TAK1 in DC proliferation.

Finally, we determined whether TAK1 regulated DC development by acting on DC precursors. One of the earliest precursor populations identified for DCs is MDPs (47–49). Because CD11c-Cre mice do not allow deletion in MDPs, we crossed Map3k7^flox/null mice with Rosa26-Cre-ER^T2 mice (a Cre-ER fusion gene was recombined into the ubiquitously expressed Rosa26 locus) to generate Map3k7^flox/null Rosa26-Cre-ER^T2 mice (called “Map3k7^CreER mice” hereafter). Three days after treatment of WT and Map3k7^CreER mice with tamoxifen in vivo, the percentage of MDPs, defined by Lin^–Sca-1^–CSF1R(CD115)⁺ in Map3k7^CreER mice was significantly decreased compared with that in WT mice (Fig. 2D). Next, we cultured WT and Map3k7^CreER BM cells with FLT3L in the presence of 4-hydroxytamoxifen (4-OHT). After 4 d of culture, Map3k7^CreER BM cells showed considerable defects to up-regulate CSF1R, a defining molecule for MDPs (49) (Fig. 2E). These data indicate an important role of TAK1 in the generation of MDPs, thereby contributing to DC development. Altogether, TAK1 is critical for the survival and development but not proliferation of DCs.

TAK1 Is Essential for Maintaining DC Survival by Integrating Prosurvival Receptor Signals.

To directly investigate the effects of TAK1 on the homeostasis of mature DCs, we analyzed DCs in WT and Map3k7^CreER mice after short-term treatment with tamoxifen. Acute systemic deletion of TAK1, although lacking cell type-specific precision, allowed us to exclude the compensatory effects that might have developed due to the continuous loss of TAK1 in DCs or precursors. Following in vivo tamoxifen-mediated TAK1 deletion, splenic cDCs were markedly reduced, with a more prominent reduction in the CD8⁺ subset. The pDC population was also greatly reduced (Fig. 3A). Accordingly, caspase activation in DCs was elevated in these cells (Fig. 3B). The effect of TAK1 deletion on DC maintenance was further tested in vitro. BM cells from WT or Map3k7^CreER mice (CD45.2⁺) were mixed with an equal number of WT (CD45.1⁺) spike cells, and after 7 d of culture with FLT3L to derive DCs, 4-OHT was added. After culturing for 6 additional days, CD11c⁺ cells from Map3k7^CreER BM cells were selectively reduced (Fig. 3C), associated with enhanced apoptosis (Fig. 3D), compared with WT or spike-derived cells. These results demonstrate that one essential mechanism for the prosurvival function of TAK1 is through the maintenance of mature DC survival.

Fig. 3.

TAK1 is essential for the maintenance of mature DCs. (A) Flow cytometry of splenic cDC, pDC, CD8⁺ cDC, and CD11b⁺ cDC populations in WT and Map3k7^CreER mice after 3 d of tamoxifen treatment. (Right) The proportion and cell numbers of the splenic DC populations. (B) Caspase activity in splenic DCs from tamoxifen-treated WT and Map3k7^CreER mice, assessed with FITC-VAD-FMK. (Right) The proportion of apoptotic VAD-FMK⁺ splenic DCs. (C and D) BM cells from WT or Map3k7^CreER mice (CD45.2⁺) were cultured with WT CD45.1⁺ BM cells at a 1:1 ratio in the presence of FLT3L, and 0.5 μM 4-OHT was added at day 7. The distribution (C) and apoptosis (D) of CD45.1^– and CD45.1⁺ populations in the gated CD11c⁺ cells were analyzed by flow cytometry at the indicated time points. Data are representative of three independent experiments. Data represent the mean ± SEM.

The life span of DCs is determined by signals from pathogens and T cells that are transduced through TLRs, CD40, and receptor activator of nuclear factor-κB (RANK) (9, 10). To test the intrinsic requirement of TAK1 in mediating these prosurvival receptor signals, we generated FLT3L-derived DCs from WT and Map3k7^DC BM cells and treated purified DCs with LPS, α-CD40, or RANKL for 2 d. In the absence of treatment, TAK1-deficient DCs had more spontaneous death compared with WT cells. LPS treatment resulted in enhanced survival of WT cells, but this effect was substantially reduced in Map3k7^DC DCs. Moreover, TAK1-deficient DCs remained nearly completely unresponsive to the survival effects induced by α-CD40 or RANKL stimulation (SI Appendix, Fig. S2). These findings identify TAK1 as an important sensor that links signaling activities of extracellular receptors and survival of DCs.

TAK1-Dependent Pro- and Antiapoptotic Signaling Mechanisms in DCs.

We dissected the biochemical and molecular mechanisms by which TAK1 regulates DC survival. We first measured the effects of TAK1 deficiency on the activity of NF-κB, which is important for DC survival (50). Constitutive NF-κB activity, assessed by IκBα phosphorylation, was considerably diminished in freshly isolated Map3k7^DC splenic DCs (Fig. 4A), indicating a role for TAK1 to mediate NF-κB activation in DCs in vivo. Activity of AKT (also known as protein kinase B, PKB), another antiapoptotic kinase in DCs (51), was also diminished in TAK1-deficient DCs. Accordingly, phosphorylation of the AKT downstream target Foxo1 (Ser256), which exerts a potent inhibitory effect on Foxo1 activity, was decreased (Fig. 4A). The AKT-Foxo axis is important for the expression of the proapoptotic molecule Bim (52). Indeed, Bim expression was up-regulated in TAK1-deficient DCs, whereas the level of the antiapoptotic Bcl2 was not altered (Fig. 4A). In addition, production of reactive oxygen species (ROS), which is under the control of TAK1 signaling in other cell types (53), was undisturbed in TAK1-deficient DCs (SI Appendix, Fig. S3). Therefore, TAK1 regulates the activities of the NF-κB pathway and the AKT-Foxo-Bim signaling axis, and the interplay of these pathways likely contributes to the physiological function of TAK1 in mediating DC survival.

Fig. 4.

TAK1 regulates the activities of NF-κB and AKT-Foxo signaling and the expression of apoptotic factors and immune response genes. (A) Western blot analyses of p-IκB, p-AKT, p-Foxo1, Bim, and Bcl-2 expression in splenic DCs from WT and Map3k7^DC mice. Numbers below lanes indicate band intensity relative to that of β-actin (loading control). (B) WT and Map3k7^CreER mice were treated with tamoxifen for 3 d and RNA of splenic DCs was analyzed for the comparison of expression profiles. A subset of genes differentially regulated in WT and TAK1-deficient DCs (false discovery rate < 0.05) is shown. Normalized gene expression is displayed. (Scale bar shows SD from the mean.) (C) WT and Map3k7^CreER mice were treated with tamoxifen for 3 d and RNA of splenic DCs was analyzed for the expression of the indicated genes. Data are representative of three independent experiments. Data represent the mean ± SEM.

To further understand the function of TAK1 in DCs, we used a functional genomics approach to analyze TAK1-dependent gene signature. To obviate compensatory effects due to sustained loss of TAK1, we purified splenic DCs from WT and Map3k7^CreER mice after acute tamoxifen treatment and used microarrays to compare their gene expression profiles. A total of 550 probe sets showed equal or greater than twofold change (with false discovery rate <0.05) between WT and TAK1-deficient DCs. TAK1-deficient DCs showed up-regulated levels of several proapoptotic factors including Apaf-1, Bim (encoded by Bcl2l11), Caspase 6, and Gadd45a and reduced expression of transcription factors Nfkb2 and Relb. In addition, expression of several cytokines, chemokines, and their receptors was altered in TAK1-deficient DCs (Fig. 4B). Real-time PCR analysis confirmed the altered expression of selected genes (Fig. 4C). Interestingly, IL-10 and the IL-10 target gene SOCS3 were found to be significantly up-regulated in DCs from tamoxifen-treated Map3k7^CreER mice (Fig. 4 B and C). DCs from Map3k7^DC mice showed a similar increase in the expression of IL-10 and SOCS3, as well as proapoptotic Bim and Gadd45a (SI Appendix, Fig. S4). Furthermore, elevated STAT3 phosphorylation was observed in DCs from Map3k7^DC and tamoxifen-treated Map3k7^CreER mice (SI Appendix, Fig. S5), thus indicating an active autocrine IL-10 signaling in TAK1-deficient DCs. Therefore, TAK1 orchestrates a prosurvival program in DCs by regulating expression of apoptotic molecules and further links them with expression of immune response genes.

TAK1 Functions in DCs to Promote T-Cell Priming and T_reg Generation.

DCs are the most potent antigen-presenting cells (APCs) to activate naive T cells. To investigate the role of TAK1 signaling in DCs to mediate T-cell–dependent immune responses, we transferred antigen-specific CD8⁺ T cells from OT-I TCR-transgenic mice (specific for the OVA_257–264 peptide) into WT and Map3k7^DC mice, followed by immunization with the cognate antigen. T cells isolated from Map3k7^DC hosts contained fewer donor-derived antigen-specific T cells (Fig. 5A). We obtained similar results for antigen-specific CD4⁺ T-cell responses using OT-II TCR-transgenic T-cells (specific for OVA_323–339) (Fig. 5B). Moreover, OT-II T cells activated with TAK1-deficient DCs in vitro were substantially impaired in their expansion (SI Appendix, Fig. S6A). These findings demonstrate that TAK1 signaling in DCs is required for the activation of antigen-specific naive T cells.

Fig. 5.

TAK1 signaling in DCs is required for T-cell priming and T_reg generation. (A and B) Antigen-specific OT-I CD8⁺ (CD45.1⁺) (A) or OT-II CD4⁺ T cells (Thy1.1⁺) (B) were transferred into WT and Map3k7^DC mice followed by antigen immunization in the presence of IFA. At day 4 after immunization, DLN cells were examined for the percentages of donor T cells. (C) Proportion of Foxp3⁺ nT_reg cells among CD4⁺ T cells in the thymus, spleen, PLNs, and MLNs of WT and Map3k7^DC mice. (Right) The proportion of Foxp3⁺ cells in CD4⁺ T cells of WT and Map3k7^DC mice. (D) WT and Map3k7^DC mice were transferred with naive OT-II CD4⁺ T cells (Thy1.1⁺) and immunized as described in B, and DLN cells were examined for the percentage of Foxp3⁺ cells among donor cells. (E) After adoptive transfer of naive OT-II CD4⁺ T cells, WT and Map3k7^DC mice were fed with water supplemented with OVA protein. Five days later, the spleen and PLN cells were examined for the percentages of donor CD4⁺ T cells (Thy1.1⁺) and of Foxp3⁺ cells among donor cells. Data are representative of two to four independent experiments. Data represent the mean ± SEM.

In addition to T-cell priming, DCs have been recently shown to promote T_reg cell generation (17, 54–57). T_reg cells either develop in the thymus, known as naturally occurring T_reg (nT_reg) cells, or are induced from naive T cells in the periphery under subimmunogenic antigen stimulation, known as induced T_reg (iT_reg) cells (58). Whereas nT_reg cells were undisturbed in the thymus of Map3k7^DC mice, they were considerably reduced in peripheral lymph organs (Fig. 5C). Given the critical importance of TAK1 signaling in T_reg cell generation (37, 38), it remained possible that the reduction of nT_reg cells could result from an off-target deletion of TAK1 in T cells in Map3k7^DC mice. However, in mixed chimeras as described above (Fig. 1D), the nT_reg population derived from Map3k7^DC BM cells developed normally (SI Appendix, Fig. S7), indicating a non-cell–autonomous defect triggered by the abnormality of DCs.

We next tested whether TAK1 regulated induction of iT_reg cells. We adoptively transferred naive OT-II CD4⁺ T cells into WT and Map3k7^DC hosts, followed by s.c. antigen immunization or administration of the antigen in the drinking water. In both models, donor cells developed into a considerably smaller Foxp3⁺ population in Map3k7^DC recipients relative to WT hosts (Fig. 5 D and E), indicating a role of TAK1 in DCs to mediate induction of antigen-specific iT_reg cells. This result was further confirmed by the impaired ability of TAK1-deficient DCs to mediate iT_reg induction in vitro (SI Appendix, Fig. S6B). We conclude that TAK1 function in DCs is critical for both naive T-cell priming and T_reg generation.

TAK1 Deficiency in DCs Disrupts Homeostasis of T Cells and Myeloid Cells.

How DCs affect homeostasis of the immune system remains incompletely understood. We therefore tested whether defective DC survival in Map3k7^DC mice influenced T-cell homeostasis. Map3k7^DC mice had reduced proportions of T cells, especially CD8⁺ T cells, in the thymus, spleen, and peripheral lymph nodes (PLNs) (Fig. 6A). Moreover, splenic T cells from Map3k7^DC mice were partially impaired to produce IFN-γ and IL-2 upon acute polyclonal stimulation (Fig. 6B), indicative of altered homeostasis of T cells in the steady state.

Fig. 6.

Map3k7^DC mice show diminished conventional T cells but expanded myeloid cells. (A) Distribution of CD4⁺ and CD8⁺ T cells in the thymus, spleen, and PLNs of WT and Map3k7^DC mice. (Right) The proportion of CD4⁺ and CD8⁺ T cells in WT and Map3k7^DC mice. (B) Expression of IFN-γ and IL-2 in splenic CD4⁺ and CD8⁺ T cells was determined by intracellular cytokine staining after stimulation with PMA and ionomycin. (C) Spleen sections from WT and Map3k7^DC mice were analyzed by H&E, Masson's trichrome, and reticulin staining for fibrosis and by anti-CD3 and anti-Mac2 immunohistochemistry. (D) Flow cytometry of myeloid cells (Gr1⁺CD11b⁺) in the spleen, PLNs, and MLNs of WT and Map3k7^DC mice. Data are representative of at least three independent experiments. Data represent the mean ± SEM.

Despite diminished DC and T-cell populations, the spleen and PLN sizes of Map3k7^DC mice were markedly increased (SI Appendix, Fig. S8A). Histological analysis showed that the structure of the secondary lymphoid organs was disrupted, associated with altered distribution of CD3⁺ T cells. In addition, the spleen of Map3k7^DC mice showed severe fibrosis, detected by trichrome and reticulin staining (Fig. 6C). Moreover, increased numbers of Mac2-reactive myeloid cells were evident in the mutant spleen and PLNs (Fig. 6C and SI Appendix, Fig. S8B). Flow cytometry analysis of spleen, MLNs, and PLNs in Map3k7^DC mice revealed a marked increase in Gr1⁺CD11b⁺ myeloid cells that likely corresponded to neutrophils and Gr1⁺ inflammatory monocytes (Fig. 6D) (59). Analysis of additional markers in Map3k7^DC lymphoid organs confirmed the expansion of neutrophils (Ly6G⁺Ly6C⁺) and monocytes (CD115⁺CD11b⁺), with the latter population mainly consisting of the Ly6C⁺ inflammatory subset rather than the Ly6C^– subset (SI Appendix, Fig. S9) (59). Flow cytometry and blood counts identified a similar shift toward neutrophils and monocytes in peripheral blood of Map3k7^DC mice (SI Appendix, Fig. S10). Further, myeloid cell infiltration was also observed in various nonlymphoid organs from Map3k7^DC mice, including the liver, lung, kidney, and colon (SI Appendix, Fig. S11). In contrast, Map3k7^DC BM contained largely normal populations of neutrophils and monocytes (SI Appendix, Fig. S12). These results collectively indicate the development of a myeloid proliferative syndrome in Map3k7^DC mice.

Myeloid Proliferation Results from Loss of TAK1 in DCs Independently of Lymphocytes.

We dissected the cellular mechanisms that caused defective homeostasis of myeloid cells in Map3k7^DC mice. Such a disorder could reflect a direct role of TAK1 in myeloid cells (for example, by nonspecific deletion) or could be due to dysregulated functions of T cells, especially T_reg cells, or triggered by a mechanism that directly sensed the abnormality of DCs in vivo. To distinguish these possibilities, we analyzed the mixed BM chimeras composed of Map3k7^DC donor and spike cells, as described above (Fig. 1D). No myeloid expansion was observed in the mixed chimeras that contained normal DC populations derived from the spike cells (Fig. 7A), suggesting that a normal DC population is important to prevent myeloid expansion.

Fig. 7.

Myeloid expansion in Map3k7^DC mice results from altered DC–myeloid cell crosstalk independently of lymphocytes. (A) Flow cytometry of spike BM-derived and WT or Map3k7^DC BM-derived myeloid cells in the mixed chimeras (Fig. 1D). (B) Flow cytometry of myeloid cells in the spleen, PLNs, MLNs, and thymus of Rag1^−/− and Map3k7^DC Rag1^−/− mice. (C) Analysis of FLT3L levels in the serum from WT and Map3k7^DC mice. Each symbol represents an individual mouse and short horizontal lines indicate the mean.

We next determined whether myeloid proliferation in Map3k7^DC mice resulted from altered T-cell function or tolerance. In particular, depletion of DCs has been implicated in the development of autoimmune diseases that is dependent upon dysregulated autoreactive T cells and/or defective T_reg-mediated immune suppression (17, 20). However, T cells in Map3k7^DC mice did not appear to be activated, indicated by the largely normal expression of the homeostasis markers CD62L and CD44 and the absence of elevated serum autoantibodies (SI Appendix, Fig. S13). To further dissect the contribution of T cells, we crossed Map3k7^DC mice onto the alymphoid Rag1^−/− background. Notably, Rag1 deficiency elevated the population of myeloid cells compared with that in immunocompetent WT mice (Fig. 7B; compare with Fig. 6D), probably triggered by the lymphopenic environment. However, deletion of TAK1 in DCs on the same Rag1^−/− background further expanded the myeloid populations including neutrophils and Ly6C⁺ inflammatory monocytes (Fig. 7B and SI Appendix, Fig. S14). Therefore, defective control of lymphoid and myeloid populations in Map3k7^DC mice was uncoupled after depletion of lymphocytes, which revealed an important role for TAK1 signaling in DCs to maintain myeloid homeostasis in a manner independently of lymphocytes.

We further explored the mechanism by which lack of DCs triggered myeloid expansion. Depletion of DCs has recently been shown to increase the levels of FLT3L, a key growth factor for myeloid cell generation (21, 60). Indeed, the serum concentration of FLT3L was significantly elevated in Map3k7^DC mice compared with the WT controls (Fig. 7C). Thus, FLT3L was up-regulated in the absence of a normal DC population that likely contributed to the abnormal myeloid expansion.

Discussion

Homeostasis of DCs is essential for the generation of adaptive immunity and the maintenance of immune tolerance. Here we have identified TAK1 as a critical regulator of DC survival and homeostasis. Using DC-specific and acute deletion systems, we have established that TAK1 is required to maintain DC populations by promoting the survival of DCs and generation of their precursors. TAK1 integrates prosurvival receptor signals and in turn activates NF-κB and AKT-Foxo pathways while controlling expression of Bim and other proapoptotic molecules. TAK1 signaling in DCs facilitates T-cell priming and generation of T_reg cells, indicating a role of TAK1 to mediate both the immunogenic and tolerogenic activities of DCs. Moreover, loss of this active control mechanism in DCs has profound effects on the homeostasis of T cells and myeloid populations, and additional analysis reveals that TAK1 acts in DCs to control myeloid cell-mediated inflammation in a lymphocyte-independent manner. Therefore, a TAK1-dependent checkpoint in DC survival orchestrates immune function and homeostasis.

DC homeostasis is dependent upon the interplay of three main processes: development from precursors, division in situ, and apoptotic cell death. Combining tissue-specific and inducible deletion systems, we found that both acute and sustained loss of TAK1 resulted in an extensive apoptotic death of DCs. This result contrasted with the observations in neuronal cells in which short-term inhibition of TAK1 protects neurons from apoptosis, whereas prolonged inhibition is not protective (61). Therefore, our findings indicate a direct effect of TAK1 to actively maintain DC survival. Moreover, TAK1 contributed to DC development by maintaining the MDP population. However, TAK1 was dispensable for DC division, which differed from a critical requirement of TAK1 in clonal expansion of lymphocytes (33, 37–40). Therefore, TAK1 has a unique role in DCs to selectively regulate their survival and development.

Among DC populations, the CD8⁺ and CD8^– cDC subsets efficiently present antigens to CD8⁺ and CD4⁺ T cells, respectively (62). The loss of CD8⁺ cDCs was more profound relative to the CD8^– subset in Map3k7^DC mice. However, the remaining CD8^– cDCs probably exhibited qualitative changes, reflected by their up-regulated CD11b levels on a per cell basis (Fig. 1A). Accordingly, both CD8⁺ and CD4⁺ T-cell responses were diminished in response to antigen challenge, in agreement with a requirement of TAK1 in the contact hypersensitivity response (63). Deletion of TAK1 was also incompatible with the survival of pDCs, and a more severe reduction of pDCs upon acute deletion of TAK1 than DC-specific TAK1 deficiency probably reflected the significant but incomplete deletion efficiency in pDCs in the CD11c-Cre line (42). Finally, the CD103⁺ DCs in the nonlymphoid organs were as sensitive to TAK1 deficiency as CD8⁺ cDCs in the lymphoid organs, which provides added genetic evidence supporting the notion that these two populations are developmentally and functionally linked (8, 43–45). Therefore, TAK1 is critically required for the survival of all major DC subsets.

The molecular mechanisms in DC survival remain to be fully established. Among the best understood pathways for the survival of DCs are the Bcl-2 family members, as previous work has implicated a pivotal role for Bcl-2, Bcl-xL, and Bim in DC survival (9). How these factors are regulated by the intracellular signaling network is not well defined. Here we found an elevated Bim expression in TAK1-deficient DCs, which likely resulted from the altered AKT-Foxo signaling axis (52). In addition, TAK1 was required for NF-κB activity in DCs. Moreover, TAK1-deficient DCs showed an enhanced autocrine IL-10 signaling, which likely further contributed to the increased cell death, given the important role of IL-10 in promoting DC apoptosis (64). Taken together, TAK1 affects signal transduction and gene expression of both proapoptotic and antiapoptotic pathways and further links them with expression of immune response genes, thereby orchestrating a prosurvival program in DCs. Consistent with this notion, TAK1 integrates multiple prosurvival receptor signals in DCs.

As the most potent type of APCs, DCs are essential for the initiation and propagation of adaptive immune responses, but how DCs are involved in the maintenance of nT_reg cell homeostasis remains under debate. Map3k7^DC mice contained a greatly reduced number of nT_reg cells in the periphery under steady state. These results differ from the earlier reports excluding a role for DCs to regulate nT_reg cells (20, 21), but are consistent with more recent studies implicating a requirement for DCs to maintain the nT_reg population (17, 27, 65). Interestingly, despite defective iT_reg generation and nT_reg homeostasis, T cells in Map3k7^DC mice exhibited no signs of prominent activation. Instead, we found reduced populations of CD4⁺ and CD8⁺ T cells and diminished production of IFN-γ and IL-2 in T cells from Map3k7^DC mice. Our results are in agreement with a recent DC ablation study in lupus-prone MRL/lpr mice that reveals an important role for DCs to maintain conventional T cells and their ability to produce IFN-γ (65). Thus, TAK1 acts in DCs to orchestrate T-cell priming and homeostasis but not self-tolerance.

Unexpectedly, deficiency of TAK1 in DCs results in a prominent myeloid proliferative disease characterized by expansion of neutrophils and Ly6C⁺ inflammatory monocytes, suggesting that DCs provide a negative signal to restrain myeloid expansion and inflammation under physiological conditions. The myeloid defects largely recapitulate the phenotypes of mice with constitutive loss of cDCs (20, 21), although the precise mechanism involved remains in debate. In particular, dysregulated autoreactive T cells and impaired T_reg cells have been suggested as important mechanisms for excessive inflammation (17, 20). However, deletion of TAK1 in DCs disrupted myeloid homeostasis even on a Rag1^−/− background. Although our results do not exclude the contribution of T_reg or conventional T cells, they provide definitive evidence that DC signaling can directly impact myeloid cell homeostasis independently of lymphocytes, thus establishing a previously unappreciated mechanism of DC-mediated active control of myeloid expansion and inflammation. The crosstalk between DCs and myeloid cells is likely mediated by FLT3L, given the key role of FLT3L in myeloid cell expansion (21, 60), although this action does not appear to affect myeloid populations in the BM. Recently, expansion of myeloid populations and increases of serum FLT3L levels have been reported in mice with DC-selective deletion of A20, a negative regulator of NF-κB signaling (66, 67). However, unlike DCs lacking TAK1, A20-deficient DCs show enhanced responses to CD40 and RANK-mediated survival signals and trigger profound activation of T cells and B cells and development of autoimmunity (66, 67). Therefore, immune homeostasis under steady state is dependent upon molecular signals in DCs, especially a delicate threshold of NF-κB signaling.

The life span of DCs is important for the strength of adaptive immunity (10, 68, 69). Our results have identified a TAK1-dependent checkpoint in DC survival that influences the magnitude and quality of adaptive immune responses, as well as homeostasis of the immune system. In Drosophila TAK1 has a crucial function in mounting host defense reactions (30, 31). Not only does the mammalian immune system retain this function for innate defense responses (32, 33), but also DCs have evolved to acquire this evolutionarily conserved pathway to regulate their life span and further imprint innate and adaptive immunity. This molecular pathway in DCs may be explored for the development of DC-based therapeutic strategies.

Materials and Methods

Mice and BM Chimeras.

C57BL/6, CD45.1, Thy1.1, Rag1^−/−, OT-I, and OT-II mice were purchased from the Jackson Laboratory. Foxp3^gfp mice were kindly provided by A. Rudensky (Memorial Sloan-Kettering Cancer Center, New York, NY) (70). Floxed Map3k7 (Map3k7^flox/null) mice were bred with CD11c-Cre or Rosa26-Cre-ER^T2 mice (37, 42, 46) and have been backcrossed to the C57BL/6 background for at least eight generations. WT controls were in the same genetic background and included Cre⁺ mice to account for Cre effects. For in vivo tamoxifen treatment, WT and Map3k7^CreER mice were injected i.p. with 1 mg per mouse of tamoxifen (Sigma) for 3 consecutive days before further analysis. For mixed BM experiments, BM cells from WT or Map3k7^DC CD45.2.2⁺ mice were mixed with cells from CD45.1.2⁺ mice at a 1:1 ratio and transferred into lethally irradiated (11 Gy, split dose) CD45.1.1⁺ mice, as described previously (71). Animal protocols were approved by Institutional Animal Care and Use Committee of St. Jude Children's Research Hospital.

Cell Purification and Cultures.

Mouse spleens were digested with Collagenase D (Roche), and DCs (CD11c⁺TCR^–CD19^–DX5^–) were sorted on a Reflection cell sorter (i-Cyt). DCs from nonlymphoid organs were isolated as described in ref. 8. Lymphocytes were sorted for naive T cells (CD4⁺CD62L^hiCD44^loCD25^–). For DC culture, BM cells were cultured in medium containing 10% FBS and mouse FLT3L (200 ng/mL); CD11c⁺ cells were sorted at day 7, washed extensively, and treated with 10 μg/mL LPS, 10 μg/mL α-CD40, 1 μg/mL RANKL, or mock for 2 d for the analysis of cell survival. For the mixed culture, WT or Map3k7^CreER BM cells were cultured with CD45.1⁺ BM cells at a 1:1 ratio in the presence of FLT3L. From day 7, 0.5 μM 4-OHT (Sigma-Aldrich) was added to each well to delete TAK1. For DC–T-cell coculture, splenic DCs and OT-II T cells (Foxp3^GFP) were mixed at a 1:10 ratio in the presence of 1 μg/mL OVA_323–339 peptide and cultured for 5 d, followed by flow cytometry analysis.

Antigen Challenge.

Antigen-specific T cells from OT-I (CD45.1⁺; 1 × 10⁶) or OT-II TCR-transgenic mice (Thy1.1⁺; 2 × 10⁶) were sorted and transferred into WT and Map3k7^DC mice intravenously. Twenty-four hours later, the mice were injected s.c. with OVA_257–264 or OVA_323–339 (5 μg) in the presence of incomplete Freund's adjuvant (IFA; Difco). At day 4 after immunization, draining lymph node (DLN) cells were isolated for further analyses (71). In the model of oral antigen stimulation, after adoptive transfer of naive OT-II T cells, mice were fed with water supplemented with 20 mg/mL OVA protein (Grade VI; Sigma-Aldrich) for a total of 5 d (71).

Flow Cytometry.

Flow cytometry was performed as described previously (46, 71, 72). For intracellular cytokine detection, the cells were stimulated for 5 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of monensin before staining according to the manufacturer's instructions (BD Biosciences). For caspase activity detection, cells were stained with CaspACE FITC-VAD-FMK in situ marker according to the manufacturer's instructions (Promega). For TUNEL staining, freshly isolated splenocytes were used according to the protocol supplied (BD Biosciences). ROS were measured by incubation for 30 min at 37 °C with 10 μM CM-H2DCFDA (Invitrogen). For BrdU labeling, mice were injected i.p. with 1 mg BrdU (5-bromo-2-deoxyuridine); 24 h later, mice were killed and BrdU incorporation was analyzed according to the manufacturer's instructions (BD Biosciences) (46).

RNA and Protein Analyses.

Real-time PCR analysis was done with primers and probe sets from Applied Biosystems as described in refs. 46 and 71. Microarray analysis was performed as described previously (46, 72), and the results have been deposited in the GEO database (GSE34417). The heat maps were generated to show relative expression of genes after z-transformation, which subtracts the mean of each from each individual value for that gene and then divides by the SD. This method sets all of the genes into the same scale with a mean of 0 and SD of 1. The scale bar shows the distance from the mean expression as SD units. Immunoblot was performed as described in refs. 46 and 71, using the following antibodies: p-STAT3, p-AKT (Ser473), p-Foxo1 (Ser256), and p-IκBα (all from Cell Signaling Technology); Bcl-2 (Santa Cruz); Bim (Abcam); and β-actin (Sigma). The serum levels of FLT3L and autoantibodies were measured by ELISA as described in ref. 73.

Histopathology and Immunohistochemistry.

The spleen and PLNs were processed as described previously (72) and stained with hematoxylin and eosin (H&E), Masson's trichrome, and reticulin stains for the visualization of collagen deposition. Immunohistochemistry was performed as described previously (72). Peripheral blood samples were collected for blood counts.

Statistical Analysis.

P values were calculated using Student's t test. P values <0.05 were considered significant.