To balance immunity and tolerance, the endogenous pool of Foxp3+ regulatory T (Treg) cells is tightly controlled, but the underlying mechanisms of this control remain poorly understood. Here we show that the number of Treg cells is negatively regulated by the kinase Lkb1 in dendritic cells (DCs). Conditional knockout of the Lkb1 gene in DCs leads to excessive Treg cell expansion in multiple organs and dampens antigen-specific T cell immunity. Lkb1-deficient DCs are capable of enhancing, compared with wild-type DCs, Treg cell proliferation via cell-cell contact involving the IKK/IKBα-independent activation of the NF-κB/OX40L pathway. Intriguingly, treating wild-type mice with lipopolysaccharide selectively depletes Lkb1 protein in DCs, resulting in Treg cell expansion and suppressed inflammatory injury upon subsequent challenge. Loss of Lkb1 does not obviously upregulate proinflammatory molecules expression on DCs. We thus identify Lkb1 as a regulatory switch in DCs for controlling Treg cell homeostasis, immune response and tolerance.
Foxp3+ Treg cells, either derived from the thymus (natural Treg cells, nTreg cells) or induced from conventional T (Tcon) cells in the periphery (induced Treg cells, iTreg cells), play pivotal roles in suppressing immune responses1,2. Under steady-state conditions, the number of Treg cells in each organ is maintained at a constant threshold level to ensure self-tolerance while allowing the efficient initiation of defensive responses2. During a variety of inflammatory processes, Treg cells usually increase in number through proliferation or de novo generation, and this increase might serve as critical negative feedback to restrain inflammatory injuries1,3,4. However, despite the earlier recognition of IL-2 produced by effector T cells as a basic factor in the maintenance of the Treg cell pool1,2, our knowledge of how the number of Treg cells is specifically regulated in a wide range of tissues and under various immune conditions is still very limited.
Originally known as sentinels of the immune system required for initiating defensive responses, DCs critically maintain immune homeostasis not only through suppressing the activation or inducing the unresponsiveness/apoptosis of self-reactive T cells but also by promoting the generation, maintenance and/or expansion of Treg cells5,6,7. It is widely accepted that DCs can induce the de novo generation of iTreg cells via mechanisms dependent on antigen presentation to induce a distinct T cell receptor (TCR) signal8, costimulation via B7-CTLA49 or BTLA-HVEM10 ligation, and TGF-β11 and retinoic acid12,13,14 production. Nevertheless, how DCs maintain or expand the existing pool of Treg cells to balance immunity and tolerance in vivo is unclear.
Liver kinase B1 (Lkb1) is a serine-threonine kinase that was first identified as a tumour suppressor whose mutation is responsible for Peutz Jeghers syndrome15,16. Previous studies have highlighted a prominent role played by Lkb1 in the immune system; Lkb1 has been identified as a critical regulator of T cell development, activation, and metabolism17. Our recent work indicates that Lkb1 epigenetically stabilizes Foxp3 expression and promotes suppressive functions in Treg cells18. Lkb1 also inhibits the activation and inflammatory functions of innate macrophages19. However, the specific role played by Lkb1 in DCs, which are central to immune regulation, has not yet been studied.
In this study, using mice with the Lkb1 gene conditionally deleted in DCs, we find that the expression of Lkb1 is a feedforward factor in DCs rather than other immune cell types, required for restraining the steady-state Treg cell numbers in multiple organs, thereby allowing the efficient initiation of antigen-specific immune responses. We further evaluate whether ectopic Lkb1 downregulation in DCs contributes to increases in the Treg cell population during inflammatory processes. Indeed, we find that after challenging wild-type (WT) mice with Escherichia coli (E. coli)-derived lipopolysaccharide (LPS), Lkb1 protein is selectively depleted in DCs in a negative-feedback manner, resulting in the expansion of Treg cells that is necessary for protecting the host from inflammatory injury responses induced by lethal re-challenge doses. Mechanistically, the loss of Lkb1 activates a regulatory transcriptional programme in DCs, including the upregulation of Ox40l gene expression, to stimulate Treg cell proliferation. Thus, we provide data indicating that the homeostasis of Treg cells and the strength of immunosuppression are dynamically controlled by an Lkb1 regulatory switch in DCs, in a feedback manner, to ensure immune equivalence.
Lkb1 deletion in DCs leads to Treg pool enlargement
To investigate the role of Lkb1 in DCs, we generated a line of mice with Lkb1 conditionally deleted in DCs by crossing Cd11cCre mice with Lkb1f/f mice. We analysed the phenotype of DCs in the spleen and lymph nodes (LNs). No significant differences in the population size or cell surface expression of activation markers, including major histocompatibility complex (MHC) class II molecules and co-stimulatory molecules CD80 and CD86, were observed between DCs from Lkb1f/f and Cd11cCreLkb1f/f mice (Supplementary Fig. 1a-c), indicating that Lkb1 has little effect on DC maintenance and activation.
Since the major functions of DCs are to preserve steady-state T cell homeostasis and induce foreign antigen-specific T cell responses20, we sought to determine the phenotypical and functional alterations in T cells from Lkb1f/f and Cd11cCreLkb1f/f mice. Cd11cCreLkb1f/f mice had higher percentages of CD4+Foxp3- and CD8+Foxp3- T cells that exhibited a CD44hiCD62Llo effector/memory phenotype (Supplementary Fig. 2a). However, despite a slight elevation in IL-2 and IL-17 production, the IL-4 and IFN-γ levels were not significantly increased in CD4+Foxp3- or CD8+Foxp3- T cells from Cd11cCreLkb1f/f mice (Supplementary Fig. 2b), indicating that excessive effector T cell differentiation was not present in Cd11cCreLkb1f/f mice. Indeed, the Cd11cCreLkb1f/f mice exhibited a normal body weight (Supplementary Fig. 2c) and did not display any signs of autoimmunity (Supplementary Fig. 2d). These results indicate that Cd11cCreLkb1f/f mice are devoid of autoimmune diseases, even in the presence of a slightly activated T cell phenotype. Surprisingly, there were no significant differences in the proliferation of CFSE-labelled CD4+ and CD8+ T cells sorted from OT-II and OT-I transgenic mice after being co-cultured with Lkb1f/f and Cd11cCreLkb1f/f DCs loaded with the respective antigenic peptide in vitro (Supplementary Fig. 2e), suggesting that Lkb1 does not affect the DC function of priming antigen-specific T cell responses.
Strikingly, the percentage and absolute number of Foxp3+ Treg cells were greatly increased in the spleen and LNs from Cd11cCreLkb1f/f mice compared with those from Lkb1f/f mice, as was the expression of Ki67, an indicator of cell proliferation (Fig. 1a–d). However, no significant difference in the apoptosis of Treg cells from Lkb1f/f mice and Cd11cCreLkb1f/f was observed (Supplementary Fig. 3a, b). There were also significantly higher frequencies of Treg cells in peripheral non-lymphoid organs, including the blood, bone marrow (BM), lungs, liver, kidneys and brain, from Cd11cCreLkb1f/f mice than in those from Lkb1f/f mice (Fig. 1e, f). However, the Treg cell frequency was not significantly elevated in the thymus in Cd11cCreLkb1f/f mice (Supplementary Fig. 3c, d), suggesting that the enlarged Treg cell compartment in the periphery was not caused by developmental dysregulation in the thymus. Treg cells from Cd11cCreLkb1f/f mice displayed a CD44hiCD62Llo activated phenotype and expressed higher levels of activation markers CD73 and ICOS (Fig. 1g). In addition, Treg cells from Cd11cCreLkb1f/f mice expressed high levels of Nrp1 and Helios21,22,23, which are characteristic markers of nTreg cells (Fig. 1g), suggesting that these expanded Treg cells were mainly derived from the thymus rather than Tcon cells in the periphery. Thus, the Lkb1 deficiency in DCs caused strong Treg cell expansion in the periphery. However, Lkb1 seemed not to directly affect the suppressor function of Treg cells, since there was no difference in the suppressive capacity of Treg cells from Lkb1f/f and Cd11cCreLkb1f/f mice (Fig. 1h).
Cd11cCreLkb1f/f mice lack Lkb1 in all cells expressing CD11c, which include DCs and macrophages. To determine whether Lkb1-deficient macrophages contribute to the phenotype of the mice, we examined Treg and Tcon cells in the spleen and LNs of Lkb1f/f and LysMCreLkb1f/f mice, which carry the specific deletion of Lkb1 in myeloid-derived cells, including macrophages. We found that the Lkb1 deficiency in macrophages did not affect the percentages of Treg cells or the activation of CD4+ Foxp3− and CD8+ Foxp3− T cells (Supplementary Fig. 4a-d). These results further confirm that expansion of the Treg compartment was mainly caused by the deletion of Lkb1 in DCs.
Impaired immune responses due to increased Treg cell number
Treg cells play a predominant role in suppressing immune responses in vivo. We next determined the impact of the increased Treg cell number on antigen-specific T cell responses using an experimental autoimmune encephalomyelitis (EAE) mouse model. We observed lower clinical scores, reflecting less severe EAE, in Cd11cCreLkb1f/f mice than in WT littermates (Fig. 2a). It is well known that EAE is mediated by encephalitogenic Th1 and Th17 cells, which produce pro-inflammatory cytokines IFN-γ and IL-17, respectively24. Thus, we analysed the populations of CD4+IFN-γ+ T (Th1) and CD4+IL-17+ T (Th17) cells in the spleen and brain. In Cd11cCreLkb1f/f mice, the Th1 cell populations, but not the Th17 populations, were significantly decreased in the spleen and brain (Fig. 2b, c). The levels of IL-17-producing T cells were slightly increased in Cd11cCreLkb1f/f mice under steady-state conditions (Supplementary Fig. 2b), which indicates that Lkb1-deficient DCs might intrinsically have an increased capacity for stimulating Th17 differentiation. There was no difference in the extent of Th17 cell development between the Lkb1f/f and Cd11cCreLkb1f/f mice in the EAE model, suggesting that enhanced Th17 differentiation might in some manner counteract the enhanced suppressor function of the expanded Treg population. However, significantly reduced Th1 cell development and EAE disease severity were observed in Cd11cCreLkb1f/f mice, indicating that the expanded Treg cell population dominated and resulted in overall alleviation of the immune response. As Lkb1 deficiency barely affected the antigen-specific T cell priming function of DCs (Supplementary Fig. 2e), we speculated that the impaired Th1 response was most likely due to enhanced immune suppression mediated by the enlarged Treg cell compartment in Cd11cCreLkb1f/f mice. To further confirm the impact of increased Treg cell populations on antigen-specific T cell responses, we bred Foxp3DTR mice to generate Lkb1f/fFoxp3DTR and Cd11cCreLkb1f/fFoxp3DTR mice, in which Foxp3+ Treg cells could be specifically depleted with diphtheria toxin (DT) treatment (short-term DT treatment only resulted in a transient reduction in the Treg cell populations and did not lead to overt disease phenotypes or death in mice)25, and we conducted an in vivo assay of OVA antigen-induced T cell priming. Two days after DT treatment (Supplementary Fig. 5a), we adoptively transferred CFSE-labelled CD4+ T cells from OT-II transgenic mice into DT-treated Lkb1f/f, Cd11cCreLkb1f/f, Lkb1f/fFoxp3DTR and Cd11cCreLkb1f/fFoxp3DTR mice via a tail vein injection, and we then challenged these mice with indicated doses of OVA to induce an antigen-specific T cell response. Lower percentages and numbers of proliferating OT-II T cells were detected in Cd11cCreLkb1f/f mice than in Lkb1f/f mice, while OT-II T cells in Cd11cCreLkb1f/fFoxp3DTR mice treated with DT exhibited stronger proliferation and higher absolute numbers than OT-II T cells in Cd11cCreLkb1f/f mice (Fig. 2d–f). In addition, the production of IFN-γ in OT-II cells was negatively correlated with the proportion of Treg cells in vivo (Fig. 2g, h). These data suggest that the impaired T cell response in the Cd11cCreLkb1f/f mice was due to the expanded Treg cell compartment.
Lkb1-deficient DCs promote Treg cell proliferation
Although IL-2 plays a role in promoting Treg cell proliferation, the increased production of IL-2 in CD4+Foxp3− T cells in Cd11cCreLkb1f/f mice was slight (Supplementary Fig. 2b) and could not explain the extremely high proportion of Treg cells among CD4+ T cells. In addition, as the most prominent driving factor of effector T cell responses, the elevation of IL-2 alone could not account for the defective T cell response in the Cd11cCreLkb1f/f mice. Therefore, we postulated that Lkb1-deficient DCs could directly promote Treg cell proliferation. Since splenic DCs could facilitate ubiquitous or tissue-restricted self-antigen-mediated TCR signalling to induce the polyclonal expansion of Treg cells26,27, we established a co-culture system to assess the direct impact of DCs on Treg cells18. To determine the direct effect of Lkb1-deficient DCs on Treg cell expansion, we sorted splenic DCs (CD11c+MHCII+) from Lkb1f/f and Cd11cCreLkb1f/f mice and co-cultured them with CFSE-labelled Treg cells (CD4+CD25+ population containing more than 95% Foxp3+ cells) sorted from B6 (CD45.1+) mice in the presence of exogenous IL-2 (Supplementary Fig. 5b). The Treg cells displayed markedly increased proliferation and higher numbers of CD4+Foxp3+ cells when co-cultured with Lkb1-deficient DCs than when co-cultured with WT DCs (Fig. 3a–c). We also confirmed this result with Treg cells (CD4+YFP+) from Foxp3YFP-Cre mice (Supplementary Fig. 5c-e). Next, we established a mixed BM chimaera mouse model to acquire WT and Lkb1-deficient DCs from the same environment. After co-culturing these cells with CFSE-labelled Treg cells in the presence of exogenous IL-2 for 4 days, we observed the same result, Lkb1-deficient DCs could induce stronger Treg cell expansion (Fig. 3d–f). These results indicate that Lkb1-deficient DCs could directly promote Treg cell expansion independent of other environmental elements. We next used Transwell plates to explore whether this effect of DCs was dependent on direct cell–cell contact or soluble factors secreted from DCs; in this way, Treg cells and DCs could be physically separated but share the same medium in close proximity. Indeed, we found that enhanced Treg cell proliferation only occurred when direct contact was made with Lkb1-deficient DCs (Fig. 3g), suggesting that Lkb1-deficient DCs promoted Treg cell proliferation in a contact-dependent manner. To determine whether Lkb1-deficient DCs could induce Treg cell proliferation in vivo, we transferred DCs sorted from Lkb1f/f or Cd11cCreLkb1f/f mice together with Treg cells into irradiated NSG mice. After 3 days, we detected that ~85% of the transferred cells maintained Foxp3 expression (Supplementary Fig. 5f), and a markedly greater absolute number of divided Treg cells was observed after co-transfer with Lkb1-deficient DCs than co-transfer with WT DCs (Fig. 3h–j). These results indicate that Lkb1 depletion in DCs was a direct driver of Treg cell proliferation.
DC OX40L upregulation contributes to Treg cell proliferation
AMPK is a well-known downstream Lkb1 target critical for regulating metabolism28. To determine whether AMPK is involved in Lkb1-deficient DC function, we generated Cd11cCreAMPKα1f/fAMPKα2f/f mice with the specific depletion of AMPK in DCs. However, there were no significant differences in the percentage of Treg cells or the activation of Tcon cells between AMPKα1f/fAMPKα2f/f and Cd11cCreAMPKα1f/fAMPKα2f/f mice (Supplementary Fig. 6a-d), suggesting that Lkb1 function in DCs is independent of AMPK activation.
To elucidate the molecular mechanism by which Lkb1 controls DC function, we profiled the transcriptome of DCs sorted from Lkb1f/f and Cd11cCreLkb1f/f mice. We found more than 500 transcripts that were significantly (P < 0.05) up- or downregulated (1.5-fold or more) in Lkb1-deficient DCs compared with WT DCs and thus constituted the Lkb1-deficient DC-specific transcriptional signature (Supplementary Data 1). Since Lkb1-deficient DCs promoted Treg cell proliferation in a contact-dependent manner, we first screened the significantly altered pathways related to cell adhesion through gene set enrichment analysis (GSEA) and noted that the gene set of cell-cell adhesion was enriched in Lkb1-deficient DCs (Fig. 4a). Among the genes upregulated in Lkb1-deficient DCs are several that have been previously implicated in immune adhesion, including the transcripts of Ox40l (Tnfsf4)29,30,31, Cd4032, Fas33 and Pdcd134 (Fig. 4b, Supplementary Data 1). The differential expression of these molecules at the mRNA and protein levels was evaluated by real-time PCR and flow cytometry, respectively. Lkb1 deficiency moderately increased the protein expression of OX40L and CD40 but barely affected that of Fas and PD1 (Fig. 4c, d and Supplementary Fig. 1c). However, recent studies have shown that transgenic mice with constitutive CD11c-specific CD40 signalling had low Treg cell frequencies35,36, suggesting that the expansion of Treg cells was unlikely due to CD40 upregulation on Lkb1-deficient DCs. We also examined the expression of OX40L on different subpopulations of DCs and found that OX40L was marginally expressed on pDCs and CD8+ cDCs from the spleen and LNs. However, the expression of OX40L on CD11b+ DCs from the spleen and LNs was higher in Cd11cCreLkb1f/f mice than in Lkb1f/f mice (Supplementary Fig. 7a, b). OX40L is a member of the TNF superfamily that has been implicated in DC-T cell interactions29,30,31. The higher expression of OX40 on Treg cells than on naive and activated CD4+ T cells (Supplementary Fig. 8a, b) suggests that OX40L-OX40 interactions might preferentially promote Treg cell proliferation.
Pre-blocking OX40L with a neutralizing antibody significantly hampered the increased proliferation and absolute number of Treg cells when co-cultured with Lkb1-deficient DCs but not when co-cultured with WT DCs (Fig. 4e–g). Since the co-stimulatory molecules CD80 and CD86 expressed on DCs have been suggested to be strong stimulators of Treg cell proliferation37, we took CD80 and CD86 as positive controls and found that blocking CD80 and/or CD86 on both WT and Lkb1-deficient DCs suppressed Treg proliferation (Supplementary Fig. 9a, b), which is consistent with the comparable expression of CD80 and CD86 on DCs from Lkb1f/f and Cd11cCreLkb1f/f mice (Supplementary Fig. 1c). The effect of blocking OX40L on DCs from Cd11cCreLkb1f/f mice was obvious, indicating that it plays a significant role in enhancing Treg cell expansion.
To confirm this result in vivo, DCs sorted from Lkb1f/f or Cd11cCreLkb1f/f mice were pre-incubated with anti-OX40L neutralizing antibody or isotype control and then transferred with CFSE-labelled Treg cells from B6 mice into irradiated NSG mice. After 3 days, we detected significantly greater proliferation and absolute cell number of Treg cells after co-transfer with Lkb1-deficient DCs than after co-transfer with WT DCs, and these increases could be mitigated by blocking OX40L (Fig. 4h–j). These results indicate that increased OX40L expression was involved in the superior Treg cell-stimulating effect of Lkb1-deficient DCs. However, when Tcon cells from B6 mice were co-cultured with WT and Lkb1-deficient DCs, we also observed increased Teff cell (CD44hiCD62Llow) proliferation that could be restrained with anti-OX40L neutralizing antibody (Supplementary Fig. 10a-c). Although Lkb1-deficient DCs have a slightly increased capacity for promoting Tcon cell survival compared with that of WT DCs, blocking OX40L did not have significant effect on the survival of Tcon cells (Supplementary Fig. 10d, e). This phenomenon indicates that the upregulated expression of OX40L on Lkb1-deficient DCs also contributes to the increased proportion of polyclonal Teff cells through enhancing proliferation.
Lkb1 restrains NF-κB signalling to suppress OX40L expression
We next explored the signalling pathways through which Lkb1 regulates Ox40l gene expression. Analysis of the cis-elements of the Ox40l gene revealed several conserved NF-κB binding sites (Supplementary Table 1). NF-κB signalling plays critical roles in regulating immune responses38. The NF-κB p65 transcription factor is a major component of the NF-κB family, and its phosphorylation marks the activation of the NF-κB pathway. Indeed, the level of phosphorylated NF-κB p65 was higher in Lkb1-deficient DCs than in WT DCs with or without LPS treatment (Fig. 5a and Supplementary Fig. 15a-g). In addition, more p65 was accumulated in the nucleus of Lkb1-deficient DCs (Fig. 5b and Supplementary Fig. 15h-j). Surprisingly, the depletion of Lkb1 did not result in the upstream activation of IKKα/β or IκBα38, indicating that Lkb1 deficiency in DCs activated NF-κB signalling distinct from the canonical NF-κB signalling pathway. In addition, the NF-κB inhibitor SC75741 decreased the mRNA and protein levels of OX40L in Lkb1-deficient DCs (Fig. 5c–e). Meanwhile, compared with untreated DCs, Lkb1-deficient DCs pre-treated with the NF-κB inhibitor showed smaller increases in the proliferation and number of Treg cells (Fig. 5f, g). We also confirmed these results in vivo by co-transferring DCs from Lkb1f/f and Cd11cCreLkb1f/f mice pre-treated with the NF-κB inhibitor with CFSE-labelled Treg cells into irradiated NSG mice (Fig. 5h, i). Furthermore, chromatin immunoprecipitation (ChIP) assays showed more NF-κB p65 binding on the Ox40l promotor in Lkb1-deficient DCs than in WT DCs (Fig. 5j), suggesting a direct effect of NF-κB signalling on promoting Ox40l transcription. These results suggest that an unusual form of IKKα/β- and IκBα-independent NF-κB activation drove the upregulation of Ox40l in Lkb1-deficient DCs and that this specifically activated NF-κB signalling promoted Treg cell proliferation at least partially via OX40L.
SIRT1 (sirtuin-1) has been found to interact with the Lkb1/AMPK complex and inhibit NF-κB signalling to regulate innate immunity defences39,40. To investigate whether SIRT1 was involved in Lkb1- and LPS-mediated signalling in DCs, we examined the protein level of SIRT1 in Lkb1-deficient DCs and DCs from LPS-treated mice. The total protein level of SIRT1 was diminished in DCs from LPS-treated mice but not in Lkb1-deficient DCs (Supplementary Figs. 11a and 15o, p). Since SIRT1 negatively regulates NF-κB p65 activation38,39, the downregulation of SIRT1 may contribute to the activation of NF-κB p65 in DCs from LPS-treated mice. However, SIRT1 is not reduced in Lkb1-deficient DCs, suggesting that Lkb1 loss provokes NF-κB hyperactivation independent of SIRT1. These results indicate that SIRT1 might be involved in the activation process of DCs from LPS-treated mice rather than mediating the Lkb1 effect in DCs. In addition, SIRT1 has been found to interact with the Lkb1/AMPK complex to regulate the downstream signalling39,41. However, in our study, Lkb1 controlled DC function independent of AMPK. These results collectively suggest that Lkb1 function in DCs is possibly independent of SIRT1.
LPS induces Treg cell expansion via depleting Lkb1 in DCs
The above results show the extreme enlargement of the Treg cell compartment in mice with the Lkb1 gene conditionally deleted in DCs. Considering the abundance of evidence demonstrating increased frequencies of Treg cells during certain immune responses, including those to bacterial infection42,43,44,45, we next sought to determine whether the expression of Lkb1 would be decreased in DCs to contribute to the augmentation of Treg cells under these conditions. We intraperitoneally treated mice with LPS, a Toll-like receptor (TLR) agonist derived from gram-negative bacteria, which is capable of triggering strong immune activation. Interestingly, we found that the Lkb1 protein level was greatly reduced in DCs (Fig. 6a and Supplementary 15k, l) but not in T and B lineage cells (Supplementary Fig. 12a and 15q-t). Despite a significant reduction in the Lkb1 protein level in DCs, no obvious change in the Lkb1 mRNA level was observed under LPS stimulation (Fig. 6b), suggesting that LPS might deplete Lkb1 protein via a post-transcriptional mechanism.
As expected, we observed elevated percentages and absolute numbers of Treg cells in mice under LPS stimulation (Fig. 6c, d). The majority of these Treg cells expressed high levels of Nrp1 and Helios (Fig. 6e), indicating that they were mostly nTreg cells. To determine the impact of the LPS-induced increase in Treg cells on the prognosis of an immune response, we pre-treated mice with or without a low dose of LPS before a lethal dose of LPS. The LPS pre-treatment protected Lkb1f/f mice from the lethal LPS challenge, but this protective effect was significantly abolished in Lkb1f/fFoxp3DTR mice treated with DT (Fig. 6f). These findings indicate the critical role of increased Treg cell populations in mediating protection against LPS-mediated inflammatory injury.
As LPS could affect the phenotype and function of multiple cell types, to confirm that the expansion of the Treg cell compartment was directly caused by DCs in LPS-treated mice, we sorted DCs from mice treated with or without LPS, incubated them with OX40L neutralizing antibody or isotype control, and co-cultured them with CFSE-labelled Treg cells for 4 days. Flow cytometry analysis showed that LPS-modified DCs promoted stronger Treg cell proliferation than did control DCs and that blocking with anti-OX40L antibody could restrain the proliferation of Treg cells in the LPS group, but not in the control group (Fig. 6g–i). These results suggest that the Treg cell expansion in the mice was directly caused by LPS-programmed DCs and that this effect was at least partially mediated by OX40L. To evaluate whether the Lkb1 protein reduction in DCs alone would contribute to the protective effect under LPS stimulation, we administered a lethal dose of LPS to Lkb1f/f, Cd11cCreLkb1f/f and Cd11cCreLkb1f/fFoxp3DTR mice treated with DT and analysed the survival rate. The survival rate of the Cd11cCreLkb1f/f mice was significantly higher than that of the other two groups of mice (Fig. 6j), but the Cd11cCreLkb1f/fFoxp3DTR mice treated with DT, which had reduced Treg cell populations, displayed significantly reduced survival. However, there was no statistically significant difference in the survival rate between the Lkb1f/fFoxp3DTR and Cd11cCreLkb1f/fFoxp3DTR mice treated with DT when challenged with a lethal LPS dose (Supplementary Fig. 13a). Thus, the LPS-induced Lkb1 depletion in DCs was directly involved in promoting Treg cell expansion to enhance immunosuppression. To further explore how Lkb1 deletion-induced Treg expansion would protect the mice from LPS toxicity, we examined the pro-inflammatory cytokine levels and T cell effector function of Lkb1f/f and Cd11cCreLkb1f/f mice in response to LPS. The mRNA levels of Tnf and Ifnγ were lower in the lungs of the Cd11cCreLkb1f/f mice than in those of the Lkb1f/f mice (Supplementary Fig. 14a). We also examined the production of Th-associated cytokines by T cells in Lkb1f/f and Cd11cCreLkb1f/f mice and found lower IFN-γ production in T cells from the lungs and spleen of Cd11cCreLkb1f/f mice (Supplementary Fig. 14b, c). These results indicate that the reduced production of TNF and IFN-γ in the lung tissue and the decreased numbers of IFN-γ-producing T cells might be part of the mechanisms contributing to the alleviated LPS toxicity.
E. coli induces Treg cell expansion by depleting Lkb1 in DCs
Because LPS is a major pathogenic factor derived from gram-negative bacteria, we further tested whether infection with the gram-negative bacteria E. coli could recapitulate the effect of LPS. Consistently, the decrease in Lkb1 protein was also observed in DCs from mice with an intraperitoneal E. coli infection (Fig. 7a and Supplementary Fig. 15m, n), along with an increased frequency and absolute number of Treg cells (Fig. 7b, c). These Treg cells were mostly nTreg cells given their high expression of Nrp1 and Helios (Fig. 7d). E. coli pre-treatment protected mice from secondary lethal E. coli challenge. Surprisingly, reducing the Treg cell population with DT did not abolish the protective effect of the E. coli pre-treatment (Fig. 7e), indicating that mechanisms other than Treg cells predominately mediated the protection from a secondary infection. As gram-negative bacteria, E. coli contain more complex virulence factors and have been shown to induce immune tolerance by multiple mechanisms46, which might override the protective effect of the bacteria pre-treatment-induced increase in the Treg cell population. To further determine whether Lkb1 depletion in DCs alone is sufficient to mediate tolerance to an acute primary lethal E. coli challenge, we administered lethal amounts of E. coli to Lkb1f/f, Cd11cCreLkb1f/f, and Cd11cCreLkb1f/fFoxp3DTR mice treated with DT. The Cd11cCreLkb1f/f mice showed a significantly higher survival rate than the Lkb1f/f mice upon lethal E. coli challenge, and reducing the Treg cell population in the Cd11cCreLkb1f/fFoxp3DTR mice through DT treatment resulted in poor survival (Fig. 7f), indicating that conditional knockout of the Lkb1 gene in DCs increases the Treg cell number and the tolerance of the host to primary bacterial infections. Finally, Lkb1f/fFoxp3DTR and Cd11cCreLkb1f/fFoxp3DTR mice treated with DT showed comparable survival rates when challenged with a lethal dose of E. coli (Supplementary Fig. 13b). These results indicate that the Lkb1 depletion-induced expansion of the Treg cell population promotes tolerance to primary bacterial infections but is dispensable for establishing tolerance to secondary infections.
Lkb1 discriminates regulatory from inflammatory programmes
While LPS is a well-known pathogenic factor that acts on DCs to promote inflammation, here, we show that LPS also depletes Lkb1 in DCs to induce Treg cell expansion and immunosuppression. To understand the molecular basis that differentiates the regulatory and pro-inflammatory functions, we further profiled the transcriptome of DCs sorted from mice treated with or without LPS for comparison with the aforementioned transcriptional signature of Lkb1-deficient DCs (Supplementary Data 1 and 2). GSEA showed that pathways related to inflammatory responses, inflammatory effector processes, immune responses and acute immune responses were significantly upregulated in DCs from LPS-treated mice, while no significant enrichment in these gene sets appeared in Lkb1-deficient DCs (Fig. 8a). A substantial proportion of the upregulated transcripts in DCs from LPS-treated mice were not significantly altered in Lkb1-deficient DCs, including genes encoding the pro-inflammatory cytokines Il1α, Il27, Il15 and Il1f9, the costimulation molecule Cd80, and the chemokines Cxcl11, Cxcl9, Cxcl10 and Ccl5, which was confirmed by real-time PCR (Fig. 8b, c and Supplementary Data 2). However, Ox40l, the upregulated transcript in Lkb1-deficient DCs, was also increased in DCs from LPS-treated mice, and real-time PCR and flow cytometry further confirmed that LPS could upregulate OX40L expression in DCs (Fig. 8d–f). These results demonstrate that Lkb1 operated as a “regulatory switch” to discriminate the regulatory from the pro-inflammatory transcriptional programme in DCs.
Along with tolerogenic DCs that are thought to be associated with developmental immaturity45,47,48, the concept of “regulatory DCs”, which are proposed to evolve under certain immune conditions and exhibit a superior immunosuppressive capacity, has been considered for decades10,45, but the underlying mechanism that governs the specification of this distinctive lineage is still elusive. In this study, we demonstrate that a deficiency in Lkb1, achieved through either artificial gene deletion or bacteria/LPS-induced protein depletion, establishes the regulatory function of DCs to augment Treg cell expansion under either steady-state or inflammatory conditions. These findings indicate a mechanism underlying this regulatory functional specification.
An increasing body of evidence suggests that bacterial infection not only induces the activation of effector cells to eliminate pathogens but also elicits immunosuppression that involves iTreg cell de novo generation1,45,49. In our study, judging from the positive expression of Nrp1 and Helios, nTreg but not iTreg cells are major components of the enlarged Treg cell pool in mice intraperitoneally challenged with bacteria or LPS. We showed that bacteria and LPS pre-treatment provided protection against a lethal secondary infection. LPS pre-treatment-induced Lkb1 depletion promoted Treg expansion to protect mice from a secondary lethal challenge. However, E. coli pre-treatment-induced protection from the secondary lethal infection was largely independent of Treg expansion, indicating that other tolerance mechanisms might exist, which may override the effect of Treg cell expansion during bacterial infection. Nonetheless, functional genomic analysis revealed that the Lkb1 deficiency established a regulatory transcriptional programme in DCs without interfering with the pro-inflammatory transcriptional programme, thus providing a unique molecular basis for bacterial infection-induced tolerance.
Despite the well-recognized role of OX40L–OX40 interactions in promoting effector T cell responses29,30, the impact of OX40L–OX40 interactions on Treg cells remains controversial. Some reports have suggested that OX40 favours Treg cell suppression via increasing expansion27,50, while others have demonstrated that OX40 represss Treg cell suppressive function due to Treg cell exhaustion and a lack of IL-231. Our study defines the role of OX40L–OX40 interactions in promoting Treg cell proliferation driven by Lkb1-deficient DCs. Although the upregulation of OX40L on Lkb1-deficient DCs was moderate, it had a significant impact on Treg cell proliferation, as blocking OX40L on Lkb1-deficient DCs reduced Treg cell proliferation to an extent similar to that of blocking both CD80 and CD86, which are known as strong stimulators of Treg cell proliferation37. However, blocking OX40L on Lkb1-deficient DCs could only partially rescue the enhanced proliferation of Treg cells, suggesting that other molecules might also contribute to Treg cell proliferation. Indeed, Lkb1 also suppressed the expression of other cell membrane molecules, including semaphorins, which are involved in cell adhesion and communication51,52, and CCL22, which is thought to promote Treg cell chemotaxis53,54. The impact of these molecules on Treg cells within the complex immune microenvironment cannot be excluded. Therefore, we propose that Lkb1 might control a number of genes in DCs to govern the magnitude of Treg cell expansion in vivo.
We found that NF-κB p65 activation induced Ox40l expression in Lkb1-deficient DCs and that the inhibition of NF-κB signalling in Lkb1-deficient DCs reduced the proliferation of Treg cells, suggesting that NF-κB activation in Lkb1-deficient DCs promotes Treg cell proliferation at least via the upregulation of OX40L. NF-κB is a well-known mediator of TLR-triggered pro-inflammatory immune responses38. Therefore, it was surprising that Lkb1 deficiency-triggered p65 activation promoted the regulatory but not the pro-inflammatory function of DCs. Indeed, the conditional deletion of Lkb1 did not lead to the activation of IKKα/β or the degradation of IκBα. This unusual Lkb1 regulation of NF-κB signalling is in contrast with the results of previous studies showing that Lkb1 regulates IKKα/β activation in other immune cell types;18,19 thus, this might be a unique mechanism for discriminating the regulatory programme from the pro-inflammatory programme in DCs. In addition to OX40L, we cannot exclude the possibility that Lkb1-controlled NF-κB signalling might regulate other molecules to affect Treg cell homeostasis, which awaits future clarification.
Lkb1 also plays intrinsic roles in Treg cells. Our previous work demonstrates that Lkb1 promotes Treg cell Foxp3 expression and suppressive function18. In addition, Lkb1 was recently shown to programme Treg cell metabolic and functional fitness55. Intriguingly, here, we show that Lkb1 operates in DCs to negatively regulate Treg cell expansion and immunosuppression. Furthermore, LPS treatment selectively depleted Lkb1 in DCs but not in Treg cells to potentiate immunosuppression. Surprisingly, the conditional deletion of Lkb1 in monocytes/macrophages, which share certain functions (e.g., antigen presentation, inflammatory mediator production) with DCs, did not lead to Treg cell expansion. Together, these results highlight an environment- and cell type-conditioned function of Lkb1 in the orchestration of immunity versus tolerance.
In conclusion, these findings indicate that the abundance and immunosuppression strength of Treg cells are dynamically governed, in both a feedforward and feedback manner, by the Lkb1 “regulatory switch” in DCs to maintain immune equilibrium. Our study also revises the concept of “regulatory DCs” by providing evidence supporting a unifying model of regulatory and inflammatory programmes that can co-evolve but are separately instructed by different signals in the same DCs during maturation/activation. These findings provide insight into the sophisticated DC-Treg cell interactions that are fundamental for controlling immune equilibrium. Given that dysregulated Treg cell pools are involved in the pathogenesis of various cancerous and autoimmune diseases, our work may provide therapeutic targets for the clinical treatment of these Treg cell-related immune diseases.
All animals were maintained in specific pathogen-free barrier facilities and used in accordance with protocols approved by the Institutional Animal Care and User Committee at the Institute of Hematology, Chinese Academy of Medical Sciences. C57BL/6, Lkb1f/f, AMPKα1f/f, AMPKα2f/f, Cd11cCre, Foxp3YFP-Cre, LysMCre, Foxp3DTR and NOD/scid IL2Rgnull (NSG) mice were purchased from Jackson Laboratories. All mice had been backcrossed with C57BL/6 mice for at least seven generations. Lkb1f/f, AMPKα1f/f and AMPKα2f/f mice were crossed with Cd11cCre, LysMCre to generate Cd11cCreLkb1f/f, Cd11cCreAMPKα1f/fAMPKα2f/f, and LysMCreLkb1f/f mice, respectively. Cd11cCreLkb1f/f mice were crossed with Foxp3DTR mice to generate Cd11cCreLkb1f/fFoxp3DTR mice. All mice were used when 6–8 weeks old unless otherwise noted. The sample size was selected to maximize the chance of uncovering a mean difference with statistical significance. No statistical methods were used to predetermine the sample size. The experiments were not randomized, and the investigators were not blinded to group allocation during the experiments or outcome assessments.
Cell purification and flow cytometry
For the analysis of cell surface markers, single-cell suspensions were prepared from spleen and LN samples for staining with APC-Cy7-anti-CD4 (100413, Biolegend), APC-Cy7-anti-CD45.1 (110715, Biolegend), APC-anti-CD45.2 (109813, Biolegend), APC-anti-CD4 (100411, Biolegend), APC-anti-MHC II (107613, Biolegend), APC-anti-CD62L (104411, Biolegend), APC-anti-CD304 (neuropilin-1) (145205, Biolegend), PerCP-anti-CD80 (104721, Biolegend), PerCP-anti-CD45.2 (109825, Biolegend), PerCP-anti-CD44 (103035, Biolegend), PerCP-anti-CD8α (100731, Biolegend), FITC-anti-CD62L (104405, Biolegend), FITC-anti-CD279 (PD1) (135213, Biolegend), FITC-anti-CD45.1 (110705, Biolegend), PE-Cy7-anti-CD11c (117317, Biolegend), PE-Cy7-CD45.1 (110729, Biolegend), PE-Cy7-CD86 (105013, Biolegend), PE-Cy7-anti-CD8α (100721, Biolegend), PE-Cy7-anti-CD45.2 (109829, Biolegend), PE-Cy7-anti-CD278 (ICOS) (25–9942–80, eBioscience), PE-anti-CD25 (101903, Biolegend), PE-anti-CD252 (Ox40l) (12-5905-81, eBioscience), PE-anti-CD40 (124609, Biolegend), PE-anti-CD95 (Fas) (152607, Biolegend), and PE-anti-CD11c (117307, Biolegend). These antibodies were obtained from eBioscience or Biolegend. CD4+ T cells, CD8+ T and CD11c+ DCs were purified with Dynabeads Untouched Mouse CD4 and CD8 Cell Kits (11416D, 11417D, Invitrogen) and CD11c MicroBeads (130-108-338, Miltenyi Biotec), respectively. The indicated Treg cell populations were sorted from purified CD4+ T cells using a FACSAria III system (BD Biosciences), and the sorted populations were >98% pure unless otherwise specified. Intracellular staining with PE-Foxp3 (12-5773-82, eBioscience), APC-anti-Helios (137221, Biolegend), APC-anti-Ki67 (652405, Biolegend), and antibodies to cytokines, including APC-anti-IL-17 (506915, Biolegend), FITC-anti-IL-2 (503805, Biolegend), and PE-anti-IFN-γ (505807, Biolegend) (spleen cells were stimulated with phorbol myristate acetate (PMA, 50 ng ml-1) and ionomycin (500 ng ml−1) (Sigma-Aldrich) for 4 h before analysis of the cytokine expression in the indicated populations), were performed with Foxp3 staining kits (72-5775-40, eBioscience). Cell surface staining was mostly performed at 4 °C for 30 min. OX40L on splenic DCs was stained at room temperature for 2–3 h. Flow cytometry data were acquired on an LSR II, LSRFortessa (BD Biosciences) or FACSCanto II (BD Biosciences) system and analysed with FlowJo software (Tree Star). Gating strategies are described in Supplemental Table 3.
Treg cell proliferation
CD4+CD25+ Treg cells sorted from B6 mice were confirmed to consist of more than 95% of Foxp3+ cells and labelled using CFSE Cell Proliferation Kits (Invitrogen) for 8 min. at room temperature and then washed twice with phosphate-buffered saline (PBS). For the in vitro study, CFSE-labelled Treg cells were co-cultured with CD11c+ cells purified from Cd11cCreLkb1f/f and Lkb1f/f mice in the presence or absence of recombinant murine IL-2 (100 ng/ml, Biolegend) for 4 days. For the in vivo study, to exclude the influence of DCs from the host, we used NSG mice as recipient mice, whose DCs are defective; 2 × 106 CFSE-labelled Treg cells were transferred together with 1 × 106 DCs purified from Lkb1f/f or Cd11cCreLkb1f/f mice into sublethally (2 Gy) irradiated NSG mice by tail vein injection. After 3 days, the spleen was removed, and the proliferation of CD4+Foxp3+ Treg cells was analysed by flow cytometry. In some experiments, DCs were treated with OX40L (R&D, MAB1236 and Bioxcell, BE0033), CD80 (R&D, AF740), and CD86 (R&D, AF-441-NA) neutralizing antibodies or their isotype controls at a concentration of 30 μg/ml at 4 °C for 1.5 h. In some experiments, DCs are sorted from Lkb1f/f or Cd11cCreLkb1f/f mice treated with or without NF-κB inhibitor SC75741(Selleck, S7273). Mixed BM chimaera mouse models were established by co-transferring equal numbers of total BM cells (1 × 107) from Lkb1f/f (CD45.1+CD45.2+) and Cd11cCreLkb1f/f (CD45.2+) mice together into lethally irradiated (8 Gy) B6 (CD45.1+) mice, followed by reconstitution for at least 6 weeks.
Treg cell suppression
CD4+CD25-CD44loCD62Lhi naïve T (Tn) cells sorted from CD45.1+ mice were labelled with CFSE and used as responder cells (Tresp). Tresp cells (5 × 104) were cultured for 3 days with DCs (1 × 105) in the presence or absence of the indicated numbers of CD4+CD25+ Treg cells sorted from Lkb1f/f or Cd11cCreLkb1f/f mice.
Annexin V and PI staining was performed using an apoptosis detection kit (Biolegend) according to the manufacturer’s instruction to determine the apoptosis of Treg cells in the spleen from Lkb1f/f and Cd11cCreLkb1f/f mice.
T cell proliferation and activation
CD4+ and CD8+ T cells were sorted from OT-II and OT-I transgenic mice, labelled with CFSE, and then co-cultured with Cd11cCreLkb1f/f and Lkb1f/f DCs loaded with the respective antigen peptide (OVA 323-339 and OVA257–264, Sigma-Aldrich) for 4 days. For the in vivo study, Lkb1f/f, Cd11cCreLkb1f/f and Cd11cCreLkb1f/fFoxp3DTR mice treated were with diphtheria toxin (DT) for 2 days; then, CFSE-labelled CD4+ T cells were transferred into these mice via tail vein injection. After 1 day, the mice were challenged by a subcutaneous injection of 20μg of OVA protein (A5503, Sigma-Aldrich) with 100 μl of Complete Freund's adjuvant (CFA). In addition, 3 days later, the LNs were removed and the cell populations were analysed by flow cytometry.
DCs were sorted from Lkb1f/f and Cd11cCreLkb1f/f mice and treated as indicated, and then the cells were lysed with RIPA buffer or nuclear/cytosol fractionation reagent (Bio Vision) supplemented with protease and phosphatase inhibitors. The protein concentration in the extract was measured by BCA assay. The same amount of protein for each sample was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred by electroblotting and membranes blocked in 5% milk/Tris buffered saline with Tween 20 (TBS-T) for 1 h. Membranes were incubated with appropriate antibodies overnight at 4 °C, then exposed to secondary antibodies for 2 h at room temperature and developed using ECL Western Blotting Substrate. Western blot was carried out with antibodies against Lkb1 (1:500), phospho-IKKα/β (Ser176/180, 1:500), phospho-IκBα (Ser32, 1:500), phospho-NF-κB p65 (Ser536, 1:500), IKKα (1:500), IκBα (1:500), NF-κB p65 (1:500) and GAPDH (1:1000). These antibodies were purchased from Cell signalling Technology, USA. Uncropped scans of all the blots were provided in the Supplementary Figure 15.
Microarray and quantitative real-time PCR
DCs were sorted from the spleen of Lkb1f/f mice, LPS-treated mice, and Cd11cCreLkb1f/f mice for RNA extraction with TRIzol reagent (Invitrogen). Total RNA was reverse-transcribed, amplified, labelled, and hybridized to Mouse Genome 2.0 arrays (Affymetrix). The microarray data sets were analysed using Agilent GeneSpring GS 11 software and GSEA. RNA from different samples was obtained in the same manner as for the microarray analysis, and real-time PCR was performed with SYBR Green PCR Master Mix (ABI). The sequences of the primer pairs used are listed in Supplementary Table 2.
The concentrations of IgG in the serum of Lkb1f/f and Cd11cCreLkb1f/f mice were determined by sandwich ELISA (Biolegend), according to the manufacturer's instructions.
Chromatin immunoprecipitation (ChIP)
ChIP assays were performed using ChIP kits (Active Motif) according to the manufacturer’s protocols. Precipitated DNA and input DNA were assessed by real-time PCR using the primers listed in Supplementary Table 2.
Lkb1f/f and Cd11cCreLkb1f/f female mice (8–10 weeks old) were immunized subcutaneously (s.c.) with MOG35–55 emulsified in CFA and were administered pertussis toxin (PT) intraperitoneally (i.p.) after 0 and 2 days. In our experiments, the EAE symptoms usually started between days 14 and 28. Disease symptoms were regularly monitored and scored as follows: 0: no clinical signs; 1: flaccid tail; 2: hind limb weakness or abnormal gait; 3: complete hind limb paralysis; 4: complete hind limb paralysis + forelimb weakness or paralysis; 5: moribund or deceased (0–5 graduations with 0.5 for intermediate scores).
Injection of LPS and bacteria
LPS (Sigma-Aldrich) or E. coli provided by Prof. Yuanfu Xu (Chinese Academy of Medical Sciences) in 200 μl of PBS was administered by tail vein or intraperitoneal injection. Bacteria were stored in 20% glycerol at −80 °C. For expansion, bacteria were cultured on LB agar through plated streaking overnight. One colony of bacteria was selected, suspended in 4 ml of LB and incubated for 12 h (200 revolutions/min, 37 °C). The suspension was then centrifuged at 8000 rpm for 5 min. The pellet was washed twice with PBS and then resuspended in PBS. The determined concentration of the suspension was adjusted to the desired dose using PBS.
An unpaired two-tailed Student's t-test (for the comparison of two groups) or two-way ANOVA (for the comparison of more than two groups) were performed using Prism (GraphPad) to calculate the statistical significance of differences in the mean values as indicated by the P value. P values < 0.05 were considered statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001.
The data that support the findings of this study are available from the corresponding author on reasonable request. The microarray data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession number GSE117286. A reporting summary for this Article is available as a Supplementary Information file.
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This work was supported by the National Basic Research Program of China (2015CB964402), the National Natural Science Foundation of China (81670107, 81870090, 81370104 and 81421002), the Recruitment Program of Global Youth Experts, the CAMS Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-1–003, and 2017-I2M-1–015), and the Tianjin Science Funds for Distinguished Young Scholars. We thank Prof. Yuanfu Xu, the State Key Laboratory of Experimental Hematology, the Institute of Hematology and the Hospital of Blood Disease, Chinese Academy of Medical Sciences, for providing E. coli.