Graphical AbstractHighlights d LKB1-deficient T reg cells produce Th1 and Th17 cytokines d The loss of LKB1 compromises the mevalonate pathway in T reg cells d Mevalonate or GGPP treatment restores function and stability in LKB1-deficient T reg cells d LKB1-mediated regulation of T reg cells is independent of AMPK SUMMARY The function of regulatory T (T reg ) cells depends on lipid oxidation. However, the molecular mechanism by which T reg cells maintain lipid metabolism after activation remains elusive. Liver kinase B1 (LKB1) acts as a coordinator by linking cellular metabolism to substrate AMP-activated protein kinase (AMPK). We show that deletion of LKB1 in T reg cells exhibited reduced suppressive activity and developed fatal autoimmune inflammation. Mechanistically, LKB1 induced activation of the mevalonate pathway by upregulating mevalonate genes, which was essential for T reg cell functional competency and stability by inducing T reg cell proliferation and suppressing interferon-gamma and interleukin-17A expression independently of AMPK. Furthermore, LKB1 was found to regulate intracellular cholesterol homeostasis and to promote the mevalonate pathway. In agreement, mevalonate and its metabolite geranylgeranyl pyrophosphate inhibited conversion of T reg cells and enhanced survival of LKB1-deficient T reg mice. Thus, LKB1 is a key regulator of lipid metabolism in T reg cells, involved in optimal programming of suppressive activity, immune homeostasis, and tolerance.

Abbreviations: A770041, a selective inhibitor of Lck; Ag, antigen; BMMC, bone marrow-derived mast cells; CCL3, C-C motif chemokine ligand 3; DNP-HSA, dinitrophenyl-human serum albumin; eMIP, a 69-amino acid variant of human CCL3; ERK/Erk1/2, extracellular signal-regulated kinase 1/2; Fexo, fexofenadine; GPCR, G protein-coupled receptor; HSP, heat shock proteins; HSP70, heat shock protein 70; IgE, immunoglobulin E; IKK, IκB kinase; IL-6, interleukin-6; IRF, interferon regulatory factor; JNK, c-Jun N-terminal kinase; KIT, c-kit receptor; KO, knockout; LAT, linker for activation of T cells; Lck, lymphocyte-specific protein tyrosine kinase; LTC 4 , leukotriene C 4 ; MAPK, mitogen-activated protein kinase; M-dose, micromolar doses; MyD88, myeloid differentiation factor 99; N-dose, nanomolar doses; NF-κB, nuclear factor-kappa B; NK, natural killer; PCA, passive cutaneous anaphylaxis or anaphylactic; PGD 2 , prostaglandin D 2 ; Phospho-Lck, phosphorylated Lck; Phospho-Syk, phosphorylated Syk; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLCγ, phospholipase Cγ; SCF, stem cell factor; SHP-1, Src homology region 2 domain-containing phosphatase-1; Syk, spleen tyrosine kinase; TIR domain, Toll/interleukin-1 receptor homology domain; TIRAP, TIR domain-containing adaptor protein; TLR4, Toll-like receptor 4; TNF-α, tumor necrosis factor-α; TRAF6, TNF receptor-associated factor 6; β-Hex, β-hexosaminidase. AbstractBackground: Signal transduction pathways mediated by various receptors expressed on mast cells are thought to be complex, and inhibitory signals that turn off activating signals are not known. Methods: Upstream signaling cascades mediated by several known receptors in bonemarrow-derived mast cells that lead to degranulation and mediator release were studied by immunoblotting and immunoprecipitation. Small interfering RNAs and knockout mice were used to confirm findings. Results: All ligands tested including IgE/Ag, SCF, HSP70, CCL3, and its valiant eMIP induced phosphorylation of linker for activation of T cells (LAT), which triggered their receptor-mediated downstream signaling cascades that controlled degranulation and mediator release. Phosphorylation of lymphocyte-specific protein kinase (Lck) was induced by each ligand, which commonly played an indispensable role in LAT phosphorylation. In contrast, phosphorylation of spleen tyrosine kinase was additionally induced in cells stimulated only with IgE/Ag and SCF, which is also associated with LAT phosphorylation in part. Degranulation and mediator release induced by IgE/Ag, SCF, or HSP70 were enhanced by nanomolar doses of CCR1 ligands CCL3 and eMIP via enhanced LAT phosphorylation. On the other hand, micromolar doses of CCR1 ligand inhibited degranulation and mediator release from mast cells stimulated with IgE/Ag, SCF, or HSP70 by de-phosphorylation of phosphorylated Lck with Src homology region 2 domain-containing phosphatase-1.

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CD4+ T cells are essential in inflammation and autoimmune diseases. Interferon-γ (IFN-γ) secreting T helper (Th1) and IL-17 secreting T helper (Th17) cells are critical for several autoimmune diseases. To assess the inhibitory effect of a given compound on autoimmune disease, we screened many compounds with an in vitro Th differentiation assay. BJ-3105, a 6-alkoxypyridin-3-ol analog, inhibited IFN-γ and IL-17 production from polyclonal CD4+ T cells and ovalbumin (OVA)-specific CD4+ T cells which were activated by T cell receptor (TCR) engagement. BJ-3105 ameliorated the experimental autoimmune encephalomyelitis (EAE) model by reducing Th1 and Th17 generation. Notably, Th cell differentiation was significantly suppressed by BJ-3105 treatment without inhibiting in vitro proliferation of T cells or inducing programmed cell death. Mechanistically, BJ-3105 inhibited the phosphorylation of JAK and its downstream signal transducer and activator of transcription (STAT) that is critical for Th differentiation. These results demonstrated that BJ-3105 inhibits the phosphorylation of STAT in response to cytokine signals and subsequently suppressed the differentiation of Th cell responses.

Recent studies emphasize on developing immune tolerance by an interim administration of various immunosuppressive drugs. In this study, a robust protocol is reported for local immunomodulation using a single‐dose of FK506 microspheres and clodronate liposomes (mFK+CLO) in a xenogeneic model of islet transplantation. Surprisingly, the single‐dose treatment with mFK+CLO induce tolerance to the islet xenograft. The recipient mice display tolerogenic dendritic cells (tDCs) with decreased antigen presenting ability and T cell activation capacity. Furthermore, a reduced percentage of CD4+ and CD8+ T cells and an impaired differentiation of naïve CD4+ T cells into interferon‐γ producing Th1 and interleukin‐17 producing Th17 cells are observed. In addition, the immunosuppressive protocol leads to the generation of Foxp3+ regulatory T cells (Tregs) which are required for the long‐term graft survival. The enhanced generation of tDCs and Tregs by the single treatment of mFK+CLO cause xenograft tolerance, suggesting a possible clinical strategy which may pave the way towards improving therapeutic outcomes of clinical islet transplantation.

CD4 T cells differentiate into distinct effector subsets upon antigenic stimulation. Cytokines, and micro-environmental factors present during T-cell priming, direct differentiation of naïve CD4 T cells into pro-inflammatory Th1 and Th17 cells. From extensive screening of 2,4,5-trimethylpyridin-3-ol derivatives with various functional groups at C(6)-position, BJ-2266, a 6-thioureido-derivative, showed potent inhibitory activity on in vitro T helper (Th)-cell differentiation. This compound inhibited IFN-γ and IL-17 production from polyclonal CD4 T cells and ovalbumin (OVA)-specific CD4 T cells that were activated by T-cell receptor (TCR) engagement. We assessed the inhibitory effect of BJ-2266 in experimental autoimmune encephalomyelitis (EAE). Our results suggest that BJ-2266 treatment significantly suppresses EAE disease progression with reduced generation of Th1 and Th17 cells. Notably, Th-cell differentiation was significantly suppressed by BJ-2266 treatment with no effect on apoptosis, activation and proliferation of activated T cells. Furthermore, adoptive transfer of BJ-2266 treated MOG-reactive Th1 and Th17 cells led to a lower EAE disease score and better clinical recovery from EAE. The underlying mechanism of BJ-2266 effect involved the inhibition of JAK/STAT phosphorylation that is critical for Th-cell differentiation. We conclude that BJ-2266 regulates the JAK/STAT pathway in response to cytokine signals and subsequently suppresses the differentiation of Th-cell responses.

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