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.

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Background: Nuclear receptor subfamily 4 group A member 1 (NR4A1), an orphan nuclear receptor, has been implicated in several biological events such as metabolism, apoptosis, and inflammation. Recent studies indicate a potential role for NR4A1 in mast cells, yet its role in allergic responses remains largely unknown.Objectives: The aim of this study was to clarify the role of NR4A1 in mast cell activation and anaphylaxis. Methods: To evaluate the function of NR4A1 in mast cells, the impacts of siRNA knockdown, gene knockout, adenoviral overexpression, and pharmacological inhibition of NR4A1 on FcεRI signaling and effector functions in mouse bone marrowderived mast cells (BMMCs) in vitro and on anaphylactic responses in vivo were evaluated. Results: Knockdown or knockout of NR4A1 markedly suppressed degranulation and lipid mediator production by FcεRI-crosslinked BMMCs, while its overexpression augmented these responses. Treatment with a NR4A1 antagonist also blocked mast cell activation to a similar extent as NR4A1 knockdown or knockout. Moreover, mast cell-specific NR4A1-deficient mice displayed dampened anaphylactic responses in vivo. Mechanistically, NR4A1 promoted FcεRI signaling by counteracting the liver kinase B1 (LKB1)/adenosine monophosphate-activated protein kinase (AMPK) axis. Following FcεRI crosslinking, NR4A1 bound to the LKB1/AMPK complex and sequestered it in the nucleus, thereby promoting FcεRI downstream signaling pathways. Silencing or knockout of LKB1/AMPK largely abrogated the effect of NR4A1 on mast cell activation. Additionally, NR4A1 facilitated spleen tyrosine kinase activation independently of LKB1/AMPK.

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.

CD4+ T cells are the central for the mammalian adaptive immune system. Naïve CD4+ T cells mainly differentiate in to pro-inflammatory Th1, Th2 and Th17 cells upon antigenic stimulation. IFN-γ secreting Th1 cells and IL-17 secreting Th17 cells are found to play key roles in autoimmune diseases like multiple sclerosis (MS) and ulcerative colitis (UC). In this study we found NTG-A-009, 6-aminopyridin-3-ol, has great inhibitory effect on in vitro differentiation of Th1 and Th17 cells without affecting regulatory T cells. Moreover, NTG-A-009 had no effect on CD4+ T cell proliferation and viability. In vivo treatment has shown that NTG-A-009 has ameliorated experimental autoimmune encephalomyelitis (EAE) and dextran sulfate sodium (DSS) induced colitis through the inhibition of Th1 and Th17 cells differentiation. Mechanistically, NTG-A-009 suppressed Th1 and Th17 cells differentiation via the modulation of JAK/STAT signaling pathway. Thus, our data demonstrated that NTG-A-009 ameliorated inflammation through the inhibition of Th1 and Th17 cells generation making it a potential therapeutic candidate for the treatment of inflammatory diseases.