Conventional T (Tcon) cells and Foxp3(+) T-regulatory (Treg) cells are thought to have differing metabolic requirements, but little is known of mitochondrial functions within these cell populations in vivo. In murine studies, we found that activation of both Tcon and Treg cells led to myocyte enhancer factor 2 (Mef2)-induced expression of genes important to oxidative phosphorylation (OXPHOS). Inhibition of OXPHOS impaired both Tcon and Treg cell function compared to wild-type cells but disproportionally affected Treg cells. Deletion of Pgc1α or Sirt3, which are key regulators of OXPHOS, abrogated Treg-dependent suppressive function and impaired allograft survival. Mef2 is inhibited by histone/protein deacetylase-9 (Hdac9), and Hdac9 deletion increased Treg suppressive function. Hdac9(-/-) Treg showed increased expression of Pgc1α and Sirt3, and improved mitochondrial respiration, compared to wild-type Treg cells. Our data show that key OXPHOS regulators are required for optimal Treg function and Treg-dependent allograft acceptance. These findings provide a novel approach to increase Treg function and give insights into the fundamental mechanisms by which mitochondrial energy metabolism regulates immune cell functions in vivo.

Tec family nonreceptor tyrosine kinases are expressed by hematopoietic cells, activate phospholipase C (PLC)γ, and regulate cytoskeletal rearrangement, yet their role in FcγR-induced signaling and phagocytosis remains unknown. We demonstrate in this study that Bruton’s tyrosine kinase (Btk) and Tec, the only Tec kinases expressed by RAW 264.7 cells, are activated throughout phagocytosis. Activated Btk and Tec kinase accumulate at an early stage at the base of phagocytic cups and inhibition of their activity by the specific inhibitor LFM-A13 or expression by small interfering RNA significantly inhibited FcγR-induced phagocytosis. Similarly, a significant role for these kinases in phagocytosis was found in primary macrophages. FcγR-induced activation of Mac-1, which is required for optimal phagocytosis, was markedly inhibited and our findings suggest that the roles of kinases Btk and Tec in Mac-1 activation account for their functions in the early stages of phagocytosis. Initial activation of PLCγ2, the predominant PLC isoform in RAW 264.7 cells, is dependent on Syk. In contrast, a late and prolonged activation of PLCγ2 was dependent on Btk and Tec. We found accumulation of diacylglycerol (DAG), a PLCγ product, in phagosome membranes, and activated Btk, but not Tec, colocalized with phagosomal DAG. Inhibition of Tec family kinase activity increased the level of DAG in phagosomes, suggesting a negative regulatory role for Btk. Tec, in contrast, clustered at sites near phagosome formation. In summary, we elucidated that Tec family kinases participate in at least two stages of FcγR-mediated phagocytosis: activation of Mac-1 during ingestion, and after phagosome formation, during which Btk and Tec potentially have distinct roles.

e T-regulatory (Treg) cells are important to immune homeostasis, and Treg cell deficiency or dysfunction leads to autoimmune disease. A histone/protein acetyltransferase (HAT), p300, was recently found to be important for Treg function and stability, but further insights into the mechanisms by which p300 or other HATs affect Treg biology are needed. Here we show that CBP, a p300 paralog, is also important in controlling Treg function and stability. Thus, while mice with Treg-specific deletion of CBP or p300 developed minimal autoimmune disease, the combined deletion of CBP and p300 led to fatal autoimmunity by 3 to 4 weeks of age. The effects of CBP and p300 deletion on Treg development are dose dependent and involve multiple mechanisms. CBP and p300 cooperate with several key Treg transcription factors that act on the Foxp3 promoter to promote Foxp3 production. CBP and p300 also act on the Foxp3 conserved noncoding sequence 2 (CNS2) region to maintain Treg stability in inflammatory environments by regulating pCREB function and GATA3 expression, respectively. Lastly, CBP and p300 regulate the epigenetic status and function of Foxp3. Our findings provide insights into how HATs orchestrate multiple aspects of Treg development and function and identify overlapping but also discrete activities for p300 and CBP in control of Treg cells. Eukaryotic transcription is tightly regulated by histone modifications, including acetylation of lysine residues of histone tails (1). Acetylation is controlled by the competing actions of histone/ protein acetyltransferases (HATs) and histone/protein deacetylases (HDACs), which also regulate the acetylation of many nonhistone proteins. Three main families of HATs are reported: CBP/ p300, GNAT (GCN5/PCAF), and MYST (2). CBP and p300 have high sequence homology (1) but share little sequence similarity with other HAT enzymes (3). CBP (KAT3A) and p300 (KAT3B) function as transcriptional coactivators, acetyltransferases, and ubiquitin ligases and interact with multiple transcription factors (4). Changes resulting in homozygous CBP or p300 knockout mice or CBP/p300 doubly heterozygous mice are invariably lethal (5), consistent with the widespread roles of these molecules in cell growth and development. CBP-and p300-deficient embryos have phenotypes that are partially distinct from those of p300 and CBP heterozygous null mice (5, 6), suggesting they are not interchangeable and have different substrate specificities. For example, CBP and p300 exhibit distinct specificities on acetylating histones (7,8), and they can acetylate the same lysines with different degrees of efficiency (9). Biologically, CBP promotes but p300 inhibits transcription of the antiapoptotic survivin gene (10). Likewise, loss of CBP, but not loss of p300, increases the proportion of CD8 singly positive thymocytes and the likelihood of development of T cell lymphoma (11,12). Lastly, retinoic acid-induced transcriptional upregulation of the p21Cip1 cell cycle inhibitor required p300 but did not require CBP, whereas the rev...

ObjectiveAutoantibody and inflammatory cytokines play crucial roles in the development of systemic lupus erythematosus (SLE); however, the regulation of their production warrants further investigation. This study aimed to investigate the role of basophil activation in the development of SLE based on studies in patients with SLE and spontaneous lupus-prone MRL-lpr/lpr mice.MethodsThe phenotypes of peripheral basophils and the production of autoantibody and interleukin (IL)-17 in patients with SLE were determined by flow cytometry and enzyme-linked immunosorbent assay, and also their correlations were investigated by statistical analysis. Thereafter, the effect of basophils on autoantibody production by B cells and Th17 differentiation in SLE were evaluated in vitro. Finally, the effect of basophil depletion on the development of autoimmune disorders in spontaneous lupus-prone MRL-lpr/lpr mice was examined.ResultsThe decreased numbers and an increased activation of peripheral basophils were found to be correlated with increased autoantibody production and disease activity in patients with SLE. Correspondingly, in vitro coculture studies showed that basophils obtained from patients with SLE promoted autoantibody production by SLE B cells and promoted Th17 differentiation from SLE naïve CD4+ T cells. The decrease of peripheral basophils in patients with SLE might be due to their migration to lymph nodes post their activation mediated by (autoreactive) IgE as supported by their increased CD62L and CCR7 expressions and accumulation in the lymph nodes of MRL-lpr/lpr mice. Furthermore, an increased activation of peripheral basophils was identified in MRL-lpr/lpr mice. Importantly, basophil-depleted MRL-lpr/lpr mice exhibited an extended life span, improved renal function, and lower serum levels of autoantibodies and IL-17, while basophil-adoptive-transferred mice exhibited the opposite results.ConclusionThese finding suggest that basophil activation-dependent autoantibody and IL-17 production may constitute a critical pathogenic mechanism in SLE.

Histone/protein deacetylases (HDACs) are frequently upregulated in human malignancies and have therefore become therapeutic targets in cancer therapy. However, inhibiting certain HDAC isoforms can have pro-tolerogenic effects on the immune system, which could make it easier for tumor cells to evade the host immune system. Therefore, a better understanding of how each HDAC isoform affects immune biology is needed to develop targeted cancer therapy. Here, we studied the immune phenotype of HDAC5−/− mice on a C57BL/6 background. While HDAC5−/− mice replicate at expected Mendelian ratios and do not develop overt autoimmune disease, their T-regulatory (Treg) cells show reduced suppressive function in vitro and in vivo. Likewise, CD4+ T-cells lacking HDAC5 convert poorly to Tregs under appropriately polarizing conditions. HDAC5−/− Tregs show increased acetylation of Foxo1, which is deacetylated by HDAC5 and important for maintaining the Treg cell phenotype. To test if this attenuated Treg formation and suppressive function translated into improved anti-cancer immunity, we inoculated HDAC5−/− mice and littermate controls with a lung adenocarcinoma cell line. Cumulatively, lack of HDAC5 did not lead to better anti-cancer immunity. We found that CD8+ T cells missing HDAC5 had a reduced ability to produce the cytokine, IFN-γ, in vitro and in vivo, which may offset the benefit of weakened Treg function and formation. Taken together, targeting HDAC5 weakens suppressive function and de-novo induction of Tregs, but also reduces the ability of CD8+ T cells to produce IFN-γ.

Induced Foxp3+ T-regulatory cells (iTreg) are essential to gastrointestinal immune homeostasis and loss of the ability to develop iTregs may lead to autoimmune colitis. We previously showed a role for Sirtuin-1 (Sirt1) in control of Treg function and hypothesized that targeting of Sirt1 might enhance iTreg development and thereby represent a potential therapy for inflammatory bowel disease (IBD). We adoptively transferred CD4+CD25−Foxp3− T effector (TE) cells from wild-type (C57BL/6) or fl-Sirt1/CD4cre mice into B6/Rag1−/− mice and monitored the mice until they lost 10-15% of their weight. Adoptive transfer of TE cells lacking Sirt1 to B6/Rag1−/− mice resulted in a 2.8-fold increase in iTreg formation compared to mice receiving wild-type TE cells and correlated with attenuated colitis and reduced weight loss (1.04±1.4% vs. 13.97±2.2%, respectively, p<0.001). In a second model of IBD, we used pharmacologic Sirt1 targeting of mice receiving multiple cycles of dextran sodium sulfate (DSS) in their drinking water, alternated with fresh water. Likewise, wild-type mice receiving cyclic DSS and a Sirt1 inhibitor, EX-527, had reduced weight loss (5.8±5.9% vs. 13.2±6.9%, p=0.03) and increased iTreg formation compared to controls. Sirt1 appears a promising target for pharmacologic therapy of IBD as a result of promoting iTreg development.

Collybistin promotes submembrane clustering of gephyrin and is essential for the postsynaptic localization of gephyrin and ␥-aminobutyric acid type A (GABA A ) receptors at GABAergic synapses in hippocampus and amygdala. Four collybistin isoforms are expressed in brain neurons; CB2 and CB3 differ in the C terminus and occur with and without the Src homology 3 (SH3) domain. We have found that in transfected hippocampal neurons, all collybistin isoforms (CB2 SH3؉ , CB2 SH3؊ , CB3 SH3؉ , and CB3 SH3؊ ) target to and concentrate at GABAergic postsynapses. Moreover, in nontransfected neurons, collybistin concentrates at GABAergic synapses. Hippocampal neurons co-transfected with CB2 SH3؊ and gephyrin developed very large postsynaptic gephyrin and GABA A receptor clusters (superclusters). This effect was accompanied by a significant increase in the amplitude of miniature inhibitory postsynaptic currents. Co-transfection with CB2 SH3؉ and gephyrin induced the formation of many (supernumerary) non-synaptic clusters. Transfection with gephyrin alone did not affect cluster number or size, but gephyrin potentiated the clustering effect of CB2 SH3؊ or CB2 SH3؉ . Co-transfection with CB2 SH3؊ or CB2 SH3؉ and gephyrin did not affect the density of presynaptic GABAergic terminals contacting the transfected cells, indicating that collybistin is not synaptogenic. Nevertheless, the synaptic superclusters induced by CB2 SH3؊ and gephyrin were accompanied by enlarged presynaptic GABAergic terminals. The enhanced clustering of gephyrin and GABA A receptors induced by collybistin isoforms was not accompanied by enhanced clustering of neuroligin 2. Moreover, during the development of GABAergic synapses, the clustering of gephyrin and GABA A receptors preceded the clustering of neuroligin 2. We propose a model in which the SH3؊ isoforms play a major role in the postsynaptic accumulation of GABA A receptors and in GABAergic synaptic strength.A fundamental issue in the GABAergic synapse field is to understand the mechanisms that regulate the postsynaptic clustering of GABA A 3 receptors (GABA A Rs) and GABAergic synaptic strength during inhibitory synapse formation. Collybistin (CB) is a cytoplasmic protein that binds to gephyrin, helping the latter to cluster and translocate to the submembranous compartment (1-4). CB is a guanine nucleotide exchange factor (GEF) that catalyzes GDP-GTP exchange on the small GTPase Cdc42 of the Rho family (5). CB is essential for the initial synaptic localization and maintenance of gephyrin and GABA A Rs at GABAergic synapses in the hippocampus and amygdala (6, 7).In adult rat or mouse brain, two alternative spliced forms are expressed, CB2 and CB3, which are identical except for the C termini (3). There is also CB1 with a different C terminus, but this isoform is not expressed in neurons or in the adult brain. In humans, CB3 is called hPEM2. However, CB2 has not been detected in humans (3). There are also splice variants of CB2 and CB3 (or hPEM2) with or without an Src homology 3 (SH3) domain (1, 3). Alt...

We have found that the γ2 subunit of the GABAA receptor (γ2-GABAAR) specifically interacts with protocadherin γ-C5 (Pcdh-γC5) in the rat brain. The interaction occurs between the large intracellular loop of the γ2-GABAAR and the cytoplasmic domain of Pcdh-γC5. In brain extracts, Pcdh-γC5 co-immunoprecipitates with GABAARs. In co-transfected HEK293 cells, Pcdh-γC5 promotes the transfer of γ2-GABAAR to the cell surface. We have previously shown that in cultured hippocampal neurons, endogenous Pcdh-γC5 forms clusters, some of which associate with GABAergic synapses. Overexpression of Pcdh-γC5 in hippocampal neurons increases the density ofγ2-GABAAR clusters but has no significant effect on the number of GABAergic contacts that these neurons receive, indicating that Pcdh-γC5 is not synaptogenic. Deletion of the cytoplasmic domain of Pcdh-γC5 enhanced its surface expression but decreased the association with both γ2-GABAAR clusters and presynaptic GABAergic contacts. Cultured hippocampal neurons from the Pcdh-γ triple C-type isoform knockout (TCKO) mouse (Pcdhgtcko/tcko) showed plenty of GABAergic synaptic contacts, although their density was reduced compared with sister cultures from wild type and heterozygous mice. Knocking down Pcdh-γC5 expression with shRNA decreased γ2-GABAAR cluster density and GABAergic innervation. The results indicate that although Pcdh-γC5 is not essential for GABAergic synapse formation or GABAAR clustering, i) Pcdh-γC5 regulates the surface expression of GABAARs via cis-cytoplasmic interaction with γ2-GABAAR; and ii) Pcdh-γC5 plays a role in the stabilization and maintenance of some GABAergic synapses.