Foxp3⁺ cells upregulate CD69 after TCR activation in an antigen-non-specific manner.

<p>(A) Gating strategy on CD4⁺ enriched T cells after overnight culture without stimulation. Cells are gated in the lymphocyte gate based on forward versus side scatter, then doublets are excluded based on forward scatter height versus amplitude and live cells are gated as negative for the dead cell marker. Finally Foxp3⁺ and Foxp3^- CD4⁺ T cells are gated using CD4 and Foxp3 expression. This gating strategy was applied throughout the analyses unless otherwise stated. (B) CD69 expression on Foxp3^- (top row) and Foxp3⁺ (bottom row) CD4⁺ cells before and after overnight stimulation with or without anti-CD3 stimulation. Staining is representative of at least three independent experiments. (C) CD69 expression on Foxp3^- (left) and Foxp3⁺ (right) enriched CD4⁺ T cells from either wild-type (white bars) or OT-II (black bars) donor mice, after overnight culture with BMDCs. Unstim, control without BMDC; /, unpulsed BMDC; OVA, OVA-pulsed BMDC, anti-CD3, anti-CD3-coated BMDC. Data are representative of four independent experiments with each 3 replicates per group. (D) Normalized MHCII KO BMDC-induced CD69 expression compared to control BMDC-induced CD69. The graphs show the frequency of CD69⁺ cells among CD4⁺ Foxp3^- (left) and Foxp3⁺ (right) T cells after stimulation with MHCII KO BMDC, normalized to CD4⁺ Foxp3^- and Foxp3⁺ T cells stimulated with wild type BMDC for each given condition. 100% represents the same CD69 expression by cells stimulated with control or MHCII KO BMDC. Data show mean + SD, *p ≤ 0.05; **p ≤ 0.01; *** p ≤ 0.001, ns, non significant. Data are representative of three independent experiments with each n = 3 per group.</p

Foxp3⁺ cells upregulate Nur77 after TCR activation in an antigen-specific manner.

<p>(A) Time course of Nur77 (black bars) and CD69 expression (white bars) on Foxp3⁺ T-cells among sorted CD25⁺ CD4⁺ splenocytes stimulated with plate-bound αCD3 and 1U/ml IL-2. Data are representative of 3 independent experiments with n ≥ 3. (B) Nur77 expression on Foxp3^- (left) and Foxp3⁺ (right) among enriched CD4⁺ T-cells from either wild-type (white bars) or OT-II (black bars) mice, co-cultured overnight with unstimulated, OVA pulsed or αCD3 coated BMDCs. Data are representative of two independent experiments, with each n = 3 per group. unstim, control without BMDC; /, unpulsed BMDC; OVA, OVA-pulsed BMDC, anti-CD3, anti-CD3-coated BMDC. Data show mean + SD, *p ≤ 0.05, *** p ≤ 0.001; ns, non significant. Data are representative of four independent experiments each with n = 3 per group.</p

Nur77 intensity is not modulated by cytokines.

<p>(A) Nur77 expression on Foxp3^- (white bars) and Foxp3⁺ (black bars) CD4⁺ T cells from the culture in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137393#pone.0137393.g002" target="_blank">Fig 2B</a>. Data are representative of at least two independent experiments. (B) Representative plots of Nur77 versus CD69 expression on Foxp3⁺ among enriched CD4⁺ T cells, stimulated for 4 hours (top row) or 16 hours (bottom row) without additional cytokines (/), with IFN-α, TNF-α, or plate-bound αCD3. Data are representative of two independent experiments. (C) Nur77 expression on Foxp3⁺ cells among sorted CD25⁺ CD4⁺ splenocytes stimulated with plate-bound αCD3 with low (1U/ml) or high (1000U/ml) IL-2 for 6 hours. Data are representative of four independent experiments. Data show mean + SD, ns, non significant.</p

Foxp3⁺ cells preferentially upregulate CD69 in response to soluble factors.

<p>(A) CD69 expression on Foxp3^- (left) and Foxp3⁺ (right) enriched CD4⁺ T cells after overnight incubation with or without BMDC-conditioned culture medium. /, control medium; OVA, medium supplemented with OVA; BMDC SN, supernatant from BMDCs cultured overnight with fresh medium; BMDC OVA SN, supernatant from BMDCs cultured overnight with medium supplemented with OVA. Data are representative of at least two independent experiments. (B) CD69 expression on Foxp3^- (white bars) and Foxp3⁺ (black bars) among enriched CD4⁺ T cells cultured overnight without additional cytokines (/) or with IL-1β, IFN-α or TNF-α. Data are representative of at least two independent experiments. (C) CD69 expression on Foxp3⁺ among enriched CD4⁺ T cells cultured overnight without additional cytokines (/) or with IL-33, IL-4, IL-12, IL-27, IL-6, IFN-γ, or GM-CSF. (D) Representative FACS plot and response to stimulation of sorted CD4⁺ Foxp3^RFP+ cells. Right: representative FACS plot showing the purity of Foxp3^RFP+ cells after sort. Cells are gated on forward and side scatter and doublets and dead cells are excluded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137393#pone.0137393.g001" target="_blank">Fig 1A</a>. Plot shows CD4 versus intracellular Foxp3 staining. Left, CD69 expression on Foxp3⁺ among enriched CD4⁺ T cells (white bars) or Foxp3^RFP+ sorted (black bars) CD4⁺ T cells, cultured overnight without additional cytokines (/) or with IFN-α or TNF-α (left). (E) CD69 expression on Foxp3⁺ among enriched CD4⁺ splenocytes from TNFR1 KO, Myd88 KO and ctrl C57BL/6 or from IFNAR1 KO and control 129 mice, cultured overnight with control medium (/) or with supernatant from OVA stimulated BMDCs (BMDC OVA SN). (F) CD69 expression on Foxp3⁺ among enriched CD4⁺ T cells of IFNAR1 <i>KO</i> and control mice cultured overnight with control medium (unstim) or with supernatant from OVA stimulated BMDCs (BMDC OVA SN) with or without addition of different concentrations of blocking anti-TNF-α antibody. Data are representative from two independent experiments. Data show mean + SD, *** p ≤ 0.001 compared to unstimulated control, unless comparison indicated by line below the stars, n ≥ 3 per group.</p

PNOCARC Neurons Promote Hyperphagia and Obesity upon High-Fat-Diet Feeding

Calorie-rich diets induce hyperphagia and promote obesity, although the underlying mechanisms remain poorly defined. We find that short-term high-fat-diet (HFD) feeding of mice activates prepronociceptin (PNOC)-expressing neurons in the arcuate nucleus of the hypothalamus (ARC). PNOCARC neurons represent a previously unrecognized GABAergic population of ARC neurons distinct from well-defined feeding regulatory AgRP or POMC neurons. PNOCARC neurons arborize densely in the ARC and provide inhibitory synaptic input to nearby anorexigenic POMC neurons. Optogenetic activation of PNOCARC neurons in the ARC and their projections to the bed nucleus of the stria terminalis promotes feeding. Selective ablation of these cells promotes the activation of POMC neurons upon HFD exposure, reduces feeding, and protects from obesity, but it does not affect food intake or body weight under normal chow consumption. We characterize PNOCARC neurons as a novel ARC neuron population activated upon palatable food consumption to promote hyperphagia

TFEB induces mitochondrial itaconate synthesis to suppress bacterial growth in macrophages

Successful elimination of bacteria in phagocytes occurs in the phago-lysosomal system, but also depends on mitochondrial pathways. Yet, how these two organelle systems communicate is largely unknown. Here we identify the lysosomal biogenesis factor transcription factor EB (TFEB) as regulator for phago-lysosome-mitochondria crosstalk in macrophages. By combining cellular imaging and metabolic profiling, we find that TFEB activation, in response to bacterial stimuli, promotes the transcription of aconitate decarboxylase (Acod1, Irg1) and synthesis of its product itaconate, a mitochondrial metabolite with antimicrobial activity. Activation of the TFEB-Irg1-itaconate signalling axis reduces the survival of the intravacuolar pathogen Salmonella enterica serovar Typhimurium. TFEB-driven itaconate is subsequently transferred via the Irg1-Rab32-BLOC3 system into the Salmonella-containing vacuole, thereby exposing the pathogen to elevated itaconate levels. By activating itaconate production, TFEB selectively restricts proliferating Salmonella, a bacterial subpopulation that normally escapes macrophage control, which contrasts TFEB's role in autophagy-mediated pathogen degradation. Together, our data define a TFEB-driven metabolic pathway between phago-lysosomes and mitochondria that restrains Salmonella Typhimurium burden in macrophages in vitro and in vivo

Mitochondrial priming by CD28

T cell receptor (TCR) signaling without CD28 can elicit primary effector T cells, but memory T cells generated during this process are anergic, failing to respond to secondary antigen exposure. We show that, upon T cell activation, CD28 transiently promotes expression of carnitine palmitoyltransferase 1a (Cpt1a), an enzyme that facilitates mitochondrial fatty acid oxidation (FAO), before the first cell division, coinciding with mitochondrial elongation and enhanced spare respiratory capacity (SRC). micro-RNA-33 (miR33), a target of thioredoxin-interacting protein (TXNIP), attenuates Cpt1a expression in the absence of CD28, resulting in cells that thereafter are metabolically compromised during reactivation or periods of increased bioenergetic demand. Early CD28-dependent mitochondrial engagement is needed for T cells to remodel cristae, develop SRC, and rapidly produce cytokines upon restimulation-cardinal features of protective memory T cells. Our data show that initial CD28 signals during T cell activation prime mitochondria with latent metabolic capacity that is essential for future T cell responses

Discrete Determinants in ArfGAP2/3 Conferring Golgi Localization and Regulation by the COPI Coat

From yeast to mammals, two types of GTPase-activating proteins, ArfGAP1 and ArfGAP2/3, control guanosine triphosphate (GTP) hydrolysis on the small G protein ADP-ribosylation factor (Arf) 1 at the Golgi apparatus. Although functionally interchangeable, they display little similarity outside the catalytic GTPase-activating protein (GAP) domain, suggesting differential regulation. ArfGAP1 is controlled by membrane curvature through its amphipathic lipid packing sensor motifs, whereas Golgi targeting of ArfGAP2 depends on coatomer, the building block of the COPI coat. Using a reporter fusion approach and in vitro assays, we identified several functional elements in ArfGAP2/3. We show that the Golgi localization of ArfGAP3 depends on both a central basic stretch and a carboxy-amphipathic motif. The basic stretch interacts directly with coatomer, which we found essential for the catalytic activity of ArfGAP3 on Arf1-GTP, whereas the carboxy-amphipathic motif interacts directly with lipid membranes but has minor role in the regulation of ArfGAP3 activity. Our findings indicate that the two types of ArfGAP proteins that reside at the Golgi use a different combination of protein–protein and protein–lipid interactions to promote GTP hydrolysis in Arf1-GTP