Increased macrophage uptake of apoptotic cells induced by leptin promotes proliferation of self antigen-reactive T cells.

<p>TAMRA-labeled apoptotic bodies containing OVA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112826#s2" target="_blank">Methods</a>) were co-cultured with CFSE-labeled macrophages for 2 h, followed by staining with APC-labeled anti-CD11b Ab and flow cytometry. (A) Frequency of TAMRA⁺CFSE⁺ CD11b⁺ cells (macrophages positive for OVA-containing apoptotic bodies) in OVA-TCR transgenic mice (DO11.10) after culture in the presence of scalar doses of leptin. (B) Flow cytometry staining with clonotype-specific KJ1.26 Ab (OVA-specific TCR) 48 h after co-incubation of OVA-immunized DO11.10 (OVA-TCR transgenic) mouse splenocytes with macrophages containing OVA-loaded apoptotic bodies (TAMRA⁺). *p<0.05, **p<0.01 vs control (n = 5).</p

Leptin modulates the uptake of apoptotic cells <i>in vitro</i> from lupus macrophage.

<p>Peritoneal macrophages (MΦ) from NZB/W lupus mice (A–B) or leptin-receptor-deficient (non-autoimmune) <i>db/db</i> mice (C–D) were co-cultured <i>ex vivo</i> with 1×10⁷ CFSE-labeled apoptotic cells (A, C) or necrotic cells (B, D) in the presence of scalar doses of leptin (<i>x</i> axis). After 2 h, cells were stained with PE-labeled anti-mouse CD11b Ab and the labeled phagocytosed material within macrophages (PE-positive) was visualized as co-staining in flow cytometry. Additional details are reported in the Methods. *p<0.01 vs no (0) leptin.</p

Leptin does not influence peritoneal macrophage recruitment.

<p>Total number of peritoneal macrophages recovered three days after i.p. thioglycolate in NZB/W mice in that had been injected i.p. with vehicle (control) or leptin. Further details can be found in the Methods. p not significant.</p

Leptin promotes phagocytosis by lupus macrophages <i>in vivo</i>.

<p>NZB/W mice were injected i.p. with thioglycolate prior to injection of either vehicle (A–C) or 2 µg/g leptin (D–F) at 12-h intervals. After 72 h, 1×10⁶ CFSE-positive apoptotic cells were injected i.p. After 30 min, uptake of apoptotic cells by CD11b⁺ macrophages (PE) in peritoneal fluid of recipient animals was visualized <i>ex vivo</i> by confocal microscopy as colocalization of PE-positive macrophages (red) and CFSE-positive (green) apoptotic bodies within the same cell (yellow). Representative of three experiments (n = 6 per group). Original magnification: 10x. (G) Cumulative flow cytometry of peritoneal macrophages costaining for apoptotic cells. *p<0.01 vs control.</p

Effects of leptin on cAMP levels in macrophages.

<p>cAMP was measured as intracellular (A) and in supernatant (B) of NZB/W macrophages cultured 1 h with scalar doses of leptin (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112826#s2" target="_blank">Methods</a> for further details). Representative of three experiments in triplicate. *p<0.05 vs control (0)</p

Multiple pathway analysis in HaCaT cells exposed to resveratrol, UVB or both: earlier time point (15 minutes).

<p>HaCaT cells were pretreated with 25 and 100 µM resveratrol for 2 and 24 hours prior irradiation with UVB (10, 40, and 100 mJ/cm²). Cell lysates were collected 15′ after UVB irradiation. Phosphorylated or not phosphorylated protein levels were analyzed by western blot. The figure shows representative blots analyzing the levels of the phosphorylated forms of ERK1/2, p38, p53, AKT, and S6, and the levels of BAX, and Bcl2.GAPDH was used as loading control.</p

Resveratrol, UVB or both and HaCaT cells proliferation.

<p>Resveratrol (25 and 100 µM) was added to HaCaT cells for 2 hours (panel A) or 24 hours (panel B) and withdrawn prior to irradiation with UVB (10, 20, 40, and 100 mJ/cm²). Cell count was performed after 48 hours in culture in standard medium. Graphs report mean values ± standard deviation of three independent measurement for each experimental point. RSV: resveratrol; * p<0.05.</p

Relative contribution of apoptosis and autophagy to resveratrol enhanced UVB-induced HaCaT cells death.

<p>(A) HaCaT cells were pretreated for 24 hours with 25 µM resveratrol followed by 2 hours in the presence or absence of pan caspase inhibitor Z-VAD (50 µM) prior to irradiation with UVB (30 mJ/cm²). Cell count was performed after additional 24 hours of culture in standard medium. Graphs report mean values ± standard deviation of three independent measurements for each experimental point. RSV: resveratrol; * p<0.05. (B) “Total” represents the difference of cell number between UVB-irradiated HaCaT cells and resveratrol pre-treated and UVB-irradiated HaCaT cells; “Apoptotic” represent the difference of cell number between resveratrol/Z-VAD pre-treated/UVB-irradiated HaCaT cells and resveratrol pre-treated and UVB-irradiated HaCaT cells; Both “Total” and “Apoptotic” have been reported as percentage of “Total”(100% and 45% respectively). “Non-apoptotic” (55%) represent the difference of the percentages between “Total” and “Apoptotic”.</p

Cleavage of caspase 8 and PARP in HaCaT cells exposed to resveratrol, UVB or both.

<p>HaCaT cells were treated with resveratrol (25 and 100 µM, 2 and 24 hours), UVB (10, 40, and 100 mJ/cm² alone or pretreated for 2 or 24 hours with resveratrol (25 and 100 µM) prior irradiation with UVB (10, 40, and 100 mJ/cm²). Lysates were collected 4 hours and 30′ after irradiation and resveratrol withdrawal and challenged with anti-caspase 8 and anti PARP antibodies recognizing the cleaved forms of the two proteins (panel A), or with anti-Beclin 1. Panel B reports the densitometric analysis of caspase 8 cleaved band and Beclin 1 at both 2 and 24 hours prior irradiation. The same lysates were challenged with anti γ-H2AX, recognizing the phosphorylated form of the histone (Panel D). GAPDH was used as loading control.</p

Analysis of effects of UVB irradiation on autophagosomes and lysosomes compartments and on microtubule cytoskeleton in HaCaT cells untreated or pretreated with resveratrol.

<p>(A) HaCaT cells, in control conditions (CTR) or upon different treatments (RSV, UVB, RSV/UVB) were stained with monodansylcadaverin (MDC, in green) or with lysotracker (lysot., in red) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080728#s2" target="_blank">materials and methods</a>. Serial confocal sections were collected. The 3D reconstruction (black and white panels) is shown. Bar, 6 µm. (B) HaCaT cells, grown on coverslips, were loaded with lysotracker (in red), fixed and stained with a specific antibody against tubulin and revealed by FITC-conjugated secondary antibodies (green). Nuclei were labeled with DAPI (blue). Serial confocal sections were collected. Bar, 8 µm. (C) Lc3-I to Lc3-II conversion analysis in HaCaT cells exposed to resveratrol, UVB or both. HaCaT cells were pretreated for 24 hours with 25 µM resveratrol in the presence or the absence of NH₄Cl (20 mM) and leupeptin (100 µM) and then irradiated with UVB (10, 40, and 100 mJ/cm²). Cell lysates were collected 15′ (panel A) or 4h30′ (panel B) after UVB irradiation and resveratrol withdrawal. A representative blot analyzing the levels of Lc3 I to Lc3 II conversion is shown. GAPDH was used as loading control.</p