The immunoregulatory role of alpha enolase in dendritic cell function during Chlamydia infection

Abstract Background We have previously reported that interleukin-10 (IL-10) deficient dendritic cells (DCs) are potent antigen presenting cells that induced elevated protective immunity against Chlamydia. To further investigate the molecular and biochemical mechanism underlying the superior immunostimulatory property of IL-10 deficient DCs we performed proteomic analysis on protein profiles from Chlamydia-pulsed wild-type (WT) and IL-10−/− DCs to identify differentially expressed proteins with immunomodulatory properties. Results The results showed that alpha enolase (ENO1), a metabolic enzyme involved in the last step of glycolysis was significantly upregulated in Chlamydia-pulsed IL-10−/− DCs compared to WT DCs. We further studied the immunoregulatory role of ENO1 in DC function by generating ENO1 knockdown DCs, using lentiviral siRNA technology. We analyzed the effect of the ENO1 knockdown on DC functions after pulsing with Chlamydia. Pyruvate assay, transmission electron microscopy, flow cytometry, confocal microscopy, cytokine, T-cell activation and adoptive transfer assays were also used to study DC function. The results showed that ENO1 knockdown DCs had impaired maturation and activation, with significant decrease in intracellular pyruvate concentration as compared with the Chlamydia-pulsed WT DCs. Adoptive transfer of Chlamydia-pulsed ENO1 knockdown DCs were poorly immunogenic in vitro and in vivo, especially the ability to induce protective immunity against genital chlamydia infection. The marked remodeling of the mitochondrial morphology of Chlamydia-pulsed ENO1 knockdown DCs compared to the Chlamydia-pulsed WT DCs was associated with the dysregulation of translocase of the outer membrane (TOM) 20 and adenine nucleotide translocator (ANT) 1/2/3/4 that regulate mitochondrial permeability. The results suggest that an enhanced glycolysis is required for efficient antigen processing and presentation by DCs to induce a robust immune response. Conclusions The upregulation of ENO1 contributes to the superior immunostimulatory function of IL-10 deficient DCs. Our studies indicated that ENO1 deficiency causes the reduced production of pyruvate, which then contributes to a dysfunction in mitochondrial homeostasis that may affect DC survival, maturation and antigen presenting properties. Modulation of ENO1 thus provides a potentially effective strategy to boost DC function and promote immunity against infectious and non-infectious diseases

The emerging role of ASC in dendritic cell metabolism during Chlamydia infection.

Chlamydia trachomatis is a bacterial agent that causes sexually transmitted infections worldwide. The regulatory functions of dendritic cells (DCs) play a major role in protective immunity against Chlamydia infections. Here, we investigated the role of ASC in DCs metabolism and the regulation of DCs activation and function during Chlamydia infection. Following Chlamydia stimulation, maturation and antigen presenting functions were impaired in ASC-/- DCs compared to wild type (WT) DCs, in addition, ASC deficiency induced a tolerant phenotype in Chlamydia stimulated DCs. Using real-time extracellular flux analysis, we showed that activation in Chlamydia stimulated WT DCs is associated with a metabolic change in which mitochondrial oxidative phosphorylation (OXPHOS) is inhibited and the cells become committed to utilizing glucose through aerobic glycolysis for differentiation and antigen presenting functions. However, in ASC-/- DCs Chlamydia-induced metabolic change was prevented and there was a significant effect on mitochondrial morphology. The mitochondria of Chlamydia stimulated ASC-/- DCs had disrupted cristae compared to the normal narrow pleomorphic cristae found in stimulated WT DCs. In conclusion, our results suggest that Chlamydia-mediated activation of DCs is associated with a metabolic transition in which OXPHOS is inhibited, thereby dedicating the DCs to aerobic glycolysis, while ASC deficiency disrupts DCs function by inhibiting the reprogramming of DCs metabolism within the mitochondria, from glycolysis to electron transport chain

Effect of ASC on DCs glycolytic pathway.

<p>To determine the influence of ASC on DC glycolysis DCs. A). DCs were stimulated with <i>C</i>. <i>muridarum</i> for 30 minutes and real-time ECAR was measured in a live DCs using an XFe-96 analyzer. Vertical lines indicate addition of glucose (glycolysis substrate), oligomycin (ATP synthase inhibitor), and 2-DG (glycolysis inhibitor). Graphs in this figure represent the mean ± SD of three independent experiments. B). Culture supernatants from WT and ASC^-/- DCs stimulated at a ratio of 1:5 with <i>C</i>. <i>muridarum</i> at 0, 1 and 2 hours were collected, and their pyruvate concentration in μM/ml was determined. Data were calculated as the mean ± SD for triplicate cultures from each experiment (*, P < 0.05). Control groups included uninfected DCs and culture media. n = 12 pooled from three independent experiments. In ASC^-/- DCs, the pyruvate production was markedly reduced compared to WT DCs after <i>Chlamydia</i> stimulation. C). Western blotting was used to analyze HEK II, ENO-1 and PDH in lysates from <i>C</i>. <i>muridarum</i> stimulated WT and ASC^-/- BMDCs after 0, 1 and 2 hours. The expression levels of the glycolytic enzymes HEK II and ENO-1 in WT and ASC^-/- DCs were unchanged before or after <i>C</i>. <i>muridarum</i> stimulation. PDH expression was downregulated during <i>C</i>. <i>muridarum</i> stimulation of ASC^-/- DCs.</p

ASC regulates DCs antigen processing events <i>in vitro</i> and i<i>n vivo</i>.

<p>WT and ASC^-/- DCs were γ-irradiated and stimulated with UV-inactivated <i>C</i>. <i>muridarum</i> and then co-cultured with splenic T cells from immune animals. A). T cell proliferation was determined using Cell Signaling XTT Cell Viability Kit protocol. Results are expressed as cell viability ratio, which is the ratio between the absorbance values of stimulated and non-stimulated cells and the bars represent the mean and SD of three independent experiments. B). The supernatants were collected and cytokines amounts determined using a 23 plex multiplex assay from Bio-Rad. The concentration of the cytokines for each sample was obtained by extrapolation from a standard calibration curve generated simultaneously. Data was calculated as the mean and SD for triplicate cultures in each experiment. The results were from 2 independent experiments and are reported as mean cytokine concentrations (pg/ml) ± SD. Results were compared using Student’s t-test. *, P<0.05. C). Isolated DCs from WT and ASC^-/- mice were stimulated with <i>C</i>. <i>muridarum</i> and adoptively transferred into naïve WT mice. Mice were then infected intravaginally with live <i>C</i>. <i>muridarum</i> one week after the adoptive transfer. Infection was monitored by periodic cervicovaginal swabbing every 3 days for 2 weeks and then once every week. The <i>C</i>. <i>muridarum</i> IFUs was determined using standard methods with anti-chlamydial antibodies from Bio-Rad. The experiment was repeated three times.</p

Effect of ASC on DCs mitochondrial respiration and morphology.

<p>We analyzed the effect of <i>Chlamydia</i> on WT and ASC^-/- DCs mitochondrial respiration and morphology using XF extracellular flux analyzer assay and electron transmission microscopy (TEM) respectively. A). WT and ASC^-/-DCs were stimulated with <i>C</i>. <i>muridarum</i>. After 24 h, mitochondrial function was assessed. Real-time changes in oxygen consumption rates (OCRs) in a live cell were analyzed using extracellular flux analysis. Where indicated, OCRs were analyzed in response to 1 μM oligomycin, 1.5 μM fluoro-carbonyl cyanide phenylhydrazone (an uncoupling agent that disrupts ATP synthesis by transporting hydrogen ions through a cell membrane before they can be used to provide the energy for oxidative phosphorylation), and 100 nM rotenone plus 1 μM antimycin A that in combination shuts down mitochondrial respiration and enables the calculation of non-mitochondrial respiration driven by processes outside the mitochondria (all from Sigma-Aldrich), as indicated. Graphs in this figure represent mean values ± SD of three independent experiments. B). The WT and ASC^-/- DCs were infected with <i>C</i>. <i>muridarum</i> at a ratio of 1:5 for 2 hours. A JEOL 1200EX transmission electron microscope was used to examine mitochondrial changes in the fixed DC slices. Magnification used was 30,000X; scale bar indicates 400nm. The ultrastructure of the tubular mitochondria was well defined in unstimulated WT, ASC^-/- and <i>C</i>. <i>muridarum</i> stimulated WT DCs. <i>C</i>. <i>muridarum</i> stimulated ASC^-/- DCs did not have well-defined mitochondrial morphology and their cristae appear degraded. The experiment was repeated three times.</p

ASC deficiency results in higher number of IFUs following an acute <i>Chlamydia</i> infection.

<p>The WT and ASC^-/- mice were infected with <i>C</i>. <i>muridarum</i> and swabbed intravaginally every three days for two weeks and once a week for the last two weeks. <i>C</i>. <i>muridarum</i> IFUs was determined to monitor the course of the infection. Each data point depicts the mean ± SD of the individual numbers of recoverable IFUs from each mouse per group of 8 mice, at the indicated days post infection; expressed as Log₁₀ IFU/ml ± SD. The results showed that <i>C</i>. <i>muridarum</i> infection took a longer time to clear in ASC^-/- mice. The experiment was repeated three times.</p

Deficiency of ASC induces changes in DCs maturation and cytokine profile.

<p>A). WT and ASC^-/- DCs stimulated at a ratio of 1:5 with <i>C</i>. <i>muridarum</i> for 2 hours were stained with antibodies against maturation and activation markers (CD80, CD103, MHC II, CD40, CD14, TLR4, and CD11c) conjugated with mAbs to FITC, APC and PE. The cells 10,000 events were analyzed by flow cytometry. For each sample, at least 100,000 events were collected. Data was analyzed with GuavaSoft 2.7. B). Culture supernatants from WT and ASC^-/- DCs stimulated at a ratio of 1:5 with <i>C</i>. <i>muridarum</i> for 2 hours were collected and cytokines were analyzed using a Luminex machine. Cytokine concentration was extrapolated from a standard calibration curve. The mean and SD of all replicate cultures were calculated. Experiments were repeated two times. *, P<0.05.</p

Snail transcription factor negatively regulates maspin tumor suppressor in human prostate cancer cells

<p>Abstract</p> <p>Background</p> <p>Maspin, a putative tumor suppressor that is down-regulated in breast and prostate cancer, has been associated with decreased cell motility. Snail transcription factor is a zinc finger protein that is increased in breast cancer and is associated with increased tumor motility and invasion by induction of epithelial-mesenchymal transition (EMT). We investigated the molecular mechanisms by which Snail increases tumor motility and invasion utilizing prostate cancer cells.</p> <p>Methods</p> <p>Expression levels were analyzed by RT-PCR and western blot analyses. Cell motility and invasion assays were performed, while Snail regulation and binding to maspin promoter was analyzed by luciferase reporter and chromatin immunoprecipitation (ChIP) assays.</p> <p>Results</p> <p>Snail protein expression was higher in different prostate cancer cells lines as compared to normal prostate epithelial cells, which correlated inversely with maspin expression. Snail overexpression in 22Rv1 prostate cancer cells inhibited maspin expression and led to increased migration and invasion. Knockdown of Snail in DU145 and C4-2 cancer cells resulted in up-regulation of maspin expression, concomitant with decreased migration. Transfection of Snail into 22Rv1 or LNCaP cells inhibited maspin promoter activity, while stable knockdown of Snail in C4-2 cells increased promoter activity. ChIP analysis showed that Snail is recruited to the maspin promoter in 22Rv1 cells.</p> <p>Conclusions</p> <p>Overall, this is the first report showing that Snail can negatively regulate maspin expression by directly repressing maspin promoter activity, leading to increased cell migration and invasion. Therefore, therapeutic targeting of Snail may be useful to re-induce expression of maspin tumor suppressor and prevent prostate cancer tumor progression.</p