TSA-mediated antitumor activity requires tumor expression of IRF-8.

<p>CMS4 (<i>A</i>) or IRF-8-deficient CMS4-shRNA (<i>B</i>) tumor cells were injected SQ into BALB/c mice. When tumor size was palpable (∼30 mm³), mice were treated with six daily peritumoral injections of TSA or vehicle control. * <i>P</i><0.01 or ** <i>P</i><0.001, based on comparing the control to TSA-treated mice at the indicated time points in panel <i>A</i>. Data were not significant in panel <i>B</i>.</p

HDACi enhances IRF-8 expression in tumor cells.

<p>(<i>A</i>) CMS4 cells were treated with TSA, IFN-γ (100 U/ml) or a combination of both at the indicated concentrations and then analyzed by real-time PCR (top). Representative RT-PCR is shown in the bottom panel, which shares the same treatment labels. Data in top panel are presented as fold-change (shown above each bar) of the treated samples relative to the vehicle-treated controls. (<i>B</i>) Similar to <i>A</i>, except that CMS4 cells were treated with DP (25 ng/ml) instead of TSA. (<i>C</i>) CMS4-met.sel cells were treated with TSA (500 nM), IFN-γ or a combination of both and then analyzed real-time PCR (top) or RT-PCR (bottom), as in <i>A</i>. (<i>D</i>) Similar to <i>C</i>, except that CMS4 cells were treated with DP instead of TSA. All data are expressed as the mean ± SEM of triplicate determinations (shown above each bar). *<i>P</i><0.05, based on comparing the single agent treatment to the vehicle-treated control. **<i>P</i><0.05, based on comparing the combination regimen to the single treatment counterparts.</p

TSA-mediated IRF-8 transcription is STAT1-dependent.

<p>(<i>A</i>) STAT1 mRNA levels in CMS4 or CMS4.met.sel cells after the indicated treatments, as in Fig. 1. <i>(A)</i> Top, real-time PCR. Data presented as fold-change, as in Fig. 1. (<i>A</i>) Bottom, RT-PCR. *<i>P</i><0.05, based on comparing the single agent treatment to the vehicle-treated control. **<i>P</i><0.05, based on comparing the combination regimen to the single treatment counterparts. (<i>B</i>) CMS4 or CMS4.met.sel cells were transfected with an IRF-8 promoter reporter construct, followed by treatment with the indicated agents for 6 hr. Results are reported as the mean ± SEM of the fold-change relative to the vehicle-treated cells from three separate experiments. *<i>P</i><0.05, based on comparing treatment to matched vehicle control. No activity was observed using the pGL3 vector lacking a promoter. (<i>C</i>) Similar to <i>B</i>, except that CMS4 cells were silenced for STAT1 expression. *<i>P</i><0.05, based on comparing the indicated treatment group to the matched vehicle-treated control. **<i>P</i><0.05, based on comparing the STAT1-deficient groups to their matched STAT1-expressing vector controls. (<i>D</i>) Phosphorylated STAT1 (pSTAT1) and total STAT1 protein levels in CMS4.met.sel cells after treatment with the indicated treatments (TSA, 500 nM; IFN-γ, 200 U/ml) for 15 min, as measured by Western blot. This experiment is representative of one of three with similar results. (<i>E</i>) Similar to <i>D</i>, except that acetylated STAT1 and total STAT1 levels were measured by IP-Western blot (i.e., IP with anti-STAT1 antibody, followed by Western blot with anti-acetyl-lysine antibody) after treatment with or without TSA (500 nM for 6 hr). Band intensities were quantified, and the data presented as fold-change of TSA-treated vs. untreated samples (mean ± SEM of triplicate experiments). *<i>P</i><0.05, based on the TSA-treated group relative to the matched vehicle-treated control.</p

Role for IRF-8 in the HDACi-mediated antitumor response.

<p>Engagement of the IFN-γ receptor (IFN-γR) by IFN-γ and/or the uptake of HDACi, such as TSA, induce IRF-8 transcription. While activation of IRF-8 in both cases is STAT1-dependent, the mechanisms by which this is achieved may be distinct and involve phosphorylated and unphosphorylated modifications. In regard to the latter, the precise nature of interactions remains to be fully detailed. IRF-8 expression, in turn, is known to affect apoptosis by regulating genes associated with both extrinsic and intrinsic pathways of cell death (not illustrated).</p

TSA treatment enhances Fas-mediated tumor cell death through an IRF-8-dependent mechanism.

<p>(<i>A</i>) Control or IRF-8-deficient CMS4-shRNA cells were exposed to recombinant mouse FasL (100 ng/ml) after treatment with TSA (100 nM), IFN-γ (200 U/ml), a combination of both or a vehicle control, and cell death measured by a flow-based assay. (<i>B</i>) IRF-8-mutant CMS4-K79E and CMS4-vector control cells were exposed to FasL after treatment with TSA (20 nM) and/or IFN-γ as in <i>A</i>. Data in <i>A</i> and B are expressed as mean ± SEM of six or three independent experiments, respectively. *<i>P</i><0.05, based on comparing the indicated treatment group to the FasL only control. **<i>P</i><0.05, based on comparing the IRF8-deficient to its matched IRF-8-expressing vector controls.</p

TSA enhances IRF-8 expression in a human tumor cell line model of varying malignant potential.

<p>SW480 (<i>A</i>) or SW620 (<i>B</i>) cells were treated with TSA (500 nM), IFN-γ (100 U/ml) or a combination of both and then analyzed by real-time PCR, as in Fig. 1. Data in <i>B</i> are presented as fold-change of the treated samples relative to the vehicle-treated controls. Data expressed as the mean ± SEM of triplicate determinations. *<i>P</i><0.05, based on comparing the single agent treatment to the vehicle-treated control. **<i>P</i><0.05, based on comparing the combination regimen to the single treatment counterparts.</p

NET formation in young and old trauma patients and volunteers.

<p>NET formation in PMN isolated from young healthy volunteers (n = 10, <b>A and E</b>), young trauma patients (n = 7, <b>B and F</b>), old healthy volunteers (n = 6, <b>C and G</b>), and old trauma patients (n = 5, <b>D and H</b>) was evaluated. <b>A-D</b>: PMN were immediately fixed after attachment. <b>E-H</b>: PMN were stimulated with 20 nM PMA for 3–4 h.</p

Plasma mtDNA levels are higher in the elderly.

<p><b>A</b>. MtDNA concentration was assessed in plasma obtained from subjects from four groups: 1. Young healthy (n = 11, average age 23 years); 2. Elderly healthy (n = 7, average age 72 years); 3. Young trauma (n = 9, average age 37 years); and 4. Elderly trauma (n = 6, average age 83 years). All pairs showed significant difference except between old healthy vs. young trauma; p<0.001, One Way ANOVA, followed by Tukey’s Test. <b>B</b>. ISS is shown for young and older trauma patients listed in <b>A</b>. p = 0.164 (Student’s t-test).</p

Effect of NADPH oxidase in local and systemic accumulation of MDSCs in tumor-bearing mice.

<p>A) Representative quantification of MDSCs. Splenocytes from WT and p47<i>^phox−/−</i> mice at day 42 and 90 after MOSEC administration were analyzed for MDSC accumulation. Gating on myeloid (CD11b⁺) cells, the proportion of monocytic MDSCs (R1; Ly6C⁺Ly6G⁻) and granulocytic MDSCs (R2; Ly6G⁺Ly6C^Low) significantly increased at day 90 versus day 42. All gates were set based on isotypes. This approach was used to quantify MDSCs in PECs, lymph nodes, and spleens. B) Proportion of MDSCs in myeloid PECs on day 42 and 90. The proportion with granulocytic and monocytic MDSC markers was greater in advanced (day 90) versus early (day 42) stage tumor burden in both genotypes. C) In draining lymph nodes, there was a trend toward increased monocytic MDSC accumulation in p47<i>^phox−/−</i> versus WT mice at day 42 but not at day 90. There was no effect of NADPH oxidase on granulocytic MDSC accumulation at either time point. D) In spleens, there was an increased accumulation of MDSCs, particularly granulocytic MDSCs, in mice with advanced versus early disease, but no effect of mouse genotype. Data (± SEM) are from at least 3 mice per genotype per time point, and are representative of 3 separate experiments. Comparison between genotypes: p = NS.</p

Peritoneal and splenic granulocytic MDSCs from tumor-bearing mice suppress T cell proliferation independently of NADPH oxidase.

<p>Ly6G-enriched PECs and splenocytes from MOSEC-bearing WT and p47<i>^phox−/−</i> mice (day 90) were co-cultured with splenocytes from non-tumor-bearing WT mice (E∶T ratio: 1∶1). A) Ly6G-enriched PECs completely suppressed anti-CD3/B7.1-stimulated CD4⁺ and CD8⁺ T cell proliferation. PECs from 3 mice per genotype were evaluated. B) In Ly6G-enriched splenocytes, the majority of cells had a granulocytic morphology (arrows), and 86% of CD11b⁺ cells expressed granulocytic MDSC markers (Ly6G⁺Ly6C^low). C) Ly6G-enriched splenocytes from WT and p47<i>^phox−/−</i> mice modestly suppressed anti-CD3/B7.1-stimulated CD4⁺ and CD8⁺ T cell proliferation. N = 3 mice per genotype were used in this experiment, and results are representative of 3 experiments. Comparison between genotypes: p = NS.</p