Journal: Investigative ophthalmology & visual science

Article Title: Nicotinamide Riboside Mitigates Retinal Degeneration by Suppressing Damaged DNA-Stimulated Microglial Activation and STING-Mediated Pyroptosis.

doi: 10.1167/iovs.66.4.14

Figure Lengend Snippet: FIGURE 4. NR mitigated damaged DNA-stimulated cGAS-STING pathway. (A) Representative microscopic images of dsDNA immunopositivity (in red) and DAPI-counterstained nuclei (in blue). Scale bar: 50 μm. The boxed areas within the images were magnified and displayed in the right panels (arrowheads). Scale bar: 10 μm. (B) Colocalization of Tom20 (green), dsDNA (red), and DAPI (blue) expressions in retinas of each group (arrows). Scale bar: 10 μm. (C) Heatmap of cGAS and STING from RNA sequencing of naive and LIRD retinas with or without NR treatment (n = 4/group). (D) Colocalization of cGAS (red) and Iba1-positive microglia (green) expression in the retinas of each group (arrows). Scale bar: 30 μm. (E) Quantification of cGAS-positive microglia of each group in the ONL at three days after light exposure (n = 4–6/group). (F) Colocalization of STING (red) and Iba1-positive microglia (green) expression in the retinas of each group (arrows). Scale bar: 30 μm. (G) Quantification of STING-positive microglia of each group in the ONL at three days after light exposure (n = 5–6/group). NR treatment significantly decreased the expression of cGAS and STING compared with the PBS-treated group, ****P < 0.0001.

Article Snippet: Iba-1 Rabbit 1:500 Abcam* ab178846 Iba-1 Goat 1:800 Abcam* ab5076 CD68 (FA-11) Rat 1:200 Thermo† 14-0681-82 dsDNA Mouse 1:200 Santa Cruz‡ sc-58749 Tom20 (D8T4N) Rabbit 1:200 Cell Signaling Technology§ 42406 Cgas Mouse 1:200 Santa Cruz‡ sc-515777 STing Rabbit 1:200 Proteintech║ 19851-1-AP NLRP3 Rabbit 1:200 Abmart¶ P60622R3 Casepase-1 Mouse 1:100 Santa Cruz‡ sc-56036 IL-1β Mouse 1:100 Cell Signaling Technology§ 12242 GSDMD Mouse 1:200 Santa Cruz† sc-393581 Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) Goat 1:400 Jackson# 111-545-003 Goat Anti-Rat IgG H&L (Alexa Fluor 594) Goat 1:300 Jackson# 112-505-175 Donkey Anti-Rabbit IgG H&L (Alexa Fluor 488) Donkey 1:400 Thermo† A21206 Donkey Anti-Mouse IgG H&L (Alexa Fluor 555) Donkey 1:400 Thermo† A31570 Donkey Anti-goat IgG H&L (Alexa Fluor 568) Donkey 1:400 Thermo† A11057 * Abcam, Cambridge, MA, USA.

Techniques: RNA Sequencing, Expressing

Journal: Journal for immunotherapy of cancer

Article Title: Adding liposomal doxorubicin enhances the abscopal effect induced by radiation/αPD1 therapy depending on tumor cell mitochondrial DNA and cGAS/STING.

doi: 10.1136/jitc-2022-006235

Figure Lengend Snippet: Figure 4 Doxil and RT induce IFN-I which is produced on cGAS/STING activation (A) Flow cytometric determination of γH2AX in MC38 cells treated with or without RT, Doxil or both. (B) ELISA results showing IFNβ1 protein secreted by B16-CD133 (left) and MC38 (right) tumor cells treated with free doxorubicin or Doxil. (C) ELISA results showing IFNβ1 protein secreted by B16- CD133/MC38 cells treated with Doxil or RT with or without inhibitors of cGAS, STING, or TBK1. P values were calculated between the non-inhibitor-treated group and the inhibitor-treated groups. (D) Expression of cGAS and STING in control Cgas+/+, Sting+/+ and in Cgas−/− or Sting−/− B16-CD133 and MC38 cells as determined by Western blot; β-ACTIN was used as a protein loading control (one of two independent Western blots with similar results is shown). (E) ELISA results showing IFNβ1 protein secreted by Doxil or RT-treated B16-CD133 and MC38 cells with KO of either cGAS or STING. P values were calculated between control (Cgas+/+, Sting+/+) and Cgas−/− or Sting−/− cells. (F) Growth of Sting−/− or Cgas −/− MC38 tumors for primary/ irradiated and secondary/non-irradiated tumor in untreated, RT + Doxil or triple-treated mice, n=5–9 mice per group. Data are presented as means with SEM. P values (ns, not significant; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) were determined by two-tailed t-test (B, F) one-way ANOVA with Dunnett test (C, E) and log-rank test (F right). In (F) the comparison time points for tumor volume measurement, that is, the final time point at which no mouse of the compared groups had yet reached the experimental end point, are indicated. ANOVA, analysis of variance; cGAS, cyclic GMP-AMP synthase; KO, knockout; RT, radiotherapy; STING, stimulator of interferon genes.

Article Snippet: Antibodies: Translocase of outer mitochondrial membrane 20, TOM20 (D8T4N; #42406; RRID: AB_2687663), STING (D2P2F; #13647), cGAS (D3O8O; #31659), β-ACTIN (13E5; #4970) were purchased from Cell Signaling.

Techniques: Produced, Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Control, Western Blot, Irradiation, Two Tailed Test, Comparison, Knock-Out

Journal: Journal for immunotherapy of cancer

Article Title: Adding liposomal doxorubicin enhances the abscopal effect induced by radiation/αPD1 therapy depending on tumor cell mitochondrial DNA and cGAS/STING.

doi: 10.1136/jitc-2022-006235

Figure Lengend Snippet: Figure 7 Graphical summary. Local tumor RT can induce and locally recruit tumor-specific CD8+ T cells by inducing ICD mediators such as IFN-I, the related CXCL10, and ATP, and by reducing the tumor mass. DCs take up tumor antigen in the irradiated tumor and transport it to the tumor-draining lymph nodes (TDLNs) where tumor-specific CD8+ T cells are primed or stimulated. The activated T cells upregulate PD-1 and the CXCL10-interacting chemokine receptor CXCR3. Combination with αPD-1 can enhance local and systemic tumor-specific T cell responses. However, RT + αPD-1 is often not sufficient to induce a strong abscopal effect. Additional systemic Doxil can strongly increase the RT/αPD-1-induced abscopal effect by several mechanisms. First, Doxil can induce the T cell-attracting chemokine CXCL10 directly in the tumor cells which is an immunomodulatory property. Moreover, in contrast to many other chemotherapeutics, doxorubicin/Doxil is immunogenic, that is, it can induce tumor-specific T cells. We show that Doxil enhances DC antigen cross presentation which is important for inducing CD8+ T cells. However, the antitumor T cell immunity in the non-irradiated tumor induced by Doxil/αPD1 was not as strong as that after hRT/Doxil/αPD-1 triple therapy which caused complete abscopal responses in a substantial proportion of mice. We furthermore show evidence that Doxil stimulates the mtDNA/cGAS/STING/IFN-I axis which is crucial for DC cross- presentation-mediated induction of tumor antigen-specific CD8+ T cells. Doxil-induced cytosolic mtDNA release was correlated with downregulation of expression of the nucleic acid-binding mitochondrial membrane protein TFAM. This figure was created with BioRender. cGAS, cyclic GMP-AMP synthase; DC, dendritic cell; mtDNA, mitochondrial DNA; RT, radiotherapy; STING, stimulator of interferon genes; TFAM, mitochondrial transcription factor A.

Article Snippet: Antibodies: Translocase of outer mitochondrial membrane 20, TOM20 (D8T4N; #42406; RRID: AB_2687663), STING (D2P2F; #13647), cGAS (D3O8O; #31659), β-ACTIN (13E5; #4970) were purchased from Cell Signaling.

Techniques: Irradiation, Expressing, Binding Assay, Membrane