EMBO/soda-nel huggingface dataset:
Zinc inhibits lethal shock by reducing microbe-induced interferon signature in intestinal epithelium (2020)
Figure 3

Figure 3: Transcriptional downregulation of ISRE/IRF genes in the intestinal epithelium by zinc part 1.

RNA-seq experiment of GRWT and GRDim mice which received normal drinking water or drinking water containing 25mM ZnSO4 for 1 week. Ileum was sampled and RNA-seq performed (N=5-6 per group). Genes downregulated by zinc compared to water control animals (LFC<0.8 and p<0.05) were considered.

(A) HOMER transcription factor motif analysis of genes downregulated by zinc in GRWT (left panel) and in GRDim mice (right panel).

(B) Overlap between the collection of genes, in blue, upregulated in GRDim mice (water) compared to GRWT mice (water), in pink, genes downregulated by zinc in GRDim mice and, in green, the genes downregulated by zinc in GRWT mice. The core of these three collections contains 48 genes which are listed.

(C) Heat map of a selection of the 48 genes of Fig 3B.
Zinc inhibits lethal shock by reducing microbe-induced interferon signature in intestinal epithelium (2020)
Figure 4

Figure 4: Transcriptional downregulation of ISRE/IRF genes in the intestinal epithelium by zinc part 2.

(A-B) Expression levels (RT-qPCR data) of Mlkl and Zbp1, from the collection of 48, in ileum biopsy samples, showing the reducing effects of zinc in both GRWT and GRDim mice, and the higher expression in GRDim compared to GRWT (N=4-6/group).

(C-E) RT-qPCR data of Mlkl, Zbp1 and Ripk3, in ileum biopsy samples of C57BL/6J mice or different TLR full knockout mice. (N=4/23 per group)

(F-G) RT-qPCR data of Mlkl and Zbp1, in ileum biopsy samples of C57BL/6J mice treated with normal drinking water or 25 mM ZnSO4 in the drinking water for 1 week, either or not pretreated with antibiotics (AB) for three weeks. (N=5 per group)

Data information: Data is shown as mean +/- SD. In A,B, F,G p-values were analyzed with a two-way ANOVA test and in C-E with a one-way ANOVA test.
Zinc inhibits lethal shock by reducing microbe-induced interferon signature in intestinal epithelium (2020)
Figure 5

Figure 5: Zinc protects Paneth cells from TNF-induced cell death and prevents TNF-induced bacterial invasion of spleens and mesenteric lymph nodes.

(A-B) C57BL/6J mice received control or 25 mM ZnSO4 drinking water for 7 days and were injected i.p. with 50 µg TNF, solved in 200 µL sterile PBS, per 20 g bodyweight or with PBS only. 6h later, sections through ileum were made and stained with (A) Hematoxylin & Eosin and MMP7, a Paneth cell marker. Pictures at magnification of 20x were taken and one representative picture of each condition is shown. Arrows point to crypts and their (ab)normal occurrence (see text). Scale bars: 50 μm

(C) Transmission electron microscopy (TEM) images of ileum crypts show protective effects of zinc therapy on the level of TNF induced effects on Paneth cells. C57BL/6J mice (N=2 /group) were put on normal drinking water or on 25 mM ZnSO4 water for 7 days. Mice were given 50 µG TNF or PBS and 8h later, ileum samples were taken and prepared for TEM. Figure shows ileal crypts at a magnification of 1000x. In each of the 4 conditions, 2 Paneth cells are selected in red area (see text).

(D-F) RT-qPCR measurement of mRNA levels of Defa6 and Lyz2, two Paneth cell markers, and Ripk3, a TNF-induced cell-death gene, in ileum of the experiment in A.

Data information: Data is shown as mean +/- SD. D (N=10/15 per group), E (N=10 per group), F (N=5/6 per group). P-values were analyzed with a Two-way ANOVA test.
Zinc inhibits lethal shock by reducing microbe-induced interferon signature in intestinal epithelium (2020)
Figure 6

Figure 6: Zinc modulates bacterial colonization and composition as a mechanism to protect against TNF.

(A-B) C57BL/6J mice received control or 25 mM ZnSO4 drinking water for 7 days and were injected i.p. with 50 µg TNF, solved in 200 µL sterile PBS, per 20 g bodyweight or with PBS only (N=5). 18h after TNF challenge, spleen and MLNs were isolated and lysates prepared and plated on TSA plates. Colonies were counted and expressed as bacterial colonies per ml of lysate. 40 colonies per plate were prepared for Maldi Tof identification and the relative amounts of bacterial species found on the plates are mentioned underneath the figure. Data are shown as means +/- SD. p-value was analyzed by means of a Student's t-test (unpaired, two-tailed).

(C-D) C57BL/6J mice received control or 25 mM ZnSO4 drinking water for 7 days. Then semi-solid ileum slurry was isolated, weighed, mixed, diluted and normalized and was plated on TSA plates. After randomly picking 110 and 102 colonies from plates of H20 treated mice and ZnSO4 treated mice resp., their identity was determined by MALDI-TOF and expressed in a pie diagram. Chi2-tests were performed.

(E) Impact of zinc concentration in bacterial TSA plates on the growth of colonies of 4 bacterial species commonly found in spleen and MLNs of mice injected with TNF (see A-B). Pure colonies of Staphylococcus nepalensis (orange), Staphylococcus sciuri (red), Eschericchia coli (purple) and Enterococcus faecalis (blue) were grown in LB medium, then diluted and plated on TSA plates containing increasing concentrations of ZnCL2. 24h later, the amounts of colonies were counted, and the amounts found in the 0 mM ZnCl2 condition expressed as 100%. Data are shown as mean +/- SD pooled from three independent experiments.

(F-I) Monocolonization experiment. Mice (N=5 all groups) were treated with H20 or ZnSO4 for 7 days, after which the ZnSO4 was removed and replaced by H20. 12h and 24h later, the mice were transplanted with PBS, 108 Staphylococcus sciuri or 108 S. nepalensis (in 100 ul), and 3h later a lethal dose of TNF (35 µg/mouse) was injected, and 24h later body temperature (insert in F) and lethality (F) recorded and estimated by one-way ANOVA and Chi2-tests resp. Survival significance was tested towards the H20 control group. In a second experiment (G-I) (N=4 all groups) with S. sciuri colonization, Zbp1 and Stat1, two key ISRE genes, and Mt2, a Zn-induced gene, were measured in ileum biopsies by RT-qPCR.

Data information: Data is shown as mean+/- SD. In A,B and F p-values were analyzed with one-way ANOVA test and in G-I by means of a Student's t-test (unpaired, one-tailed). For the survival curve in F, p-value was analyzed with a Chi2-test.
Zinc inhibits lethal shock by reducing microbe-induced interferon signature in intestinal epithelium (2020)
Figure 7

Figure 7: Additive protection of zinc and dexamethasone against TNF-induced lethal inflammation.

(A) C57BL/6J mice received 25 mM ZnSO4 or control water for 7 days. Mice were injected i.p. with 100, 200 or 500 µg dexamethasone (Dex, rapidexon), solved in sterile PBS, 30 min before a 50 µg TNF/20 g bodyweight i.p. challenge. STAT1+/+ and STAT1-/- mice were challenged with equal TNF dose, as controls. No mice died later than 120 h after TNF injection. Data show combined results of 2 experiments.

(B-D) Mice (N=4 per group) treated as in (A) and 6h after challenge, ileum was taken and RT-qPCR performed to study gene expression of cell death ISRE genes Mlkl and Zbp1 as well as Stat1.

(E) Hypothesis of the mechanism of interference of zinc in TNF-induced cell death in ileum (Paneth) cells. See text for more details.

Data information: For the survival curve in A, p-value was analyzed with a Chi2-test and compared to the PBS group. In B-D Pairwise analysis between groups with or without zinc were performed using Student's t-test (unpaired, one-tailed).

The paper explained

PROBLEM

Zinc has therapeutic effects, mainly in intestinal infections and diarrhea. The mechanism of this zinc effect is poorly known. Zinc also protects against intestinal effects induced by the cytokine tumor necrosis factor (TNF), which plays a key role in Crohn's disease. Since zinc was previously shown to protect in the TNF model in mice, we applied this model to investigate zinc's mode of action.

RESULTS

By applying RNA-seq in ileum biopsies in mice treated with water or ZnSO4 in the drinking water, we found that zinc causes strong downregulation of a large group of genes, known as ISRE/IRF genes. These are thought to (confirmed here) to be induced in intestinal epithelium by gut microbes. Zinc lowers these genes, by actively and directly modulating the composition of the gut microbes, and our data suggest that mainly Staphylococci would be targeted by zinc. The reduction in ISRE/IRF genes is important, because several of these genes play an important role in killing certain epithelial cells (known as Paneth cells), when TNF is present. Because of the effects of zinc on bacteria and ISRE/IRF genes in these cells, they resist killing by TNF. How this leads to survival of the animals is not yet clear, but since zinc also reduces the escape of bacteria from the gut to other organs, the impact of zinc on the gut microbes or on the Paneth cell's death appears responsible as well. Mice with extremely high ISRE/IRF gene expression (e.g. because they have no adrenals) or extremely low ISRE/IRF genes, because they have no gut microbes, are not susceptible to the beneficial effects of zinc.

IMPACT

Now that the mechanism of zinc is unfolded, more precise zinc targeting or zinc replacing strategies in intestinal diseases where TNF is implicated (Crohn's disease) can be considered.
AIFM1 is a component of the mitochondrial disulfide relay that
drives complex I assembly through efficient import of NDUFS5 (2022)
Figure 3

Figure 3. NDUFS5 import and oxidation are impaired in AIFM1 knockout.

A. In organello import assay with NDUFS5, NDUFB7 and as control the matrix protein SOD2. In vitro translated radioactive proteins were incubated with mitochondria isolated from HEK293 and AIFM1 knockout cells. Non-imported proteins were removed by treatment with Proteinase K. Imported proteins were analyzed by reducing SDS-PAGE and autoradiography. Signals were quantified using ImageQuantTL and the amount of imported protein was plotted. NDUFS5 import is strongly affected by loss of AIFM1. Import of NDUFB7 is less affected and SOD2 import remained unaffected. This is despite the fact that the core domains important for oxidative folding of NDUFS5 an NDUFB7 are very similar. N = 3 replicates; error bars indicate SD.

B. Oxidation kinetics of endogenous NDUFB7 and NDUFS5. HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1-HA were analyzed by pulse-chase experiments coupled to redox state determination. To this end, cells were incubated with 35S-Met for 5 min (pulse). After removal of 35S-Met, cells were left in "cold" Met for different chase times. Further oxidation was stopped by addition of trichloroacetic acid (TCA). Free thiols but not thiols in disulfide bonds were modified using mmPEG24 and then MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP), and analyzed by non-reducing SDS-PAGE and autoradiography. Occurrence of oxidized NDUFB7 was slightly delayed in AIFM1 knockout cells. NDUFS5 levels dropped rapidly and only little oxidized NDUFS5 could be observed. N = 4 biological replicates for NDUFB7 and NDUFS5. Error bars indicate SD.

C. Synthesis of NDUFS5 and NDUFB7 in wild type and AIFM1 knockout cells. Cells were incubated with 35S-Met for 5 min (pulse), and then MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP), and analyzed by non-reducing SDS-PAGE and autoradiography. NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells.

D. Interaction between endogenous MIA40/CHCHD4 and its substrates in wild type and AIFM1 knockout cells. Cells were subjected to native immunoprecipitation (IP) against endogenous MIA40/CHCHD4. Precipitates were analyzed by SDS-PAGE and immunoblotting against the indicated proteins. In AIFM1 knockout cell interaction between MIA40/CHCHD4 and its substrates is strongly affected with the interaction between MIA40/CHCHD4 and NDUFS5 almost completely abolished. This is despite the fact that NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells, indicating that lowered steady state levels of these proteins are consequence of a lacking interaction with MIA40/CHCHD4 rather than the cause. Quantification using Image lab. Data from 2 experiments were combined and standard deviations are presented. Asterisk indicates background band; orange arrow indicates signal of NDUFS5 and NDUFB7 in AIFM1 KO cells, respectively.
AIFM1 is a component of the mitochondrial disulfide relay that
drives complex I assembly through efficient import of NDUFS5 (2022)
Figure 4

Figure 4. MIA40/CHCHD4 is present in a stable long-lived complex with AIFM1.

A. Assessment of MIA40/CHCHD4 variant-AIFM1 interaction. The indicated MIA40/CHCHD4-Strep variants were affinity precipitated (AP) under native conditions after stopping thiol-disulfide exchange reactions by NEM incubation. Precipitates were tested for AIFM1, ALR and MIA40/CHCHD4 by reducing SDS-PAGE and immunoblotting. 1% of the total lysate was loaded as input control. M, mock control not expressing MIA40-Strep. Both, wildtype MIA40/CHCHD4 and MTS-MIA40/CHCHD4 but not MIA40/CHCHD4∆1-40 variants coprecipitated AIFM1. All mitochondria-localized variants precipitate ALR indicating redox functionality. Arrowhead indicates endogenous MIA40/CHCHD4.

B,C. Emetine-chase experiments to assess the stability of the AIFM1/CHCHD4 complex. HEK293 cells stably expressing MIA40/CHCHD4-Strep (B) or AIFM1 knockout cells stably expressing AIFM1-HA (C) were treated for the indicated times with the ribosome inhibitor emetine. Then, cells were lysed under native conditions (triton X-100), and MIA40/CHCHD4-Strep (B) or AIFM1-HA (C) were precipitated. Precipitates were analysed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 and AIFM1 interact over a period of at least 6-8 hours (B,C), while the two MIA40/CHCHD4 substrates, NDUFB7 and AK2, are quickly released from MIA40/CHCHD4 (B, gray box). AP, affinity precipitation; asterisk, background band. N = 3 biological replicates.

D,E. Gel filtration analysis to assess the in vitro reconstituted AIFM1-MIA40/CHCHD4 complex. Purified MIA40/CHCHD4 and AIFM1 were incubated in the presence of NADH and subjected to gel filtration analysis. Elution was monitored by following the absorption at 280 nm (D). Eluted fractions were subjected to SDS-PAGE and TCE staining (E). AIFM1 and full-length MIA40/CHCHD4 migrate together indicating formation of a complex. MIA40/CHCHD4 was present in excess over AIFM1 resulting in the presence of free MIA40/CHCHD4. AIFM1 and ∆1-40 MIA40/CHCHD4 do not migrate together emphasizing the importance of the N-terminal amino acids in MIA40 for interaction wth AIFM1.

F. Isothermal Titration Calorimetry (ITC) analysis of AIFM1 and MIA40/CHCHD4. The partners bind to each other with a KD of approximately 0.18 µM and a stoichiometry between AIFM1 and MIA40/CHCHD4 of 2 to 1. Data shown was normalized by the background signal of titrating MIA40/CHCHD4 into buffer.
AIFM1 is a component of the mitochondrial disulfide relay that
drives complex I assembly through efficient import of NDUFS5 (2022)
Figure 6

Figure 6. In AIFM1 knockout cells, impaired OMM translocation of NDUFS5 results in its rapid proteasomal degradation.

A. Oxidation kinetics of NDUFB7 and NDUFS5 in the presence of the proteasome inhibitor MG132. HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1-HA were analysed by pulse-chase experiments coupled to redox state determination. Levels and occurrence of oxidized NDUFB7 was not changed in the presence of MG132. Conversely, NDUFS5 levels were strongly increased. In AIFM1 knockout cells, large amounts of NDUFS5 accumulated in their reduced state indicating impaired oxidation-dependent protein import. The fraction of oxidized protein accumulating is slightly larger than in the absence of MG132. N = 1 biological replicate for NDUFB7 and 2 biological replicates for NDUFS5.

B. Model. The AIFM1-MIA40/CHCHD4 complex mediates proper oxidation-dependent protein import and insertion of the four disulfide relay substrates into complex I. In the absence of AIFM1, monomeric soluble MIA40/CHCHD4 is still capable of importing many of its substrates. It fails however to accumulate NDUFS5. This protein is not properly oxidized and as a consequence, it becomes rapidly degraded by the proteasome. Lack of NDUFS5 results in stalling of complex I assembly and lowered levels of complex I.
Anti-BDCA2 monoclonal antibody inhibits plasmacytoid dendritic cell activation through Fc-dependent and Fc-independent mechanisms. (2015)
Figure 1

Anti-BDCA2 mAb inhibits IFNα production by pDCs and induces internalization of BDCA2 on the surface of pDCs

A, B Whole blood or PBMC were treated with increasing concentrations of 24F4A and stimulated with CpG-A for 16 h at 37°C. Anti-BDCA2-mediated IFNα inhibition in whole-blood assays (A). IFNα levels were detected using human IFNα ELISA. Graph depicts average of duplicate wells of one representative donor (n = 10). IC50 of 24F4A-mediated IFNα inhibition (B) in whole blood (circles) (n = 10) and PBMC (n = 18) from healthy human donors (squares) or SLE patients (triangles) (n = 11). Horizontal bar represents the mean IC50 for each sample type. Error bars represent SD of IC50 between donors.

C Anti-BDCA2-mediated internalization in whole-blood assays. Whole blood was treated with increasing concentrations of 24F4A for 16 h. Mean fluorescence intensity (MFI) of BDCA2 was determined with a non-cross-blocking anti-BDCA2 mAb (2D6). Shown is a representative plot of 10 experiments conducted.

D The IC50 of 24F4A-mediated IFNα inhibition was compared to the EC50 of 24F4A-induced BDCA2 internalization.
Anti-BDCA2 monoclonal antibody inhibits plasmacytoid dendritic cell activation through Fc-dependent and Fc-independent mechanisms. (2015)
Figure 2

24F4A induces rapid internalization of BDCA2 resulting in trafficking to LAMP1<sup>+</sup> compartments and sustained IFNα inhibition

A Whole blood was treated with the indicated concentrations of 24F4A or the IgG1 isotype control antibody for 0, 1, 3, 6, 9, and 24 h. FACS analysis was performed to determine BDCA2 levels on pDCs. Fluorescence minus one (FMO) represents background staining of BDCA2. Shown is a representative plot of three independent experiments.

B Isolated pDCs were treated with 10 μg/ml 24F4A-AF647 at 4°C or at 37°C and analyzed at the indicated time points. Subcellular localization of BDCA2 (red) compared to LAMP1 (green) was assessed using confocal microscopy. Phalloidin was used to delineate the cell membrane. Yellow represents co-localization of BDCA2 and LAMP1. Shown is a representative image of four experiments conducted.

C, D Whole blood was either treated with 24F4A for 1 h at 37°C (pre-incubation) or left untreated. PBMC were isolated from each condition. PBMC from the untreated whole blood were subsequently treated with 10 μg/ml 24F4A, and cells from all conditions were stimulated with CpG-A for 16 h. Plot represents mean of duplicate wells. Flow cytometry was used to measure BDCA2 levels (MFI). Shown is a representative donor of eight donors tested.

E Whole blood was pre-treated for 0, 1, 3, 6, and 9 h with 10 μg/ml of 24F4 or the isotype control and then stimulated with CpG-A for additional 16 h. IFNα levels were measured by ELISA. Shown is a representative plot of two independent experiments.

Source data are available online for this figure.
Anti-BDCA2 monoclonal antibody inhibits plasmacytoid dendritic cell activation through Fc-dependent and Fc-independent mechanisms. (2015)
Figure 4

The Fc region of 24F4A enhances the inhibition of immune complex-induced IFNα production by human pDCs

A–F Isolated human pDCs were treated with increasing concentrations of 24F4A (red circles), 24F4A-ef (black squares), or 10 μg/ml of the isotype control (black triangle) and stimulated with 5 μM of R848 (A and B), 1 μM of CpG-A (C and D), or immune complexes (E and F) for 16 h at 37°C. IFNα concentrations in cultured supernatants were determined using ELISA. Representative plots for each stimulation (A, C and E) and the average IC50 of 24F4A and 24F4A-ef for each stimulation condition (B, D and F) are shown (n = 3). Horizontal bars represent mean IC50, and error bars represent SD of the mean from three independent experiments.

G Isolated human pDCs were treated with 10 μg/ml of 24F4A (black line), 24F4A-ef (gray shaded histogram), or the isotype control (gray dotted line) for 10 min at 37°C. Flow cytometry was used to evaluate pSYK and pPLCγ2. Shown is a representative plot of two independent experiments.

H Isolated pDCs treated with 10 μg/ml of anti-CD32a (AT10) and stimulated with CpG-A, R848, or immune complexes. Error bars represent SD of percent inhibition of IFNα from three independent experiments.
Anti-BDCA2 monoclonal antibody inhibits plasmacytoid dendritic cell activation through Fc-dependent and Fc-independent mechanisms. (2015)
Figure 5

Treatment with 24F4A leads to a dose-dependent downmodulation of CD32a on pDCs

A–F Isolated pDCs were treated with increasing concentrations of 24F4A (red circles), 24F4A-ef (squares), Chi-6G6 (triangle), or isotype control (diamond) for 16 h at 37°C and stimulated with CpG-A. Flow cytometry was performed to evaluate the MFI of (A) BDCA2 and (C) CD32a on the surface of pDCs. Percent internalization of BDCA2 (B) and CD32a (D) on pDCs treated with 10 μg/ml of 24F4A, 24F4A-ef, Chi-6G6, or isotype control. Error bars represent SD of percent internalization of five experiments conducted. (E) Percent CD32a downmodulation on isolated pDCs treated with 10 μg/ml of an anti-CD40 mAb. Error bars represent SD of percent downmodulation of two independent experiments. FMO represents background staining of CD32a (inverted triangle). (F) Percent of maximal expression of BDCA2 or CD32a in the presence of increasing concentrations of 24F4A or Chi-6G6. Maximal expression was defined by the MFI of both BDCA2 and CD32a on untreated cells. Shown is a representative plot of five independent experiments conducted.
MET inhibition overcomes radiation resistance of glioblastoma stem-like cells (2016)
Figure 1

Figure 1. GSCs are radioresistant.

A, Cell viability of NS, GBM, and astrocyte cell lines measured 72 h after irradiation (fold vs. non-irradiated cells, ctrl). *: one-way ANOVA, P=0.0001. B, Cell viability of BT308NS and their pseudo-differentiated progeny (BT308 diff) measured 24 h after irradiation (fold vs. non-irradiated cells, ctrl). *: t-test, P=0.0006. C, Flow cytometric analysis of phosphorylated histone H2AX (H2AX) at the indicated time points after IR (5 Gy, fold vs. non-irradiated cells, ctrl). *: one-way ANOVA, P=0.03. D, Neutral comet assay performed 24 h after IR (5 Gy). Ctrl: non-irradiated cells. Comet tail’s length is proportional to DSB extent. E, Western blot showing phosphorylation of ATM (pATM) and Chk2 (pChk2), and accumulation of RAD51 after IR (5 Gy). Total ATM protein is also shown. Vinculin was used as loading control. F, qPCR of RAD51 expression measured in NS, BT308 diff, and astrocytes at the indicated time-points after IR (5 Gy, fold vs. non-irradiated cells, ctrl). Data are represented as mean ± SEM.