Journal: BMC Biology

Article Title: FGF-independent MEK1/2 signalling in the developing foetal testis is essential for male germline differentiation in mice

doi: 10.1186/s12915-023-01777-x

Figure Lengend Snippet: Summary of treatments and doses used in gonad cultures

Article Snippet: PH-797804 (p38i) , 500 nM , IC50 – p38α: 26 nM; p38β 102 nM , Phase II, NCT00559910 , MedChem Express, HY-10403 , [ ] .

Techniques: Control, Recombinant

Journal: Antiviral Research

Article Title: Inhibition of p38 signaling curtails the SARS-CoV-2 induced inflammatory response but retains the IFN-dependent antiviral defense of the lung epithelial barrier

doi: 10.1016/j.antiviral.2022.105475

Figure Lengend Snippet: SARS-CoV-2 infection activates p38 MAPK signaling and induces a pro-inflammatory cytokine response. A) Comparisons of a priori selected blood cytokine levels in COVID-19 patients with moderate/severe (IMC, n = 7) or critical (ICU, n = 16) disease and healthy controls (n = 11). Levels of IL6, CXCL8, CXCL10 and TNF were measured by multiplex proximity extension assay. Statistical significance was determined using non-parametric Mann-Whitney-U-test. **p < 0.01, ***p < 0.001, ****p < 0.0001. AU: arbitrary units, IMC: intermediate care wards, ICU: intensive care units. B) Replication kinetic in Calu-3 cells infected with SARS-CoV-2 strain hCoV-19/Germany/FI1103201/2020 at 0.01 MOI. Virus titers are expressed as plaque forming units (PFU/ml), n = 3. C) Phosphorylation of p38, MSK1 and NF-κB p65 during SARS-CoV-2 infection. Calu-3 cells were infected with SARS-CoV-2 at MOI 0.01 for 48 h or the highly pathogenic influenza A virus KAN-1 with MOI 0.001 for 48 h. Viral infections were verified by immuno-detection of the SARS-CoV-2 Nucleoprotein (N) and the IAV polymerase basic 1 (PB1) protein. Signals for total and phosphorylated p38, MSK1 and NF-κB p65 were detected by Western blot using (phospho-) specific antibodies. Quantification of p38, MSK1 and NF-κB p65 phosphorylation is provided below the blot. D) Phosphorylation and signal transduction of p38 at early time points during SARS-CoV-2 infection. Calu-3 cells were infected with SARS-CoV-2 at MOI 2 for 2 h. Signals for total and phosphorylated p38 and MSK1 were detected by Western blot using (phospho-) specific antibodies. Quantification of p38 and MSK1 phosphorylation is shown below the blot. E) Phosphorylation of p38 and MSK1 following infection with Delta S pseudotyped VSV. Vero E6 cells were infected with VSV WT or VSV expressing the spike protein of the Delta variant with MOI 1.5 for 60 min or 75 min, respectively. As a positive control, cells were exposed to 1 kJ/m 2 ultraviolet light 30 min prior to lysis. Tubulin was used as a loading control. Quantification of p38, MSK1 and TRIM28 phosphorylation is shown below the blot. F) and G) Cytokine response over time during SARS-CoV-2 infection. Calu-3 cells were infected with SARS-CoV-2 with MOI 0.01 for the indicated time points and mRNA expression of pro-inflammatory cytokines, IFNB1 and ISGs OAS1 and MX1 was determined by qRT-PCR. Statistical significance was determined using one-way ANOVA followed by Dunnett's multiple comparison test. Data are presented as n-fold gene induction over non-infected cells; bars indicate means ± SEM; n = 4.

Article Snippet: In this study, we explore the antiviral and anti-inflammatory properties of the two clinically pre-evaluated and highly selective inhibitors of the p38 MAPK α/β isoforms, PH-797804 (PH, Pfizer) ( ) and VX-702 (VX, Vertex) , in lung epithelial cell lines, three-dimensional primary human lung explants and human lung epithelial organoids to provide a pre-clinical evaluation of their therapeutic potential to dampen cytokine responses in COVID-19.

Techniques: Infection, Multiplex Assay, MANN-WHITNEY, Virus, Phospho-proteomics, Western Blot, Transduction, Expressing, Variant Assay, Positive Control, Lysis, Control, Quantitative RT-PCR, Comparison

Journal: Antiviral Research

Article Title: Inhibition of p38 signaling curtails the SARS-CoV-2 induced inflammatory response but retains the IFN-dependent antiviral defense of the lung epithelial barrier

doi: 10.1016/j.antiviral.2022.105475

Figure Lengend Snippet: p38 MAPK inhibition reduces the expression of pro-inflammatory cytokines. Calu-3 cells were pre-treated for 1 h with DMSO (control) or the inhibitors PH-797804 and VX-702 before infection with SARS-CoV-2 at MOI 0.01 for 48 h. A) Western blot analysis to determine inhibition of p38 MAPK by the inhibitors PH-797804 and VX-702. Phosphorylation of p38 (p-p38) and the downstream kinase MSK1 (p-MSK1) was determined using phospho-specific antibodies. Quantification of p38 (p-p38/p38) and MSK1 (p-MSK1/MSK1) phosphorylation levels are depicted below the blots. B) Effect of PH-797804 and VX-702 treatment on virus replication. Calu-3 cells were treated as described above. Viral titers are displayed as PFU/ml ± SEM from 3 independent experiments and effect of C) PH-797804 and D) VX-702 treatment on cytokine expression. Calu-3 cells were treated as described above and 48 h p.i. total RNA was isolated and mRNA levels of the indicated cytokines and ISGs were determined using qRT-PCR with gene specific primers. Data are displayed as n-fold induction over non-treated and non-infected cells. Bars represent mean values ± SEM from at least 3 independent replicates. Statistical significance was determined using two-way ANOVA followed by Dunnett's multiple comparison test. E) Chip-based kinase activity profiling of SARS-CoV-2-infected and PH-797804 or VX-702 inhibitor-treated cells. Calu-3 cells were infected with SARS-CoV-2 at 0.1 MOI for 24 h in the presence of DMSO, PH-797804 or VX-702 (5 μM). Serine-threonine kinase activity was evaluated using PamGene technology and differences in kinase activity in infected inhibitor-treated compared to infected and DMSO-treated cells are depicted as median kinase statistic (negative values = lower activity, positive values = higher activity), n = 3.

Article Snippet: In this study, we explore the antiviral and anti-inflammatory properties of the two clinically pre-evaluated and highly selective inhibitors of the p38 MAPK α/β isoforms, PH-797804 (PH, Pfizer) ( ) and VX-702 (VX, Vertex) , in lung epithelial cell lines, three-dimensional primary human lung explants and human lung epithelial organoids to provide a pre-clinical evaluation of their therapeutic potential to dampen cytokine responses in COVID-19.

Techniques: Inhibition, Expressing, Control, Infection, Western Blot, Phospho-proteomics, Virus, Isolation, Quantitative RT-PCR, Comparison, Activity Assay

Journal: Antiviral Research

Article Title: Inhibition of p38 signaling curtails the SARS-CoV-2 induced inflammatory response but retains the IFN-dependent antiviral defense of the lung epithelial barrier

doi: 10.1016/j.antiviral.2022.105475

Figure Lengend Snippet: p38 MAPK inhibitors PH-797804 and VX-702 reduce the inflammatory response to SARS-CoV-2. Calu-3 cells were treated for 1 h with 5 μM of PH-797804, 5 μM of VX-702 or DMSO (control) before infection with SARS-CoV-2 at MOI 0.001 for 48 h in the presence of the inhibitors. Non-infected, DMSO-treated cells were used as an additional control (non-infected control, mock). Gene expression was analyzed using the multiplexed RNA hybridization NanoString Human Host Response Panel and compared between non-treated and infected cells as well as infected and inhibitor-treated cells to determine differentially expressed genes (DEG). Statistical significance was determined using multiple testing and Benjamini and Hochberg correction. A) Principal component analysis (PCA) of relative gene expression levels from all samples. B) Heatmap representation of gene expression levels in mock (n = 6), solvent control (n = 6) and SARS-CoV-2 infected samples treated with PH-797804 (n = 3) and VX-702 (n = 4). C) Volcano plot of up- and down-regulated host response genes during SARS-CoV-2 infection; adj. p-value <0.01, <1.5-fold change (Log 2 = 0.5849625). D) Effect of inhibitor treatment on host response genes compared to infected cells using linear regression from the package LIMMA; adj. p-value <0.01, <1.5-fold change (Log 2 = 0.5849625). Comparison of mRNA expression in infected and inhibitor-treated infected cells of E) COVID-19-relevant pro-inflammatory cytokines, F) Type I, II and III interferons, G) IFN response genes normalized to the non-infected control. Data are expressed as means of Log 2 -fold changes; adj. p-value <0.01, <1.5-fold change (Log 2 = 0.5849625). *p < 0.05; **p < 0.01, ***p < 0.001.

Article Snippet: In this study, we explore the antiviral and anti-inflammatory properties of the two clinically pre-evaluated and highly selective inhibitors of the p38 MAPK α/β isoforms, PH-797804 (PH, Pfizer) ( ) and VX-702 (VX, Vertex) , in lung epithelial cell lines, three-dimensional primary human lung explants and human lung epithelial organoids to provide a pre-clinical evaluation of their therapeutic potential to dampen cytokine responses in COVID-19.

Techniques: Control, Infection, Gene Expression, Hybridization, Solvent, Comparison, Expressing

Journal: Antiviral Research

Article Title: Inhibition of p38 signaling curtails the SARS-CoV-2 induced inflammatory response but retains the IFN-dependent antiviral defense of the lung epithelial barrier

doi: 10.1016/j.antiviral.2022.105475

Figure Lengend Snippet: p38 inhibitors reduce IL6 and CXCL8 expression in ex vivo infected human lung explants A) Human lung explants were infected with 10 6 PFU/well SARS-CoV-2 for 72 h (n = 5). Infectious virus in the supernatants was determined by plaque assay. Bars indicate means ± SEM. B) Visualization of SARS-CoV-2 N protein, ACE2 and TMPRSS2 in human lung explants 48 h after ex vivo infection with 10 6 PFU/well by immunohistochemistry. C) Gene expression in human lung explants infected for 48 h with 1 × 10 6 PFU/well SARS-CoV-2 in the presence or absence of the respective p38 inhibitors VX-702 and PH-797804. Expression of pro-inflammatory cytokines and ISGs was determined by qRT-PCR. Data represent means ± SEM from at least 5 patients. Statistical significance was determined with one-way-ANOVA followed by Dunnett's multiple comparison test. *p < 0.05; **p < 0.01.

Article Snippet: In this study, we explore the antiviral and anti-inflammatory properties of the two clinically pre-evaluated and highly selective inhibitors of the p38 MAPK α/β isoforms, PH-797804 (PH, Pfizer) ( ) and VX-702 (VX, Vertex) , in lung epithelial cell lines, three-dimensional primary human lung explants and human lung epithelial organoids to provide a pre-clinical evaluation of their therapeutic potential to dampen cytokine responses in COVID-19.

Techniques: Expressing, Ex Vivo, Infection, Virus, Plaque Assay, Immunohistochemistry, Gene Expression, Quantitative RT-PCR, Comparison

Journal: Antiviral Research

Article Title: Inhibition of p38 signaling curtails the SARS-CoV-2 induced inflammatory response but retains the IFN-dependent antiviral defense of the lung epithelial barrier

doi: 10.1016/j.antiviral.2022.105475

Figure Lengend Snippet: Treatment with p38 inhibitors PH and VX maintains the antiviral type I IFN response in SARS-CoV-2 infected human lung epithelial (AT-II) organoids. Human lung stem cell organoids from three patients were treated with DMSO (control) or the inhibitors PH and VX (5 μM) after infection with SARS-CoV-2 (MOI 1). A) SARS-CoV-2-induced immune response in non-treated cells (non-infected/infected) and inhibitor-treated infected cells (non-infected/PH-797804, non-infected/VX-702) from human lung stem cell organoids. Volcano plots displaying gene expression from RNAseq analysis; adj. p-value <0.05, <1.5-fold change (Log 2 = 0.5849625). B) Replication of SARS-CoV-2 in the human lung epithelial organoids treated or non-treated with PH and VX (5 μM). C) Top 10 biological processes induced by SARS-CoV-2 infection with or without p38 inhibition. Analysis was performed using GO term enrichment for all up-regulated DEGs comparing infected control and infected treated cells with PH or VX (5 μM). D) Gene expression levels of COVID-19 relevant antiviral restriction factors. Data are displayed as n-fold induction over non-treated and non-infected organoids. Bars represent mean values ± SEM from three donors. Statistical significance was determined using one-way ANOVA followed by Dunnett's multiple comparison test. *p < 0.05; **p < 0.01, ***p < 0.001.

Article Snippet: In this study, we explore the antiviral and anti-inflammatory properties of the two clinically pre-evaluated and highly selective inhibitors of the p38 MAPK α/β isoforms, PH-797804 (PH, Pfizer) ( ) and VX-702 (VX, Vertex) , in lung epithelial cell lines, three-dimensional primary human lung explants and human lung epithelial organoids to provide a pre-clinical evaluation of their therapeutic potential to dampen cytokine responses in COVID-19.

Techniques: Infection, Control, Gene Expression, Inhibition, Comparison

Journal: Frontiers in Neurology

Article Title: Aging Increases Hippocampal DUSP2 by a Membrane Cholesterol Loss-Mediated RTK/p38MAPK Activation Mechanism

doi: 10.3389/fneur.2019.00675

Figure Lengend Snippet: DUSP2 protein levels are upregulated in the hippocampus of old mice. (A) DUSP2 levels increase in a cholesterol loss and p38MAPK activity dependent manner. Hippocampal neurons in culture were or were not treated with the p38MAPK inhibitor PH797804 (2 μM, also referred as PH) prior to cholesterol oxidase (Choox). (B) DUSP2 protein levels increase with aging in the mouse hippocampus. (C) Representative pictures show the increased expression of DUSP2 in the cortex and in the hippocampus with age. Top pictures (i-iii) show expression of DUSP2 in adult mice (7 months); lower pictures (iv-vi) show DUSP2 expression in old mice (22 months). DUSP2 is expressed in CA1 layer (iii; vi). Panels (iii) and (vi) show higher magnifications of the regions boxed in (ii) and (v) respectively. Vcx, Visual cortex; CA1, Cornu Ammonis of the hippocampus layer 1; CA3, Cornu Ammonis layer 3. Scale Bar in i, ii, iv, v, 500 μm; Scale bar in iii, vi, 20 μm. Colored bars on the right show the Look-Up-Table used to color-code the intensity of DUSP2 labeling. (D) Pictures show a magnification of CA3 layer. Scale bar represents 20 μm. Bar graphs: Values inside indicate the number of independent experiments. Data are represented as mean ± SEM. The asterisks indicate the p- values (ns, not significant; * p < 0.05).

Article Snippet: The following compounds were added to cell medium of hippocampal neurons: Cholesterol oxidase (Choox; Calbiochem ref.: 228250; 10 IU/ml); K252a (Tocris ref.: 1683; 1 μM); SB203580 (Shelleckchem ref.: S1076; 20 μM); PH797804 (Axon Medchem ref.: 1837; 2 μM); H89 (Tocris ref.: 2910; 50 μM); Chelerythrine (Tocris ref.: 1330; 10 μM); XL-184 (Tocris ref.: 5422; 1 μM); BAPTA-AM (Invitrogen ref.: B-6769, 10 μM).

Techniques: Activity Assay, Expressing, Labeling