Fig 1: Hif-1α-activated tnfa is cyclooxygenase dependent. (A) Confocal micrographs of 2 dpf caudal vein region of larvae in the TgBAC(tnfa:GFP)pd1028 line. Phenol red (PR) and dominant active Hif-1α (DA1) injected larvae were treated with DMSO, SC560 (Cox-1 inhibitor), and NS398 (Cox-2 inhibitor). Dotted lines indicate the yolk extension of the larvae where there is non-specific fluorescence. (B) Corrected fluorescence intensity levels of tnfa:GFP in larvae in (A). Mean ± SEM, n = 36 cells from six embryos representative of three independent experiments. P-values shown are: *P < 0.05, **P < 0.01, and ***P < 0.001, two way ANOVA with Bonferonni post-test adjustment. (C) Confocal micrographs of 2 dpf caudal vein region of larvae in the TgBAC(tnfa:GFP)pd1028 line. DMSO and FG4592 treated larvae were co-treated with DMSO, SC560 (Cox-1 inhibitor), and NS398 (Cox-2 inhibitor). Dotted lines indicate the yolk extension of the larvae where there is non-specific fluorescence. (D) Corrected fluorescence intensity levels of tnfa:GFP in larvae in (C). Mean ± SEM, n = 48 cells from eight embryos representative of three independent experiments. P-values shown are: *P < 0.05, **P < 0.01, and ***P < 0.001, two way ANOVA with Bonferonni post-test adjustment. (E) TNF ELISA of human monocyte derived macrophages treated with LPS and incubated in normoxia or 0.8% hypoxia with or without treatment with NS398. LPS negative controls were performed but TNF produced in these groups was below detectable levels. Mean ± SEM, n = 5–8 biological repeats from 3 to 4 donors. P-values shown are: *P < 0.05, **P < 0.01, and ***P < 0.001, two way ANOVA with Bonferonni post-test adjustment. (F) Schematic of the arachidonic pathway indicating that stabilizing Hif-1α upregulates tnfa, an effect that is blocked using the Cox1/2 inhibitors SC560/NS398.
Fig 2: Proposed mechanism on how RBLE could act as a hepatoprotective agent in liver injury model·H2O2 is transformed into hydroxyl radical (HO*) through Fenton reaction with the presence of transition metal such as iron and copper. Under normal condition, H2O2 is neutralized by Glutathione Peroxidase (GPX) enzymes activity through oxidation of GSH into GSSG. It seemed that excessive H2O2 lead to down regulation of GPX gene expression. Hydroxyl radical activates NFkB that lead to TNF-α production. TNF/TNFR engagement induces production of catalase (CAT) and superoxide dismutase (SOD) and thus increasing extracellular H2O2. Meanwhile, TNF-α also induced JNK signaling pathways, resulting in increased mitochondrial ROS and lead to activation of Caspase 9 and apoptosis executioner, Caspase3. On the other hand, high amount of HO* triggers mitochondrial permeability transition (MPT), causing mitochondria to swell, and suppress ATP production. This pathway lead to unregulated cell death, necrosis; increase inflammation; and promote cell death. Lack of ATP also affects apoptosis pathway and switch it into necrosis instead. If this condition left untreated, liver injury could take place. Treatment with RBLE could neutralized HO* in cells and thus lowering necrosis, shifted the cells death pathway to apoptosis which induce lower inflammation response instead. Subsequently, RBLE treatment could increase the survival of hepatic cells.
Fig 3: Certain strains of Lactobacillus inhibit the production of H. pylori-induced TNF and IL-6 in host macrophages. Human macrophages were infected with either H. pylori alone or in combination with lactobacilli. (A) TNF and (B) IL-6 were measured by ELISA in cell culture supernatants after 2, 4, 6, and 8 h of stimulation of THP-1-derived macrophages with Hp alone or in combination with Lactobacillus strains, L. gas, LGG, L. oris, or L. bre. H. pylori alone or in combination with L. gas or L. bre was incubated with MDMs for 8 h. (C) TNF and (D) IL-6 were determined by ELISA. The data presented are representative of results from at least three independent experiments with triplicate samples. Statistical analyses were performed using ANOVA (analysis of variance), followed by Bonferroni posttest. Error bars indicate standard deviation. *P < 0.05 compared with H. pylori alone. Unstim., unstimulated.
Fig 4: TLR7/8 agonist activates MyD88-dependent and PI3K–Akt–mTOR signaling pathways in Vδ2 T cells. (A) Two-dimensional UMAP panel showing the cluster analysis of Vδ2 T cells with relative expression of seven upregulated genes (red) and five downregulated genes (black) identified as unique to γδ T-cell activation and differentiation. scRNA-seq data from three healthy donors were analyzed. (B) Heat map showing marker gene expression hits in naive Vδ2 T cells and terminally differentiated effector memory Vδ2 T cells. (C) UMAP sets showing clusters of selected naive Vδ2 T cells and terminally differentiated effector memory Vδ2 T cells for further analysis. (D) Functional categories of significantly different hits between selected naive Vδ2 T cells and terminally differentiated effector memory Vδ2 T cells. (E) KEGG pathway analysis of phosphoprotein of significantly different hits in (D). (F) Heap map showing the ssGSEA of single cell selected. (G) UMAP showing ssGSEA score of PI3K–Akt signaling pathway-related genes. (H) Violin plots showing the ssGSEA score of PI3K–Akt signaling pathway related genes. (I) Heat map of cluster analysis of significantly different protein hits of RPPA assay analysis. (J) KEGG pathway analysis of significantly different protein hits of RPPA assay analysis. (K) Inhibition of Vδ2 T-cell expansion from PBMCs by mTOR inhibitors. PBMC was incubated with IPP or IPP plus resiquimod as well as mTOR inhibitors, Rapa or Torin. Representative flow cytometry results showing the concentrations of Vδ2 T cells. (L) Statistical analysis of K, data shown as mean±SEM. (M) Inhibition of Vδ2 T-cell expansion from PBMCs by a MyD88 inhibitor ST2825. PBMCs were incubated with IPP or IPP plus resiquimod as well as ST2825 with indicated concentrations. Representative flow cytometry results showing the concentration of Vδ2 T cells. (N) Statistical analysis of M; data are shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001. IFN, interferon; IPP, isopentenyl pyrophosphate; KEGG, Kyoto Encyclopedia of Genes and Genomes; NS, no significant difference; PBMC, peripheral blood mononuclear cell; Rapa, rapamycin; RPPA, reverse-phase protein array; scRNA-seq, single-cell RNA sequencing; ssGSEA, single-sample gene set enrichment analysis; TCR, T-cell receptor; TLR, toll-like receptor; TNF, tumour necrosis factor; Torin, torin1; UMAP, uniform manifold approximation and projection.
Fig 5: PPAR-α mediates FAO and LD accumulation in monocytes(A–D) Monocytes were isolated from young mice. (A) Western blot analysis of PPAR-α and HADHA protein levels in monocytes treated with the indicated concentrations of the PPAR-α inhibitor MK886. (B–D) Monocytes were treated with MK886 (0.5 nM). Continuous OCR values (B), ATP concentrations (C), and LD levels (D) were detected.(E–H) MSC2 cells were transfected with Crispr-cas9 lentiviral-sg Ppara knockdown vectors. The transfection efficiency was assessed by western blot analysis (E). The continuous OCR (F), ATP levels, (G) and LD levels (H) in MSC2 and Ppara knockdown cells were examined.(I–L) Western blot analysis of PPAR-α and HADHA expression (I), OCR values (J), ATP levels (K), and LD levels (L) in monocytes isolated from old mice treated with the indicated concentrations of fenofibrate.(M–P) MSC2-C4 (Ppara−/−) cells were infected with lentiviral PPARA overexpression vectors. The transfection efficiency was examined by western blot analysis (M), and the OCR values (N), ATP levels, (O), and LD concentrations (P) were determined in MSC2-C4 and MSC2-C4-OE3 (PPAR-α overexpression) cells.(Q–S) Blood monocytes were isolated from WT or Ppara−/- mice. PPAR-α protein levels were determined by western blot (Q), and LD levels (S) and Tnfa and Il6 mRNA expression was detected in monocytes isolated from 4- and 20-week-old mice. (n = 4 per group).All experiments were replicated at least three times. The data represent the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; t test.
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