Journal: Microorganisms

Article Title: Sulfate-Reducing Bacteria Induce Pro-Inflammatory TNF-α and iNOS via PI3K/Akt Pathway in a TLR 2-Dependent Manner

doi: 10.3390/microorganisms12091833

Figure Lengend Snippet: LY294002 inhibited DSV-induced activation of PI3K/Akt/TNF pathway. ( A ) RAW 264.7 cells were treated with LY at either 10 or 50 μM for 2 h before infection with DSV for 4 h. Cells were lysed and protein lysate prepared. Fifty μg of protein lysate was separated on SDS-PAGE and analyzed for p-Akt, total Akt, p-P70S6K, P70S6K, TNF-α, and iNOS, by Western blotting. Actin was used as a loading control. ( B – E ) Quantification of Western blots. ( F ) Post-infection, cell culture supernatant was collected and proteins were precipitated using ethanol precipitation. Proteins were resuspended in PBS and 50 μg was loaded on SDS-PAGE. Secreted TNF-α (s-TNF-α) was detected using anti-TNF-α antibody. Actin was used as a loading control. ( G ) Quantification of s-TNF-α. ( H ) Cells were pretreated with or without LY 50 μM followed by infection with DSV for 30 min and protein samples were analyzed for p-p65 NFκB and total NFκB. ( I ) Blots were quantified with ImageJ by analyzing the ratio of p-NFκB/NFκB. Values were normalized and compared to control. Data represent Mean ± SEM from at least three independent experiments. One-way ANOVA was used to determine the statistical significance, with a post-hoc Dunnett’s test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns (non-significant).

Article Snippet: For drug treatments, cells were incubated with LY294002, a PI3K inhibitor (Cell Signaling Technology, Danvers, MA, USA; 9901) at 10 or 50 μm for 2 h followed by DSV infection for 30 min or 4 h. For TL2-C29 treatment, a TLR 2 antagonist (Invivogen, Waltham, MA, USA; inh-c29), cells were incubated with the inhibitor at 200 μm for 3 h followed by infection with DSV for 4 h.

Techniques: Activation Assay, Infection, SDS Page, Western Blot, Control, Cell Culture, Ethanol Precipitation

Journal: Microorganisms

Article Title: Sulfate-Reducing Bacteria Induce Pro-Inflammatory TNF-α and iNOS via PI3K/Akt Pathway in a TLR 2-Dependent Manner

doi: 10.3390/microorganisms12091833

Figure Lengend Snippet: Schematic representation of activation of PI3K/Akt pathway by DSV. DSV activates TLR 2 signaling which is inhibited by C29 (TL2-C29). TLR 2-mediated intracellular signaling occurs via formation of heterodimers of TLR 2 with TLR 1 or TLR 6 (TLR 1/6) that allows recognition of diverse sets of pathogen-associated patterns. TLR 2 activation further leads to the activation of PI3K/Akt as indicated by phosphorylation of Akt at Thr308 and Ser473 positions which is required for a complete activation of Akt. This step is inhibited by PI3K inhibitor LY294002. Once activated, Akt further induces transcription factor NFκB. Upon activation, NFκB induces expression of proinflammatory TNF-α and iNOS.

Article Snippet: For drug treatments, cells were incubated with LY294002, a PI3K inhibitor (Cell Signaling Technology, Danvers, MA, USA; 9901) at 10 or 50 μm for 2 h followed by DSV infection for 30 min or 4 h. For TL2-C29 treatment, a TLR 2 antagonist (Invivogen, Waltham, MA, USA; inh-c29), cells were incubated with the inhibitor at 200 μm for 3 h followed by infection with DSV for 4 h.

Techniques: Activation Assay, Phospho-proteomics, Expressing

Journal: Cancers

Article Title: Lactococcus lactis subsp. cremoris C60 Upregulates Macrophage Function by Modifying Metabolic Preference in Enhanced Anti-Tumor Immunity

doi: 10.3390/cancers16101928

Figure Lengend Snippet: C60 upregulates glycolysis-associated metabolism and immune function via TLR signaling in IT macrophage. Intragastric (i.g.) administration of saline or HK-C60 and B16-OVA inoculation were performed in the mice following protocol represented in A. IT macrophages were isolated from tumor and used for experiments. ( A ) TLR2 and ( B ) TLR4 expressions in IT macrophages analyzed by flow cytometry. ( C ) Glucose uptake assay. IT macrophages were cultures with vehicle, HK-C60 or fC60-CWE, and 2-NBDG MFI was analyzed by flow cytometry. ( D ) TNF-α production assay. IT macrophages were cultures with vehicle, HK-C60 or fC60-CWE, and TNF-α MFI was analyzed by flow cytometry. ( E , F ) Glucose uptake assay with TLR inhibition. IT macrophages were isolated from HK-C60 supplemented mice, and treated with vehicle, TL2-C29 (TLR2 inhibitor) or SsnB (TLR4 inhibitor). 2-NBDG MFI was measured in the macrophages with HK-60 ( E ) or fC60-CWE ( F ) stimulation by flow cytometry. ( G , H ) TNF-α production with TLR inhibition. The IT macrophages were cultured in the same conditions represented in E,F. The TNF-α MFI was measured by flow cytometry. ( I – K ) Tumor growth in TLR-deficient mice and IT macrophage functional assay. Intragastric (i.g.) administration of HK-C60 and B16-OVA inoculation was performed in the WT, LTR2-KO or TLR4-KO mice following protocol represented in A. The tumor volumes were measured after 7 and 14 days of post tumor inoculation. IT macrophages were isolated from tumor and subjected to ATP and glucose uptake assay, and tumor antigen specific CD8+ T cells were also analyzed in the tumor isolated leukocytes. ( I ) Tumor volumes of B16-OVA inoculated mice. n = 10 in each group. ( J ) ATP concentration measured by luminescence probe. ( K ) 2-NBDG MFI measured by flow cytometry. ( L ) Percentage of tumor antigen specific CD8+ T cells analyzed by flow cytometry. Fold expression change of MFI was calculated by following the molecule expression in the target sample vs. control sample (as a base = 1) in flow cytometry analysis. The cumulative data were shown as mean ± SD of six samples. Student t -test or one-way ANOVA was used to analyze data for significant differences. Values of * p < 0.05 or ** p < 0.001 were regarded as significant. ns: not significant.

Article Snippet: Moreover, some cultures were pre-treated with vehicle (DMSO), TL2-C29 (TLR2 inhibitor, 10 μM; InvivoGen, San Diego, CA, USA), or Sparstolonin B (SsnB, TLR4 inhibitor, 100 μM; Sigma-Aldrich) at 37 °C for 60 min, then subsequently subjected to a glucose uptake assay in the presence of HK-C60 (5.0 × 10 7 CFU/mL) or CWE (isolated from 5.0 × 10 7 CFU/mL) at 37 °C for 60 min.

Techniques: Saline, Isolation, Flow Cytometry, Inhibition, Cell Culture, Functional Assay, Concentration Assay, Expressing

Journal: bioRxiv

Article Title: Buprenorphine Induces Human Fetal Membrane Sterile Inflammation

doi: 10.1101/2024.11.22.624850

Figure Lengend Snippet: Human FM explants from 6-7 patients were treated with no treatment (NT) or buprenorphine (Bup, 40μg/ml) in the presence of media or (A) the p65 NFκB inhibitor, BAY11-7085; (B) the p38 MAPK inhibitor, SB203580; (C) the ERK inhibitor, SCH77298; or (D) the JNK inhibitor, SP600125. After 48hrs, cell-free supernatants were collected and measured by ELISA for IL-6, IL-8, IL-1β, G-CSF, PGE2, MMP1, and MMP9. * p <0.05 relative to NT/media or NT/inhibitor controls, or as otherwise indicated.

Article Snippet: For some experiments, FMs were treated with NT or buprenorphine in the presence or absence of inhibitors to: TLR4 (LPS-RS, 10μg/ml) (Invivogen, San Diego, CA); TLR2 (TL2-C29, 50μM) (Invivogen, San Diego, CA); p38 MAPK (SB203580, 1μM); p65 NFκB (BAY11-7085, 10μM) (Invivogen, San Diego, CA); ERK (SCH77298, 10nM) (Selleck Chemicals, Houston, TX); JNK (SP600125, 25μM) (Millipore Sigma, Burlington, VT), and NLRP3 (MCC950, 10μM) (Invivogen, San Diego, CA).

Techniques: Enzyme-linked Immunosorbent Assay