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

Journal: Experimental & Molecular Medicine

Article Title: IL33-induced neutrophil extracellular traps (NETs) mediate a positive feedback loop for synovial inflammation and NET amplification in rheumatoid arthritis

doi: 10.1038/s12276-024-01351-7

Figure Lengend Snippet: a TLR1 to TLR10 mRNA levels in RA FLSs ( n = 5) treated with 1 ng/μg spon-NETs. b , c Levels of IL-33 and CXCL8 secreted from RA FLSs ( n = 3) preincubated with 20 μM TLR2 antagonist TL2-C29, 1 μM TLR4 antagonist CLI-095 and 3 μM TLR9 antagonist ODN TTAGGG (ODN A151) for 1 h prior to the addition of 1 ng/μL spon-NETs for 24 h. d GSEA of the NF-κB and MAPK (ERK1/2) signaling pathways. e , f Representative western blots showing the levels of phosphorylated ERK1/2, p38, IκBα and p65 in RA neutrophils incubated with 1 ng/μL spon-NETs for 24 h. g , h Levels of IL-33/CXCL8 secreted from RA FLSs ( n = 3) preincubated with the ERK inhibitor PD98059 (1 μM) or the p38 inhibitor SB203580 (10 μM) or the NF-κB inhibitor BAY11-7082 (10 μM) for 1 h prior to the addition of 1 ng/μL spon-NETs for 24 h. i Flowchart of the animal experimental procedures created using BioRender.com. DBA/1J mice received 1.5 mg/mouse type II collagen mAbs via the tail vein on Day 0. On Day 3, 50 µg of LPS was injected via the tail vein to synergize with the mAbs for arthritis development, and intra-articular injections of 3 μM TLR9 antagonist ODN 2088 were administered into the knee joint. The mice were also administered 1 µg of IL-33 intraperitoneally for five consecutive days. j Inflammatory scores of IL-33-induced CAIA mice treated with a TLR9 antagonist ( n = 5) and control mice ( n = 5). k Radiological scores of bone erosion in IL-33-induced CAIA mice treated with a TLR9 antagonist and controls. l citH 3 -DNA levels in the peripheral serum of CAIA model mice treated with a TLR9 antagonist and control mice. m Representative immunofluorescence staining of joint tissue from IL-33-induced CAIA mice treated with a TLR9 antagonist. All the data are presented as the means ± SEMs/SD. Asterisks represent significant differences compared with the reference group, without asterisks. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The inhibitors used included the TLR2 antagonist TL2-C29 (20 μM, Invitrogen), the TLR4 antagonist CLI-095 (1 μM, InvivoGen), the TLR9 antagonist ODN TTAGGG (ODN A151, 3 μM, InvivoGen), the ERK inhibitor PD98059 (1 μM, Selleck), the p38 inhibitor SB203580 (10 μM, Selleck), the NF-κB inhibitor BAY11-7082 (10 μM, Selleck) and DNase I (0.1 mg/mL, Roche).

Techniques: Protein-Protein interactions, Western Blot, Incubation, Injection, Control, Immunofluorescence, Staining