Journal: Frontiers in Immunology

Article Title: Human neutrophils recognize group B streptococci via formylated peptide receptors and toll-like receptor 8

doi: 10.3389/fimmu.2026.1828994

Figure Lengend Snippet: TLR8 is required for GBS-induced cytokine production. Neutrophils were treated with the TLR8 inhibitor CU-CPT9a (0.75, 1.50 and 3.00μM) or the TLR2 inhibitor TL2-C29 (5, 10 and 25μM) before stimulation with live [MOI of 5, (A, B) ] or heat-killed GBS [25 μg/ml, (C, D) ]. IL-8 (A, C) and TNF-α (B, D) were measured in 24h culture supernatants. Escherichia coli lipopolysaccharide (LPS; 10 ng/mL) was included as a positive control. (E) Effect of pretreatment with the TLR8 CU-CPT9a inhibitor (3μM) on the release of reactive oxygen species after stimulation with live GBS (MOI 100). Data are expressed as means ± standard deviations from five independent experiments, each performed in duplicate. *p < 0.05 and **p < 0.01, as determined by the Mann-Whitney test; ns, not significant.

Article Snippet: The contribution of FPRs and TLRs to IL-8 and ROS production induced by live bacteria was evaluated by pretreating neutrophils for 1 hour at 37 °C in 5% CO 2 with the selective FPR2 antagonist WRW4 (Abcam), the pan-FPR inhibitor Boc-2 (GenScript), the selective TLR8 inhibitor CU-CPT9a (InvivoGen) or the selective TLR2 inhibitor TL2-C29 (InvivoGen) at the indicated concentrations prior to stimulation with heat-killed (HK) or live bacteria.

Techniques: Positive Control, MANN-WHITNEY

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: DSV induced activation of PI3K/Akt/TNF pathway in a time-dependent manner. ( A ) RAW 264.7 cells (8 × 10 5 ) were infected with DSV at MOI 20 for 5, 15, 30, 120, or 240 min. Cells were lysed and protein lysate was prepared. Fifty μg of protein lysate was separated on SDS-PAGE and analyzed for the expression of p-Akt, total Akt, TNF-α, iNOS, p-IκB, total IκB, p-NFκB p65, and total p65 NFκB by Western blotting. Original blots were cut at different molecular weights to enable probing of as many proteins as possible in the same blot. Actin was used as a loading control. ( B – G ) Quantification of Western blots. Blots were quantified with Fiji ImageJ by analyzing the ratio of protein of interest/Actin. For phosphorylated proteins, the ratio of phosphorylated/total protein was calculated. Data represent Mean ± SEM from at least three independent experiments. Values were normalized to control. One-way ANOVA was used to determine the statistical significance. Values were compared to control, with a post-hoc Dunnett’s test. * p < 0.05, ** p < 0.01, *** p < 0.001.

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, Expressing, Western Blot, Control

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: Live and heat-killed DSV, but not bacterial culture supernatant, significantly induced PI3K/Akt/TNF/iNOS pathway. ( A ) RAW 264.7 cells were treated with live or heat-killed (HK) DSV (MOI 20) or bacterial culture supernatant (Sup) for 240 min (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, TNF-α, and iNOS by Western blotting. Actin was used as a loading control. ( B – D ) Quantification of Western blots. Blots were quantified with Fiji ImageJ by analyzing the ratio of protein of interest/Actin. For phosphorylated Akt, the ratio of p-Akt/total Akt was calculated. Data represent Mean ± SEM from at least three independent experiments. Values were normalized to control. One-way ANOVA was used to determine the statistical significance. Values were compared to live DSV. * p < 0.05, 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: SDS Page, Western Blot, Control

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: Comparison of effects of DSV and Lactobacillus plantarum on PI3K/Akt/TNF/iNOS pathway. ( A ) RAW 264.7 cells were treated with DSV or Lacto (MOI 20) 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, TNF-α, and iNOS by Western blotting. Lanes were spliced in the original blot and combined. Actin was used as a loading control. ( B – D ) Quantification of Western blots. Blots were quantified with ImageJ by analyzing the ratio of protein of interest/Actin. For phosphorylated Akt, the ratio of p-Akt/total Akt was calculated. Data represent Mean ± SEM from at least three independent experiments. Values were normalized and compared to Control. One-way ANOVA was used to determine the statistical significance. * p < 0.05, *** p < 0.001, 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: Comparison, SDS Page, Western Blot, Control

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: Activation of PI3K/Akt pathway by DSV was dependent on TLR 2 signaling ( A ) RAW 264.7 cells were treated with TLR 2 signaling inhibitor C29 at 200 μM for 3 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. ( G ) Blots were quantified with ImageJ by analyzing the ratio of protein of interest/Actin. For phosphorylated proteins, the ratio of phosphorylated/total protein was calculated. Data represent Mean ± SEM from at least three independent experiments. Values were normalized to control. A two-tailed t -test was used to compare DSV and DSV + C29. * p < 0.05, ** p < 0.01.

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, Two Tailed Test

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: 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 NET–DNA levels produced from RA neutrophils incubated with various concentrations of IL-33 for 4 h. b Dynamic observation of NET formation in RA neutrophils induced with 100 ng/mL IL-33 using a live cell workstation. c Representative immunofluorescence staining and scanning electron microscopy images of NET formation induced by 100 ng/mL IL-33 in RA neutrophils. d Cellular ROS levels in neutrophils stimulated with 100 ng/mL IL-33, 40 nM PMA, or PBS for 2 h. e NET–DNA levels produced by RA neutrophils pretreated with 10 μM DPI, 10 μM Cl-amidine or 20 μM sivelestat for 1 h prior to stimulation with 100 ng/mL IL-33 or 40 nM PMA for 4 h. f Coimmunoprecipitation assay showing interactions among ST2, MyD88, and TRAF6 in RA neutrophils treated with 100 ng/mL IL-33 for 1 h. g Representative western blots showing ERK1/2, p38, IκBα and p65 phosphorylation levels in RA neutrophils incubated with 100 ng/mL IL-33 for 4 h. h NET–DNA levels induced from RA or HD neutrophils pretreated with 0.2 mg/mL ST2 polyclonal neutralizing antibody for 1 h and then stimulated with 100 ng/mL IL-33 or 40 nM PMA for 4 h. i ST2 mRNA levels in peripheral blood neutrophils from RA patients ( n = 18) and HDs ( n = 18) with or without incubation with 100 ng/mL IL-33 for 12 h. j Flow cytometric analysis of the proportion of CD66b+ST2+ cells among peripheral blood neutrophils from RA patients ( n = 18) and HDs ( n = 18) with or without incubation with 100 ng/mL IL-33 for 12 h. k , l Fold change in the ST2 mRNA level and the proportion of CD66b+ST2+ cells among RA ( n = 18) or HD ( n = 18) neutrophils incubated with 100 ng/mL IL-33 for 12 h. All the data are presented as the means ± SDs/SEMs. Asterisks represent significant differences compared with the reference group, which is without asterisks. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant.

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: Produced, Incubation, Immunofluorescence, Staining, Electron Microscopy, Co-Immunoprecipitation Assay, Western Blot, Phospho-proteomics

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

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

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: bioRxiv

Article Title: α-Synuclein aggregates induce mitochondrial damage and trigger innate immunity to drive neuron–microglia communication

doi: 10.1101/2025.06.23.661105

Figure Lengend Snippet: 𝜶 -Syn-induced intercellular connections are inflammation-dependent. (a) Representative phalloidin-stained images of neuronal cells (top panels) and microglial cells (bottom panels) treated with α-Syn aggregates, the STING inhibitor H-151, or both to assess for intercellular connections. (b-c) Quantification of the percentage of connected cells in different conditions for neuronal cells (b) and microglial cells (c). N=3 independent experiments, n=20-21 fields of views. Statistical significance was analyzed using Brown-Forsythe and Welch One-Way ANOVA and Dunnett’s T3 multiple comparison. ns: p>0.05, ****p<0.0001. (d) Representative phalloidin-stained images of neuronal cells (top panels) and microglial cells (bottom panels) treated with α-Syn aggregates, the NF-κB inhibitor JSH-23, or both to assess for intercellular connections. (e-f) Quantification of the percentage of connected cells in different conditions for neuronal cells (e) and microglial cells (f). N=3 independent experiments, n=18-21 fields of views. Statistical significance was analyzed using Brown- Forsythe and Welch One-Way ANOVA and Dunnett’s T3 multiple comparison. ns: p>0.05, ****p<0.0001. (g) Representative phalloidin-stained images of neuronal cells (top panels) and microglial cells (bottom panels) treated with the STING activator diABZI alone, or alongside H-151 or JSH-23 to assess for intercellular connections. (h-i) Quantification of the percentage of connected cells in different conditions for neuronal cells (h) and microglial cells (i). N=3 independent experiments, n=20-22 fields of views. Statistical significance was analyzed using Brown-Forsythe and Welch One-Way ANOVA and Dunnett’s T3 multiple comparison. ns: p>0.05, ****p<0.0001. Data in all the graphs are represented as median and quartiles, with mean percentage mentioned within the graphs. (j) Schematic of α-Syn-induced inflammatory regulators of intercellular connections (left panel), and effect of STING activation on such connections, depicting a gate-keeping activity of NF-κB in the process.

Article Snippet: For inflammatory modulations, following reagents were used with associated treatment concentration and durations: STING activator – diABZI (SelleckChem – compound 3, S8796; 2.5 μM for 2h towards the end of experiment), STING inhibitor – H-151 (Invivogen InvitroFit TM , inh-h151; 0.5 μg/mL for 16h for TNT assessment experiments, and 24h for α-Syn transfer experiments), NF-κB inhibitor – JSH-23 (Sigma, J4455; 10 μM for 16h for TNT assessment experiments, and 24h for α-Syn transfer experiments), human cGAS inhibitor – G140 (Invivogen InvitroFit TM , inh-g140; 10 μM for 16h), Bcl-2 inhibitor – ABT-737 (Sigma, 197333; 10 μM for 6h, 12h, or 24h) co-treated with pan-caspase inhibitor – Q-VD-OPh (Non-O- methylated, Sigma, 551476, 10 μM), human IL-1α (Sigma, I2778; 3 ng/mL for 24h), human IL-1β (Sigma, H6291; 20 ng/mL for 24h), human TNF-α (Merck Millipore, 654205; 50 ng/mL for 24h), TLR4 inhibitor – TAK-242 (Tocris, 6587; 10 μM for 16h), TLR2 inhibitor – TL2-C29 (Invivogen InvitroFit TM , inh-C29; 100 μM for 16h), lysosomotropic agent LLOMe (Sigma, L7393; 1 mM for 1h towards the end of experiment), mitochondrial electron transport chain uncoupler CCCP (Sigma, C2759; 20 μM for 2h).

Techniques: Staining, Comparison, Activation Assay, Activity Assay

Journal: bioRxiv

Article Title: α-Synuclein aggregates induce mitochondrial damage and trigger innate immunity to drive neuron–microglia communication

doi: 10.1101/2025.06.23.661105

Figure Lengend Snippet: 𝜶 -Syn spread to microglia via TNTs. (a) Representative confocal images of neuronal cells loaded with α-Syn (donors) in co-culture for 24h with microglial cells (acceptors) in the presence of the STING activator diABZI alone, or alongside the STING inhibitor H-151. Cyan asterisks highlight acceptor microglial cells positive for α- Syn. (b) Quantification of the percentage of acceptor microglial cells positive for α-Syn. N=3 independent experiments, n=25-26 fields of views. Statistical significance was analyzed using Kruskal Wallis test with Dunn’s multiple comparison. ns: p>0.05, ***p<0.001. (c) Quantification of the number of α-Syn aggregates per acceptor microglial cell as in (b). n=106 cells for control, 177 cells for diABZI-treated group, and 92 cells for diABZI+H-151 co-treated group. Statistical significance was analyzed using Kruskal Wallis test with Dunn’s multiple comparison. ns: p>0.05, *p<0.05. (d) Quantification of the extent of α-Syn transfer via secretion (CM: conditioned media). N=3 independent experiments, n=15 fields of views. Statistical significance was analyzed using Kruskal Wallis test with Dunn’s multiple comparison. ns: p>0.05. (e) Representative confocal images of neuronal cells loaded with α- Syn (donors) in co-culture for 24h with microglial cells (acceptors) in the presence of pro- inflammatory cytokines alone (upper panels) or co-treated with the NF-κB inhibitor JSH-23 (lower panels). Cyan asterisks highlight acceptor microglial cells positive for α-Syn. (f) Quantification of the percentage of acceptor microglial cells positive for α-Syn. N=3 independent experiments, n=24-25 fields of views. Statistical significance was analyzed using Two-Way ANOVA with Šídák’s multiple comparison. *p<0.05, **p<0.01, ****p<0.0001. (g) Quantification of the number of α-Syn aggregates per acceptor microglial cell as in (f). n=126 (Control –JSH-23), 124 (control +JSH-23), 227 (IL-1α –JSH-23), 163 (IL-1α +JSH-23), 223 (IL-1𝛽 –JSH-23), 144 (IL-1𝛽 +JSH-23), 188 (TNFα –JSH-23), and 134 (TNFα +JSH-23) cells. Statistical significance was analyzed using Two-Way ANOVA with Šídák’s multiple comparison. ns: p>0.05, ***p<0.001, ****p<0.0001. (h) Quantification of the extent of α-Syn transfer via secretion (CM: conditioned media). N=3 independent experiments, n=14-16 fields of views. Statistical significance was analyzed using Two-Way ANOVA with Šídák’s multiple comparison. ns: p>0.05, *p<0.05. Data in all the graphs are represented as median and quartiles. Mean values are mentioned within the graphs.

Article Snippet: For inflammatory modulations, following reagents were used with associated treatment concentration and durations: STING activator – diABZI (SelleckChem – compound 3, S8796; 2.5 μM for 2h towards the end of experiment), STING inhibitor – H-151 (Invivogen InvitroFit TM , inh-h151; 0.5 μg/mL for 16h for TNT assessment experiments, and 24h for α-Syn transfer experiments), NF-κB inhibitor – JSH-23 (Sigma, J4455; 10 μM for 16h for TNT assessment experiments, and 24h for α-Syn transfer experiments), human cGAS inhibitor – G140 (Invivogen InvitroFit TM , inh-g140; 10 μM for 16h), Bcl-2 inhibitor – ABT-737 (Sigma, 197333; 10 μM for 6h, 12h, or 24h) co-treated with pan-caspase inhibitor – Q-VD-OPh (Non-O- methylated, Sigma, 551476, 10 μM), human IL-1α (Sigma, I2778; 3 ng/mL for 24h), human IL-1β (Sigma, H6291; 20 ng/mL for 24h), human TNF-α (Merck Millipore, 654205; 50 ng/mL for 24h), TLR4 inhibitor – TAK-242 (Tocris, 6587; 10 μM for 16h), TLR2 inhibitor – TL2-C29 (Invivogen InvitroFit TM , inh-C29; 100 μM for 16h), lysosomotropic agent LLOMe (Sigma, L7393; 1 mM for 1h towards the end of experiment), mitochondrial electron transport chain uncoupler CCCP (Sigma, C2759; 20 μM for 2h).

Techniques: Co-Culture Assay, Comparison, Control

Journal: bioRxiv

Article Title: α-Synuclein aggregates induce mitochondrial damage and trigger innate immunity to drive neuron–microglia communication

doi: 10.1101/2025.06.23.661105

Figure Lengend Snippet: 𝜶 -Syn spread to microglia via TNTs. (a) Representative confocal images of neuronal cells loaded with α-Syn (donors) in co-culture for 24h with microglial cells (acceptors) in the presence of the STING activator diABZI alone, or alongside the STING inhibitor H-151. Cyan asterisks highlight acceptor microglial cells positive for α- Syn. (b) Quantification of the percentage of acceptor microglial cells positive for α-Syn. N=3 independent experiments, n=25-26 fields of views. Statistical significance was analyzed using Kruskal Wallis test with Dunn’s multiple comparison. ns: p>0.05, ***p<0.001. (c) Quantification of the number of α-Syn aggregates per acceptor microglial cell as in (b). n=106 cells for control, 177 cells for diABZI-treated group, and 92 cells for diABZI+H-151 co-treated group. Statistical significance was analyzed using Kruskal Wallis test with Dunn’s multiple comparison. ns: p>0.05, *p<0.05. (d) Quantification of the extent of α-Syn transfer via secretion (CM: conditioned media). N=3 independent experiments, n=15 fields of views. Statistical significance was analyzed using Kruskal Wallis test with Dunn’s multiple comparison. ns: p>0.05. (e) Representative confocal images of neuronal cells loaded with α- Syn (donors) in co-culture for 24h with microglial cells (acceptors) in the presence of pro- inflammatory cytokines alone (upper panels) or co-treated with the NF-κB inhibitor JSH-23 (lower panels). Cyan asterisks highlight acceptor microglial cells positive for α-Syn. (f) Quantification of the percentage of acceptor microglial cells positive for α-Syn. N=3 independent experiments, n=24-25 fields of views. Statistical significance was analyzed using Two-Way ANOVA with Šídák’s multiple comparison. *p<0.05, **p<0.01, ****p<0.0001. (g) Quantification of the number of α-Syn aggregates per acceptor microglial cell as in (f). n=126 (Control –JSH-23), 124 (control +JSH-23), 227 (IL-1α –JSH-23), 163 (IL-1α +JSH-23), 223 (IL-1𝛽 –JSH-23), 144 (IL-1𝛽 +JSH-23), 188 (TNFα –JSH-23), and 134 (TNFα +JSH-23) cells. Statistical significance was analyzed using Two-Way ANOVA with Šídák’s multiple comparison. ns: p>0.05, ***p<0.001, ****p<0.0001. (h) Quantification of the extent of α-Syn transfer via secretion (CM: conditioned media). N=3 independent experiments, n=14-16 fields of views. Statistical significance was analyzed using Two-Way ANOVA with Šídák’s multiple comparison. ns: p>0.05, *p<0.05. Data in all the graphs are represented as median and quartiles. Mean values are mentioned within the graphs.

Article Snippet: For inflammatory modulations, following reagents were used with associated treatment concentration and durations: STING activator – diABZI (SelleckChem – compound 3, S8796; 2.5 𝜇M for 2h towards the end of experiment), STING inhibitor – H-151 (Invivogen InvitroFit TM , inh-h151; 0.5 𝜇g/mL for 16h for TNT assessment experiments, and 24h for α-Syn transfer experiments), NF-κB inhibitor – JSH-23 (Sigma, J4455; 10 𝜇M for 16h for TNT assessment experiments, and 24h for α-Syn transfer experiments), human cGAS inhibitor – G140 (Invivogen InvitroFit TM , inh-g140; 10 𝜇M for 16h), Bcl-2 inhibitor – ABT-737 (Sigma, 197333; 10 𝜇M for 6h, 12h, or 24h) co-treated with pan-caspase inhibitor – Q-VD-OPh (Non-O- methylated, Sigma, 551476, 10 𝜇M), human IL-1α (Sigma, I2778; 3 ng/mL for 24h), human IL-1𝛽 (Sigma, H6291; 20 ng/mL for 24h), human TNF-α (Merck Millipore, 654205; 50 ng/mL for 24h), TLR4 inhibitor – TAK-242 (Tocris, 6587; 10 𝜇M for 16h), TLR2 inhibitor – TL2-C29 (Invivogen InvitroFit TM , inh-C29; 100 𝜇M for 16h), lysosomotropic agent LLOMe (Sigma, L7393; 1 mM for 1h towards the end of experiment), mitochondrial electron transport chain uncoupler CCCP (Sigma, C2759; 20 𝜇M for 2h).

Techniques: Co-Culture Assay, Comparison, Control

Journal: bioRxiv

Article Title: α-Synuclein aggregates induce mitochondrial damage and trigger innate immunity to drive neuron–microglia communication

doi: 10.1101/2025.06.23.661105

Figure Lengend Snippet: (a) Representative confocal images of mitochondria-labeled control (upper panels) or α-Syn-loaded (bottom panels) neuronal cells (donors) in co-culture for 24h with microglial cells (acceptors). (b) Quantification of the percentage of microglial cells receiving MitoDsRed+ mitochondria in both co-cultures and conditioned media (secretion) experiments. N=3 independent experiments, n=35 (N MitoDsRed + M co-culture), 47 (N MitoDsRed α-Syn + M co- culture), 21 (N MitoDsRed + M secretion) and 22 (N MitoDsRed α-Syn + M secretion) fields of views. Statistical significance was analyzed using Two-Way ANOVA with Uncorrected Fisher’s LSD multiple comparison. ns: p>0.05, ****p<0.0001. (c) Quantification of the number of mitochondrial particles received per acceptor microglia as in (b). N=74 (N MitoDsRed + M co- culture), 185 (N MitoDsRed α-Syn + M co-culture), 30 (N MitoDsRed + M secretion), and 34 (N MitoDsRed α- Syn + M secretion) cells. Statistical significance was analyzed using Two-Way ANOVA with Uncorrected Fisher’s LSD multiple comparison. ns: p>0.05, ***p<0.001. (d) Representative confocal images of neuronal cell-derived MitoDsRed+ mitochondria in microglial cells. (e-f) Quantification of the proportion of transferred mitochondria from naïve neuronal cells (e) or α- Syn-burdened neuronal cells (f) that remain isolated in microglial cells, or overlap with the host network. Total number of mitochondrial particles analyzed is mentioned within the pie-chart. (g) Quantification of the relative health of transferred mitochondria (Cytochrome c level, indicative of outer membrane permeabilization state of mitochondria) from naïve (N MitoDsRed + M) or α-Syn-burdened (N MitoDsRed α-Syn + M) neuronal cells. N=3 independent experiments, n=50 transferred mitochondrial particles analyzed. Statistical significance was tested using Mann-Whitney test. *p<0.05. (h) Representative confocal image, and orthogonal views of dashed boxes, highlighting encapsulation of MitoDsRed+ mitochondrial particles derived from α-Syn-burdened neuronal cells within LAMP1+ lysosomes of microglial cells. (i) Quantification of overlap between MitoDsRed+ mitochondria and LAMP1+ lysosomes in acceptor microglial cells. Data in violin plots are represented as median and quartiles, with mean values mentioned within the graphs.

Article Snippet: For inflammatory modulations, following reagents were used with associated treatment concentration and durations: STING activator – diABZI (SelleckChem – compound 3, S8796; 2.5 𝜇M for 2h towards the end of experiment), STING inhibitor – H-151 (Invivogen InvitroFit TM , inh-h151; 0.5 𝜇g/mL for 16h for TNT assessment experiments, and 24h for α-Syn transfer experiments), NF-κB inhibitor – JSH-23 (Sigma, J4455; 10 𝜇M for 16h for TNT assessment experiments, and 24h for α-Syn transfer experiments), human cGAS inhibitor – G140 (Invivogen InvitroFit TM , inh-g140; 10 𝜇M for 16h), Bcl-2 inhibitor – ABT-737 (Sigma, 197333; 10 𝜇M for 6h, 12h, or 24h) co-treated with pan-caspase inhibitor – Q-VD-OPh (Non-O- methylated, Sigma, 551476, 10 𝜇M), human IL-1α (Sigma, I2778; 3 ng/mL for 24h), human IL-1𝛽 (Sigma, H6291; 20 ng/mL for 24h), human TNF-α (Merck Millipore, 654205; 50 ng/mL for 24h), TLR4 inhibitor – TAK-242 (Tocris, 6587; 10 𝜇M for 16h), TLR2 inhibitor – TL2-C29 (Invivogen InvitroFit TM , inh-C29; 100 𝜇M for 16h), lysosomotropic agent LLOMe (Sigma, L7393; 1 mM for 1h towards the end of experiment), mitochondrial electron transport chain uncoupler CCCP (Sigma, C2759; 20 𝜇M for 2h).

Techniques: Labeling, Control, Co-Culture Assay, Comparison, Derivative Assay, Isolation, Membrane, MANN-WHITNEY, Encapsulation

Journal: bioRxiv

Article Title: α-Synuclein aggregates induce mitochondrial damage and trigger innate immunity to drive neuron–microglia communication

doi: 10.1101/2025.06.23.661105

Figure Lengend Snippet: (a) Representative super-resolution SIM images of α-Syn-burdened MitoDsRed+ neuronal cells in co-culture with microglial cells for 24h. Insets i, ii, and iii highlight transferred mitochondrial particles that are mtDNA+, mtDNA-, and have mtDNA extruding (white arrowhead) from the matrix, respectively. (b) Quantification of the proportion of transferred mitochondria from control or α-Syn-burdened neuronal cells that are positive or negative for mtDNA. N=3 independent experiments, numbers of mitochondrial particles analyzed are mentioned within the bars. Error bars represent SEM. (c) Representative confocal images of STING translocation to GM130+ Golgi in microglial cells when co-cultured for 24h with naïve (left panels) or α-Syn-burdened (right panels) neuronal cells. (d) Quantification of mean STING fluorescence intensity on GM130+ Golgi in microglial cells. N=3 independent experiments, n=100 cells. Statistical significance was analyzed using Mann-Whitney test. **p<0.01. (e) Representative confocal images of nuclear translocation of NF-κB in microglial cells when co- cultured for 24h with naïve (left panels) or α-Syn-burdened (right panels) neuronal cells. (f) Quantification of mean nuclear NF-κB fluorescence intensity in microglial cells. N=3 independent experiments, n=100 cells. Statistical significance was analyzed using Mann- Whitney test. ****p<0.0001. (g) Representative confocal images of nuclear translocation of IRF3 in microglial cells when co-cultured for 24h with naïve (left panels) or α-Syn-burdened (right panels) neuronal cells. (h) Quantification of mean nuclear IRF3 fluorescence intensity in microglial cells. N=3 independent experiments, n=100 cells. Statistical significance was analyzed using Mann-Whitney test. ****p<0.0001. Data in violin graphs are represented as median and quartiles. Mean values are mentioned within the graphs.

Article Snippet: For inflammatory modulations, following reagents were used with associated treatment concentration and durations: STING activator – diABZI (SelleckChem – compound 3, S8796; 2.5 𝜇M for 2h towards the end of experiment), STING inhibitor – H-151 (Invivogen InvitroFit TM , inh-h151; 0.5 𝜇g/mL for 16h for TNT assessment experiments, and 24h for α-Syn transfer experiments), NF-κB inhibitor – JSH-23 (Sigma, J4455; 10 𝜇M for 16h for TNT assessment experiments, and 24h for α-Syn transfer experiments), human cGAS inhibitor – G140 (Invivogen InvitroFit TM , inh-g140; 10 𝜇M for 16h), Bcl-2 inhibitor – ABT-737 (Sigma, 197333; 10 𝜇M for 6h, 12h, or 24h) co-treated with pan-caspase inhibitor – Q-VD-OPh (Non-O- methylated, Sigma, 551476, 10 𝜇M), human IL-1α (Sigma, I2778; 3 ng/mL for 24h), human IL-1𝛽 (Sigma, H6291; 20 ng/mL for 24h), human TNF-α (Merck Millipore, 654205; 50 ng/mL for 24h), TLR4 inhibitor – TAK-242 (Tocris, 6587; 10 𝜇M for 16h), TLR2 inhibitor – TL2-C29 (Invivogen InvitroFit TM , inh-C29; 100 𝜇M for 16h), lysosomotropic agent LLOMe (Sigma, L7393; 1 mM for 1h towards the end of experiment), mitochondrial electron transport chain uncoupler CCCP (Sigma, C2759; 20 𝜇M for 2h).

Techniques: Co-Culture Assay, Control, Translocation Assay, Cell Culture, Fluorescence, MANN-WHITNEY