Journal: Journal of Translational Medicine

Article Title: Mitochondrial DNA induces nucleus pulposus cell pyroptosis via the TLR9-NF-κB-NLRP3 axis

doi: 10.1186/s12967-023-04266-5

Figure Lengend Snippet: Knockdown or inhibition of TLR9 activation reduced NPC pyroptosis under OS. Knockdown by siRNA or inhibition of TLR9 activation by E6446 with 100 µM H 2 O 2 -treated the NPCs for 24 h. a Agar gelatin electrophoresis images of TLR9 cDNA in the negative control, si-Scrambled, and si-TLR9 groups. b RT-qPCR quantitative analysis of TLR9 cDNA after siRNA knockdown in the negative control, si-Scrambled, and si-TLR9 groups. c Western blotting images of the expression levels of TLR9 and NF-κB after siRNA knockdown in the negative control, si-Scrambled, and si-TLR9 groups. d , e Quantitative analysis of the protein level of TLR9 and NF-κB after siRNA knockdown in the negative control, si-Scrambled, and si-TLR9 groups. f , g Immunofluorescence images of TLR9 and NF-κB in the negative control, si-Scrambled, and si-TLR9 groups. h Western blotting images of NLRP3, ASC, pro caspase-1, cleaved caspase-1, GSDMD, and cleaved GSDMD in the negative control, si-Scrambled, and si-TLR9 groups. i Fluorescence images of Calcein AM/PI staining in the negative control, si-Scrambled, and si-TLR9 groups. j TEM images of the NPCs in the negative control, si-Scrambled, and si-TLR9 groups. k Quantitative analysis of the expression levels of NLRP3, ASC, pro caspase-1, cleaved caspase-1, GSDMD, and cleaved GSDMD in the negative control, si-Scrambled, and si-TLR9 groups. l Quantitative analysis of IL-1β by ELISA in the negative control, si-Scrambled, and si-TLR9 groups. m Western blotting images of NLRP3, ASC, pro caspase-1, cleaved caspase-1, GSDMD, and cleaved GSDMD in the E6446- or PBS-treated NPCs. n Fluorescence images of Calcein AM/PI staining in the E6446- or PBS-treated NPCs. o TEM images of the NPCs in the E6446- or PBS-treated NPCs. p Quantitative analysis of the expression levels of NLRP3, ASC, pro caspase-1, cleaved caspase-1, GSDMD, and cleaved GSDMD in the E6446- or PBS-treated NPCs. q Quantitative analysis of IL-1β by ELISA in the E6446- or PBS-treated NPCs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: To explore the effect of the inhibition of TLR9 activation in NPCs under OS, NPCs were treated with 5 µM E6446 (T4206, TargetMol) and 100 µM H 2 O 2 for 24 h. To explore the effect of blocking mPTP from opening in NPCs under OS, NPCs were administered with 100 µM H 2 O 2 and 10 µM cyclosporine (CsA) (59865-13-3, Sigma-Aldrich) for 24 h. E6446 is a specific TLR9 antagonist and inhibits DNA-TLR9 interaction [ ].

Techniques: Knockdown, Inhibition, Activation Assay, Electrophoresis, Negative Control, Quantitative RT-PCR, Western Blot, Expressing, Immunofluorescence, Fluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Medicine

Article Title: Mitochondrial DNA induces nucleus pulposus cell pyroptosis via the TLR9-NF-κB-NLRP3 axis

doi: 10.1186/s12967-023-04266-5

Figure Lengend Snippet: Inhibit TLR9 activation improved IVDD in vivo. A rat punctured IVDD model on Co7/8 and Co8/9 discs, and the punctured Co8/9 disc and injected with E6446 (10 µM), while punctured Co7/8 disc and injected with the same amount of PBS. a Representative X-ray and MRI T2 weighted images of a rat tail. b The disc height index (DHI) was measured in the negative control, puncture + PBS injection, and puncture + E6446 injection groups (n = 5). c The Pfirrmann grade was evaluated in the negative control, puncture + PBS injection, and puncture + E6446 injection groups (n = 5). d Representative images of the H&E, safranin O-fast green, and Masson staining of rat discs in the negative control, puncture + PBS injection, and puncture + E6446 injection groups. e – g Representative fluorescence images of TLR9, NF-κB, and NLRP3 in the negative control, puncture + PBS injection, and puncture + E6446 injection groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: To explore the effect of the inhibition of TLR9 activation in NPCs under OS, NPCs were treated with 5 µM E6446 (T4206, TargetMol) and 100 µM H 2 O 2 for 24 h. To explore the effect of blocking mPTP from opening in NPCs under OS, NPCs were administered with 100 µM H 2 O 2 and 10 µM cyclosporine (CsA) (59865-13-3, Sigma-Aldrich) for 24 h. E6446 is a specific TLR9 antagonist and inhibits DNA-TLR9 interaction [ ].

Techniques: Activation Assay, In Vivo, Injection, Negative Control, Staining, Fluorescence

Journal: Journal of Translational Medicine

Article Title: Mitochondrial DNA induces nucleus pulposus cell pyroptosis via the TLR9-NF-κB-NLRP3 axis

doi: 10.1186/s12967-023-04266-5

Figure Lengend Snippet: Mitochondrial DNA induces nucleus pulposus cell pyroptosis via the TLR9-NF-κB-NLRP3 axis. Graphical abstract of the critical roles of mtDNA via mPTP opening release into the cytoplasm and TLR9-NF-κB-NLRP3 axis-dependent NPCs pyroptosis in IVDD. a In IVDD, mtDNA via mPTP opening release into the cytoplasm triggered TLR9-NF-κB-NLRP3 axis-dependent NPCs pyroptosis. b The antagonist of mPTP opening (CsA) and TLR9 (E6446) mitigated NPCs pyroptosis and IVDD by inhibiting the TLR9-NF-κB-NLRP3 axis

Article Snippet: To explore the effect of the inhibition of TLR9 activation in NPCs under OS, NPCs were treated with 5 µM E6446 (T4206, TargetMol) and 100 µM H 2 O 2 for 24 h. To explore the effect of blocking mPTP from opening in NPCs under OS, NPCs were administered with 100 µM H 2 O 2 and 10 µM cyclosporine (CsA) (59865-13-3, Sigma-Aldrich) for 24 h. E6446 is a specific TLR9 antagonist and inhibits DNA-TLR9 interaction [ ].

Techniques:

Journal: International Journal of Biological Sciences

Article Title: Neutrophil Recruitment via Hepatocyte IL-1α Drives NETs-Mediated AIM2 Hepatocyte Apoptosis in Alcohol-associated steatohepatitis

doi: 10.7150/ijbs.121255

Figure Lengend Snippet: TLR9 acts downstream of IL-1α to mediate NET formation and hepatocyte apoptosis in ASH. (A) Volcano plot of RNA-seq data showing differentially expressed genes in neutrophils treated with IL-1α compared to untreated controls. TLR9 was among the significantly upregulated genes (red dots, n = 3 per group). (B) KEGG pathway enrichment analysis of 46 upregulated genes identified Toll-like receptor signaling as the most enriched pathway. (C) Experimental timeline. Mice were pair-fed or fed an ethanol-containing Lieber-DeCarli diet for 10 days. E6446 (a TLR9 inhibitor, 20 mg/kg, orally [p.o.]) was administered 24 and 15 hours prior to euthanasia. (D, E) Serum ALT and AST levels were significantly reduced in EtOH-fed mice treated with E6446 compared to untreated ASH mice (n = 6 per group). (F, G) Representative H&E and Oil Red O staining of liver sections showing improved histology and reduced lipid accumulation in the E6446 group. Scale bar = 100 μm. (H) Immunofluorescence staining of liver tissues showing reduced intrahepatic NET formation (MPO: red; CitH3: green; DAPI: blue) after E6446 treatment. Original images, scale bar = 50 μm; magnified insets, scale bar = 10 μm. (I, J) Representative TUNEL staining and quantification of TUNEL-positive areas showed a reduction in hepatocyte apoptosis in E6446-treated mice (n = 6 per group). Scale bar = 100 μm. (K) Western blot and densitometric quantification of hepatic TLR9, CitH3, and Cle-CASP3 levels. E6446 treatment significantly reduced expression of all three proteins (n = 6 per group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. Statistical comparisons were performed using one-way ANOVA followed by Tukey's multiple comparisons test.

Article Snippet: To evaluate the effect of NET-DNA and IL-1α signaling, mice were treated with DNase I (10 U in 100 μL 0.9% NaCl, Intraperitoneal injection (i.p.); Cat. No. EN0521, Thermo Fisher Scientific/Fermentas, USA) or the TLR9 inhibitor E6446 (20 mg/kg, Oral administration (p.o.); Cat. No. S6719, Selleck, China) at 24 and 15 hours prior to tissue harvest.

Techniques: RNA Sequencing, Staining, Immunofluorescence, TUNEL Assay, Western Blot, Expressing

Journal: International Journal of Biological Sciences

Article Title: Neutrophil Recruitment via Hepatocyte IL-1α Drives NETs-Mediated AIM2 Hepatocyte Apoptosis in Alcohol-associated steatohepatitis

doi: 10.7150/ijbs.121255

Figure Lengend Snippet: Schematic model depicting the intercellular signaling axis between ethanol-injured hepatocytes and neutrophils during ASH progression. Ethanol exposure triggers IL-1α release from hepatocytes, acting as a key damage-associated molecular pattern (DAMPs). This hepatocyte-derived IL-1α upregulates TLR9 expression in recruited neutrophils (①), thereby promoting a phenotypic switch toward a SiglecF⁺ NET-prone state (②). Enhanced NET formation follows (③), releasing extracellular NET-DNA, which is subsequently internalized by hepatocytes. Intracellular NET-DNA activates the cytosolic DNA sensor AIM2, leading to caspase-3 cleavage and hepatocyte apoptosis. This positive feedback loop sustains sterile hepatic inflammation and accelerates ASH pathogenesis.

Article Snippet: To evaluate the effect of NET-DNA and IL-1α signaling, mice were treated with DNase I (10 U in 100 μL 0.9% NaCl, Intraperitoneal injection (i.p.); Cat. No. EN0521, Thermo Fisher Scientific/Fermentas, USA) or the TLR9 inhibitor E6446 (20 mg/kg, Oral administration (p.o.); Cat. No. S6719, Selleck, China) at 24 and 15 hours prior to tissue harvest.

Techniques: Derivative Assay, Expressing, Sterility

Journal: Journal of Virology

Article Title: African swine fever virus infection enhances CD14-dependent phagocytosis of porcine alveolar macrophages to promote bacterial uptake and apoptotic body-mediated viral transmission

doi: 10.1128/jvi.00690-25

Figure Lengend Snippet: The upregulated CD14 in bystander PAMs facilitates viral transmission by enabling the phagocytosis of more ASFV-containing apoptotic bodies. ( A ) The supernatants from PAMs infected with ASFV (MOI 1) at 3, 6, 12, and 24 hpi were collected, filtered through a 0.1 µm PVDF filter to remove ASFV, and then added to untreated PAMs for culturing for 3 h. The relative level of CD14 mRNA in PAMs was analyzed by qPCR (mean of three independent experiments ± SD). ( B ) The ASFV-removed supernatants from PAMs at 12 and 24 hpi were cultured with untreated PAMs for 3 h, respectively, and then infected with E. coli -EGFP (MOI 10) for 2 h. The “positive CTRL group” represents PAMs that were infected with unfiltered ASFV before bacterial infection. Flow cytometry was used to detect the phagocytic rate of PAMs (mean of three independent experiments ± SD). hpt, hours post-treatment. ( C ) PAMs were incubated with different cytokines at a concentration of 1 ng/mL (20 ng/mL for GM-CSF) for 3 h. The relative level of CD14 mRNA in PAMs was analyzed by qPCR (mean of four independent experiments ± SD). ( D ) The supernatants from PAMs infected with ASFV (MOI 1) at 12 and 24 hpi were collected, filtered through a 0.1 µm PVDF filter to remove ASFV, and extracted viral DNA. The level of CP204L was analyzed by qPCR, and the copy number was calculated using a standard curve (mean of three independent experiments ± SD). ( E ) PAMs were treated for 3 h with the ASFV-removed supernatant that was treated with DNase (4 U/100 µL) at 37°C for 30 min. The expression of CD14 was detected by flow cytometry (mean of three independent experiments ± SD). ( F, G ) PAMs were pre-treated with the TLR9 inhibitor E6446 (10 µM) for 1 h, and then with extracted viral DNA (24 hpi) for 3 h. The DNA extracted from the filtered supernatant of CTRL PAMs was used as the control. All groups were infected with E. coli -EGFP (MOI 10) for 2 h. CD14 MFI ( F ) and phagocytosis of E. coli -EGFP ( G ) were analyzed by flow cytometry (mean of three independent experiments ± SD). ( H ) PAMs were infected with ASFV-mCherry (MOI 2) for 24 hpi. ApoBDs were observed by IFA. Nuclei were stained with DAPI. Scale bar, 100 µm. ( I ) PAMs were infected with ASFV-mCherry (MOI 2) for 24 hpi. Isolated ApoBDs were observed by IFA. Phosphatidylserine (PS) was stained with Annexin V-Alexa 488 at room temperature for 15 min. The arrows point to only a subset of representative ApoBDs containing ASFV-mCherry. Scale bar, 25 µm. ( J ) PAMs were treated with siCD14 for 24 h, then incubated with ASFV-removed supernatant for another 3 h, and subsequently infected with ApoBDs-mCherry for 1 h. The DNA was extracted, and the level of mCherry was analyzed by qPCR (mean of three independent experiments ± SD). ( K ) PAMs were treated with siCD14 for 24 h, then incubated with ASFV-removed supernatant for another 3 h, and subsequently infected with ApoBDs-mCherry for 12 h. The infection efficiency of PAMs was observed by IFA. Nuclei were stained with DAPI. Representative data from multiple experiments are shown. Scale bar, 100 µm. ( L ) Calculate the percentage of infected PAMs (mean ± SD of 10 samples). ( M ) PAMs were treated with siCD14 for 24 h, then incubated with ASFV-removed supernatant for another 3 h, and subsequently infected with ApoBDs-mCherry for 12 h. The titer of ASFV in PAMs was detected using TCID 50 (mean of three independent experiments ± SD). ( N ) The relative levels of CD14 mRNA in PAMs at 3 hpi were analyzed by qPCR. The control inoculum was harvested from PAMs that had been induced to apoptosis by UV irradiation for 60 min. The supernatant and ApoBDs were isolated from it using the same method as the ASFV inoculum. The supernatant was used as the control for infection with ASFV particles, and the ApoBDs were used as the control for infection with ApoBDs (containing ASFV) (mean of three independent experiments ± SD).

Article Snippet: NF-κB inhibitor BAY 11-7082 (HY-13453), cGAS inhibitor RU.521 (HY-114180), STING inhibitor C176 (HY-112906), TLR9 inhibitor E6446 (HY-12756), and bacterial adhesion inhibitor Sibofimloc (HY-12820) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Transmission Assay, Infection, Cell Culture, Flow Cytometry, Incubation, Concentration Assay, Expressing, Control, Staining, Isolation, Irradiation

Journal: Journal of Virology

Article Title: African swine fever virus infection enhances CD14-dependent phagocytosis of porcine alveolar macrophages to promote bacterial uptake and apoptotic body-mediated viral transmission

doi: 10.1128/jvi.00690-25

Figure Lengend Snippet: Model of ASFV infection enhances the phagocytosis of PAMs. In the early stages of ASFV internalization, the viral dsDNA released into the cytoplasm is recognized by the cGAS-STING pathway, inducing the downstream nuclear translocation of NF-κB p65. This ultimately activates the transcription and expression of CD14 in PAMs. Free viral DNA released from infected PAMs enters bystander PAMs and enhances CD14 expression via the TLR9 pathway. The high expression of CD14 on the cell membrane surface facilitates phagocytosis in PAMs and induces a stronger transcription of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α). The upregulation of CD14 in bystanders enhances the phagocytosis of ApoBDs-containing ASFV, which benefits the transmission of the virus.

Article Snippet: NF-κB inhibitor BAY 11-7082 (HY-13453), cGAS inhibitor RU.521 (HY-114180), STING inhibitor C176 (HY-112906), TLR9 inhibitor E6446 (HY-12756), and bacterial adhesion inhibitor Sibofimloc (HY-12820) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Infection, Translocation Assay, Expressing, Membrane, Transmission Assay, Virus