tlr9 agonist odn1668  (InvivoGen)

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicles Derived From Streptococcus anginosus Aggravate Lupus Nephritis by Triggering TLR2‐MyD88‐NF‐κB Signalling in NK Cells

doi: 10.1002/jev2.70134

Figure Lengend Snippet: Reduced frequency yet heightened cytotoxicity of peripheral blood NK cells in SLE patients. (a) Representative flow cytometry plots depicting gating strategy for TBNK cell populations. (b) Quantitative analysis of TBNK subpopulations in SLE patients ( n = 54) versus healthy controls (HCs; n = 43). (c) Correlation of peripheral blood NK cell percentage and SLEDAI score in SLE patients. (d) Intracellular expression of Granzyme B and TNF‐α in primary human NK cells following stimulation with PMA (10 ng/mL), ionomycin (1 µg/mL) and brefeldin A (BFA; 1 µg/mL) for 12 h, analysed by flow cytometry. (e) Quantification of granzyme B and TNF‐α expression. (f) Serum concentration of IL‐6, IL‐17A and TNF‐α in the serum of HC and SLE ( n = 12). (g) The cytotoxicity of NK against K‐562 cells at varying effector: target cell ratios. NK cells were purified from PBMC. NK cells and target cells were co‐cultured with tumour cells for 4 h. (h) Correlation between NK cell percentage and SLEDAI score in SLE patients ( n = 12). (i) Correlation of NK cell cytotoxicity with Streptococcus anginosus abundance in SLE patients ( n = 12). (j) Surface expression of TLR2, TLR4, TLR6, TLR7 and TLR9 on human NK cells. (k) Quantitative analysis of TLR receptor mean fluorescence intensity (MFI) on NK cells ( n = 6). Data are shown as mean ± SD, with individual data points representing biological replicates (average of technical duplicates). Statistical comparisons were performed using one‐way ANOVA with Tukey post‐tests. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ns indicates not significant.

Article Snippet: TLR2 inhibitor (C29, Cat: HY‐100461), TLR4 (Stepharine, HY‐N9347), TLR7 (Enpatoran, HY‐134581A), TLR9 (AT791, HY‐124603) and MyD88 inhibitor (MyD88‐IN‐1, Cat: HY‐149992) were obtained from MedChemExpress (MCE, USA).

Techniques: Flow Cytometry, Expressing, Concentration Assay, Purification, Cell Culture, Fluorescence

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