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tlr9 inhibitor e6446  (MedChemExpress)


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    MedChemExpress tlr9 inhibitor e6446
    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 <t>TLR9</t> inhibitor <t>E6446</t> (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).
    Tlr9 Inhibitor E6446, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "African swine fever virus infection enhances CD14-dependent phagocytosis of porcine alveolar macrophages to promote bacterial uptake and apoptotic body-mediated viral transmission"

    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

    Journal: Journal of Virology

    doi: 10.1128/jvi.00690-25

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

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



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    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 <t>TLR9</t> inhibitor <t>E6446</t> (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).
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    FIGURE 1 Innate immune responses to AAV serotypes in human blood. (A–D) Whole blood from healthy human donors was stimulated with AAV6-GFP or AAV9-GFP at 0,3,10, and 30 E10 vg or <t>TLR9</t> agonist CpGC. Scatter plots of secreted IFN-a and IL-6 in response to AAV6-GFP (A, B) or AAV9-GFP (C, D). Data in (A, C) were combined from two independent experiments (n=8 donors). Data in (B, D) represent one independent experiment with (n=4). (E, F) Whole blood from healthy human donors was stimulated with mammalian cell-derived AAV9-HEK carrying a human transgene at 10 E10 vg or TLR9 agonist CpGC. Scatter plots of secreted IFN-a (E) and IL-6 (F) in response to AAV9-HEK. P-value was determined based on a one-way ANOVA nonparametric multiple comparison test with Dunn method, *P <0.05, and **P <0.01. “Unstim” indicates unstimulated.
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    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).

    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

    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.

    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

    Primer sequences for qPCR.

    Journal: Life

    Article Title: Osteogenic CpG Oligodeoxynucleotide, iSN40, Inhibits Osteoclastogenesis in a TLR9-Dependent Manner

    doi: 10.3390/life14121572

    Figure Lengend Snippet: Primer sequences for qPCR.

    Article Snippet: A TLR9 inhibitor, dihydrochloride (E6446) (MedChem Express, Monmouth Junction, NJ, USA), was dissolved in dimethyl sulfoxide.

    Techniques: Sequencing

    iSN40 inhibits osteoclastogenesis in a TLR9-dependent manner. ( A ) Representative images of the TRAP staining of RAW264.7 cells treated with 30 ng/mL RANKL, 0.3 μM ODN, and 1 μM E6446 for 6 days. Scale bar, 100 μm. ( B ) The number of nuclei in TRAP + osteoclasts was quantified. No TRAP + cells were observed in the iSN40/E6446(−) and CpG-2006/E6446(−) groups. NS, no significant difference, ** p < 0.01 vs. each E6446(−) group. n = 4 fields. ( C ) qPCR results of RAW264.7 cells treated with 30 ng/mL RANKL, 0.3 μM ODNs, and 1 μM E6446 for 24 h. ** p < 0.01 vs. control/E6446(−), †† p < 0.01 vs. iSN40/E6446(−), ‡‡ p < 0.01 vs. CpG-2006/E6446(−). n = 3.

    Journal: Life

    Article Title: Osteogenic CpG Oligodeoxynucleotide, iSN40, Inhibits Osteoclastogenesis in a TLR9-Dependent Manner

    doi: 10.3390/life14121572

    Figure Lengend Snippet: iSN40 inhibits osteoclastogenesis in a TLR9-dependent manner. ( A ) Representative images of the TRAP staining of RAW264.7 cells treated with 30 ng/mL RANKL, 0.3 μM ODN, and 1 μM E6446 for 6 days. Scale bar, 100 μm. ( B ) The number of nuclei in TRAP + osteoclasts was quantified. No TRAP + cells were observed in the iSN40/E6446(−) and CpG-2006/E6446(−) groups. NS, no significant difference, ** p < 0.01 vs. each E6446(−) group. n = 4 fields. ( C ) qPCR results of RAW264.7 cells treated with 30 ng/mL RANKL, 0.3 μM ODNs, and 1 μM E6446 for 24 h. ** p < 0.01 vs. control/E6446(−), †† p < 0.01 vs. iSN40/E6446(−), ‡‡ p < 0.01 vs. CpG-2006/E6446(−). n = 3.

    Article Snippet: A TLR9 inhibitor, dihydrochloride (E6446) (MedChem Express, Monmouth Junction, NJ, USA), was dissolved in dimethyl sulfoxide.

    Techniques: Staining, Control

    TLR9-independent pro-osteogenic and TLR9-dependent anti-osteoclastogenic effects of iSN40, iSN40-GC, and iSN41. CpG motifs are underlined.

    Journal: Life

    Article Title: Osteogenic CpG Oligodeoxynucleotide, iSN40, Inhibits Osteoclastogenesis in a TLR9-Dependent Manner

    doi: 10.3390/life14121572

    Figure Lengend Snippet: TLR9-independent pro-osteogenic and TLR9-dependent anti-osteoclastogenic effects of iSN40, iSN40-GC, and iSN41. CpG motifs are underlined.

    Article Snippet: A TLR9 inhibitor, dihydrochloride (E6446) (MedChem Express, Monmouth Junction, NJ, USA), was dissolved in dimethyl sulfoxide.

    Techniques:

    FIGURE 1 Innate immune responses to AAV serotypes in human blood. (A–D) Whole blood from healthy human donors was stimulated with AAV6-GFP or AAV9-GFP at 0,3,10, and 30 E10 vg or TLR9 agonist CpGC. Scatter plots of secreted IFN-a and IL-6 in response to AAV6-GFP (A, B) or AAV9-GFP (C, D). Data in (A, C) were combined from two independent experiments (n=8 donors). Data in (B, D) represent one independent experiment with (n=4). (E, F) Whole blood from healthy human donors was stimulated with mammalian cell-derived AAV9-HEK carrying a human transgene at 10 E10 vg or TLR9 agonist CpGC. Scatter plots of secreted IFN-a (E) and IL-6 (F) in response to AAV9-HEK. P-value was determined based on a one-way ANOVA nonparametric multiple comparison test with Dunn method, *P <0.05, and **P <0.01. “Unstim” indicates unstimulated.

    Journal: Frontiers in immunology

    Article Title: Essential role of pre-existing humoral immunity in TLR9-mediated type I IFN response to recombinant AAV vectors in human whole blood.

    doi: 10.3389/fimmu.2024.1354055

    Figure Lengend Snippet: FIGURE 1 Innate immune responses to AAV serotypes in human blood. (A–D) Whole blood from healthy human donors was stimulated with AAV6-GFP or AAV9-GFP at 0,3,10, and 30 E10 vg or TLR9 agonist CpGC. Scatter plots of secreted IFN-a and IL-6 in response to AAV6-GFP (A, B) or AAV9-GFP (C, D). Data in (A, C) were combined from two independent experiments (n=8 donors). Data in (B, D) represent one independent experiment with (n=4). (E, F) Whole blood from healthy human donors was stimulated with mammalian cell-derived AAV9-HEK carrying a human transgene at 10 E10 vg or TLR9 agonist CpGC. Scatter plots of secreted IFN-a (E) and IL-6 (F) in response to AAV9-HEK. P-value was determined based on a one-way ANOVA nonparametric multiple comparison test with Dunn method, *P <0.05, and **P <0.01. “Unstim” indicates unstimulated.

    Article Snippet: Blood samples were incubated with TLR9 inhibitor E6446 (Selleckchem.com, Cat#S0716) at three concentrations: 0.2, 2, and 20 μM for 60 minutes.

    Techniques: Derivative Assay, Comparison

    FIGURE 4 Inhibition of TLR9 suppressed Type IFN response to AAV. Whole blood samples from healthy human donors identified previously as responders to AAV9 or AAV6 vectors were incubated with E6466 inhibitor at three concentrations 0.2, 2, or 10 µM for one hour prior to stimulation with AAV9-GFP or AAV6-GFP at 10 E10 vg for 24 hours. (A) Scatter plots and (B) line plot of concentration-dependent E6466 inhibition of IFN-a response AAV9- GFP. (C) Scatter plots and (D) line plot of concentration-dependent E6466 inhibition of IFN-a response to AAV6-GFP. Data represent mean ± SEM and representative of two independent experiments (n=4 per experiment). P-value was determined based one-way ANOVA with nonparametric multiple comparison Wilcoxon method, *P <0.05.

    Journal: Frontiers in immunology

    Article Title: Essential role of pre-existing humoral immunity in TLR9-mediated type I IFN response to recombinant AAV vectors in human whole blood.

    doi: 10.3389/fimmu.2024.1354055

    Figure Lengend Snippet: FIGURE 4 Inhibition of TLR9 suppressed Type IFN response to AAV. Whole blood samples from healthy human donors identified previously as responders to AAV9 or AAV6 vectors were incubated with E6466 inhibitor at three concentrations 0.2, 2, or 10 µM for one hour prior to stimulation with AAV9-GFP or AAV6-GFP at 10 E10 vg for 24 hours. (A) Scatter plots and (B) line plot of concentration-dependent E6466 inhibition of IFN-a response AAV9- GFP. (C) Scatter plots and (D) line plot of concentration-dependent E6466 inhibition of IFN-a response to AAV6-GFP. Data represent mean ± SEM and representative of two independent experiments (n=4 per experiment). P-value was determined based one-way ANOVA with nonparametric multiple comparison Wilcoxon method, *P <0.05.

    Article Snippet: Blood samples were incubated with TLR9 inhibitor E6446 (Selleckchem.com, Cat#S0716) at three concentrations: 0.2, 2, and 20 μM for 60 minutes.

    Techniques: Inhibition, Incubation, Concentration Assay, Comparison