Journal: Molecular Medicine

Article Title: Deficiency of mindin reduces renal injury after ischemia reperfusion

doi: 10.1186/s10020-022-00578-2

Figure Lengend Snippet: Mindin deficiency inhibited TLR4/JNK/NF-κB signaling activation in renal IR injury A TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα measured by western blot analysis in kidneys of WT and mindin KO mice induced by IR or not. B – E Quantitative analysis of TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα protein expression. *P < 0.05 versus WT Sham or KO Sham. # P < 0.05 versus WT IR

Article Snippet: Mindin protein (HY-P70142, MedChemExpress) and TLR4 protein (HY-P73586, MedChemExpress) were used to study the protein binding in CM5 electronic chips by biomolecular interaction instrument (Biacore T2000).

Techniques: Activation Assay, Western Blot, Expressing

Journal: Molecular Medicine

Article Title: Deficiency of mindin reduces renal injury after ischemia reperfusion

doi: 10.1186/s10020-022-00578-2

Figure Lengend Snippet: Mindin overexpression promoted production of inflammatory mediators and activation of TLR4/JNK/ NF-κB signaling after HR in vitro. A Representative photomicrographs showing the immunofluorescence of GFP in adGFP and adMindin cells. B Mindin protein expression in adGFP and adMindin cells. C – E Protein levels of TNF-α and MCP-1 in the media of cultured TECs by Western blot. *P < 0.05 versus adGFP Con or adMindn Con. # P < 0.05 versus adGFP HR. F TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα measured by western blot analysis in adGFP and adMindin cells induced by HR or not. G – J Quantitative analysis of TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα protein expression. *P < 0.05 versus adGFP Con or adMindn Con. # P < 0.05 versus adGFP HR. K Quantitative analysis of TLR4, JNK, P65, TNF-α and MCP-1 mRNA expression in mindin-overexpressed HK-2 cells. ***P < 0.001 versus adGFP HR group

Article Snippet: Mindin protein (HY-P70142, MedChemExpress) and TLR4 protein (HY-P73586, MedChemExpress) were used to study the protein binding in CM5 electronic chips by biomolecular interaction instrument (Biacore T2000).

Techniques: Over Expression, Activation Assay, In Vitro, Immunofluorescence, Expressing, Cell Culture, Western Blot

Journal: Molecular Medicine

Article Title: Deficiency of mindin reduces renal injury after ischemia reperfusion

doi: 10.1186/s10020-022-00578-2

Figure Lengend Snippet: Mindin knockdown inhibited the activation of TLR4/JNK/ NF-κB signaling after HR in vitro. A TLR4, Mindin, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα measured by western blot analysis in si-Mindin, si-TLR4 and si-JNK cells induced by HR respectively. B – F Quantitative analysis of TLR4, Mindin, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα protein expression. * P < 0.05, ** P < 0.01 versus HR. G Quantitative analysis of TLR4, JNK, P65, TNF-α and MCP-1 mRNA expression in si-Mindin, si-TLR4 and si-JNK cells induced by HR respectively. ***P < 0.001 versus control HR group

Article Snippet: Mindin protein (HY-P70142, MedChemExpress) and TLR4 protein (HY-P73586, MedChemExpress) were used to study the protein binding in CM5 electronic chips by biomolecular interaction instrument (Biacore T2000).

Techniques: Knockdown, Activation Assay, In Vitro, Western Blot, Expressing, Control

Journal: Molecular Medicine

Article Title: Deficiency of mindin reduces renal injury after ischemia reperfusion

doi: 10.1186/s10020-022-00578-2

Figure Lengend Snippet: Co-immunoprecipitation of TLR4 and mindin in HK-2 cells. Lysates of HK-2 cells were incubated with anti-TLR4 antibody and immune complexes were precipitated by protein A/G beads. The TLR4 complex was determined TLR4 and mindin proteins in the complex. The whole cell lysate was used to detect TLR4 and mindin by Western blot

Article Snippet: Mindin protein (HY-P70142, MedChemExpress) and TLR4 protein (HY-P73586, MedChemExpress) were used to study the protein binding in CM5 electronic chips by biomolecular interaction instrument (Biacore T2000).

Techniques: Immunoprecipitation, Incubation, Western Blot

Journal: Molecular Medicine

Article Title: Deficiency of mindin reduces renal injury after ischemia reperfusion

doi: 10.1186/s10020-022-00578-2

Figure Lengend Snippet: Binding assy of mindin to TLR4 proteins by SPR. Different concen-trations of TLR4 protein (3.125, 6.25, 12.5, 25.0, 50.0, 100.0 nM) were performed to analyse the binding and dissociation rate constants between mindin and TLR4 proteins. The binding constant of them was calculated to evaluate protein binding

Article Snippet: Mindin protein (HY-P70142, MedChemExpress) and TLR4 protein (HY-P73586, MedChemExpress) were used to study the protein binding in CM5 electronic chips by biomolecular interaction instrument (Biacore T2000).

Techniques: Binding Assay, Protein Binding

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 2. Upregulation of HMGB1, TLR4, pIKBα and GFAP in astrocytes and brain microvessel endothelial cells in MCAO rats. (A, B and D) Compared with the sham group, the expression of HMGB1, TLR4, pIKBα and GFAP was potentiated in the cerebral cortex tissues of MCAO rats. The scale bars represent 60 and 30 μm in (A) and (B) respectively. (C and E) Consistently, the HMGB1 (red) in astrocytes in MCAO group translocated from nucleus (Blue color represents DAPI) to the cytoplasm (Green color represents GFAP). The expression of TLR4 (yellow) and pIKBα (yellow) in brain microvessel endothelial cells (Green color represents VIII factor) in MCAO group was potentiated respectively. The expression of GFAP (green) in astrocytes (Blue color represents DAPI) in MCAO group was also increased. The scale bars represent 10 μm. (D and E) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques: Expressing

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 8. HMGB1 contributed to upregulation of P-gp via TLR4/NF-kB pathway. (A) The relationship among HMGB1 and changes of TLR4, TIRAP, NF-kB and P-gp in rBMECs after OGD was investigated using related positive agents. (B) Changes of TLR4 (green) and TIRAP (red) after OGD and treatment with positive agents in rBMECs. (C) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group; #P < 0.05 vs. OGD group. The scale bars represent 10 μm.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques:

Journal: Microbiome

Article Title: Ochratoxin A induces liver inflammation: involvement of intestinal microbiota

doi: 10.1186/s40168-019-0761-z

Figure Lengend Snippet: OTA promotes liver inflammation. a Relative mRNA expressions of TLR4, MYD88, IKBα, IL-6, and TNF-α in the liver after OTA oral gavage ( n = 6, mean with SEM). b Relative protein abundances of TLR4, MYD88, p-IKBα, p-IKBα/IKBα, and p-p65 in the liver after OTA oral gavage ( n = 6, mean with SEM). c Effect of OTA on levels of liver cytokines, including IL-1β, IL-6, TNF-α, IL-8, IL-10, and IFN-γ ( n = 6, mean with SEM). d Representative images of H&E-stained liver sections in CON and OTA group (magnification × 400, scale bar 100 μm, n = 6). e Statistical analysis of the percentage of inflammatory cells in different groups shown in d ( n = 6, mean with SEM). f Effect of OTA on serum levels of AST, ALT, ALP, and LDH ( n = 6, mean with SEM). g Serum LPS level with or without OTA treatment ( n = 6, mean with SEM). h Effect of OTA on serum levels of IL-1β, IL-6, TNF-α, and IL-10 ( n = 6, mean with SEM). Data in a , b , c , e , f , g , and h were analyzed with unpaired t test, * P < 0.05; ** P < 0.01, *** P < 0.001, P <0.0001

Article Snippet: Proteins were loaded onto the SDS-PAGE gel (BioRad) and electrophoresed and analyzed by WB using antibodies against TLR4 (BA1717, Boster, Wuhan, China), MYD88 (abs135682, Absin, Shanghai, China), IKBα (D120138, Sangon Biotech, Shanghai, China), p-IKBα (D151548, Sangon Biotech, Shanghai, China), p-p65 (HZ4902812, TW reagent, Shanghai, China), TJP-1 (mAb13663, Cell Signaling, USA), Occludin (ab167161, Abcam, USA), and actin (60008, Proteintech, USA).

Techniques: Staining

Journal: Microbiome

Article Title: Ochratoxin A induces liver inflammation: involvement of intestinal microbiota

doi: 10.1186/s40168-019-0761-z

Figure Lengend Snippet: OTA fails to promote liver inflammation in antibiotics-treated ducks. a Liver LPS level in different groups ( n = 6, mean with SEM). b Relative mRNA expressions of TLR4, MYD88, IKBα, IL-6, and TNF-α in the liver of antibiotics-treated ducks with or without OTA ( n = 6, mean with SEM). c Levels of IL-1β, IL-6, TNF-α, IL-8, IL-10, and IFN-γ in the liver of antibiotics-treated ducks with or without OTA ( n = 6, mean with SEM). d Representative images of H&E-stained liver sections (magnification × 400; scale bar 100 μm; n = 6). e Statistical analysis of the percentage of inflammatory cells in different groups shown in d ( n = 6, mean with SEM). f Effect of OTA on serum levels of AST, ALT, ALP and LDH in antibiotics-treated ducks ( n = 6, mean with SEM). g Serum LPS level with or without OTA treatment in antibiotics-treated ducks ( n = 6, mean with SEM). h Effect of OTA on serum levels of IL-1β, IL-6, TNF-α, and IL-10 ( n = 6, mean with SEM). Data were analyzed with unpaired t test. n.s., not significant

Article Snippet: Proteins were loaded onto the SDS-PAGE gel (BioRad) and electrophoresed and analyzed by WB using antibodies against TLR4 (BA1717, Boster, Wuhan, China), MYD88 (abs135682, Absin, Shanghai, China), IKBα (D120138, Sangon Biotech, Shanghai, China), p-IKBα (D151548, Sangon Biotech, Shanghai, China), p-p65 (HZ4902812, TW reagent, Shanghai, China), TJP-1 (mAb13663, Cell Signaling, USA), Occludin (ab167161, Abcam, USA), and actin (60008, Proteintech, USA).

Techniques: Staining

Journal: Microbiome

Article Title: Ochratoxin A induces liver inflammation: involvement of intestinal microbiota

doi: 10.1186/s40168-019-0761-z

Figure Lengend Snippet: OTA-originated microbiota induces liver inflammation. a Liver LPS level in FMT (CON) and FMT (OTA) ducks. b Relative mRNA expressions of TLR4, MYD88, IKBα, IL-6, and TNF-α in the liver after FMT ( n = 6, mean with SEM). c Relative protein abundance of TLR4, MYD88, p-IKBα, p-IKBα/IKBα, and p-p65 in the liver after FMT ( n = 6, mean with SEM). d Effect of FMT on the liver levels of IL-1β, IL-6, TNF-α, and IL-10 ( n = 6, mean with SEM). e Representative H&E-stained liver sections. f Statistical analysis of the percentage of inflammatory cells in different groups shown in e ( n = 6, mean with SEM). g Serum levels of AST, ALT, ALP, and LDH in different groups ( n = 6, mean with SEM). h Serum LPS level in different FMT groups ( n = 6, mean with SEM). i Serum levels of IL-1β, IL-6, and TNF-α after FMT ( n = 6, mean with SEM). FMT (CON): ducks received the CON group fecal microbiota. FMT (OTA): ducks received the OTA group fecal microbiota. Data were analyzed with unpaired t test, n = 6. * P < 0.05; ** P < 0.01, *** P < 0.001, **** P <0.0001

Article Snippet: Proteins were loaded onto the SDS-PAGE gel (BioRad) and electrophoresed and analyzed by WB using antibodies against TLR4 (BA1717, Boster, Wuhan, China), MYD88 (abs135682, Absin, Shanghai, China), IKBα (D120138, Sangon Biotech, Shanghai, China), p-IKBα (D151548, Sangon Biotech, Shanghai, China), p-p65 (HZ4902812, TW reagent, Shanghai, China), TJP-1 (mAb13663, Cell Signaling, USA), Occludin (ab167161, Abcam, USA), and actin (60008, Proteintech, USA).

Techniques: Quantitative Proteomics, Staining

Journal: European journal of immunology

Article Title: Gx-50 reduces β-amyloid-induced TNF-α, IL-1β, NO, and PGE2 expression and inhibits NF-κB signaling in a mouse model of Alzheimer's disease.

doi: 10.1002/eji.201545855

Figure Lengend Snippet: Figure 5. Effect of gx-50 on TLR4 and recruited proteins in primary microglia and APP-Tg mice. (A) mRNA of TLR4 in primary microglia pretreated with gx-50 in the absence or presence of Aβ42 was detected by real-time PCR. GAPDH was used as internal control. Fold changes were calculated with the 2−Ct method. (B) Western blot was performed to detect the protein of TLR4 in primary microglia and APP-Tg mice (n = 8 mice per group). (C) TLR4 downstream proteins, MyD88 and TRAF6, were measured by Western blot analysis in primary microglia and APP-Tg mice (n = 8 mice per group). GAPDH was used as internal control. In the histogram, the control group and APP−/saline group were standardized to onefold. Data are shown as mean ± SD and pooled from three independent experiments with 20 or 32 mice per experiment, *p < 0.05, **p < 0.01 (one-way ANOVA/Dunnett’s test).

Article Snippet: After blocking with 5% skimmed milk at room temperature for 1 h, the membranes were incubated with antibodies against COX2 (1:500; Proteintech, USA), iNOS (1:500; Abcam, USA), p65, phosphop38, phospho-ERK1/2, phospho-JNK, phospho-IκB (1:1000; CST, USA), TLR4 (1:200, Biorbyt, UK), MyD88 (1:500; Bioworld, USA), TRAF6 (1:500; Proteintech), or GAPDH (1:2000; CST) at 4°C overnight.

Techniques: Real-time Polymerase Chain Reaction, Control, Western Blot, Saline

Journal: European journal of immunology

Article Title: Gx-50 reduces β-amyloid-induced TNF-α, IL-1β, NO, and PGE2 expression and inhibits NF-κB signaling in a mouse model of Alzheimer's disease.

doi: 10.1002/eji.201545855

Figure Lengend Snippet: Figure 6. Effect of gx-50 on TLR4 knockdown and overexpression in BV2 cells. TLR4 was knocked down and overexpression by transfection with TLR4-siRNA and pEGFP-N1-TLR4 plasmid, respectively. After transfection, BV2 microglial cells were pretreated with gx-50 for 30 min and then incubated with Aβ42. Supernatants and cells were collected for ELISA and Western blot analysis. (A) The secretion of IL-1β was examined by ELISA. (B) The expression of TRAF6 was measured by Western blot. (C) The expression of MyD88 and TRAF6 was measured by Western blot. GAPDH was used as internal control. In the histogram, the control group was standardized to onefold. Data are shown as mean ± SD and pooled from three independent experiments with four samples per experiment * p < 0.05, **p < 0.01 (one-way ANOVA/Dunnett’s test).

Article Snippet: After blocking with 5% skimmed milk at room temperature for 1 h, the membranes were incubated with antibodies against COX2 (1:500; Proteintech, USA), iNOS (1:500; Abcam, USA), p65, phosphop38, phospho-ERK1/2, phospho-JNK, phospho-IκB (1:1000; CST, USA), TLR4 (1:200, Biorbyt, UK), MyD88 (1:500; Bioworld, USA), TRAF6 (1:500; Proteintech), or GAPDH (1:2000; CST) at 4°C overnight.

Techniques: Knockdown, Over Expression, Transfection, Plasmid Preparation, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control

Journal: European journal of immunology

Article Title: Gx-50 reduces β-amyloid-induced TNF-α, IL-1β, NO, and PGE2 expression and inhibits NF-κB signaling in a mouse model of Alzheimer's disease.

doi: 10.1002/eji.201545855

Figure Lengend Snippet: Figure 7. Schematic showing the anti- inflammatory effect of gx-50 via a mechanism involving a TLR4-meditated NF-κB /MAPK signaling cascade. Aβ initiates the inflam- mation by triggering the activation of TLR4, which subsequently recruits MyD88 and TRAF6, leading to the activation of NF-κB and MAPK pathway. The activation of NF-κB or MAPK directly controls the gene expression of pro-inflammatory mediators. However, gx-50 inhibits the TLR4 signal pathway and also shows direct disaggregation on Aβ to interfere with further inflammatory responses or neuronal damage.

Article Snippet: After blocking with 5% skimmed milk at room temperature for 1 h, the membranes were incubated with antibodies against COX2 (1:500; Proteintech, USA), iNOS (1:500; Abcam, USA), p65, phosphop38, phospho-ERK1/2, phospho-JNK, phospho-IκB (1:1000; CST, USA), TLR4 (1:200, Biorbyt, UK), MyD88 (1:500; Bioworld, USA), TRAF6 (1:500; Proteintech), or GAPDH (1:2000; CST) at 4°C overnight.

Techniques: Activation Assay, Gene Expression

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: Primer used for Real-time qPCR.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Sequencing

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: Cell surface level of TLR4 in BV-2 cells treated with chestnut leaf or spiny bur extracts and LPS. BV-2 cells were pre-treated (or not) with spiny bur (SB) or leaf (L) extracts (0.5 mg/mL) for 3 h, incubated with or without LPS (0.5 μg/mL) for a total of 24 h, then subjected to flow cytometric analysis of the immunostaining for TLR4 receptor. ( a ) Representative plots of untreated BV-2 cells ( b ) Representative plots of BV-2 cells not activated with LPS ( c ) Representative plots of LPS-stimulated BV-2 cells ( d ) Relative quantification is expressed as MFI, median fluorescence intensity. Results are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by Fisher’s LSD test following one-way ANOVA. ° p < 0.05 significantly different from control cells; * p < 0.05 significantly different from LPS-treated cells.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Incubation, Immunostaining, Fluorescence, Control

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: RT-PCR analysis of TLR4 gene expression in BV-2 cells treated with chestnut extracts. BV-2 cells were treated with spiny bur or leaf extracts (0.5 mg/mL) for 3 h, then stimulated (or not) with LPS (0.5 µg/mL) for a total of 24 h. At the end of incubation, RNA was extracted from cells and samples were subjected to RT-PCR analysis using a specific primer for TLR4, as explained in the Materials and Methods section. The relative mRNA content of TLR4 was normalised to ( a ) untreated control and ( b ) untreated LPS control. Relative quantification is obtained as reported in the Materials and Methods section; results are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by Fisher’s LSD test following one-way ANOVA. ° p < 0.05 significantly different from control cells; * p < 0.05 significantly different from LPS-treated cells.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Control

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: RT-PCR analysis of TLR4 and CD14 gene expression in BV-2 cells treated with different chestnut leaf extract fractions. BV-2 cells were treated for 3 h with 50 μg/mL of fractions at different polarity (L Fr.), then stimulated with 0.5 μg/mL LPS for a total of 24 h. At the end of incubation, RNA was extracted from cells and samples were subjected to RT-PCR analysis using a specific primer for TLR4 ( a ) and CD14 ( b ), as reported in the Materials and Methods section. The relative mRNA contents of TLR4 and CD14 were normalised to LPS-treated cells (red bars). Relative quantification is obtained as described in the Materials and Methods section, and results are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by Fisher’s LSD test following one-way ANOVA. * p < 0.05 significantly different from LPS-treated cells.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation

Journal: Frontiers in Oral Health

Article Title: Blockade of connexin43-containing hemichannel attenuates the LPS-induced inflammatory response in human dental pulp cells by inhibiting the extracellular flux of ATP and HMGB1

doi: 10.3389/froh.2024.1496819

Figure Lengend Snippet: Schematic representation of the potential roles and mechanisms of Cx43 in LPS-induced inflammation in hDPCs. LPS interacts with TLR-4 on the cell membrane, leading to the phosphorylation and subsequent degradation of IκBα, which in turn facilitates the nuclear translocation of NF-κB, ultimately triggering inflammation in hDPCs. Additionally, LPS stimulates the activity of Cx43 hemichannels (HCs), promoting the extracellular release of ATP and HMGB1 through these channels. The released ATP may then bind to P2X7 receptors, further exacerbating LPS-induced inflammation via autocrine and paracrine pathways. Concurrently, the activation of Cx43 HCs by LPS may initiate their fusion with vesicles containing HMGB1 on the plasma membrane. This fusion results in the displacement of the Cx43 HC-HMGB1 complex from the cytoskeleton, allowing for the subsequent release of HMGB1 into the extracellular space. This released HMGB1 may then activate TLR-4 on neighboring cells, further promoting LPS-induced inflammation. Dashed lines in the schematic represent hypothetical inferences.

Article Snippet: The main experimental reagents used in this study included LPS from Sigma (USA), reverse transcription kit and SYBR Premix Ex TaqTM Kit from Novagen (China), phosphorylated NF-κB antibody (p-NF-κB) from CST (USA), NF-κB antibody from Abcam (USA), Toll-like receptor 4 (TLR-4) from Santa (USA), Gap19, Gap26, and HMGB1 from MCE (USA), ATP reagent and ATP detection kit from Bi Yun Tian (China), ELISA kit from Wuhan Sevier (China), and ethidium bromide (EB) and LY from Sigma (USA).

Techniques: Membrane, Phospho-proteomics, Translocation Assay, Activity Assay, Activation Assay, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Lactoferrin Retargets Human Adenoviruses to TLR4 to Induce an Abortive NLRP3-Associated Pyroptotic Response in Human Phagocytes

doi: 10.3389/fimmu.2021.685218

Figure Lengend Snippet: TLR4 engagement and signalling involved in HAdV-lactoferrin DC uptake and maturation. (A) HAdV-lactoferrin complexes were incubated with recombinant TLR4/MD-2 for 30 min. and then added to DCs. Uptake was quantified 24 h post-incubation by flow cytometry (n =11, statistical analyses by two-tailed paired t-test); (B) DCs were pre-treated for 1 h with TAK-242, HAdV-lactoferrin complex uptake was analysed 24 h post-incubation by flow cytometry (n = 6, statistical analyses by two-tailed paired t-test); (C) IL-1β release following pre-treatment with TAK-242 (n ≥ 3 statistical analyses by t-test); (D) TNF levels 24 h post-incubation ± TAK-242 (n ≥ 3, statistical analyses by two-tailed paired t-test); (E) Percent infection following inhibition with Pepinh-TRIF (n ≥ 6, statistical analyses by two-tailed paired t-test); (F) Percent infection following inhibition with oxPAPC (n ≥ 6, statistical analyses by two-tailed paired t-test); (G) IL-1β release from DCs incubated with HAdV-lactoferrin ± oxPAPC and Pepinh-TRIF (n = 3, statistical analyses by two-tailed paired t-test).

Article Snippet: Fluorescence intensity was assessed at 24 h. TLR4 surface expression level was assessed with an anti-TLR4 antibody (Miltenyi Biotech) after 4 or 24 h. DC maturation at 24 h post-incubation was assessed by measuring CD86 surface expression level with an anti-CD86 antibody (clone 2331, BD Biosciences) or by measuring dextran (4.4 kDa) uptake (dextran TRITC, Sigma).

Techniques: Incubation, Recombinant, Flow Cytometry, Two Tailed Test, Infection, Inhibition

Journal: Frontiers in Immunology

Article Title: Lactoferrin Retargets Human Adenoviruses to TLR4 to Induce an Abortive NLRP3-Associated Pyroptotic Response in Human Phagocytes

doi: 10.3389/fimmu.2021.685218

Figure Lengend Snippet: TLR4-mediated HAdV-lactoferrin uptake in DCs and IL-1β release Lactoferrin binds to HAdV capsid and retargets the capsid toward TLR4 complex on the cells surface. Following TLR4 engagement, its TIR domain recruits MyD88 and TIRAP, which bridge TLRs to IRAK and MAPK family members that activate NF-kB, AP-1, and IRF. This priming event initiates transcription of genes coding for inflammasome components (e.g., NLRP3 and IL-1β). Under prototypic conditions, DCs detect a second perturbation (signal 2) that induced ROS release (mitochondrial stress) or K + efflux (perturbations of cellular integrity), and/or cathepsin B release from lysosome rupture. These pathways do not appear to be activated by HAdV-lactoferrin complexes. In addition to TLR4 pathways, the RIPK1-RIPK3 pathway is activated through an autocrine-TNF release. During inflammasome formation, pro-caspase-1 auto-activation induces cleavage of pro-IL-1β and likely GSDMD, which will initiate, but not complete, the loss of plasma membrane integrity via pore formation, allowing IL-1β release. Twenty-four hours post-challenge, DC membrane integrity is intact, consistent with the involvement of ESCRT-III complex and repairing GSDMD pores.

Article Snippet: Fluorescence intensity was assessed at 24 h. TLR4 surface expression level was assessed with an anti-TLR4 antibody (Miltenyi Biotech) after 4 or 24 h. DC maturation at 24 h post-incubation was assessed by measuring CD86 surface expression level with an anti-CD86 antibody (clone 2331, BD Biosciences) or by measuring dextran (4.4 kDa) uptake (dextran TRITC, Sigma).

Techniques: Activation Assay, Clinical Proteomics, Membrane