Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Mapping of the TLR4/MD2/RAGE-interacting motifs of HMGB1 or PTX3. (A, B) Schematic diagram of the domain of HMGB1 (A) and PTX3 (B) (top). GST-tagged truncates covering TLR4-, RAGE-, or MD2-binding domain of HMGB1 or PTX3 were constructed. Bottom panel (A, B) depicts GST-pull down of the indicated tagged receptors (HA-TLR4, Myc-RAGE, and His-MD2) with transiently expressed GST-tagged truncates. HEK293T cells transfected with the GST-tagged constructs in combination with HA-TLR4, Myc-RAGE or His-MD2 are indicated followed by GST-pull down. Representative western blots of WCLs and eluates after GST-pull down are shown, using GAPDH as a loading control in WCLs. Based on pulldown binding results, TLR4-, MD2-, and RAGE-targeted peptides were constructed and designated as H-T, H-R, P-T, or P-M peptides (indicated in panels A, B). (C) BMDMs were incubated with H-T, H-R, P-T, or P-M peptide for the indicated concentrations for 48 h, and then cell viability was measured with cell viability assay. Data shown are the means ± SD of three experiments (D) Cell-free immunoprecipitation assay using H-T, H-R, P-T, or P-M peptides. Mix 5 μg of alarmin protein (rHMGB1 or rPTX3) with 5 μg of each indicated tagged receptors protein (HA-TLR4, Myc-RAGE, and His-MD2), and mix as indicated to confirm the competitive inhibition effect of peptides was treated for 4 h. They were immunoprecipitated with α Flag for 2 h, separated by SDS-PAGE, and evaluated by immunoblotting. (E) Competition between rHMGB1 (top) or PTX3 (bottom) and the constructed peptides for their respective receptors. After treatment with rHMGB1 (top) or PTX3 (bottom) (10 μg/mL, 3 h) together with increasing amounts of the indicated peptides, BMDMs were used for immunoprecipitation (IP) with α TLR4 or α RAGE and IB with indicated antibodies. (F) Decrease of LPS-induced cytokine production by H-T, P-T, or P-M peptide and rHMGB1-induced cytokine production by H-R peptide. TNF- α , IL-6, IL-12p40, and IL-10 levels in the supernatant of LPS (100 ng/mL, 18 h) together with increasing amounts of the indicated peptides-treated BMDMs were determined by ELISA. The data shown represent at least three independent experiments ( n ≥ 3), bars denote mean ± SEM (∗∗∗ P < 0.001, ∗∗∗∗ P < 0.001), and significance was measured by two-way ANOVA. n.s = not significant.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Binding Assay, Construct, Transfection, Western Blot, Control, Incubation, Viability Assay, Immunoprecipitation, Inhibition, SDS Page, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Extracellular HMGB1 and PTX3 contribute to inflammatory responses by interacting with TLR4 or RAGE in murine macrophages. (A, B) Release of HMGB1 (A) or PTX3 (B) from murine macrophages after stimulation with LPS. LPS-induced HMGB1 (A) or PTX3 (B) release from murine macrophages. Immunoblots (IB) for HMGB1 and PTX3 in the supernatant and cell lysates in LPS-treated BMDMs at indicated time points after LPS (100 ng/mL). (C, D) Interaction between HMGB1 (C) or PTX3 (D) with its receptors. HEK293T cells were transfected with constructs, and cell lysates were immunoprecipitated with a flag-specific antibody and were analyzed by immunoblotting with the indicated antibodies. BMDMs were incubated with the rFlag-HMGB1 (C) or rFlag-PTX3 (D) 10 μg/mL for 3 h, and cell lysates were immunoprecipitated with α TLR4 or α RAGE (C, right), and α TLR4 or α MD-2 (D, right). Immunoprecipitants and WCL were analyzed by immunoblotting with the indicated antibodies. (E) TNF- α , IL-6, IL-12p40, and IL-10 levels in the supernatant of LPS (100 ng/mL, 18 h) or recombinant alarmins (rHMGB1 or rPTX3, each (2, 5 μg/mL))-treated BMDMs determined by ELISA. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Inhibitory effects of TMR-Lipo on TLR4/RAGE-mediated responses. (A) LPS-primed BMDMs (100 ng/mL LPS with 12 h) were treated with TMR-Lipo (0.1, 1, 5 μmol/L) for 12 h, followed by co-IP with α TLR4, α RAGE, or α MD2 and IB with indicated antibodies. (B) Schematic representation of TLR4 and RAGE signaling pathway and therapeutic targeting of TMR-Lipo (C) BMDMs were treated with TMR-Lipo as indicated concentrations (2 h), followed by treatment with LPS (100 ng/mL, 8 h). The amounts of HMGB1, PTX3, MyD88, IRAK, TRAF6, NF- κ B, KRAS, and p-p38 were measured by IB in the whole cell lysates. GAPDH was used as a loading control. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗ P < 0.01, ∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA. n.s = not significant. (D) BMDMs were treated with TMR-Lipo (0.1, 0.5, 5, 20, or 100 μmol/L) in the presence LPS for 18 h. The secretion levels of TNF- α , IL-6, IL-12p40 and IL-10 were measured by ELISAs. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗ P < 0.01, ∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA. n.s = not significant.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Co-Immunoprecipitation Assay, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: TMR-Lipo-Abs protects mice from CLP-induced polymicrobial sepsis. (A–C) Schematic design of mid-grade (A) or high-grade (B, C) sepsis mouse model induced by cecal ligation and punctuation (CLP) depending on the position of the cecal ligation (top). The survival of CLP mice treated with the indicated therapy was monitored for 10 days (A) or 72 h (B, C); mortality was measured for n = 10 mice per group (bottom). Statistical differences compared with the PBS or TMR-Lipo-Abs-treated mice are indicated (log-rank test). The data are representative of three independent experimental replicates with similar results. (D) Serum cytokine levels. The data shown represent at least three independent experiments ( n ≥ 3), significance was measured by Ordinary one-way ANOVA (TNF- α , ∗∗ P = 0.0010 and ∗∗∗ P = 0.0002, IL-6, ∗∗ P = 0.0057 and ∗∗∗ P = 0.0004 and IL12p40, both ∗∗∗∗ P < 0.0001). n.s = not significant. (E) IB identification and comparison of HMGB1 and PTX3 expression in spleen, lung, and liver cells of CLP mice after different treatment as a DAMP indicator for tissue damage. (F) Representative H&E staining of the lung, spleen, and liver from 3 mice per group. Scale bar, 100 μm.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Ligation, Comparison, Expressing, Staining

Journal: Human Vaccines & Immunotherapeutics

Article Title: The cationic liposome CCS/C adjuvant induces immunity to influenza independently of the adaptor protein MyD88

doi: 10.1080/21645515.2020.1750247

Figure Lengend Snippet: TLR2 and TLR4 do not contribute to antiviral immunity induced by F-HA and CCS/C-HA

Article Snippet: All animal studies were approved by the Institutional Animal Care and Use Committees of the Hebrew University. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Spleen Vaccine ( n = 5) Incubation Treatment Proliferation ( SI a ) IFN γ ( pg/ml ) IL-2 ( pg/ml ) IL-5 ( pg/ml ) Con A 0.25 µg HA/ml 8.2 >1000 70 ± 13 152 ± 21 PBS 0.5 µg HA/ml 1.4 0 3 ± 1 0 0.05 µg HA/ml 1.5 0 2 ± 0.4 0 F-HA WT 0.5 µg HA/ml 2 182 ± 10 75 ± 28 439 ± 79 0.05 µg HA/ml 1.5 154 ± 18 57 ± 20 391 ± 62 CCS/C-HA WT 0.5 µg HA/ml 4 400 ± 22 96 ± 21 660 ± 59 0.05 µg HA/ml 2.4 422 ± 65 22 ± 13 620 ± 28 F-HA Tlr2 −/- 0.5 µg HA/ml 1.8 0 63 ± 11 547 ± 28 0.05 µg HA/ml 1.8 0 53 ± 13 368 ± 25 CCS/C-HA Tlr2 −/- 0.5 µg HA/ml 3.2 593 ± 62 590 ± 29 292 ± 45 0.05 µg HA/ml 5 581 ± 112 342 ± 29 217 ± 25 F-HA Tlr4 −/- 0.5 µg HA/ml 0.8 29 ± 18 85 ± 17 325 ± 57 0.05 µg HA/ml 1 0 61 ± 6 200 ± 23 CCS/C-HA Tlr4 −/- 0.5 µg HA/ml 3.3 417 ± 26 316 ± 38 306 ± 41 0.05 µg HA/ml 2.6 463 ± 18 318 ± 42 302 ± 35 Open in a separate window C57BL/6 (WT), Tlr2 −/- and Tlr4 −/- female mice ( n = 3–5) were vaccinated on d 0 and 28 with PBS only or F-HA vs. CCS/C-HA (Aventis Pasteur Vaxigrip 2008–09, 3 μg HA).

Techniques:

Journal: Human Vaccines & Immunotherapeutics

Article Title: The cationic liposome CCS/C adjuvant induces immunity to influenza independently of the adaptor protein MyD88

doi: 10.1080/21645515.2020.1750247

Figure Lengend Snippet: Proliferative response and cytokine production by spleen cells following two doses of F-HA vs. CCS/C-HA vaccination in WT, Tlr2-/- , and Tlr4-/- mice

Article Snippet: All animal studies were approved by the Institutional Animal Care and Use Committees of the Hebrew University. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Spleen Vaccine ( n = 5) Incubation Treatment Proliferation ( SI a ) IFN γ ( pg/ml ) IL-2 ( pg/ml ) IL-5 ( pg/ml ) Con A 0.25 µg HA/ml 8.2 >1000 70 ± 13 152 ± 21 PBS 0.5 µg HA/ml 1.4 0 3 ± 1 0 0.05 µg HA/ml 1.5 0 2 ± 0.4 0 F-HA WT 0.5 µg HA/ml 2 182 ± 10 75 ± 28 439 ± 79 0.05 µg HA/ml 1.5 154 ± 18 57 ± 20 391 ± 62 CCS/C-HA WT 0.5 µg HA/ml 4 400 ± 22 96 ± 21 660 ± 59 0.05 µg HA/ml 2.4 422 ± 65 22 ± 13 620 ± 28 F-HA Tlr2 −/- 0.5 µg HA/ml 1.8 0 63 ± 11 547 ± 28 0.05 µg HA/ml 1.8 0 53 ± 13 368 ± 25 CCS/C-HA Tlr2 −/- 0.5 µg HA/ml 3.2 593 ± 62 590 ± 29 292 ± 45 0.05 µg HA/ml 5 581 ± 112 342 ± 29 217 ± 25 F-HA Tlr4 −/- 0.5 µg HA/ml 0.8 29 ± 18 85 ± 17 325 ± 57 0.05 µg HA/ml 1 0 61 ± 6 200 ± 23 CCS/C-HA Tlr4 −/- 0.5 µg HA/ml 3.3 417 ± 26 316 ± 38 306 ± 41 0.05 µg HA/ml 2.6 463 ± 18 318 ± 42 302 ± 35 Open in a separate window C57BL/6 (WT), Tlr2 −/- and Tlr4 −/- female mice ( n = 3–5) were vaccinated on d 0 and 28 with PBS only or F-HA vs. CCS/C-HA (Aventis Pasteur Vaxigrip 2008–09, 3 μg HA).

Techniques: Incubation

Journal: EBioMedicine

Article Title: Macrophage-derived myeloid differentiation protein 2 plays an essential role in ox-LDL-induced inflammation and atherosclerosis

doi: 10.1016/j.ebiom.2020.102706

Figure Lengend Snippet: MD2 is essential for ox-LDL-induced TLR4 dimerization and activation via direct interaction with ox-LDL ( a ) Macrophages from wildtype mice were incubated with 50 µg/mL ox-LDL for indicated time periods. TLR4 was immunoprecipitated (IP) and MD2 was detected by immunoblotting (IB). Lower panel showing the densitometric quantification of MD2-TLR4 association [ n = 4]. ( b ) Interaction between MD2 and TLR4 was confirmed by co-immunoprecipitation assay in HEK-293T cells transfected with MD2-His and TLR4-Flag expressing plasmids. Lower panel showing the densitometric quantification [ n = 4]. ( c ) Dimerization of TLR4 was assessed by co-immunoprecipitation assay in HEK-293T cells transfected with Flag- and HA-tagged TLR4 and His-tagged MD2 plasmids. Cells were exposed to 50 µg/mL ox-LDL for 30 min. Samples were immunoprecipitation (IP) with anti-HA followed by immunoblotting (IB) with anti-Flag. Lower panel showing the densitometric quantification. [ n = 4]. ( d ) Primary macrophages from wildtype (WT) mice and MD2 −/− mice (KO) were incubated with 50 µg/mL ox-LDL for 30 min. TLR4 was immunoprecipitated (IP) and MyD88 was detected by immunoblotting (IB). Lower panel showing the densitometric quantification [ n = 4]. ( e ) Interaction between MyD88 and TLR4 was detected by co-immunoprecipitation assay in HEK-293T cells transfected with TLR4-HA, MyD88-Flag, and MD2-His expressing plasmids. Cells were treated with or without 50 µg/mL ox-LDL for 30 min. Lower panel showing the densitometric quantification [ n = 3]. ( f ) Macrophages isolated from wildtype mice were exposed to 50 μg/mL ox-LDL or LDL for 30 min. Interaction between ApoB100, MD2, and TLR4 was assessed by co-immunoprecipitation. Right panel showing the densitometric quantification [ n = 3]. ( g ) Interaction between ApoB100, MD2, and TLR4 was detected by co-immunoprecipitation in HEK-293T cells transfected with TLR4-HA and MD2-His expressing plasmids. Cells were treated with or without 50 µg/mL ox-LDL for 30 min. Right panel showing the densitometric quantification [ n = 4]. ( h ) Primary macrophages were treated with 50 µg/mL ox-LDL or LDL, and then were stained for MD2 (green) and ApoB100 (red). DAPI (blue) was used to counterstain. Lower panels show higher magnification. ( i ) Macrophages isolated from wildtype (WT) and Md2 −/− ( Md2 KO) mice were incubated with 50 µg/mL ox-LDL for 30 min. Interaction between TLR4 and ApoB100 was assessed by co-immunoprecipitation. Lower panel showing the densitometric quantification [ n = 3] ( j ) Macrophages isolated from WT and Tlr4 −/− ( Tlr4 KO) mice were incubated with 50 µg/mL ox-LDL for 30 min. Interaction between MD2 and ApoB100 was assessed by co-immunoprecipitation. Lower panel showing the densitometric quantification [ n = 3]. ( k ) The interaction between MD2 and ox-LDL was determined using a cell-free assay. rhMD2 was immobilized and DiI-labeled ox-LDL was added. Binding was determined by relative fluorescence intensity (RFI), normalized to blanks without rhMD2 immobilization [ n = 4]. ( l ) Representative immunofluorescence staining of MD2 (red) and ApoB100 (green) in aortic sinus of Apoe −/− mice maintained on HFD. Tissues were counterstained with DAPI (blue) [scale bar = 50 μm]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To assess TLR4 dimerization upon ox-LDL exposure, HEK-293 cells were transfected with pCMV-HA-TLR4, pCMV-Flag-TLR4 and pCMV-His-MD2 (Sino Biological) using polyethylenimine (PEI; Sigma-Aldrich).

Techniques: Activation Assay, Incubation, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Transfection, Expressing, Isolation, Staining, Cell-Free Assay, Labeling, Binding Assay, Fluorescence, Immunofluorescence

Journal: EBioMedicine

Article Title: Macrophage-derived myeloid differentiation protein 2 plays an essential role in ox-LDL-induced inflammation and atherosclerosis

doi: 10.1016/j.ebiom.2020.102706

Figure Lengend Snippet: MD2 in macrophages is not required for ox-LDL uptake ( a ) Primary macrophages isolated from widetype (WT) and Md2 −/− (KO) mice were incubated with 50 µg/mL DiI-labeled ox-LDL for indicated time periods. DiI-ox-LDL uptake was detected as mean fluorescence intensity by flow cytometry [ n = 4]. ( b ) Oil Red O staining of WT and KO macrophages incubated with 50 µg/mL ox-LDL for 24 h. Right panel showing quantification of area highlighted by Oil Red O staining [ n = 4]. ( c ) HEK-293T cells transfected with MD2-His-plasmid alone or co-transfected with MD2-His-and TLR4-HA plasmids were cultured with 50 µg/mL DiI-labeled ox-LDL for 30 min. DiI-ox-LDL uptake was detected by flow cytometry. Flow histograms showing mean fluorescence intensity (MFI) values. ( d ) The interaction between ApoB100, MD2, and TLR4 was assessed by co-immunoprecipitation in macrophages from wildtype mice. Cells were pretreated with or without LDL-uptake inhibitor Dynasore at 80 µM for 1 h prior to exposure of cells to 50 µg/mL ox-LDL for 30 min. ( e ) Immunofluorescence staining of macrophages from wildtype mice for MD2 (green) and ApoB100 (red). Cells were treated as indicated in panel D. Cells were counterstained with DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To assess TLR4 dimerization upon ox-LDL exposure, HEK-293 cells were transfected with pCMV-HA-TLR4, pCMV-Flag-TLR4 and pCMV-His-MD2 (Sino Biological) using polyethylenimine (PEI; Sigma-Aldrich).

Techniques: Isolation, Incubation, Labeling, Fluorescence, Flow Cytometry, Staining, Transfection, Plasmid Preparation, Cell Culture, Immunoprecipitation, Immunofluorescence