Journal: Nature Communications

Article Title: Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response

doi: 10.1038/s41467-019-09453-x

Figure Lengend Snippet: 25HC interacts with α5β1 and αvβ3 integrins. a , b Biotinylation reactions were performed in the presence of either 25HC or DMSO (control). Cell lysates collected from BMDMs ( a ) or RAW 264.7 macrophages ( b ) incubated with biotinylated 25HC (biotin-25HC) or DMSO at 4 °C were precipitated (PPT) with avidin–agarose and subsequently immuno-blotted (IB) with either α5 integrin ( a ) or αv integrin ( b ) antibody. Total lysates were also blotted with α5 integrin ( a ), αv integrin ( b ), and actin ( a , b ) antibodies. c , d Biotin-25HC and DMSO (control) were incubated (PPT; precipitated) with avidin–agarose beads. 25HC conjugated beads were then incubated either with purified α5β1 integrin ( c ) or αvβ3 integrin ( d ). The avidin–agarose bound complex was subsequently immuno-blotted with either α5 integrin ( c ) or αv integrin ( d ) antibody. e , f Protein G-agarose beads were conjugated with either control IgG or antibody (Ab) specific for α5 integrin ( e ) and αv integrin ( f ). The beads were then incubated with either purified α5β1 integrin ( e ) or αvβ3 integrin ( f ) protein. Control beads (control IgG + purified integrin protein) and integrin protein bound beads (integrin-specific antibody + purified integrin protein) was incubated with tritiated 3 H-25HC. Bead-bound radioactivity (count per minute; CPM) was measured by scintillation counter. g Cell lysate collected from RAW 264.7 macrophages incubated with biotin-25HC, biotinylated 27-hydroxycholesterol (biotin-27HC), biotinylated 4β-hydroxycholesterol (biotin-4βHC) or DMSO (control) at 4 °C was precipitated (PPT) with avidin–agarose and subsequently immuno-blotted with α5 integrin antibody. Total cell lysate was also blotted with α5 integrin and β-actin antibodies. h Surface plasmon resonance (SPR) analyses were performed by injecting increasing concentrations of 25HC (16 nM, 40 nM, 160 nM, 640 nM, 1.6 µM) into both the αvβ3 integrin-immobilized and control surface of the CM5 chips. Each background-corrected sensorgram is a representative of replicate runs. i Binding of radioactive 3 H-25HC to immobilized αvβ3 integrin protein in the presence or absence of purified chemokine domain of fractalkine protein was assessed as described in the “methods” section. Immunoblot images are representative from three independent experiments. The radioactive values (mean ± standard deviation) are representative from two independent experiments. * p ≤ 0.05 using a Student’s t -test

Article Snippet: Purified human αvβ3 integrin protein (Yo proteins AB, Huddinge, Sweden) was covalently immobilized on a flow cell of CM5 chip (carboxy-methylated dextran coated) in 10 mM sodium acetate buffer, pH 4.0, using EDC/NHS amine coupling chemistry at 25 °C.

Techniques: Incubation, Avidin-Biotin Assay, Purification, Radioactivity, SPR Assay, Binding Assay, Western Blot, Standard Deviation

Journal: Nature Communications

Article Title: Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response

doi: 10.1038/s41467-019-09453-x

Figure Lengend Snippet: Modeling identifies specific interactions between αvβ3 integrin and 25HC. a Ectodomain structure of αvβ3 integrin containing ‘RGD’’-binding site (site I) (blue) and site-II (red) residues highlighted by a layer of solvent molecule of radius 1.4 Å. The site-II-bound 25HC molecule is presented as a surface model in green and Mn 2+ ions from metal-ion-dependent ligand-binding site (MIDAS) and adjacent to MIDAS (ADMIDAS) sites of βI domain, β-propeller domain and genu knee region are presented in cyan. b Potential interacting residues for molecular recognition of αvβ3 integrin by 25HC were identified in a docking simulation. H-bonds are highlighted in dashed lines in red. c , d Ser399 of β-propeller (red), Ser162 (blue) and Ala263 (black) of βI domain make stable H-bond interactions with 3- and 25-hydroxyl groups of 25HC, respectively. The H-bond distances ( c ) and H-bond angles ( d ) are plotted against MD simulation time (200 ns). e The root-mean-square deviations (RMSD) measured for specificity-determining loop (SDL), α1- and α7 helices during the entire 200 ns long simulation. The SDL region undergoes significant conformational change (RMSD of ~6 Å) upon 25HC binding. f The conformational change in the SDL is accompanied by disruption of H-bond interaction between Tyr122-Thr182 residues, as well as change in the β-propeller blade that interacts with SDL. Movement of the regions from the beginning 0 ns (blue) to the end 200 ns (red) is indicated by arrows. g Correlation matrices (as heat maps) indicating correlated (red) or anti-correlated motions (blue) of α- and β-subunits of αvβ3 integrin in unbound and 25HC-bound states. h , i The distance between the amide NH of Q120 of the β-propeller domain and backbone carbonyl oxygen of P169 of the SDL. Breakage of this electrostatic interaction in the 25HC-bound protein (blue) during the simulation, around ~30 ns simulation causes significant increase in the distance, which remain intact in the unbound protein (magenta) during the entire 200 ns simulation time

Article Snippet: Purified human αvβ3 integrin protein (Yo proteins AB, Huddinge, Sweden) was covalently immobilized on a flow cell of CM5 chip (carboxy-methylated dextran coated) in 10 mM sodium acetate buffer, pH 4.0, using EDC/NHS amine coupling chemistry at 25 °C.

Techniques: Binding Assay, Ligand Binding Assay

Journal: Nature Communications

Article Title: Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response

doi: 10.1038/s41467-019-09453-x

Figure Lengend Snippet: 25HC activates αvβ3 integrin in epithelial cell focal adhesions and macrophage podosomes. a Human lung epithelial cells (A549 cells) or differentiated THP-1 macrophages were subjected to various treatments and immunostained as indicated. In untreated (UT) A549 and THP-1 cells (first row), αvβ3 integrin, immunostained using the antibody LM609, was present in paxillin-positive focal adhesions and podosomes, respectively. In contrast, active αvβ3 integrin staining, indicated using the antibody AP5 (second and third row), was found almost exclusively in cells treated for 2 h with 0.5 µM 25HC compared with DMSO-treated controls. Merged images show the co-localization of αvβ3 integrin (total or activated) with focal adhesions/podosomes. Bar, 10 µm. b Quantification of the number of focal adhesions (FAs) and AP5 + FAs in A549 cells (three experiments, >12 cells per experiment; n ≥ 30). c Quantification of the number of podosomes and AP5 + podosomes in THP-1 cells (three experiments, 25 cells per experiment; n ≥ 70). Two- and One-tailed Wilcoxon rank-sum tests were performed to evaluate differences between total and AP5 + FA/podosome counts, respectively. Graphs depict mean ± SEM. * p ≤ 0.05

Article Snippet: Purified human αvβ3 integrin protein (Yo proteins AB, Huddinge, Sweden) was covalently immobilized on a flow cell of CM5 chip (carboxy-methylated dextran coated) in 10 mM sodium acetate buffer, pH 4.0, using EDC/NHS amine coupling chemistry at 25 °C.

Techniques: Staining, One-tailed Test

Journal: Nature Communications

Article Title: Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response

doi: 10.1038/s41467-019-09453-x

Figure Lengend Snippet: α5β1 and αvβ3 integrins regulate 25HC-mediated proinflammatory response. a , c – g α5β1 and αvβ3 integrin-deficient cells were treated with 50 µM 25HC for 8 h to evaluate NFκB activation and proinflammatory response. a TNF secretion from 25HC-treated NR-9456 macrophages in the presence of either control IgG or α5β1 integrin blocking antibody (Ab). b Western blot analyses of α5 integrin protein expression in THP-1 cells transfected with either control siRNA (con siRNA) or α5 integrin-specific siRNA (α5 siRNA). c RT-PCR analyses of TNF expression in 25HC-treated THP-1 cells transfected with either control siRNA (con siRNA) or α5 integrin-specific siRNA (α5 siRNA). d Western blot and densitometric analyses of phospho-IκB status in 25HC-treated wild-type (WT) and α5 integrin knockout (KO) HAP1 cells. e TNF secretion from 25HC-treated WT and β3 integrin-deficient (β3 +/− cells) BMDMs. f , g TNF ( f ) and IL-6 ( g ) secretion from 25HC-treated THP-1 macrophages in the presence of either control IgG or αvβ3 integrin blocking antibody (Ab). RT-PCR images are representative from two independent experiments. The ELISA values (mean ± standard deviation) are representative from two or three independent experiments ( n = 4). * p ≤ 0.05 using a Student’s t -test. The densitometric quantification values for phospho-IκB (p-IκB) immunoblot represent the ratio of phospho-IκB:actin and the fold-induction was calculated after normalizing with the control 0 min group. The densitometric values represent the mean ± standard deviation from three independent studies. * p ≤ 0.05 using a Student’s t -test

Article Snippet: Purified human αvβ3 integrin protein (Yo proteins AB, Huddinge, Sweden) was covalently immobilized on a flow cell of CM5 chip (carboxy-methylated dextran coated) in 10 mM sodium acetate buffer, pH 4.0, using EDC/NHS amine coupling chemistry at 25 °C.

Techniques: Activation Assay, Blocking Assay, Western Blot, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Knock-Out, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Nature Communications

Article Title: Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response

doi: 10.1038/s41467-019-09453-x

Figure Lengend Snippet: The 25HC-integrin-FAK signaling network is required for optimal Nod2 response. a IL-6 in serum of mice injected (via intraperitoneal route) with 25HC (50 mg/kg; 4 h) ( n = 5). b , c , e , g – m Cells were treated with 25 µg/ml of MDP for 8 h and d , f mice were injected intraperitoneally with 20 mg/kg of MDP for 4 h. C25H expression, 25HC production, FAK activation, and proinflammatory response were assessed. b RT-PCR analyses of C25H expression in MDP-treated wild-type (WT) and C25H knockout (KO) BMDMs. c 25HC levels in the medium supernatant of MDP-treated BMDMs. d 25HC levels in the serum of WT mice treated with MDP ( n = 4). e IL-6 secretion from WT and C25H KO BMDMs treated with MDP. f IL-6 levels in the serum of WT and C25H KO mice treated with MDP ( n = 4). g Western blot and densitometric analyses of FAK activation (phospho-FAK, Tyr397) status in MDP-treated THP-1 macrophages. h IL-6 production from MDP-treated WT and FAK KO MEFs (mouse embryo fibroblasts). i IL-6 secretion from MDP-treated NR-9456 macrophages in the presence of either control IgG or α5β1 integrin blocking antibody (Ab). j RT-PCR analyses of TNF expression in MDP-treated THP-1 cells transfected with either control siRNA (con siRNA) or α5 integrin-specific siRNA (α5 siRNA). k TNF secretion from MDP-treated WT and β3 integrin-deficient (β3 +/− cells) BMDMs. l TNF secretion from MDP-treated THP-1 macrophages in the presence of either control IgG or αvβ3 integrin blocking antibody. m Western blot and densitometric analyses of FAK activation (phospho-FAK, Tyr925) status in MDP-treated WT and C25H KO BMDM. RT-PCR images are representative from two independent experiments. The ELISA values (mean ± standard deviation) are representative from two or three independent experiments ( n = 4). * p ≤ 0.05 using a Student’s t -test. The densitometric quantification values for phospho-FAK (p-FAK) immunoblot represent the ratio of phospho-FAK:actin and the fold-induction was calculated after normalizing with the control untreated (UT) group. The densitometric values represent the mean ± standard deviation from three independent studies. * p ≤ 0.05 using a Student’s t- test

Article Snippet: Purified human αvβ3 integrin protein (Yo proteins AB, Huddinge, Sweden) was covalently immobilized on a flow cell of CM5 chip (carboxy-methylated dextran coated) in 10 mM sodium acetate buffer, pH 4.0, using EDC/NHS amine coupling chemistry at 25 °C.

Techniques: Injection, Expressing, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Knock-Out, Western Blot, Blocking Assay, Transfection, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Nature Communications

Article Title: Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response

doi: 10.1038/s41467-019-09453-x

Figure Lengend Snippet: The 25HC-integrin-FAK signaling network regulates proinflammatory response during virus infection. a 25HC levels in the medium supernatant of BMDMs infected with human respiratory syncytial virus (RSV). b TNF production from wild-type (WT) and C25H knockout (KO) BMDMs infected with RSV. c Western blot and densitometric analyses of FAK activation (phospho-FAK, Tyr925) status in RSV-infected BMDMs. d Western blot and densitometric analyses of FAK activation (phospho-FAK, Tyr925) status in influenza A virus (IAV)-infected BMDMs. e IL-6 production from RSV-infected WT and FAK KO MEFs. f IL-6 production from IAV-infected WT and FAK KO MEFs. g TNF production from RSV-infected BMDMs in the presence of either control IgG or α5β1 integrin blocking antibody (Ab). h IL-6 production from IAV-infected BMDMs in the presence of either control IgG or α5β1 integrin blocking antibody. i TNF secretion from IAV-infected WT and β3 integrin-deficient (β3 +/− cells) BMDMs. j IL-6 secretion from IAV-infected THP-1 cells in the presence of either control IgG or αvβ3 integrin blocking antibody. k IL-6 in the lungs of mice injected (via intratracheal or I.T route) with 25HC (5 mg/kg; 6 h) in the presence of either IgG or β1 integrin blocking antibody (Ab) administered to the mice via I.T route ( n = 4). l IL-6 in the lungs of mice injected (via I.T route) with 25HC (5 mg/kg; 6 h) in the presence of either vehicle (control) or FAK inhibitor (PND-1186) administered to the mice via I.T route ( n = 4). The ELISA values (mean ± standard deviation) are representative from two or three independent experiments ( n = 4). * p ≤ 0.05 using a Student’s t -test. The densitometric quantification values for phospho-FAK (p-FAK) immunoblot represent the ratio of phospho-FAK:actin and the fold-induction was calculated after normalizing with the control mock-infected group. The densitometric values represent the mean ± standard deviation from three independent studies. * p ≤ 0.05 using a Student’s t -test

Article Snippet: Purified human αvβ3 integrin protein (Yo proteins AB, Huddinge, Sweden) was covalently immobilized on a flow cell of CM5 chip (carboxy-methylated dextran coated) in 10 mM sodium acetate buffer, pH 4.0, using EDC/NHS amine coupling chemistry at 25 °C.

Techniques: Infection, Knock-Out, Western Blot, Activation Assay, Blocking Assay, Injection, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Nature Communications

Article Title: Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response

doi: 10.1038/s41467-019-09453-x

Figure Lengend Snippet: A schematic model showing regulation of proinflammatory response by 25HC-integrin-FAK signaling network. Nod2 activation and virus (RSV and IAV) infection triggers expression of C25H, which results in production of 25HC. Extracellular 25HC activates integrin-FAK-NFκB signaling. Thus, 25HC links PRR (Nod2) pathway with integrin-FAK-NFκB signaling to confer optimal proinflammatory response following Nod2 activation and virus infection. INTG, integrin; FAK, focal adhesion kinase

Article Snippet: Purified human αvβ3 integrin protein (Yo proteins AB, Huddinge, Sweden) was covalently immobilized on a flow cell of CM5 chip (carboxy-methylated dextran coated) in 10 mM sodium acetate buffer, pH 4.0, using EDC/NHS amine coupling chemistry at 25 °C.

Techniques: Activation Assay, Infection, Expressing

Journal:

Article Title: Characterization of Entry and Infection of Monocytic THP-1 cells by Kaposi's Sarcoma Associated Herpesvirus (KSHV): Role of Heparan Sulfate, DC-SIGN, Integrins and Signaling

doi: 10.1016/j.virol.2010.07.012

Figure Lengend Snippet: A. KSHV internalization in the presence of heparin and mannan. 1×106 THP-1 cells mock treated or pre-incubated with the indicated concentrations of mannan at 4°C were infected with 10 KSHV DNA copies per cell or heparin pretreated KSHV (100ug/ml). At different time points, uninternalized virus was removed by washing and trypsinization. Infected and mock infected cells were washed and internalized viral copy numbers were determined by real-time DNA PCR as described in the figure 1C/D legend. The data are represented as the percent inhibition of KSHV DNA internalized in comparison with cells incubated with virus alone. Each reaction was done in duplicate and each bar represents the mean ± SD of the results of three experiments. B. KSHV internalization in THP1 cells upon blocking DC-SIGN. THP1 cells incubated with mouse IgG or mouse anti-DC-SIGN mAb at 4° C for 1 h. The washed cells were incubated with KSHV (10 genome copies/cell) at 4° C for 1 h followed by at 37° C for 1 h. Uninternalized virus was removed by washing and trypsinization, internalized viral copy numbers were determined by real-time DNA PCR as described in the figure 1C/D legend. The data are represented as the percent inhibition of KSHV DNA internalization in comparison with IgG control. C. KSHV gene expression in the presence of soluble integrins. 1×106 THP-1 cells were infected with 30 KSHV DNA copies per cell of untreated virus or with virus pretreated with 10μg/ml of soluble α3β1, αvβ3, αvβ5, α5β1 and α4β7 integrins for 1 h at 37° C. At 2 h p.i. uninternalized virus was removed by washing and trypsinization and the infected cells were further incubated. At 24 h p.i., the cells were washed, RNA was isolated, ORF73 gene expression was examined as described under the figure 2B legend. ORF73 gene expression was represented as percent inhibition compared to control untreated KSHV infection.

Article Snippet: Rabbit anti-integrin α3 and β1 and soluble human integrins α3β1, αvβ3, αvβ5 and α5β1 were from Upstate Biotechnology, Lake Placid, N.Y. Recombinant human integrin α4β7 was from R&D systems, Minneapolis, MN.

Techniques: Incubation, Infection, Inhibition, Blocking Assay, Expressing, Isolation

Journal:

Article Title: Characterization of Entry and Infection of Monocytic THP-1 cells by Kaposi's Sarcoma Associated Herpesvirus (KSHV): Role of Heparan Sulfate, DC-SIGN, Integrins and Signaling

doi: 10.1016/j.virol.2010.07.012

Figure Lengend Snippet: THP-1 cells were infected with 10 KSHV DNA copies per cell for 5 and 10 min. The cells were fixed in 2 % paraformaldehyde for 10 min, permeabilized with 0.2% Triton X-100 for 5 min and blocked. Infected and uninfected cells were stained with anti-KSHV gB and anti-α3β1/αvβ3/αvβ5/α5β1/αvβ6 integrin antibodies and visualized by confocal microscopy. KSHV-gB and integrins were visualized by incubation with Alexa-488 secondary antibody (green) and Alexa-594 secondary antibody (red), respectively. The arrows indicate the areas of KSHV co-localization with integrins. A. KSHV+ α3β1; B. KSHV+ αvβ3; C. KSHV+ αvβ5; D. KSHV+ α5β1; E. KSHV+ αvβ6.

Article Snippet: Rabbit anti-integrin α3 and β1 and soluble human integrins α3β1, αvβ3, αvβ5 and α5β1 were from Upstate Biotechnology, Lake Placid, N.Y. Recombinant human integrin α4β7 was from R&D systems, Minneapolis, MN.

Techniques: Infection, Staining, Confocal Microscopy, Incubation