Journal: Frontiers in pharmacology

Article Title: Activation of Platelet NLRP3 Inflammasome in Crohn's Disease.

doi: 10.3389/fphar.2021.705325

Figure Lengend Snippet: FIGURE 1 | The expression of ASC and NLRP3 in platelets from the healthy group and patients with active Crohn’s disease (CD). (A,B) The mRNA levels of NLRP3 (A) and ASC (B) were determined by RT-PCR in platelets from the healthy group (n 17) and patients with active CD (n 17). (C) Western blotting was used to determine the protein levels of NLRP3 and ASC in platelets from patients with active CD and the healthy group. (D,E) The relative protein quantification of NLRP3 (D) and ASC (E) in platelets from the healthy group (n 38) and patients with active CD (n 38). Health, the healthy group; CD, the patients with active CD. Data are presented as mean ± SD, *p < 0.05, ***p < 0.001.

Article Snippet: The platelets were then incubated with goat anti-NLRP3 antibody (Novus, NB100-41104, 1:100), mouse anti-ASC antibody (Santa Cruz, sc-271054, 1:100), and rabbit anti-CD41 antibody (Abcam, ab134131, 1:300) at 4°C overnight, followed by incubation with 1:200 dilutions of relative secondary antibody for 1 h. A confocal laser scanner microscope (Leica TCS SP8, Solms, Germany) was used to obtain images that were then processed using LAS AF Lite software (Leica, Germany).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in pharmacology

Article Title: Activation of Platelet NLRP3 Inflammasome in Crohn's Disease.

doi: 10.3389/fphar.2021.705325

Figure Lengend Snippet: FIGURE 2 | Activation of NLRP3 inflammasome in platelets from patients with active Crohn’s disease (CD). (A) Co-immunoprecipitation of ASC with NLRP3 in platelets from the healthy group and patients with active CD (i). Quantification of the interaction between ASC and NLRP3 in platelets was below (n 3), (ii). (B) Immunofluorescence staining showed colocalization of ASC (green) and NLRP3 (red) in platelets from patients with active CD (i). The experiment was repeated three times. Intensity traces were plotted below (ii). Scale bars were 20 μm. Health, the healthy group; CD, the patients with active Crohn’s disease. Data are presented as mean ± SD, ***p < 0.001.

Article Snippet: The platelets were then incubated with goat anti-NLRP3 antibody (Novus, NB100-41104, 1:100), mouse anti-ASC antibody (Santa Cruz, sc-271054, 1:100), and rabbit anti-CD41 antibody (Abcam, ab134131, 1:300) at 4°C overnight, followed by incubation with 1:200 dilutions of relative secondary antibody for 1 h. A confocal laser scanner microscope (Leica TCS SP8, Solms, Germany) was used to obtain images that were then processed using LAS AF Lite software (Leica, Germany).

Techniques: Activation Assay, Immunoprecipitation, Staining

Journal: Frontiers in pharmacology

Article Title: Activation of Platelet NLRP3 Inflammasome in Crohn's Disease.

doi: 10.3389/fphar.2021.705325

Figure Lengend Snippet: FIGURE 4 | Increased ROS generation in platelets from patients with active Crohn’s disease (CD). (A) We explored the ROS generation by flow cytometry in platelets from the healthy group (n 26) and patients with active CD (n 26). The relative quantification was at right. (B) The NLRP3 protein levels were positively correlated with ROS levels in platelets from patients with active CD (n 26). Health, the healthy group; CD, the patients with active CD. Data are presented as mean ± SD, ***p < 0.001.

Article Snippet: The platelets were then incubated with goat anti-NLRP3 antibody (Novus, NB100-41104, 1:100), mouse anti-ASC antibody (Santa Cruz, sc-271054, 1:100), and rabbit anti-CD41 antibody (Abcam, ab134131, 1:300) at 4°C overnight, followed by incubation with 1:200 dilutions of relative secondary antibody for 1 h. A confocal laser scanner microscope (Leica TCS SP8, Solms, Germany) was used to obtain images that were then processed using LAS AF Lite software (Leica, Germany).

Techniques: Cytometry

Journal: Frontiers in pharmacology

Article Title: Activation of Platelet NLRP3 Inflammasome in Crohn's Disease.

doi: 10.3389/fphar.2021.705325

Figure Lengend Snippet: FIGURE 5 | Correlation between the platelet NLRP3 protein levels and P-selectin exposure or fibrinogen binding in patients with active Crohn’s disease (CD). (A) P-selectin exposure was assessed by FCM in platelets from the healthy group (n 26) and patients with active CD (n 26). The relative quantification was at right. (B) Fibrinogen binding was measured by FCM in platelets from the healthy group (n 26) and patients with active CD (n 26). The relative quantification was at right. (C) The NLRP3 protein levels were positively correlated with P-selectin exposure in platelets from patients with active CD (n 26). (D) The NLRP3 protein levels were positively correlated with fibrinogen binding in platelets from patients with active CD (n 26). Health, the healthy group; CD, the patients with active CD. Data are presented as mean ± SD, ***p < 0.001.

Article Snippet: The platelets were then incubated with goat anti-NLRP3 antibody (Novus, NB100-41104, 1:100), mouse anti-ASC antibody (Santa Cruz, sc-271054, 1:100), and rabbit anti-CD41 antibody (Abcam, ab134131, 1:300) at 4°C overnight, followed by incubation with 1:200 dilutions of relative secondary antibody for 1 h. A confocal laser scanner microscope (Leica TCS SP8, Solms, Germany) was used to obtain images that were then processed using LAS AF Lite software (Leica, Germany).

Techniques: Binding Assay

Journal: Frontiers in pharmacology

Article Title: Activation of Platelet NLRP3 Inflammasome in Crohn's Disease.

doi: 10.3389/fphar.2021.705325

Figure Lengend Snippet: FIGURE 6 | Schematic representation of inflammasome activation in active Crohn’s disease (CD). In active CD, the bacteria derived DAMPs and pathogen- associated molecular patterns (PAMPs) could enter platelets and induce generation of intracellular ROS. Elevated ROS consequently stimulates the assembly of NLRP3 inflammasome. Followed by activation of NLRP3 inflammasome, pro-caspase-1 is cleaved into active caspase-1, and consequently induces generation of interleukin- 1β, which results in platelet hyperactivity. In the healthy group, there is no formation of the NLRP3 inflammasome.

Article Snippet: The platelets were then incubated with goat anti-NLRP3 antibody (Novus, NB100-41104, 1:100), mouse anti-ASC antibody (Santa Cruz, sc-271054, 1:100), and rabbit anti-CD41 antibody (Abcam, ab134131, 1:300) at 4°C overnight, followed by incubation with 1:200 dilutions of relative secondary antibody for 1 h. A confocal laser scanner microscope (Leica TCS SP8, Solms, Germany) was used to obtain images that were then processed using LAS AF Lite software (Leica, Germany).

Techniques: Activation Assay, Bacteria, Derivative Assay