Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Adipose-Derived Mesenchymal Stem Cells Ameliorating Pseudomonas aeruginosa –induced Acute Lung Infection via Inhibition of NLRC4 Inflammasome

doi: 10.3389/fcimb.2020.581535

Figure Lengend Snippet: Stimulation of NLRC4 inflammasome expression induced by PA. (A, C) in vivo , compared with the control group, the expression of Caspase-1 (p20) protein, IL-18 and IL-1β were significantly increased in the PA-infected group of WT mice; Caspase-1 was scarcely expressed in each group of N4 −/− mice, and the expression of IL-18 and IL-1β was slightly higher in the PA-infected group than in the control group; Conversely, Caspase-1 and IL-18, IL-1β expression were significantly increased in PA-infected group of N3 −/− mice than in control group. In addition, the expression of Caspase-1 (p20) protein, IL-18 and IL-1β in PA+N4 −/− mice was significantly lower than that in PA+WT group; Compared with PA+WT group, the expression of IL-1β in PA+N3 −/− group was significantly increased, the expression of IL-18 and Caspase-1 (p20) protein were not significantly different. (D) In vitro , IL-1β expression level showed the highest when PA MOI was 1:1 and time was 4 h. (B, E, F) In vitro , results showed the same trend as in vivo experiments. Data were shown as mean ± SD. ASC, adipose tissue-derived mesenchymal stem cells; N4 −/− , NLRC4 −/− ; N3 −/− , NLRP3 −/− .

Article Snippet: NLRC4 was detected with rabbit anti-NLRC4 monoclonal antibody (ECM Biosciences, China). β-actin was detected with rabbit anti-β-actin monoclonal antibody (Abmart, Chin).

Techniques: Expressing, In Vivo, Control, Infection, In Vitro, Derivative Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Adipose-Derived Mesenchymal Stem Cells Ameliorating Pseudomonas aeruginosa –induced Acute Lung Infection via Inhibition of NLRC4 Inflammasome

doi: 10.3389/fcimb.2020.581535

Figure Lengend Snippet: Inhibition of NLRC4 inflammasome activation by ASCs in PA-induced acute pulmonary infection. (A) In vivo , mice were infected by PA and treated with ASC or L929 cells. (B) In vitro , human THP-1 cells or mouse peritoneal macrophages were stimulated with PA for 4 h. The ASC were added to macrophages in transwell coculture system. (C, E, F) NLRC4 and Caspase-1 activation were both markedly suppressed in macrophages by treated or transwell coculture with ASC as assessed by Western blot. (D, G, H, J) Consistent with the reduction in NLRC4 and Caspase-1 activity, the levels of IL-1β and IL-18 in BALF, lung or the supernatants were all significantly reduced in ASC treatment group than in PA infection group. (I) PA infection induced an increase in NLRC4 mRNA in lung as measured by real time RT-PCR. Treated with ASC could affect the transcript levels of NLRC4 in lung, while NLRC4 transcripts were significantly reduced by ASC. However, NLRP3 transcripts has not been promoted by PA. Data were shown as mean ± SD. (K) THP-1 cells were subjected to PA stimulation and ASC co-culture in vitro , and the phagocytic rate of macrophages in each group was measured by flow cytometry analysis to observe the phagocytic ability of macrophages. Grouping: THP-1 untreated group (THP-1 group), ASC simple co-culture group, PA simple stimulation group and PA stimulation + ASC co-culture group. The results showed that in THP-1 cells, the phagocytic rate of macrophages in PA-simple stimulation group was increased compared to THP-1 untreated group and ASC simple co-culture group, but the phagocytic rate of PA stimulation + ASC co-culture group was significantly higher than that of PA-simple stimulation group. The phagocytosis rate in ASC simple co-culture group was not significantly different from that in THP-1 untreated group. Ma, macrophages; ASC, adipose tissue-derived mesenchymal stem cells.

Article Snippet: NLRC4 was detected with rabbit anti-NLRC4 monoclonal antibody (ECM Biosciences, China). β-actin was detected with rabbit anti-β-actin monoclonal antibody (Abmart, Chin).

Techniques: Inhibition, Activation Assay, Infection, In Vivo, In Vitro, Western Blot, Activity Assay, Quantitative RT-PCR, Co-Culture Assay, Flow Cytometry, Derivative Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Adipose-Derived Mesenchymal Stem Cells Ameliorating Pseudomonas aeruginosa –induced Acute Lung Infection via Inhibition of NLRC4 Inflammasome

doi: 10.3389/fcimb.2020.581535

Figure Lengend Snippet: Activation of NLRC4 inflamasome induced by PA aggravated inflammatory damage in lung. (A) The survival rate of PA+NLRC4 −/− group was significantly improved compared with PA+WT group and PA+NLRP3 −/− group, n = 30 for all groups. (B–D) BALF total inflammatory cell count and bacterial load in BALF and lung in PA+NLRC4 −/− group were all markedly decreased. N4 −/− , NLRC4 −/− ; N3 −/− , NLRP3 −/− .

Article Snippet: NLRC4 was detected with rabbit anti-NLRC4 monoclonal antibody (ECM Biosciences, China). β-actin was detected with rabbit anti-β-actin monoclonal antibody (Abmart, Chin).

Techniques: Activation Assay, Cell Counting

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Adipose-Derived Mesenchymal Stem Cells Ameliorating Pseudomonas aeruginosa –induced Acute Lung Infection via Inhibition of NLRC4 Inflammasome

doi: 10.3389/fcimb.2020.581535

Figure Lengend Snippet: Effect of STC-1 on NLRC4 inflammasome activation induced by PA. (A–C) Recombinant human (rh) STC-1, (rh) TSG-6 and (rh) IL-1RA were directly and respectively added to THP-1 at PA priming step. The dose from left to right was in turn 10ng/ml、100ng/ml、200ng/ml in each recombinant group. The results demonstrated that (rh) STC-1, (rh) TSG-6 and (rh) IL-1RA dose independently decreased NLRC4 inflammasome activation as well as IL-1β and IL-18 secretion, while the inhibitory effect of (rh) STC-1was the most obvious. (D, E) The expression levels of STC-1 in supernatant of ASC and THP-1 were respectively examined. We found that the expression level of STC-1 in ASC supernatant in Ad-MSC co-culture group was 2017 pg/ml, and the expression of STC-1 in PA-stimulated + ASC co-culture group was significantly increased compared to ASC co-culture group (3000pg/ml). However, in THP-1 supernatant, the expression level of STC-1 in THP-1 untreated group was only 304 pg/ml. The expression level of STC-1 in PA +ASC co-culture group was significantly higher than PA stimulation group (800 pg/ml). (F, G) in vivo experiments, the expression level of STC-1 in PA+ASC treatment group was significantly higher than that in PA+PBS group. Data were shown as mean ± SD. ASC, adipose tissue-derived mesenchymal stem cells.

Article Snippet: NLRC4 was detected with rabbit anti-NLRC4 monoclonal antibody (ECM Biosciences, China). β-actin was detected with rabbit anti-β-actin monoclonal antibody (Abmart, Chin).

Techniques: Activation Assay, Recombinant, Expressing, Co-Culture Assay, In Vivo, Derivative Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Adipose-Derived Mesenchymal Stem Cells Ameliorating Pseudomonas aeruginosa –induced Acute Lung Infection via Inhibition of NLRC4 Inflammasome

doi: 10.3389/fcimb.2020.581535

Figure Lengend Snippet: ASCs inhibit the activation of NLRC4 inflammasome in macrophages induced by PA and reduce inflammatory damage. PA infection can activate NLRC4 inflammasome in macrophage. NLRC4 Inflammasome activation leads to the cleavage of proCaspase-1 to activated Caspase-1 that induces the processing of pro-IL-1β and pro-IL-18, which are released as mature IL-1β and IL-18, leading to inflammatory damage in mouse lung tissue; ASC can negatively regulate the NLRC4 inflammasome in macrophages primarily by secreting STC‐1 in response to crosstalk with activated macrophages through PA infection, which can alleviate inflammatory damage in lung tissue of mice. ASC, adipose tissue-derived mesenchymal stem cells.

Article Snippet: NLRC4 was detected with rabbit anti-NLRC4 monoclonal antibody (ECM Biosciences, China). β-actin was detected with rabbit anti-β-actin monoclonal antibody (Abmart, Chin).

Techniques: Activation Assay, Infection, Derivative Assay

Journal: Cell Death & Disease

Article Title: Sensing soluble uric acid by Naip1-Nlrp3 platform

doi: 10.1038/s41419-021-03445-w

Figure Lengend Snippet: A Uric acid levels measured in the serum of humans, mice and rhesus monkeys. IL-1β Elisa of B monocyte-derived macrophages collected from healthy people, C murine bone marrow-derived macrophages and D monocyte-derived macrophages collected from rhesus monkeys, all stimulated under different conditions. In B – D , LPS was added for 1 h at 100 ng/mL and the media were posteriorly changed. MSU (100 μg/mL) and sUA (200 μΜ) were added for 6 h. Each coloured dot represents a different individual. E IL-1β Elisa of BMDM derived from Naip1 −/− , Naip2 −/− , Naip5 −/− , ΔNaip −/− and Nlrc4 −/− mice under different stimulus. F IL-1β Elisa of human THP1 cells virally transduced with plasmids carrying Naip1, Naip5, Naip6, Nlrc4 and empty vector using lentivirus constructs and posteriorly stimulated under different conditions. In E , F , LPS was added for 1 h at 100 ng/mL and the media were posteriorly changed. sUA (200 μΜ) was added for 6 h and nigericin (10 μΜ) was added for 90 min. In A , n = 5 for each analysed species. Triangle refers to human’s sample, Circle refers to murine’s sample and Square refers to rhesus’ sample. In B , C , we collected cells from eight different individuals; in D , we collected cells from seven different individuals. In E , F , data are plotted as the median of a triplicate of three to four independent experiments. * p < 0.05 and *** p < 0.001; n.s. not significant.

Article Snippet: Plasmids for GFP-tagged mNaip1 (#60200), Naip5 (#60205), Naip6 (#60202), Nlrc4 (#60199) and empty vector (#60206) were purchased from Addgene (Watertown, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Transduction, Plasmid Preparation, Construct

Journal: The Journal of Biological Chemistry

Article Title: Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4 phosphorylation

doi: 10.1074/jbc.RA119.010648

Figure Lengend Snippet: NLRC4-related pathway was activated in the gingival tissue of diabetic mice and macrophage exposed to high glucose for 24 h. A, Western blotting analysis showing IRF8, p-NLRC4, and NLRC4 specific immunoreactivity in the gingival tissue of the N and D groups. The O.D. values of IRF8, p-NLRC4, and NLRC4 levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus N group. B, representative of immunohistochemical staining of IRF8 and NLRC4 on the gingival sections from the N group and D group. Scale bar, 50 μm. The percentage of positive cells was calculated and is represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus N group. C, the expression levels of IRF8, p-NLRC4, and NLRC4 in macrophage were analyzed by Western blotting. The O.D. values of IRF8, NLRC4, and p-NLRC4 levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus 30 mm glucose for 6 h. #, p < 0.05; ##, p < 0.01 versus 30 mm glucose for 24 h. The black vertical line represents the splicing border, because there is a protein marker between the 6- and 24-h samples in one band. D, IRF8 and NLRC4 levels were measured by immunofluorescence staining. Scale bar, 50 μm. The fluorescence intensity is represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus 5 mm glucose for 24 h. E, the expression levels of Caspase-1, cleaved Caspase-1, and NF-κB were analyzed by Western blotting in vivo. The O.D. values of Caspase-1, cleaved Caspase-1, and NF-κB levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus N group. Fig. 1C and panels A and E in Fig. 3 use the same band of β-actin because of the identical protein sample in same Western blotting experiment. F, the expression levels of Caspase-1, cleaved Caspase-1, and NF-κB were analyzed by Western blotting in vitro. The O.D. values of Caspase-1, cleaved Caspase-1, and NF-κB levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus 30 mm glucose for 6 h. #, p < 0.05; ##, p < 0.01 versus 30 mm glucose for 24 h. The black vertical line represents the splicing border, because there is a protein marker between the 6- and 24-h samples in one band. Figs. 2B and ​and33 (C and F) use the same band of β-actin because of the identical protein sample in same Western blotting experiment.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against β-actin (1:1000; ta-09; ZSGB-BIO), p-NLRC4 (1:500; NM5491; ECMbiosciences), NLRC4 (1:1000; ab99860; Abcam), IRF8 (1:1000; sc-365042; Santa Cruz), Caspase-1 (1:1000; ET1608–69; Huabio), NF-κB p65 (1:1000; sc-514451; Santa Cruz), p16 (1:1000; sc-166760; Santa Cruz), and p21 (1:1000; sc-166630; Santa Cruz) followed by either secondary anti-rabbit (1:5000; 1706515; Bio-Rad) or anti-mouse (1:5000; ZB-2305; ZSGB-BIO) antibody for 1 h at room temperature.

Techniques: Western Blot, Immunohistochemical staining, Staining, Expressing, Marker, Immunofluorescence, Fluorescence, In Vivo, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4 phosphorylation

doi: 10.1074/jbc.RA119.010648

Figure Lengend Snippet: High glucose was incapable of inducing cellular senescence and SASP in in IRF8−/− or NLRC4−/− macrophage exposed to 30 mm glucose for 24 h. A, the activity of SA–β-gal was determined in control, NLRC4−/−, and IRF8−/− macrophage exposed to 5 and 30 mm glucose for 24 h. Scale bar, 100 μm. The rate of SA–β-gal–positive cells is represented in bar histograms. The data are means ± S.D. (n = 3). **, p < 0.01 versus control CRISPR/Cas9 plasmid (30 mm glucose). B, p16 and p21 levels were measured by immunofluorescence staining. Scale bar, 50 μm. The fluorescence intensity is represented in bar histograms. The data are means ± S.D. (n = 3). **, p < 0.01 versus control CRISPR/Cas9 plasmid (30 mm glucose). C, the change of SASP-associated factors is displayed in the heat map. D, Western blotting analysis showing IRF8, p-NLRC4, NLRC4, Caspase-1, cleaved Caspase-1, NF-κB, p16, and p21 specific immunoreactivity. The O.D. values of these proteins' levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus control CRISPR/Cas9 plasmid (30 mm glucose).

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against β-actin (1:1000; ta-09; ZSGB-BIO), p-NLRC4 (1:500; NM5491; ECMbiosciences), NLRC4 (1:1000; ab99860; Abcam), IRF8 (1:1000; sc-365042; Santa Cruz), Caspase-1 (1:1000; ET1608–69; Huabio), NF-κB p65 (1:1000; sc-514451; Santa Cruz), p16 (1:1000; sc-166760; Santa Cruz), and p21 (1:1000; sc-166630; Santa Cruz) followed by either secondary anti-rabbit (1:5000; 1706515; Bio-Rad) or anti-mouse (1:5000; ZB-2305; ZSGB-BIO) antibody for 1 h at room temperature.

Techniques: Activity Assay, Control, CRISPR, Plasmid Preparation, Immunofluorescence, Staining, Fluorescence, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4 phosphorylation

doi: 10.1074/jbc.RA119.010648

Figure Lengend Snippet: Metformin ameliorated the burden of senescent cells in gingival tissue and the SASP in serum of diabetic mice. A, all protocols were performed strictly according to the procedure. The diabetic mice were treated with the metformin (300 mg/kg body weight, everyday) from weeks 9 to 17. B, fasting blood glucose levels were determined at sacrifice (week 17) among control mice (N group), diabetic mice (D group), and diabetic mice treated with metformin (DM group). The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus D group. C, representative of immunohistochemical staining of p16, p21, IRF8, and NLRC4 on the gingival sections from the N, D, and DM groups. Scale bar, 50 μm. The percentage of positive cells was calculated and is represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus the D group. D, the SASP factors in the serum of the N, D, and DM groups were determined at sacrifice (week 17) by a Luminex assay customization tool and shown in the heat map. E, the gingival tissues of the N, D, and DM group mice were stained for immunofluorescence using an F4/80 antibody targeting macrophages (red) and a p16 antibody (green). The nuclei were stained with DAPI (blue). Scale bar, 50 μm. F, IRF8, p-NLRC4, NLRC4, Caspase-1, cleaved Caspase-1, NF-κB, p16, and p21 in the gingival tissue of the N, D, and DM groups were analyzed by Western blotting. The O.D. values of these proteins' levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus D group.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against β-actin (1:1000; ta-09; ZSGB-BIO), p-NLRC4 (1:500; NM5491; ECMbiosciences), NLRC4 (1:1000; ab99860; Abcam), IRF8 (1:1000; sc-365042; Santa Cruz), Caspase-1 (1:1000; ET1608–69; Huabio), NF-κB p65 (1:1000; sc-514451; Santa Cruz), p16 (1:1000; sc-166760; Santa Cruz), and p21 (1:1000; sc-166630; Santa Cruz) followed by either secondary anti-rabbit (1:5000; 1706515; Bio-Rad) or anti-mouse (1:5000; ZB-2305; ZSGB-BIO) antibody for 1 h at room temperature.

Techniques: Control, Immunohistochemical staining, Staining, Luminex, Immunofluorescence, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4 phosphorylation

doi: 10.1074/jbc.RA119.010648

Figure Lengend Snippet: Metformin attenuated high glucose–induced cellular senescence and SASP in macrophage exposed to high glucose (30 mm) for 24 h. A, the activity of SA–β-gal were determined in macrophage treated with low glucose (5 mm) (N), high glucose (30 mm) (HG), and high glucose (30 mm) + metformin (10 mm) (HGM). Scale bar, 100 μm. The rate of SA–β-gal–positive cells was calculated and is represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus HG. B and E, p16, p21, IRF8, and NLRC4 levels were measured by immunofluorescence staining. Scale bar, 50 μm. The fluorescence intensity is represented in bar histograms. The data are means ± S.D. (n = 3). **, p < 0.01 versus HG. C, the change of SASP factors are displayed in the heat map. D, the expression levels of IRF8, p-NLRC4, NLRC4, Caspase-1, cleaved Caspase-1, NF-κB, p16, and p21 in N, HG, and HGM were analyzed by Western blotting. The O.D. values of these proteins' levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus HG.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against β-actin (1:1000; ta-09; ZSGB-BIO), p-NLRC4 (1:500; NM5491; ECMbiosciences), NLRC4 (1:1000; ab99860; Abcam), IRF8 (1:1000; sc-365042; Santa Cruz), Caspase-1 (1:1000; ET1608–69; Huabio), NF-κB p65 (1:1000; sc-514451; Santa Cruz), p16 (1:1000; sc-166760; Santa Cruz), and p21 (1:1000; sc-166630; Santa Cruz) followed by either secondary anti-rabbit (1:5000; 1706515; Bio-Rad) or anti-mouse (1:5000; ZB-2305; ZSGB-BIO) antibody for 1 h at room temperature.

Techniques: Activity Assay, Immunofluorescence, Staining, Fluorescence, Expressing, Western Blot