Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 was upregulated and positively correlated with NET formation in renal transplant patients. ( A ) Paired analysis of serum IL-33 levels, ( B ) paired analysis of serum MPO-DNA levels, ( C ) paired analysis of serum citH3 levels in renal transplant patients preoperatively and postoperatively ( n = 20). ( D ) Correlation analysis for postoperative serum IL-33 level and serum MPO-DNA level (Spearman r = 0.6892, P = 0.0008). ( E ) Correlation analysis of postoperative serum IL-33 level and serum citH3 level (Spearman r = 0.6486, P = 0.002). * P < 0.05, ** P < 0.01

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques:

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 and NETs were elevated following renal I/R in mice. ( A - B ) Serum IL-33 levels ( A ) and renal tissue homogenate IL-33 levels ( B ) after renal I/R ( n = 6). ( C - D ) Representative blots ( C ) and statistical analysis ( D ) of IL-33 protein expression levels in renal tissues after I/R ( n = 6). ( E - F ) Representative images ( E ) and statistical analysis ( F ) of IL-33 immunohistochemistry in renal tissues after I/R ( n = 6). Scale bar was 50 μm. ( G - H ) IL-33 expression and distribution among the groups (blue staining indicate DAPI for nuclei), IL-33 (red) and CD31 (green) ( n = 6). Scale bar was 50 μm. (I-J) Serum MPO-DNA (I) and citH3 levels ( J ) following renal I/R ( n = 6). ( K - L ) Representative blots ( K ) and statistical analysis ( L ) of citH3 protein expression levels in renal tissues after I/R ( n = 6). ( M - N ) NET formation in in the indicated groups (blue indicate DAPI staining), MPO (red) and citH3 (green) ( n = 6). Scale bar was 20 μm. ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 promoted NET generation during renal IRI. ( A ) A diagram showing WT mice receiving renal IRI or sham surgery following intraperitoneal injection of rIL-33 (10 µg per mouse) or vehicle (PBS). ( B - C ) Serum MPO-DNA ( B ) and citH3 levels ( C ) of mice in each group ( n = 6). ( D - E ) Representative blots ( D ) and statistical analyses ( E ) of citH3 protein expression levels in renal tissues in the indicated groups ( n = 6). ( F - G ) NET formation in the indicated groups through DAPI staining (blue), MPO (red) and citH3 (green) ( n = 6). Scale bar was 20 μm. ( H - I ) Serum Cr ( H ) and BUN levels ( I ) in the indicated groups ( n = 6). ( J - K ) Representative blots ( J ) and statistical analysis ( K ) of KIM-1 protein expression levels in renal tissues in the indicated groups ( n = 6). ( L - N ) Renal tissues from mice in the indicated groups stained with HE and KIM-1 ( L ), followed by score of tubular injury ( M ) and quantitative analysis of KIM-1 ( N ) ( n = 6). Scale bar was 50 μm. * P < 0.05, ** P < 0.01

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: Injection, Expressing, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 exacerbated renal IRI by inducing NET formation. ( A ) A diagram showing the time node of intraperitoneal injection of GSK484 (4 mg/kg) and rIL-33 (10 µg per mouse) in WT mice before renal I/R. (B-C) Serum MPO-DNA ( B ) and citH3 levels ( C ) in the indicated groups ( n = 6). ( D - E ) Representative blots ( D ) and statistical analysis ( E ) of citH3 expression levels in renal tissues of the indicated groups ( n = 6). ( F - G ) NET formation in the indicated groups as determined using the DAPI (blue), MPO (red) and citH3 (green) staining ( n = 6). Scale bar was 20 μm. ( H - I ) Serum Cr ( H ) and BUN levels ( I ) in the indicated groups ( n = 6). ( J - K ) Representative blots ( J ) and statistical analysis ( K ) of KIM-1 protein expression levels in renal tissues from the indicated groups ( n = 6). ( L - N ) Renal tissues of mice in the indicated groups stained with HE and KIM-1 ( L ), followed by score of tubular injury ( M ) and quantitative analysis of KIM-1 ( N ) ( n = 6). Scale bar was 50 μm. ** P < 0.01, *** P < 0.001

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: Injection, Expressing, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: Blocking IL-33 improved renal IRI by reducing NET formation. ( A ) The diagram of WT mice receiving renal IRI or sham surgery after intraperitoneal injection of anti-IL-33 monoclonal antibody (Anti-IL-33) (10 µg per ounce) or vehicle (IgG2b Isotype Control). ( B - C ) Serum MPO-DNA ( B ) and citH3 levels ( C ) of mice in the indicated groups ( n = 6). (D-E) Representative blots ( D ) and statistical analysis ( E ) of citH3 protein expression levels in renal tissues in the indicated groups ( n = 6). ( F - G ) NET formation in the indicated groups following DAPI (blue), MPO (red) and citH3 (green) staining ( n = 6). Scale bar was 20 μm. ( H - I ) Serum Cr ( H ) and BUN levels ( I ) in the indicated group ( n = 6). ( J - K ) Representative blots ( J ) and statistical analysis ( K ) of KIM-1 protein expression levels in renal tissues in the indicated groups ( n = 6). ( L - N ) Renal tissues of mice in the indicated groups subjected to HE and KIM-1 staining ( L ), followed by score of tubular injury ( M ) and statistical analysis of KIM-1 ( N ) ( n = 6). Scale bar was 50 μm. ** P < 0.01, *** P < 0.001

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: Blocking Assay, Injection, Control, Expressing, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 stimulated NET generation in vitro. ( A - B ) Neutrophils incubated with PBS, PMA (100nM) and various concentrations of rIL-33 (20, 60, 100 ng/mL) for 4 h. rIL-33 stimulation increased MPO-DNA ( A ) and citH3 levels ( B ) in the neutrophil culture medium in a dose-dependent manner relative to the PBS group ( n = 3). ( C - D ) Relative to the PBS group, rIL-33 activated neutrophils to increase citH3 protein in a dose-dependent manner ( n = 3). ( E ) Compared with the PBS group, rIL-33 increased the expression of ST2 mRNA on neutrophils ( n = 3). ( F ) Representative scanning electron microscopy graphs of neutrophils treated with PBS or rIL-33 (100ng/mL) for 4 h. Scale bar was 10 μm. ( G ) The NET formation as indicated by DAPI (blue), MPO (red) and citH3 (green) staining, was comparable between rIL-33 (100ng/mL) and PMA group. Scale bar was 50 μm. ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: In Vitro, Incubation, Expressing, Electron Microscopy, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 induced NET formation via ST2/PI3K/Akt and ST2/PAD4 signaling pathways. Mouse bone marrow-derived neutrophils were treated with PBS or rIL-33 (100ng/mL) for 4 h, followed by RNA sequencing ( n = 3). ( A - B ) Volcano plot ( A ) and heatmap ( B ) showing differential gene expression between PBS and rIL-33 groups. ( C ) GO enrichment analysis of DEGs. ( D ) KEGG pathway enrichment analysis of DEGs. Bone marrow-derived neutrophils from WT and ST2 KO mice were treated with PBS or rIL-33 (100ng/mL) for 4 h. The production of MPO-DNA ( E ) and citH3 ( F ) in the cell culture medium of ST2 KO neutrophils treated with IL-33 was markedly decreased relative to the IL-33-treated WT neutrophils ( n = 3). ( G ) Confocal microscopy was conducted to examine NET formation (co-localization of DAPI, MPO and citH3) in each group. ( H - I ) The expression of PI3K, p-PI3K, Akt, p-Akt and citH3 in WT and ST2 KO neutrophils stimulated by rIL-33 as determined by Western blot ( n = 3). ( J - K ) WT neutrophils were treated with 10µM LY294002 (PI3K inhibitor) or 10µM MK2206 (Akt inhibitor) for 24 h, and 100ng/mL rIL-33 was added on the 20th h to stimulate WT neutrophils for 4 h. The expression of PI3K, p-PI3K, Akt, p-Akt and citH3 in neutrophils was determined by Western blot ( n = 3). ( L ) Gene expression of Padi1, Padi2, Padi4 and Padi6 in neutrophils stimulated with rIL-33 ( n = 3). The gene expression data were expressed as log 2 (FPKM + 1). ( M - N ) PAD4 protein expression levels and quantitative analysis in WT and ST2 KO neutrophils treated with rIL-33 were quantified by Western blots ( n = 3). ( O - P ) WT neutrophils were pretreated with GSK484 (PAD4 inhibitor) for 30 min after treatment with 100ng/mL rIL-33 for 4 h. The protein expression of citH3 in neutrophils was determined by Western blot ( n = 3). ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques: Protein-Protein interactions, Derivative Assay, RNA Sequencing, Gene Expression, Cell Culture, Confocal Microscopy, Expressing, Western Blot

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: Schematic diagram of the mechanism by which IL-33 exacerbated renal IRI by enhancing NET formation during renal I/R

Article Snippet: Mice were intraperitoneally injected with recombinant IL-33 (rIL-33) (10ug per mouse, MedChemExpress, America) or PBS immediately following exposure to renal ischemia.

Techniques:

Journal: Clinical & Translational Immunology

Article Title: Blockade of IL‐33 signalling attenuates osteoarthritis

doi: 10.1002/cti2.1187

Figure Lengend Snippet: Increasing IL‐33 levels exacerbates OA in vivo . (a) protein and (b) mRNA expression of cartilage‐degrading proteases (MMP‐13, ADAMTS‐5 and MMP‐3) and chondrogenic markers (COL2A1, SOX‐9 and Aggrecan) in isolated human chondrocytes from Non‐OA ( n = 20) and OA patients ( n = 20) treated with either PBS (vehicle control) or rIL‐33 (30 ng mL −1 , 24 h). (c) MMP‐13 and (d) MMP‐3 protein level in cell media supernatants obtained from isolated human chondrocytes from Non‐OA ( n = 12) and OA patients ( n = 12) treated with either PBS (vehicle control) or rIL‐33 (30 ng mL −1 ; 24 h). (e, f) OARSI scoring of cartilage tissue, (g) synovitis scoring and (h) osteophyte maturity scoring of sham‐operated ( n = 20) or DMM‐operated ( n = 20) WT mice (12 weeks post‐surgery end timepoint) treated intraperitoneally with either PBS (vehicle control) or rIL‐33 (33 μg kg −1 ; daily for 12 weeks post‐surgery). (i) von Frey pain assessment of sham‐operated ( n = 20) or DMM‐operated ( n = 20) WT mice (12 weeks post‐surgery end timepoint) treated intraperitoneally with either PBS (vehicle control) or rIL‐33 (33 μg kg −1 ; daily for 12 weeks post‐surgery). (j) mRNA expression of cartilage‐degrading proteases (MMP‐13, ADAMTS‐5 and MMP‐3) and chondrogenic markers (COL2A1, SOX‐9 and Aggrecan) in whole knee joints of DMM‐operated ( n = 20) WT mice (12 weeks post‐surgery end timepoint) treated intraperitoneally with either PBS (vehicle control) or rIL‐33 (33 μg kg −1 ; daily for 12 weeks post‐surgery). All RT‐qPCR gene expressions were normalised to the endogenous level of 18 s in respective groups. Data are expressed as mean ± S.E.M. with unpaired 2‐tailed Student’s t ‐tests ( c, d ), two‐way analysis of variance followed by the Tukey‐Kramer test ( f, g, h ), or repeated measures 2‐way ANOVA with Bonferroni’s post hoc tests was used to compare groups at each time point (i; DMM PBS vs DMM rIL‐33). n indicates the number of human specimens or mice per group. NS = non‐significant. P < 0.01, P < 0.001 or P < 0.0001 are represented as **, *** or ****, respectively.

Article Snippet: For experiments which increased IL‐33 levels in vivo , mouse rIL‐33 (Enzo Life Sciences, Farmingdale) was administered intraperitoneally (i.p) on a daily basis at 33 μg kg −1 for 12 weeks post‐surgery.

Techniques: In Vivo, Expressing, Isolation, Quantitative RT-PCR

Journal: Clinical & Translational Immunology

Article Title: Blockade of IL‐33 signalling attenuates osteoarthritis

doi: 10.1002/cti2.1187

Figure Lengend Snippet: Neutralising ST2 attenuates OA. (a, b) OARSI scoring of cartilage tissue, (c) synovitis scoring and (d) osteophyte maturity scoring of sham‐operated ( n = 20) or DMM‐operated ( n = 20) WT mice (12 weeks post‐surgery end timepoint) treated intraperitoneally with either IgG1 (vehicle control; 50 µg per mouse; daily for 12 weeks post‐surgery) or αST2 (50 µg per mouse; daily for 12 weeks post‐surgery). (e) von Frey pain assessment of sham‐ ( n = 20) or DMM‐ ( n = 20) operated WT mice (12 weeks post‐surgery end timepoint) treated intraperitoneally with either IgG1 (vehicle control; 50 µg per mouse; daily for 12 weeks post‐surgery) or αST2 (50 µg per mouse; daily for 12 weeks post‐surgery). (f) mRNA expression of cartilage‐degrading proteases (MMP‐13, ADAMTS‐5 and MMP‐3) and chondrogenic markers (COL2A1, SOX‐9 and Aggrecan) in whole knee joints of DMM‐operated ( n = 20) treated intraperitoneally with either IgG1 (vehicle control; 50 µg per mouse; daily for 12 weeks post‐surgery) or αST2 (50 µg per mouse; daily for 12 weeks post‐surgery). (g) protein and (h) mRNA expression of cartilage degrading proteases (MMP‐13, ADAMTS‐5 and MMP‐3) and chondrogenic markers (COL2A1, SOX‐9 and Aggrecan) in isolated human chondrocytes from Non‐OA ( n = 20) and OA patients ( n = 20) treated with rIL‐33 (30 ng mL −1 , 24 h) and IgG1 (vehicle control; 3 μg mL −1 , 24 h) or αST2 (3 μg mL −1 , 24 h). (i) MMP‐13 (j) MMP‐3 protein level in cell media supernatants obtained from isolated human chondrocytes from Non‐OA ( n = 12) and OA patients ( n = 12) treated with rIL‐33 (30 ng mL −1 ; 24 h) and IgG1 (vehicle control; 3 μg mL −1 , 24 h) or αST2 (3 μg mL −1 , 24 h). All RT‐qPCR gene expressions were normalised to the endogenous level of 18 s in respective groups. Data are expressed as mean ± S.E.M. with two‐way analysis of variance followed by the Tukey‐Kramer test (b, c, d) or repeated measures 2‐way ANOVA with Bonferroni’s post hoc tests was used to compare groups at each time point ( e ; DMM IgG1 control mice vs DMM αST2 mice) or with unpaired 2‐tailed Student’s t ‐tests (i, j) . n indicates the number of human specimens or mice per group. NS = non‐significant. P < 0.001 or P < 0.0001 are represented as *** or ****, respectively.

Article Snippet: For experiments which increased IL‐33 levels in vivo , mouse rIL‐33 (Enzo Life Sciences, Farmingdale) was administered intraperitoneally (i.p) on a daily basis at 33 μg kg −1 for 12 weeks post‐surgery.

Techniques: Expressing, Isolation, Quantitative RT-PCR

Journal: Clinical & Translational Immunology

Article Title: Blockade of IL‐33 signalling attenuates osteoarthritis

doi: 10.1002/cti2.1187

Figure Lengend Snippet: Neutralising IL‐33 attenuates OA. (a, b) OARSI scoring of cartilage tissue, (c) synovitis scoring and (d) osteophyte maturity scoring of sham‐operated ( n = 20) or DMM‐operated ( n = 20) WT mice (12 weeks post‐surgery end timepoint) treated intraperitoneally with either IgG1 (vehicle control; 15 µg per mouse; daily for 12 weeks post‐surgery) or αIL‐33 (15 µg per mouse; daily for 12 weeks post‐surgery). (e) von Frey pain assessment of sham‐operated ( n = 20) or DMM‐operated ( n = 20) WT mice (12 weeks post‐surgery end timepoint) treated intraperitoneally with either IgG1 (vehicle control; 15 µg per mouse; daily for 12 weeks post‐surgery) or αIL‐33 (15 µg per mouse; daily for 12 weeks post‐surgery). (f) mRNA expression of cartilage degrading proteases (MMP‐13, ADAMTS‐5 and MMP‐3) and chondrogenic markers (COL2A1, SOX‐9 and Aggrecan) in whole knee joints of DMM‐operated ( n = 20) treated intraperitoneally with either IgG1 (vehicle control; 15 µg per mouse; daily for 12 weeks post‐surgery) or αIL‐33 (15 µg per mouse; daily for 12 weeks post‐surgery). (g) protein and (h) mRNA expression of cartilage degrading proteases (MMP‐13, ADAMTS‐5 and MMP‐3) and chondrogenic markers (COL2A1, SOX‐9 and Aggrecan) in isolated human chondrocytes from Non‐OA ( n = 20) and OA patients ( n = 20) treated with rIL‐33 (30 ng mL −1 , 24 h) and IgG1 (vehicle control; 10 μg mL −1 , 24 h) or αIL‐33 (10 μg mL −1 , 24 h). (i) MMP‐13 ( j) MMP‐3 protein level in cell media supernatants obtained from isolated human chondrocytes from Non‐OA ( n = 12) and OA patients ( n = 12) treated with rIL‐33 (30 ng mL −1 ; 24 h) and IgG1 (vehicle control; 10 μg mL −1 , 24 h) or αIL‐33 (10 μg mL −1 , 24 h). All RT‐qPCR gene expressions were normalised to the endogenous level of 18 s in respective groups. Data are expressed as mean ± S.E.M. with two‐way analysis of variance followed by the Tukey‐Kramer test ( b, c, d ) or repeated measures 2‐way ANOVA with Bonferroni’s post hoc tests was used to compare groups at each time point ( e ; DMM IgG1 control mice vs DMM αIL‐33 mice) or with unpaired 2‐tailed Student’s t ‐tests ( i, j ). n indicates the number of human specimens or mice per group. NS = non‐significant. P < 0.0001 is represented as ****.

Article Snippet: For experiments which increased IL‐33 levels in vivo , mouse rIL‐33 (Enzo Life Sciences, Farmingdale) was administered intraperitoneally (i.p) on a daily basis at 33 μg kg −1 for 12 weeks post‐surgery.

Techniques: Expressing, Isolation, Quantitative RT-PCR

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 promoted NET generation during renal IRI. ( A ) A diagram showing WT mice receiving renal IRI or sham surgery following intraperitoneal injection of rIL-33 (10 µg per mouse) or vehicle (PBS). ( B - C ) Serum MPO-DNA ( B ) and citH3 levels ( C ) of mice in each group ( n = 6). ( D - E ) Representative blots ( D ) and statistical analyses ( E ) of citH3 protein expression levels in renal tissues in the indicated groups ( n = 6). ( F - G ) NET formation in the indicated groups through DAPI staining (blue), MPO (red) and citH3 (green) ( n = 6). Scale bar was 20 μm. ( H - I ) Serum Cr ( H ) and BUN levels ( I ) in the indicated groups ( n = 6). ( J - K ) Representative blots ( J ) and statistical analysis ( K ) of KIM-1 protein expression levels in renal tissues in the indicated groups ( n = 6). ( L - N ) Renal tissues from mice in the indicated groups stained with HE and KIM-1 ( L ), followed by score of tubular injury ( M ) and quantitative analysis of KIM-1 ( N ) ( n = 6). Scale bar was 50 μm. * P < 0.05, ** P < 0.01

Article Snippet: Neutrophils were seeded in 6-well plates (2 × 10[ ] cells per well) and incubated with Phorbol 12-myristate 13-acetate (PMA, 100 nM, Sigma-Aldrich, America) or rIL-33 (20, 60, 100 ng/mL, MedChemExpress, America) for 4 h. This was followed by treatment with LY294002 (10μM, MedChemExpress, America) for 24 h and stimulation with rIL-33 (100ng/mL, MedChemExpress, America) for 4 h after 20 h. Similarly, neutrophils were treated with MK-2206 (10μM, MedChemExpress, America) for 24 h and incubated with rIL-33 (100ng/mL, MedChemExpress, America) for 4 h after 20 h. They were pretreated with GSK484 (100μM, MedChemExpress, America) for 30 min and stimulated with rIL-33 (100ng/mL, MedChemExpress, America) for 4 h. The supernatant was removed, the samples were washed 2 ml cold PBS and then centrifuged at 1000 g for 10 min at 4°C as previously described [ ].

Techniques: Injection, Expressing, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 exacerbated renal IRI by inducing NET formation. ( A ) A diagram showing the time node of intraperitoneal injection of GSK484 (4 mg/kg) and rIL-33 (10 µg per mouse) in WT mice before renal I/R. (B-C) Serum MPO-DNA ( B ) and citH3 levels ( C ) in the indicated groups ( n = 6). ( D - E ) Representative blots ( D ) and statistical analysis ( E ) of citH3 expression levels in renal tissues of the indicated groups ( n = 6). ( F - G ) NET formation in the indicated groups as determined using the DAPI (blue), MPO (red) and citH3 (green) staining ( n = 6). Scale bar was 20 μm. ( H - I ) Serum Cr ( H ) and BUN levels ( I ) in the indicated groups ( n = 6). ( J - K ) Representative blots ( J ) and statistical analysis ( K ) of KIM-1 protein expression levels in renal tissues from the indicated groups ( n = 6). ( L - N ) Renal tissues of mice in the indicated groups stained with HE and KIM-1 ( L ), followed by score of tubular injury ( M ) and quantitative analysis of KIM-1 ( N ) ( n = 6). Scale bar was 50 μm. ** P < 0.01, *** P < 0.001

Article Snippet: Neutrophils were seeded in 6-well plates (2 × 10[ ] cells per well) and incubated with Phorbol 12-myristate 13-acetate (PMA, 100 nM, Sigma-Aldrich, America) or rIL-33 (20, 60, 100 ng/mL, MedChemExpress, America) for 4 h. This was followed by treatment with LY294002 (10μM, MedChemExpress, America) for 24 h and stimulation with rIL-33 (100ng/mL, MedChemExpress, America) for 4 h after 20 h. Similarly, neutrophils were treated with MK-2206 (10μM, MedChemExpress, America) for 24 h and incubated with rIL-33 (100ng/mL, MedChemExpress, America) for 4 h after 20 h. They were pretreated with GSK484 (100μM, MedChemExpress, America) for 30 min and stimulated with rIL-33 (100ng/mL, MedChemExpress, America) for 4 h. The supernatant was removed, the samples were washed 2 ml cold PBS and then centrifuged at 1000 g for 10 min at 4°C as previously described [ ].

Techniques: Injection, Expressing, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 stimulated NET generation in vitro. ( A - B ) Neutrophils incubated with PBS, PMA (100nM) and various concentrations of rIL-33 (20, 60, 100 ng/mL) for 4 h. rIL-33 stimulation increased MPO-DNA ( A ) and citH3 levels ( B ) in the neutrophil culture medium in a dose-dependent manner relative to the PBS group ( n = 3). ( C - D ) Relative to the PBS group, rIL-33 activated neutrophils to increase citH3 protein in a dose-dependent manner ( n = 3). ( E ) Compared with the PBS group, rIL-33 increased the expression of ST2 mRNA on neutrophils ( n = 3). ( F ) Representative scanning electron microscopy graphs of neutrophils treated with PBS or rIL-33 (100ng/mL) for 4 h. Scale bar was 10 μm. ( G ) The NET formation as indicated by DAPI (blue), MPO (red) and citH3 (green) staining, was comparable between rIL-33 (100ng/mL) and PMA group. Scale bar was 50 μm. ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Neutrophils were seeded in 6-well plates (2 × 10[ ] cells per well) and incubated with Phorbol 12-myristate 13-acetate (PMA, 100 nM, Sigma-Aldrich, America) or rIL-33 (20, 60, 100 ng/mL, MedChemExpress, America) for 4 h. This was followed by treatment with LY294002 (10μM, MedChemExpress, America) for 24 h and stimulation with rIL-33 (100ng/mL, MedChemExpress, America) for 4 h after 20 h. Similarly, neutrophils were treated with MK-2206 (10μM, MedChemExpress, America) for 24 h and incubated with rIL-33 (100ng/mL, MedChemExpress, America) for 4 h after 20 h. They were pretreated with GSK484 (100μM, MedChemExpress, America) for 30 min and stimulated with rIL-33 (100ng/mL, MedChemExpress, America) for 4 h. The supernatant was removed, the samples were washed 2 ml cold PBS and then centrifuged at 1000 g for 10 min at 4°C as previously described [ ].

Techniques: In Vitro, Incubation, Expressing, Electron Microscopy, Staining

Journal: Inflammation

Article Title: Interleukin-33 Promotes Neutrophil Extracellular Trap Formation To Aggravate Renal Ischemia-Reperfusion Injury Through ST2/PI3K/Akt and ST2/PAD4 Pathways

doi: 10.1007/s10753-025-02364-8

Figure Lengend Snippet: IL-33 induced NET formation via ST2/PI3K/Akt and ST2/PAD4 signaling pathways. Mouse bone marrow-derived neutrophils were treated with PBS or rIL-33 (100ng/mL) for 4 h, followed by RNA sequencing ( n = 3). ( A - B ) Volcano plot ( A ) and heatmap ( B ) showing differential gene expression between PBS and rIL-33 groups. ( C ) GO enrichment analysis of DEGs. ( D ) KEGG pathway enrichment analysis of DEGs. Bone marrow-derived neutrophils from WT and ST2 KO mice were treated with PBS or rIL-33 (100ng/mL) for 4 h. The production of MPO-DNA ( E ) and citH3 ( F ) in the cell culture medium of ST2 KO neutrophils treated with IL-33 was markedly decreased relative to the IL-33-treated WT neutrophils ( n = 3). ( G ) Confocal microscopy was conducted to examine NET formation (co-localization of DAPI, MPO and citH3) in each group. ( H - I ) The expression of PI3K, p-PI3K, Akt, p-Akt and citH3 in WT and ST2 KO neutrophils stimulated by rIL-33 as determined by Western blot ( n = 3). ( J - K ) WT neutrophils were treated with 10µM LY294002 (PI3K inhibitor) or 10µM MK2206 (Akt inhibitor) for 24 h, and 100ng/mL rIL-33 was added on the 20th h to stimulate WT neutrophils for 4 h. The expression of PI3K, p-PI3K, Akt, p-Akt and citH3 in neutrophils was determined by Western blot ( n = 3). ( L ) Gene expression of Padi1, Padi2, Padi4 and Padi6 in neutrophils stimulated with rIL-33 ( n = 3). The gene expression data were expressed as log 2 (FPKM + 1). ( M - N ) PAD4 protein expression levels and quantitative analysis in WT and ST2 KO neutrophils treated with rIL-33 were quantified by Western blots ( n = 3). ( O - P ) WT neutrophils were pretreated with GSK484 (PAD4 inhibitor) for 30 min after treatment with 100ng/mL rIL-33 for 4 h. The protein expression of citH3 in neutrophils was determined by Western blot ( n = 3). ns no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Neutrophils were seeded in 6-well plates (2 × 10[ ] cells per well) and incubated with Phorbol 12-myristate 13-acetate (PMA, 100 nM, Sigma-Aldrich, America) or rIL-33 (20, 60, 100 ng/mL, MedChemExpress, America) for 4 h. This was followed by treatment with LY294002 (10μM, MedChemExpress, America) for 24 h and stimulation with rIL-33 (100ng/mL, MedChemExpress, America) for 4 h after 20 h. Similarly, neutrophils were treated with MK-2206 (10μM, MedChemExpress, America) for 24 h and incubated with rIL-33 (100ng/mL, MedChemExpress, America) for 4 h after 20 h. They were pretreated with GSK484 (100μM, MedChemExpress, America) for 30 min and stimulated with rIL-33 (100ng/mL, MedChemExpress, America) for 4 h. The supernatant was removed, the samples were washed 2 ml cold PBS and then centrifuged at 1000 g for 10 min at 4°C as previously described [ ].

Techniques: Protein-Protein interactions, Derivative Assay, RNA Sequencing, Gene Expression, Cell Culture, Confocal Microscopy, Expressing, Western Blot