Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: MIIP in CRC cells inhibits neutrophil infiltration and NETs formation through IL‐8. (A) Human cytokine array of the CM collected from SW620 cells with vector control and MIIP overexpression. (B) ELISA quantification of secreted IL‐8 protein levels in CM derived from three CRC cell lines with differential MIIP expression ( n = 3). (C) Volcano plot of DEGs from RNA‐seq analysis of CRC cell lines with varying levels of MIIP expression ( n = 3 per group). (D) CXCL8 mRNA expression in human CRC cells with different levels of MIIP expression was tested by RT–qPCR ( n = 3). (E) Quantification of neutrophils that migrated to CRC CM with or without recombinant human IL‐8 protein (rhIL‐8). Quantification of neutrophils that migrated to the CRC CM with or without anti‐IL‐8 neutralizing antibody (N‐IL‐8) ( n = 3). (F–H) Representative images and statistical quantification of IF staining ( n = 5) and MPO‐DNA ELISA ( n = 3) for NETs formation after neutrophils were co‐cultured with the CM from SW620 cells treated with recombinant IL‐8 protein and with the CM from SW480 and HCT116 cells treated with an anti‐IL‐8 neutralizing antibody. Scale bar, 50 µm. All the data are shown as the mean ± SD; * , p < 0.05; ** , p < 0.01; *** , p < 0.001. NS, not significant. Statistical significance was assessed using Student's t‐test and two‐way ANOVA in (B, D, E, G, and H).

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques: Plasmid Preparation, Control, Over Expression, Enzyme-linked Immunosorbent Assay, Derivative Assay, Expressing, RNA Sequencing, Analysis, Quantitative RT-PCR, Recombinant, Staining, Cell Culture

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: MIIP in CRC cells decreases neutrophil infiltration and NETs formation through NF‐κB p65/IL‐8 signaling. (A) Bubble plot of the enriched KEGG pathways showing that the NF‐κB signaling pathway was enriched in HCT116 cells (marked with a red border). (B) GSEA revealed that MIIP knockdown in HCT116 cells significantly activated the NF‐κB signaling pathway. (C) Western blotting analysis of key proteins involved in the NF‐κB p65 signaling pathway in human CRC cells with different levels of MIIP expression. (D) Representative images of H&E and IHC staining for MIIP, p‐p65, IL‐8, and Ly6G in tumors from the cecum of an orthotopic CRC mouse model between the MIIP‐overexpressing group and the control group (n = 5 per group). Scale bar, 50 µm. (E) Statistical analysis of Ly6G + neutrophils and the expression of p‐p65 and IL‐8 in different groups. (F) Representative images and statistical quantification of IF staining for MPO (red), Cit‐H3 (green), and DAPI (blue) in tumor tissue from the cecum and comparisons between the two groups ( n = 5). Scale bar, 25 µm. (G) Western blotting of NF‐κB p65 and NF‐κB p‐p65 in CRC cells treated with or not treated with an NF‐κB inhibitor (Bay 11–7082, 10 µM). (H) RT‐qPCR of CXCL8 mRNA in CRC cells treated or not treated with an NF‐κB inhibitor ( n = 3). (I) ELISA of IL‐8 in CM from CRC cells treated or not treated with an NF‐κB inhibitor ( n = 3). (J) Quantification of neutrophils that migrated to the CM from CRC cells treated or not treated with an NF‐κB inhibitor ( n = 3). (K–M) ELISA of MPO‐DNA and representative images and statistical quantification of IF staining for NETs formation after neutrophils were cultured with CM from SW480 and HCT116 cells treated or not treated with an NF‐κB inhibitor ( n = 3–5). Scale bar, 50 µm. All the data are shown as the mean ± SD; * , p < 0.05; ** , p < 0.01; *** , p < 0.001. NS, not significant. Statistical significance was assessed using Student's t‐test and the Mann‐Whitney U test in (E); the Mann‐Whitney U test in (F); and two‐way ANOVA in (H, I, J, K, and M).

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques: Knockdown, Western Blot, Analysis, Expressing, Immunohistochemistry, Control, Staining, Tissue, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, MANN-WHITNEY

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: MIIP modulates the NF‐κB p65/IL‐8 signaling pathway ‐mediated neutrophil infiltration and NETs formation through interactions with PKR. (A) IP–MS revealed PKR as a MIIP‐interacting protein. (B) The binding structure of MIIP and PKR was predicted using HDOCK molecular docking software. (C) Endogenous interaction between MIIP and PKR were detected via Co‐IP in HCT116 cells. (D) 293T cells were cotransfected with MIIP‐Flag and PKR‐HA, and reverse and forward Co‐IP analyses were performed. (E) Co‐IP assays were used to determine the PKR‐binding region of MIIP. (F) Co‐IP assays revealed that the PKR binding site for MIIP encompasses its IKKβ‐binding domain. (G) The expression of PKR was determined by Western blotting in CRC cells with different levels of MIIP expression. (H) Co‐IP was performed to examine the effect of MIIP expression on the interaction between PKR and IKKβ. (I–M) Double‐stable transgenic CRC cell lines were used in a rescue assay to demonstrate that the regulatory effects of MIIP on the NF‐κB p65‐mediated IL‐8 signaling pathway (I–K) and NETs formation (L, M) were dependent on PKR ( n = 3–5). All the data are shown as the mean ± SD; * , p < 0.05; ** , p < 0.01; *** , p < 0.001. NS, not significant. Statistical significance was assessed using two‐way ANOVA in (J–M).

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques: Protein-Protein interactions, Binding Assay, Docking Assay, Software, Co-Immunoprecipitation Assay, Expressing, Western Blot, Transgenic Assay, Rescue Assay

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: Schematic illustration shows that MIIP inhibits colorectal cancer progression by decreasing TANs infiltration and NETs formation through the PKR/NF‐κB p65/IL‐8 axis, providing potential therapeutic strategies for colorectal cancer.

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques:

Journal: Military Medical Research

Article Title: The cellular response capacity (CRC) as a novel immunomonitoring approach in sepsis

doi: 10.1016/j.mmr.2026.100010

Figure Lengend Snippet: Concentration-dependent change in the humoral inflammatory response following incubation with Escherichia coli ( E. coli ) in the ex vivo whole blood model. a Absolute plasma concentrations of IL-6, IL-8, and MMP9 determined by enzyme-linked immunosorbent assay. b Normalized values and EC 50 curve fit by BuC=0% and 50 000 CFU/ml E. coli= 100%, respectively, for IL-6, IL-8, and MMP9 as indicated by EC 50 (%) on the respective Y-axis. BuC indicates buffer control after 60 min incubation; numbers on the X-axis indicate E. coli bacteria in concentrations of 2000 to 50 000 CFU/ml after 60 min incubation; LPS indicates lipopolysaccharide (LPS) 100 ng/ml after 60 min incubation. Values are shown as median and interquartile range. n =8. Statistical analysis was performed using the Kruskal-Wallis test with Dunn’s post-hoc test, comparing all shown concentrations of E. coli bacteria and 100 ng/ml LPS with BuC. P -values are indicated above the respective data points. ⁎ P <0.05, ⁎⁎ P <0.01, ⁎⁎⁎ P <0.001. CFU. Colony-forming units; IL. Interleukin; MMP9. Matrix metallopeptidase 9.

Article Snippet: For the samples of the ex vivo whole blood model, the plasma concentrations of matrix metallopeptidase 9 (MMP9, #DY911, R&D Systems, Minneapolis, USA), IL-6 (#555220, BD Biosciences, San Jose, USA), and IL-8 (#DY208, R&D Systems) were measured in citrate-anticoagulated plasma using enzyme-linked immunosorbent assay according to the respective manufacturer’s instructions.

Techniques: Concentration Assay, Incubation, Ex Vivo, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Control, Bacteria

Journal: Military Medical Research

Article Title: The cellular response capacity (CRC) as a novel immunomonitoring approach in sepsis

doi: 10.1016/j.mmr.2026.100010

Figure Lengend Snippet: Diagnostic performance for the detection of bacteremia, analyzing the neutrophil phenotype by determining the median fluorescence intensity (MFI) and the cellular response capacity (CRC) in comparison with traditional markers of humoral inflammation (IL-6, IL-8, MMP9). a Comparison of receiver operating characteristic (ROC) at 10,000 CFU/ml Escherichia coli ( E. coli ) with the respective 95% confidence interval (CI) and P -value, and half-maximal effective concentration (EC 50 ) as a function of the E. coli concentration. b Detailed comparison of the EC 50 as a function of the E. coli concentration. c Exemplary comparison of EC 50 curve fit after normalization as indicated by EC 50 (%) on the respective Y-axis to BuC=100% and 50 000 CFU/ml E. coli =0% for the humoral marker IL-6 (the IL-6 values were multiplied by −1 before EC 50 calculation to facilitate comparability with the CRC) and the change in neutrophil phenotype represented by CD11b CRC. BuC indicates buffer control after 60 min incubation; numbers on the X-axis of c indicate E. coli bacteria in concentrations of 2000 to 50 000 CFU/ml after 60 min incubation. Values are shown as median and interquartile range. n =8. Statistical analysis was performed using the Kruskal-Wallis test with Dunn’s post-hoc test, evaluating the EC 50 of IL-8, MMP9, the MFI, and CRC of CD10, CD11b, and CD62L in comparison to the EC 50 of IL-6. P -values are indicated above the respective data points. ⁎ P <0.05. CFU. Colony-forming units; IL. Interleukin; MMP9. Matrix metallopeptidase 9.

Article Snippet: For the samples of the ex vivo whole blood model, the plasma concentrations of matrix metallopeptidase 9 (MMP9, #DY911, R&D Systems, Minneapolis, USA), IL-6 (#555220, BD Biosciences, San Jose, USA), and IL-8 (#DY208, R&D Systems) were measured in citrate-anticoagulated plasma using enzyme-linked immunosorbent assay according to the respective manufacturer’s instructions.

Techniques: Diagnostic Assay, Fluorescence, Comparison, Concentration Assay, Marker, Control, Incubation, Bacteria

Journal: Military Medical Research

Article Title: The cellular response capacity (CRC) as a novel immunomonitoring approach in sepsis

doi: 10.1016/j.mmr.2026.100010

Figure Lengend Snippet: Clinical specifications and parameters over all time points of the sepsis cohort. a Suspected infection cause of sepsis. b Distribution of the individual score points of the Sequential Organ Failure Assessment (SOFA) score. c Total SOFA score. d-h Traditional and humoral markers of inflammation: leukocytes and neutrophil-lymphocyte ratio ( d ), C-reactive protein (CRP) and procalcitonin (PCT) ( e ), interleukin-6 (IL-6) and interleukin-8 (IL-8) ( f ), serum amyloid A (SAA) and calprotectin ( g ), matrix metallopeptidase 9 (MMP9) and myeloperoxidase (MPO) ( h ). Values are shown as median and interquartile range. n =14. CNS. Central nervous system; HV. Healthy volunteers.

Article Snippet: For the samples of the ex vivo whole blood model, the plasma concentrations of matrix metallopeptidase 9 (MMP9, #DY911, R&D Systems, Minneapolis, USA), IL-6 (#555220, BD Biosciences, San Jose, USA), and IL-8 (#DY208, R&D Systems) were measured in citrate-anticoagulated plasma using enzyme-linked immunosorbent assay according to the respective manufacturer’s instructions.

Techniques: Infection

Journal: Chembiochem

Article Title: Functional Heterogeneity of the Autism Spectrum Disorder‐Associated Gut Microbial Ecosystem Revealed by Fecal Lipopolysaccharides and Bacterial Proteomic Profiling

doi: 10.1002/cbic.70557

Figure Lengend Snippet: Effects of fecal LPS on HMC3 microglia cell viability and activation. (A) Bright‐field image of HMC3 cells (40× magnification, scale bar: 20 µm). (B) HMC3 microglia cell viability after 24 h exposure to increasing concentrations (0.1, 1, 10, 100 ng/mL) of the pooled fecal LPS preparations obtained from the ASD and NASD cohorts. Cell viability was expressed as % relative to non‐stimulated (NS) cells and assessed by MTT assay (570 nm). NS cells were also considered as negative control. (C,D) Quantification of (C) IL‐8 and (D) IL‐6 levels following LPSs treatment, measured by DuoSet‐ELISA kit. Three independent experiments ( n = 3) were performed, with each experimental condition tested in triplicate wells within each experiment. Statistical significance was assessed by ordinary one‐way ANOVA followed by Tukey's multiple‐comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus NS; # p < 0.05, ### p < 0.001, #### p < 0.0001, versus LPS.

Article Snippet: Cytokine levels (pg/mL) in HMC3 supernatants were measured using DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA) for human IL‐8 (#DY208) and IL‐6 detection (#DY206).

Techniques: Activation Assay, MTT Assay, Negative Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: MIIP in CRC cells inhibits neutrophil infiltration and NETs formation through IL‐8. (A) Human cytokine array of the CM collected from SW620 cells with vector control and MIIP overexpression. (B) ELISA quantification of secreted IL‐8 protein levels in CM derived from three CRC cell lines with differential MIIP expression ( n = 3). (C) Volcano plot of DEGs from RNA‐seq analysis of CRC cell lines with varying levels of MIIP expression ( n = 3 per group). (D) CXCL8 mRNA expression in human CRC cells with different levels of MIIP expression was tested by RT–qPCR ( n = 3). (E) Quantification of neutrophils that migrated to CRC CM with or without recombinant human IL‐8 protein (rhIL‐8). Quantification of neutrophils that migrated to the CRC CM with or without anti‐IL‐8 neutralizing antibody (N‐IL‐8) ( n = 3). (F–H) Representative images and statistical quantification of IF staining ( n = 5) and MPO‐DNA ELISA ( n = 3) for NETs formation after neutrophils were co‐cultured with the CM from SW620 cells treated with recombinant IL‐8 protein and with the CM from SW480 and HCT116 cells treated with an anti‐IL‐8 neutralizing antibody. Scale bar, 50 µm. All the data are shown as the mean ± SD; * , p < 0.05; ** , p < 0.01; *** , p < 0.001. NS, not significant. Statistical significance was assessed using Student's t‐test and two‐way ANOVA in (B, D, E, G, and H).

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques: Plasmid Preparation, Control, Over Expression, Enzyme-linked Immunosorbent Assay, Derivative Assay, Expressing, RNA Sequencing, Analysis, Quantitative RT-PCR, Recombinant, Staining, Cell Culture

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: MIIP in CRC cells decreases neutrophil infiltration and NETs formation through NF‐κB p65/IL‐8 signaling. (A) Bubble plot of the enriched KEGG pathways showing that the NF‐κB signaling pathway was enriched in HCT116 cells (marked with a red border). (B) GSEA revealed that MIIP knockdown in HCT116 cells significantly activated the NF‐κB signaling pathway. (C) Western blotting analysis of key proteins involved in the NF‐κB p65 signaling pathway in human CRC cells with different levels of MIIP expression. (D) Representative images of H&E and IHC staining for MIIP, p‐p65, IL‐8, and Ly6G in tumors from the cecum of an orthotopic CRC mouse model between the MIIP‐overexpressing group and the control group (n = 5 per group). Scale bar, 50 µm. (E) Statistical analysis of Ly6G + neutrophils and the expression of p‐p65 and IL‐8 in different groups. (F) Representative images and statistical quantification of IF staining for MPO (red), Cit‐H3 (green), and DAPI (blue) in tumor tissue from the cecum and comparisons between the two groups ( n = 5). Scale bar, 25 µm. (G) Western blotting of NF‐κB p65 and NF‐κB p‐p65 in CRC cells treated with or not treated with an NF‐κB inhibitor (Bay 11–7082, 10 µM). (H) RT‐qPCR of CXCL8 mRNA in CRC cells treated or not treated with an NF‐κB inhibitor ( n = 3). (I) ELISA of IL‐8 in CM from CRC cells treated or not treated with an NF‐κB inhibitor ( n = 3). (J) Quantification of neutrophils that migrated to the CM from CRC cells treated or not treated with an NF‐κB inhibitor ( n = 3). (K–M) ELISA of MPO‐DNA and representative images and statistical quantification of IF staining for NETs formation after neutrophils were cultured with CM from SW480 and HCT116 cells treated or not treated with an NF‐κB inhibitor ( n = 3–5). Scale bar, 50 µm. All the data are shown as the mean ± SD; * , p < 0.05; ** , p < 0.01; *** , p < 0.001. NS, not significant. Statistical significance was assessed using Student's t‐test and the Mann‐Whitney U test in (E); the Mann‐Whitney U test in (F); and two‐way ANOVA in (H, I, J, K, and M).

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques: Knockdown, Western Blot, Analysis, Expressing, Immunohistochemistry, Control, Staining, Tissue, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, MANN-WHITNEY

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: MIIP modulates the NF‐κB p65/IL‐8 signaling pathway ‐mediated neutrophil infiltration and NETs formation through interactions with PKR. (A) IP–MS revealed PKR as a MIIP‐interacting protein. (B) The binding structure of MIIP and PKR was predicted using HDOCK molecular docking software. (C) Endogenous interaction between MIIP and PKR were detected via Co‐IP in HCT116 cells. (D) 293T cells were cotransfected with MIIP‐Flag and PKR‐HA, and reverse and forward Co‐IP analyses were performed. (E) Co‐IP assays were used to determine the PKR‐binding region of MIIP. (F) Co‐IP assays revealed that the PKR binding site for MIIP encompasses its IKKβ‐binding domain. (G) The expression of PKR was determined by Western blotting in CRC cells with different levels of MIIP expression. (H) Co‐IP was performed to examine the effect of MIIP expression on the interaction between PKR and IKKβ. (I–M) Double‐stable transgenic CRC cell lines were used in a rescue assay to demonstrate that the regulatory effects of MIIP on the NF‐κB p65‐mediated IL‐8 signaling pathway (I–K) and NETs formation (L, M) were dependent on PKR ( n = 3–5). All the data are shown as the mean ± SD; * , p < 0.05; ** , p < 0.01; *** , p < 0.001. NS, not significant. Statistical significance was assessed using two‐way ANOVA in (J–M).

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques: Protein-Protein interactions, Binding Assay, Docking Assay, Software, Co-Immunoprecipitation Assay, Expressing, Western Blot, Transgenic Assay, Rescue Assay

Journal: Advanced Science

Article Title: MIIP Inhibits Colorectal Cancer Progression by Modulating Neutrophils Infiltration and Neutrophil Extracellular Trap Formation

doi: 10.1002/advs.78063

Figure Lengend Snippet: Schematic illustration shows that MIIP inhibits colorectal cancer progression by decreasing TANs infiltration and NETs formation through the PKR/NF‐κB p65/IL‐8 axis, providing potential therapeutic strategies for colorectal cancer.

Article Snippet: In the rescue assay, neutralizing antibodies against IL‐8 (10 mg/mL, MAB208, R&D, USA; RRID: AB_2249110) or IL‐8 protein (10 ng/mL, HY‐P7224, MCE, USA) were added to the CRC CM in the lower chamber for the rescue assay.

Techniques:

Journal: Frontiers in Immunology

Article Title: ANKRD22 contributes to NF-κB-associated inflammatory responses in alveolar type II epithelial cells during PM 2.5 -triggered airway inflammation

doi: 10.3389/fimmu.2026.1894350

Figure Lengend Snippet: ANKRD22 inhibition mitigated PM 2.5 -induced inflammation in A549 cells. (A, B) A549 cells were transfected with ANKRD22 siRNA then stimulated with PM 2.5 , mRNA expression (A) and protein levels (B) of ANKRD22 were determined by qRT-PCR and Western blot. (C–G) mRNA expression levels of IL6 (C) , IL8 (D) , IL1B (E) , CXCL1 (F) , and CXCL2 (G) in A549 cells were detected by qRT-PCR. (H, I) ELISA measurement of IL-6 (H) and IL-8 (I) protein levels in A549 cells culture supernatants. Multiple comparisons were performed using one-way ANOVA followed by Bonferroni’s post hoc test. Experimental data analysis results are expressed as mean ± SEM. Data are representative of three biological replicates with similar results (n = 3 per group) ; * P < 0.05, ** P < 0.01, *** P < 0.001. ns, non-significant.

Article Snippet: In vitro experiment, the culture supernatants from A549 cells were collected, and the protein level of IL-6 (RK00004, ABclonal, China) and IL-8 (RK00011, ABclonal, China) were measured by ELISA.

Techniques: Inhibition, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Analysis