Journal: Frontiers in Microbiology

Article Title: Klebsiella pneumoniae infection induces an S100A8/A9-mediated autocrine loop in human airway epithelium to amplify inflammation

doi: 10.3389/fmicb.2026.1768140

Figure Lengend Snippet: Extracellular S100A8/A9 functions as a potent pro-inflammatory stimulus for primary HBE cells. (A) Primary HBE cells were infected with WT K. pneumoniae (MOI 100) for the indicated time points. Relative mRNA expression of TLR4 was determined by qRT-PCR, with data calibrated to GAPDH and shown as fold induction over the baseline (0 h). (B) Representative western blot showing total TLR4 protein levels. β -Actin served as the housekeeping protein. (C) Quantification of TLR4 protein expression from three independent replicates, with values standardized to β-Actin. (D,E) HBE cells were treated for 24 h with the specified doses of endotoxin-free recombinant human S100A8/A9 (rS100A8/A9). The release of (D) IL-8 and (E) IL-6 was detected by ELISA. Data are presented as mean ± SEM of three independent experiments. * p < 0.05 versus the untreated control (A,C,D,E) .

Article Snippet: The concentrations of secreted S100A8/A9 heterodimer, IL-8, and IL-6 were quantified by commercial Quantikine ELISA kits (R&D Systems, Cat# DS8900 for S100A8/A9, D8000C for IL-8, and D6050B for IL-6).

Techniques: Infection, Expressing, Quantitative RT-PCR, Western Blot, Recombinant, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Microbiology

Article Title: Klebsiella pneumoniae infection induces an S100A8/A9-mediated autocrine loop in human airway epithelium to amplify inflammation

doi: 10.3389/fmicb.2026.1768140

Figure Lengend Snippet: Epithelial-derived S100A8/A9 is functionally required for the full inflammatory response to K. pneumoniae infection. Primary HBE cells were transfected with control siRNA (siCtrl) or S100A9 siRNA (siA9). (A) Knockdown efficiency was confirmed 48 h post-transfection by qRT-PCR analysis of S100A9 mRNA levels following a 12-h infection with WT K. pneumoniae (MOI 100), negative values represent fold reduction calculated as the negative inverse of 2 −ΔΔCt following the Schmittgen and Livak protocol. (B) Functional knockdown was confirmed by measuring S100A8/A9 protein secretion by ELISA in supernatants from infected cells at 24 h. (C,D) Control and S100A9 -deficient cells were infected with WT K. pneumoniae (MOI 100) for 24 h, and the secretion of cytokines were quantified by ELISA. Data are presented as mean ± SEM of three independent experiments. * p < 0.05 vs. Kp + siCtrl (A,B) or uninfected cells (C,D) , # p < 0.05 vs. Kp + siCtrl (C,D) .

Article Snippet: The concentrations of secreted S100A8/A9 heterodimer, IL-8, and IL-6 were quantified by commercial Quantikine ELISA kits (R&D Systems, Cat# DS8900 for S100A8/A9, D8000C for IL-8, and D6050B for IL-6).

Techniques: Derivative Assay, Infection, Transfection, Control, Knockdown, Quantitative RT-PCR, Functional Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: Klebsiella pneumoniae infection induces an S100A8/A9-mediated autocrine loop in human airway epithelium to amplify inflammation

doi: 10.3389/fmicb.2026.1768140

Figure Lengend Snippet: TLR4 is the essential receptor mediating S100A8/A9-induced inflammation in HBE cells during K. pneumoniae infection. (A,B) HBE cells were pre-incubated with neutralizing antibodies against TLR4 (10 μg/mL), RAGE (100 μg/mL), or an isotype control (Iso Ctrl) for 1 h, followed by stimulation with recombinant human S100A8/A9 (20 μg/mL) for 24 h. Levels of (A) IL-8 and (B) IL-6 in supernatants were determined by ELISA. (C,D) HBE cells were pre-treated with the indicated antibodies for 1 h and subsequently infected with WT K. pneumoniae (MOI 100) for 24 h. Secretion of (C) IL-8 and (D) IL-6 was quantified by ELISA. All results are expressed as mean ± SEM of three independent experiments. * p < 0.05 compared with the indicated groups.

Article Snippet: The concentrations of secreted S100A8/A9 heterodimer, IL-8, and IL-6 were quantified by commercial Quantikine ELISA kits (R&D Systems, Cat# DS8900 for S100A8/A9, D8000C for IL-8, and D6050B for IL-6).

Techniques: Infection, Incubation, Control, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: Klebsiella pneumoniae infection induces an S100A8/A9-mediated autocrine loop in human airway epithelium to amplify inflammation

doi: 10.3389/fmicb.2026.1768140

Figure Lengend Snippet: The S100A8/A9 autocrine loop drives NF-κB activation and subsequent cytokine release in HBE cells. (A) Representative immunoblots showing degradation of IκBα in primary HBE cells stimulated with rS100A8/A9 (20 μg/mL) for 60 min. β-Actin served as a loading control. (B) Densitometric quantification of IκBα protein levels from (A) . (C) Representative immunofluorescence images showing subcellular localization of the p65 subunit (green) at 60 min post-stimulation with rS100A8/A9. Nuclei were counterstained with DAPI (blue). (D) HBE cells were transfected with control siRNA (siCtrl) or siRNA targeting S100A9 (siS100A9) and subsequently infected with wild-type K. pneumoniae (MOI 100). Representative immunoblots of IκBα levels are shown at 60 min post-infection. (E) Densitometric quantification of IκBα degradation from (D) . (F) Representative immunofluorescence images showing p65 localization (green) in siCtrl- or siS100A9-transfected HBE cells at 60 min post- K. pneumoniae infection. Nuclei were stained with DAPI (blue). (G) Secretion of IL-8 and IL-6 by HBE cells. Cells were pre-treated with or without the NF-κB inhibitor BAY 11–7,082 (10 μM) for 60 min, followed by stimulation with rS100A8/A9 (20 μg/mL) for 24 h. Cytokine levels in supernatants were measured by ELISA. All graphs display mean ± SEM, * p < 0.05 compared with the untreated control (B,E,G) ; # p < 0.05 compared with siCtrl + Kp or the S100A8/A9-stimulated group without inhibitor (E,G) .

Article Snippet: The concentrations of secreted S100A8/A9 heterodimer, IL-8, and IL-6 were quantified by commercial Quantikine ELISA kits (R&D Systems, Cat# DS8900 for S100A8/A9, D8000C for IL-8, and D6050B for IL-6).

Techniques: Activation Assay, Western Blot, Control, Immunofluorescence, Transfection, Infection, Staining, Enzyme-linked Immunosorbent Assay

Journal: Autophagy

Article Title: DCN released from ferroptotic cells ignites AGER-dependent immune responses

doi: 10.1080/15548627.2021.2008692

Figure Lengend Snippet: DCN mediates inflammation caused by ferroptotic cells. (A) Ferroptotic cells lacking Dcn, Atg5, or Mcoln1 do not elicit the production of the pro-inflammatory TNF and IL6 cytokine by 1 × 10 6 BMDMs ( n = 3 biologically independent samples; * P < 0.05, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (B,C) Anti-DCN neutralizing antibody (1 mg/ml) inhibits ferroptotic cells, but not late apoptotic cell- or necroptotic cell-induced TNF and IL6 release in BMDMs ( n = 3 biologically independent samples; * P < 0.05, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (D,E) Lack of Dcn in late apoptotic or necroptotic cells fails to affect the production of TNF and IL6 by BMDMs ( n = 3 biologically independent samples; data are presented as means ± SD).

Article Snippet: Commercially available ELISA kits were used to measure the concentrations or activity of HMGB1 (Shino Test Corporation, ST51011), DCN (R&D Systems, DY143, DY1060), IL6 (R&D Systems, M6000B, D6050), TNF (R&D Systems, MTA00B, DTA00D), NFKB (Active Motif, 43,296), MPO (Abcam, ab105136), LDH (Abcam, ab102526), trypsin (Abcam, ab102531), CTSB (Abcam, ab119585), and AMY (Abcam, ab102523) in the indicated samples.

Techniques:

Journal: Autophagy

Article Title: DCN released from ferroptotic cells ignites AGER-dependent immune responses

doi: 10.1080/15548627.2021.2008692

Figure Lengend Snippet: AGER mediates the response to DCN. (A) Western blot analysis of protein expression by BMDMs after transfection with the indicated shRNAs. (B) ELISA analysis of TNF and IL6 release in the indicated gene knockdown BMDMs following treatment with ferroptotic MEFs ( n = 3 biologically independent samples; * P < 0.05 versus control shRNA group, one-tailed t test; data are presented as means ± SD). (C) Lack of Ager in BMDMs blocks ferroptotic cell-induced the production of the pro-inflammatory TNF and IL6 cytokines ( n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (D) Anti-AGER neutralizing antibody (Ab; 1 mg/ml), but not anti-TLR4 neutralizing antibody (1 mg/ml), inhibits ferroptotic cell-induced TNF and IL6 release in BMDMs ( n = 3 biologically independent samples; * P < 0.05, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (E, F) Lack of Ager in BMDMs inhibits DCN-induced TNF and IL6 release in the absence or presence of HMGB1 ( n = 3 biologically independent samples; * P < 0.05 versus WT group, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (G, H) Analysis of NFKB activity and TNF release in the indicated BMDMs following treatment with ferroptotic cells or DCN in the absence or presence of the NFKB pathway inhibitor BMS-345541 ( n = 3 biologically independent samples; * P < 0.05 versus WT group, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (I) His-tag affinity pull-down analysis of the binding of DCN to AGER.

Article Snippet: Commercially available ELISA kits were used to measure the concentrations or activity of HMGB1 (Shino Test Corporation, ST51011), DCN (R&D Systems, DY143, DY1060), IL6 (R&D Systems, M6000B, D6050), TNF (R&D Systems, MTA00B, DTA00D), NFKB (Active Motif, 43,296), MPO (Abcam, ab105136), LDH (Abcam, ab102526), trypsin (Abcam, ab102531), CTSB (Abcam, ab119585), and AMY (Abcam, ab102523) in the indicated samples.

Techniques: Western Blot, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Knockdown, Control, shRNA, One-tailed Test, Activity Assay, Binding Assay

Journal: Autophagy

Article Title: DCN released from ferroptotic cells ignites AGER-dependent immune responses

doi: 10.1080/15548627.2021.2008692

Figure Lengend Snippet: Inhibiting the DCN-AGER pathway protects against cerulein-induced acute pancreatitis. WT and pancreatic gpx4 −/− mice received anti-DCN Ab (20 mg/kg; monoclonal rat IgG2A; R&D Systems, 161026), anti-AGER Ab (20 mg/kg; monoclonal rat IgG2A; R&D Systems, 175410), control IgG (20 mg/kg; R&D Systems) or liproxsatin-1 (10 mg/kg) by i.p. injection 2 h after completion of the cerulein-induced pancreatitis protocol. (A) Pancreatic sections were stained with H&E at 16 h (bar: 100 μm). (B–G) Serum AMY (amylase), pancreatic trypsin activity, pancreatic MPO activity, serum DCN, serum TNF, and serum IL6 were assayed at 8 and 16 h ( n = 5 mice/group; * P < 0.05 versus IgG group, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD).

Article Snippet: Commercially available ELISA kits were used to measure the concentrations or activity of HMGB1 (Shino Test Corporation, ST51011), DCN (R&D Systems, DY143, DY1060), IL6 (R&D Systems, M6000B, D6050), TNF (R&D Systems, MTA00B, DTA00D), NFKB (Active Motif, 43,296), MPO (Abcam, ab105136), LDH (Abcam, ab102526), trypsin (Abcam, ab102531), CTSB (Abcam, ab119585), and AMY (Abcam, ab102523) in the indicated samples.

Techniques: Control, Injection, Staining, Activity Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: MiR-449a antagonizes EMT through IL-6-mediated trans -signaling in laryngeal squamous cancer

doi: 10.1016/j.omtn.2024.102140

Figure Lengend Snippet: miR-449a directly targets IL-6R and switches off IL-6 trans -signaling (A) The putative miR-449a binding sites in IL-6R were predicted using online bioinformatics tools. The interaction between miR-449a and IL-6R was verified using a dual luciferase reporter assay. ∗p < 0.05; ∗∗p < 0.001; ∗∗∗p < 0.0005; ∗∗∗∗p < 0.0001 Student’s t test vs. control group. Each experiment was repeated three times. Bars indicate SDs. (B) RIP assay of the enrichment of Ago2 on IL-6R and miR-449a transcripts normalized to a negative control irrelevant IgG antibody (CTR) in HNO-210 cell line overexpressing miR-449a. (C) Levels of sIL-6R and IL-6R in HNO-210 cell culture medium by quantitative ELISA (left and right) and expression levels of sIL-6R in HNO-210 cells by qRT-PCR (last panel). The experiments were repeated three times in triplicate. Bars, SDs. (D) Western blots analysis of transduced HNO-210 cell models in different experimental conditions: (i) 0.1% serum starvation, (ii) Hyper IL-6 stimulation (20 ng/mL for 30 min), (iii) sgp130fc treatment (1,000 ng/mL for 1 h), and sgp130fc pretreatment followed by Hyper IL-6 stimulation. miR-449a modulation of IL-6 trans -signaling mediated by pStat3 Tyr705 – pERK (Thr202; Tyr204)—pAKT Ser473. Vinculin was used as loading control. The experiments were repeated at least three times giving always similar results. Columns represent the intensity of the different bands evaluated as arbitrary units. Bars are SDs. ANOVA ∗p < 0.01; ∗∗p < 0.05; ∗∗∗p < 0.005; ∗∗∗∗p < 0.0001. (E) Relative expression levels of SNAI2, E-cadherin, and N-cadherin of transduced HNO-210 cell models in different experimental conditions: (i) 0.1% serum starvation, (ii) Hyper IL-6 stimulation (20 ng/mL for 30 min), (iii) sgp130fc treatment (1,000 ng/mL for 1 h) and sgp130fc pretreatment followed by Hyper IL-6 stimulation. ANOVA ∗<0.01 ∗∗p < 0.05; ∗∗∗p < 0.005; ∗∗∗∗p < 0.0001. All data are representative of three independent experiments.

Article Snippet: Human IL-6R and IL-6 cytokine in cell culture supernatants were quantified using IL-6R and IL-6 Quantikine ELISA Kits (R&D Systems Cat# DR600, D6050).

Techniques: Binding Assay, Luciferase, Reporter Assay, Control, Negative Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot