Journal: Immunity, Inflammation and Disease

Article Title: Interleukin‐35 Ameliorates Obesity‐Associated Impairment of Endothelium‐Dependent Vasodilation via the STAT3‐MAPK/ERK‐eNOS Signaling Axis

doi: 10.1002/iid3.70564

Figure Lengend Snippet: The Impact of IL‐35 on Serum Lipid Profiles. (A) Serum lipid profiles including TG, TC, LDL‐C, and HDL‐C, n = 7. Data are mean ± SD. ns, not significant; *** p < 0.001, **** p < 0.0001.

Article Snippet: HUVECs were assigned to the following groups: control, PA (100 μM), PA + IL‐35 (50 ng/mL), PA + IL‐35 (100 ng/mL), PA + IL‐35 + sorafenib (5 μmol/L, Raf inhibitor, MCE, USA), PA + IL‐35 + SCH772984 (0.5 μmol/L, ERK inhibitor, MCE, USA), PA + IL‐35 + si‐NC (50 nM), and PA + IL‐35 + si‐STAT3 (50 nM), with a total treatment duration of 24 h. PA, IL‐35 and inhibitors were added simultaneously for the control, PA, PA + IL‐35, PA + IL‐35+sorafenib and PA + IL‐35 + SCH772984 groups.

Techniques:

Journal: Immunity, Inflammation and Disease

Article Title: Interleukin‐35 Ameliorates Obesity‐Associated Impairment of Endothelium‐Dependent Vasodilation via the STAT3‐MAPK/ERK‐eNOS Signaling Axis

doi: 10.1002/iid3.70564

Figure Lengend Snippet: IL‐35 Subunit EBI3 is Downregulated in both Obese Mouse Aortas and PA‐Treated HUVECs, and Restored by Exogenous IL‐35. (A, B) Immunofluorescence of EBI3 (red) with CD31 (green) and DAPI (blue) in mouse aortas, with semi‐quantification of fluorescence intensity, n = 3 (scale bar = 50 µM). (C, D) Immunofluorescence of IL‐12A (red) with CD31 (green) and DAPI (blue) in mouse aortas, with semi‐quantification of fluorescence intensity, n = 3 (scale bar = 50 µM). (E) Serum IL‐35 concentrations, n = 8. (F, H) Representative Western blot bands of EBI3 and IL‐12A in aortic tissues (F) and densitometric analysis (H, n = 4). (G, I) Representative Western blot bands of EBI3 and IL‐12A in HUVECs (G) and densitometric analysis (I, n = 4). Data are mean ± SD. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: HUVECs were assigned to the following groups: control, PA (100 μM), PA + IL‐35 (50 ng/mL), PA + IL‐35 (100 ng/mL), PA + IL‐35 + sorafenib (5 μmol/L, Raf inhibitor, MCE, USA), PA + IL‐35 + SCH772984 (0.5 μmol/L, ERK inhibitor, MCE, USA), PA + IL‐35 + si‐NC (50 nM), and PA + IL‐35 + si‐STAT3 (50 nM), with a total treatment duration of 24 h. PA, IL‐35 and inhibitors were added simultaneously for the control, PA, PA + IL‐35, PA + IL‐35+sorafenib and PA + IL‐35 + SCH772984 groups.

Techniques: Immunofluorescence, Fluorescence, Western Blot, Analysis

Journal: Immunity, Inflammation and Disease

Article Title: Interleukin‐35 Ameliorates Obesity‐Associated Impairment of Endothelium‐Dependent Vasodilation via the STAT3‐MAPK/ERK‐eNOS Signaling Axis

doi: 10.1002/iid3.70564

Figure Lengend Snippet: IL‐35 Restores eNOS Ser1177 phosphorylation and nitric oxide production to enhance endothelium‐dependent relaxation in obesity. (A) Concentration‐response curves of endothelium‐dependent relaxation to ACh and comparison of maximal relaxation (Emax) in mouse aortic rings, n = 4. (B) Concentration‐response curves of endothelium‐independent relaxation to SNP and comparison of Emax, n = 4. (C, D) Western blot bands of p‐eNOS (Ser1177) and total eNOS in mouse aortic tissues (C) and HUVECs (D). (E, F) Densitometric analysis of total eNOS and p‐eNOS (Ser1177)/total eNOS in aortic tissues (E, n = 4) and HUVECs (F, n = 4). (G) NO fluorescence intensity in HUVECs, scale bar = 50 μM. (H) Semi‐quantification of NO fluorescence intensity in HUVECs, n = 3. Data are mean ± SD. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: HUVECs were assigned to the following groups: control, PA (100 μM), PA + IL‐35 (50 ng/mL), PA + IL‐35 (100 ng/mL), PA + IL‐35 + sorafenib (5 μmol/L, Raf inhibitor, MCE, USA), PA + IL‐35 + SCH772984 (0.5 μmol/L, ERK inhibitor, MCE, USA), PA + IL‐35 + si‐NC (50 nM), and PA + IL‐35 + si‐STAT3 (50 nM), with a total treatment duration of 24 h. PA, IL‐35 and inhibitors were added simultaneously for the control, PA, PA + IL‐35, PA + IL‐35+sorafenib and PA + IL‐35 + SCH772984 groups.

Techniques: Phospho-proteomics, Concentration Assay, Comparison, Western Blot, Analysis, Fluorescence

Journal: Immunity, Inflammation and Disease

Article Title: Interleukin‐35 Ameliorates Obesity‐Associated Impairment of Endothelium‐Dependent Vasodilation via the STAT3‐MAPK/ERK‐eNOS Signaling Axis

doi: 10.1002/iid3.70564

Figure Lengend Snippet: IL‐35 induces the MAPK/ERK signaling pathway, thereby enhancing eNOS Ser1177 phosphorylation and NO production in endothelial cells. (A) Volcano plot of eNOS‐interacting proteins in HUVECs (PA vs. PA + IL‐35) by IP‐mass spectrometry. Red dots: upregulated in PA + IL‐35 group (fold change > 1.5, p < 0.05); MAPK3 (ERK1) is highlighted. (B) STRING‐based PPI network of top 20 binding proteins. ERK1 is the only direct interactor with eNOS. (C) Western blot bands of p‐cRAF, p‐MEK, p‐ERK, p‐eNOS (Ser1177), and total proteins in HUVECs. (D–G) Densitometric analysis of p‐cRAF, p‐MEK, p‐ERK, and p‐eNOS (Ser1177) normalized to total proteins, n = 3. (H, I) NO fluorescence intensity in HUVECs (H, scale bar = 50 μM) with semi‐quantification (I, n = 3). Data are mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: HUVECs were assigned to the following groups: control, PA (100 μM), PA + IL‐35 (50 ng/mL), PA + IL‐35 (100 ng/mL), PA + IL‐35 + sorafenib (5 μmol/L, Raf inhibitor, MCE, USA), PA + IL‐35 + SCH772984 (0.5 μmol/L, ERK inhibitor, MCE, USA), PA + IL‐35 + si‐NC (50 nM), and PA + IL‐35 + si‐STAT3 (50 nM), with a total treatment duration of 24 h. PA, IL‐35 and inhibitors were added simultaneously for the control, PA, PA + IL‐35, PA + IL‐35+sorafenib and PA + IL‐35 + SCH772984 groups.

Techniques: Phospho-proteomics, Mass Spectrometry, Binding Assay, Western Blot, Analysis, Fluorescence

Journal: Immunity, Inflammation and Disease

Article Title: Interleukin‐35 Ameliorates Obesity‐Associated Impairment of Endothelium‐Dependent Vasodilation via the STAT3‐MAPK/ERK‐eNOS Signaling Axis

doi: 10.1002/iid3.70564

Figure Lengend Snippet: IL‐35 activates the STAT3‐MAPK/ERK‐eNOS signaling axis to promote eNOS phosphorylation and NO production in endothelial cells. (A) Western blot bands of p‐STAT3, p‐eNOS (Ser1177), p‐cRAF, p‐MEK, p‐ERK, and corresponding total proteins in HUVECs. (B–I) Densitometric analysis of p‐STAT3, STAT3, p‐eNOS (Ser1177), eNOS, p‐cRAF, p‐MEK, and p‐ERK normalized to total proteins or β‐tubulin, n = 3. (J–K) NO fluorescence intensity in HUVECs (J, scale bar = 50 μM) with semi‐quantification (K, n = 3). Data are mean ± SD. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: HUVECs were assigned to the following groups: control, PA (100 μM), PA + IL‐35 (50 ng/mL), PA + IL‐35 (100 ng/mL), PA + IL‐35 + sorafenib (5 μmol/L, Raf inhibitor, MCE, USA), PA + IL‐35 + SCH772984 (0.5 μmol/L, ERK inhibitor, MCE, USA), PA + IL‐35 + si‐NC (50 nM), and PA + IL‐35 + si‐STAT3 (50 nM), with a total treatment duration of 24 h. PA, IL‐35 and inhibitors were added simultaneously for the control, PA, PA + IL‐35, PA + IL‐35+sorafenib and PA + IL‐35 + SCH772984 groups.

Techniques: Phospho-proteomics, Western Blot, Analysis, Fluorescence

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: Regenerative Therapy

Article Title: Prednisolone attenuates the immunomodulatory capacity and reshapes the functional phenotype of canine adipose-derived mesenchymal stromal cells

doi: 10.1016/j.reth.2026.101184

Figure Lengend Snippet: Effects of prednisolone on macrophage polarization and regulatory T (Treg) cell induction mediated by cADSCs. (a) Representative flow cytometry plots showing cluster designation (CD) 80 and CD206 expression in CD14 + DH82 cells. Cells cultured alone under control conditions or stimulated with interleukin (IL)-4/IL-13 or lipopolysaccharide (LPS)/interferon (IFN)-γ are shown, together with DH82 cells co-cultured with cADSCs pretreated with prednisolone at the indicated concentrations (0–100 μM) under LPS/IFN-γ stimulation. (b) Quantitative analysis of macrophage subsets, defined as CD14 + CD80 + CD206 - , CD14 + CD80 − CD206 + , CD14 + CD80 + CD206 + , and CD14 + CD80 − CD206 - cells. IL-4/IL-13 stimulation was included as an M2-polarizing control, whereas LPS/IFN-γ stimulation induced M1-like polarization. (c) Representative flow cytometry plots showing CD25 and Foxp3 expression in CD4 + T cells after co-culture of PBMCs with cADSCs pretreated with prednisolone. Red boxes indicate CD25 + Foxp3 + Treg cells. (d) Quantification of CD25 + Foxp3 + Treg cells within the CD4 + T-cell population. Data are presented as mean ± SD from three independent experiments using cADSCs derived from three independent donors. * p < 0.05 vs. control or PBMCs alone; † p < 0.05 vs. LPS/IFN-γ.

Article Snippet: To induce macrophage polarization, either a combination of 50 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) and 20 ng/mL recombinant canine interferon (IFN)-γ (R&D Systems), or a combination of 40 ng/mL recombinant human interleukin (IL)-4 (Thermo Fisher Scientific) and 40 ng/mL recombinant human IL-13 (Thermo Fisher Scientific), was added to the cultures, followed by incubation at 37 °C in 5% CO 2 for 72 h. At the end of the co-culture period, cells were collected, washed with FACS buffer, and incubated on ice with canine Fc receptor binding inhibitor.

Techniques: Flow Cytometry, Expressing, Cell Culture, Control, Analysis, Co-culture Assay, Derivative Assay