Journal: Frontiers in Immunology

Article Title: Age-related increase of mitochondrial content in human memory CD4+ T cells contributes to ROS-mediated increased expression of proinflammatory cytokines

doi: 10.3389/fimmu.2022.911050

Figure Lengend Snippet: Capacity of cytokine production in memory CD4 + T cell. Cells were activated with PMA/ionomycin for 5 h, adding BFA for the last 3 hours for intracellular cytokine expression. (A) Representative example of cytokine expression in memory CD4 + T cells from young and aged donors after stimulation with PMA/ionomycin. (B) Frequencies in % and geometric mean fluorescence intensity (gMFI) of IL-2, IL-4, IL-10, IL-17, IFN-γ, and TNF-α producing memory CD4 + T cells (mean ± SEM; n = 7).

Article Snippet: Incubation was conducted by dissolving 1x 10 6 cells/ml in RPMI 1640 medium (Thermo Fisher Scientific) with 5% human AB serum (Sigma Aldrich) and 1% penicillin/streptomycin (Thermo Fisher Scientific) at 37˚C in a 5% CO 2 atmosphere Cells were collected, washed, fixed, permeabilized (Inside staining kit, Miltenyi Biotec) and stained with a combination of monoclonal antibodies including anti-human-IFNγ (PERCP-Cy5.5, Biolegend, clone 4SB3, Cat.# 502526), anti-human-IL-2 (APC-vio770, Miltenyi Biotec, clone N7.48 A, Cat.# 130-097-011), anti-human-IL-4 (PE, Miltenyi Biotec, clone 7A3-3, Cat.# 130-091-647), anti-human-IL-17A (FITC, Miltenyi Biotec, clone CZ8-23G1, Cat.# 130-094-520), and anti-human-TNF-α (PE-Vio770 (Miltenyi Biotec, clone cA2, Cat.# 130-096-755).

Techniques: Expressing, Fluorescence

Journal: PLOS ONE

Article Title: Anti-inflammatory effect of the combined treatment of LMT-28 and kaempferol in a collagen-induced arthritis mouse model

doi: 10.1371/journal.pone.0302119

Figure Lengend Snippet: (A) Arthritis scores of mice were measured every 3 days starting at 21 days after the first immunization. (B) DBA1/J mice showed deformation and swelling of the toe and ankle joints. (C) Paraffin-embedded sections of CIA mice joint were stained with hematoxylin and eosin. (D) Histological analysis was performed based on the analysis criteria: inflammation, synovial hyperplasia, pannus formation, and erosion of cartilage and bone. (E) The protein levels of IL-6, IL-1β and IL-17 in CIA mouse serum were measured using ELISA. Data are presented as mean ± SEM. # p <0.05 vs. normal group, * p <0.05, ** p <0.01, and *** p <0.005 vs. CIA control group.

Article Snippet: The cells were incubated with anti-IL-17A-PE antibody (Miltenyi Biotec) or anti-mouse FOXP3 antibody (Invitrogen, Waltham, MA, USA) diluted in 1× permeabilization buffer (eBioscience) for 30 min at 4°C in the dark.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Bioactive Materials

Article Title: Engineering a halloysite nanotube-enhanced hydrogel 3D skin model for modulated inflammation and accelerated wound healing

doi: 10.1016/j.bioactmat.2024.11.013

Figure Lengend Snippet: Viability and functionality assessment of HFFs in HNT-modified hydrogel (a) Representative images and percentage of HFFs positive for vimentin by IF staining on days 0, 3, and 7 in natural hydrogel and 1 μg/mL HNT-based hydrogel. Data are represented as mean ± SD. ∗ P < 0.05 (two-way ANOVA analysis). Scale bar = 20 μm. (b) Relative expression levels of talin2, plasminogen, fibronectin, and paxillin assessed by RT-PCR tests in cultured HFFs on day 3. Each dot represents one independent experiment. Data are represented as mean ± SD. ns, not significant, ∗ P < 0.05; ∗∗ P < 0.01 (unpaired Student's t-test). (c) Representative images and percentage of HFFs positive for vimentin by IF staining in natural hydrogel and 1 μg/mL HNT-based hydrogel. The cells were stimulated with cytokines (TNF-α, IL-17A, and IL-22) in different concentrations (0, 25, 50, 100 ng/mL). Scale bar = 100 μm. (d) Representative 3D images from the top view of HFFs positive for vimentin in I collagen-based hydrogel, natural hydrogel, and 1 μg/mL HNT-based hydrogel on days 1, 3 and 7. The cells were stimulated without or with cytokines (TNF-α, IL-17A, and IL-22, 50 ng/mL for each cytokine). Scale bar = 100 μm. (e) Relative expression levels of vimentin in HFFs in I collagen-based hydrogel, natural hydrogel, and 1 μg/mL HNT-based hydrogel on days 1, 3, and 7 without cytokine stimulation. Data are represented as mean ± SD. ∗ P < 0.05 (two-way ANOVA analysis). (f) Relative expression levels of vimentin in HFFs in I collagen-based hydrogel, natural hydrogel, and 1 μg/mL HNT-based hydrogel on days 1, 3, and 7 with cytokine (TNF-α, IL-17A, and IL-22, 50 ng/mL for each) stimulation. Data are represented as mean ± SD. ∗∗ P < 0.01 (two-way ANOVA analysis).

Article Snippet: Subsequently, the samples were incubated overnight at 4 °C with primary antibodies, including rabbit anti-Ki-67 (catalog #34330, CST, USA), keratin (K) 5 (catalog # 71536, CST, USA), vimentin (catalog # 10366-1-AP, Proteintech, China), fibronectin (catalog # 15613-1-AP, Proteintech, China), K17 (catalog # 17516-1-AP, Proteintech, China), mouse anti-K10 (catalog # ab9025, Abcam, UK), CD34 (catalog # 31120-1-AP, Proteintech, China), or IL-17A (catalog # 66148-1-Ig, Proteintech, China).

Techniques: Modification, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture

Journal: Bioactive Materials

Article Title: Engineering a halloysite nanotube-enhanced hydrogel 3D skin model for modulated inflammation and accelerated wound healing

doi: 10.1016/j.bioactmat.2024.11.013

Figure Lengend Snippet: Inflammatory bioactivity of modified hydrogel and its potential for cell proliferation, migration, and adhesion. (a) Schematic of RNA-seq of the HFFs in hydrogel group. Four groups were set as group A (without HNT), group B (with HNT of 1 μg/mL), group C (without HNT, stimulated with TNF-α, IL-17A, and IL-22, 50 ng/mL) and group D (with HNT of 1 μg/mL, stimulated with with TNF-α, IL-17A, and IL-22, 50 ng/mL). (b) Heatmap of relative expression levels of genes associated with inflammation, proliferation and migration in group B (with HNT) compared to group A (without HNT). (c) KEGG pathway analysis of enriched genes showing the relevant functional terms in group D compared to group B. (d) Specific inflammatory clusters of genes related to KEGG analysis showing the relevant functional terms in group D compared to group B. (e) KEGG pathway analysis of enriched genes showing the relevant functional terms in group A compared to group C as well as group D compared to group B. (f) Venn diagram of differentially expressed genes overlapped in the four groups. (g) Representative 3D images of K17 expression in upper-layer from the top and side-view of the modified 3D skin model. (h) Relative expression levels of K17 in 0 μg/mL and 1 μg/mL HNT-based hydrogel with cytokine (TNF-α, IL-17A, and IL-22, 50 ng/mL) stimulation. Each dot represents one independent experiment. Data are represented as mean ± SD. ∗ P < 0.05 (unpaired student's t-test).

Article Snippet: Subsequently, the samples were incubated overnight at 4 °C with primary antibodies, including rabbit anti-Ki-67 (catalog #34330, CST, USA), keratin (K) 5 (catalog # 71536, CST, USA), vimentin (catalog # 10366-1-AP, Proteintech, China), fibronectin (catalog # 15613-1-AP, Proteintech, China), K17 (catalog # 17516-1-AP, Proteintech, China), mouse anti-K10 (catalog # ab9025, Abcam, UK), CD34 (catalog # 31120-1-AP, Proteintech, China), or IL-17A (catalog # 66148-1-Ig, Proteintech, China).

Techniques: Modification, Migration, RNA Sequencing, Expressing, Functional Assay

Journal: Mediators of Inflammation

Article Title: Analysis of Th17 and Tc17 Frequencies and Antiviral Defenses in Gut-Associated Lymphoid Tissue of Chronic HIV-1 Positive Patients

doi: 10.1155/2015/395484

Figure Lengend Snippet: Activation markers, IFN- γ and IL-17A expression in CD4 + and CD8 + T cells subsets in blood and in gut. Panel (a): representative flow cytometry plots outlining CD38 and HLA-DR expression on naïve, effector, and central memory CD4 + and CD8 + lymphocytes derived from peripheral blood mononuclear cells (PBMCs) and gut-associated lymphoid tissue (GALT). Panel (b): representative flow cytometry plots outlining IFN- γ and IL-17A intracitoplasmatic expression on naïve, effector, and central memory CD4 + (Th1 and Th17) and CD8 + (Tc1 and Tc17) lymphocytes derived from PBMCs and GALT.

Article Snippet: Cultured cells were fixed, permeabilized (BD Cytofix/Cytoperm, Becton Dickinson, San Jose, CA, USA), and stained with combinations of fluorochrome-labeled monoclonal antibodies: CD3-PerCP, CD4-APC-Vio770, CD8-FITC, CD45RO-PE-Vio770, CD27-VioBlue, IFN- γ -APC, and IL-17A-PE (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activation Assay, Expressing, Flow Cytometry, Derivative Assay