th17 cell differentiation  (Galectin Therapeutics)

Journal: International Journal of Molecular Sciences

Article Title: A Narrative Review on the Multifaceted Roles of Galectins in Host–Pathogen Interactions During Helicobacter pylori Infection

doi: 10.3390/ijms26157216

Figure Lengend Snippet: Molecular pathways of Helicobacter pylori -induced immune activation, immune evasion, and modulation by galectins within the gastric mucosa. H. pylori colonization begins with bacterial motility mediated by flagella, enabling the bacterium to penetrate the gastric mucus layer and adhere to epithelial cells via outer membrane proteins and lipopolysaccharides. The bacterium deploys the type IV secretion system to translocate the CagA effector protein into host epithelial cells, where CagA activates NF-κB signaling. This leads to the upregulation of IL-8 production, driving neutrophil recruitment to the infection site, which exacerbates mucosal inflammation and tissue injury. Simultaneously, VacA toxin enters epithelial cells through endocytosis, where it disrupts mitochondrial membranes, inducing apoptosis of epithelial cells, contributing to mucosal damage. VacA also targets lysosomal membranes, leading to lysosomal dysfunction and oxidative stress, which impairs intracellular pathogen clearance. The immune system responds via Th1 and Th17 polarization, producing IFN-γ and IL-17, respectively, which drive chronic inflammation but are insufficient for bacterial eradication due to immune evasion strategies. H. pylori counters these defenses by promoting the expansion of regulatory T cells (Tregs), partly facilitated by VacA- and CagA-mediated suppression of co-stimulatory signals, which collectively inhibit Th1/Th17 responses and promote immune tolerance within the mucosa. Red arrows mean inhibition, green and black arrows mean activation.

Article Snippet: Galectin-9 facilitates IgA class switching indirectly by supporting Th17 cell differentiation [ ].

Techniques: Activation Assay, Membrane, Infection, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Paeoniflorin Directly Targets ENO1 to Inhibit M1 Polarization of Microglia/Macrophages and Ameliorates EAE Disease

doi: 10.3390/ijms26083677

Figure Lengend Snippet: Figure 3. PF reduces the percentages of M1 microglia/macrophages and pathogenic T cells in the CNS of EAE mice. The CNS mononuclear cells were isolated from the EAE-PBS and EAE-PF group mice on day 16 post immunization. (A,B) Flow cytometry analysis of CD45int CD11b+ microglia cells and CD45high CD11b+ macrophages in CNS from the two group mice (n = 4). (C) Flow cytometry analysis of CD86+ or CD206+ cells in CD11b+ cells from the two group mice (n = 4). (D) The mRNA expression levels of cytokines and chemokines in CNS mononuclear cells from the two group mice were determined by qPCR (n = 4). (E) The percentage of CD4+ T cells and T-helper cells such as Th1, Th17, and Treg cells in the CNS were analyzed by flow cytometry (n = 4). Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001; ns, no significance.

Article Snippet: For T cell differentiation, 2 × 105 CD4+ T cells were cultured with corresponding medium for 72 h: Th1 differentiation medium was supplemented with the same concentration of anti-CD3e/CD28 mAbs, anti-IL-4 (10 μg/mL), IL-12 (10 ng/mL), as well as IL-2 (10 ng/mL); Th17 differentiation media were mixed according to the CellXVivo Th17 Cell Differentiation Kit (R&D Systems, Inc., Minneapolis, MN, USA); for Treg cell differentiation, the same concentration of anti-CD3e/CD28 mAbs, anti-IL-4 (10 μg/mL), and anti-IFN-γ (10 μg/mL), TGF-β (15 ng/mL), IL-2 (10 ng/mL) were included.

Techniques: Isolation, Flow Cytometry, Expressing

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: (A) Treatment with the α2B/2C agonist (AGN-762) after the development of chronic DED effectively ameliorates the disease, assessed by corneal fluorescein staining (CFS) score. ****, p < 0.0001 vs untreated or vehicle group; n = 30 eyes (15 mice) /group from two independent experiments. (B) Corneal inflammation was assessed at the end of treatment by quantifying mature MHC-II + CD11b + cells using flow cytometry and protein levels of IL-1β and IL-6 using ELISA. The upper panel shows the gating strategy for flow cytometry analysis. (C) Conjunctival inflammation was assessed by quantifying infiltrating CD4 + T cells using flow cytometry and protein levels of IL-17A, IL-17F, and IFN-γ using ELISA. The upper panel of the flow plots shows the gating strategy for flow cytometry analysis. (D) Draining lymph node (DLN) Th17 response was determined by flow cytometry. The upper panel shows the gating strategy for flow cytometry analysis. (E) The suppressive function of Treg cells on cell proliferation was quantified using the BrdU incorporation assay and compared with the proliferative responses in the absence of Treg (0 % suppression). For flow cytometry of corneal and conjunctival tissues, samples were pooled from 5 eyes in each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; data shown were from one representative experiment out of two performed and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Article Snippet: Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, BrdU Incorporation Assay

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: (A) A 7-day treatment with the α2B/2C agonist (AGN-762) in chronic DED (day 21 – 28) effectively maintains therapeutic efficacy for months (day 28 – 56), and suppresses disease exacerbation in treated animals upon re-challenged with desiccating stress (day 56 – 63). Disease severity was assessed by corneal fluorescein staining (CFS) score. ***, p < 0.001; ****, p < 0.0001 vs untreated or vehicle group; n = 20 eyes (10 mice) /group from two independent experiments. (B) Corneal inflammation was assessed at day 63 by quantifying mature MHC-II + CD11b + cells using flow cytometry and protein levels of IL-1β and IL-6 using ELISA. The same gating strategy for flow cytometry analysis was used as shown in . (C) Conjunctival inflammation was assessed at day 63 by quantifying protein levels of IL-17A, IL-17F, and IFN-γ using ELISA. (D) Draining lymph node Th17 response was determined by flow cytometry at day 63. The same gating strategy for flow cytometry analysis was used as shown in . (E) The suppressive function of Treg cells on cell proliferation was quantified using the BrdU incorporation assay and compared with the proliferative responses in the absence of Treg (0 % suppression). For flow cytometry of corneal and conjunctival tissues, samples were pooled from 5 eyes in each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., not significant; data shown were from one representative experiment out of two performed and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Article Snippet: Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, BrdU Incorporation Assay

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: (A) Flow cytometry plots show the purity of freshly sorted Teff cells defined as CD4 + CD25 − Foxp3 − cells that were subjected to cell cultures in vitro. (B) Flow cytometric analysis of CD4 + CD25 − Teff after 24 h culturing with AGN-762, with representative dot plots shown (the upper panel shows the gating strategy), and frequencies of IL-17A + IFN-γ − Th17, IL-17A + IFN-γ + Th17/1, and IL-17A − IFN-γ + Th1 summarized in the bar graphs. (C) ELISA assay on the culture supernatants for the levels of IL-17A and IFN-γ. Data shown were from one representative experiment out of two performed. *, p < 0.05; data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Article Snippet: Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: (A) Treg cells were isolated from DED mice and in vitro cultured with 1 μM AGN-762 or vehicle for 24 h. The freshly sorted effector CD4 + T cells from DED (Teff) along with normal Treg, DED-Treg pre-treated with the vehicle, or DED-Treg pre-treated with AGN-762 were adoptively transferred to Rag1 −/− mice, which were subsequently exposed to desiccating stress for 6 days. Disease severity was assessed by corneal fluorescein staining (CFS) score, and summarized from two independent experiments. n = 10–14 eyes (5–7 mice) /group. (B) Flow cytometric analysis of T cell response in the draining lymph nodes of Rag1 −/− recipients at day 6, with representative dot plots shown on the left (the upper panel shows the gating strategy) and frequencies of IL-17-producing CD4 + T cells (including both IL-17A + IFN-γ − Th17 and IL-17A + IFN-γ + Th17/1) summarized from two independent experiments on the right. *, p < 0.05; **, p < 0.01; data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Article Snippet: Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Isolation, In Vitro, Cell Culture, Staining

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: (A) DED-Treg cells were pre-treated with 1 μM AGN-762 or vehicle for 24 h and then subjected to suppressive function assay in the presence of anti-IL-10 neutralizing antibody or isotype IgG control. (B) Treg cells were isolated from DED mice and in vitro cultured with 1 μM AGN-762 for 24 h. The freshly sorted effector CD4 + T cells from DED (Teff) along with DED-Treg pre-treated with AGN-762 were adoptively transferred to Rag1 −/− mice, which were subsequently exposed to desiccating stress for 6 days. The recipients received treatment with anti-IL-10 antibody or control IgG immediately after adoptive transfer (day 0) as well as at day 3. Disease severity was assessed by corneal fluorescein staining (CFS) score, and summarized from two independent experiments. n = 16 eyes (8 mice) /group (C) Flow cytometric analysis of T cell response in the draining lymph nodes of Rag1 −/− recipients at day 6, with representative dot plots shown on the left and frequencies of total T cells and IL-17-producing CD4 + T cells (including both IL-17A + IFN-γ − Th17 and IL-17A + IFN-γ + Th17/1) summarized from two independent experiments on the right. *, p < 0.05; **, p < 0.01; data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test (A) or unpaired t test (B and C).

Article Snippet: Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Functional Assay, Control, Isolation, In Vitro, Cell Culture, Adoptive Transfer Assay, Staining