Journal: Frontiers in Immunology

Article Title: Cytokine-driven glycosphingolipid metabolism modulates endoplasmic reticulum calcium homeostasis in primary human renal mesangial cells

doi: 10.3389/fimmu.2025.1688916

Figure Lengend Snippet: hRMCs secrete many cytokines/chemokines in response to proinflammatory cytokine stimuli. Triplicate wells were treated with IFNα (100 ng/ml), IFNγ (100 ng/ml), TNFα (100 ng/ml), IL-1β, (25 ng/ml), mix of all 4 cytokines (cytokine mix), or vehicle (dH2O) as indicated on the graphs. The media was collected 24h post-stimulation and cell viability measured by the alamar blue assay. For (A, B) , media was screened using a 71-plex cytokine/chemokine array. Each point represents an independent experiment, one with lot 17544 and one with lot 20057. Cytokine concentrations were normalized to cell viability and are presented relative to vehicle-treated samples. The 8 most highly expressed factors (A) and next 8 most highly expressed factors (B) based on expression levels in response to the cytokine mix are presented. “+” = high off the standard curve for one of the experiments. For (C–G) , IL-6 levels measured by ELISA in 5 independent experiments each using a different lot of cells: (C) lot 20018; (D) lot 20057; (E) lot 17544; (F) lot HK31; (G) lot HK32. Concentrations were normalized to cell viability. Media from each triplicate well was assayed independently and the mean +SD is presented. P-values were determined by unpaired T-test between vehicle and each stimulant.

Article Snippet: Purchased reagents and kits include: recombinant, animal-free human IL-1β (#AF-200-01B), IFNγ (#AF-300-02), IFNα (#01-672-628), and TNFα (#AF-300-01A) (Fisher Scientific, Waltham, MA), Alamar blue (#A50101 Invitrogen, Waltham, MA), eliglustat hemitartrate (#50-187–3104 Fisher Scientific/MedChemexpress), and anti-αSMA monoclonal antibody (17H19L35, #701457 Invitrogen); Alexa Fluor Plus 488-conjugated goat anti-rabbit IgG (H+L), highly cross-adsorbed secondary antibody (#A32731 Invitrogen); Fluoromount-G with DAPI (#00-4959-52, Invitrogen) (Fisher Scientific); and Rhodamine Phalloidin (#PHDR1, Cytoskeleton, Inc., Denver, CO).

Techniques: Alamar Blue Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Cytokine-driven glycosphingolipid metabolism modulates endoplasmic reticulum calcium homeostasis in primary human renal mesangial cells

doi: 10.3389/fimmu.2025.1688916

Figure Lengend Snippet: HexCers levels increase in response to IL-1β and reduced by eliglustat in hRMCs. For (A, B) , all 6 lots of cells were grown to ~90% confluency in duplicate, trypsinized, counted, and HexCers (A) and LacCers (B) measured in 1x10^6 cells by high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). Major chain lengths expressed in the cells are presented. Bars are mean +SD of duplicate wells. For (C–H) , hRMCs lots 17544, 12445, 20057, and 20018 were treated in quadruplicate with vehicles, IL-1β, or IL-1β +20 nM eliglustat for 24h (C, F) , 48h (D, G) , or 72h (E, H) . Media was collected, cell viability measured by the alamar blue assay, and cells collected. Quadruplicates within each experiment were pooled and HexCers in cells (C–E) and media (F–H) measured as in (A, B) . HexCers measured in the culture media alone (was not incubated with cells) was subtracted from the media levels in (F–H) . Levels were then normalized to cell viability and fold change calculated setting vehicle-treated to 1 for each experiment (hRMC lot). Fold change for the three lots were averaged and mean +SD presented. P-values were determined by two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Purchased reagents and kits include: recombinant, animal-free human IL-1β (#AF-200-01B), IFNγ (#AF-300-02), IFNα (#01-672-628), and TNFα (#AF-300-01A) (Fisher Scientific, Waltham, MA), Alamar blue (#A50101 Invitrogen, Waltham, MA), eliglustat hemitartrate (#50-187–3104 Fisher Scientific/MedChemexpress), and anti-αSMA monoclonal antibody (17H19L35, #701457 Invitrogen); Alexa Fluor Plus 488-conjugated goat anti-rabbit IgG (H+L), highly cross-adsorbed secondary antibody (#A32731 Invitrogen); Fluoromount-G with DAPI (#00-4959-52, Invitrogen) (Fisher Scientific); and Rhodamine Phalloidin (#PHDR1, Cytoskeleton, Inc., Denver, CO).

Techniques: High Performance Liquid Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy, Alamar Blue Assay, Incubation

Journal: Frontiers in Immunology

Article Title: Cytokine-driven glycosphingolipid metabolism modulates endoplasmic reticulum calcium homeostasis in primary human renal mesangial cells

doi: 10.3389/fimmu.2025.1688916

Figure Lengend Snippet: IL-1β upregulates expression of genes in ER stress pathways in hRMCs. hRMCs lots 20018, 12445, and 17544 were treated with vehicle, 25 ng/ml IL-1β, or 25 ng/ml IL-1β +20 nM eliglustat for 24h, 48h, or 72h in quadruplicate and RNA sequencing performed. (A–B) UpSet plots showing shared and unique differentially expressed genes between IL-1β and untreated (A) , and IL-1β +eliglustat and untreated (B) samples across different time points. (C, D) Volcano plots displaying significantly upregulated (purple) and downregulated (orange) genes following IL-1β treatment (C) and IL-1β +eliglustat treatment (D) . Highlighted genes are associated with immune response and ER functions. (E) Representative network dendrogram showing correlation patterns between detected co-expression modules, treatments, and time points, and below, dot plots with standard errors (SEs) illustrating the association of selected modules. SEs are calculated based on the module eigengenes across samples. Dots represent the mean eigengene expression for each module. (F, G) Network visualization of the top 200 connections ranked by weighted topological overlap for the salmon (F) and floralwhite (G) modules. Node size corresponds to the number of edges (degree). Enriched pathways for biological process are shown below each module. CTRL= vehicle-treated.

Article Snippet: Purchased reagents and kits include: recombinant, animal-free human IL-1β (#AF-200-01B), IFNγ (#AF-300-02), IFNα (#01-672-628), and TNFα (#AF-300-01A) (Fisher Scientific, Waltham, MA), Alamar blue (#A50101 Invitrogen, Waltham, MA), eliglustat hemitartrate (#50-187–3104 Fisher Scientific/MedChemexpress), and anti-αSMA monoclonal antibody (17H19L35, #701457 Invitrogen); Alexa Fluor Plus 488-conjugated goat anti-rabbit IgG (H+L), highly cross-adsorbed secondary antibody (#A32731 Invitrogen); Fluoromount-G with DAPI (#00-4959-52, Invitrogen) (Fisher Scientific); and Rhodamine Phalloidin (#PHDR1, Cytoskeleton, Inc., Denver, CO).

Techniques: Expressing, RNA Sequencing

Journal: Frontiers in Immunology

Article Title: Cytokine-driven glycosphingolipid metabolism modulates endoplasmic reticulum calcium homeostasis in primary human renal mesangial cells

doi: 10.3389/fimmu.2025.1688916

Figure Lengend Snippet: Cytokine treatment increases cytosolic Ca 2+ and reduces ER Ca 2+ store capacity, and inhibiting GSL synthesis restores ER Ca 2+ stores in primary hRMCs. (A) Representative images of hRMC lot 20018 loaded with Fluo-8 AM under control conditions (top) and after cytokine treatment (bottom) with the cytokine mix of TNFα, IFNα, IFNγ, and IL-1β for 24 h. Scale bar = 50 µm. (B) Quantification of basal cytosolic Ca 2+ levels, shown as percent of mean control values. Data represent mean +SEM of 4 independent experiments (n ≥ 57 cells); ***p< 0.001 by unpaired t-test. (C) Representative Ca 2+ response trace from Fluo-8 AM–loaded male hRMCs (n ≥ 10 cells) under control (black) and cytokine-treated (red) conditions using Thapsigargin (TG; 500 nM) to assess ER Ca 2+ store capacity. (D) Quantification of TG-induced Ca 2+ release in hRMCs treated with vehicle (control), IL-1β alone, or the cytokine mix for 24 h. Both IL-1β and cytokine mix significantly reduced ER Ca 2+ release amplitude compared to vehicle. Bars represent mean +SEM (N = 3 independent experiments; n ≥ 30 cells). Changes in Ca 2+ amplitude in response to TG were measured in (E) HK31 hRMCs and (F) 20018 hRMCs after treatment with cytokine mix with or without eliglustat (1 nM) for 24 hr. Bars represent mean +SEM of 3 independent experiments (n ≥ 30 cells per group). ***p< 0.001, **p< 0.01 vs. control by one-way ANOVA with Tukey’s post hoc test. ns, not significant.

Article Snippet: Purchased reagents and kits include: recombinant, animal-free human IL-1β (#AF-200-01B), IFNγ (#AF-300-02), IFNα (#01-672-628), and TNFα (#AF-300-01A) (Fisher Scientific, Waltham, MA), Alamar blue (#A50101 Invitrogen, Waltham, MA), eliglustat hemitartrate (#50-187–3104 Fisher Scientific/MedChemexpress), and anti-αSMA monoclonal antibody (17H19L35, #701457 Invitrogen); Alexa Fluor Plus 488-conjugated goat anti-rabbit IgG (H+L), highly cross-adsorbed secondary antibody (#A32731 Invitrogen); Fluoromount-G with DAPI (#00-4959-52, Invitrogen) (Fisher Scientific); and Rhodamine Phalloidin (#PHDR1, Cytoskeleton, Inc., Denver, CO).

Techniques: Control

Journal: Frontiers in Immunology

Article Title: Cytokine-driven glycosphingolipid metabolism modulates endoplasmic reticulum calcium homeostasis in primary human renal mesangial cells

doi: 10.3389/fimmu.2025.1688916

Figure Lengend Snippet: Eliglustat does not significantly impact IL-6, IL-8, MCP-1 release in response to IL-1β or cytokine mix at 24 hours post-treatment. hRMC lots 20057, 20018, HK31, and HK32 were treated with: (A–C) IL-1β, IL-1β plus 1 nM, 10 nM, or 100 nM eliglustat, or vehicles in triplicate; or (D–F) cytokine mix, cytokine mix plus 1 nM, 10 nM, or 100 nM eliglustat, or vehicles in triplicate. Media was collected after 24h, cell viability measured by alamar blue, and IL-6 (A, D) , IL-8 (B, E) , or MCP-1 (C, F) measured in the media by ELISA. Data is the mean of the triplicate wells +SD.

Article Snippet: Purchased reagents and kits include: recombinant, animal-free human IL-1β (#AF-200-01B), IFNγ (#AF-300-02), IFNα (#01-672-628), and TNFα (#AF-300-01A) (Fisher Scientific, Waltham, MA), Alamar blue (#A50101 Invitrogen, Waltham, MA), eliglustat hemitartrate (#50-187–3104 Fisher Scientific/MedChemexpress), and anti-αSMA monoclonal antibody (17H19L35, #701457 Invitrogen); Alexa Fluor Plus 488-conjugated goat anti-rabbit IgG (H+L), highly cross-adsorbed secondary antibody (#A32731 Invitrogen); Fluoromount-G with DAPI (#00-4959-52, Invitrogen) (Fisher Scientific); and Rhodamine Phalloidin (#PHDR1, Cytoskeleton, Inc., Denver, CO).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Cytokine-driven glycosphingolipid metabolism modulates endoplasmic reticulum calcium homeostasis in primary human renal mesangial cells

doi: 10.3389/fimmu.2025.1688916

Figure Lengend Snippet: Summary schematic of results. IL-1β and TNFα, and a mix of cytokines (IL-1β, TNFα, IFNα, and IFNγ) elicited the highest response from hRMCs as measured by secretion of multiple cytokines. IL-1β or the mix of cytokines increased GSLs HexCers and LacCers, upregulated proinflammatory pathways and genes/pathways associated with ER-related functions, increased cytosolic Ca 2+ , and decreased ER Ca 2+ capacity. Eliglustat, an inhibitor of GlcCer synthase that catalyzes the generation of GlcCer from ceramide, reduced HexCers and LacCers and restored ER Ca 2+ capacity, but did not have an effect on cytokine secretion in response to IL-1β or the mix of cytokines. Thus, eliglustat likely inhibits or reverses cytokine-induced ER stress in hRMCs. Figure created using BioRender.com .

Article Snippet: Purchased reagents and kits include: recombinant, animal-free human IL-1β (#AF-200-01B), IFNγ (#AF-300-02), IFNα (#01-672-628), and TNFα (#AF-300-01A) (Fisher Scientific, Waltham, MA), Alamar blue (#A50101 Invitrogen, Waltham, MA), eliglustat hemitartrate (#50-187–3104 Fisher Scientific/MedChemexpress), and anti-αSMA monoclonal antibody (17H19L35, #701457 Invitrogen); Alexa Fluor Plus 488-conjugated goat anti-rabbit IgG (H+L), highly cross-adsorbed secondary antibody (#A32731 Invitrogen); Fluoromount-G with DAPI (#00-4959-52, Invitrogen) (Fisher Scientific); and Rhodamine Phalloidin (#PHDR1, Cytoskeleton, Inc., Denver, CO).

Techniques: