Journal: Frontiers in Immunology

Article Title: Immune and ionic mechanisms mediating the effect of dexamethasone in severe COVID-19

doi: 10.3389/fimmu.2023.1143350

Figure Lengend Snippet: Changes in ion channel expression with disease progression and dexamethasone treatment. Fold change in mRNA abundance of Kv1.3 ( KCNA3 , A ), KCa3.1 ( KCNN4 , B ), Orai ( ORAI1 , C ) and Stim1 ( STIM1 , D ) in PBMCs from healthy donors (n=4), and patients with mild COVID-19 (n=4), severe COVID-19 (Severe, n=3), and severe COVID-19 + dexamethasone (Dexa, n=4) was determined by RT-qPCR. Data are normalized to healthy donor PBMCs. Each sample was run in triplicate. 18S rRNA was used as the housekeeping gene. Bars represent means ± SD, and each symbol represents an individual patient. Data were analyzed by one-way analysis of variance (ANOVA) (P < 0.001 in A , P = 0.4270 in B , P=0.0003 in C, and P=0.0414 in D ), and post hoc testing was performed by the Holm-Sidak method.

Article Snippet: Cells were stained for surface guinea pig anti- Kv1.3 (Alomone Labs) and Orai1 (ATTO-633, Alomone Labs) primary antibodies overnight at 4°C in the dark.

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Immune and ionic mechanisms mediating the effect of dexamethasone in severe COVID-19

doi: 10.3389/fimmu.2023.1143350

Figure Lengend Snippet: Effect of dexamethasone on Kv1.3 channel abundance. (A) Kv1.3 ( KCNA3 ), Orai1 ( ORAI1 ), and Stim1 ( STIM1 ) mRNA expression in PBMCs from healthy donors (n=4) treated with 0.1 μM and 1μM dexamethasone or vehicle for 48 h was determined by RT-qPCR. Data were normalized to vehicle-treated control. Significance was determined by one-way analysis of variance (ANOVA) for KCNA3 (P < 0.001), ORAI1 (P=0.005), and by ANOVA on ranks for STIM1 (p=0.630). Post-hoc testing was performed by the Holm-Sidak method. (B) Kv1.3 and Orai1 abundance in immune cell subsets from healthy donor PBMCs treated with 1 μM dexamethasone for 48 h. (Left) Representative flow cytometry histograms showing Kv1.3 (top) and Orai1 (bottom) abundance in the presence or absence of dexamethasone treatment in CD3 + T cell subsets. (Right) Mean fluorescence intensity (MFI) of Kv1.3 and Orai1 in immune cell subsets from three healthy donors treated in vitro with 1 μM dexamethasone or vehicle for 48 h. Significance determined by paired t-test. means ± SD, and each symbol represents an individual donor. .

Article Snippet: Cells were stained for surface guinea pig anti- Kv1.3 (Alomone Labs) and Orai1 (ATTO-633, Alomone Labs) primary antibodies overnight at 4°C in the dark.

Techniques: Expressing, Quantitative RT-PCR, Control, Flow Cytometry, Fluorescence, In Vitro

Journal: Frontiers in Immunology

Article Title: Immune and ionic mechanisms mediating the effect of dexamethasone in severe COVID-19

doi: 10.3389/fimmu.2023.1143350

Figure Lengend Snippet: Effect of dexamethasone on Kv1.3 channel function, Ca 2+ signaling and cytokine production. (A) Inhibition of Kv1.3 currents in CD8 + T cells treated with 1 μM dexamethasone (Dexa) for 24 and 48 h. Representative Kv1.3 currents are shown on the left, and percentage inhibition of Kv1.3 currents by dexamethasone in n=4 donors is shown on the right. (B) Representative Ca 2+ response (shown as a ratio of Indo-1 fluorescence at 400 and 480 nm) recorded in activated healthy donor CD8 + T cells treated with either 1μM dexamethasone, 10 nM ShK-Dap22, or vehicle for 48 h are shown on the left. Cells were loaded with Indo-1 ratiometric dye, and the fluorescence was recorded by flow cytometry. Indo-1 loaded cells were first exposed to thapsigargin (arrow) in 0 mM Ca 2+ , followed by 2 mM Ca 2+ , which yields a rapid influx of Ca 2+ (see Materials and Methods). Data are representative of independent experiments performed in CD8 + T cells isolated from n=3 healthy donors. The average fold change in peak Ca 2+ levels in n = 3 healthy donors are shown on the right. Significance was determined by one-way analysis of variance (ANOVA, p=0.0309), and post hoc testing was performed by Tukey’s test. (C) IFN-γ release determined by ELISA in the supernatant of activated CD8 + T cells from n=4 healthy donors and n=3 severe COVID-19 patients. Significance was determined by unpaired t test (D) Percent inhibition of IFN-γ secretion as compared to vehicle treated cells after treatment of activated healthy donor (n=4) CD8 + T cells with either 0.1 μM and 1μM dexamethasone, 10 nM and 100 nM ShK-Dap22, or vehicle for 48 h. Significance was determined by one-way analysis of variance (ANOVA, p=0.9390). (E) Percent inhibition of IFN-γ secretion compared to vehicle treated cells after treatment of activated severe COVID-19 patient (n=3) CD8 + T cells with either 1μM dexamethasone, 10 nM ShK-Dap22, or vehicle for 48 h. Significance was determined by paired-t test. Activation of CD8 + T cells in panels (B–E) was done for 48 h with plate bound anti-CD3 and anti-CD28 antibodies. Bars represent means ± SD, and each symbol represents an individual.

Article Snippet: Cells were stained for surface guinea pig anti- Kv1.3 (Alomone Labs) and Orai1 (ATTO-633, Alomone Labs) primary antibodies overnight at 4°C in the dark.

Techniques: Inhibition, Fluorescence, Flow Cytometry, Isolation, Enzyme-linked Immunosorbent Assay, Activation Assay