Fig. 6: Simulated microgravity induces distinct modifications to immune cell cytoskeletal morphology and cytokine production. | Nature Communications

Fig. 6: Simulated microgravity induces distinct modifications to immune cell cytoskeletal morphology and cytokine production.

From: Single-cell analysis identifies conserved features of immune dysfunction in simulated microgravity and spaceflight

Fig. 6

A Super-resolution microscopy analysis of actin in 2D for cell area (left), intensity (middle), and texture as punctate over diffuse index (PDI, variance/mean, right) between 25 hours of 1G or simulated uG. Dots represent individual PBMCs (n = 159 cells for 1G, and n = 154 cells for uG) from 4 independent donors. Donors were male (25 years old), and females (35, 38, and 46 years old). One outlier for actin intensity and actin PDI from each condition is removed based on Grubbs’ test. Two-tailed Welch’s t test was used for all comparisons. ***p ≤ 0.001. Data are plotted as mean ± standard error of the mean (SEM) and source data are provided with this paper. B Representative super-resolution microscopy images (2D left, 3D right) of PBMCs from 1G and simulated uG (25 hours) from donor 1 (35 yr F) and donor 2 (25 yr M; 2 of total 4 donors from A are shown here). 3D images better highlight changes to overall cell shape and actin protrusions in simulated uG. Scale bar = 2 μm and 1 μm, respectively. C Sixteen-channel granularity spectrum measurement of PBMCs stimulated with TLR7/8 agonist (9 hours stimulation, 16 hours conditioning prior to stimulation) from 1G (pink line) and uG (brown line) minus the corresponding unstimulated cells (25 hours total culture). The effect of simulated microgravity on unstimulated granularity spectrum is plotted in gray. Asterisks compare pink vs brown lines only. P values generated from unpaired two-tailed t test. n = 3 donors tested from A, 35-year-old female sample was not used. **p ≤ 0.01, *p ≤ 0.05. Data are plotted as mean ± SEM, and source data are provided with this paper. D Super-resolution microscopy analysis of 3D actin surface area (left, 1G n = 179 cells and uG n = 194 cells) and actin spike length (right, 1G n = 162 cells and uG n = 165 cells) between 25 hours of 1G or simulated uG. Dots represent individual PBMCs from 4 independent donors. Donors were male (25 years old), and females (35, 38, and 46 years old). Two-tailed Welch’s t test was used to calculate p values. *p ≤ 0.05, **p ≤ 0.01. Data are plotted as mean ± SEM and source data are provided with this paper. E G-LISA levels of active GTP-bound Cdc42 in PBMCs either unstimulated (25 hours) or treated with TLR7/8 agonist (9 hours + 16 hours conditioning) from 1G and simulated uG. n = 7, donors were male (25 years old), and females (38, 46, 25, 27, 26, and 40 years old). Two-tailed paired parametric t test was used to calculate p values, *p ≤ 0.05, ***p ≤ 0.001. Data are plotted as mean ± SEM and source data are provided with this paper. F ELISA levels of secreted IFNs by PBMCs treated with TLR7/8 agonist (16 hours conditioning + 9 hours stimulation) from 1G and simulated uG. n = 9, donors were male (36 years old), and females (33, 25, 38, 46, 27, 25, 26, and 40 years old). Two-tailed paired parametric t test was used, *p ≤ 0.05. Data are plotted as mean ± SEM, and source data are provided with this paper. G ELISA level of secreted ILs by PBMCs exposed to 25 hours simulated uG and 1G. n = 10 for IL-8 and n = 11 for IL-6, donors were females (32, 25, 38, 46, 25, 27, 26, 40 years old) and males (36, 33, 26 years old); 38-year-old female sample was not used for IL-8. Two-tailed paired parametric t test was used, *p ≤ 0.05. Data are plotted as mean ± SEM and source data are provided with this paper.

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