Human Primary Immune Cells Exhibit Distinct Mechanical Properties that Are Modified by Inflammation

Abstract

T lymphocytes are key modulators of the immune response. Their activation requires cell-cell interaction with different myeloid cell populations of the immune system called antigen-presenting cells (APCs). Although T lymphocytes have recently been shown to respond to mechanical cues, in particular to the stiffness of their environment, little is known about the rigidity of APCs. In this study, single-cell microplate assays were performed to measure the viscoelastic moduli of different human myeloid primary APCs, i.e., monocytes (Ms, storage modulus of 520 +90/-80 Pa), dendritic cells (DCs, 440 +110/-90 Pa), and macrophages (MPHs, 900 +110/-100 Pa). Inflammatory conditions modulated these properties, with storage moduli ranging from 190 Pa to 1450 Pa. The effect of inflammation on the mechanical properties was independent of the induction of expression of commonly used APC maturation markers, making myeloid APC rigidity an additional feature of inflammation. In addition, the rigidity of human T lymphocytes was lower than that of all myeloid cells tested and among the lowest reported (Young's modulus of 85 ± 5 Pa). Finally, the viscoelastic properties of myeloid cells were dependent on both their filamentous actin content and myosin IIA activity, although the relative contribution of these parameters varied within cell types. These results indicate that T lymphocytes face different cell rigidities when interacting with myeloid APCs in vivo and that this mechanical landscape changes under inflammation.

Figure 1

(A) Expression of surface markers by APCs (indicated above histograms) analyzed by flow cytometry for DCs and MPHs either untreated or treated with cytokines or LPS. Gray histograms: isotype control (nonspecific binding of fluorescent antibody); open histograms: binding of surface-marker-specific antibody; black line: mean fluorescence intensity for untreated cells. The shift of open histograms to the right side of the black line indicates increased expression of the surface marker. (B) Production of IL-2 from CD4⁺ T lymphocytes cultured overnight with autologous DCs or MPHs in the presence or absence of TSST-1 superantigen (0–10 ng/mL). Results were obtained from four different donors (values from each donor were normalized according to median).

Figure 2

(A) Schematic view of the microplates experiment. Oscillations are applied at the base of the cantilever and the cell’s response at the tip is sampled via an optical sensor. The storage modulus ($G_0^{\prime }$) and loss modulus ($G_0^{″}$) are then derived from phase-shift and amplitude ratios between input and output signals. (B) Bright-field image of a MPH during an experiment; scale bar: 10 μm. (C) Storage ($G_0^{\prime }$) and loss ($G_0^{″}$) moduli for human Ms, DCs, and MPHs (^∗p < 0.05, ^∗∗p < 0.01, Mann-Whitney U test for each condition; N, number of cells tested, from at least three different donors). For values see Table 1.

Figure 3

Storage ($G_0^{\prime }$) and loss ($G_0^{″}$) moduli of DCs and MPHs after treatment with different inflammation factors. Cells were incubated with IFNγ (light gray), TNFα/PGE₂ (dark gray), or LPS (black) for 24 h before testing (^∗p < 0.05, ^∗∗p < 0.01, according to Mann-Whitney U test compared with untreated cells; N, number of cells tested, from at least three different donors). For values, see Table 1.

Figure 4

Measurement of the apparent Young’s modulus of primary CD4⁺ T cells and Jurkat T cells. (A) A cell was caught and squeezed between the plates by applying a displacement d on the rigid plate. The displacement of the flexible plate δ was optically measured and the length before and after compression was measured to derive the apparent static Young modulus E_app. (B) For primary CD4⁺ T cells, the data points (+) of the apparent Young’s modulus followed a log-normal distribution whose mean E_app (mean = 85 ± 5 Pa, solid line) matches the equivalent Young’s modulus E_eq (mean = 90 ± 10 Pa, dashed line) of Jurkat cells obtained through calculation. (C) Comparison of stepwise compression experiments (E_app data points in dark circles, mean = 70 +30/−20 Pa in dashed line) and dynamic mechanical analysis ($G_0^{\prime }$ data points in white circles, mean = 80 +70/−40 Pa in solid line) for Jurkat cells.

Figure 5

(A) Summary of elasticity values (equivalent Young’s modulus E_eq) for the immune cells tested. (B) Stiffness scale for cells and tissues. To see this figure in color, go online.

Figure 6

(A and B) Plots of the equivalent Young’s modulus E_eq versus (A) total F-actin and (B) total myosin IIA heavy chain contents measured in confocal microscopy images. Pearson correlation coefficients were calculated (r_actin = 0.83, r_myosin = 0.67).

Figure 7

(A) Ms, DCs, and MPHs were subjected to 5 μM of blebbistatin for 15 min before dynamic mechanical testing was performed and the effect on storage ($G_0^{\prime }$) and loss ($G_0^{″}$) moduli (^∗∗p < 0.01, ^∗∗∗p < 0.001, according to Mann-Whitney U test compared with untreated; N, number of cells tested, from at least three different donors) was determined. (B) Blebbistatin-induced percentage decrease of the storage modulus $G_0^{\prime }$ as compared with that of untreated cells. (C) Comparison of the ratio of total myosin IIA heavy chain content and total F-actin content for Ms, MPHs, and DCs. (D) Quantification of western blot bands for the phosphorylated Ser-19 of the myo-RLC (⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001, ⁺⁺⁺⁺p < 0.0001, unpaired t-test with Welch’s correction for each condition; N, number of cells tested, from four donors; n_D, number of donors).