Mitofusin-2 regulates leukocyte adhesion and β2 integrin activation

Abstract

Neutrophils are critical for inflammation and innate immunity, and their adhesion to vascular endothelium is a crucial step in neutrophil recruitment. Mitofusin-2 (MFN2) is required for neutrophil adhesion, but molecular details are unclear. Here, we demonstrated that β₂ -integrin-mediated slow-rolling and arrest, but not PSGL-1-mediated cell rolling, are defective in MFN2-deficient neutrophil-like HL60 cells. This adhesion defect is associated with reduced expression of fMLP (N-formylmethionyl-leucyl-phenylalanine) receptor FPR1 as well as the inhibited β₂ integrin activation, as assessed by conformation-specific monoclonal antibodies. MFN2 deficiency also leads to decreased actin polymerization, which is important for β2 integrin activation. Mn²⁺ -induced cell spreading is also inhibited after MFN2 knockdown. MFN2 deficiency limited the maturation of β2 integrin activation during the neutrophil-directed differentiation of HL60 cells, which is indicated by CD35 and CD87 markers. MFN2 knockdown in β2-integrin activation-matured cells (CD87^high population) also inhibits integrin activation, indicating that MFN2 directly affects β2 integrin activation. Our study illustrates the function of MFN2 in leukocyte adhesion and may provide new insights into the development and treatment of MFN2 deficiency-related diseases.

Keywords: CD87; HL60; Mitoifusin-2; Neutrophil adhesion; actin polymerization; neutrophil maturation; β2 integrin activation.

FIGURE 1. Mitofusin-2 knockdown reduces the adhesion of neutrophil-like HL60 cells.

(A) Western blot showing protein expression of mitofusin-2 (MFN2) in HL60 cells transfected with control (CT) or MFN2 knockdown (KD) with shRNA. Tubulin is an internal control. (B) Mean ± SD of MFN2 versus tubulin expression from n = 5 independent western blot experiments. (C) Mean ± SD of MFN2 expression from n = 8 independent intracellular staining flow cytometry experiments. (D) Tracks of Control or MFN2 KD HL60 cells rolling on a substrate of P-selectin or P-selectin/ICAM-1 under a wall shear stress of 6 dyn/cm². The recording time of rolling was 60 s. The rolling paths (line) and ending positions (circle) of n = 15 cells from 3 individual experiments per group are shown. (E and F) A cumulative histogram (E) and a bar graph (F, mean ± SD, n = 45 cells from 3 individual experiments per group) showing the rolling velocity of CT or MFN2 KD HL60 cells rolling on the substrate of P-selectin with or without ICAM-1 under a wall shear stress of 6 dyn/cm². (G and H) The number of arrested CT or MFN2 KD HL60 cells on the substrate of P-selectin/ICAM-1 with or without the stimulation of fMLP (100 nM) under a wall shear stress of 6 dyn/cm² (G) and 2 dyn/cm² (H). Mean ± SD, n = 3 individual records. n.s., non-significant (P > 0.05). *P < 0.05, ****P < 0.0001 by paired Student’s t-tests (B and C) or 2-way ANOVA followed by Tukey’s multiple comparisons tests (F and H). HL60 cells were pre-differentiated with 1.3% DMSO for 7 days

FIGURE 2. Mitofusin-2 knockdown reduces fMLP receptor expression as well as receptor-dependent and independent β₂ integrin activation in neutrophil-like HL60 cells.

(A) Mean ± SD of surface FPR1 expression on Control or MFN2-knockdown (MFN2 KD) HL60 cells from n = 3 individual experiments. (B-I) Mean ± SD of overall surface CD18 (β₂ integrins, B,C), CD11a (α_L integrins, D,E), CD11b (α_M integrins, F,G), CD11c (α_X integrins, H,I) expression on Control or MFN2 KD HL60 cells stimulated with FPR1-dependent fMLP (100 nM, at RT for 20 min, closed bars, B, D, F, H), receptor-independent PMA (100 nM, at RT for 20 min, gray bars, C, E, G, I), or vehicle control (open bars) from n = 3 individual experiments. (J-U) Mean ± SD of high-affinity (H⁺, mAb24 staining, J-M) and extended (E⁺, KIM127 staining, N-Q) β₂ integrin as well as H⁺ α_M integrin (CBRM1/5 staining, R-U) expression on Control or MFN2 KD HL60 cells stimulated with FPR1-dependent fMLP (100 nM, at RT for 20 min, closed bars, J, L, N, P, R, T), receptor-independent PMA (100 nM, at RT for 20 min, gray bars, K, M, O, Q, S, U) or vehicle control (open bars) from n = 3 individual experiments. In L,M; P,Q; and T,U, the MFI of mAb24, KIM127, and CBRM1/5 is normalized to overall expression of CD18 (B,C), CD18 (B,C), and CD11b (F,G), respectively. n.s., non-significant (P > 0.05). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s t-test (A) or 2-way ANOVA followed by Tukey’s multiple comparisons test (B-U). HL60 cells were pre-differentiated with 1.3% DMSO for 7 days

FIGURE 3. Mitofusin-2 knockdown eliminates actin polymerization in neutrophil-like HL60 cells after receptor-dependent and independent stimulation.

(A) Representative images of F-actin (phalloidin-AF568) in Control or MFN2-knockdown (MFN2 KD) HL60 cells stimulated with FPR1-dependent fMLP (100 nM, at RT for 20 min), receptor-independent PMA (100 nM, at RT for 20 min), or vehicle control. The scale bar is 10 μm. (B) Mean ± SD of phalloidin MFI quantified from n = 45 cells in 6 independent records per group. n.s., non-significant (P > 0.05) *P < 0.05, ****P < 0.0001 by 1-way ANOVA followed by Tukey’s multiple comparisons test. HL60 cells were pre-differentiated with 1.3% DMSO for 7 days

FIGURE 4. Mitofusin-2 knockdown inhibited Mn²⁺-induced integrin-outside-in-signaling-dependent spreading of neutrophil-like HL60 cells.

(A) Representative total internal reflection fluorescence images of spreading in Control or MFN2-knockdown (MFN2 KD) HL60 cell footprints pretreated with anti-CD18 blocking antibody or isotype control. Cells were stained with CellTracker Orange CMRA. Spreading was induced by 1 μM Mn²⁺ on coated ICAM-1. The scale bar is 10 μm. (B) Mean ± SD of the cell footprint area of n = 122 (control, isotype), n = 114 (control, CD18 Ab), n = 118 (MFN2 KD, isotype), and n = 134 (MFN2 KD, CD18 Ab) cells, respectively, from 6 independent records per group. n.s., non-significant (P > 0.05) ***P < 0.001, ****P < 0.0001 by 1-way ANOVA followed by Tukey’s multiple comparisons test. HL60 cells were pre-differentiated with 1.3% DMSO for 7 days

FIGURE 5. The maturation of β₂ integrin activation during the DMSO-induced differentiation of HL60 cells.

(A) Contour plots showing expression of high-affinity (H⁺, mAb24 staining) and extended (E⁺, KIM127 staining) β₂ integrins on HL60 cells (upper panels) or primary human neutrophils (lower panel) stimulated with fMLP (1 mM, right panels) or vehicle control (left panels). HL60 cells were pre-differentiated with 1.3% DMSO for indicated days. (B,C) Expression of mAb24 (B) and KIM127 (C) on HL-60 cells after different days of DSMO-induced differentiation with or without fMLP stimulation. (D) The percentage of mAb24⁺KIM127⁺ HL-60 cells after different days of DSMO-induced differentiation with or without fMLP stimulation. (E) Expression of overall CD18 (conformation unspecific) on HL-60 cells after different days of DSMO-induced differentiation with or without fMLP stimulation. (F) FPR1 expression on HL-60 cells after different days of DSMO-induced differentiation. Mean ± 95%CI for n = 3 individual experiments in (B-G), **P < 0.01, ***P < 0.001, ****P < 0.0001 by comparing fMLP stimulated cells and vehicle control using 2-way ANOVA followed by Sidak’s multiple comparisons test

FIGURE 6. Mitofusin-2 knockdown impairs expression of β₂-integrin-activation maturation markers during the DMSO-induced differentiation of HL60 cells.

(A,B) Representative histograms showing expression of CD35 (A) and CD87 (B) on HL60 cells pre-differentiated for 5 days (dark gray) or not (light gray). (C-D) Expression of CD35 (C), and CD87 (D) on HL-60 cells after different days of DSMO-induced differentiation. Mean ± 95%CI for n = 3 individual experiments. (E-H) Contour plots showing expression of high-affinity (H⁺, mAb24 staining) β₂ integrins on pre-differentiated HL60 cells with different expression of CD35 (E), CD87 (F), and overall CD18 (G) stimulated with fMLP (100 nM, right panels) or vehicle control (left panels). (H) The percentage of mAb24⁺ cells in the low-expression or high-expression population of different markers upon fMLP stimulation. (I-K) Expression of CD35 (I) and CD87 (J-K) on Control or MFN2-knockdown (MFN2 KD) HL60 cells after pre-differentiated for 5 (I-J) or 7 (K) days. Mean ± SD for n = 3 (I) or 9 (J-K) individual experiments, *P < 0.05, ****P < 0.0001 by Student’s t-test

FIGURE 7. Mitofusin-2 knockdown limits nucleus segmentation during the DMSO-induced differentiation of HL60 cells.

(A) Representative bright field (BF) and nucleus (anti-Histone H3-AF647) images of Control or MFN2-knockdown (MFN2 KD) HL60 cells before and after DMSO-induced differentiation. (B) Representative electron microscopy images of Control or MFN2 KD HL60 cells before and after the DMSO-induced differentiation. Scale bars are 10 μm. (C and D) Mean ± SD of nucleus segment number of n = 36 (control undifferentiated), n = 34 (control differentiated), n = 47 (MFN2 KD undifferentiated), and n = 24 (MFN2 KD differentiated) cells from 7, 9, 6, and 11 independent records, respectively, per group. n.s., non-significant (P > 0.05). *P < 0.05, **P < 0.01, ****P < 0.0001 by 1-way ANOVA followed by Tukey’s multiple comparisons test. HL60 cells were pre-differentiated with 1.3% DMSO for 7 days

FIGURE 8. Mitofusin-2 knockdown inhibits β₂ integrin activation and adhesion of HL60 cells at the same maturation level.

(A) Mean ± SD of surface CD87 expression on CD87^high Control and MFN2-knockdown (MFN2 KD) HL60 cells from n = 6 individual experiments. (B) Mean ± SD of nucleus segment number of CD87^high Control (n = 21) and MFN2 KD (n = 19) HL60 cells from 9 and 11 independent records, respectively. (C and D) Mean ± SD of MFN2 and FPR1 expression on CD87^high Control and MFN2 KD HL60 cells from n = 3 individual experiments. (E and F) Mean ± SD of overall surface CD18 (β₂ integrins) expression on CD87^high control or MFN2 KD HL60 cells stimulated with FPR1-dependent fMLP (100 nM, at RT for 20 min, closed bars, E), receptor-independent PMA (100 nM, at RT for 20 min, gray bars, F), or vehicle control (open bars) from n = 3 individual experiments. (G-N) Mean±SD of high-affinity (H⁺, mAb24 staining, G-J) and extended (E⁺, KIM127 staining, K-N) β₂ integrin expression on CD87^high control or MFN2 KD HL60 cells stimulated with FPR1-dependent fMLP (100 nM, at RT for 20 min, closed bars, G, I, K, M), receptor-independent PMA (100 nM, at RT for 20 min, gray bars, H, J, L, N), or vehicle control (open bars) from n = 3 individual experiments. In (I and J) and (M and N), the MFI of mAb24 and KIM127 is normalized to overall expression of CD18 (E and F). (O and P) A cumulative histogram (O) and a bar graph (P, mean ± SD, n = 46 cells from 3 individual experiments per group) showing the rolling velocity of CD87^high Control or MFN2 KD HL60 cells rolling on the substrate of P-selectin+ICAM-1 under a wall shear stress of 6 dyn/cm². (Q) The number of arrested CD87^high Control or MFN2 KD HL60 cells on the substrate of P-selectin/ICAM-1 with or without the stimulation of fMLP (100 nM) under a wall shear stress of 6 dyn/cm². Mean ± SD, n = 17 individual records. n.s., non-significant (P > 0.05). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s t-test (A-D, P) or 2-way ANOVA followed by Tukey’s multiple comparisons test (E-N, Q). HL60 cells were pre-differentiated with 1.3% DMSO for 7 days

FIGURE 9. Schematics showing the function of MFN2 in HL60 adhesion.

(A) During the neutrophil-directed differentiation of HL60 cells, β₂ integrin activation increases in response to fMLP stimulation, which is critical for cell adhesion. We call this process the maturation of β₂ integrin activation. Expression of β₂ integrins, fMLP receptor FPR1, CD87, and CD35 also increase during the neutrophil-directed differentiation of HL60 cells. The maturation of β₂ integrin activation is associated with the expression of β₂ integrins, CD87, and CD35. Thus, the expression of β₂ integrins, CD87, and CD35 can serve as markers for the maturation of β₂ integrin activation. (B) Knockdown of MFN2 inhibited the maturation of β₂ integrin activation during the neutrophil-directed differentiation of HL60 cells, causing less expression of FPR1, CD87, and CD35, less activation of β₂ integrins, and less cell adhesion underflow. (C) In β₂ integrin activation matured cells (CD87^high in this study), the loss of MFN2 expression decreases β₂ integrin