IL-6 enhances CD4 cell motility by sustaining mitochondrial Ca2+ through the noncanonical STAT3 pathway

Abstract

Interleukin 6 (IL-6) is known to regulate the CD4 T cell function by inducing gene expression of a number of cytokines through activation of Stat3 transcription factor. Here, we reveal that IL-6 strengthens the mechanics of CD4 T cells. The presence of IL-6 during activation of mouse and human CD4 T cells enhances their motility (random walk and exploratory spread), resulting in an increase in travel distance and higher velocity. This is an intrinsic effect of IL-6 on CD4 T-cell fitness that involves an increase in mitochondrial Ca²⁺ Although Stat3 transcriptional activity is dispensable for this process, IL-6 uses mitochondrial Stat3 to enhance mitochondrial Ca²⁺-mediated motility of CD4 T cells. Thus, through a noncanonical pathway, IL-6 can improve competitive fitness of CD4 T cells by facilitating cell motility. These results could lead to alternative therapeutic strategies for inflammatory diseases in which IL-6 plays a pathogenic role.

Keywords: CD4 T cells; STAT3; interleukin-6; mitochondrial calcium; motility.

Fig. 1.

IL-6 increases motility of mouse CD4 T cells. (A and B) CD4 T cells were activated with anti-CD3/CD28 mAbs in the presence or absence of IL-6 (50 ng/mL). After 48 h, cells were harvested, washed, and loaded onto chambers on a temperature-controlled stage. Live images were captured using a confocal microscope (bright field) every 30 s for 10 min. Distance (A) and velocity (B) of individual cells are shown. (C) CD4 T cells were activated as in A for 48 h, washed, and cells were seeded into transwell chambers in medium alone. After 5 h, cells that migrated in the wells were counted (n = 3). (D) CD4 T cells were activated as in A, and after 48 h they were washed and placed into transwell chambers in the presence of CCL19 (100 ng/mL) that was added to the lower chamber. After 5 h, cells that had migrated in the wells were counted. (E) CCR7 expression by CD4 T cells activated for 48 h in the absence or presence of IL-6 as determined by flow cytometry analysis. (F and G) CD4 T cells were activated as in A for 48 h, washed, and loaded onto chambers on a temperature-controlled stage in the presence of CCL19 (100 ng/mL). Live microscopy images were captured as described in A. Distance (F) and velocity (G) for individual cells are shown. Error bars show ± SD; *P < 0.05, as determined by Student’s t test. Results are representative of two to three experiments.

Fig. 2.

IL-6–induced motility of mouse CD4 T cells during activation requires mitochondrial Ca²⁺. (A) CD4 T cells were activated with anti-CD3/CD28 mAbs in the presence of IL-6 for 48 h, and during the last 4 h, they were treated with (10 μM) CGP-37157 or vehicle. Cells were washed and placed into transwell chambers in medium alone. Migrated cells were counted after 5 h (n = 3). (B) CD4 T cells were activated for 48 h in the presence of IL-6, and for the last 18 h, they were treated with (10 μM) Ru360 or vehicle. Cells were then washed and placed in transwell chambers as in A. (C and D) CD4 T cells were activated in the presence of IL-6, treated with Ru360 or vehicle, washed, and loaded onto chambers on a temperature-controlled stage, in medium alone. Live images were captured under a confocal microscope. Distance (C) and velocity (D) of individual cells are shown. (E) Expression of MCU in CD4 T cells activated in the absence or presence of IL-6 as determined by Western blot analysis. β-actin was used as a control. (F) Expression of MCU in freshly isolated CD4 T cells from cMCU KO mice by Western blot analysis. (G) Mitochondrial Ca²⁺ in CD4 cells from WT and cMCU KO mice activated in the presence of IL-6 for 48 h was determined by staining with Rhod-2 AM and flow cytometry analysis. (H) CD4 T cells from WT and cMCU KO mice were activated with anti-CD3/CD28 mAbs in the presence of IL-6 for 48 h, washed, and placed into transwell chambers in medium alone. Migrated cells were counted after 5 h (n = 3). (I and J) CD4 T cells from WT mice were stained with carboxyfluorescein succinimidyl ester (CFSE) or CellTrace Violet and activated in the absence or presence of IL-6, respectively. After 48 h, cells were washed and coinjected i.v. (5 × 10⁶ cells total) at equal ratio into WT recipient mice (n = 5). After 16 h, the presence of donor cells in the spleen was examined by flow cytometry analysis (I). The number of each labeled donor cell type is shown (J). (K) CD4 T cells from WT or cMCU KO mice were stained with CFSE or CellTrace Violet, respectively, activated in the presence of IL-6 for 48 h, washed, and injected into WT recipient mice (n = 4) as described in J. After 16 h, CD4 T cells were analyzed by flow cytometry. Numbers of CD4 T cells labeled with CFSE or CellTrace Violet in the spleen are shown. (L–N) CD4 T cells were activated with anti-CD3/CD28 mAbs in the absence (MED) or presence of IL-6 (50 ng/mL) for 48 h, cytospinned, stained for actin (L, red), vinculin (M, green), or phospho-MLC (P-Ser19, red) (N), and visualized at the confocal microscope. DAPI (blue) was used as a nuclear marker. Error bars show ± SD; *P < 0.05, as determined by Student’s t test. Results are representative of two or three experiments.

Fig. 3.

Impaired CD4 T-cell motility in Stat3 S⁷²⁷A KI mice. (A) CD4 T cells from WT and mut-Stat3 mice were activated with anti-CD3/CD28 mAbs in the presence or absence of IL-6 for 48 h, washed, and placed into transwell chambers in medium alone. Migrated cells were counted after 5 h (n = 3). (B and C) WT and Stat3 S⁷²⁷A KI CD4 T cells were activated in the presence or absence of IL-6 for 48 h, and MMP was determined by tetramethylrhodamine, ethyl ester (TMRE) staining (B). Percentage of TMRE^high cells as defined in B is shown (n = 3) (C). (D and E) Mitochondrial Ca²⁺ in CD4 T cells from WT and Stat3 S⁷²⁷A KI mice activated as in B was assayed by staining with Rhod-2 AM and flow cytometry analysis (D). Percentage of TMRE ^high cells as defined in D is shown (E) (n = 3). (F) CD4 T cells from WT and Stat3 S⁷²⁷A KI mice were activated as in A and placed into transwell chambers in medium alone. Migrated cells were counted after 5 h (n = 3). (G) CD4 T cells from WT or Stat3 S⁷²⁷A KI mice were stained with CFSE or CellTrace Violet, respectively, activated in the presence of IL-6 for 48 h, washed, and injected into WT recipient mice (n = 4). After 16 h, CD4 T cells were analyzed by flow cytometry. Numbers of CD4 T cells labeled with CFSE or CellTrace Violet in the spleen are shown. (H and I) WT and Stat3 S⁷²⁷A KI CD4 T cells were activated as in A in the presence of IL-6 for 48 h, cytospinned, stained for actin (H) or phospho-MLC (I), and visualized at the confocal microscope. DAPI (blue) was used as a nuclear marker. Error bars show ± SD; *P < 0.05, as determined by Student’s t test. Results are representative of two to three experiments.

Fig. 4.

IL-6 increases human CD4 T-cell motility through mitochondrial Ca²⁺. (A) Isolated CD4 RA and CD4 RO cells from healthy individuals were activated with anti-human CD3 and CD28 Abs and IL-6 production after 24 h of activation was determined by ELISA (n = 7). (B and C) Isolated CD4 RA and CD4 RO cells were activated with anti-CD3/CD28 mAbs for 48 h, stained for TMRE (B) or Rhod2-AM (C), and analyzed by flow cytometry. (D and E) Isolated CD4 RO cells from healthy volunteers were activated with anti-CD3/CD28 mAbs in the absence or presence of an anti–IL-6 Ab (10 µg/mL). After 48 h, cells were stained with TMRE (D) or Rhod2-AM (E) and analyzed by flow cytometry. (F and G) Isolated CD4 RA cells from healthy volunteers were activated with anti-CD3/CD28 mAbs in the absence or presence of a human IL-6 (50 ng/mL). After 48 h, cells were stained with TMRE (F) or Rhod2-AM (G) and analyzed by flow cytometry. (H) CD4 RA were activated in the presence or absence of IL-6 for 48 h, washed, and cells were placed into transwell chambers in medium alone. Migrated cells were counted after 5 h (n = 3). (I) CD4 RA were activated in the presence IL-6 for 48 h, treated with Ru360 or vehicle for the last 24 h, washed, and placed into transwell chambers in medium alone. Migrated cells were counted after 5 h (n = 3). (J) CD4 RA were activated in the presence IL-6 for 48 h, treated with atovaquone or vehicle for the last 20 h, washed, and placed into transwell chambers in medium alone. Migrated cells were counted after 5 h (n = 3). (K) Isolated CD4 RO were activated in the presence or absence of anti–IL-6 for 48 h, washed ,and cells were and placed into transwell chambers in medium alone. Migrated cells were counted after 5 h (n = 3). (L) CD4 RO were activated for 48 h, treated with atovaquone or vehicle for the last 20 h, washed, and placed into transwell chambers in medium alone. Migrated cells were counted after 5 h (n = 3). Error bars show ± SD; *P < 0.05, as determined by Student’s t test. Results are representative of two to three experiments.