Physical exercise mitigates chronic psychological stress‐induced vascular inflammation via the BDNF–Kif4–TARM1 axis

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  FIGURE 1. Voluntary exercise reduces stress‐induced anxiety‐like behaviours and systemic inflammation. (A) Experimental schematic. Male C57BL/6 wild‐type mice were exposed to CRS (2 h/day) and performed voluntary wheel running (1 h/day) for 14 days. (B) ELISA was performed to measure plasma corticosterone levels (n = 8). (C, D) Representative locomotion tracks of mice in the OFT and EPM tests are shown. (E, F) Complete blood cell count in peripheral blood, including the numbers of leukocytes and monocytes (n = 8). (G, H) Flow cytometry analysis of peripheral blood monocyte phenotypes. Representative flow cytometry plots and quantification of CD11b+Ly6Chigh monocytes (n = 5). (I) Plasma concentrations of IL‐6 and IL‐1β quantified by ELISA (n = 8). (J, K) Flow cytometry analysis of BMDM polarization. Gating and quantification of M1 (F4/80+CD86+) and M2 (F4/80+CD206+) macrophages (n = 6). (L) qRT‐PCR analysis of proinflammatory (IL‐6, IL‐1β, Nos2, CCL2 and TNF‐α) and anti‐inflammatory (Arg1, CD206, Retnlb and MGL) gene expression in BMDMs (n = 4). The data are presented as the means ± SDs. p‐values were determined using one‐way ANOVA.

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  FIGURE 2. Targeting Kif4 enables the exercise‐mediated attenuation of stress‐induced inflammatory monocytes. (A) Volcano plots of DEGs for the CRS vs. control and CRS+EX vs. CRS comparisons; (B) Venn diagrams of overlapping DEGs: upregulated DEGs in the CRS vs. control group and downregulated DEGs in the CRS+EX vs. CRS group (left panel); downregulated DEGs in the CRS vs. control group and upregulated DEGs in the CRS+EX vs. CRS group (right panel). (C) Heatmap showing overlapping DEGs. (D, E) qRT‐PCR analysis of Kif4 expression in peripheral blood monocytes (n = 6) and BMDMs (n = 5). (F) Western blot analysis of Kif4 protein expression in peripheral blood monocytes. (G) Experimental schematic. Wild‐type mice received tail vein injections of scrambled plasmid, AAV‐shKif4, or AAV‐Kif4. Fourteen days after the injection, mice in all groups were subjected to daily restraint stress (2 h/day) for 14 consecutive days. (H) qRT‐PCR analysis of Kif4 expression in peripheral blood monocytes (n = 5). (I) Flow cytometry analysis of CD11b+Ly6Chigh monocytes in peripheral blood. (J) Plasma IL‐6 and IL‐1β levels measured by ELISA (n = 5). (K, L) Gating strategy and quantification of BMDMs (n = 5). (M)…

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  FIGURE 3. Kif4 promotes the microtubule‐dependent delivery of TARM1 to the plasma membrane, activating macrophage inflammation. (A) Venn diagram showing the overlap between Kif4‐interacting proteins in BMDMs and membrane proteins upregulated in OE‐Kif4 BMDMs compared with control BMDMs. (B) Schematic representation of molecular docking between the Kif4 (green) and TARM1 (blue) proteins, ΔiG = −10.4). (C) Co‐IP assay validating the interaction between Kif4 and TARM1. (D, E) Western blot analysis of TARM1 protein levels in membrane fractions isolated from RAW264.7 or Kif4 knockout RAW264.7 cells (n = 3). (F) Immunofluorescence staining showing that TARM1 (green) localized to the cell membrane in RAW264.7 or Kif4 knockout RAW264.7 cells. Scale bar = 5 µm. (G, H) Western blot analysis of TARM1 protein levels in membrane fractions isolated from THP‐1 or Kif4 knockout THP‐1 cells (n = 3). (I) Immunofluorescence staining showing that TARM1 (green) localized to the cell membrane in THP‐1 or Kif4 knockout THP‐1 cells. Scale bar = 20 µm. (J) Immunofluorescence staining showing Kif4 (purple), α‐tubulin (red; microtubules) and TARM1 (green) colocalization in BMDMs. Scale bar = 20 µm. (K, L) Flow…

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  FIGURE 4. Monocyte surface TARM1 contributes to endothelial dysfunction. (A, B) Representative images of THP‐1 cells adhering to HUVECs and quantification of adhesion after 24 h of coculture with TARM1‐Fc‐ and/or OE‐Kif4‐treated THP‐1 cells (n = 5). Scale bar = 100 µm. (C, D) qRT‐PCR analysis of ICAM‐1 and VCAM‐1 expression in HUVECs following 24 h of coculture with TARM1‐Fc‐ and/or OE‐Kif4‐treated THP‐1 cells (n = 5). (E, F) Western blot analysis of ICAM‐1 and VCAM‐1 protein levels in HUVECs following 24 h of coculture with TARM1‐Fc‐ and/or OE‐Kif4‐treated THP‐1 cells (n = 3). (G, H) HUVEC migration following 12/24 h of coculture with TARM1‐Fc‐ or OE‐Kif4‐treated THP‐1 cells (n = 3). Scale bar = 200 µm. (I, J) Immunofluorescence staining for ROS in HUVECs after 24 h of coculture with TARM1‐Fc‐ and/or OE‐Kif4‐treated THP‐1 cells (n = 6). Scale bar = 100 µm. (K, L) JC‐1 staining and analysis of the mitochondrial membrane potential in HUVECs following 24 h of coculture with TARM1‐Fc‐ and/or OE‐Kif4‐treated BMDMs (n = 6). The data are presented as the means ± SDs. p‐values were determined using one‐way ANOVA.

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  FIGURE 5. BDNF–TrkB–STAT3 signalling suppresses the Kif4–TARM1 axis to inhibit macrophage inflammation. (A) Plasma BDNF levels measured by ELISA in wild‐type mice after 14 days of voluntary exercise (1 h/day) and restraint stress (2 h/day) (n = 5). p‐values were determined using one‐way ANOVA. (B) qRT‐PCR analysis of BDNF expression in the skeletal muscle, heart, hypothalamus and spleen of wild‐type mice exposed to voluntary exercise and restraint stress (n = 4). p‐values were determined using one‐way ANOVA. (C) Immunofluorescence staining for BDNF (red) in the hypothalamus of C57BL/6 wild‐type mice after voluntary exercise and restraint stress. Scale bar = 50 µm. 3 V, third ventricle. (D) qRT‐PCR analysis of Kif4 mRNA expression in BMDMs treated with rBDNF (0, 5, 25, 50, 100 or 200 ng/mL) (n = 3). p‐values were determined using two‐sample t‐tests. (E) Western blot analysis of TrkB, p‐TrkB, STAT3, p‐STAT3 and Kif4 protein levels in BMDMs treated with rBDNF (100 ng/mL). (F) ChIP‒PCR analysis of p‐STAT3 binding to four Kif4 promoter regions (n = 3). p‐values were determined using two‐sample t‐tests. (G) Western blot analysis of TARM1 protein levels in membrane fractions isolated from BMDMs…

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  FIGURE 6. ANA‐12 inhibits the exercise‐mediated suppression of stress‐induced inflammatory monocytes. (A) Experimental schedules. Wild‐type mice received tail vein injections of AAV‐shKif4 or scrambled shRNA as a control. Starting at 14 days post‐injection, the model mice were subjected to daily intraperitoneal injections of ANA‐12 (0.5 mg/kg) or saline concurrently with daily restraint stress (2 h/day) and voluntary wheel running (1 h/day) for 14 days. (B, C) Representative locomotion tracks of model mice in the open‐field test (OFT) and elevated plus maze (EPM) test are shown. (D) Western blot analysis of TrkB, p‐TrkB, STAT3, p‐STAT3 and Kif4 protein levels in BMDMs from model mice. (E) Immunofluorescence staining showing that TARM1 (green) localized to the cell membrane in the BMDMs of the model mice. Scale bar = 20 µm. (F) Plasma IL‐6 and IL‐1β levels measured by ELISA (n = 5). (G) Representative flow cytometry plots of CD11b+Ly6Chigh monocytes in peripheral blood. (H) Flow cytometry gating strategy for M1 (F4/80+CD86+) and M2 (F4/80+CD206+) BMDMs from model mice. (I) qRT‐PCR analysis of inflammatory and anti‐inflammatory gene expression in BMDMs from model mice (n = 5). The data are…

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  FIGURE 7. The BDNF–Kif4–TARM1 axis is involved in the exercise‐mediated reduction in vascular inflammation and plaque stabilization in model mice. (A) Experimental schedules. ApoE−/− mice were fed an HFD for 12 weeks to establish an atherosclerosis model. At week 9 of the HFD, the mice were intravenously injected with AAV‐shKif4 (Kif4 knockdown), AAV‐Kif4 (Kif4 overexpression), or scrambled control AAV. Beginning at week 11 of the HFD, the mice were subjected to daily restraint stress (2 h/day) and voluntary exercise (1 h/day) for 2 weeks. (B) Immunofluorescence staining of atherosclerotic plaques showing CD68+ macrophages (green), Kif4 (red) and DAPI (blue). Scale bar = 50 µm. (C) Plasma BDNF levels in atherosclerotic mice measured by ELISA (n = 10). (D) Representative flow cytometry plots of CD11b+Ly6Chigh monocytes in peripheral blood. (E) Flow cytometry gating strategy for M1 (F4/80+CD86+) and M2 (F4/80+CD206+) BMDMs from atherosclerotic mice. (F) Representative images of Oil Red O staining of total aortic plaques. Scale bar = 5 mm. (G) Analysis of Oil Red O staining to determine the total aortic plaque area (n = 10). (H) Representative images of aortic atherosclerotic plaques. Scale…

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  FIGURE 8. The anti‐inflammatory effect of regular exercise on psychological stress in CAD patients involves the BDNF–Kif4–TARM1 axis. (A) Experimental schematic. CAD patients enrolled at the 3‐month outpatient follow‐up visit were categorized into four groups (n = 30 patients/group) on the basis of the following parameters: PSS‐10 scores (low vs. high) and regular exercise habits (≥3–5 aerobic sessions/week). (B, C) Representative flow cytometry plots and quantification of CD14highCD16low monocytes in the peripheral blood of CAD patients (n = 30). (D) Plasma IL‐6 and IL‐1β levels quantified by ELISA (n = 30). (E) Plasma BDNF levels in CAD patients measured by ELISA (n = 30). (F) Analysis of the correlation between plasma BDNF levels and PSS‐10 scores (n = 120). (G) qRT‐PCR analysis of Kif4 expression in peripheral blood monocytes from CAD patients (n = 30). (H) Analysis of the correlation between Kif4 expression levels and PSS‐10 scores (n = 120). (I, J) Western blot analysis of Kif4 protein levels in peripheral blood monocytes from CAD patients (n = 3). (K, L) Western blot analysis of TARM1 protein levels in membrane fractions isolated from the peripheral blood monocytes of patients with…