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. 2023 May 8;15(5):e17198.
doi: 10.15252/emmm.202217198. Epub 2023 Mar 10.

Macrophage DCLK1 promotes atherosclerosis via binding to IKKβ and inducing inflammatory responses

Zhuqi Huang  1   2 , Sirui Shen  1   2 , Xue Han  2   3 , Weixin Li  2 , Wu Luo  2 , Liming Lin  2 , Mingjiang Xu  2 , Yi Wang  2 , Weijian Huang  1 , Gaojun Wu  1 , Guang Liang  1   2   3
Affiliations

Macrophage DCLK1 promotes atherosclerosis via binding to IKKβ and inducing inflammatory responses

Zhuqi Huang et al. EMBO Mol Med. .

Abstract

Atherosclerosis is a chronic inflammatory disease with high morbidity and mortality rates worldwide. Doublecortin-like kinase 1 (DCLK1), a microtubule-associated protein kinase, is involved in neurogenesis and human cancers. However, the role of DCLK1 in atherosclerosis remains undefined. In this study, we identified upregulated DCLK1 in macrophages in atherosclerotic lesions of ApoE-/- mice fed an HFD and determined that macrophage-specific DCLK1 deletion attenuates atherosclerosis by reducing inflammation in mice. Mechanistically, RNA sequencing analysis indicated that DCLK1 mediates oxLDL-induced inflammation via NF-κB signaling pathway in primary macrophages. Coimmunoprecipitation followed by LC-MS/MS analysis identified IKKβ as a binding protein of DCLK1. We confirmed that DCLK1 directly interacts with IKKβ and phosphorylates IKKβ at S177/181, thereby facilitating subsequent NF-κB activation and inflammatory gene expression in macrophages. Finally, a pharmacological inhibitor of DCLK1 prevents atherosclerotic progression and inflammation both in vitro and in vivo. Our findings demonstrated that macrophage DCLK1 promotes inflammatory atherosclerosis by binding to IKKβ and activating IKKβ/NF-κB. This study reports DCLK1 as a new IKKβ regulator in inflammation and a potential therapeutic target for inflammatory atherosclerosis.

Keywords: DCLK1; IKKβ; atherosclerosis; inflammation; macrophage.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. DCLK1 is up‐regulated in macrophages in atherosclerotic lesion
  1. A

    Volcano plot analysis of DEGs up‐regulated (red) or down‐regulated (blue) in atherosclerotic aortas compared to normal aortas from datasets GSE94044 and GSE137581. FC, fold change.

  2. B, C

    Western blot analysis (B) and densitometric quantification (C) of DCLK1 in aortas of LFD and HFD‐fed ApoE−/− mice. β‐actin was used as the loading control (n = 10 biological replicates).

  3. D

    mRNA levels of DCLK1 in aortas of LFD and HFD‐fed ApoE−/− mice were determined by RT‐qPCR. The values were normalized to Rn18s (n = 10 biological replicates).

  4. E

    Representative immunofluorescence staining of CD31 (green) and DCLK1 (red) in aortic roots (scale bar = 25 μm).

  5. F

    Representative immunofluorescence staining of α‐SMA (green) and DCLK1 (red) in aortic roots (scale bar = 25 μm).

  6. G

    Representative immunofluorescence staining of F4/80 (green) and DCLK1 (red) in aortic roots (scale bar = 25 μm). White arrows indicate co‐location of F4/80 and DCLK1.

  7. H, I

    Time‐course of DCLK1 induction in response to oxLDL in mouse primary peritoneal macrophages (MPMs). MPMs were exposed to oxLDL (50 μg/mL) for indicated time. Western blot analysis (H) and densitometric quantification (I) of DCLK1 were shown. GAPDH was used as the loading control (n = 3 biological replicates).

Data information: Data were shown as mean ± SEM; *P < 0.05; ns, not significant, two‐tailed unpaired Student's t‐test. Source data are available online for this figure.
Figure 2
Figure 2. Macrophage‐specific DCLK1 deletion reduces atherosclerotic plaques in HFD‐fed ApoE−/− mice
The animal experiment using ApoE−/−DCLK1f/f and ApoE−/−DCLK1MCKO mice fed with or without HFD was described in the Materials and Methods section.
  1. A, B

    Representative en face Oil Red O staining (A) and quantification (B) of Oil Red O‐positive plaque lesion area in aortas. Plaque area was defined as percentage of total surface area of the aorta (n = 10 biological replicates).

  2. C, D

    Representative images (C) and quantification (D) of plaque lesion area in aortic arches (n = 10 biological replicates). The plaque area was quantified by the proportion of plaque area to aortic arches area.

  3. E, F

    Representative images of Oil Red O staining (E) and quantification (F) of atherosclerotic lesion in aortic roots (scale bar = 250 μm, n = 10 biological replicates). Plaque area was quantified by the proportion of plaque area to aortic root area.

  4. G, H

    Representative images of Masson's Trichrome staining (G) and quantification (H) for collagen deposition in aortic roots (scale bar = 25 μm, n = 10 biological replicates).

Data information: Data were shown as mean ± SEM; *P < 0.05; ns, not significant, two‐tailed unpaired Student's t‐test. Source data are available online for this figure.
Figure 3
Figure 3. Macrophage‐specific DCLK1 deletion alleviates aortic inflammation and inflammatory cell infiltration in atherosclerotic lesions
The animal experiment using ApoE−/−DCLK1f/f and ApoE−/−DCLK1MCKO mice fed with or without HFD was described in the Materials and Methods section.
  1. A, B

    Representative immunofluorescence staining images (A) and quantification (B) of F4/80 (green) in aortic roots. Tissues were counterstained with DAPI (blue). F4/80 area was quantified by the proportion of F4/80‐positive area to aortic root area. Scale bar = 250 μm, n = 10 biological replicates.

  2. C–F

    Representative immunohistochemistry staining images and quantification of Ly6G (C, D) and Ly6C (E, F) in aortic roots (scale bar = 25 μm, n = 10 biological replicates).

  3. G–I

    Scatter diagram (G) and quantification (H‐I) of neutrophil and monocyte in plasma measured by an automated blood cell analyzer (n = 10 biological replicates). SFL, side fluorescence; SSC, side scatter.

  4. J, K

    Protein (J) and mRNA (K) levels of inflammatory cytokines TNF‐α and IL‐6 in serum and aortas. The values of mRNA levels were normalized to Rn18s (n = 10 biological replicates).

  5. L

    mRNA levels of proinflammatory chemokines and adhesion molecules in aortas (n = 10 biological replicates). The values were normalized to Rn18s.

Data information: Data were shown as mean ± SEM; *P < 0.05; ns, not significant, two‐tailed unpaired Student's t‐test. Source data are available online for this figure.
Figure 4
Figure 4. DCLK1 deletion attenuates inflammatory response in macrophages via inhibiting NF‐κB activation
  1. A, B

    Mouse primary peritoneal macrophages (MPMs) isolated from DCLK1f/f and DCLK1MCKO mice were challenged with oxLDL (50 μg/ml) for 24 h. Protein levels of TNF‐α (A) and IL‐6 (B) were analyzed using ELISA (n = 3 biological replicates).

  2. C, D

    MPMs isolated from DCLK1f/f and DCLK1MCKO mice were challenged with oxLDL (50 μg/ml) for 6 h. mRNA levels of Tnf‐α (C) and Il‐6 (D) were determined via RT‐qPCR (n = 3 biological replicates). The values were normalized to β‐actin.

  3. E, F

    MPMs isolated from DCLK1f/f and DCLK1MCKO mice were challenged with DiI‐oxLDL (50 μg/ml) for 24 h. Fluorescence staining (E) of DiI‐oxLDL (red) in MPMs. Cells were counterstained with DAPI (blue, scale bar = 25 μm). Flow cytometry analysis (F) of DiI‐oxLDL in MPMs.

  4. G

    MPMs isolated from DCLK1f/f and DCLK1MCKO mice were challenged with oxLDL (50 μg/ml) for 6 h. Total RNA was sequenced to identify differentially expressed genes (DEGs). Volcano plot analysis of DEGs up‐regulated in DCLK1MCKO + oxLDL group compared to DCLK1f/f + oxLDL group (red) and down‐regulated in DCLK1MCKO + oxLDL group compared to DCLK1f/f + oxLDL group (blue). FC, fold change.

  5. H

    Gene‐set enrichment analysis (GSEA) of signaling pathways enriched in DCLK1f/f + oxLDL versus DCLK1MCKO + oxLDL group.

  6. I

    NF‐κB signaling pathway is enriched in GSEA of DCLK1f/f + oxLDL versus DCLK1MCKO + oxLDL group. (J‐K) MPMs isolated from DCLK1f/f and DCLK1MCKO mice were challenged with oxLDL (50 μg/ml) for 1 h.

  7. J, K

    Western blot analysis (J) and densitometric quantification (K) of p‐p65 and IκBα. GAPDH and p65 were used as loading controls (n = 3 biological replicates).

  8. L, M

    MPMs were treated as described in panel (J). Western blot analysis (L) and densitometric quantification (M) of p65 in nucleus and cytoplasm. GAPDH was used as the loading control for cytosolic fractions. Lamin B1 was used as the loading control for nuclear fractions (n = 3 biological replicates).

  9. N, O

    MPMs were treated as described in panel (J). Representative immunofluorescence staining images (N) and quantification (O) of NF‐κB p65 (red) translocating into nucleus in MPMs. Cells were counterstained with DAPI (blue). Scale bar = 25 μm, n = 3 biological replicates.

  10. P, Q

    Representative immunofluorescence staining images (P) and quantification (Q) of p‐p65 (red) in aortic roots. Tissues were counterstained with DAPI (blue). Scale bar = 100 μm, n = 10 biological replicates. p‐p65 area was quantified by the proportion of p‐p65 positive area to plaque area.

Data information: Data were shown as mean ± SEM; *P < 0.05; ns, not significant, two‐tailed unpaired Student's t‐test. Source data are available online for this figure.
Figure 5
Figure 5. DCLK1 directly interacts with IKKβ to promote phosphorylation of IKKβ at S177/181
  1. A

    Schematic illustration of quantitative proteomic screen to identify proteins binding to DCLK1.

  2. B

    293T cells were transfected with Flag‐DCLK1 plasmid for 24 h. Control cells were transfected with empty vector (EV). Levels of Flag were measured by Western blot (n = 3 biological replicates).

  3. C

    MS/MS spectrum of the peptide showing DLKPENIVLQQGEQR from DCLK1.

  4. D

    Co‐immunoprecipitation of DCLK1 and IKKβ in 293T cells transfected with Flag‐DCLK1. Flag‐DCLK1 was immunoprecipitated by anti‐Flag antibody. IgG, immunoglobulin G.

  5. E

    Co‐immunoprecipitation of DCLK1 and IKKβ in MPMs challenged with oxLDL (50 μg/ml) for 1 h. DCLK1 was immunoprecipitated by anti‐DCLK1 antibody.

  6. F

    MPMs isolated from DCLK1f/f and DCLK1MCKO mice were challenged with oxLDL (50 μg/ml) for 1 h. Western blot analysis and densitometric quantification of p‐IKKβ. GAPDH and IKKβ were used as loading controls (n = 3 biological replicates).

  7. G

    293T cells were transfected with IKKβ siRNA (si‐IKKβ) for 24 h, while control cells were transfected with negative control (NC) siRNA. Levels of IKKβ protein were measured by Western blot (n = 3 biological replicates).

  8. H, I

    293T cells were co‐transfected with Flag‐DCLK1 and si‐IKKβ for 24 h. Western blot analysis (H) and densitometric quantification (I) of IκBα, p‐IKKβ and p‐p65. GAPDH, IKKβ and p65 were used as loading controls (n = 3 biological replicates).

  9. J

    293T cells were co‐transfected with Flag‐DCLK1 and si‐IKKβ for 48 h. Protein levels of TNF‐α and IL‐6 were analyzed using ELISA (n = 3 biological replicates).

Data information: Data were shown as mean ± SEM; *P < 0.05; ns, not significant, two‐tailed unpaired Student's t‐test. Source data are available online for this figure.
Figure 6
Figure 6. Pharmacological inhibitor of DCLK1 mitigates NF‐κB and inflammatory response in macrophages
  1. A

    The chemical structure of DCLK1‐IN‐1.

  2. B

    MPMs were pretreated with DCLK1‐IN‐1 (5 and 10 μM) or vehicle (DMSO, 1‰) for 1 h, followed by exposure of oxLDL (50 μg/ml) for 24 h. Protein levels of TNF‐α and IL‐6 were analyzed using ELISA (n = 3 biological replicates).

  3. C

    MPMs were pretreated with DCLK1‐IN‐1 (5 and 10 μM) or vehicle (DMSO, 1‰) for 1 h, followed by exposure of oxLDL (50 μg/ml) for 6 h. mRNA levels of Tnf‐α and Il‐6 were determined via RT‐qPCR (n = 3 biological replicates). The values were normalized to β‐actin.

  4. D, E

    MPMs were pretreated with DCLK1‐IN‐1 (5 and 10 μM) or vehicle (DMSO, 1‰) for 1 h, followed by exposure of oxLDL (50 μg/ml) for 1 h. Western blot analysis (D) and densitometric quantification (E) of IκBα, p‐IKKβ and p‐p65. GAPDH, IKKβ and p65 were used as loading controls (n = 3 biological replicates).

  5. F, G

    MPMs were treated as described in panel (E). Western blot analysis (F) and densitometric quantification (G) of p65 in nucleus and cytoplasm. GAPDH was used as the loading control for cytosolic fractions. Lamin B1 was used as the loading control for nuclear fractions (n = 3 biological replicates).

  6. H, I

    MPMs were treated as described in panel (D). Representative immunofluorescence staining images (H) and quantification (I) of NF‐κB p65 (red) translocating into nucleus in MPMs. Cells were counterstained with DAPI (blue). Scale bar = 25 μm, n = 3 biological replicates. Data information: Data were shown as mean ± SEM; *P < 0.05, two‐tailed unpaired Student's t‐test.

Source data are available online for this figure.
Figure 7
Figure 7. Pharmacological inhibitor of DCLK1 prevents atherosclerotic progression and inflammation in mice
The animal experiment using HFD‐fed ApoE−/− mice treated with or without DCLK1‐IN‐1 was described in the Materials and Methods section.
  1. A, B

    Representative en face Oil Red O staining (A) and quantification (B) of Oil Red O‐positive plaque lesion area in aortas. Plaque area was defined as percentage of total surface area of the aorta (n = 10 biological replicates).

  2. C, D

    Representative images (C) and quantification (D) of plaque lesion area in aortic arches (n = 10 biological replicates). The plaque area was quantified by the proportion of plaque area to aortic arches area.

  3. E

    Representative images of Oil Red O staining of atherosclerotic lesion in aortic roots (scale bar = 250 μm).

  4. F

    Representative images of Masson's Trichrome staining for collagen deposition in aortic roots (scale bar = 25 μm).

  5. G

    Representative immunofluorescence staining images of F4/80 (green) in aortic roots. Tissues were counterstained with DAPI (blue). Scale bar = 250 μm.

  6. H, I

    Representative immunohistochemistry staining images of Ly6G (H) and Ly6C (I) in aortic roots (scale bar = 25 μm).

  7. J

    Representative immunofluorescence staining images of p‐p65 (red) in aortic roots. Tissues were counterstained with DAPI (blue). Scale bar = 100 μm.

  8. K–N

    Protein (K, L) and mRNA (M, N) levels of inflammatory cytokines TNF‐α and IL‐6 in serum and aortas. The values of mRNA levels were normalized to Rn18s (n = 10 biological replicates).

Data information: Data were shown as mean ± SEM; *P < 0.05, two‐tailed unpaired Student's t‐test. Source data are available online for this figure.
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