Neurogenic differentiation 2 promotes inflammatory activation of macrophages in doxorubicin-induced myocarditis via regulating protein kinase D

Abstract

Background: Although it has been established that protein kinase D (PKD) plays a crucial role in various diseases, its precise role in myocarditis remains elusive.

Methods: To investigate PKD's involvement in myocarditis, we established a mouse model of myocarditis using doxorubicin (DOX) to assess cardiac function, observe pathological changes, and quantify inflammatory cytokine levels in heart tissues. Additionally, macrophages were isolated from heart tissues of both control and DOX-treated groups to assess PKD expression and inflammatory cytokines in these macrophages. We explored the molecular mechanism of Neurogenic Differentiation 2 (NeuroD2) in myocarditis by utilizing NeuroD2 overexpression plasmids and NeuroD2 small interfering RNA (siRNA). Furthermore, we conducted dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays to investigate the interaction between NeuroD2 and PKD.

Results: We observed significant upregulation of PKD in macrophages and heart tissues induced by DOX. The administration of a PKD inhibitor reduced inflammatory cytokine levels, improved cardiac function, and mitigated pathological changes in myocarditis-afflicted mice. Mechanistically, we found upregulated expression of NeuroD2 in both macrophages and heart tissues exposed to DOX. NeuroD2 could directly target PKD, enhancing the NLRP3/NF-κB signaling pathway and exacerbating macrophage inflammation.

Conclusions: Our study demonstrates that NeuroD2 can directly bind to the PKD promoter, potentially promoting inflammatory activation of macrophages in DOX-induced myocarditis via the NLRP3/NF-κB pathway. This suggests that the NeuroD2/PKD axis may hold promise as a potential therapeutic approach for treating DOX-induced myocarditis.

Keywords: Inflammatory activation; Macrophages; Myocarditis; NLRP3/NF-κB pathway; NeuroD2; PKD.

Fig. 1

Protein kinase D is highly expressed in the heart tissues of mice induced by doxorubicin. (A-E) On day 21 of doxorubicin (DOX) injection, echocardiography was conducted to detect the cardiac function of mice. LVEDd (B), LVESd (C), LVFS (D), and LVEF (E) were measured. (F) Hematoxylin and Eosin (H&E) staining of pathological changes in heart tissues in each group. (G) Mice heart tissue were subjected to immunofluorescence staining for macrophage marker CD68. (H-I) The mRNA expressions and cytokine levels of TNF-α, IL-6, IL-18, and IL-1β in heart tissues were measured by quantitative real-time reverse transcription polymerase chain reaction (qPCR) and Enzyme-linked immunosorbent assay (ELISA), respectively. (J) PKD, phosphorylated p65 (p-p65), p65, and NLRP3 expression in macrophages from mice heart tissues was examined using qRT-PCR analysis and western blotting, respectively. Values were presented as the mean ± SD of triplicate experiments. Compared with the control group, *P < 0.05, **P < 0. 01, ***P < 0. 001

Fig. 2

Protein kinase D inhibitor CID755673 improves inflammation and cardiac function in mice with myocarditis. (A-B) Mice with myocarditis treated with or without PKD inhibitor CID755673, the expression of PKD was detected by qPCR analysis and western blotting. (C-G) The cardiac function (C), LVEDd (D), LVESd (E), LVFS (F), and LVEF (G) were explored by echocardiography. (H) H&E staining for inflammatory cell infiltration in each group. (I) Immunofluorescence staining for the macrophage marker CD68 was performed on mouse heart tissue. (J-K) The levels of TNF-α, IL-6, IL-18, and IL-1β in mice heart tissues were measured by qPCR (J) and ELISA (K). (L) The protein levels of NLRP3, p65, and p-p65 in different groups. (M) The relative expression of NLRP3 was measured by qPCR analysis. Values were presented as the mean ± SD of triplicate experiments. Compared with DOX group, *P < 0.05, **P < 0. 01, ***P < 0. 001

Fig. 3

Protein kinase D is involved in the DOX-induced inflammatory activation of macrophages. Macrophages were isolated from the heart tissues of mice in different groups. (A) The purity of macrophages was determined by a FACSAria II flow cytometer. (B-C) The mRNA expressions and protein levels of PKD were detected using qPCR analysis and western blotting. (D-G) qPCR and ELISA assay were carried out to measure the levels of TNF-α, IL-6, IL-18, and IL-1β in macrophages. (H) The protein levels of NLRP3, p-p65, and 65 in each group. (I) The expression of NLRP3 was analyzed in macrophage using qPCR analysis. Values were presented as the mean ± SD of triplicate experiments. Compared with the control group, *P < 0.05, **P < 0. 01, ***P < 0. 001; Compared with DOX + DMSO group, ^#P < 0.05, ^##P < 0. 01, ^###P < 0. 001

Fig. 4

Functional interaction between Neurogenic Differentiation 2 and protein kinase D. Macrophages were isolated from normal tissues and treated with 1µmol/L DOX for 12 h. (A) The binding site of PKD and Neurod2 was predicted by the JASPAR database. (B-C) qPCR analysis and western blotting were performed to detect the expression of Neurod2 in macrophages and tissues induced by DOX. (D-E) Macrophages isolated from the heart of normal mice were co-transfected with Neurod2 overexpression plasmid, and siNeurod2. qPCR and western blotting were performed to test the expression of Neurod2 mRNA and protein, respectively. (F-G) The levels of PKD in each group were explored by qPCR and western blotting. (H-I) The binding ability between PKD and Neurod2 was confirmed by dual-luciferase reporter assay (H) and CHIP assay (I). Values were presented as the mean ± SD of triplicate experiments. Compared with the control group or si-NC group, *P < 0.05, **P < 0. 01, ***P < 0. 001; Compared with the pc-NC group, ^#P < 0.05, ^##P < 0. 01, ^###P < 0. 001

Fig. 5

The overexpression of Protein kinase D can reverse the impact of neurogenic differentiation 2 on doxorubicin-induced inflammatory activation of macrophages. Macrophages isolated from normal tissue and treated with 1µmol/L DOX for 12 h. (A-B) Macrophages were co-transfected with PKD overexpression plasmid and si-Neurod2. qPCR was used to test the mRNA expressions of PKD and NLRP3. (C) The protein levels of PKD, p-p65, p65, and NLRP3 were detected. (D-G) The levels of TNF-α, IL-6, IL-18, and IL-1β in each group were measured by qPCR and ELISA. Values were presented as the mean ± SD of triplicate experiments. Compared with the DOX group, *P < 0.05, **P < 0. 01, ***P < 0. 001; Compared with DOX + pc-PKD group, ^#P < 0.05, ^##P < 0. 01, ^###P < 0. 001

Fig. 6

PKD-mediated macrophage inflammasome activation in heart tissue. The combination of NeuroD2 and PKD promoter could enhance NLRP3/NF-κB signaling,