Sectm1a deficiency aggravates inflammation-triggered cardiac dysfunction through disruption of LXRα signalling in macrophages

Abstract

Aims: Cardiac dysfunction is a prevalent comorbidity of disrupted inflammatory homeostasis observed in conditions such as sepsis (acute) or obesity (chronic). Secreted and transmembrane protein 1a (Sectm1a) has previously been implicated to regulate inflammatory responses, yet its role in inflammation-associated cardiac dysfunction is virtually unknown.

Methods and results: Using the CRISPR/Cas9 system, we generated a global Sectm1a-knockout (KO) mouse model and observed significantly increased mortality and cardiac injury after lipopolysaccharide (LPS) injection, when compared with wild-type (WT) control. Further analysis revealed significantly increased accumulation of inflammatory macrophages in hearts of LPS-treated KO mice. Accordingly, ablation of Sectm1a remarkably increased inflammatory cytokines levels both in vitro [from bone marrow-derived macrophages (BMDMs)] and in vivo (in serum and myocardium) after LPS challenge. RNA-sequencing results and bioinformatics analyses showed that the most significantly down-regulated genes in KO-BMDMs were modulated by LXRα, a nuclear receptor with robust anti-inflammatory activity in macrophages. Indeed, we identified that the nuclear translocation of LXRα was disrupted in KO-BMDMs when treated with GW3965 (LXR agonist), resulting in higher levels of inflammatory cytokines, compared to GW3965-treated WT-cells. Furthermore, using chronic inflammation model of high-fat diet (HFD) feeding, we observed that infiltration of inflammatory monocytes/macrophages into KO-hearts were greatly increased and accordingly, worsened cardiac function, compared to WT-HFD controls.

Conclusion: This study defines Sectm1a as a new regulator of inflammatory-induced cardiac dysfunction through modulation of LXRα signalling in macrophages. Our data suggest that augmenting Sectm1a activity may be a potential therapeutic approach to resolve inflammation and associated cardiac dysfunction.

Keywords: Cardiac function; Cardiac inflammation; Inflammation; LXR; Macrophage.

Graphical abstract

Figure 1

Kinetics of LPS-stimulated gene expression of Sectm1a. (A and B) Gene expression of Sectm1a was measured in WT BMDMs treated with indicated doses of LPS (A) for 24 h or with LPS (10 ng/mL) for indicated time points (B) (n = 3). (C and D) WT mice were i.p. injected with LPS (10 mg/kg of BW), Sectm1a mRNA levels in whole blood (C) and spleen (D) were determined at various time points (n = 3–5) (* and ^# P < 0.05; data are presented as mean ± SEM; Student’s t-test).

Figure 2

Sectm1a deficiency aggravates LPS-induced systemic inflammation and mortality. (A) Products of qRT-PCR were run on agarose gel to validate that Sectm1a KO model was successfully generated. (B) WT and Sectm1a KO mice injected i.p. with LPS (10 mg/kg) were monitored for survival up to 72 h post-treatment. n = 10–20. (C–E) Plasma cytokine levels (C: IL-6; D: TNFα; E: IL-1β) of WT and Sectm1a KO mice were measured with ELISA 12 h after LPS injection (number of serum samples: n = 9–12 for C; n = 7 for D; n = 6–10 for E) (*P < 0.05; data are presented as mean ± SEM; Student’s t-test).

Figure 3

Sectm1a deficiency enhances cardiac inflammation and dysfunction. 12 h after injecting mice with LPS (10 mg/kg) (A and B) cardiac function was determined by echocardiography (n = 5–9). (C–J) Representative flow cytometry plots and quantification of cardiac macrophage marker expression showed more macrophage accumulation (F4/80+) with inflammatory phenotype (CCR2+, MHC-II+, CD206-) in the heart of KO mice 12 h after LPS injection (n = 4). Gating strategy is shown in Supplementary material online, Figure S3A. (K–M) cytokine levels in the myocardium were measured by ELISA (n = 3–4) (*P < 0.05; data are presented as mean ± SEM; Student’s t-test).

Figure 4

Lack of Sectm1a augments LPS-induced inflammation via skewing BMDMs towards pro-inflammatory phenotype. BMDMs were isolated from WT and Sectm1a KO mice allowed to differentiate for 7 days (A) representative images of mature BMDMs and flow cytometry analyses showed no differences on cell morphology and differentiation. (B–E) After treating BMDMs with LPS (10 ng/mL), cytokine levels: TNFα (B), IL-1β (C), IL-6 (D), and MCP-1 (E) from cell culture supernatant were measured using ELISA at 12- and 24-h time points (n = 4–6). (F) Representative flow cytometry plots and quantification of macrophage marker expression revealed stronger inflammatory phenotype, as evidenced by increased CD38+ and lowered CD206+ expression, in KO BMDMs 6 h after LPS treatment (n = 3 dishes of BMDMs, each from separate mouse). (G) Western blotting of phosphorylated p65 and IkBα in BMDMs with or without LPS stimulation (10 ng/mL, 30 min.) (n = 3 dishes of BMDMs for isolation of proteins, each dish from separate mouse) (Scale bar, 200 µm; *P < 0.05; ^#P < 0.05 when comparing WT-LPS to KO-LPS; data are presented as mean ± SEM; Student’s t-test).

Figure 5

Gene expression profile in Sectm1a KO BMDMs determined by high-throughput RNA-seq. (A and B) Heatmap (A) and volcano plot (B) of the overall gene expression alteration in BMDMs isolated from WT and Sectm1a KO mice (n = 3 per genotype). (C) Heatmap showing the top 20 most significantly down-regulated genes in Sectm1a KO BMDMs. (D and E) Volcano plot (D) and heat-map (E) of all LXR-related genes that were differentially expressed in Sectm1a KO BMDMs. (F) expression of most common LXR-target genes in Sect1ma KO BMDMs were validated using qRT-PCR. n = 3 for each genotype (*P < 0.05; data are presented as mean ± SEM; Student’s t-test).

Figure 6

LXR agonist fails to rescue LPS-induced inflammation and cardiac dysfunction in Sectm1a KO model. (A) Gene expression of LXRα and target genes in WT and Sectm1a-KO BMDMs after 3 h of LPS (10 ng/mL) treatment was measured using qRT-PCR (n = 3). (B) Graphic scheme of treatment protocol. BMDMs from WT and Sectm1a KO mice were first treated with LXR agonist, GW3965 (2 µm, 12 h) followed by LPS stimulation (10 mg/mL, up to 48 h). (C) Immunofluorescent staining of BMDMs with LXRα after 12 h GW3965 and 30 min LPS treatment. DNA was stained with DAPI (blue). (D–G) After treating BMDMs with GW3965 for 12 h, cytokine levels in cell culture supernatant was determined by ELISA at indicated time points post-LPS treatment (n = 4–5). (H and I) WT and Sectm1a KO mice received three injection of GW3965 (30 mg/kg of BW, once daily, DMSO used as control), 6 h after last GW3965 injection, all mice received LPS injection (10 mg/kg) and underwent echocardiography measurement to assess cardiac function (n = 4–7) (Scale bar, 10 µm; *P < 0.05 when comparing WT-DMSO to WT-GW, ^#P < 0.05 when comparing WT-GW to KO-GW; data are presented as mean ± SEM; two-way ANOVA).

Figure 7

Lack of Sectm1a promotes HFD-induced cardiac inflammation and dysfunction. (A) WT BMDMs were treated with indicated doses of palmitate (A) for 24 h and (B) RAW264.7 macrophages were treated with 0.5 mM palmitate for indicated time points, then gene expression of Sectm1a was measured with qRT-PCR (n = 3). (C) WT and Sectm1a KO BMDMs were treated with palmitate (0.5 mM, 24 h), and cytokine levels in cell culture supernatant were measured using ELISA (n = 7–8). (D and E) Cardiac function was determined by echocardiography after WT and Sectm1a KO mice were fed with HFD for 20 weeks (n = 10 per group). (F–M) Representative flow cytometry plots and quantification of cardiac macrophage marker expression showed more monocytes (Ly6C+) and macrophage accumulation (F4/80+) with inflammatory phenotype (CCR2+, CD301-) in the heart of KO mice 5 weeks after HFD feeding (n = 6). FS, fractional shortening; LVID;d, left ventricular internal diameter at diastole; LVID;s, left ventricular internal diameter at systole (*P < 0.05; data are presented as mean ± SEM; Student’s t-test).