SENP3 facilitates M1 macrophage polarization via the HIF-1α/PKM2 axis in lipopolysaccharide-induced acute lung injury

Abstract

M1/M2 macrophage polarization plays a pivotal role in the development of acute lung injury (ALI). The hypoxia-inducible factor-1α/pyruvate kinase M2 (HIF-1α/PKM2) axis, which functions upstream of macrophage polarization, has been implicated in this process. The function of HIF-1α is known to be tightly regulated by SUMOylation. Upregulation of SUMO-specific peptidase 3 (SENP3), a deSUMOylation enzyme, is induced by reactive oxygen species (ROS), which are abundantly produced during ALI. To explore the links between SENP3, macrophage polarization, and lung injury, we used mice with Senp3 conditional knockout in myeloid cells. In the lipopolysaccharide (LPS)-induced ALI model, we found that in vitro and in vivo SENP3 deficiency markedly inhibited M1 polarization and production of pro-inflammatory cytokines and alleviated lung injury. Further, we demonstrated that SENP3 deficiency suppressed the LPS-induced inflammatory response through PKM2 in a HIF-1α-dependent manner. Moreover, mice injected with LPS after PKM2 inhibitor (shikonin) treatment displayed inhibition of M1 macrophage polarization and reduced lung injury. In summary, this work revealed that SENP3 promotes M1 macrophage polarization and production of proinflammatory cytokines via the HIF-1α/PKM2 axis, contributing to lung injury; thus, SENP3 may represent a potential therapeutic target for ALI treatment.

Keywords: HIF-1α; Macrophage; PKM2; SENP3; SUMOylation; acute lung injury.

Figure 1

SENP3 deficiency in macrophages alleviated LPS-induced acute lung injury and lung inflammation. Notes: Senp3 fl/fl (n = 12) and Senp3 cKO mice (n = 12) were intratracheally injected with LPS (5 mg/kg) for 24 h. (A) Hematoxylin and eosin staining. (B) Lung injury score (n = 6). (C) Wet-to-dry ratio assessment (n = 6). BALF (D) IL-6, (E) TNF-α, (F) IL-10, and (G) IL-4 levels were assessed using ELISA (n = 6). The graphs show the means ± SDs, and data shown in B–G are representative of three independent experiments. Values of p < 0.05 were considered statistically significant, and data marked with a one (*), two (**) and three (***) asterisks indicate p values of <0.05, < 0.01 and <0.001, respectively. SENP3, SUMO-specific peptidase 3; LPS, lipopolysaccharide; BALF, bronchoalveolar lavage fluid; IL, interleukin; TNF-α, tumor necrosis factor α.

Figure 2

SENP3 facilitates alveolar macrophage M1 polarization and expression of pro-inflammatory genes. Notes: Senp3 fl/fl (n = 6) and Senp3 cKO mice (n = 6) were intratracheally injected with LPS (5 mg/kg) and BALF macrophages were assessed using flow cytometry (A-G). (A) Gate strategy for alveolar macrophages in BALF. (B) CD11b (+) F4/80 (+) cells of the total BALF cells. (C) Frequency of CD11b (+) F4/80 (+) cells. (D) Gated cells were analyzed for CD80 and CD206 expression. Upper left quadrants: CD80 (-) CD206 (+) cells (M2 phenotype); lower right quadrants: CD80(+) CD206 (-) cells (M1 phenotype); upper right quadrants: CD80 (+) CD206 (+) cells (mixed M1/M2 phenotype); lower left quadrants: control. (E) Percentage of CD80 (+) CD206 (-) cells out of all CD11b (+) F4/80 (+) cells. (F) Percentage of CD80 (+) CD206 (+) cells out of all CD11b (+) F4/80 (+) cells. BMDMs were isolated from SENP3 fl/fl (n = 6 per group) and SENP3 cKO mice (n = 6 per group) and stimulated with LPS (100 ng/mL) for 24 h, and the transcription levels of (G) iNOs, (H) IL-6, (I) TNF-α, and (J) IL-10 were measured using qRT-PCR (n = 3 per group). The graphs show the means ± SDs, and the data shown in A–J are representative of three independent experiments. Values of p < 0.05 were considered statistically significant, and data marked with a one (*), two (**) and three (***) asterisks indicate p values of <0.05, < 0.01 and <0.001, respectively. SENP3, SUMO-specific peptidase 3; LPS, lipopolysaccharide; BALF, bronchoalveolar lavage fluid; IL, interleukin; TNFα, tumor necrosis factor α; iNOS, inducible nitric oxide synthase.

Figure 3

SENP3 inhibited PKM2 transcription in a HIF-1α-dependent manner and the PKM2 inhibitor shikonin dampened the LPS-induced macrophage inflammatory response. Notes: BMDMs were isolated from Senp3 fl/fl (n = 6) and Senp3 cKO mice (n = 6) and stimulated with LPS (100 ng/mL) for the indicated time. (A) The mRNA expression of key glycolytic enzymes was analyzed using qRT-PCR. (B) SENP3, HIF-1α, and PKM2 levels were determined using immunoblotting at the indicated time points. (C) Effects of the HIF-1α inhibitor PX-478 and HIF-1α agonist FG-4592 on the PKM2 levels of BMDMs from Senp3 fl/fl and Senp3 cKO mice. (D, E) Cells were treated with the PKM2 inhibitor shikonin, after which the transcription levels of IL-6 and TNF-α were measured using qRT-PCR. The graphs show the means ± SDs, and the data shown in A–E are representative of three independent experiments. Values of p < 0.05 were considered statistically significant, and data marked with a one (*), two (**) and three (***) asterisks indicate p values of <0.05, < 0.01 and <0.001, respectively. SENP3, SUMO-specific peptidase 3; LPS, lipopolysaccharide; IL, interleukin; TNF-α, tumor necrosis factor α; HIF-1α, hypoxia-inducible factor-1α; BMDMs, bone marrow-derived macrophages; PKM2, pyruvate kinase M2.

Figure 4

The PKM2 inhibitor shikonin alleviated the LPS-induced macrophage inflammation and lung injury. Notes: (A–F) Male C57BL/6 mice were administered shikonin (8 mg/kg) or PBS intraperitoneally every 8 h with a single dose of LPS (5 mg/kg) or PBS at 6 h after the first treatment (n = 6 per group). (A) Hematoxylin and eosin staining of lung tissues of the four groups was assessed 24 h after LPS injection. (B) Lung injury score assessment (n = 6, LPS injected group; and n = 3, control group). (C-D) The levels of IL-6 and TNF-α in BALF 24 h after LPS injection, as assessed via ELISA (n = 6, LPS injected group; and n = 3, control group). (E) CD80 (+) CD206 (-) cells of F4/80 (+) cells from BALF. (F) Percentage of CD80 (+) CD206 (-) cells out of all CD11b (+) F4/80 (+) cells. The graphs show the means ± SDs, and the data shown in A–F are representative of three independent experiments. Values of p < 0.05 were considered statistically significant, and data marked with a one (*), two (**) and three (***) asterisks indicate p values of <0.05, < 0.01 and <0.001, respectively. LPS, lipopolysaccharide; IL, interleukin; TNF-α, tumor necrosis factor α; BALF, bronchoalveolar lavage fluid; PKM2, pyruvate kinase M2.