Administration of GDF3 Into Septic Mice Improves Survival via Enhancing LXRα-Mediated Macrophage Phagocytosis

Abstract

The defective eradication of invading pathogens is a major cause of death in sepsis. As professional phagocytic cells, macrophages actively engulf/kill microorganisms and play essential roles in innate immune response against pathogens. Growth differentiation factor 3 (GDF3) was previously implicated as an important modulator of inflammatory response upon acute sterile injury. In this study, administration of recombinant GDF3 protein (rGDF3) either before or after CLP surgery remarkably improved mouse survival, along with significant reductions in bacterial load, plasma pro-inflammatory cytokine levels, and organ damage. Notably, our in vitro experiments revealed that rGDF3 treatment substantially promoted macrophage phagocytosis and intracellular killing of bacteria in a dose-dependent manner. Mechanistically, RNA-seq analysis results showed that CD5L, known to be regulated by liver X receptor α (LXRα), was the most significantly upregulated gene in rGDF3-treated macrophages. Furthermore, we observed that rGDF3 could promote LXRα nuclear translocation and thereby, augmented phagocytosis activity in macrophages, which was similar as LXRα agonist GW3965 did. By contrast, pre-treating macrophages with LXRα antagonist GSK2033 abolished beneficial effects of rGDF3 in macrophages. In addition, rGDF3 treatment failed to enhance bacteria uptake and killing in LXRα-knockout (KO) macrophages. Taken together, these results uncover that GDF3 may represent a novel mediator for controlling bacterial infection.

Keywords: CD5L; LXRα; growth differentiation factor 3; macrophage; phagocytosis; sepsis.

Figure 1

Administration of rGDF3 attenuates polymicrobial sepsis-induced mortality and organ injury in mice. (A) Graphic illustration of experimental design: WT mice were received rGDF3 (20 μg/kg, or BSA as control) through the tail-vein injection, followed by CLP surgery. (B) Kaplan–Meier survival curves were generated to compare mortality between 2 groups, significance was determined by log-rank (Mantel-Cox) test (*p < 0.05; n = 20 per group). (C,D) Representative images of lung sections with hematoxylin and eosin (H&E) staining from both (C) BSA- and (D) rGDF3-treated mice at 24 h after CLP surgery (Scale bar, 50 μm). (E) The lung injury scores were assessed as described in section Materials and Methods (n = 5). (F,G) The wet weight to dry weight ratios of (F) lung and (G) spleen in mice treated with BSA vesicle or rGDF3 at 24 h post-CLP were quantified (n = 7–8). (H,I) Serum levels of (H) alanine aminotransferase (ALT), a biochemical marker of liver injury, and (I) creatinine (Cr), a biochemical marker of kidney injury, in both groups were measured using ELISA. Data are representative of two independent experiments. Results are presented as mean ± SEM and analyzed by Student's t-test (*p < 0.05).

Figure 2

Administration of rGDF3 decreases bacterial burden and cytokine levels in response to CLP. (A–D) The bacterial burden in both blood (A, C) and PLF (B, D) were compared between BSA- and rGDF3-treated mice at 20 h after CLP surgery. (E–H) Cytokine levels in serum [(E): IL-6, (F): TNF-α] and PLF [(G): IL-6, (H): TNF-α] in mice were measured at 20 h after CLP surgery using ELISA assays (n = 9–10 per group). CFU, colony-forming unit. Data are representative of two independent experiments. All results are presented as mean ± SEM and analyzed by Student's t-test (*p < 0.05).

Figure 3

rGDF3 treatment promotes phagocytic activity of macrophages. (A,B) After stimulating BMDMs (A) and RAW264.7 macrophages (B) with different doses of rGDF3, the phagocytic capacity was assessed by adding Red fluorescence–conjugated pHrodo E. coli BioParticles. (C–F) Representative confocal images of phagocytosis assay in BMDMs (C) and RAW 264.7 macrophages (E) with E. coli BioParticles after rGDF3 treatment (scale bar, 10 μm). The mean fluorescence intensity in BMDMs (D) and RAW 264.7 macrophages (F) was quantified. (G,H) Representative flow cytometry plot (G) and histogram (H) showing PM (F4/80+) phagocytosis of E. coli BioParticles after rGDF3 treatment. Similar results were obtained in another separated experiment. All results are shown as mean ± SEM and analyzed by Student's t-test (*p < 0.05).

Figure 4

rGDF3 treatment promotes macrophage phagocytosis and intracellular killing of live bacteria. (A) Graphic illustration of experimental design: Gentamicin protection assay was used to test the phagocytic and bactericidal activities of BMDMs and RAW 264.7 macrophages using live E. coli. After 1 h of infection, Gentamicin was added to the cell culture medium. After 30 min, cell lysate was extracted with serial dilution, then plated on LB agar plates. The CFUs were measured as an indicator for phagocytosis capacity of BMDMs (B) and RAW 264.7 cells (E). In addition, 6 h after the gentamicin was added, the CFUs isolated from BMDMs (C) and RAW 264.7 cells (F) were determined to assess the number of bacteria that remained inside macrophages. The killing percentages of BMDMs (D) and RAW264.7 macrophages (G) were calculated as described in section Materials and Methods (n = 6–9). Data are representative of three independent experiments. All results are shown as mean ± SEM and analyzed by Student's t-test (*p < 0.05).

Figure 5

Gene expression profile in BMDMs treated with rGDF3 by high-throughput RNA sequencing. (A) Heatmap of the differentially expressed genes from BMDMs treated with BSA or rGDF3 (20 ng/mL) (n = 3). (B) The altered expression of CD5L, Bcl2a1b, and MRPS18C gene in rGDF3-treated BMDMs was validated by qRT-PCR (n = 6). (C) Expression of CD5L and Bcl2a1b in GW3965 (1 μmol/L)-treated BMDMs was determined by qRT-PCR (n = 6) at 18 h post-treatment. (D) Expression of CD5L and Bcl2a1b in BMDMs were measured at 18 h post-GSK2033 (2 μmol/L) treatment (n = 6). (E) Graphic scheme of the LXRα-CD5L signal cascade in macrophages. Representative images (F) and quantification (G) of immunofluorescence staining for LXRα (green) in BMDMs at 18 h after rGDF3 (20 ng/mL) or GW3965 (1 μmol/L) treatment (Scale bar, 10 μm; n = 5). Data are representative of two (B–D,F,G) independent experiments. All results are shown as mean ± SEM and analyzed by Student's t-test (*p < 0.05).

Figure 6

Inhibition or deficiency of LXRα abolishes rGDF3-mediated effects on macrophage phagocytosis and bacterial killing activities. (A) BMDMs were treated with DMSO (0.005%, vesicle control), rGDF3, GW3965, and GSK2033+rGDF3 for 18 h, then phagocytic capacity was assessed using red E. coli BioParticles. (B) Representative confocal images of phagocytosis assay in BMDMs treated with DMSO, rGDF3, GW3965, and GSK2033 + rGDF3 with red E. coli BioParticles (Scale bar, 10 μm), and (C) their quantifications of the mean fluorescence intensity in each group. (D) Graphic scheme of experimental design for BMDMs isolated from LXRα-KO mice and following treatment. (E) The altered expression of CD5L gene in rGDF3-treated BMDMs isolated from LXRα-KO mice was validated by qRT-PCR (n = 4). (F) Phagocytic capacity of WT- and LXRα-KO-BMDMs after rGDF3 treatment was assessed by adding E. coli BioParticles. (G–I) Gentamicin protection assay was used to test the phagocytic and bactericidal activities of WT- and LXRα-KO-BMDMs. (G) The CFUs were measured as an indicator for phagocytosis capacity of BMDMs. (H) 6 h after the gentamicin was added, the bacterial residue remained inside macrophages were assessed. (I) The killing percentages of LXRα-KO BMDMs were calculated (n = 6). Similar results were obtained in other two independent experiments. All results are shown as mean ± SEM and analyzed by Student's t-test (*p < 0.05).

Figure 7

Therapeutic effects of rGDF3 in WT mice upon CLP surgery. A single dose of rGDF3 (100 μg/kg) or BSA vehicle was injected at 1 h post-CLP. (A) The bacterial burden in blood was compared between BSA- and rGDF3-treated mice at 24 h after CLP surgery. (B,C) Cytokine levels in sera [(B): IL-6, (C): TNF-α] of BSA- and rGDF3-treated mice were measured at 24 h post-CLP surgery using ELISA kits (n = 6). (D) The bacterial burden in PLF was compared between BSA- and rGDF3-treated mice at 24 h post-CLP surgery. (E,F) Cytokine levels in PLF [(E): IL-6, (F): TNF-α] of BSA- and rGDF3-treated mice were measured at 24 h post-CLP surgery (n = 6). (G,H) The wet weight to dry weight ratios of lung (G) and spleen (H) in BSA- and rGDF3-treated mice at 24 h post-CLP were quantified (n = 6). Similar results were obtained in another separated experiment. All results are presented as mean ± SEM and analyzed by Student's t-test (*p < 0.05). (I) Kaplan–Meier survival curves were generated to compare mortality between two groups, significance was determined by log-rank (Mantel-Cox) test (*p < 0.05; n = 16).

Figure 8

Scheme depicting that administration of GDF3 into septic mice improves survival via enhancing macrophage phagocytosis, which is mediated by activation of the LXRα-CD5L pathway.