Increased pro-SFTPB in HDL promotes the pro-inflammatory transition of HDL and represents a sign of poor prognosis in ARDS patients

Abstract

Background: Acute respiratory distress syndrome (ARDS) is causatively associated with excessive alveolar inflammation involving deregulated pro-inflammatory macrophage polarization. High-density lipoprotein (HDL) showed critical anti-inflammatory roles by modulating macrophage function, and its adverse transition to pro-inflammation has an important role in the pathogenesis of ARDS. However, the relationship between HDL protein constituents and functional remodeling is unknown in ARDS.

Methods: Proteomic techniques were applied to examine the protein profile changes in HDL from septic-ARDS patients versus HDL from healthy controls across two distinct cohorts: a discovery cohort (8 patients and 8 healthy controls) and a validation cohort (22 patients and 10 healthy controls). The changed components significantly associated with prognosis were identified. Luminex assessed the levels of 38 plasma cytokines and chemokines. The in vitro constructed pro-SFTPB enriched HDL was applied to confirm the effect on M1 polarization of THP1-derived macrophage.

Results: 18 proteins were validated from 102 changed HDL proteins identified in the discovery cohort, including HDL particle components, such as apolipoproteins, pro-inflammatory substances known as serum amyloid As (SAAs), and anti-oxidative proteins like paraoxonases (PONs). Among these proteins, only the increase of pro-SFTPB in HDL was significantly associated with poor prognosis of ARDS patients. Notably, HDL-pro-SFTPB level was correlated with plasma pro-inflammatory cytokines and chemokines levels. The correlation assay of pro-SFTPB with other HDL components showed that it was positively and negatively correlated with SAA2 and PON3, respectively. Furthermore, the in vitro studies confirmed that the pro-SFTPB enriched HDL significantly promoted M1 polarization of monocyte-derived macrophages.

Conclusions: The increase of HDL-pro-SFTPB promotes HDL pro-inflammatory transition during septic ARDS, leading to exacerbated progression of ARDS through enhancing M1 macrophage polarization. HDL-pro-SFTPB could be a useful prognostic biomarker for septic ARDS.

Keywords: ARDS; HDL proteomics; Macrophage; Pro-SFTPB; Sepsis.

Fig. 1

The HDL from ARDS patients indicated an adverse functional transition. A The A-HDL treatment (50 μg/ml, 12 h) induced M1 polarization of THP1-derived macrophage by qPCR assay of M1/M2-associated markers (n = 4 per group). *p < 0.05; M1 marker: TNF-α, IL-1β and IL-8; M2 marker: MRC-1. B The study design encompassed a series of experiments including proteomic analysis on the discovery cohort, targeting protein MS quantification on the validation cohort, and subsequent in vitro confirmation. ARDS, acute respiratory distress syndrome; HDL, high-density lipoprotein; N-HDL, HDL from normal subjects; A-HDL, HDL from ARDS patients; TNF, tumor necrosis factor; IL-, interleukin-; MRC-1, mannose receptor C-type 1; HC, healthy controls; DIA-MS, data-independent acquisition mass spectrometry

Fig. 2

The pro-inflammatory HDL from ARDS patients showed marked changes in protein profile by proteomic analysis. The plasma samples from 8 ARDS patients and 8 healthy controls were collected. The plasma mixture from 2 subjects with similar ages and clinical situations was used for the isolation procedure to improve the quality. A The DEPs between N-HDL and A-HDL by DIA-MS proteome analysis. There was significant specificity between N-HDL and A-HDL by PCA. B, C The volcano plot and heatmap showed that a total of 384 proteins were identified with 53 upregulated and 49 downregulated proteins between A-HDL and N-HDL. The blue and red-colored proteins denoted the 18 HDL proteins with the most significant fold-changes. D The Gene Ontology analysis on these DEPs and the most affected processes were presented. ARDS, acute respiratory distress syndrome; HDL, high-density lipoprotein; N-HDL, HDL from normal subjects; A-HDL, HDL from ARDS patients; DEPs, differentially expressed proteins; DIA-MS, data-independent acquisition mass spectrometry; PCA, principal component analysis; SFTPB, pulmonary surfactant-associated protein B; SAA, serum amyloid A; Apo, apolipoprotein; HPR, haptoglobin-related protein; PON, paraoxonase; SERPIN, serpin family A member; IGFBP3, insulin-like growth factor-binding protein 3; LBP, lipopolysaccharide-binding protein; LPA, apolipoprotein(a); SA100A8, S100 calcium-binding protein A8; LGALS3BP, lectin, galactoside-binding, soluble 3 binding protein; ITIH4, inter-alpha-trypsin inhibitor 4

Fig. 3

The altered HDL proteins identified in the discovery cohort were confirmed in the validation cohort. A Flowchart of ARDS patient enrollment along with healthy controls. B The significantly changed HDL constituent proteins in A-HDL versus N-HDL in the validation cohort. ARDS, acute respiratory distress syndrome; HDL, high-density lipoprotein; N-HDL, HDL from normal subjects; A-HDL, HDL from ARDS patients; pro-SFTPB, pro-pulmonary surfactant-associated protein B; Apo, apolipoprotein; SAA, serum amyloid A; PON, paraoxonase; LBP, lipopolysaccharide binding protein; SERPINA5, serpin family A member 5; LGALS3BP, lectin galactoside-binding soluble 3 binding protein; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 4

Pro-SFTPB in HDL is associated with the prognosis of ARDS patients. A Only pro-SFTPB in HDL showed a significant difference between survivors (n = 12) and non-survivors (n = 10). B ROC curves for pro-SFTPB in HDL and SOFA score in prediction of the 28-day mortality in ARDS patients. ARDS, acute respiratory distress syndrome; HDL, high density lipoprotein; Pro-SFTPB, pro-pulmonary surfactant-associated protein B; Apo, apolipoprotein; SAA, serum amyloid A; PON, paraoxonase; LBP, lipopolysaccharide binding protein; SERPINA5, serpin family A member 5; LGALS3BP, lectin galactoside-binding soluble 3 binding protein; ROC, receiver operating characteristic; AUC, area under the curve; CI, confidence interval; SOFA, sequential organ failure assessment; *p < 0.05

Fig. 5

Scatter diagram for the relationship between pro-SFTPB and SAA2 and PON3 in HDL. pro-SFTPB, pro-pulmonary surfactant-associated protein B; SAA2, serum amyloid A-2 protein; PON3, serum paraoxonase 3; HDL, high density lipoprotein

Fig. 6

Pro-SFTPB enriched HDL induced M1 macrophage polarization. A Schematic diagram of HDL-pro-SFTPB (PS-HDL) synthesis. B Immunoblotting of HDL-pro-SFTPB showed that HDL successfully carried pro-SFTPB. C The PS-HDL treatment (100 μg/ml, 12 h) induced M1 polarization of THP-1 derived macrophage by qPCR assay of M1/M2-associated markers. (n = 5 per group). D The phagocytic capability of THP-1 derived macrophages by quantifying the uptake of labeled E. coli particles after HDL and PS-HDL treatments (100 μg/ml, 12 h), the percentage of particle-positive cells was presented as bar figure (n = 4 per group). E intracellular ROS release in THP-1 derived macrophages 30 min after HDL exposure was quantified via the changes in DCF fluorescence (n = 5 per group). HDL, high density lipoprotein; Pro-SFTPB, pro-pulmonary surfactant-associated protein B; C-H: control HDL; PS-HDL, Pro-SFTPB enriched HDL; TNF, tumor necrosis factor; IL-, interleukin-; MRC-1, mannose receptor C-type 1; ROS, reactive oxygen species; **p < 0.01, ***p < 0.001, ****p < 0.0001