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. 2020 Aug 17;8(8):290.
doi: 10.3390/biomedicines8080290.

The Heat Shock Protein 27 Immune Complex Enhances Exosomal Cholesterol Efflux

Chunhua Shi  1 , Daiana Alvarez-Olmedo  1 , Yuan Zhang  1 , Badal S B Pattar  1 , Edward R O'Brien  1
Affiliations

The Heat Shock Protein 27 Immune Complex Enhances Exosomal Cholesterol Efflux

Chunhua Shi et al. Biomedicines. .

Abstract

Previously, we demonstrated that Heat Shock Protein 27 (HSP27) reduces the inflammatory stages of experimental atherogenesis, is released by macrophage (MΦ) exosomes and lowers cholesterol levels in atherosclerotic plaques. Recently, we discovered that natural autoantibodies directed against HSP27 enhance its signaling effects, as HSP27 immune complexes (IC) interact at the cell membrane to modulate signaling. We now seek to evaluate the potential role of the HSP27 IC on MΦ exosomal release and cholesterol export. First, in human blood samples, we show that healthy control subjects have 86% more exosomes compared to patients with coronary artery disease (p < 0.0001). Treating human THP-1 MΦ with rHSP27 plus a validated anti-HPS27 IgG antibody increased the abundance of exosomes in the culture media (+98%; p < 0.0001) as well as expression of Flotillin-2, a marker reflective of exosomal release. Exosome cholesterol efflux was independent of Apo-A1. THP-1 MΦ loaded with NBD-labeled cholesterol and treated with the HSP27 IC showed a 22% increase in extracellular vesicles labeled with NBD and a 95% increase in mean fluorescent intensity. In conclusion, exosomal abundance and secretion of cholesterol content increases in response to HSP27 IC treatment, which may represent an important therapeutic option for diseases characterized by cholesterol accumulation.

Keywords: Heat Shock Protein 27; antibody; exosome; immune complex.

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Conflict of interest statement

EOB is the Scientific Cofounder of Pemi31 Therapeutics Inc., a start-up company that is developing HSP27 immunotherapeutics. EOB and CS have equity interests in Pemi31 Therapeutics Inc.

Figures

Figure 1
Figure 1
Serum exosome levels in coronary artery disease (CAD) patients are diminished compared with healthy controls. Exosome abundance was assessed by ELISA and quantified by optical density using arbitrary units obtained after background subtraction (A.U.). (A) ELISA performed with anti-MHC primary (capture) antibody and anti-CD81 antibody to detect exosomes in the purified fractions (n = 4) from THP1 MΦ. Horizontal axis labels: Exosome = Purified exosome fraction; Upper = the upper fraction without exosomes after ultracentrifugation; Medium = cell culture medium (only) as negative control. (B) Detection of serum exosomes in CAD patients and healthy (control) subjects. Anti-CD81 was applied as the capture antibody and anti-MHC-II was used as the developing antibody to quantify exosomes in each sera sample. Analysis was performed via two-way ANOVA (n = 4 subjects per group, with each subject’s sample measured in triplicate). (C) Detection of exosomes in the sera of CAD patients and healthy individuals (control). Anti-CD81 was applied as the capture antibody and anti-MHC-II was used as the developing antibody to quantify exosomes in each sera sample. The data were analyzed using a Mann-Whitney test and plotted to note the exosome distribution in each population. Exosome levels were downregulated in CAD patients.
Figure 2
Figure 2
HSP27 IC Modulate Cholesterol Efflux and Expression of FLOT2. (A) MΦ exosome release was assayed in the media by ELISA. An anti-CD81 antibody was used to capture exosomes and anti-MHC-II/biotin antibody was used to detect the secondary exosomal biomarker (MHC-II). The HSP27 IC increased the release of exosomes by 98% compared to rC1 + PAb (p < 0.0001). (B) Western blot analysis of Flotillin-2 (FLOT2) expression in THP-1 MΦ after 24 h treatment with PBS, rHSP27, PAb or rHSP27 + PAb, as indicated. (C) Densitometry quantification of the FLOT2/β-actin. FLOT2 expression was enhanced by 1028% after treatment with the HSP27 IC (only). Each band was measured with ImageJ, and the data set was analyzed via one-way ANOVA. The bars represent the means of three independent experiments ± SEM (p < 0.0001).
Figure 3
Figure 3
Cholesterol can be Exported in Exosomes Independent of Apo-A1. (A) Analysis of NBD cholesterol in exosome fraction by fast protein liquid chromatography (FPLC) and ELISA. The exosome fraction was loaded into the FPLC and the signal of NBD cholesterol was recorded by its fluorescent signaling (485 nm/535 nm). CD81 was measured with the ELISA method. This involved each fraction being coated on an ELISA plate and the anti-CD81 monoclonal antibody was used to detect its concentration (O.D. 450 nm). (B) NBD cholesterol was transported outside the cell in both the HDL and exosome fractions, but did not require Apo-A1, thereby indicating that both pathways are used for cholesterol efflux. HDL alone and NBD cholesterol in the media were used as the controls. Protein standards were loaded to indicate each fraction’s molecular weight. 2000 kDa = 20 nm diameter. (C) Colocalization of NBD cholesterol (FITC signal) and CD81 (PE-Texas Red-A), using flow cytometry in purified exosome samples derived from THP-1 MΦ. The exosome fractions were then scanned by flow cytometry detecting NBD cholesterol (FITC). The experiments were performed in triplicate. Representative graph of the EVs percentage immunolabeled for: (i) background control (0.0%), (ii)) NBD cholesterol (1.1%) and (iii) CD81 (28.0%). NBD cholesterol and the exosome biomarker CD81 colocalized by both FPLC separation and flow cytometry techniques. The results confirm that cholesterol can be transported out of the cell, through exosomal pathways, in the presence and absence of HDL and Apo-A1.
Figure 4
Figure 4
HSP27 IC Modulates Cholesterol Efflux Via Exosomes. (A) Effects of rHSP27 alone or the HSP27 IC on cholesterol efflux in THP-1 MΦ. NBD cholesterol assayed in the media. The HSP27 IC increased NBD cholesterol efflux to the medium by 16% after 24 h of incubation (p < 0.0001). (B) Fluorescence-activated cell sorting (FACS) analysis of NBD cholesterol secretion through extracellular vesicles from THP-1 MΦ. The medium was centrifuged for 30 min at 10,000g × and the supernatant was used for FACS. The exosome fractions were then scanned by flow cytometry to the NBD cholesterol signal (FITC). The graphs are representative of 3 separate experiments: (i) NC = negative control (0.23 +/− 0.06%), (ii) rHSP27 (33.5 +/− 1.5%), (iii) rC1 (33.6 +/− 0.4%), (iv) PBS (34.3 +/− 1.5%), (v) rHSP27 + PAb (57.6 +/− 0.7%) and rC1 + PAb (36.0 +/− 1.2%). (C) Statistical analysis of the percentage of extracellular vesicles positive for NBD cholesterol. The HSP27 IC increased the number of NBD cholesterol positive extracellular vesicles by 22% (p < 0.0001), in comparison to the control treatment [rC1 + PAb]. (D) Statistical analysis of mean fluorescente intensity (MFI; measured as NBD cholesterol). Treatment with the HSP27 IC resulted in a 95% increase in the amount of NBD cholesterol transported by the extracellular vesicles (p < 0.0001 vs. [rC1 + PAb]).
Figure 5
Figure 5
Working model for HSP27 Immune Complex Altered Signaling and Transport (ICAST) modulation of cholesterol efflux. The HSP27 Immune Complex Altered Signaling and Transport unit (or exosomes containing HSP27 + anti-HSP27 antibody) trigger intracellular signaling that modulates the release of cholesterol loaded exosomes. For example, expression of Flotillin-2 is increased, and the cholesterol loaded into exosomes, as well as the number of exosomes released is enhanced. Once the exosomes are released to the extracellular space, they may act via autocrine or paracrine signaling, or proceed to cholesterol disposal via yet to be elucidated mechanisms.
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