Extracellular vesicles are associated with C-reactive protein in sepsis

Abstract

There is increasing evidence that C-reactive protein (CRP) can mediate inflammatory reactions following the transformation of functionally inert pentameric CRP (pCRP) into its structural isoform pCRP* and into monomeric CRP (mCRP). This conversion can occur on the membranes of apoptotic or activated cells or on extracellular vesicles (EVs) shed from the cell surface. Here, we characterized the association of CRP with EVs in plasma from sepsis patients using flow cytometry, and found highly elevated levels of total EV counts and CRP⁺ EVs as compared to healthy individuals. We further assessed the ability of PentraSorb CRP, an extracorporeal device for the adsorption of CRP, to deplete free CRP and CRP⁺ EVs. Treatment of septic plasma with the adsorbent in vitro resulted in almost complete removal of both, free CRP and CRP⁺ EVs, while total EV counts remained largely unaffected, indicating the detachment of CRP from the EV surface. EVs from septic plasma elicited a release of interleukin-8 from cultured human monocytes, which was significantly reduced by adsorbent treatment prior to EV isolation. Our findings provide evidence that CRP⁺ EVs exhibit pro-inflammatory characteristics and can contribute to the spreading of inflammation throughout the circulation on top of their pro-coagulant activity.

Figure 1

Characterization of plasma samples from sepsis patients and healthy individuals. (a) Plasma from sepsis patients and (b) plasma from healthy individuals was stained with anti-CRP-FITC and Anx5-PE to identify CRP-carrying EVs (CRP⁺ EVs) using flow cytometry. Detergent lysis with Triton X-100 abolished the EV cloud, confirming the presence of intact vesicles. Total EV counts (c), and the association of EVs with CRP (d) were determined in plasma from sepsis patients (n = 30) as compared to healthy individuals (n = 5); (e) Levels of plasma CRP in sepsis patients vs. healthy individuals. Data are given as mean ± SD and were compared using a Mann–Whitney test; *p < 0.05, ***p < 0.001. (f) EVs were pelleted from plasma by centrifugation and both EV-depleted supernatant and the EV pellet (15 µg protein each) were separated by SDS-PAGE under reducing conditions and probed for CRP by Western blotting. Human CRP was used as positive control (0.5, 0.1, 0.05 µg).

Figure 2

Depletion of CRP and CRP⁺ extracellular vesicles from plasma from sepsis patients. Plasma from sepsis patients (6 patients; n = 3 for each patient) was incubated with (+) or without (−) PentraSorb for 30 and 60 min to deplete CRP, as described in the Methods section. EV counts (a), CRP-carrying (CRP⁺) EVs (b), as well as plasma CRP (c) were quantified at baseline (0 min), and after 30 and 60 min. The decrease of plasma EV counts over time was mainly due to unspecific binding of EVs by the adsorbent, as a comparable drop in EV counts was observed for the non-functionalized adsorbent matrix (non-functionalized agarose), as shown in Supplementary Fig. S2. CRP⁺ EVs and plasma CRP were efficiently depleted by PentraSorb treatment. (d) Summary of data as mean of all six patients for EV counts, CRP⁺ EVs, as well as plasma CRP. Data are given as mean ± SD and were compared using a two-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Stimulation of monocytes with extracellular vesicles. (a) Human primary monocytes were isolated from peripheral blood mononuclear cells by negative depletion, as described in the Methods section. Cells were stained with anti-CD14-PE and anti-CD45-PB, monocytes were identified as CD45⁺CD14⁺ cells, and their purity was calculated in relation to all leukocytes (CD45⁺ cells). (b) EVs were enriched by centrifugation from plasma from septic patients without (left) or with (right) PentraSorb treatment. (c) Monocytes were stimulated with EVs from septic plasma, which was either pre-treated with PentraSorb (sepsis + A) or left untreated (sepsis -A). IL-8 was quantified in the supernatant by ELISA. Incubation of monocytes without EVs (-EVs) or with EVs from healthy individuals (healthy) served as control. n = 5 (-EVs); n = 7 (sepsis -A); n = 7 (sepsis + A); n = 4 (healthy). Data were compared using an unpaired t-test with or without Welch correction. Data are given as mean ± SD; *p < 0.05, **p < 0.01. Part (c) created with BioRender.com (Academic Subscription).