Burn Injury Induces Proinflammatory Plasma Extracellular Vesicles That Associate with Length of Hospital Stay in Women: CRP and SAA1 as Potential Prognostic Indicators

Abstract

Severe burn injury is a devastating form of trauma that results in persistent immune dysfunction with associated morbidity and mortality. The underlying drivers of this immune dysfunction remain elusive, and there are no prognostic markers to identify at-risk patients. Extracellular vesicles (EVs) are emerging as drivers of immune dysfunction as well as biomarkers. We investigated if EVs after burn injury promote macrophage activation and assessed if EV contents can predict length of hospital stay. EVs isolated early from mice that received a 20% total body surface area (TBSA) burn promoted proinflammatory responses in cultured splenic macrophages. Unbiased LC-MS/MS proteomic analysis of early EVs (<72 h post-injury) from mice and humans showed some similarities including enrichment of acute phase response proteins such as CRP and SAA1. Semi-unbiased assessment of early human burn patient EVs found alterations consistent with increased proinflammatory signaling and loss of inhibition of CRP expression. In a sample of 50 patients with large burn injury, EV SAA1 and CRP were correlated with TBSA injury in both sexes and were correlated with length of hospital stay in women. These findings suggest that EVs are drivers of immune responses after burn injury and their content may predict hospital course.

Keywords: biomarkers; burn injury; extracellular vesicles; sepsis; trauma.

Figure 1

Overall Translational Experimental Design. (1) Mice undergo a 20% total body surface area (TBSA) burn injury, and EVs isolated within 72 h after injury. (2) Mouse burn EVs are tested for pro-inflammatory capacity in mouse splenic macrophages. (3) Mouse burn EVs are assessed for protein content using LC-MS/MS proteomic analysis. (4) EVs are isolated from human burn patients with severe burn injury. (5) Proteomic and miRNAomic analysis of human EVs is performed. (6) Proteomic profiles of human and mouse burn EVs are compared, and promising candidates identified. (7) Promising candidates were measured in a larger cohort of human burn patients and associated with length of hospital stay.

Figure 2

Plasma extracellular vesicles (EVs) after burn injury promote pro-inflammatory signaling in macrophages. Adult mice underwent a 20% total body surface area (TBSA) burn injury and plasma EVs were collected 48 h after injury. EVs from burn injury mice (Burn EV) or sham injured mice (Sham EV) were applied (3 × 10⁷/well) to splenic macrophages +/− LPS (100 ng/mL) for 24 h. (A–D) Media cytokines were measured by multiplex. Burn EVs cause robust increases in pro-inflammatory (A) IL-6, (B) MCP-1, (C) IL-12p70, and (D) IFN¦Ã in macrophage media. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. sham EVs, t-test. (E,F) Macrophage lysates were harvested, mRNA isolated, and gene changes measured by NanoString. (E) Volcano plot of several genes significantly induced by burn-EVs relative to sham EVs. (F) nSolver pathway analysis scores of changes in measured immune pathways. Burn EVs significantly altered TLR signaling, cytokine signaling, host-pathogen interactions, NFkB signaling, and the innate immune system in macrophages relative to sham EVs.

Figure 3

Proteomic changes in mouse plasma EVs after burn injury. Adult mice underwent a 20% total body surface area (TBSA) burn injury and plasma EVs were collected 72 h after injury and protein content measured by LC-MS/MS. (A) Differentially expressed protein peptides in burn vs. sham EVs (LFQ ratio, p-value Student’s t-test, n = 3 per group). (B) Characteristics of proteins that are increased in burn EVs relative to controls. (C) Characteristics of proteins that are decreased in burn EVs relative to controls. (D) ELISA measurement of SAA1 in plasma EVs from sham and burn mice. SAA1 protein was robustly increased in burn-EVs compared to sham. ** p < 0.01, t-test, n = 4 sham, 8 burn.

Figure 4

Proteomic changes in human burn patient plasma EVs. Plasma EVs from human burn patients with severe burn injury were isolated within 72 h of admission. Protein content was measured by LC-MS/MS. (A) EV number was measured by NTA. Human burn patients showed an increase in circulating plasma EVs compared to healthy donors. ** p < 0.01, n = 3/group. Inset shows representative images of EVs captured by the ParticleMetrix^TM machine. (B) NTA size analysis shows the majority of EVs are in the 100–400 µm diameter size range, with burn EVs showing increased numbers in the MV size range. (C) Differentially expressed protein peptides in burn vs. healthy donor EVs (LFQ ratio, p-value Student’s t-test, n = 3 per group). (D) Characteristics of proteins that are increased in burn EVs relative to healthy individuals. (E) Characteristics of proteins that are decreased in burn EVs relative to healthy individuals. (F) Comparison of robust changes between human burn and mouse burn EVs. Both SAA1 and coagulation proteins were increased across species.

Figure 5

Proinflammatory miRNA signature in EVs from human burn patients by NanoString. Plasma EVs were isolated from human burn injury patients during the first 48 h of admission or healthy controls and miRNA content assessed by NanoString. (A) NanoString assessment of human burn patient EVs revealed that 26 miRNAs were significantly altered, 22 of which showed reduced expression. n = 3 per group. (B) Predicted outcomes of changes in differentially expressed miRNAs. Directional changes in 11 of the miRNAs would promote inflammation, while reductions in 5 miRNAs that target CRP would promote CRP expression, and reductions in 4 miRNAs that target metabolic and insulin signaling could alter cellular metabolism.

Figure 6

Assessment of human plasma EVs from human burn patients. Plasma EVs were isolated from 50 human burn patients within the first 72 h of admission. (A) Assessment of EV sign distribution by nanoparticle tracking analysis (NTA) found the majority of EVs were within the MV size range. (B) Western blot on EVs isolated from 4 human burn patients stained for MV marker Annexin-A1. (C) EV concentration was positively correlated with size of burn injury. (D) No correlation between EV concentration and length of hospital stay was found across subjects.

Figure 7

Association of EV CRP and SAA-1 in EVs with length of hospital stay in severe burn injury. EVs were isolated from 50 human burn patients and levels of CRP (A–D) and SAA1 (E–H) measured by ELISA and their association with length of stay (LOS) in the hospital were determined. (A) CRP levels from MV-depleted plasma showed no association with severity of burn injury. (B) CRP levels in EVs from human burn patients showed a positive correlation with severity of burn injury. r = 0.32, * p < 0.05. (C) CRP levels in EVs of male human burn patients was not related to LOS. (D) CRP concentration in EVs from female burn patients was positively correlated with hospital LOS (r = 0.56, * p < 0.05). (E) SAA1 protein concentration in MV-depleted plasma showed no association with severity of burn injury. (F) SAA1 concentrations in EVs from human burn patients showed a positive correlation with severity of burn injury. r = 0.33, * p < 0.05. (G) SAA1 concentrations in EVs of male human burn patients was not related to LOS. (H) SAA1 concentration in EVs from female burn patients was positively correlated with hospital LOS (r = 0.58, * p < 0.05). (I) Concentrations of CRP and SAA1 in EVs were significantly correlated with each other. r = 0.39, * p < 0.02.