Prospective study and validation of early warning marker discovery based on integrating multi-omics analysis in severe burn patients with sepsis

Abstract

Background: Early detection, timely diagnosis and rapid response are essential for case management and precautions of burn-associated sepsis. However, studies on indicators for early warning and intervention have rarely been conducted. This study was performed to better understand the pathophysiological changes and targets for prevention of severe burn injuries.

Methods: We conducted a multi-center, prospective multi-omics study, including genomics, microRNAomics, proteomics and single-cell transcriptomics, in 60 patients with severe burn injuries. A mouse model of severe burn injuries was also constructed to verify the early warning ability and therapeutic effects of potential markers.

Results: Through genomic analysis, we identified seven important susceptibility genes (DNAH11, LAMA2, ABCA2, ZFAND4, CEP290, MUC20 and ENTPD1) in patients with severe burn injuries complicated with sepsis. Through plasma miRNAomics studies, we identified four miRNAs (hsa-miR-16-5p, hsa-miR-185-5p, hsa-miR-451a and hsa-miR-423-5p) that may serve as early warning markers of burn-associated sepsis. A proteomic study indicated the changes in abundance of major proteins at different time points after severe burn injury and revealed the candidate early warning markers S100A8 and SERPINA10. In addition, the proteomic analysis indicated that neutrophils play an important role in the pathogenesis of severe burn injuries, as also supported by findings from single-cell transcriptome sequencing of neutrophils. Through further studies on severely burned mice, we determined that S100A8 is also a potential early therapeutic target for severe burn injuries, beyond being an early warning indicator.

Conclusions: Our multi-omics study identified seven susceptibility genes, four miRNAs and two proteins as early warning markers for severe burn-associated sepsis. In severe burn-associated sepsis, the protein S100A8 has both warning and therapeutic effects.

Keywords: Early warning; Integrating multi-omics analysis; Marker; Sepsis; Severe burn.

Figure 1

Multi-omics landscape of severe burn patients. (a) Overview of the experimental design. (b) The number of samples for genomics, microRNAomics, proteomics and scRNA-seq analyses. WES whole exome sequencing, DIA data-independent acquisition, PRM parallel reaction monitoring, scRNA-seq single-cell transcriptome sequencing, C control, NS burn group without sepsis, S burn group with sepsis

Figure 2

Genomic analysis of patients with severe burn injuries. Genomic profiles. Top: counts of the mutated genes in each patient; bottom: group assignment for 40 patients. Mutation frequency is shown by the bar chart in the left panel and mutation type is shown by the bar chart in the right panel. NS burn group without sepsis, S burn group with sepsis

Figure 3

MicroRNAomics analysis of patients with severe burn injuries. (a) Heatmap of differential miRNAs in healthy controls and severe burn injuries. (b) Heatmap of differential proteins severe burn patients with sepsis or non-sepsis. (c) GO analysis diagram of NS/S group compared with control (C) group. The size of the circle represents the amount of protein enrichment and deeper color indicates greater functional significance. (d) GO analysis diagram of S group compared with NS group. The size of the circle represents the amount of protein enrichment and deeper color indicates greater functional significance. C control, NS burn group without sepsis, S burn group with sepsis, GO gene ontology

Figure 4

Similarities and differences of severe burn patients at different time points. (a) PCA score plot of severe burn patients [days (D) 0, 1, 3, 5, 7]. (b) Heatmap of differential proteins in healthy controls and severe burn injuries. (c) GO analysis diagram of different groups. The size of the circle represents the amount of protein enrichment and deeper color indicates greater functional significance. (d) Venn diagram of differential proteins on days (D) 1, 3, 5 and 7 of severe burn patients compared with healthy controls (group C). (e) Heatmap of common differential proteins in severe burn injuries compared with healthy controls. (f) Reactome enrichment analyses of common differential proteins. DIA data-independent acquisition, C control, GO gene ontology

Figure 5

Differences between severe burn patients with and without sepsis. (a–d) PCA score plot of severe burns with sepsis and non-sepsis on days 1, 3, 5 and 7. (e, f) Trends in associated proteins at three time points (C, Day 1, Day 3) for severe burn injuries. Numbers 0, 1, 3, 5 represent uptrends, numbers 2, 4, 6, 7 represent downtrends. (g) Heatmap of differential proteins on day 1 of severe burn patients with sepsis and non-sepsis. (h) GO analysis diagram of two groups. The size of the circle represents the enrichment of the protein and the larger the value on the abscissa the greater the functional significance. DIA data-independent acquisition, C control, NS burn group without sepsis, S burn group with sepsis

Figure 6

Proteomic revalidation of expanded samples by PRM assay. (a, b) PRM method was used to quantify 26 proteins of 45 patients on Burn-day 1 and Burn-day 3. (c, d) ROC curves of S100A8 protein on Burn-day 1 and Burn-day 3. (e, f) ROC curves of SERPINA10 protein on Burn-day 1 and Burn-day 3. ^*p < 0.05, ^**p < 0.01. NS burn group without sepsis, S burn group with sepsis, AUC area under the curve, PRM parallel reaction monitoring

Figure 7

scRNA-seq analyses of healthy controls and severe burn injuries. (a) Heatmap of differential genes in subsets of neutrophils in healthy controls and severely burn injuries (days 1–3). Yellow represents high expression level, purple represents low expression level and black represents moderate expression level. (b) Heatmap of s100-related gene expression of neutrophils in healthy controls and severely burn injuries (days 1–3). (c, d) Violin plot of the genes S100a8 and S100a9 in different subsets of neutrophils

Figure 8

Therapeutic effect of S100A8 inhibitor on severely burned mice. (a) Schematic diagram of experimental design and sample collection strategy. Mice were scalded on 30% of their body area and then injected with PAQ at different time points. Blood and organ samples were collected at various points. (b) Expression of S100A8 in peripheral blood plasma of different groups of mice. (c) Survival rate change in severe burn mouse model. C57BL6/J mice with severe burn injury were randomized in three groups: sham group (Sham, n = 12), severe burn injury group (Burn, n = 16) and severe burn injury with PAQ group (Burn+PAQ, n = 16). (d) The proportion of peripheral blood neutrophils in each group on the first day of surgery. (e) CD177 expression of neutrophils in each group on the first day of operation. (f) Flow cytometry of the expression of seven inflammatory factors in peripheral blood plasma (IL-2, IL-4, IL-6, IFN-γ, TNF, IL-17A and IL-10). (g, h) The mean fluorescence intensity of inflammatory factors IL-6 and TNF in each group. (i) Histopathology of mouse lung tissue in each group (Hematoxylin–eosin staining, scale bar: = 100 μm). Data represent means ± SD (n ≥ 3) of two independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001, ns = not statistically significant compared with the control group. PAQ paquinimod, MFI mean fluorescence intensity, IL Iterleukin, IFN-γ interferon-γ, TNF tumor necrosis factor