Two New Neutrophil Subsets Define a Discriminating Sepsis Signature

Abstract

Rationale: Sepsis is the leading cause of death in adult ICUs. At present, sepsis diagnosis relies on nonspecific clinical features. It could transform clinical care to have immune-cell biomarkers that could predict sepsis diagnosis and guide treatment. For decades, neutrophil phenotypes have been studied in sepsis, but a diagnostic cell subset has yet to be identified. Objectives: To identify an early, specific immune signature of sepsis severity that does not overlap with other inflammatory biomarkers and that distinguishes patients with sepsis from those with noninfectious inflammatory syndrome. Methods: Mass cytometry combined with computational high-dimensional data analysis was used to measure 42 markers on whole-blood immune cells from patients with sepsis and control subjects and to automatically and comprehensively characterize circulating immune cells, which enables identification of novel, disease-specific cellular signatures. Measurements and Main Results: Unsupervised analysis of high-dimensional mass cytometry data characterized previously unappreciated heterogeneity within the CD64⁺ immature neutrophils and revealed two new subsets distinguished by CD123 and PD-L1 (programmed death ligand 1) expression. These immature neutrophils exhibited diminished activation and phagocytosis functions. The proportion of CD123-expressing neutrophils correlated with clinical severity. Conclusions: This study showed that these two new neutrophil subsets were specific to sepsis and detectable through routine flow cytometry by using seven markers. The demonstration here that a simple blood test distinguishes sepsis from other inflammatory conditions represents a key biological milestone that can be immediately translated into improvements in patient care.

Keywords: CD123; PD-L1; diagnosis; neutrophils; sepsis.

Figure 1.

Study design. (A) Blood samples from patients with sepsis (S) (n = 17) or NIC, post–cardiothoracic surgery patients with inflammation (n = 12) were enrolled in the discovery cohort of the study; in addition, blood samples were obtained from HDs (n = 11), and BM biopsy specimens were obtained from orthopedic surgery patients (n = 5). Immunostaining targeting 42 parameters was performed, and results were analyzed by using mass cytometry. (B) A computational “discovery strategy” was used to identify S-specific subsets, (C) a “computational validation” analysis was used to check whether the identified S-specific subsets were strategy-dependent, and (D) an additional “expert-driven validation” was used to define a small set of markers to gate on the S-specific neutrophil subsets. (E) A second independent validation cohort, including patients with S (n = 24) and NIPs (n = 18), was used for the “biological validation” of these S-specific neutrophil subsets through conventional flow cytometry. BM = bone marrow; HD = healthy donor; NIC = noninfected control; NIP = noninfected patient; PD-L1 = programmed death ligand 1.

Figure 2.

Identification of Sepsis (S) Day 1 (D1)–specific neutrophils by using a discovery analysis strategy. (A) t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis was performed on neutrophils from all samples, with cells being organized along t-SNE-1–2 and t-SNE-2–2 axes according to per-cell expression of CD11b, CD66b, CD16, CD10, CD64, CD123, and PD-L1 (programmed death ligand 1). Cell density for the concatenated file of each group is shown on a black to yellow heat scale for each group time point. (B) The heatmap shows sample clustering (columns) according to nodes’ log₂-transformed cell proportion and centered around the mean proportion of all samples’ nodes (rows). Samples and the mean-centered, log₂-transformed node cell proportion were arranged according to complete-linkage hierarchical clustering. Heat intensity (from blue to yellow) reflects the mean-centered log₂-transformed cell proportion of each sample’s node. (C) The heatmap shows the characterization of cell nodes identified using the Spanning-tree Progression Analysis of Density-normalized Events (SPADE) algorithm (columns) according to the mean expression of seven markers (rows). Markers were arranged according to complete-linkage hierarchical clustering, and nodes were preordered according to the heatmap node order in B. Heat intensity (from blue to red) reflects the mean expression of each marker for each node. (D) Four groups of nodes were back-viewed on a t-SNE1–2/t-SNE2–2 map. (E) The cell abundance of each meta-cluster subset (CD10⁻CD64⁺CD16⁺PD-L1⁺ cell subset in red, CD10⁻CD64⁺CD16^lowCD123⁺ cell subset in blue, and CD10⁻CD64⁺ cell subset in green) was presented as the cell proportion among the total neutrophils of each group of samples. A nonparametric, two-tailed Mann-Whitney test was used to compare differences in the cellular abundance of cell subsets between noninfected control (NIC)-D1 and S-D1 samples (see Methods). Sample sizes were as follows: HD = 11, BM = 5, NIC = 12, and S = 17. BM = bone marrow; HD = healthy donor.

Figure 3.

Validation of Sepsis (S) Day 1 (D1)–specific neutrophil subsets by using a second computational strategy. As a first step, Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) analysis was performed on all samples’ neutrophils, and cells were organized along UMAP-1 and UMAP-2 axes according to per-cell expression of CD11b, CD66b, CD16, CD10, CD64, CD123, and PD-L1 (programmed death ligand 1). As a second step, FlowSOM clustering was done to separate neutrophil subsets into 50 clusters. MEM was then used to quantify the enriched features of the 50 clusters. Protein enrichment was reported on a +10 to −10 scale, in which +10 indicates that the protein’s expression was especially enriched and −10 indicates that the protein’s expression was excluded from those cells, relative to the other neutrophil clusters. (A) Among these clusters, two meta-clusters were identified as being phenotypically identical to the strategy 1 S-specific neutrophils: clusters 18 and 19 (in red), composed of CD10⁻CD64⁺PD-L1⁺ neutrophils, and clusters 6 and 7 (in blue), composed of CD10⁻CD64⁺CD16^lowCD123⁺ neutrophils. (B) The cell abundance of each meta-cluster subset (the CD10⁻CD64⁺CD16⁺PD-L1⁺ cell subset in red and the CD10⁻CD64⁺CD16^lowCD123⁺ cell subset in blue) was presented as the cell proportion among the total neutrophils of each group of samples. A nonparametric, two-tailed Mann-Whitney test was used to compare differences in the cellular abundance of cell subsets between NIC-D1 and S-D1 samples (see Methods). Sample sizes were as follows: HD = 11, BM = 5, NIC = 12 ,and S = 17. (C) Each meta-cluster of cells (red and blue) was back-viewed on both a UMAP-1/UMAP-2 map and a t-SNE1–2/t-SNE2–2 map. BM = bone marrow; HD = healthy donor; MEM = marker enrichment modeling; NIC = noninfected control; t-SNE = t-Distributed Stochastic Neighbor Embedding.

Figure 4.

Sepsis (S) Day 1 (D1)–specific neutrophil subsets validated by using expert gating and correlated with severity scores. (A) An expert gating strategy with a seven-marker set allowed for the selection of a CD10⁻CD64⁺PD-L1⁺ cell subset (in red) and a CD10⁻CD64⁺CD16^lowCD123⁺ cell subset (in blue), which were back-viewed on both discovery (t-SNE1–2/t-SNE2–2) and validation (uniform Manifold Approximation and Projection for Dimension Reduction [UMAP]-1/UMAP-2) maps. (B) The two neutrophil subsets are significantly more abundant in the blood of patients with S collected at D1 after admission to the ICU than in the blood collected at D1 or D7 from noninfected control (NIC) post–cardiothoracic surgery patients or healthy donors (HDs). (C) Correlation between the log₁₀-transformed frequency of the CD10⁻CD64⁺PD-L1⁺ neutrophil subset (in red) or the CD10⁻CD64⁺CD16^lowCD123⁺ neutrophil subset (in blue) and Simplified Acute Physiology Score II (SAPS II) score (green squares) or Sequential Organ Failure Assessment (SOFA) score (purple squares). (D) The area under the receiver operating characteristic curve (AUROC) obtained by using only the CD123⁺ neutrophil subset. (E) The AUROC obtained by the using CD123⁺PD-L1⁺ neutrophil subsets. (F and G) The AUROCs obtained by using the SOFA (F) and SAPS II (G) clinical scores. A nonparametric, two-tailed Mann-Whitney test was used to compare the cellular abundance of cell subsets between S-D1 samples and NIC-D1, NIC-D7, or HD samples. A nonparametric, two-tailed Wilcoxon signed rank test was used to compare cellular abundance between the two matched groups: S-D1 and S-D7. Linear regression lines and Spearman rank correlation were used to assess the relationship between neutrophil subset frequency and severity scores (see the Methods). Spearman r coefficients and two-tailed P value are presented. Sample sizes were as follows: HD = 11, BM = 5, NIC = 12, and S = 17. BM = bone marrow; CI = confidence interval; PD-L1 = programmed death ligand 1; Std. = standard; t-SNE = t-Distributed Stochastic Neighbor Embedding.

Figure 5.

Nonneutrophil cell analysis identifies sepsis (S) immune hallmarks. (A) The lymphocyte and monocyte numbers and the intensity of mHLA-DR were obtained from nonneutrophil computational analysis and presented for each group. (B) Neutrophil numbers were obtained previously from the computational separation of neutrophils from nonneutrophils and were used to calculate the neutrophil/lymphocyte ratio. (C and D) Cell numbers for the main immune-cell subsets that were differentially abundant in the S group as compared with the healthy donor (HD) and NIC groups (C) and in the S group as compared with the HD group only (D). D1 = Day 1; mDC = mococyte-derived dendritic cells; mHLA-DR = HLA-DR expression on monocytes; NIC = noninfected control.

Figure 6.

Sepsis-specific neutrophils are detectable by conventional cytometry and discriminate infected from noninfected patients. (A) The gating strategy applied on fluorescent flow cytometry data of three patients with sepsis from the validation cohort. The overlay of FMT-stained control samples and the FP-stained samples of each representative patient showed an increase of sepsis-specific neutrophil subsets and a decrease of CD10 expression by neutrophils (CD14⁻CRTH2⁻CD15⁺ cells). (B–E) The AUROCs obtained by using only the CD123⁺ neutrophil subset (B), the two CD123⁺ and PD-L1⁺ neutrophil subsets (C), or the SOFA (D) and SAPS II (E) clinical scores. AUROC = area under the receiver operating characteristic curve; CI = confidence interval; FMT = full-minus-two panel; FP = full panel; FSC = forward scatter; PD-L1 = programmed death ligand 1; S = sepsis sample; SAPS II = Simplified Acute Physiology Score II; SOFA = Sequential Organ Failure Assessment; SSC = side scatter; Std. = standard.

Figure 7.

Staphylococcus aureus– and zymosan-specific activation and phagocytosis are impaired in sepsis immature neutrophils. To address the phagocytic capacities of sepsis immature (CD64⁺CD10⁻) neutrophils, 100 μl of blood was incubated with 20 μl or 40 μl of beads coated with S. aureus or zymosan, respectively, coupled with pH acidification–sensitive fluorochrome. After 1 hour of incubation at 37°C (positive control [PC]) or 4°C (negative control [NC]), cells were stained and analyzed by using flow cytometry. (A) Gating strategy of CD15⁺CD14⁻CD3⁻CD19⁻ neutrophils from healthy donors (HDs), Sepsis Day 1 (S-D1) samples, and bone marrow (BM) samples. Cells were separated into two gates on the basis of CD10 expression and phagocytosis marker intensity (S. aureus or zymosan), and PC cells (red dots) were overlaid onto NC cells (blue dots). The proportions of total phagocytic neutrophils were presented for the three groups. t-SNE analysis organized cells along the t-SNE axes according to per-cell expression of five proteins and phagocytosis fluorescence. (B and C) The cell expression of CD11b after S. aureus (B) or zymosan (C) stimulation for one representative individual from the HD group and one representative individual from the S-D1 group stimulated at +4°C (NC) and +37°C (PC) is shown on a heat scale. (D and E) For each individual, the PC/NC mean fluorescence intensity (MFI) ratios for CD66b and CD11b and after S. aureus (D) or zymosan (E) stimulation in each group were plotted in histograms. CD10⁻ cells have less phagocytic capacity, whether measured by using the MFI or a proportion. Stimulated CD10⁻ cells exhibit a lower level of expression of CD11b and CD66b. A nonparametric, two-tailed Mann-Whitney test was used to compare differences in the cellular abundance of cell subsets and in the MFI ratios (see the Methods). Sample sizes were as follows: HD = 4, S-D1 = 6 and BM = 3; PD-L1 = programmed death ligand 1; t-SNE = t-Distributed Stochastic Neighbor Embedding.