The prognostic impact of SIGLEC5-induced impairment of CD8+ T cell activation in sepsis

Abstract

Background: Sepsis is associated with T-cell exhaustion, which significantly reduces patient outcomes. Therefore, targeting of immune checkpoints (ICs) is deemed necessary for effective sepsis management. Here, we evaluated the role of SIGLEC5 as an IC ligand and explored its potential as a biomarker for sepsis.

Methods: In vitro and in vivo assays were conducted to both analyse SIGLEC5's role as an IC ligand, as well as assess its impact on survival in sepsis. A multicentre prospective cohort study was conducted to evaluate the plasmatic soluble SIGLEC5 (sSIGLEC5) as a mortality predictor in the first 60 days after admission in sepsis patients. Recruitment included sepsis patients (n = 346), controls with systemic inflammatory response syndrome (n = 80), aneurism (n = 11), stroke (n = 16), and healthy volunteers (HVs, n = 100).

Findings: SIGLEC5 expression on monocytes was increased by HIF1α and was higher in septic patients than in healthy volunteers after ex vivo LPS challenge. Furthermore, SIGLEC5-PSGL1 interaction inhibited CD8⁺ T-cell proliferation. Administration of sSIGLEC5r (0.8 mg/kg) had adverse effects in mouse endotoxemia models. Additionally, plasma sSIGLEC5 levels of septic patients were higher than HVs and ROC analysis revealed it as a mortality marker with an AUC of 0.713 (95% CI, 0.656-0.769; p < 0.0001). Kaplan-Meier survival curve showed a significant decrease in survival above the calculated cut-off (HR of 3.418, 95% CI, 2.380-4.907, p < 0.0001 by log-rank test) estimated by Youden Index (523.6 ng/mL).

Interpretation: SIGLEC5 displays the hallmarks of an IC ligand, and plasma levels of sSIGLEC5 have been linked with increased mortality in septic patients.

Funding: Instituto de Salud Carlos III (ISCIII) and "Fondos FEDER" to ELC (PIE15/00065, PI18/00148, PI14/01234, PI21/00869), CDF (PI21/01178), RLR (FI19/00334) and JAO (CD21/00059).

Keywords: Biomarker; HIF1α; Immune checkpoint; SIGLEC5; Sepsis; T-cell exhaustion.

Fig. 1

LPS induces SIGLEC5 expression on human monocytes under Hypoxia Inducible Factor 1 alpha (HIF1α) control. (a) Normalized SIGLEC5 RNA expression on the main immune cells in the blood from the Monaco database. (b) SIGLEC5 expression on the cell surface of a panel of blood circulating immune populations stimulated (grey filled line) or not (black clear line) with lipopolysaccharide (LPS), including neutrophils, T lymphocytes, classical monocytes (CD14⁺CD16^–), intermediate monocytes (CD14⁺CD16⁺) and non-classical monocytes (CD14^–CD16⁺); MFI of LPS-stimulated or not conditions were indicated into the plots. A fluorescence minus one (FMO) was used as negative control of SIGLEC5 expression represented in dotted line. (c) SIGLEC5 expression on monocytes (left panel, n = 35) and neutrophils (right panel, n = 14) from HVs challenged or not with 10 ng/mL of LPS for 16 h. (d) Fold change of SIGLEC5 expression on monocytes, neutrophils and B cells from HVs challenged stimulated with LPS vs. control without stimulation. (e) Hypoxia Response Elements (HRE) in the human SIGLEC5 sequence (Ensembl, ENSG00000268500) based on the consensus sequence [5′-ACGTG-3′]. Sequences and positions are shown. (f) A Chromatin immunoprecipitation (ChIP) assay was conducted on: control monocytes (white column), LPS-stimulated monocytes for 16 h (grey filled column), HIF1α-transfected monocytes (burgundy filled column), all of them immunoprecipitated with an antibody against HIF1α, and HIF1α-transfected monocytes immunoprecipitated with an unspecific IgG antibody (burgundy filled and lined column). HREs band intensities of PCR products normalized to one of the negative controls (n = 4). (g) Relative expression (mRNA) by RT-qPCR of HIF1α (left panel) and SIGLEC5 (right panel) on CD14⁺ cells from HVs nucleofected with pHIF1α (burgundy filled column) or a control plasmid (white column) (n = 6). (h) Representative histogram overlay of SIGLEC5 expression (left panel) on CD14⁺ cells nucleofected with pControl (white histogram) or pHIF1α (burgundy histogram) and MFI of SIGLEC5 (right panel) on CD14⁺ cells after 16 h of nucleofection with pControl (white column) or pHIF1α (burgundy filled column) (n = 4). (i) Representative histogram overlay of SIGLEC5 on CD14⁺ cells pre-treated (grey thickest line) or not (grey thinnest line) with a specific inhibitor (PX-478) of HIF1α for 3 h, and then challenged with LPS for 16 h (left panel), and MFI of SIGLEC5 on CD14⁺ cells pre-treated (grey filled columns) or not (black clear column) with a specific HIF1α inhibitor (PX-478) for 3 h, and then challenged (grey filled and thickest line) or not (grey filled and thinnest line) with LPS for 16 h (right panel) (n = 7). Data shown as mean ± SEM. (c, d, and g–i) Paired t-test (∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001). (f) One-way ANOVA test (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

Fig. 2

SIGLEC5 on monocytes impairs CD8⁺T cell proliferation, which is restored after blocking the SIGLEC5/PSGL1 axis. (a) Scheme of the proliferation assay of monocytes stimulated of not with LPS for 16 h in presence of a blocking antibody against SIGLEC5 (α-SIGLEC5) or an unspecific IgG, and then co-cultured with autologous lymphocytes stimulated or not with PWD (2.5 μg/mL) for 5 days. (b) Proliferation levels (CFSE^dim) of CD8⁺ cells from HVs, stimulated or not with PWD and co-cultured for 5 days with autologous monocytes pre-challenged (grey filled columns) or not (white columns) with 10 ng/mL of LPS for 16 h. In some indicated conditions, a blocking antibody against SIGLEC5 (α-SIGLEC5) or an unspecific IgG was added (n = 3, left panel). Representative histogram overlay of proliferation levels (CFSE^dim) of PWD-stimulated CD8⁺ cells from HVs, and co-cultured for 5 days with autologous monocytes pre-challenged with 10 ng/mL of LPS for 16 h, in presence or not of a blocking antibody against SIGLEC5, α-SIGLEC5 (right panel). (c) Scheme of the proliferation assay of monocytes nucleofected or not with a SIGLEC5 expression vector (pSIGLEC5) and then co-cultured with autologous lymphocytes stimulated or not with PWD (2.5 μg/mL) for 5 days. (d) MFI of SIGLEC5 on CD14⁺ cells 16 h after nucleofection with either an expression vector of SIGLEC5, (pSIGLEC5), or a control vector, pControl (n = 4) (left panel). Representative histogram overlay of SIGLEC5 expression on CD14⁺ cells 16 h after nucleofection with either an expression vector of SIGLEC5, pSIGLEC5 (blue histogram), or a control vector, pControl (white histogram) (right panel). (e) Proliferation levels (CFSE^dim) of CD8⁺ cells from HV, stimulated or not with PWD and co-cultured for 5 days with autologous monocytes pre-nucleofected (blue filled column) or not (white column) with an expression vector of SIGLEC5 (pSIGLEC5) (n = 4) (left panel). Representative histogram overlay of proliferation levels (CFSE^dim) of PWD-stimulated CD8⁺ cells from HVs, and co-cultured for 5 days with autologous monocytes pre-nucleofected (blue filled line) or not (white line) with pSIGLEC5 (right panel). (f) Scheme of the binding assay of recombinant sSIGLEC5r-FC to CD8⁺ T cells. (g) Quantification of sSIGLEC5r (sSIGLEC5r-FC) binding to CD8⁺ T cells from HVs pre-treated or not with Neuraminidase (NM, 0.01 U/mL) for 24 h (n = 6). Binding was revealed with α-FC-FITC and analyzed by flow cytometry, using the secondary antibody alone as control (left panel). Representative histogram overlay of sSIGLEC5r-FC binding to CD8⁺ cells from HVs pre-treated or not with NM (0.01 U/mL) for 24 h and unspecific control using the antibody α-FC-FITC only (vehicle, right panel). (h) Proliferation levels (CFSE^dim) of CD8⁺ T cells from HVs in the presence or not of PWD and co-cultured for 5 days with autologous monocytes pre-nucleofected with an expression vector of SIGLEC5 (pSIGLEC5, blue filled columns) or an empty vector (pControl, white columns) (n = 6). In some conditions, a blocking antibody against PSGL1 (α-PSGL1) was added. Data shown as mean ± SEM. (b, d, e, g, and h) Paired t-test (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

Fig. 3

Soluble SIGLEC5 impairs CD8⁺T cell proliferation. (a) Proliferation levels (percentage of CFSE^dim cells) (left panel) and linear regression (right panel) of PWD-stimulated CD4⁺ (orange) and CD8⁺ (mocha) cells from HVs in presence of different concentrations of human recombinant sSIGLEC5r for 5 days (n = 5). (b) Representative histograms overlays of the proliferating CFSE^dim PWD-stimulated CD4⁺ (left panel) and CD8⁺ (right panel) cells from HVs in presence (filled line) or not (clear line) of human sSIGLEC5r for 5 days. (c) Proliferation levels (percentage of CFSE^dim cells) of PWD-stimulated CD4⁺ (orange) and CD8⁺ (mocha) T cells from HVs in presence or not of the highest concentration of sSIGLEC5r used (1000 ng/mL) for 5 days (n = 5). (d) Percentage of apoptotic (PI^–/AnnexinV⁺) CD8⁺ T cells isolated from HV and incubated (filled column) or not (clear column) with sSIGLEC5r for 24 h at 500 and 1000 ng/mL (n = 5). (e) Soluble SIGLEC5 (sSIGLEC5) levels in supernatants of monocytes (n = 14) and neutrophils (n = 6) treated (grey filled column) or not (white column) with 10 ng/mL of LPS for 16 h. (f) Representative histogram overlay of SIGLEC5 expression on LPS-stimulated CD14⁺ cells in absence (grey filled) or presence (wine filled) of a pan-inhibitor of metalloproteinases (GM6001) added 6 h before the LPS stimulation for 16 h. (g) MFI of SIGLEC5 on CD14⁺ cells from HVs exposed to LPS (grey), GM6001 (orange), and combinations (wine) for the indicated times (n = 3). (h) sSIGLEC5 levels in the supernatants of monocytes from HVs exposed to LPS (grey), GM6001 (orange) and combinations (wine) for indicated times (n = 3). A control without any stimulation is represented in white. Data shown as mean ± SEM. (a, left panel) Two-way ANOVA test (∗∗p < 0.01). (a, right panel) Spearman linear regression test (∗p < 0.05). (c–e) Paired t-test (∗p < 0.05; ∗∗p < 0.01). (g and h) One-way ANOVA test (∗∗p < 0.01; ∗∗∗p < 0.001).

Fig. 4

SIGLEC5 reduces survival in endotoxemia mouse models in a CD8α-dependent manner. (a) Kaplan–Meier estimation of survival from CLP-mice injected or not with sSIGLEC5r (χ² = 33.20, p < 0.0001 for all four groups comparison: Sham, Sham + sSIGLEC5r, CLP + saline and CLP + sSIGLEC5r; χ² = 7.591, p = 0.0059, hazard ratio [HR] CLP plus sSIGLEC5r vs. CLP groups comparison of 8.302; 95% CI, 1.842–37.42) (n = 7 per group). (b) Kaplan–Meier estimation of survival from CLP-WT or CLP-Rag1^−/− mice injected or not with sSIGLEC5r (χ² = 0.064, p = n.s., Hazard ratio [HR] Rag1^−/− CLP vs. Rag1^−/− CLP + sSIGLEC5 of 0.828, 95% CI, 0.191–3.582) (n = 5 per group in WT mice and n = 7 per group in Rag1^−/− mice). (c) Acute lung injury evaluated on Haematoxylin/Eosin-stained lung sections of Sham or CLP-mice in the background wild-type or Rag1^−/− injected or not with sSIGLEC5r (n = 7 per group). (d) Kaplan–Meier estimates of survival from mice injected with sSIGLEC5r and/or LPS (χ² = 5.026, p = 0.0250, HR of 6.019, 95% CI, 1.253–28.91) (n = 12 per group). (e) Flow cytometry-gating strategy for CD4⁺ and CD8α⁺ T cells in blood from control and CD8α-depleted mice at day 1 and 6 after injection of a depleting CD8α antibody (20 μg/mice). (f) Kaplan–Meier estimates of survival from mice injected with LPS (20 mg/kg) with or without CD8α depletion (χ = 0.242, p = 0.6227, HR of 0.920, 95% CI, 0.413–2.049) or injected with LPS and sSIGLEC5r with or without CD8α depletion (χ² = 9.731, p = 0.0018, HR of 14.21, 95% CI, 2.681–75.29) (n = 12 per group). (g) Acute lung injury evaluated on Haematoxylin/Eosin stained lung sections of the endotoxemia mouse model with LPS injection (20 mg/mL), CD8α-depleted or not, and injected or not with sSIGLEC5r (n = 12 per group). (a, b, and d–f) Kaplan–Meier estimation of survival test (∗p < 0.05; ∗∗p < 0.01). (c and g) Data shown as mean ± SEM. Paired t-test (ns, non-significant; ∗∗p < 0.01).

Fig. 5

Septic patients overexpress SIGLEC5 on monocytes, and blockade of the SIGLEC5/PSGL1 axis restores CD8⁺T cell proliferation. (a) Flow cytometry gating strategy for CD14⁺ cells from both HVs and septic patient (left panels). Representative histogram overlay of SIGLEC5 expression on CD14⁺ cells from a HVs (white line) and a septic patient (violet filled line); a dotted line was used as isotype control (right panel). (b) MFI of SIGLEC5 on CD14⁺ cells from HVs (grey clear box, n = 35) and septic patients (violet filled box, n = 38). (c) SIGLEC5 expression on monocytes from HVs (grey) and septic patients (violet) challenged or not with 10 ng/mL of LPS for 16 h. (d) SIGLEC5 expression on monocytes from hemoculture-positive (n = 9) and –negative (n = 19) septic patients challenged or not with 10 ng/mL of LPS for 16 h (Violet dots: Hemoculture-negative, violet squares: Hemoculture-positive with gram-negative bacteria, violet squares green filled: Hemoculture-positive with gram-positive bacteria). (e) sSIGLEC5r-FC binding quantification on CD8⁺ T cells from patients with sepsis (n = 4) (left panel) and a representative histogram overlay (right panel). (f) Proliferation levels (CFSE^dim) of CD8⁺ T cells from HVs and patients with sepsis in the presence (green filled column) or not (white column) of PWD for 5 days. (g) Proliferation levels (CFSE^dim) of CD8⁺ T cells from patients with sepsis in the presence or not of PWD and co-cultured for 5 days with autologous monocytes pre-challenged (grey filled column) or not (white column) with 10 ng/mL of LPS for 16 h in the presence of anti-SIGLEC5 blocking antibody and/or anti-PSGL1 blocking antibody (n = 6). Data shown as mean ± SEM. (b–d and f) Unpaired t-test (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). (c–e and g) Paired t-test (∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

Fig. 6

Soluble SIGLEC5 classified septic patients on admission. (a) Soluble SIGLEC5 (sSIGLEC5) levels in plasma from HVs (grey clear dots, n = 100) and septic patients (sepsis in violet clear dots and shock septic in purple filled dots, n = 346). (b) sSIGLEC5 levels in plasma from HVs (n = 100) and patients with sepsis (n = 188), septic shock (n = 158), SIRS (n = 80), aneurysm (n = 11) and stroke (n = 16). (c) sSIGLEC5 levels in plasma from septic patients classified according to their hospitalization (n = 234) or their admission to ICU (n = 112). (d) sSIGLEC5 levels in plasma from septic patients hospitalized survivors (n = 166), hospitalized exitus (n = 68), admitted in ICU survivors (n = 62) and admitted in ICU exitus (n = 50). (e) sSIGLEC5 levels in plasma from patients with sepsis (violet clear dots) and septic shock (purple filled dots) classified according to their outcome after 60 days as Survivors (n = 228) and Exitus (n = 118). (f) Septic patients were dichotomized according to the optimal cut-off, estimated by Youden index for plasmatic sSIGLEC5 concentration to be 523.6 ng/mL. Kaplan–Meier survival curves from diagnosis to day 60 according to baseline plasmatic sSIGLEC5 (χ² = 33.20, p < 0.0001) are shown. Data shown as mean ± SEM. (a and c–e) Unpaired t-test (∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). (b) One-way ANOVA test (∗∗∗∗p < 0.0001). (f) Kaplan–Meier estimation of survival from septic patients according Youden index (∗∗∗∗p < 0.0001).