CXCR4+ PD-L1+ neutrophils are increased in non-survived septic mice

Abstract

The dysregulated host response to infections can lead to sepsis, a complex disease characterized by a spectrum of clinical phenotypes. Using scRNA-seq, we analyzed the immune cell of survived and non-survived CLP-septic mice to gain insights into the immunological mechanisms by which neutrophils contribute to the hyperinflammatory phenotype. Our findings reveal that non-survived mice exhibit increased frequencies of immature CXCR4⁺ PD-L1⁺ neutrophils in the bloodstream, accompanied by an accumulation of trafficking-specific CXCR4⁺ PD-L1⁺ neutrophils into the lungs. The IFN-gamma and LPS promote the PD-L1 expression on neutrophils and an activation profile associated with inflammation and organ damage. Notably, abrogating the IFN-gamma reduced susceptibility to CLP-sepsis and diminished CXCR4⁺ PD-L1⁺ neutrophils frequency. This study provides insights into the immune cell activation profiles associated with the worsening of the CLP-sepsis, and the CXCR4⁺ PD-L1⁺ neutrophils population highlighted here represents a promising target for therapeutic modulation in clinical sepsis hyperinflammatory phenotype.

Keywords: Biological sciences; Immune response; Immunology; Natural sciences.

Graphical abstract

Figure 1

A cohort of mice with the same genetic characteristics exhibits a heterogeneous host response to polymicrobial sepsis (A) survival rate of homogeneous mice after the induction of CLP-sepsis followed by antibiotic treatment (n = 10). (B) Bacteremia (C) the plasmatic concentration of cytokines, and (D) the plasmatic concentration of alanine aminotransferase (ALT) and blood urea nitrogen (BUN) in control, survived, and non-survived mice after CLP-sepsis. (E) Schematic of the experimental approach used to perform single-cell RNA sequencing from magnetic isolated CD45⁺ leukocytes from the blood of survived (n = 4), non-survived (n = 5), and control (n = 2) mice after 12 h of CLP-sepsis induction. (F) Dimensionality reduction of the multiplexed samples (n = 10). (G) UMAP visualization of leukocytes isolated from controls, survived and non-survived after 12 h of CLP-sepsis induction, colored by each cluster. (H) Log-normalized expression per cell of genes used for the leukocyte population identification. (I) Percentage of leukocyte populations in controls, survived and non-survived CLP-septic mice. (J) Flow cytometry gating strategy used to identify the different subpopulations of leukocytes isolated from mice after 12 h of the CLP-sepsis induction. (K) Percentage of leukocyte populations observed in control, survived, and non-survived septic mice. The scRNA-seq data is from one experiment. The data are representative of two independent experiments with similar results. The data are shown as means or means ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 2

scRNA-seq identification of the B and T subpopulations activation signature of non-survived CLP-septic mice (A) Schematic of the dimensionality reduction analysis specific for T cell populations identified in Figure 1. (B) UMAP visualization of T cells isolated from controls (n = 2), survived (n = 4), and non-survived (n = 4) after 12 h of CLP-sepsis induction, colored by each cluster. (C) Log-normalized expression per cell of genes used for the T cells subpopulations identification. (D) Percentage of T cells subpopulations identified in control, survived, and non-survived septic mice. (E) Significantly enriched biological processes for the set of significantly upregulated and downregulated genes of the subpopulation of T and B cells in the non-survived septic mice compared with survived. The data are from one experiment. The data are shown as means. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 3

The activation signature of monocytes related to the CLP-sepsis outcome (A) Significantly enriched biological processes for the set of significantly upregulated and downregulated genes of the monocytes identified in Figure 1 in the non-survived septic mice compared with survived. (B) (left) Flow cytometry gating strategy to identify populations of immature monocytes isolated from mice after 12 h of the CLP-sepsis induction, and (right) the frequency of PD-L1+ CXCR4+ monocytes observed in controls (n = 3), survived (n = 5) and non-survived septic mice (n = 5). The scRNA-seq data is from one experiment. The flow cytometry data is representative of two independent experiments with similar results. IThe data are shown as means ± s.e.m, and each data point represents one mouse. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 4

The CXCR4+ PD-L1+ neutrophil subpopulation is increased in non-survived septic mice (A) Schematic of the dimensionality reduction analysis specific for neutrophils identified in Figure 1. (B) UMAP visualization of neutrophils isolated from controls (n = 2), survived (n = 4), and non-survived (n = 4) after 12 h of CLP-sepsis induction, colored by each cluster. (C) Log-normalized expression per cell of genes used for the neutrophils subpopulations identification. (D) Percentage of neutrophils subpopulations identified in control, survived, and non-survived septic mice. (E) (left) flow cytometry gating strategy to identify the population of CXCR4⁺ neutrophils from the blood of mice after 12 h of the CLP induction and (right) the percentage of CXCR4⁺ neutrophils populations observed in control (n = 3), survived (n = 6), and non-survived (n = 5) CLP-septic mice. (F and G) Significantly enriched biological processes for the set of significantly upregulated and downregulated genes of the subpopulation of neutrophils in the non-survived septic mice compared with survived. (H) Expression of selected genes in the populations of neutrophils identified in Figure 4B. (I) (left) flow cytometry gating strategy to identify the population of PD-L1⁺ neutrophils from the blood of mice after 12 h of the CLP induction and (right) the percentage of PD-L1⁺ neutrophils populations observed in control (n = 3), survived (n = 7), and non-survived (n = 5) CLP-septic mice. (J) Expression of CXCR4 in the subpopulations of neutrophils identified in Figure 4I. The scRNA-seq data is from one experiment. The flow cytometry data is representative of two independent experiments with similar results. The data are shown as means ± s.e.m, and each data point represents one mouse. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 5

Non-survived septic mice exhibit an increased accumulation of neutrophils PD-L1+ CXCR4+ in the lungs (A and B) (left) Flow cytometry gating strategy to identify neutrophils (Ly6G⁺) and (right) percentage and the number of neutrophils from A, lungs and B, peritoneal cavity after 12 h of the CLP induction in controls, predicted to survive, and predicted to non-survive CLP-septic mice. (C and D) (left) Flow cytometry gating strategy to identify neutrophils (Ly6G⁺) and (right) percentage and number of PD-L1⁺ CXCR4⁺ neutrophils from C, lungs and D, peritoneal cavity after 12 h of the CLP induction in controls, predicted to survive, and predicted to non-survive CLP-septic mice. (E) Expression of CXCR2 and CCR2 in the subpopulations of neutrophils identified in Figure 4I. The data is representative of two independent experiments with similar results. The data are shown as means ± s.e.m, and each data point represents one mouse. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 6

IFN-gamma induces PD-L1 expression on neutrophils and increases the susceptibility of mice to the CLP-sepsis (left) flow cytometry representation, and (right) the median of fluorescence intensity (MFI) of CXCR4 on (A), neutrophils from bone marrow, and (B) neutrophils from blood. (left) flow cytometry representation, and (right) the median of fluorescence intensity (MFI) of (C), PD-L1, (D) CXCR4, (E) CD11b, (F) CD63, (G) Phosphatidylserine in CXCR4⁺ neutrophils isolated from bone marrow after 4 h of stimulation with LPS and IFN-gamma. Evaluation of (H), IL-6, TNF-alpha, and IL-10 concentration, and (I), NETs concentration in CXCR4⁺ neutrophils isolated from bone marrow after 4 h of stimulation with LPS and IFN-gamma. Evaluation of (J), IFN-gamma concentration, and (K) NETs concentration in plasma of controls, survived and non-survived mice after 24 h of CLP-sepsis. (L) Survival rate of WT and IFN-gamma deficient mice after the induction of CLP-sepsis (n = 22). (M) Plasmatic concentration of NETs in WT and IFN-gamma deficient mice after 24 h of CLP-sepsis. (N) (left) flow cytometry gating strategy, and (right) the frequency of the PD-L1⁺ neutrophils after 24 h of CLP-sepsis induction in WT and IFN-gamma deficient mice. (O) Plasmatic concentration of IL-6, TNF-alpha, and IL-10 in WT and IFN-gamma deficient mice after 24 h of CLP-sepsis. The data are representative of two independent experiments with similar results. The data are shown as means ± s.e.m, and each data point represents one mouse. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test.