Myeloid S1PR1 is associated with systemic neutrophil expansion and redistribution.

(A-D) Counts of white blood cells (WBCs), neutrophils, monocytes, and lymphocytes in peripheral blood from LysM-S1pr1 TG (n = 6), S1pr1fsf (n = 6), LysM-S1pr1 KO (n = 4), and S1pr1f/f (n = 5) mice. Total blood cell counts were measured using the HemaVet system. (E) Representative blood smear images of neutrophils in wild-type (WT), LysM-S1pr1 KO, and TG mice. (F) Images and weights of spleens from S1pr1fsf(n = 9) and LysM-S1pr1 TG (n = 8) mice. (G) Flow cytometric quantification of splenic neutrophil frequency (CD45+CD11b+Ly6G+) in S1pr1fsf (n = 4) and LysM-S1pr1 TG (n = 6) mice. (H) Spleen/body weight ratio in vehicle-or FTY720-treated S1pr1fsf and LysM-S1pr1 TG mice (n ≥ 4 mice per group). (I) Representative H&E images of spleens after FTY720 or vehicle treatment. (J and K) Neutrophil counts in the lung (J) (n ≥ 7 mice per group) and liver (K) (n = 3 mice per group). (L) Plasma alanine transaminase (ALT, U/L) level (n = 3 mice per group). (M) Total protein in BALF (n = 3 mice per group). (N) Bone marrow neutrophil frequency among WBCs in S1pr1fsf(n = 4) and LysM-S1pr1 TG (n = 5) mice. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with multiple comparisons, or two-tailed unpaired Student’s t test).

Sustained S1PR1 signaling alters neutrophil phenotypes.

(A) Flow cytometric analysis of surface and total S1PR1 staining on splenic neutrophils (n ≥ 3 mice per group). (B) ImageStream analysis showing intracellular localization of S1PR1 in individual splenic neutrophils (n ≥ 3 mice per group). (C) Giemsa staining of isolated bone marrow (BM) and splenic (Spleen) neutrophils; segmented nuclei indicated by white arrows. Scale bar = 20 μm. (D) Quantification of segmented nuclei in BM and splenic neutrophils (n = 3 mice per group). (E and F) Flow cytometric analysis of CXCR2+ (E) and CXCR4+ (F) neutrophils in BM (n ≥ 4 mice per group), spleen (n ≥ 4 mice per group), and blood (n ≥ 3 mice per group). (G and H) Mean fluorescence intensity (MFI) of CD62L (G) and CD101 (H) on neutrophils (CD11b+Ly6G+) in BM, spleen, and blood. Quantification on the right. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (two-tailed unpaired Welch’s t test).

S1PR1 overexpression prolongs neutrophil survival.

(A) Time course of peritoneal neutrophil numbers in LysM-S1pr1 TG (n = 3) and control (n = 3) mice. (B) Flow cytometry of apoptotic splenic neutrophils (n ≥ 5 mice per group) stained with Annexin V and 7-AAD. Annexin V+7-AAD−cells were classified as early apoptotic, and Annexin V+7-AAD+ cells as late apoptotic/necrotic. (C) Lactate dehydrogenase (LDH) leakage assay was performed on bone marrow (BM) neutrophils (n ≥ 3 mice per group) after 24 h of ex vivo culture. (D) In vivo survival of neutrophils (n ≥ 3 mice per group) was assessed by tracking fluorescently labeled neutrophils in the peritoneal cavity at 0, 24, 48, and 72 h post-transfusion. (E) Oxygen consumption rate (OCR) of splenic neutrophils (n ≥ 3 mice per group) was measured by Seahorse assay. Cells (2 × 105/well) were sequentially challenged with the ATP synthase inhibitor oligomycin, the uncoupler FCCP, and the complex I/II inhibitors rotenone/antimycin A. (F) Mitochondrial mass (left) and membrane potential (right) in splenic neutrophils (n ≥ 10 mice per group) were measured by mean fluorescence intensity (MFI) of MitoTracker Green and MitoTracker Red, respectively. (G) Confocal microscopy of mitochondria (Tom20, green) and nuclei (blue) in isolated splenic neutrophils. (H) Quantification of mitochondrial intensity, size, and count in BM and splenic neutrophils (n ≥ 3 mice per group). Data are mean ± SD. *p < 0.05, **p < 0.01 (two-tailed unpaired Student’s t test).

S1PR1hi neutrophils exhibit a prosurvival, metabolic switch, and anti-inflammatory gene expression program.

(A) Uniform manifold approximation and projection (UMAP) plot of peritoneal cell clusters. (B) Dot plot showing expression of selected marker genes across cell subsets identified by single-cell RNA sequencing. Dot size represents the percentage of cells expressing the indicated gene within each cluster, and color intensity reflects the average normalized expression level. (C) UMAP plot of peritoneal neutrophils clustered into G5a, G5b, and G5c. (D) Dot plot showing representative marker genes distinguishing G5a, G5b, and G5c neutrophil subsets. (E) Increased G5c and reduced G5a/b clusters in LysM-S1pr1 TG neutrophils. (F) Flow cytometric validation of G5a (IFIT1−CXCR4lo), G5b (IFIT1+), and G5c (IFIT1−CXCR4hi) in the spleen. (G-I) Violin plots of per-cell module scores for apoptosis (G), autophagy (H), and mitophagy (I) in neutrophils. (J) Mitochondrial biogenesis module score per neutrophil. (K-O) Violin plots show per-cell module scores for inflammatory response (K), TNFα signaling via NFkB (L), NADPH oxidative stress (M), reactive oxygen species (N), and neutrophil degranulation (O). Control cells are in blue and LysM-S1pr1 TG cells in red. Dots mark group medians. Black brackets indicate Wilcoxon rank-sum tests performed at the cell level with Benjamini–Hochberg FDR correction; the bracket label reports the FDR p-value. Scores were computed as standardized module scores from curated gene sets; higher scores indicate stronger pathway-level expression. This cell-level analysis is exploratory.

Decreased ROS production in LysM-S1pr1 TG neutrophils.

(A) Phagocytosis of pHrodo-E. coli bioparticles was assessed in thioglycollate-induced peritoneal neutrophils at 0, 30, and 60 minutes (n = 4 mice per group). pHrodo+ cells were counted as phagocytosed neutrophils, and the ratio of pHrodo+ to total neutrophils was reported. (B) fMLP-induced ROS were measured by luminol-based chemiluminescence (n = 3 mice per group). The area under the curve (AUC) was plotted on the right. (C and D) Mean fluorescence intensity (MFI) of dichlorodihydrofluorescein diacetate (DCFDA) staining for intracellular ROS in splenic (C) and BM neutrophils (D) was plotted (n ≥ 3 mice per group). (E) Bacterial burden in the lung after intraperitoneal (IP) bacterial challenge, quantified as CFU per gram of tissue. (F) Bacterial burden in bronchoalveolar lavage fluid (BALF) after intratracheal (IT) bacterial challenge, quantified as CFU. (G) Proportion of neutrophils (CD11b+Ly6G+) among CD45+ cells in BALF after IT bacterial challenge. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed, unpaired Student’s t test).

LysM-S1pr1 TG mice are protected against H1N1-induced lung injury.

(A) Schematic of the high-dose H1N1 (LD50) model over 21 days. (B) Survival curve of LysM-S1pr1 TG (n = 20) and control (n = 18) mice in response to H1N1 infection. Survival curves were compared using the Gehan–Breslow–Wilcoxon test. (C) Body weight loss in LysM-S1pr1 TG (n = 8) and control (n = 8) mice. (D) Blood oxygensaturation (SpO2) in LysM-S1pr1 TG (n = 9) and control (n = 11) mice post-infection. Both (C) and (D) were analyzed by two-way mixed-effects analysis with Geisser– Greenhouse correction and Šídák’s multiple-comparison test; *p < 0.05, **p < 0.01. (E) Schematic of the low-dose (0.16 LD50) H1N1 model at 3 and 7 dpi. (F) Total cell count in BALF at 3 and 7 dpi (n = 7 mice per group). (G and H) BALF neutrophil and macrophage counts at 3 and 7 dpi (n = 7 mice per group). (I-K) Expression of viral genes, including nucleoprotein (NP), polymerase acidic protein (PA), and hemagglutinin (HA), in the infected lung in control (n = 3) and LysM-S1pr1 TG (n = 4) mice. (L) Total protein in BALF in control (n = 3) and LysM-S1pr1 TG (n = 6) mice at 7 dpi. (M) IL-6 (ng/mL) secretion in BALF in control (n ≥ 4) and LysM-S1pr1 TG (n ≥ 5) mice at 3 and 7 dpi. (N) IL-10 (ng/mL) secretion in BALF in control (n ≥ 4) and LysM-S1pr1 TG (n ≥ 3) mice at 3 and 7 dpi. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired Student’s t test).

Neutrophil-intrinsic S1PR1 signaling is sufficient to improve outcomes during influenza infection.

(A) Kaplan–Meier survival curves for Mrp8-S1pr1 transgenic (TG) mice (n = 25) and Cre-negative littermate controls (n = 21) after intranasal infection with high-dose H1N1 (LD50). Survival was monitored for up to 21 days post-infection. Statistical significance was assessed using the log-rank (Mantel–Cox) and Gehan– Breslow–Wilcoxon tests. (B) Body weight change in control (n = 13) and Mrp8-S1pr1 TG (n = 21) mice after influenza infection, expressed as a percentage of initial body weight. Data represent mean ± SD. (C) Arterial oxygen saturation (SpO) measured longitudinally after infection using pulse oximetry in control (n = 13) and Mrp8-S1pr1 TG (n = 21) mice. Data represent mean ± SD. Statistical significance is denoted as *p < 0.05, **p < 0.01, ***p < 0.001.