Fig 2: eNAPRT is a risk factor for septic patients. a Western blot analysis of NAPRT in 10× concentrated HD macrophages supernatants (SN), in which apoptosis (APO) and necrosis (NECRO) were induced as described in “Methods” section. Detection of cyclophilin A in SN was used as positive control for necrosis induction. b Kaplan–Meier curves showing overall survival (OS) of mice treated intraperitoneally with LPS (25 mg/kg) with or without rNAPRT (1 mg/kg) or rNAPRT (25 mg/kg) 1–2 h prior to LPS administration. N = 6 mice/treatment group. Log-rank test shows statistical significance. c Box plots showing mRNA expression levels of TNFA evaluated by qRT-PCR in RNA from HD macrophages (n = 5) treated as follow: 30 h of pre-conditioning (preT) with LPS 10 ng/ml or rNAPRT 10 ng/ml followed by 6 h of challenging with LPS 1 µg /ml. Paired t-test. d Scatter dot plot showing eNAPRT levels in septic patients, according to outcome. e Graph representing the percentage of septic patients who survived (black bars) or died (gray bars) when dividing patients according to the eNAPRT cut-off of 15 ng/ml. f Scatter dot plots reporting LDH, creatinine and CRP levels in septic patients dividing according to the eNAPRT cut-off. Mann–Whitney test. g Kaplan–Meier curves showing overall survival (OS) of the cohort of 100 septic patients divided on the basis of eNAPRT levels. Log-rank test shows statistical significance. h Regression line showing a positive correlation between CRP (x-axis) and eNAPRT (y-axis) levels, as detected in 92 plasma from septic patients. Pearson coefficient (r) and the corresponding p-value are noted. Results are reported as box plots and dot plot, where the line in the box defines the median and the error bars define the minimum and maximum or the interquartile range. Source data are provided as a Source Data file

Fig 3: eNAPRT binds to TLR4. a Western blot analysis of p-p65 in HDs macrophages upon treatment (30 min) with rNAPRT (WT) or the G379A mutant at the concentration of 1 µg/ml (n = 5). b Confocal images showing p65 localization (green fluorescence) in human macrophages treated with rNAPRT (WT) or the G379A mutant (1 µg/ml, 30 min). Original magnification ×63, scale bar: 25 µm. c SPR measurements of TLR4 (1 µM), NAPRT (100 µM), and TLR4/NAPRT (1 µM/100 nM). The measurements were performed at 25 °C and the flux was fixed at 30 µL/min. d Western blot analysis of p-p65 in scramble (sc) or TLR4 siRNA-silenced macrophages upon treatment (30 min) with rNAPRT (1 µg/ml), rNAMPT (1 µg/ml), and LPS (2 µg/ml). Box plot on the right represents quantification obtained by ImageQuant of p-p65/p65 (n = 6 for rNAPRT and LPS, n = 4 for rNAMPT, paired t-test). e Confocal images showing p65 localization (green fluorescence) in macrophages (n = 3) transfected with a scramble control siRNA (sc) or a TLR4 siRNA (white fluorescence for TLR4 staining) and treated (30 min) with rNAPRT (1 µg/ml). Original magnification ×63, scale bar: 50 µm. f Box plots showing mRNA expression levels of IL1B and IL8 evaluated by qRT-PCR upon 15 h treatments with rNAPRT (1 µg/ml), rNAMPT (1 µg/ml), and LPS (2 µg/ml) of macrophages transfected for 72 h with a scramble (sc) control siRNA or with a TLR4 siRNA (n = 8 for rNAPRT and LPS, n = 6 for rNAMPT, paired t-test). g Western blot analysis of p-p65 in macrophages derived from TLR4-/- (n = 7) or wt (n = 5) mice treated as in d. h Box plots showing mRNA expression levels of CCL2 evaluated by qRT-PCR in macrophages derived from TLR4-/- (n = 6) or wt (n = 4) mice treated with rNAPRT (1 µg/ml), rNAMPT (1 µg/ml), and LPS (2 µg/ml) for 15 h, unpaired t-test. Results are reported as box plots, where the line in the box defines the median and the error bars define the minimum and maximum of all data. Source data are provided as a Source Data file

Fig 4: eNAPRT enhances differentiation of myeloid cells into macrophages via M-CSF secretion. a Box plots showing mRNA expression levels of CSF1 (left panel) in macrophages (n = 5) treated with rNAPRT (1 µg/ml), rNAMPT (1 µg/ml), and LPS (2 µg/ml, left panels) for 15 h (paired t-test) and M-CSF protein concentration in corresponding culture supernatants measured by ELISA (n = 8, right panel, Wilcoxon test). b Box plot representing the cell count of residual adherent cells obtained from HD PBMC preparations (n = 8) treated once at the beginning of culture with rNAPRT, rNAMPT, or LPS (all at 10 ng/ml). After 10–12 days of culture, cells were stained with Giemsa. Representative images (×10, scale bar: 200 µm, and a zoomed area in the square) are shown in c. Images were acquired using a CANON EOS 600D camera fitted to an AXIO Lab A1 ZEISS microscope. Cell count was calculated with ImageJ software (freely downloadable at http://rsbweb.nih.gov/ij/, paired t-test). d Confocal microscopy analysis of the expression of CD11b and CD68 in residual adherent cells obtained in the same conditions as in c (original magnification ×63, scale bar: 10 µm). Cells were counter-stained with Alexa568-phalloidin and DAPI. On the side, box plots indicating the relative mean fluorescence intensity of three different experiments. Mann–Whitney test. e eNAPRT protein concentration measured in macrophage supernatants by NAPRT luminex assay (n = 11) (f) and western blot performed on concentrated (×10) culture supernatants derived from two different HD macrophage preparation (SN MO). rNAPRT was loaded in the gel as control. g Confocal microscopy analysis of NAPRT protein expression in macrophages (original magnification ×63, the image represents a zoomed area). Cells were counter-stained with Alexa568-phalloidin and DAPI. Results are reported as box plots and dot plot, where the line in the box defines the median and the error bars define the minimum and maximum or the interquartile range. Source data are provided as a Source Data file

Fig 5: Structural determinants of rNAPRT and rNAMPT involved in TLR4 binding. a Western blot analysis of p-p65 in HDs macrophages upon treatments (30 min) with rNAPRT, rNAMPT, and their bacteria ortholog, PncB (n = 7) and NadV (n = 4), respectively (all at 1 µg/ml). Box plot on the right represents band quantification using ImageQuant software of p-p65/p65, Wilcoxon test. b Box plot showing relative quantification of nuclear p65 mean fluorescence intensity in human macrophages after 30 min treatments with rNAPRT, PncB, rNAMPT, and NadV (all at 1 µg/ml, at least n = 3, Mann–Whitney test). c Representative confocal microscopy images showing p65 localization (green fluorescence, original magnification ×63, scale bar: 25 µm). Cells were counter-stained with Alexa568-phalloidin and DAPI. d Superposition of dimeric human (Hs) (in pink, PDB: 4YUB) and E. faecalis (Ef, in gold, PDB: 4MZY) NAPRT in ribbon representation. The 46 amino acids insertion of HsNAPRT is highlighted in magenta. e The protein surfaces (top view) of HsNAPRT (PDB ID: 4YUB) and EfNAPRT (PDB ID: 4MZY) dimers are colored in white. The positively charged amino acids lysine and arginine are colored in cyan. The 46 amino-acid insertion of HsNAPRT is highlighted in yellow. The mouth-shaped areas of the two enzymes are contoured by a dotted oval. f Western blot analysis of p-p65 in HDs macrophages upon treatments (30 min at 1 µg/ml) with wt rNAPRT and R(65->73)A NAPRT mutant (n = 4). On the left, box plots represent band quantification using ImageQuant software of p-p65/p65, paired t-test. g Box plots showing mRNA expression levels of IL1B, IL8, and TNFA evaluated by qRT-PCR in RNA from HD macrophages (n = 8) treated with wt rNAPRT (1 µg/ml) and R(65->73)A NAPRT mutant (1 µg/ml) for 15 h, paired t-test. Results are reported as box plots, where the line in the box defines the median and the error bars define the minimum and maximum of all data. Source data are provided as a Source Data file