NAD+ protects mice from lethal bacterial infection and endotoxic shock by dampening systemic inflammation

(A) mice were treated with PBS or NAD+ prior to administration of a lethal dose of either pathogenic E. coli or LPS by intraperitoneal injection. (B) Kidneys and Livers were removed after and bacterial load was determined by counting CFU (C) Survival was monitored over 48 hours after bacterial infection and (D) LPS injection of both serotypes (n=6, 3 independent survival experiments). In addition, body temperature was monitored in the kinetics of up to 100 hours (E) Lungs, Kidneys and Livers were removed IHC was performed for H&E staining (F) Systemic levels (serum) ofIL-6, TNFα, IL-1β and IL-18 were assessed by ELISA. Column plot display mean with standard deviation (n=5). Statistical significance was determined by using Student’s T-test or One-Way-ANOVA while survival data were compared using log-rank Mantel-Cox test. Asterisks indicate p-values * = p<0.05, **= p<0.01 and *** = p<0.001, only significant values are shown.

NAD+ specifically inhibits the non-canonical inflammasome by targeting caspase 11

Bone marrow was isolated from C57BL/6 mice and BMDM were differentiated in vitro. Subsequently, BMDM were cultured in the presence of NAD+ or PBS for 2 following days. BMDMs were then primed with either Pam3CSK4 or LPS for 5-6 hours. Next primed BMDMs were stimulated for 16 hours with ATP or LPS and CTB. (A) Pro-casp-1, Pro-casp-11, Casp-11, NLRP3, Casp-1, IL-1β and GSDMD expression were determined using Western blot and (B) IL-1β secretion and LDH release were assessed in the supernatant. (C) Time dependent Caspase-1 expression was determined via active staining and assessed using a confocal microscope. (D) Cell viability and apoptosis were monitored using the IncuCyte® live microscopy system (E) LPS transfection with CTB was visualized by using FITC-coupled LPS and DAPI staining and quantified by confocal microscopy and flow cytometry. (F) Human macrophages differentiated from PBMC, was primed with Pam3CSK4 for 5-6 hours and subsequently transfected with LPS and Fugene HD Plus. Column plots display mean with standard deviation (n=6). Statistical significance was determined by using Student’s T-test or One-Way-ANOVA. Asterisks indicat p-values * = p<0.05, **= p<0.01 and *** = p<0.001, only significant values are shown.

NAD+ mediated inhibition of the non-canonical inflammasome is based on an impaired response to IFN-β

Differentiated BMDMs were cultured in the presence of NAD+ or PBS for 2 following days. BMDMs were then primed with Pam3CSK4, subsequently stimulated with LPS and CTB and RNA sequencing was performed. Unstimulated BMDMs served as controls. (A) Venn diagram plotting common gene expression between all 3 groups (B) Gene ontology enrichment analysis displaying the highest significant pathways differing when comparing NAD+ and PBS treated BMDMs (C) Expression cluster analysis of genes involved in IFN-β signaling through cluster analysis depicted in a heat map (D) Vulcano plot displaying the most significant genes up or downregulated comparing NAD+ and PBS treated BMDMs (E) Stimulated BMDMs were additionally treated with recombinant INF-β, and IL-1β and LDH release were measured. (F) Moreover, pro-Casp-1, Casp-11, NLRP3, GSDMD, (G) STAT-1 and Phospho-STAT-1 expression were assessed by Western blot.

Column plots display mean with standard deviation (n=6). Statistical significance was determined by using Student’s T-test or One-Way-ANOVA. Asterisks indicate p-values * = p<0.05, **= p<0.01 and *** = p<0.001, only significant values are shown.

Inhibitory effects of NAD+ on IFN-β downstream signaling and inflammasome activation

NAD+ inhibits STAT-1 expression and phosphorylation, thus compromising the intracellular response to IFN-β. Subsequently, stimulation of the IFNAR receptor by IFN-β leads to a decreased transcription of pro-caspase-11 as well as ISGs (IFN-inducible GTPases and GBPs). Due to diminished caspase-11 levels, non-canonical inflammasome activation through intracellular, gram-negative bacteria opsonization by GBPs is significantly inhibited.

IL-10 constitutes an additional mechanism mediating the protective capacities of NAD+ in the context of septic shock

(A) Caspase-11-/- mice were treated with NAD+ or PBS. Subsequently mice were subjected to Poly(I:C) prior to LPS injection and survival was monitored (n=5, 2 independent survival experiments). Mice treated with either NAD+ or PBS were injected with LPS and after 10 hours, Splenic frequencies of IL-10 producing (B) Macrophages and Dendritic cells (C) and CD4+ and CD8+ T cells were assessed by flow cytometry. (D) BMDMs treated with NAD+ or PBS were stimulated with LPS and CTB in the presence of IL-10 neutralizing antibodies and IL-10 receptor antagonists. Subsequently IL-1β and LDH release were assessed. (E) IL-10-/- mice treated with NAD+ or PBS were challenged with LPS and survival was monitored (n=5- 7, 2 independent survival experiments). Column plots display mean with standard deviation (n=6). Statistical significance was determined by using Student’s T-test or One Way-ANOVA while survival data were compared using log-rank Mantel-Cox test. Asterisks indicate p-values * = p<0.05, **= p<0.01 and *** = p<0.001, only significant values are shown.

NAD+ preserves ileal villi structure and reduces splenic hemorrhage during LPS induced septic shock

C57BL/6 mice were treated with PBS or NAD+ for 2 days prior to administration of a lethal dose of LPS (O55:B5 /O111:B4) by intraperitoneal injection. Ileum and Spleen were removed after 15 hours and subsequently IHC for H&E staining was performed.

NAD+ does not alter BMDM derived NF-κB expression or phosphorylation

Differentiated BMDM were cultured in the presence of 100µmol NAD+ or PBS for 2 following days. BMDMs were then primed with 1µg/ml Pam3CSK4 and subsequently stimulated with 2µg/ml LPS O111:B4 and 20µg/ml CTB. Unstimulated BMDMs served as controls. (A) P52 and p65 expression was determined using Western blot. (B) stimulated BMDMs were stained with p52, p65 and phospho-p65 and expression levels assessed using confocal microscopy.

Unstimulated BMDM cell viability and apoptosis

Differentiated BMDMs were cultured in the presence of 100µmol NAD+ or PBS for 2 following days. BMDMs were then primed with 1µg/ml Pam3CSK4, subsequently stimulated with 2µg/ml LPS O111:B4 and 20µg/ml CTB and cell viability and apoptosis were monitored for 100 hours using the IncuCyte® live microscopy system.

Neutrophils per mm2 infiltrating mice Ileum, Kidney Lung and Liver in the IHC stains.

C57BL/6 mice were treated with PBS or NAD+ for 2 days prior to administration of a lethal dose of LPS (O55:B5 /O111:B4) by intraperitoneal injection. After a 15-hour interval, the ileums, lungs, kidneys, and livers were extracted and subjected to immunohistochemical staining with hematoxylin and eosin (H&E). Neutrophil quantification was performed subsequent to the immunohistochemical staining in various tissue samples. Notably, in the Kidney, Lung, and Liver tissues, the number of neutrophils was observed to be significantly higher in the PBS-treated mice group as compared to the NAD+ treated mice group. Column plots display mean with standard deviation (n=4-5). Statistical significance was determined by using Student’s T-test or One-Way-ANOVA. Asterisks indicate p-values * = p<0.05, **= p<0.01.