Journal: Advanced Science

Article Title: scH16S‐Seq Maps Klebsiella pneumoniae ‐Associated Myeloid States and Reveals a CD38–NAD Immunometabolic Axis That Impairs Lysosomal Acidification in Sepsis

doi: 10.1002/advs.78127

Figure Lengend Snippet: CD38–NAD Modulation Restores NAD Homeostasis and Mitochondrial Bioenergetics During KP Infection. (A) Intracellular NAD + abundance in Control and KP‐infected macrophages with or without NAM treatment, CD38 inhibition with 78C, or CD38 deficiency. NAD + abundance was normalized to cell number and expressed as pmol per 10 5 cells. (B) Intracellular NAD + /NADH ratio in the indicated groups. (C) Intracellular ATP abundance in the indicated groups, normalized to cell number and expressed as pmol per 10 5 cells. (D) Representative oxygen‐consumption rate (OCR) profiles obtained by Seahorse mitochondrial stress testing. Oligomycin, FCCP, and rotenone/antimycin A (ROT/AA) were sequentially injected at the indicated time points. (E–H) Quantification of spare respiratory capacity (E), maximal respiration (F), basal respiration (G), and ATP‐linked respiration (H) derived from the Seahorse mitochondrial stress test. (I) Representative flow cytometry histograms of MitoSOX Red fluorescence in iBMDMs infected with live KP for 3 h at MOI 50, with or without 78C pretreatment (2 µM, 1 h) or NAM pretreatment (2 mM, 4 h). (J) Quantification of MitoSOX Red fluorescence intensity in the groups shown in I; representative flow cytometry gating is shown below. (K) Representative fluorescence images of MitoSOX Red and Hoechst in iBMDMs across the indicated treatment groups. Scale bars, 20 µm. (L) Representative JC‐1 flow cytometry plots assessing mitochondrial membrane potential in Control and KP‐infected iBMDMs with or without 78C or NAM pretreatment. JC‐1 red aggregates indicate polarized mitochondria, whereas increased JC‐1 green monomers indicate mitochondrial depolarization. (M) Quantification of JC‐1 red and green fractions across the indicated groups. (N) Representative JC‐1 fluorescence images showing JC‐1 red, JC‐1 green, and Hoechst signals across treatment groups. Scale bars, 20 µm. (O) Proposed model summarizing the CD38–NAD–mitochondrial bioenergetic axis.

Article Snippet: Intracellular NAD + and NADH levels were measured using an NAD + /NADH Assay Kit (WST‐8; HY‐K0313, MedChemExpress) according to the manufacturer's instructions.

Techniques: Infection, Control, Inhibition, Injection, Derivative Assay, Flow Cytometry, Fluorescence, Membrane