Journal: The FASEB Journal

Article Title: ZXDB Drives Macrophage Inflammatory Programming in Sepsis‐Induced Acute Kidney Injury by Recruiting EIF4A3 to Enhance ACACA Translation

doi: 10.1096/fj.202502962RR

Figure Lengend Snippet: ZXDB is required for pro‐inflammatory macrophage activation and metabolic reprogramming. (A, B) Flow cytometric analysis of M1‐like (CD86 + INOS + ), M2a‐like (CD206 + ARG1 + ), and M2b‐like (CD86 + IL‐10 + ) surface markers on RAW264.7 (A) and THP‐1 (B) macrophages following stimulation with LPS (100 ng/mL) for 6 h, with or without Zxdb knockdown (shZxdb). Representative plots and quantification are shown. (C, D) Western blot analysis of key M1‐like (iNOS, CD40, CD86, CD80) and M2‐like (CD206, CD163, Arg1) protein markers in RAW264.7 (C) and THP‐1 (D) cells under the same conditions. GAPDH served as the loading control. (E, F) ELISA quantification of pro‐inflammatory (TNF‐α, IFN‐γ, IL‐1β) and anti‐inflammatory (IL‐10, IL‐4, IL‐13) cytokines secreted into the supernatant of RAW264.7 (E) and THP‐1 (F) cells. (G‐J) Assessment of metabolic state via relative lactate production (G, H) and intracellular ATP levels (I, J) in RAW264.7 and THP‐1 cells. p < 0.05, * p < 0.01, ** p < 0.001.

Article Snippet: Membranes were then incubated overnight at 4°C with primary antibodies against: ZXDB (A303‐656A; Invitrogen, 1:1000), ACACA (21923‐1‐AP; Proteintech, RRID:AB_11042445, 1:1000), EIF4A3 (17 504‐1‐AP; Proteintech, RRID:AB_2097393, 1:1000), iNOS (22226‐1‐AP; Proteintech, RRID:AB_2879038, 1:1000), CD86 (13395‐1‐AP; Proteintech–, RRID:AB_2074882, 1:1000), ARG1 (16001‐1‐AP; Proteintech–, RRID:AB_2289842, 1:1000), CD206 (18704‐1‐AP; Proteintech, RRID:AB_10597232, 1:1000), GAPDH (60004‐1‐Ig; Proteintech, RRID:AB_2107436, 1:10000).

Techniques: Activation Assay, Knockdown, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: ZXDB Drives Macrophage Inflammatory Programming in Sepsis‐Induced Acute Kidney Injury by Recruiting EIF4A3 to Enhance ACACA Translation

doi: 10.1096/fj.202502962RR

Figure Lengend Snippet: ACACA mediates the pro‐inflammatory action of ZXDB on macrophages. (A) Overlap of ZXDB‐regulated genes/proteins and ACACA‐interacting proteins. (B) Protein–protein interaction network showing ACACA's association with the ZXDB network. (C) ACACA mRNA expression in THP‐1 cells, treated with LPS (100 ng/mL) +/− shZxdb for 6 h. (D) ACACA protein expression in THP‐1 cells, treated as in (C). (E–H) Cell proliferation (E, G) and apoptosis (F, H) in RAW264.7 (E, F) and THP‐1 cells (G, H) subjected to rescue experiments (LPS +/− shZxdb +/− Acaca overexpression). (I‐J) M1‐like (CD86 + INOS + ), M2a‐like (CD206 + ARG1 + ), and M2b‐like (CD86 + IL‐10 + ) macrophage populations in RAW264.7 (I) and THP‐1 cells (J) from the rescue experiments. (K‐L) Cytokine secretion (TNF‐α, IFN‐γ, IL‐1β, IL‐10, IL‐4, IL‐13) in RAW264.7 (K) and THP‐1 cell supernatants from the rescue experiments. (M‐P) Relative ATP levels (M, N) and lactate production (O, P) in RAW264.7 (M, O) and THP‐1 cells (N, P) from the rescue experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were then incubated overnight at 4°C with primary antibodies against: ZXDB (A303‐656A; Invitrogen, 1:1000), ACACA (21923‐1‐AP; Proteintech, RRID:AB_11042445, 1:1000), EIF4A3 (17 504‐1‐AP; Proteintech, RRID:AB_2097393, 1:1000), iNOS (22226‐1‐AP; Proteintech, RRID:AB_2879038, 1:1000), CD86 (13395‐1‐AP; Proteintech–, RRID:AB_2074882, 1:1000), ARG1 (16001‐1‐AP; Proteintech–, RRID:AB_2289842, 1:1000), CD206 (18704‐1‐AP; Proteintech, RRID:AB_10597232, 1:1000), GAPDH (60004‐1‐Ig; Proteintech, RRID:AB_2107436, 1:10000).

Techniques: Expressing, Over Expression

Journal: The FASEB Journal

Article Title: ZXDB Drives Macrophage Inflammatory Programming in Sepsis‐Induced Acute Kidney Injury by Recruiting EIF4A3 to Enhance ACACA Translation

doi: 10.1096/fj.202502962RR

Figure Lengend Snippet: The ZXDB‐EIF4A3 Interaction Is required for Its Pro‐inflammatory Functions. (A) Co‐IP of HA‐EIF4A3 with Flag‐ZXDB or ZXDB‐MUT in THP‐1 cells. (B) ACACA protein expression in THP‐1 cells transfected with the indicated plasmids and treated with LPS for 6 h. (C) Polysome profiling of ACACA mRNA in THP‐1 cells with Flag‐ZXDB or ZXDB‐MUT, stimulated with LPS. (D) RIP‐qPCR analysis of ACACA mRNA associated with ZXDB or ZXDB‐MUT. (E‐F) Cell proliferation (E) and apoptosis (F) in THP‐1 cells transfected and treated with LPS for 6 h. (G) M1‐like (CD86 + INOS + ), M2a‐like (CD206 + ARG1 + ) and M2b‐like (CD86 + IL‐10 + ) populations in THP‐1 cells. (H) M1‐like (iNOS, CD40, CD86, CD80) and M2‐like (CD206, CD163, ARG1) protein expression in THP‐1 cells. (I) Cytokine secretion (IFN‐γ, TNF‐α, IL‐6, IL‐1β, IL‐4, IL‐13, IL‐10) in THP‐1 cell supernatants. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were then incubated overnight at 4°C with primary antibodies against: ZXDB (A303‐656A; Invitrogen, 1:1000), ACACA (21923‐1‐AP; Proteintech, RRID:AB_11042445, 1:1000), EIF4A3 (17 504‐1‐AP; Proteintech, RRID:AB_2097393, 1:1000), iNOS (22226‐1‐AP; Proteintech, RRID:AB_2879038, 1:1000), CD86 (13395‐1‐AP; Proteintech–, RRID:AB_2074882, 1:1000), ARG1 (16001‐1‐AP; Proteintech–, RRID:AB_2289842, 1:1000), CD206 (18704‐1‐AP; Proteintech, RRID:AB_10597232, 1:1000), GAPDH (60004‐1‐Ig; Proteintech, RRID:AB_2107436, 1:10000).

Techniques: Co-Immunoprecipitation Assay, Expressing, Transfection

Journal: The FASEB Journal

Article Title: ZXDB Drives Macrophage Inflammatory Programming in Sepsis‐Induced Acute Kidney Injury by Recruiting EIF4A3 to Enhance ACACA Translation

doi: 10.1096/fj.202502962RR

Figure Lengend Snippet: Macrophage‐Specific Deletion of Zxdb Protects Against Sepsis‐Induced Acute Kidney Injury. (A) Generation strategy for myeloid‐specific Zxdb knockout (Mac‐Zxdb‐KO) mice. (B) H&E staining of kidney sections from Sham, WT SI‐AKI, and Mac‐Zxdb‐KO SI‐AKI mice. (C‐D) Serum creatinine (Scr) (C) and blood urea nitrogen (BUN) (D) levels in Sham, WT SI‐AKI, and Mac‐Zxdb‐KO SI‐AKI mice at 24 h post‐CLP. (E) KIM‐1 immunohistochemistry of kidney sections from Sham, WT SI‐AKI, and Mac‐Zxdb‐KO SI‐AKI mice. (F) TUNEL staining of kidney sections from Sham, WT SI‐AKI, and Mac‐Zxdb‐KO SI‐AKI mice. (G) Immunofluorescence of kidney sections stained for F480, CD86, and CD206. (H) Serum IL‐1β, IL‐10, and TNF‐α levels in Sham, WT SI‐AKI, and Mac‐Zxdb‐KO SI‐AKI mice at 24 h post‐CLP. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were then incubated overnight at 4°C with primary antibodies against: ZXDB (A303‐656A; Invitrogen, 1:1000), ACACA (21923‐1‐AP; Proteintech, RRID:AB_11042445, 1:1000), EIF4A3 (17 504‐1‐AP; Proteintech, RRID:AB_2097393, 1:1000), iNOS (22226‐1‐AP; Proteintech, RRID:AB_2879038, 1:1000), CD86 (13395‐1‐AP; Proteintech–, RRID:AB_2074882, 1:1000), ARG1 (16001‐1‐AP; Proteintech–, RRID:AB_2289842, 1:1000), CD206 (18704‐1‐AP; Proteintech, RRID:AB_10597232, 1:1000), GAPDH (60004‐1‐Ig; Proteintech, RRID:AB_2107436, 1:10000).

Techniques: Knock-Out, Staining, Immunohistochemistry, TUNEL Assay, Immunofluorescence

Journal: The FASEB Journal

Article Title: ZXDB Drives Macrophage Inflammatory Programming in Sepsis‐Induced Acute Kidney Injury by Recruiting EIF4A3 to Enhance ACACA Translation

doi: 10.1096/fj.202502962RR

Figure Lengend Snippet: Schematic diagram of the proposed mechanism by which ZXDB promotes M1‐like macrophage polarization and exacerbates SI‐AKI. In macrophage, ZXDB interacts with EIF4A3, promoting the translation of the ACACA gene. The resulting increase in ACACA protein expression enhances glycolysis and lactate production. This metabolic reprogramming shifts macrophage polarization toward M1‐like macrophage activation (characterized by increased Cd86, Cd80, Cd40, and iNOS) and away from an anti‐inflammatory M2‐like phenotype (characterized by Cd206, Cd163, and Arg1). The dominance of M1‐like macrophages leads to an elevated secretion of pro‐inflammatory cytokines (TNF‐α, IFN‐γ, IL‐1β) and reduced anti‐inflammatory cytokines (IL‐10, IL‐4, IL‐13), which collectively drive the pathogenesis of acute kidney injury.

Article Snippet: Membranes were then incubated overnight at 4°C with primary antibodies against: ZXDB (A303‐656A; Invitrogen, 1:1000), ACACA (21923‐1‐AP; Proteintech, RRID:AB_11042445, 1:1000), EIF4A3 (17 504‐1‐AP; Proteintech, RRID:AB_2097393, 1:1000), iNOS (22226‐1‐AP; Proteintech, RRID:AB_2879038, 1:1000), CD86 (13395‐1‐AP; Proteintech–, RRID:AB_2074882, 1:1000), ARG1 (16001‐1‐AP; Proteintech–, RRID:AB_2289842, 1:1000), CD206 (18704‐1‐AP; Proteintech, RRID:AB_10597232, 1:1000), GAPDH (60004‐1‐Ig; Proteintech, RRID:AB_2107436, 1:10000).

Techniques: Expressing, Activation Assay

Journal: Cells

Article Title: Statins Modulate Microenvironmental Cues Driving Macrophage Polarization in Simulated Periodontal Inflammation

doi: 10.3390/cells12151961

Figure Lengend Snippet: MyD88−dependent modulation of M2 polarization by simvastatin. ( A ) A schematic representation of simvastatin, MyD88 inhibitor treatment, and THP-1-macrophages (M0–M1–M2). ( B ) Cell surface antigen analysis of M2 macrophage markers CD68, CD163, and CD206 was conducted using flow cytometry. Expression levels were normalized using IgG isotype control, and values were expressed in percentage of positive cells (mean ± SD) from three independent preparations. ( C ) RT-PCR analysis and heat maps summarizing TLR and NFkB signaling from M0 (CCL2), M1 (LPS, IFNγ, TNFα), and M2 (IL4, IL10, TGFβ) macrophages. (n = 3) preparations per treatment group, and data found to be significantly different were plotted as heat maps. ( D ) Graphical summary of statin-mediated suppression of TLR and NFkB signaling.

Article Snippet: Phenotyping of macrophages was performed in 1% BSA and 3% human serum PBS according to standard methods using a panel of antibodies targeting CD68 (R and D Systems Cat# IC20401P, Minneapolis, MN, USA, RRID: http://scicrunch.org/resolver/AB_2074835 , accessed on 28 April 2023), CD163 (R and D Systems Cat# FAB1607P, RRID: http://scicrunch.org/resolver/AB_2074536 , accessed on 28 April 2023), and CD206 (R and D Systems Cat# FAB25342P, RRID: http://scicrunch.org/resolver/AB_10889015 , accessed on 28 April 2023); antibodies all from R and D Systems.

Techniques: Flow Cytometry, Expressing, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Nature

Article Title: Bacteria hijack a meningeal neuroimmune axis to facilitate brain invasion.

doi: 10.1038/s41586-023-05753-x

Figure Lengend Snippet: Fig. 3 | CGRP and RAMP1 signalling contribute to bacterial meningitis. a,b, Role of the CGRP receptor RAMP1 in bacterial meningitis. a, Bacterial load in samples collected from Ramp1 knockout (Ramp1–/–) and control littermate (Ramp1+/–, Ramp1+/+) mice 24 h after injection with S. pneumoniae (3 × 107 c.f.u.). n = 5 (Ramp1+/+) or n = 4 (Ramp1–/–, Ramp1+/–). b, Flow cytometry quantification of meningeal macrophages (Cd11b+MRC1+ gates) and neutrophils (Cd11b+Ly6G+ gates) in Ramp1 knockout (Ramp1–/–) and control (Ramp1+/+) mice 24 h after injection with S. pneumoniae (3 × 107 c.f.u.). n = 4 per group. c,d, Impact of CGRP (2 μg, intraperitoneally (i.p.)) treatment on bacterial meningitis. c, Bacterial load in samples collected 24 h after injection with S. pneumoniae (3 × 107 c.f.u.) in mice treated with CGRP or vehicle. n = 6 per group. d, Flow cytometry quantification of meningeal macrophages (Cd11b+MRC1+ gates) and neutrophils (Cd11b+Ly6G+ gates) 24 h after injection with S. pneumoniae

Article Snippet: After 24 h of infection, whole-mount dural meninges was dissected and stained for meningeal macrophages (MRC1+ cells) with anti-mouse MRC1 (5 μg ml–1; AF2535, R&D Systems) and donkey anti-goat IgG conjugated with Alexa Fluor 488 (1:500; ab150129, Abcam) as described in the Methods.

Techniques: Knock-Out, Control, Injection, Flow Cytometry

Journal: Nature

Article Title: Bacteria hijack a meningeal neuroimmune axis to facilitate brain invasion.

doi: 10.1038/s41586-023-05753-x

Figure Lengend Snippet: Fig. 5 | Meningeal macrophages are required for host defence against S. pneumoniae infection. a,b, scRNA-seq analysis of meningeal immune responses to bacterial infection. a, Left, illustration of single-cell analysis of S. pneumoniae infection. Right, UMAP visualizations of CD45+ cell types in the meninges at baseline and 24 h after injection of S. pneumoniae (meningitis, 3 × 107 c.f.u.). pDC, plasmacytoid dendritic cells. b, Volcano plot showing genes that are differentially expressed (1,468 downregulated genes; 1,185 upregulated genes) in the cluster of Mrc1+ macrophages in response to infection (baseline versus meningitis), highlighting the upregulation of chemotaxis- related genes (n = 10 pooled meninges per group). c, Whole-mount confocal images of mouse meninges (dura mater) showing meningeal macrophages (MRC1+ cells) associated with CMTPX-labelled S. pneumoniae 24 h after injection of bacteria. Scale bar, 25 μm. d,e, Depletion of meningeal MRC1+ macrophages by intracisternal injection of CLLs 3 days before infection. d, Left, illustration of experimental scheme. Right, flow cytometry analysis of meningeal macrophages. n = 4 per group. e, Bacterial loads 24 h after injection with S. pneumoniae (3 × 107 c.f.u.) in mice treated with CLLs or vehicle. n = 4 per group. f, Whole- mount confocal images of meninges (dura mater) showing the presence of macrophages (CX3CR1+ cells) near CGRP+ nerve fibres. Scale bar, 10 μm. Statistical analysis: Wilcoxon rank-sum test, dashed line, P = 0.01 (b) or unpaired two- sided t-tests (d,e). *P < 0.05, **P < 0.01, ***P < 0.001. n = biologically independent samples from mouse tissues. Each experiment was performed at least twice, and results presented are representative of 2 or more replicates. Error bars indicate the mean ± s.e.m. Box plots show the median, IQR, and minimum and maximum values. Exact P values provided in Supplementary Table 1. Illustrations in a and d were created with BioRender.com.

Article Snippet: After 24 h of infection, whole-mount dural meninges was dissected and stained for meningeal macrophages (MRC1+ cells) with anti-mouse MRC1 (5 μg ml–1; AF2535, R&D Systems) and donkey anti-goat IgG conjugated with Alexa Fluor 488 (1:500; ab150129, Abcam) as described in the Methods.

Techniques: Infection, Single-cell Analysis, Injection, Chemotaxis Assay, Bacteria, Flow Cytometry

Journal: iScience

Article Title: Aurkb deficiency disrupts microglial development, homeostasis and hinders remyelination following cuprizone-induced demyelination

doi: 10.1016/j.isci.2026.114718

Figure Lengend Snippet: Aurkb loss transiently elevates CD68 in homeostatic microglia but compromises its upregulation in an LPS-induced inflammation model (A–D) Adult Aurkb fl/fl and Cx3cr1 CreERT2/+ Aurkb fl/fl littermates (8-week-old) were i.p. injected with TAM for 5 consecutive days, followed by tissue collection at 1-month and 3-month post TAM induction. Representative immunofluorescence and quantification of CD68 in microglia at (A and B) 1-month and (C and D) 3-month post-TAM induction ( n = 5 mice per genotype per time point, Scale bars: 50 μm). (E–H) Neonatal Aurkb fl/fl and Cx3cr1 CreERT2/+ Aurkb fl/fl littermates were i.p. injected with TAM for 3 consecutive days at P1-P3, followed by tissue collection at P13. Representative immunofluorescence and quantification of (E and F) CD68 and (G and H) CD206 in microglia at P13 ( n = 6 mice per genotype, Scale bars: 50 μm). (I and J) Representative immunofluorescence and quantification of CD68 in microglia from adult Aurkb fl/fl and Cx3cr1 Cre/+ Aurkb fl/fl littermates ( n = 5 mice per genotype, Scale bars: 50 μm). The representative immunofluorescence image of the Cx3cr1 Cre/+ Aurkb fl/fl group is shared in C. (K and L) Adult Aurkb fl/fl and Cx3cr1 Cre/+ Aurkb fl/fl littermates were i.p. injected with LPS (1 mg/kg) and sacrificed at 48 h post LPS administration ( n = 5 mice per genotype, Scale bars: 50 μm). Data are presented as the mean ± SD. Two-tailed unpaired t-tests in (B, D, J, and I); two-way ANOVA with Bonferroni multiple comparisons test in (F, H); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ns, not significant compared with the Aurkb fl/fl group. See also and .

Article Snippet: The following primary antibodies were used: rabbit anti-Iba-1 antibody (1:500, Wako, Cat: 019–19741), mouse anti-Iba-1 antibody (1:400, Abcam, Cat: ab283319), rat anti-CD206 antibody (1:200, Bio-Rad, Cat: MCA2235), rat anti-BrdU antibody (1:400, Abcam, Cat: ab6326), mouse anti-phospho-Histone H3 (Ser10) antibody (1:200, Cell Signaling Technology, Cat: 9706S), mouse anti-APC (1:100, CC-1, Merck, Cat: OP80), rat anti-myelin basic protein (Mbp) monoclonal antibody (1:500, Abcam, Cat: ab7349), rabbit anti-degraded myelin basic protein (dMbp) antibody (1:2000, Millipore Sigma, Cat: AB5864), rabbit anti-Olig2 antibody (1:500, Proteintech, Cat: 13999-1-AP) and rat anti-CD68 antibody (1:500, Abcam, Cat: ab53444).

Techniques: Injection, Immunofluorescence, Two Tailed Test