Journal: Cell Reports Medicine

Article Title: Artificial exosomes synergistically reshape sepsis immune homeostasis by modulating neutrophil fate and blocking PD-1/PD-L1

doi: 10.1016/j.xcrm.2026.102819

Figure Lengend Snippet: Preparation, characterization, and interaction of AT@NV-PD1 nanoparticles with neutrophils (A) CLSM images show PD-1 (green) expression on engineered RAW 264.7 membranes (scale bars, 15 μm). (B and C) Flow cytometry quantifies PD-1 expression in primary vs. engineered cells. (D) Western blot analysis of PD-1 expression in primary and genetically engineered RAW 264.7 cells. (E and F) DLS and TEM reveal AT@NV-PD1’s hydrodynamic diameter (110–130 nm) and core-shell structure (scale bars, 50 nm). (G) CLSM demonstrates AT@NV-PD1 (red) uptake by activated neutrophils (scale bars, 10 μm). (H) Anti-PD-L1 pretreatment blocks uptake (scale bars, 10 μm). (I) Resting neutrophils show minimal uptake (scale bars, 10 μm). (J) Flow cytometry shows elevated PD-L1 on septic neutrophils. (K and L) Enhanced AT@NV-PD1 uptake in CLP-model neutrophils vs. healthy controls. (M–Q) Nanodecoys effectively neutralize inflammatory mediators (TNF-α, IL-1β, IL-6, PD-L1, LPS). All data are presented as mean ± SD ( n = 3), ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, ns, not significant.

Article Snippet: Lipopolysaccharide (LPS) , MedChemExpress (USA) , Cat. HY-D1056A1.

Techniques: Expressing, Flow Cytometry, Western Blot

Journal: Cell Reports Medicine

Article Title: Artificial exosomes synergistically reshape sepsis immune homeostasis by modulating neutrophil fate and blocking PD-1/PD-L1

doi: 10.1016/j.xcrm.2026.102819

Figure Lengend Snippet: AT@NV-PD1 regulates neutrophil fate and its mechanism in vitro (A and B) Flow cytometry analysis and quantitative results showing that AT@NV-PD1 treatment can significantly induce apoptosis in neutrophils stimulated by IFN-γ and LPS. (C) Representative immunofluorescence images of NETs; CitH3 (red), MPO (green), and DAPI (blue) staining, (scale bars, 20 μm). (D) Representative CLSM images of SYTOX Green-stained NETs. Nuclear DNA was stained with DAPI (blue), and extracellular DNA was stained with SYTOX Green (green), (scale bars, 20 μm). (E and F) Expression levels of NETs and IL-6 in neutrophils treated with different formulations, as measured by ELISA. Data are presented as mean ± standard deviation. Differences were evaluated by one-way ANOVA followed by Tukey’s multiple comparisons test, n = 5, ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗ p < 0.0001.

Article Snippet: Lipopolysaccharide (LPS) , MedChemExpress (USA) , Cat. HY-D1056A1.

Techniques: In Vitro, Flow Cytometry, Immunofluorescence, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Cell Reports Medicine

Article Title: Artificial exosomes synergistically reshape sepsis immune homeostasis by modulating neutrophil fate and blocking PD-1/PD-L1

doi: 10.1016/j.xcrm.2026.102819

Figure Lengend Snippet: AT@NV-PD1 attenuates T cell exhaustion and promotes immune function recovery and does not impair neutrophil generation in the bone marrow (A) Flow cytometry analysis of the CD4 + /CD8 + T cell ratio in peripheral blood from CLP mice 5 days after intervention with different formulations. (B and C) Representative flow cytometry plots (B) and quantitative analysis (C) of CD4 + T cells in peripheral blood. (D and E) Representative images (D) and quantification (E) of TUNEL staining for apoptosis in splenocytes from healthy control and sepsis-treated mice (scale bars, 100 μm). (F and G) Representative flow cytometry plots (F) and quantitative analysis (G) of Treg cells in peripheral blood. (H) Flow cytometry plots of CD11b + cells in peripheral blood. (I and J) Representative flow cytometry plots and quantitative analysis of M-MDSC cells in peripheral blood. (K) Mouse body weights were monitored during the experiment, including healthy mice, PBS-treated sepsis mice, and AT@NV-PD1 -treated sepsis mice (AT7519 at a dose of 5 mg/kg). (L–O) Mice were subjected to intraperitoneal (i.p.) LPS injection to establish a sepsis model, and AT@NV-PD1 treatment was administered 4 h after LPS administration. Four hours later, LPS-challenged mice were intravenously (i.v.) injected with AT@NV-PD1 (AT7519 at a dose of 5 mg/kg). Control mice received neither LPS nor AT@NV-PD1 treatment. After 72 h, all surviving mice and control (healthy) mice were challenged with LPS (i.t. [intratracheally], 10 mg/kg). At 84 h, bronchoalveolar lavage fluid (BALF) was collected to assess neutrophil counts, IL-1β, TNF-α, and IL-6 levels. All data are presented as mean ± standard deviation (SD), and differences were evaluated by one-way ANOVA followed by Tukey’s multiple comparisons test ( n = 5; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001).

Article Snippet: Lipopolysaccharide (LPS) , MedChemExpress (USA) , Cat. HY-D1056A1.

Techniques: Flow Cytometry, TUNEL Assay, Staining, Control, Injection, Standard Deviation

Journal: Cell Discovery

Article Title: Structure of the full-length human Pannexin1 channel and insights into its role in pyroptosis

doi: 10.1038/s41421-021-00259-0

Figure Lengend Snippet: a Schematic representation of hPANX1 heptamer in light blue docked into the cryo-EM density map. The middle panel shows the top view of hPANX1 with glycosylation indicated. The right panel shows a magnified view of the N-linked glycans (including two N-Acetylglucosamine and eight Mannose). b Representative images of the localization of WT and N255Q hPANX1 in THP-1-derived macrophages (MPs). The images were collected using the super-resolution STED mode. OE is the abbreviation of overexpression in all the figures. Scale bar = 5 µm. c Western blot analysis of the NLRP3 and Pro-IL-1β in WT hPANX1 overexpressing and N255Q hPANX1 overexpressing MPs. MPs were treated with LPS (1 µg/ml) and ATP (3 mM) for 24 h. GAPDH was used as a loading control. d IL-1β release from mock, WT hPANX1 overexpressing and N255Q hPANX1 overexpressing MPs. MPs were primed with LPS (1 µg/ml) overnight and stimulated with nigericin (10 µM) for 0.5, 1, or 2 h. IL-1β release was measured by ELISA. Error bars represent s.e.m. for n = 3. * P < 0.05, NS not significant (one-way ANOVA followed by Dunnett’s test, OE WT set as comparison control). e Phase-contrast images of MPs stimulated by LPS and ATP. Images were acquired with an IncusyteS3 system on day 6. f Time course of live cell confluence of MPs. Image data were collected every 2 h and analyzed with an IncusyteS3 system. Error bars represent s.d. for n = 4. *** P < 0.001, **** P < 0.0001 (one-way ANOVA followed by Dunnett’s test, OE WT set as comparison control).

Article Snippet: To differentiate THP-1 cells into macrophages (MPs), 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, P1585–1MG) was added to the growth medium and maintained for 24 or 48 h. THP-1 derived MPs were stimulated for the indicated time with 30 μM R848, pHrodo E. coli bioparticles (Life Technologies, P35361), 1 μg/ml LPS (Novus Biologicals, NBP2-25295), R848 plus bioparticles, LPS plus 3 mM ATP (Sigma-Aldrich, A2383-5G), or primed with LPS and stimulated with 10 μM nigericin (MERCK Sigma-Aldrich, 481990).

Techniques: Cryo-EM Sample Prep, Glycoproteomics, Derivative Assay, Over Expression, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Cell Discovery

Article Title: Structure of the full-length human Pannexin1 channel and insights into its role in pyroptosis

doi: 10.1038/s41421-021-00259-0

Figure Lengend Snippet: a Representative images of the localization of WT and mutant hPANX1 (D379A and S424A) in MPs. The images were collected using the super-resolution STED mode. Scale bar = 5 µm. b Western blot analysis of the NLRP3, Pro-IL-1β in WT and mutant (D379A and S424A) hPANX1 overexpressing MPs. MPs were treated with LPS (1 µg/ml) and ATP (3 mM) for 24 h. GAPDH was used as a loading control. c IL-1β release from mock, WT overexpressing and mutant (D379A and S424A) overexpressing MPs. MPs were primed with LPS (1 µg/ml) overnight and stimulated with nigericin (10 µM) for 0.5, 1, or 2 h. IL-1β release was measured by ELISA. Error bars represent s.e.m. for n = 3. * P < 0.05, ** P < 0.01, NS not significant (one-way ANOVA followed by Dunnett’s test, OE WT set as comparison control). d Phase-contrast images of MPs stimulated by LPS and ATP. Images were acquired with an IncusyteS3 system on day 6. e Time course of live cell confluence of MPs. Image data were collected every 2 h and analyzed with an IncusyteS3 system. Error bars represent s.d. for n = 4. *** P < 0.001, **** P < 0.0001 (one-way ANOVA followed by Dunnett’s test, OE WT set as comparison control).

Article Snippet: To differentiate THP-1 cells into macrophages (MPs), 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, P1585–1MG) was added to the growth medium and maintained for 24 or 48 h. THP-1 derived MPs were stimulated for the indicated time with 30 μM R848, pHrodo E. coli bioparticles (Life Technologies, P35361), 1 μg/ml LPS (Novus Biologicals, NBP2-25295), R848 plus bioparticles, LPS plus 3 mM ATP (Sigma-Aldrich, A2383-5G), or primed with LPS and stimulated with 10 μM nigericin (MERCK Sigma-Aldrich, 481990).

Techniques: Mutagenesis, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Altered Intestinal Morphology and Microbiota Composition in the Autism Spectrum Disorders Associated SHANK3 Mouse Model

doi: 10.3390/ijms20092134

Figure Lengend Snippet: Altered gut morphology in Shank3αβ knock-out (KO) mice. ( A – D ) Histological evaluation of GI tract from wild type and Shank3αβ KO mice. ( A ) Longitudinal cross sections of Shank3αβ KO mice and wild type (WT) mice were stained with hematoxylin/eosin (HE) (upper panels) and periodic acid schiff (PAS) reaction (lower panels). Exemplary images are shown. ( B – D ) Morphological analysis of ( B ) villi length and ( C ) width, and ( D ) crypt depth reveals a significantly decreased villi length (Mann-Whitney U -test, p = 0.009; n = 5 animals per group) but not width ( p = 0.534), and normal crypt depth ( p = 0.983) in Shank3αβ KO mice. ( E , F ) Immunohistochemistry was performed on 5 mice per group and 5 optic fields of view each from 3 sections per mouse were analyzed. ( E ) A slight but non-significant decrease in FABP2 signal intensity was observed in Shank3αβ KO mice compared to wild types (left panel). Significantly higher ZONULIN-1 levels were found in Shank3αβ KO mice (right panel) ( t -test, p = 0.0413). ( F ) The levels of CLAUDIN3 and lipopolysaccharide (LPS) were not significantly different between Shank3αβ KO mice and wild types in gut epithelium. ( G ) Significantly higher ZONULIN-1 levels in Shank3αβ KO mice were confirmed by western blotting using gut epithelium protein lysate ( t -test, p = 0.0434, n = 3 per group). ( H ) Protein lysate from liver tissue from WT and Shank3αβ KO mice ( n = 3 per group) were analyzed for E. coli LPS levels using Western Blotting. The results show significantly higher LPS levels in the liver of Shank3αβ β KO mice ( t -test, p = 0.0452). * p < 0.05, ** p < 0.01.

Article Snippet: Zonulin 1 antibody was purchased from Thermo Fisher Scientific (Invitrogen) (Waltham, MA, USA); Claudin3 antibody from Abcam (Berlin, Germany); FABP2 antibody from Thermo Fisher Scientific (Invitrogen); LPS antibody from Origene (Rockville, MD, USA); and IL6 antibody was purchased from Cell signaling Technologies (Danvers, MA, USA); GFAP antibody was purchased from Sigma Aldrich (St. Louis, MO, USA); Cytokeratin and Vimentin antibody from Abcam.

Techniques: Knock-Out, Staining, MANN-WHITNEY, Immunohistochemistry, Western Blot