il 18bp  (R&D Systems)

Journal: bioRxiv

Article Title: Microglia-derived IL-18 remodels hippocampal plasticity to constrain traumatic fear memory

doi: 10.64898/2026.05.04.721266

Figure Lengend Snippet: a , Schematic of PTSD model establishment. The SPS&S (Single Prolonged Stress & Shock) procedure, consisting of sequential restraint, forced swim, anesthesia, and foot shock. b–c , Contextual fear freezing in SPS&S-exposed and control mice at day 8 ( b ) and day 15 ( c ) post-stress. **P < 0.01, unpaired two-tailed t-test; n = 10 mice per group. d–e , Representative images showing IL-18-GFP fluorescence in sagittal brain sections from IL-18-GFP reporter mice at day 3 post-SPS&S. Images show whole-brain overview with magnified insets of the hippocampus and medial prefrontal cortex (mPFC) ( d ), and basolateral amygdala (BLA). IL-18-GFP specks are represented by green dots generated using IMARIS software. ( e ), scale bars,1mm (overview), 200 μm (inset, hippocampus), 100 μm (inset, mPFC and BLA). f , Quantification of mean IL-18-GFP fluorescent intensity across hippocampus, mPFC, and BLA in control and SPS&S groups. **P < 0.01, ns, not significant; two-way ANOVA with Bonferroni’s post hoc test; n = 3 mice per group. g , Schematic of the IL-18 luciferase reporter assay for quantifying IL-18 bioactivity in dissected brain regions. h , Luciferase activity in hippocampus, mPFC, and BLA from control and SPS&S mice. **P < 0.01, ns, not significant; two-way ANOVA with Bonferroni’s post hoc test; n = 3 per group. i , Schematic of hippocampal tissue collection and processing for IL-18 mRNA quantification by qPCR and protein quantification by ELISA at multiple post-stress time points. n = 3 mice per group. j , Temporal profile of hippocampal IL-18 mRNA levels from 0 to 15 days post-SPS&S. *P < 0.05, **P < 0.01 vs. 0 h; one-way ANOVA with Dunnett’s post hoc test; n = 7-11 per time point. k , Temporal profile of hippocampal mature IL-18 protein levels (normalized to total protein) from 0 to 15 days post-SPS&S. **P < 0.01 vs. 0 h; one-way ANOVA with Dunnett’s post hoc test; n = 7-11 per time point. l , Experimental timeline for intrahippocampal cannula implantation, SPS&S exposure, and daily PBS or IL-18 binding protein (IL-18BP) infusion, with fear memory assessed at days 8 and 15 post-stress. IL-18BP infusion began on day −3 to allow accumulation of antagonist before stress onset, ensuring complete neutralization of stress-induced IL-18. m–n , Contextual fear freezing at day 8 ( m ) and day 15 ( n ) in mice receiving intrahippocampal IL-18BP or PBS following SPS&S. *P < 0.05, **P < 0.01, ***P < 0.001; two-way ANOVA with Bonferroni’s post hoc test; n = 5-9 per group. o , Experimental timeline for intrahippocampal cannula implantation, SPS&S exposure, and daily IL18 infusion, with fear memory assessed at days 8 and 15 post-stress. p–q , Contextual fear freezing at day 8 ( p ) and day 15 (q ) in mice receiving intrahippocampal IL18 or PBS following SPS&S. *P < 0.05, **P < 0.01, ***P < 0.001; two-way ANOVA with Bonferroni’s post hoc test; n = 6–10 per group. Complete statistics are provided in Supplementary Table 1.

Article Snippet: IL-18 binding protein (IL-18BP; MCE) was dissolved in sterile PBS and stored at −80°C.

Techniques: Control, Two Tailed Test, Fluorescence, Generated, Software, Luciferase, Reporter Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Neutralization

Journal: bioRxiv

Article Title: Microglia-derived IL-18 remodels hippocampal plasticity to constrain traumatic fear memory

doi: 10.64898/2026.05.04.721266

Figure Lengend Snippet: a, Representative confocal images of hippocampal sections from IL-18-GFP reporter mice at day 3 post-SPS&S. Scale bars, 10 μm. b, Experimental timeline for bilateral hippocampal AAV injection (day −21), SPS&S exposure (day 1), and contextual fear memory assessment (days 8 and 15). c, Contextual fear freezing at day 8 in mice receiving cell-type-specific AAV-shRNA targeting IL-18 in microglia (Iba1-shIL18), astrocytes (GFAP-shIL18), neurons (hSyn-shIL18 and CaMKII-shIL18), or scramble control (EF1-shCon), following SPS&S or control conditions. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant Two-way ANOVA with post-hoc Bonferroni correction, n=7-11 mice per group. d, Same as c at day 15. e, Representative confocal images of hippocampal sections stained for IL-18R1. Scale bars, 10 μm. f, Experimental timeline for bilateral hippocampal AAV injection (day −21), SPS&S exposure (day 1), and contextual fear memory assessment (days 8 and 15) for IL-18R1 knockdown experiments. g, Contextual fear freezing at day 8 in mice receiving cell-type-specific AAV-shRNA targeting IL-18R1 in microglia (Iba1-shIL18R1), astrocytes (GFAP-shIL18R1), or neurons (hSyn-shIL18R1), or scramble control (EF1-shCon), following SPS&S or control conditions. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant Two-way ANOVA with post-hoc Bonferroni correction, n=8-13 mice per group. h, Same as g at day 15. Complete statistics are provided in Supplementary Table 1.

Article Snippet: IL-18 binding protein (IL-18BP; MCE) was dissolved in sterile PBS and stored at −80°C.

Techniques: Injection, shRNA, Control, Staining, Knockdown

Journal: bioRxiv

Article Title: Microglia-derived IL-18 remodels hippocampal plasticity to constrain traumatic fear memory

doi: 10.64898/2026.05.04.721266

Figure Lengend Snippet: a, Experimental timeline for infusion tube installation (day −14), SPS&S exposure (day 1), daily intrahippocampal IL-18 or PBS infusion, and tissue collection for immunostaining at day 15. b, Representative confocal images of synaptophysin (yellow) immunostaining in hippocampal CA1 from PBS- and IL-18-treated mice under control and SPS&S conditions. Scale bars, 10 μm. c, Quantification of percent area synaptophysin staining across groups. ***P < 0.001, *P < 0.05, Two-way ANOVA with post-hoc Holm-Šídák, n=9-11 fields of view (FOVs) per group, from 3 mice per group. d, Quantification of normalized synaptophysin⁺ puncta density. Two-way ANOVA with post-hoc Holm-Šídák, n=9-11 fields of view (FOVs) per group, from 3 mice per group. e, Representative confocal images of VGLUT1 (green), Homer1 (red), and DAPI (blue) co-immunostaining in hippocampal CA1 from PBS- and IL-18-treated mice under control and SPS&S conditions. Scale bars, 10 μm. f, Quantification of normalized VGLUT1–Homer1 co-localization puncta. ***P < 0.001, Two-way ANOVA with post-hoc Bonferroni correction, n=18-21 fields of view (FOVs) per group, from 3 mice per group. g, Quantification of normalized VGLUT1⁺ puncta density. ***P < 0.001, Two-way ANOVA with post-hoc Bonferroni correction, n=18-21 fields of view (FOVs) per group, from 3 mice per group. h, Representative confocal images of WFA (yellow) and DAPI (blue) staining in hippocampal CA1 from PBS- and IL-18-treated mice under control and SPS&S conditions. Scale bars, 10 μm. i, Quantification of percent area WFA staining. **P < 0.01, *P < 0.05, Two-way ANOVA with post-hoc Bonferroni correction, n=6-13 fields of view (FOVs) per group, from 3 mice per group. Data are presented as mean ± SEM. Complete statistics are provided in Supplementary Table 1.

Article Snippet: IL-18 binding protein (IL-18BP; MCE) was dissolved in sterile PBS and stored at −80°C.

Techniques: Immunostaining, Control, Staining

Journal: bioRxiv

Article Title: Microglia-derived IL-18 remodels hippocampal plasticity to constrain traumatic fear memory

doi: 10.64898/2026.05.04.721266

Figure Lengend Snippet: a, Schematic of the TRAP2 engram-labeling system. AAV9-DIO-GFP was locally injected into the hippocampus of Fos-CreER T2 mice, enabling activity-dependent fluorescent labeling of SPS&S-activated neurons upon 4-OHT administration. b, Experimental timeline. DIO-GFP AAV was injected at day −14; mice received intrahippocampal IL-18 or PBS infusion 0.5h before SPS&S at day 1 and daily after, followed by intraperitoneal 4-OHT injection 1 hour later to complete engram labeling at day1. Brains were collected at day 15 for immunostaining. c, Representative confocal images of GFP (green, engram cells) and DAPI (blue) fluorescence in hippocampal sections from control and SPS&S-exposed mice under PBS or IL-18 treatment at day 15. Scale bars, 200 μm. d, Quantification of number of eGFP⁺ cells in hippocampus. ***P < 0.001, Two-way ANOVA with post-hoc Bonferroni correction, n=13 fields of view (FOVs) per group, from 3 mice per group. e, Schematic illustrating the synaptic markers (VGLUT1, Homer1, synaptophysin) analyzed within GFP⁺ engram cells. f, Representative confocal images of synaptophysin (yellow), DAPI (blue), and GFP (engram cell, white) immunostaining in hippocampal engram cells from PBS- and IL-18-treated mice under SPS&S conditions, scale bars, 10 μm. The magnified insets. Scale bars, 5 μm. g, Quantification of synaptophysin⁺ puncta contacts in engram cells per unit area (log scale). ns, not significant; unpaired two-tailed t-test; n=9 fields of view (FOVs) per group, from 3 mice per group. The extremely low baseline of engram cells in the control group precluded reliable quantification and thus precluded valid statistical comparisons. h, Representative confocal images of VGLUT1 (green), Homer1 (red), DAPI (blue), and GFP (engram cell, white) immunostaining within hippocampal engram cells from PBS- and IL-18-treated under control and SPS&S conditions, scale bars, 10 μm. The magnified insets. Scale bars, 5 μm. i, Quantification of VGLUT1⁺/Homer1⁺ co-localized puncta contacts in engram cells per unit area (log scale). ***P < 0.001, Unpaired Student’s t-test, n=14 fields of view (FOVs) per group, from 3 mice per group. j, Quantification of VGLUT1⁺ puncta contacts in engram cells per unit area (log scale). ***P < 0.001, Unpaired t-test with Welch’s correction, n=14 fields of view (FOVs) per group, from 3 mice per group. Extremely low baseline engram counts in the control group in panels i and j preclude reliable quantification (see also panel g ). Data are presented as mean ± SEM. Complete statistics are provided in Supplementary Table 1.

Article Snippet: IL-18 binding protein (IL-18BP; MCE) was dissolved in sterile PBS and stored at −80°C.

Techniques: Labeling, Injection, Activity Assay, Immunostaining, Fluorescence, Control, Two Tailed Test

Journal: Bioactive Materials

Article Title: Bone targeted microenvironment actuated engineered exosomes for precision therapy of IBD associated bone loss

doi: 10.1016/j.bioactmat.2026.04.034

Figure Lengend Snippet: Schematic illustration of the design, bone targeting, and therapeutic mechanism of ExoBIP in IBD-associated bone loss . BMSC-derived exosomes were engineered to generate ExoBIP for bone-targeted, inflammation-triggered IL-18 neutralization: IL-18BP was first coupled to the SDSSD bone-homing peptide, and the resulting IL-18BP-SDSSD conjugate was then grafted onto the exosome surface via an MMP9-responsive cleavable linker, enabling selective IL-18BP release within the inflammatory skeletal microenvironment while preserving IL-18's gut physiology. Following systemic administration, ExoBIP preferentially accumulates in bone tissue, where elevated matrix metalloproteinases (MMPs) in the inflammatory microenvironment cleave the linker to release IL-18BP. Neutralization of IL-18 suppresses T cell activation and IFN-γ–mediated inhibition of osteogenic activity, reduces RANKL-mediated osteoclastogenesis, and restores the balance between osteoclast and osteoblast activity, thereby alleviating IBD-induced bone loss.

Article Snippet: Cells were activated with plate-bound anti-CD3ε (1 μg/mL) and soluble anti-CD28 (1 μg/mL) in the presence of 0.5 ng/mL IL-2 for 24 h. Groups included a PBS control; 20 ng/mL recombinant IL-18 (RP02521, ABclonal); 20 ng/mL recombinant IL-18BP (50206-M08H, Sino Biological); 10 μM LY294002, a PI3K pathway inhibitor (S1737, Beyotime); 25 ng/mL IFN-γ (P6137, Beyotime); 25 ng/mL RANKL; 1 μg/mL IFN-γ Antagonist (HY-P4717, MCE); and 1 μg/mL Anti-Mouse RANKL Antibody (HY-P990165, MCE); 1 μM Ruxolitinib (HY-50856, MCE), a JAK1/2 pathway inhibitor; 5 μM BAY 11-7821 (HY-13453, MCE), an NF-κB pathway inhibitor.

Techniques: Derivative Assay, Neutralization, Preserving, Activation Assay, Inhibition, Activity Assay

Journal: Bioactive Materials

Article Title: Bone targeted microenvironment actuated engineered exosomes for precision therapy of IBD associated bone loss

doi: 10.1016/j.bioactmat.2026.04.034

Figure Lengend Snippet: Persistent IL-18 accumulation after intestinal recovery drives bone loss through T cell-mediated remodeling imbalance (see also Supporting ) (A) Schematic illustration of the DSS-induced colitis model, sulfasalazine (SASP) treatment. (B) Il1 8 mRNA expression in colonic tissue measured by qPCR. (C) Representative immunofluorescence staining of IL-18 in colonic sections from Healthy, UC, and UC + Treatment mice at day 28 and day 56. (D) Serum IL-18 levels measured by ELISA at indicated time points. (E) Il1 8 mRNA expression in femoral bone tissue determined by qPCR. (F) Representative immunofluorescence staining of IL-18 in femoral bone marrow. (G) Schematic illustration of IL-18-mediated bone loss in DSS-induced UC and the effect of IL-18BP. (H) Representative micro-CT images of trabecular bone from Healthy, UC, and IL-18BP-treated mice. (I) Quantitative micro-CT analysis of bone parameters, including BMD, BV/TV. (J) Representative immunofluorescence staining of OPN and OSX in femoral bone sections. (K) Quantification of OPN-positive and OSX-positive areas in bone tissue. (L) Representative TRAP staining of femoral bone sections. (M) Quantification of osteoclast number (N.Oc/B.Pm). (N) Representative images of double fluorochrome labeling (calcein and alizarin red) in femoral bone. (O) Quantification of dynamic bone formation parameters, including mineral apposition rate (MAR). (P) Schematic illustration of the CD8 + T cell-conditioned medium system used to assess indirect effects of IL-18 on osteogenic and osteoclastogenic differentiation. (Q) Representative ALP and ARS staining of BMSCs cultured under indicated treatments. (R) Representative TRAP staining of osteoclasts differentiated under indicated conditions. (S) Quantification of ALP-positive area, ARS-positive area, and TRAP-positive area. (T) Western blot analysis of PI3K-Akt signaling in CD8 + T cells treated with IL-18 and/or IL-18BP, with corresponding densitometric analysis. For (C-D, I-O, S), data are presented as mean ± s.d. One-way ANOVA followed by Tukey's multiple-comparisons test was used for multiple groups. p < 0.05 was considered statistically significant. Six mice per group. For qPCR, ΔCt values were analyzed (equivalent to log2-transformed fold changes), fold changes (2^−ΔΔCt) are shown for visualization. p < 0.05 was considered statistically significant. n = 3 biological replicates per group.

Article Snippet: Cells were activated with plate-bound anti-CD3ε (1 μg/mL) and soluble anti-CD28 (1 μg/mL) in the presence of 0.5 ng/mL IL-2 for 24 h. Groups included a PBS control; 20 ng/mL recombinant IL-18 (RP02521, ABclonal); 20 ng/mL recombinant IL-18BP (50206-M08H, Sino Biological); 10 μM LY294002, a PI3K pathway inhibitor (S1737, Beyotime); 25 ng/mL IFN-γ (P6137, Beyotime); 25 ng/mL RANKL; 1 μg/mL IFN-γ Antagonist (HY-P4717, MCE); and 1 μg/mL Anti-Mouse RANKL Antibody (HY-P990165, MCE); 1 μM Ruxolitinib (HY-50856, MCE), a JAK1/2 pathway inhibitor; 5 μM BAY 11-7821 (HY-13453, MCE), an NF-κB pathway inhibitor.

Techniques: Expressing, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Micro-CT, Labeling, Cell Culture, Western Blot, Transformation Assay

Journal: Bioactive Materials

Article Title: Bone targeted microenvironment actuated engineered exosomes for precision therapy of IBD associated bone loss

doi: 10.1016/j.bioactmat.2026.04.034

Figure Lengend Snippet: Engineering and Characterization of an MMP-Responsive Bone-Targeted Exosome (ExoBIP) (see also Supporting ) (A) Schematic of ExoBIP construction and mechanism of action. IL-18BP was dual-functionalized with an MMP-cleavable linker (PLGVR) and a bone-targeting peptide (SDSSD) via NHS–amine chemistry. Separately, BMSC-derived exosomes (Exo) were treated with TCEP to expose surface thiol groups (-SH). The maleimide-terminated IL-18BP complex was then conjugated to the thiol-activated exosomes via thiol–maleimide coupling to form ExoBIP. Upon reaching MMP-rich bone resorption sites, the linker is cleaved, releasing IL-18BP to neutralize IL-18, while BMSC-derived exosomes preserve their inherent osteogenic potential. Some elements of this figure were created with BioRender.com (license number: YG28QKC6SI). (B) Representative TEM image of ExoBIP stained with uranyl acetate (left) and TEM image of ExoBIP bound to secondary antibody-linked gold nanoparticles (right). (C) Nanoparticle tracking analysis (NTA) and zeta potential characterization. (D) Western blot analysis of exosomal markers (CD9, CD63, CD81, TSG101) and IL-18BP conjugation. (E) Representative super-resolution confocal microscopy images of ExoBIP, with IL-18BP (red) and Exo membrane (green). (F) Line-scan analysis of Exo and IL-18BP fluorescence intensity. Signal profiles across individual particles confirmed spatial overlap and surface colocalization. (G) Protease-responsive release kinetics of IL-18BP from ExoBIP. ELISA demonstrated that recombinant MMP9 triggered sustained IL-18BP release from ExoBIP, which was abrogated by MMP inhibitor GM6001. (H) ELISA-based binding analysis showing that activity of IL-18BP released from ExoBIP is similar to the original IL-18BP. (I) Colloidal stability of ExoBIP. ExoBIP maintained stable particle size and surface charge over 7 days at 4 °C (J) Lyophilization and reconstitution of ExoBIP. Lyophilized ExoBIP powder reconstituted in PBS exhibited a distinct Tyndall effect. (K) Post-rehydration characterization of ExoBIP. Particle size and zeta potential remained stable after lyophilization, supporting storage and application feasibility. For (C, G-H, I), data are presented as mean ± s.d. Two-tailed unpaired Student's t-test was used for comparisons between two groups, and one-way ANOVA followed by Tukey's multiple-comparisons test was used for multiple groups. p < 0.05 was considered statistically significant. For C, zeta potential was measured five times; for G and H, ELISA assays were performed in triplicate; and for I, measurements were repeated three times.

Article Snippet: Cells were activated with plate-bound anti-CD3ε (1 μg/mL) and soluble anti-CD28 (1 μg/mL) in the presence of 0.5 ng/mL IL-2 for 24 h. Groups included a PBS control; 20 ng/mL recombinant IL-18 (RP02521, ABclonal); 20 ng/mL recombinant IL-18BP (50206-M08H, Sino Biological); 10 μM LY294002, a PI3K pathway inhibitor (S1737, Beyotime); 25 ng/mL IFN-γ (P6137, Beyotime); 25 ng/mL RANKL; 1 μg/mL IFN-γ Antagonist (HY-P4717, MCE); and 1 μg/mL Anti-Mouse RANKL Antibody (HY-P990165, MCE); 1 μM Ruxolitinib (HY-50856, MCE), a JAK1/2 pathway inhibitor; 5 μM BAY 11-7821 (HY-13453, MCE), an NF-κB pathway inhibitor.

Techniques: Derivative Assay, Staining, Zeta Potential Analyzer, Western Blot, Conjugation Assay, Confocal Microscopy, Membrane, Fluorescence, Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay, Activity Assay, Lyophilization, Two Tailed Test

Journal: Bioactive Materials

Article Title: Bone targeted microenvironment actuated engineered exosomes for precision therapy of IBD associated bone loss

doi: 10.1016/j.bioactmat.2026.04.034

Figure Lengend Snippet: Bone-Targeting Efficiency and In Vivo Distribution of ExoBIP (see also Supporting ) (A) Representative ex vivo fluorescence images of osteoporotic femurs incubated with Cy5-labeled exosomes for 12 h (B) Quantification of Cy5-positive area showing significantly enhanced bone-binding affinity of ExoBIP over other formulations. (C) Representative in vivo IVIS images of whole-body biodistribution over 96 h following intravenous injection, and ex vivo of major organs and bones. (D) Time course radiance quantification confirming prolonged bone retention of ExoBIP compared with controls. (E) Ex vivo fluorescence imaging quantification of major organs and bones at 24 h post-injection. (F) Representative immunofluorescence images of femoral sections labeled with Cy3. (G) Quantification of Cy3-positive area in bone tissue. (H) Immunofluorescence staining of femoral sections for IL-18BP. (I) Quantification of IL-18BP-positive area in bone tissue. For (B, E, G, I), data are presented as mean ± s.d. Two-tailed unpaired Student's t-test was used for comparisons between two groups, and one-way ANOVA followed by Tukey's multiple-comparisons test was used for multiple groups. p < 0.05 was considered statistically significant. Six mice per group were used for in vitro bone-targeting experiments in (B) and bone tissue immunofluorescence in (G-H), whereas three mice per group were used for in vivo fluorescence imaging in (D-F).

Article Snippet: Cells were activated with plate-bound anti-CD3ε (1 μg/mL) and soluble anti-CD28 (1 μg/mL) in the presence of 0.5 ng/mL IL-2 for 24 h. Groups included a PBS control; 20 ng/mL recombinant IL-18 (RP02521, ABclonal); 20 ng/mL recombinant IL-18BP (50206-M08H, Sino Biological); 10 μM LY294002, a PI3K pathway inhibitor (S1737, Beyotime); 25 ng/mL IFN-γ (P6137, Beyotime); 25 ng/mL RANKL; 1 μg/mL IFN-γ Antagonist (HY-P4717, MCE); and 1 μg/mL Anti-Mouse RANKL Antibody (HY-P990165, MCE); 1 μM Ruxolitinib (HY-50856, MCE), a JAK1/2 pathway inhibitor; 5 μM BAY 11-7821 (HY-13453, MCE), an NF-κB pathway inhibitor.

Techniques: In Vivo, Ex Vivo, Fluorescence, Incubation, Labeling, Binding Assay, Injection, Imaging, Immunofluorescence, Staining, Two Tailed Test, In Vitro