Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: NLRC4 V341A KI mice exhibit inflammasome hyperactivation. A Diagram illustrating the design and generation of NLRC4 V341A-Flag KI mice. B Mendelian inheritance analysis of NLRC4 KI offspring as indicated. C – G Six-day-old E2aCre-NLRC4 V341A fl/fl (KI) and E2aCre-NLRC4 +/+ (wild-type, WT) mice were used. C Western blot for detecting mouse NLRC4 and NLRC4-V341A-Flag expression in colon tissues of NLRC4 WT and KI mice to confirm the genotyping. D Western blot analysis of NLRC4 V341A-Flag expression in the indicated tissues probed with Flag and β-actin antibodies. SI small intestine; IECs intestinal epithelial cells; BMDMs bone marrow-derived macrophages. E Inflammasome activation in IECs from NLRC4 WT and KI mice was analyzed via western blotting with the indicated antibodies (FL GSDMD, full-length Gasdermin-D; GSDMD-N,N-terminal GSDMD. F IL-1β and IL-18 levels in colon explant cultures from NLRC4 WT and KI mice were measured via ELISA ( n = 6 per group). Colon tissues were cultured in 1 ml of medium overnight, and the supernatant was collected for analysis. G IL-1β and IL-18 levels in NLRC4 WT and KI mouse serum were measured via ELISA ( n = 10 per group). In ( F , G ), the data are shown as the mean ± SEM; ****, p < 0.0001 was determined by Student’s t test. The data are representative of at least three independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

Techniques: Western Blot, Expressing, Derivative Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: NLRC4 V341A KI mice develop autoinflammation. A Gross phenotype of NLRC4 WT (left) and NLRC4 KI (right) mice on day 6 after birth. B Body weight (left) and survival rate (right) of NLRC4 WT (NLRC4 +/+ ) and NLRC4 KI (NLRC4 KI/KI ) mice after birth were plotted ( n = 20 per group). P values were determined via two-way ANOVA and the Mantel‒Cox test. C Serum ferritin, hemoglobin and IL-6 levels in 6-day-old NLRC4 WT and NLRC4-KI mice were measured via ELISA ( n = 8 per group). D Chemical parameters of blood from NLRC4 WT and KI mice were measured as indicated. ALT, alanine aminotransferase; BUN, blood urea nitrogen; LDH, lactate dehydrogenase. Sera were pooled from 8–10 6-day-old mice per group. E Blood cells from 6-day-old NLRC4 WT and NLRC4-KI mice were counted for the indicated cellular parameters ( n = 5 per group). WBC, white blood cell; RBC, red blood cell. F Hemophagocytosis in the spleen was analyzed by flow cytometry. Splenocytes from 6-day-old NLRC4 WT and NLRC4 KI mice were first stained with fluorescence-conjugated CD11b (myeloid cells); then, the stained cells were incubated with unconjugated Ter119 antibodies to block corresponding surface antigens; and finally, the cells were permeabilized for intracellular staining of phagocytosed Ter119+ cells ( n = 3 per group). G Isolated macrophages from the spleens of 6-day-old NLRC4 WT and KI pups were analyzed for inflammatory gene expression as indicated by Q-PCR ( n = 4 per group). The data are shown as the means ± SEMs. p values were determined by Student’s t test ( C , E - G ), * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. The data are representative of three independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Fluorescence, Incubation, Blocking Assay, Isolation, Gene Expression

Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: NLRC4 V341A KI mice develop infantile enterocolitis. A Representative H&E staining of small intestine and colon tissues from 6-day-old NLRC4 WT and KI mice as indicated. B Inflammatory cell infiltration in lamina propria from colon tissues of 6-day-old NLRC4 WT and KI mice was analyzed by flow cytometry ( n = 5 per group). C Colon tissues from 6-day-old NLRC4 WT and KI mice were analyzed for inflammatory gene expression as indicated by real-time PCR ( n = 4 per group). D IL-6 and TNF-α levels in the supernatants of colon explant cultures from 6-day-old NLRC4 WT and -KI mice were measured via ELISA ( n = 6 per group). E Representative images of gross colons from 6-day-old NLRC4 WT and KI mice. F Colon diameter, colon index (colon weight/body weight), wet/dry feces ratio and stool score of 6-day-old NLRC4 WT and KI mice are shown as indicated ( n = 10 per group). The data are shown as the means ± SEMs. p values were determined by Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. The data are representative of three independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

Techniques: Staining, Flow Cytometry, Gene Expression, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: Gut barrier integrity is impaired in NLRC4 V341A KI mice. A Representative images of different histochemical stains as indicated; samples were colon (PAS staining) or otherwise distal small intestine tissues from 6-day-old NLRC4 WT and NLRC4 KI mice. Periodic acid–Schiff (PAS) staining was used for goblet cells, lysosomal staining was used for Paneth cells, TUNEL was used for cell death, and ZO-1 was used for tight junction assessment. B Western blot analysis of the type of cell death in distal small intestine tissues from 6-day-old NLRC4 WT and NLRC4 KI mice. Cleaved Gasdermin D (GSDMD-N) is indicative of pyroptosis, p-MLKL is a marker of necroptosis, and cleaved caspase 3 signifies apoptotic cell death. C Representative tight junction genes, as indicated, were analyzed via Q‒PCR. The samples were intestinal epithelial cells isolated from 6-day-old NLRC4 WT and NLRC4 KI mice ( n = 4 per group). p values were determined by Student’s t test, ** p < 0.01. The data are shown as the means ± SEMs. The data are representative of three independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

Techniques: Staining, TUNEL Assay, Western Blot, Marker, Isolation

Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: Kinetic analysis of AIFEC development in NLRC4 V341A KI pups. A Representative H&E staining of small intestine (left) and colon (right) tissues from 0-, 3-, and 6-day-old NLRC4 WT and NLRC4-KI mice. B IL-1β, IL-18 and IL-6 levels in the supernatants of colon explant cultures from NLRC4 WT and KI mice were measured via ELISA. C Colon tissues from 0-, 3-, and 6-day-old NLRC4 WT and NLRC4-KI mice were analyzed for inflammatory gene expression as indicated by Q‒PCR. D The levels of serum ferritin and IL-6 in 0-, 3-, and 6-day-old NLRC4 WT and NLRC4 KI mice were measured via ELISA. Sample size: n = 4/group. The data are shown as the means ± SEMs. p values were determined by Student’s t test, * p < 0.05, ** p < 0.01 and *** p < 0.001. The data are representative of three independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: IL18BP treatment improves survival in NLRC4 V341A KI pups with AIFEC. IL-18BP was administered to NLRC4 V341A KI pups via intraperitoneal (i.p.) injection at a dose of 8 mg/kg from day 1 to day 7 after birth. The NLRC4 WT (WT) and KI control groups received equivalent volumes of saline. Pups were monitored daily for weight and survival. A Body weights and survival rates of the indicated groups. B Representative H&E staining of colon tissues from 8-day-old and 22-day-old mice as indicated. C Stool scores of the indicated groups of mice on days 8 and 22 posttreatment. D Wet-to-dry feces ratios in the indicated groups. E Inflammatory gene expression in colon tissues from 8-day-old and 22-day-old mice was analyzed by real-time PCR. F Red blood cell (RBC) counts in blood samples from 8-day-old and 22-day-old mice. G Hemophagocytosis in the spleen was analyzed by flow cytometry, which revealed the percentage of myeloid cells that phagocytosed RBCs. H Serum levels of ferritin, IL-1β, and IL-18 in 8-day-old and 22-day-old mice were measured via ELISA. The data are presented as the means ± SEMs. Sample size: n = 5/group, except for panels E&G ( n = 4/group). Statistical significance was determined via Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001). The results are representative of at least two independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

Techniques: Injection, Control, Saline, Staining, Gene Expression, Real-time Polymerase Chain Reaction, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: TNFα blockade dramatically alleviates AIFEC in NLRC4 V341A KI pups. Infliximab was administered to NLRC4 V341A KI pups via intraperitoneal (i.p.) injection at a dose of 5 mg/kg from day 1 to day 7 after birth. The control groups received equivalent volumes of isotype control antibodies. Pups were monitored daily for weight and survival. A Body weights and survival rates of the indicated groups. B Representative H&E staining of colon tissues from 8-day-old and 22-day-old mice as indicated. C Stool scores of the indicated groups of mice on days 8 and 22 posttreatment. D Wet-to-dry feces ratios in the indicated groups. E Inflammatory gene expression in colon tissues from 8-day-old and 22-day-old mice was analyzed by real-time PCR. F Red blood cell (RBC) counts in blood samples from 8-day-old and 22-day-old mice. G Hemophagocytosis in the spleen was analyzed by flow cytometry, which revealed the percentage of myeloid cells that phagocytosed RBCs. H Serum levels of ferritin, IL-1β, and IL-18 in 8-day-old and 22-day-old mice were measured via ELISA. The data are presented as the means ± SEMs. Sample size: n = 5/group, except for panels E&G ( n = 4/group). Statistical significance was determined via Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001). The results are representative of at least two independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

Techniques: Injection, Control, Staining, Gene Expression, Real-time Polymerase Chain Reaction, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Immunology

Article Title: An animal model of NLRC4-associated autoinflammation and infantile enterocolitis reveals novel therapeutic strategies

doi: 10.1038/s41423-025-01355-x

Figure Lengend Snippet: Glucose supplementation protects NLRC4 V341A KI pups from AIFEC. Glucose was administered to NLRC4 V341A KI pups via intraperitoneal (i.p.) injection at a dose of 200 mg/kg from day 1 to day 7 after birth. The control groups received equivalent volumes of saline. Pups were monitored daily for weight and survival. A Body weights and survival rates of the indicated groups. B Representative H&E staining of colon tissues from 8-day-old and 22-day-old mice as indicated. C Stool scores of the indicated groups of mice on days 8 and 22 posttreatment. D Wet-to-dry feces ratios in the indicated groups. E Inflammatory gene expression in colon tissues from 8-day-old and 22-day-old mice was analyzed by real-time PCR. F Red blood cell (RBC) counts in blood samples from 8-day-old and 22-day-old mice. G Hemophagocytosis in the spleen was analyzed by flow cytometry, which revealed the percentage of myeloid cells that phagocytosed RBCs. H Serum levels of ferritin, IL-1β, and IL-18 in 8-day-old and 22-day-old mice were measured via ELISA. The data are presented as the means ± SEMs. Sample size: n = 5/group, except for ( E, G ) ( n = 4/group). Statistical significance was determined via Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001). The results are representative of at least two independent experiments

Article Snippet: The NLRC4 V341A-Flag flox/flox mice were generated by Cyagen Biosciences via Cyagen’s TurboKnockout ® gene targeting service through homologous recombination.

Techniques: Injection, Control, Saline, Staining, Gene Expression, Real-time Polymerase Chain Reaction, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Neurochemical Research

Article Title: ANA-12 Targets and Inhibits BDNF/TrkB Signaling to Alleviate Pain Behaviors in Rheumatoid Arthritis Mice

doi: 10.1007/s11064-025-04487-8

Figure Lengend Snippet: Changes of NLRC4 and AIM2 inflammasomes in each group. ( A , C ) Representative immunofluorescence images of NLRC4 and AIM2 in the LC. Scale bar = 50 μm. ( B , D ) Quantitative analysis of the average fluorescence intensity in the left and right LC regions of NLRC4 and AIM2. Data are presented as mean ± SD, n = 3, * P < 0.05 compared to control mice; # P < 0.05 compared to CIA mice. CIA, collagen-induced arthritis; ANA, ANA-12

Article Snippet: Anti- TrkB (Rabbit, A19832), Iba1 (Rabbit, A12391) and NLRC4 (Rabbit, A7382), antibodies were purchased from ABclonal Technology.

Techniques: Immunofluorescence, Fluorescence, Control

Journal: Neurochemical Research

Article Title: ANA-12 Targets and Inhibits BDNF/TrkB Signaling to Alleviate Pain Behaviors in Rheumatoid Arthritis Mice

doi: 10.1007/s11064-025-04487-8

Figure Lengend Snippet: The effect of ANA-12 treatment on inflammatory signaling in U251 cells upon IL-1β. ( A , B , C ) Colocalization of BDNF/NLRP3/IL-1β (green) and GFAP (red) in U251 cells. Scale bar = 50 μm. ( D ) Quantitative analysis of the immunofluorescence intensity of BDNF, NLRP3, IL-1β, and GFAP in cells. ( E ) Immunoblot analysis of GFAP and Iba-1 expression in U251 cells. ( F ) Quantitative analysis of GFAP and Iba-1 protein level. ( G ) Immunoblot analysis of BDNF, TrkB, and pTrkB expression in U251 cells. ( H ) Quantitative analysis of BDNF, pTrkB/TrkB protein level. ( I ) Immunoblot analysis of MAPK, pMAPK, NLRP3, and caspase-1 expression in U251 cells. ( J ) Quantitative analysis of pMAPK/MAPK, NLRP3, and caspase-1 protein level. ( K ) Immunoblot analysis of NLRC4 and AIM2 expression in U251 cells. ( L ) Quantitative analysis of NLRC4 and AIM2 protein level. Data are presented as mean ± SD, n = 3, * P < 0.05 compared to control groups; # P < 0.05 compared to IL-1β groups. CIA, collagen-induced arthritis; ANA, ANA-12

Article Snippet: Anti- TrkB (Rabbit, A19832), Iba1 (Rabbit, A12391) and NLRC4 (Rabbit, A7382), antibodies were purchased from ABclonal Technology.

Techniques: Immunofluorescence, Western Blot, Expressing, Control