Journal: International Journal of Molecular Sciences

Article Title: Targeting CD177: A Novel Therapeutic Strategy for NLRP3-Associated Autoinflammatory Diseases

doi: 10.3390/ijms27062841

Figure Lengend Snippet: CD177 is elevated in patients with NLRP3-AID. ( A ) Venn diagram shows the results of the transcriptome sequencing analysis. ( B ) Heatmap shows differentially expressed genes. ( C ) Flow cytometry detects the expression of CD177 in peripheral blood of NLRP3 mutant patient (PT) and healthy controls (H). ( D ) GEPIA analysis of the expression of CD177 in different immune cells in peripheral blood. ( E ) qPCR assay detects CD177 expression in peripheral blood neutrophils. ( F , G ) qPCR assay detects the expression of CD177 in neutrophils from two healthy controls (H3 and H4) treated with NLRP3 agonist (Nigericin) or NLRP3 inhibitor (CY-09). ( H – K ) qPCR assay detects the expression of IL-1β, IL-6, TNFα and IL-18 in neutrophils from HCs treated with Nigericin or CY-09. ( L ) GEPIA analysis of the expression correlation between CD177 and IL-1β. ( M ) qPCR assay examines the expression of CD177 in MCF-7 cells transfected with CD177 siRNA or negative control siRNA. ( N ) Western blot analysis of CD177 protein levels in MCF-7 cells transfected with CD177 siRNA or negative control siRNA. ( O ) The mRNA levels of IL-1β, IL-6, TNFα and IL-18 in MCF-7 cells transfected with CD177 siRNA or negative control siRNA. qPCR Data are shown as mean ± s.d. Student’s t -test was used. Statistical significance was determined as * p < 0.05; *** p < 0.001.

Article Snippet: The levels of IL-1β and IL-6 in the serum were detected using mouse-specific IL-6 (CSB-E04639m, Cusabio, Wuhan, China) and IL-1β (CSB-E08054m, Cusabio, Wuhan, China) detection kits according to the manufacturer’s instructions.

Techniques: Sequencing, Flow Cytometry, Expressing, Mutagenesis, Transfection, Negative Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Targeting CD177: A Novel Therapeutic Strategy for NLRP3-Associated Autoinflammatory Diseases

doi: 10.3390/ijms27062841

Figure Lengend Snippet: NLRP3 mutant mice exhibit phenotypic heterogeneity despite identical genotypes. ( A ) Images of wild-type ( Nlrp3 +/+ ) mice and NLRP3 mutant ( Nlrp3 L573W/+ ) mice with different inflammatory states. Notably, both mild and severe phenotypic groups are confirmed to be heterozygous for the p.L573W mutation. ( B ) Body weight of wild-type mice, NLRP3 mutant (Nlrp3 L573W/+) mice with different inflammatory states (weight measured starting from day 12, every other day). ( C ) Flow cytometry analysis of neutrophils in mouse peripheral blood ( n = 6 per group). ( D – F ) Histopathological detection of mouse tissues (skin, liver, spleen) showing varying degrees of inflammatory infiltration, Scale bars: 400 μm, 100 μm. ( G – I ) Immunohistochemical detection of mouse tissues (skin, liver, spleen) showing differences in neutrophil marker Ly6G expression, Scale bars: 200 μm, 50 μm. ( J ) Cytokine microarray in serum of wild-type mice and NLRP3 mutant mice with different inflammatory states. ( K , L ) Flow cytometry detection of IL-1β and IL-6 protein expression in mouse peripheral blood cells ( n = 6 per group). ( M – O ) qPCR detection of IL-1β, IL-6, and TNFα mRNA expression levels in mouse skin, liver, and spleen tissues ( n = 3). qPCR Data are shown as mean ± s.d. Student’s t -test was used. Statistical significance was determined as ** p < 0.01; *** p < 0.001; ns, not significant. CD177 expression is elevated in NLRP3 mutant mice and correlates with disease severity.

Article Snippet: The levels of IL-1β and IL-6 in the serum were detected using mouse-specific IL-6 (CSB-E04639m, Cusabio, Wuhan, China) and IL-1β (CSB-E08054m, Cusabio, Wuhan, China) detection kits according to the manufacturer’s instructions.

Techniques: Mutagenesis, Flow Cytometry, Immunohistochemical staining, Marker, Expressing, Microarray

Journal: International Journal of Molecular Sciences

Article Title: Targeting CD177: A Novel Therapeutic Strategy for NLRP3-Associated Autoinflammatory Diseases

doi: 10.3390/ijms27062841

Figure Lengend Snippet: Targeted IL-6 therapy is ineffective for NLRP3 mutation-induced AID. ( A – G ) Flow cytometry detection of neutrophil, IL-1β, IL-6 and CD177 protein expression in peripheral blood of NLRP3 mutant severe mice treated with or without IL-6 antibody ( n = 7). ( H – K ) qPCR detection of IL-1β, IL-6, TNFα and CD177 mRNA expression levels in skin, liver, and spleen tissues of NLRP3 mutant severe mice treated with or without IL-6 antibody ( n = 3). ( L – N ) Histopathological detection of mouse tissues (skin, liver, spleen), Scale bars: 400 μm, 100 μm. ( O – Q ) Immunohistochemical detection of Ly6G levels in mouse skin, liver, spleen tissues, Scale bars:200 μm, 50 μm. qPCR Data are shown as mean ± s.d. Student’s t -test was used. Statistical significance was determined as * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: The levels of IL-1β and IL-6 in the serum were detected using mouse-specific IL-6 (CSB-E04639m, Cusabio, Wuhan, China) and IL-1β (CSB-E08054m, Cusabio, Wuhan, China) detection kits according to the manufacturer’s instructions.

Techniques: Mutagenesis, Flow Cytometry, Expressing, Immunohistochemical staining

Journal: International Journal of Molecular Sciences

Article Title: Targeting CD177: A Novel Therapeutic Strategy for NLRP3-Associated Autoinflammatory Diseases

doi: 10.3390/ijms27062841

Figure Lengend Snippet: Targeting CD177 reverses the inflammatory phenotype of NLRP3 -AID mice. ( A , B ) Flow cytometry detection of CD177 protein expression in peripheral blood of mice treated with either IL-1β antibody or CD177 siRNA ( n = 6). ( C ) qPCR detection of CD177 mRNA expression levels in mouse skin, liver, and spleen tissues ( n = 3). ( D ) Treatment was initiated on Day 14. Body weights were recorded every two days to evaluate systemic recovery. Data are presented as mean ± SEM ( n = 6 per group). ( E ) Flow cytometry detection of neutrophils in mouse peripheral blood after treatment with either IL-1β antibody or CD177 siRNA ( n = 6). ( F – H ) Histopathological detection of mouse tissues (skin, liver, spleen), Scale bars: 400 μm, 100 μm. ( I – K ) Immunohistochemical detection of Ly6G protein expression in mouse tissues (skin, liver, spleen), Scale bars: 200 μm, 50 μm. qPCR data are shown as mean ± s.d. Student’s t -test was used. Statistical significance was determined as *** p < 0.001.

Article Snippet: The levels of IL-1β and IL-6 in the serum were detected using mouse-specific IL-6 (CSB-E04639m, Cusabio, Wuhan, China) and IL-1β (CSB-E08054m, Cusabio, Wuhan, China) detection kits according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Expressing, Immunohistochemical staining

Journal: International Journal of Molecular Sciences

Article Title: Targeting CD177: A Novel Therapeutic Strategy for NLRP3-Associated Autoinflammatory Diseases

doi: 10.3390/ijms27062841

Figure Lengend Snippet: Targeting CD177 suppresses the expression of IL-1β. ( A , B ) Flow cytometry detection of IL-1β protein expression levels in peripheral blood of mice treated with either IL-1β antibody or CD177 siRNA ( n = 6). ( C ) ELISA detection of serum IL-1β protein levels in mice treated with either IL-1β antibody or CD177 siRNA ( n = 3). ( D , E ) Flow cytometry detection of IL-6 protein expression levels in peripheral blood of mice treated with either anti-IL-1β antibody or CD177 siRNA ( n = 6). ( F ) ELISA detection of serum IL-6 protein levels in mice treated with either IL-1β antibody or CD177 siRNA ( n = 3). ( G – I ) qPCR detection of IL-6, IL-1β, and TNFα mRNA expression levels in mouse skin, liver, and spleen tissues ( n = 3). ( J ) Schematic diagram shows the role of CD177 in the development of NLRP3 mutation-related autoinflammation. qPCR Data are shown as mean ± s.d. Student’s t -test was used. Statistical significance was determined as ** p < 0.01; *** p < 0.001.

Article Snippet: The levels of IL-1β and IL-6 in the serum were detected using mouse-specific IL-6 (CSB-E04639m, Cusabio, Wuhan, China) and IL-1β (CSB-E08054m, Cusabio, Wuhan, China) detection kits according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: Advanced Science

Article Title: Loss of SOCS1 in Donor T Cells Exacerbates Intestinal GVHD by Driving a Chemokine‐Dependent Pro‐Inflammatory Immune Microenvironment

doi: 10.1002/advs.202513735

Figure Lengend Snippet: Socs1 deficiency in T cells drives effector differentiation and enhances inflammatory responses in CD8 + T cells. (A) Experimental schematic. Splenocytes from WT (littermate control; Socs1 fl/fl ) and cKO (LckCre‐ Socs1 fl/fl ) mice were isolated and sorted by FACS for CD45 + cells and subjected to single‐cell RNA sequencing (scRNA‐seq, n = 5 per group). Alternatively, CD8 + T cells from the spleen of WT and cKO mice were sorted by FACS and subjected to bulk RNA‐seq, ATAC‐seq, and CUT&Tag analyses. (B) UMAP plot of 105 040 single cells from CD45 + splenocytes colored by annotated immune cell subsets. (C) UMAP visualization of CD45 + splenocytes, split by origin. (D) Comparison of the proportions of celltypes between WT and cKO groups. (E) UMAP plot of 19 490 T cells extracted from Figure and colored by annotated T cell subsets. (F) UMAP visualization of T cells, split by origin. (G) Heatmap of Ro/e (Ratio of observed to expected) scores for T cell subtypes in WT and cKO mice. The scores, calculated from scRNA‐seq cell counts, indicate the relative enrichment (red, Ro/e > 1) or depletion (white/light orange, Ro/e < 1) of each population within each genotype. Numerical values are presented alongside a semi‐quantitative summary. (H) UMAP plot of 9159 CD8 + T cells extracted from Figure and colored by annotated T cell subsets. (I) Comparison of the proportions of indicated CD8 + T cell clusters between WT and cKO groups. (J) Representative flow cytometry plots and frequencies of naive T cells (Tn; CD44 − CD62L + ), central memory (Tcm; CD44 + CD62L + ), and effector memory (Tem; CD44 + CD62L − ) in CD8 + T cells from peripheral blood (PB, left panel) and spleen (SP, right panel) (n = 5 per group). (K) Bar plots showing the expression of perforin, GZMB, TNF‐α, IFN‐γ, IL‐2, and CD107a in CD8 + T cells from WT and cKO mice, as measured by flow cytometry (n=5 per group). (L) UMAP visualization of integrated T‐cell transcriptomes from WT (left, 25 304 cells) and cKO (right, 21 588 cells) groups. Each point represents a single cell, colored by the frequency of its corresponding TCR clonotype, highlighting clonally expanded cells. (M) Quantification of overall TCR repertoire diversity. The Gini index (top) and Shannon entropy (bottom) were calculated for the entire T‐cell population from each mouse. (N) Distribution and clonal size of T cells across identified subsets. Barplot showing the absolute cell counts (left panels) and the clonal size composition (right panels) for each T‐cell subset from WT and cKO mice. Data represent one experiment out of two independent experiments. P values were determined using two‐sided Wilcoxon rank‐sum test (D, I, M) or unpaired two‐tailed Student's t‐test (J‐K). Data represent mean ± SEM (D, I, M) or mean ± SD (J‐K). ∗ p <.05, ∗∗ p <.01 and ∗∗∗∗ p <.0001.

Article Snippet: The following kits were used: IL‐1β (Proteintech, KE10003), IL‐6 (Proteintech, KE10007), and TNF‐α (Proteintech, KE10002).

Techniques: Control, Isolation, Single Cell, RNA Sequencing, Comparison, Flow Cytometry, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Loss of SOCS1 in Donor T Cells Exacerbates Intestinal GVHD by Driving a Chemokine‐Dependent Pro‐Inflammatory Immune Microenvironment

doi: 10.1002/advs.202513735

Figure Lengend Snippet: Evolution of small intestinal immune cell composition following transplantation of Socs1 cKO CD8 + T cells. (A) Experimental schematic. Lethally irradiated BALB/c recipient mice were transplanted with splenic T cells from WT mice (WT group) or cKO mice (cKO group), along with 5 × 10 6 TCD‐BM cells from WT mice. Survival was monitored daily. Body weight and GVHD score were assessed every five days. Immune cells in PB and small intestine from WT and cKO groups were assessed on Day 7, Day 16, and Day 24 by flow cytometry. (B) Survival analysis of recipients transplanted with 1 × 10 6 , 2 × 10 6, or 3 × 10 6 splenic T cells from WT or cKO mice (n = 10 mice per group). A control group received TCD‐BM only. Median survival times for cKO groups were 36 (1 × 10 6 ), 33 (2 × 10 6 ), and 26 days (3 × 10 6 ), respectively. In the corresponding WT groups, 9/10 mice in the 1 × 10 6 group survived to the end of the observation period, with median survival times of 35 days (2 × 10 6 ), and 42.5 days (3 × 10 6 ). Data were pooled from two independent experiments (n=10 mice/group). (C‐D) GVHD score (C) and body weight changes (D) in WT and cKO recipients transplanted with 1 × 10 6 splenic T cells. (E) Proportions of CD8 + T cells and monocytes in PB, and proportions of CD8 + T cells in the IEL and LP on Day 7, 16, and 24 post‐transplantation. (F) Boxplots showing the expression of TNF‐α, IFN‐γ, perforin, and CD107a in CD8 + T cells in IEL by flow cytometry on Day 7 and 16 post‐transplantation. (G) Experimental schematic of scRNA‐seq. Lymphocytes from IEL and LP were isolated and collected from 3‐4 mice, followed by FACS sorting for CD45 + donor‐derived (H‐2 b+ H‐2 d− ) cells respectively. Sorted cells from IEL and LP were mixed in a 1:2 ratio and subsequently subjected to scRNA‐seq. (H‐I) UMAP plot of all immune cells colored by samples (H) or annotated subsets (I). (J) UMAP plot showing annotated CD8 + T cell subsets (left), with barplot illustrating the proportion of each annotated subset in recipient intestines at indicated time points between WT and cKO groups (right). (K) Violin plots comparing the expression of cytotoxic molecules and cytokine receptor genes in CD8 + T cells on Day 26 post‐transplantation between WT and cKO groups. (L) UMAP plot showing annotated myeloid cell subsets (left), with barplot illustrating the proportion of each annotated subset in recipient intestines at indicated time points between WT and cKO groups (right). (M) Violin plots comparing expression of M1‐ and M2‐associated marker genes in myeloid cells on Day 26 post‐transplantation between WT and cKO groups. Data represent three independent experiments. P values were determined using chi‐squared test (B) or unpaired two‐tailed Student's t‐test (E, F) or two‐sided Wilcoxon rank‐sum test (K, M). Differences in GVHD scores and body weight between the WT and cKO groups at each time point were analyzed using multiple unpaired two‐tailed Student's t ‐tests (C, D). Data represent mean ± SEM (C‐E). ∗ p <.05, ∗∗ p <.01, ∗∗∗ p <.001 and ∗∗∗∗ p <.0001.

Article Snippet: The following kits were used: IL‐1β (Proteintech, KE10003), IL‐6 (Proteintech, KE10007), and TNF‐α (Proteintech, KE10002).

Techniques: Transplantation Assay, Irradiation, Flow Cytometry, Control, Expressing, Isolation, Derivative Assay, Marker, Two Tailed Test