Journal: Research

Article Title: High Mobility Group Protein B1 Promotes Interferon Regulatory Factor 1 SUMOylation to Prime Trained Immunity of Circulating Monocytes and Aggravate the Progressive Synovial Inflammation in Knee Osteoarthritis

doi: 10.34133/research.1243

Figure Lengend Snippet: High mobility group protein B1 (HMGB1) serves as a specific damage-associated molecular pattern (DAMP) that stimulates circulating monocytes in knee osteoarthritis (KOA) mice to adopt the g7 phenotype and exhibit features of trained immunity. (A) Heatmap showing differences in the expression levels of inflammatory factors, chemokines, and alarmin family factors in circulating monocytes between the negative control (NC) group and the HMGB1 group in mice. (B) Gene Ontology (GO) enrichment analysis of differentially expressed genes in circulating monocytes between the NC and HMGB1 groups. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes in circulating monocytes between the NC and HMGB1 groups. (D) Gene set enrichment analysis (GSEA) of differentially expressed genes in circulating monocytes between the NC and HMGB1 groups. (E) The Cumulus heatmap shows the chromatin accessibility of circulating monocytes in the NC group and HMGB1 group mice. (F) Chromatin region occupancy in circulating monocytes of NC and HMGB1 group mice. (G) Bar graph statistics displaying the number of chromatin regions with differential accessibility (open or closed) in circulating monocytes from NC and HMGB1 group mice. (H) Chromatin accessibility of IL1A , IL1R1 , TNF , CXCL12 , CXCL3 , CXCL2 , CCL20 , and G0S2 genes in circulating monocytes of NC and HMGB1 group mice.

Article Snippet: In the in vitro experiment, HMGB1 intervention was performed by treating the cells with 40 ng/ml recombinant HMGB1 protein (HY- P70570 , MCE, USA); CCL4 intervention was performed by treating the cells with 50 ng/ml CCL4 (HY-P7257, MCE, USA); for IL-18 intervention, 1 ng/ml IL-18 (HY- P70591 , MCE, USA) was used to treat the cells.

Techniques: Expressing, Negative Control

Journal: Research

Article Title: High Mobility Group Protein B1 Promotes Interferon Regulatory Factor 1 SUMOylation to Prime Trained Immunity of Circulating Monocytes and Aggravate the Progressive Synovial Inflammation in Knee Osteoarthritis

doi: 10.34133/research.1243

Figure Lengend Snippet: High mobility group protein B1 (HMGB1) promotes interferon regulatory factor 1 (IRF1) SUMOylation via MyD88 to induce trained immunity in knee osteoarthritis (KOA) monocytes. (A) Western blotting (WB) was used to detect the expression levels of IRF1 and MyD88 proteins in peripheral blood monocytes from patients in the healthy donor (HD) and KOA groups ( n = 3). (B) WB was used to detect the expression levels of IRF1 and MyD88 proteins in peripheral blood monocytes from mice in the negative control (NC) and HMGB1 groups ( n = 3). (C) Coimmunoprecipitation (CO-IP) was used to examine the interaction between IRF1 and MyD88 proteins in peripheral blood monocytes from patients in the HD and KOA groups. (D) CO-IP was used to examine the interaction between IRF1 and MyD88 proteins in peripheral blood monocytes from mice in the NC and HMGB1 groups. (E) A laser confocal microscope was used to observe the interaction between IRF1 and MyD88 proteins in peripheral blood monocytes from mice in the NC and HMGB1 groups. (F) CO-IP was used to evaluate the SUMOylation of IRF1 in peripheral blood monocytes from patients in the HD and KOA groups. (G) CO-IP was used to evaluate the SUMOylation of IRF1 in peripheral blood monocytes from mice in the NC and HMGB1 groups. (H) CO-IP was used to compare the differences in SUMOylation levels of IRF1 in peripheral blood monocytes from wild-type and MyD88 knockout mice after HMGB1 stimulation. (I) Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in peripheral blood monocytes from wild-type and MyD88 knockout mice after HMGB1 stimulation ( n = 6). (J) Flow cytometry was used to determine the proportion of CXCR4-positive cells in peripheral blood monocytes from wild-type mice stimulated with HMGB1 ( n = 3). (K) Flow cytometry was used to determine the proportion of CXCR4-positive cells in peripheral blood monocytes from MyD88 knockout mice stimulated with HMGB1 ( n = 3). Statistical results are represented by mean ± standard error of the mean (mean ± SEM), * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: In the in vitro experiment, HMGB1 intervention was performed by treating the cells with 40 ng/ml recombinant HMGB1 protein (HY- P70570 , MCE, USA); CCL4 intervention was performed by treating the cells with 50 ng/ml CCL4 (HY-P7257, MCE, USA); for IL-18 intervention, 1 ng/ml IL-18 (HY- P70591 , MCE, USA) was used to treat the cells.

Techniques: Western Blot, Expressing, Negative Control, Co-Immunoprecipitation Assay, Microscopy, Knock-Out, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Research

Article Title: High Mobility Group Protein B1 Promotes Interferon Regulatory Factor 1 SUMOylation to Prime Trained Immunity of Circulating Monocytes and Aggravate the Progressive Synovial Inflammation in Knee Osteoarthritis

doi: 10.34133/research.1243

Figure Lengend Snippet: Inhibition of high mobility group protein B1 (HMGB1) eliminated the trained immunity of circulating monocytes and their aggregation to knee osteoarthritis (KOA) synovial tissue. (A) In vivo imaging was used to observe the locations of cell migration in the KOA group, the KOA group with transfused fluorescently labeled trained circulating monocytes, and the KOA group with transfused fluorescently labeled untrained circulating monocytes in mice ( n = 3). (B) Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in mouse peripheral serum at different time points under Nab-HMGB1 intervention ( n = 6). (C) Western blotting (WB) was used to detect the expression of IL-1β, IL-6, and TNF-α in mouse synovial tissue at different time points following Nab-HMGB1 intervention. (D) Representative WB bands showing the levels of IL-1β, IL-6, and TNF-α in peripheral circulating monocytes from mice at different time points under Nab-HMGB1 intervention ( n = 3). (E) Statistical graphs of the WB results for IL-1β, IL-6, and TNF-α levels in peripheral circulating monocytes from mice at different time points under Nab-HMGB1 intervention. (F) Representative WB bands showing the levels of IL-1β, IL-6, and TNF-α in peripheral circulating monocytes from mice at different time points under Nab-HMGB1 intervention ( n = 3). (G) Immunofluorescence was used to observe the numbers of CXCR4 + and Ly6c + cells in synovial tissue from the negative control (NC) group, KOA group, and Nab-HMGB1 group of mice. (H) The number of CXCR4 + circulating monocytes in mouse synovial tissue from the NC group, KOA group, and Nab-HMGB1 group was detected using magnetic bead sorting of mouse circulating monocytes followed by flow cytometry ( n = 3). Statistical results are represented by mean ± standard error of the mean (mean ± SEM), * P < 0.05, ** P < 0.01, *** P < 0.001. NS, not significant.

Article Snippet: In the in vitro experiment, HMGB1 intervention was performed by treating the cells with 40 ng/ml recombinant HMGB1 protein (HY- P70570 , MCE, USA); CCL4 intervention was performed by treating the cells with 50 ng/ml CCL4 (HY-P7257, MCE, USA); for IL-18 intervention, 1 ng/ml IL-18 (HY- P70591 , MCE, USA) was used to treat the cells.

Techniques: Inhibition, In Vivo Imaging, Migration, Labeling, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Immunofluorescence, Negative Control, Flow Cytometry

Journal: Research

Article Title: High Mobility Group Protein B1 Promotes Interferon Regulatory Factor 1 SUMOylation to Prime Trained Immunity of Circulating Monocytes and Aggravate the Progressive Synovial Inflammation in Knee Osteoarthritis

doi: 10.34133/research.1243

Figure Lengend Snippet: CXCR4-dependent migration of trained subsets promoted synovial inflammation in knee osteoarthritis (KOA). (A) Flow cytometry was used to assess the effects of high mobility group protein B1 (HMGB1), CCL4, and interleukin-18 (IL-18) treatments on the proportion of CXCR4-positive cells among circulating monocytes in mice ( n = 3). (B) CXCR4 + cells were isolated using flow cytometric sorting. (C) Principal component analysis (PCA) was performed on CXCR4 + and CXCR4 − cells. (D) A volcano plot displays the number of differentially expressed genes between CXCR4 + and CXCR4 − cells. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) database enrichment analysis was conducted for the differentially expressed genes between CXCR4 + and CXCR4 − cells. (F) In vivo animal fluorescence imaging demonstrates the effects of transfusing CXCR4 + cells and administering the CXCR4 receptor antagonist plerixafor on CXCR4 + cells in KOA mice ( n = 3). (G) Hematoxylin and eosin (HE) staining was used to observe synovial pathology in the KOA group, the CXCR4 + cell transfusion group, and the CXCR4 + cell transfusion plus plerixafor group. (H) Representative Western blot images show protein expression levels of IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in the synovial tissue of mice from the KOA group, CXCR4 + cell transfusion group, and CXCR4 + cell transfusion plus plerixafor group. (I) Quantitative Western blot analysis of IL-1β, IL-6, and TNF-α protein expression levels in synovial tissue from the 3 groups ( n = 3). (J) Quantitative polymerase chain reaction (PCR) analysis of IL-1β, IL-6, and TNF-α mRNA expression levels in synovial tissue from the KOA group, CXCR4 + cell transfusion group, and CXCR4 + cell transfusion plus plerixafor group ( n = 3). Statistical results are represented by mean ± standard error of the mean (mean ± SEM), ** P < 0.01, *** P < 0.001.

Article Snippet: In the in vitro experiment, HMGB1 intervention was performed by treating the cells with 40 ng/ml recombinant HMGB1 protein (HY- P70570 , MCE, USA); CCL4 intervention was performed by treating the cells with 50 ng/ml CCL4 (HY-P7257, MCE, USA); for IL-18 intervention, 1 ng/ml IL-18 (HY- P70591 , MCE, USA) was used to treat the cells.

Techniques: Migration, Flow Cytometry, Isolation, In Vivo, Fluorescence, Imaging, Staining, Western Blot, Expressing, Real-time Polymerase Chain Reaction