Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: CD163+ M2 Macrophages Promote Fibrosis in IgG4-Related Disease Via Toll-like Receptor 7/Interleukin-1 Receptor-Associated Kinase 4/NF-κB Signaling.

doi: 10.1002/art.42043

Figure Lengend Snippet: Figure 2. Identification and distribution of interleukin-1 receptor–associated kinase 4 (IRAK4) in SMGs from transgenic mice and patients with IgG4-RD. A–C, Distribution of TLR-7–related candidate molecules in SMGs from representative transgenic and knockout mice (A), from healthy controls and from patients with CS, SS, or IgG4-RD (B), and in tonsils and lymph nodes from healthy patients and SMGs from patients with IgG4-related dacryoadenitis and sialadenitis (IgG4-DS) (C). Outlined area indicates a germinal center (GC). Bars = 100 μm. D, Number of IRAK4-positive cells in SGs from healthy controls and patients with CS, SS, or IgG4-RD, measured using TissueQuest software. E, Serial sections stained with H&E or antibodies to IRAK4, CD80, CD163, CD11c, or CD123. Bars = 100 μm. F, Representative double immunostaining for IRAK4 (red), CD80 (green), CD163 (green), CD11c (green), and CD123 (green) in SMGs from patients with IgG4-RD. Counterstaining was performed with DAPI (blue). G, Number of IRAK4-expressing CD163+, CD123+, CD11c+, and CD80+ cells in SMGs from patients with IgG4-RD (n = 12), mea- sured using TissueQuest software. In D and G, data are shown as box plots. Each box represents the upper and lower interquartile range. Lines inside the boxes represent the median. Symbols represent individual subjects. * = P < 0.05; ** = P < 0.01, by Kruskal–Wallis test. See Figure 1 for other definitions.

Article Snippet: CD163+ M2 macrophages were isolated from healthy peripheral blood mononuclear cells as described in a previous report (22), and were then stimulated with 500 μM of the TLR-7 agonist loxoribine (catalog no. ALX480-097-M025; Enzo Life Sciences) and/or 1 μM of the IRAK4 inhibitor CA-4948 (Selleck) (Supplementary Figures 1 and 2, available on the Arthritis & Rheumatology website at https:// onlinelibrary.wiley.com/doi/10.1002/art.42043).

Techniques: Transgenic Assay, Knock-Out, Software, Staining, Double Immunostaining, Expressing

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: CD163+ M2 Macrophages Promote Fibrosis in IgG4-Related Disease Via Toll-like Receptor 7/Interleukin-1 Receptor-Associated Kinase 4/NF-κB Signaling.

doi: 10.1002/art.42043

Figure Lengend Snippet: Figure 3. Expression of inflammatory cytokines and Toll-like receptor 7 (TLR-7)–related candidate genes in M2 macrophages. A, Schematic illustration of the extraction of human CD163+ M2 macrophages stimulated with the TLR-7 agonist loxoribine and/or interleukin-1 receptor– associated kinase 4 (IRAK4) inhibitor CA-4948. B, Detection of CD163 and TLR-7 before and after the differentiation and selection of M2 macro- phages (day 0 and day 6) from ≥2 independent experiments, as determined by flow cytometric analysis. C, Schematic representation of TLR-7 pathways. D, Expression levels of mRNA for TLR-7–related genes in human CD163+ M2 macrophages cultured in the presence or absence of lox- oribine and/or CA-4948 (n = 6 per group). E, Production of inflammatory cytokines in CD163+ M2 macrophages stimulated in the presence or absence of loxoribine and/or CA-4948, as determined by enzyme-linked immunosorbent assay. In D and E, Data are shown as box plots. Each box represents the upper and lower interquartile range. Lines inside the boxes represent the median. Symbols represent individual subjects. * = P < 0.05; ** = P < 0.01, by Mann–Whitney U-test (D) or Kruskal–Wallis test (E). PBMC = peripheral blood mononuclear cells; MyD88 = myeloid differentiation factor 88; TRAF6 = tumor necrosis factor receptor–associated factor 6; IRF-5 = interferon (IFN) regulatory factor 5; IL-33 = interleukin-33; TGFβ = transforming growth factor β; NS = not significant.

Article Snippet: CD163+ M2 macrophages were isolated from healthy peripheral blood mononuclear cells as described in a previous report (22), and were then stimulated with 500 μM of the TLR-7 agonist loxoribine (catalog no. ALX480-097-M025; Enzo Life Sciences) and/or 1 μM of the IRAK4 inhibitor CA-4948 (Selleck) (Supplementary Figures 1 and 2, available on the Arthritis & Rheumatology website at https:// onlinelibrary.wiley.com/doi/10.1002/art.42043).

Techniques: Expressing, Extraction, Selection, Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: CD163+ M2 Macrophages Promote Fibrosis in IgG4-Related Disease Via Toll-like Receptor 7/Interleukin-1 Receptor-Associated Kinase 4/NF-κB Signaling.

doi: 10.1002/art.42043

Figure Lengend Snippet: Figure 5. Correlation between fibrosis and M2 macrophages in SMGs from patients with IgG4-RD. A, Serial sections stained with H&E, MT, or antibodies to CD163 or interleukin-1 receptor–associated kinase 4 (IRAK4). Bars = 100 μm. B, Correlation between fibrosis score and the number of CD163+ or IRAK4-positive cells in SMGs (n = 12 per group). The numbers of CD163+ cells and IRAK4-positive cells were measured using Tis- sueQuest software. Correlation coefficients and P values were determined using Spearman’s rank correlation. See Figure 1 for other definitions.

Article Snippet: CD163+ M2 macrophages were isolated from healthy peripheral blood mononuclear cells as described in a previous report (22), and were then stimulated with 500 μM of the TLR-7 agonist loxoribine (catalog no. ALX480-097-M025; Enzo Life Sciences) and/or 1 μM of the IRAK4 inhibitor CA-4948 (Selleck) (Supplementary Figures 1 and 2, available on the Arthritis & Rheumatology website at https:// onlinelibrary.wiley.com/doi/10.1002/art.42043).

Techniques: Staining, Software

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: CD163+ M2 Macrophages Promote Fibrosis in IgG4-Related Disease Via Toll-like Receptor 7/Interleukin-1 Receptor-Associated Kinase 4/NF-κB Signaling.

doi: 10.1002/art.42043

Figure Lengend Snippet: Figure 6. Schematic model of TLR-7 signaling in M2 macrophages leading to fibrosis in IgG4-RD. TLR-7 expressed on M2 macrophages rec- ognizes some viral RNAs or self RNAs released from stressed or injured cells. Activated M2 macrophages secrete fibrotic cytokines, including interleukin-1β (IL-1β), transforming growth factor β (TGFβ), and IL-33, via interleukin-1 receptor–associated kinase 4 (IRAK4)/NF-κB signaling, which leads to fibrosis. MyD88 = myeloid differentiation factor 88; TRAF6 = tumor necrosis factor receptor–associated factor 6; IRF-7 = interferon regulatory factor 7 (see Figure 1 for other definitions).

Article Snippet: CD163+ M2 macrophages were isolated from healthy peripheral blood mononuclear cells as described in a previous report (22), and were then stimulated with 500 μM of the TLR-7 agonist loxoribine (catalog no. ALX480-097-M025; Enzo Life Sciences) and/or 1 μM of the IRAK4 inhibitor CA-4948 (Selleck) (Supplementary Figures 1 and 2, available on the Arthritis & Rheumatology website at https:// onlinelibrary.wiley.com/doi/10.1002/art.42043).

Techniques: