Contribution of the appendix to ulcerative colitis by supplying pathogenic immunoglobulin G-producing cells

Figure 1 Appendiceal inflammation as skip lesions in ulcerative colitis. A: The left part shows a macroscopic image of a surgical specimen from a patient with active ulcerative colitis who underwent total proctocolectomy (a1: Rectum, a2: Ascending colon, and a3: Appendix). The middle part presents endoscopic findings of the appendiceal orifice in ulcerative colitis. The right part displays a macroscopic image of the appendiceal orifice. The black arrows indicate appendiceal orifice inflammation. The stained sections in a1, a2, and a3 correspond to b1, b2, and b3, respectively; B: Hematoxylin and eosin staining of the rectum (b1), bar = 100 μm; ascending colon (b2), bar = 100 μm; appendix (b3), bars = 500 μm (left), and 50 μm [right (enlarged view of the black square in the left panel)].

Figure 2 Single-cell landscape of appendiceal lamina propria mononuclear cells in patients with ulcerative colitis and controls. A: Hematoxylin and eosin staining of the appendix of control (left) and ulcerative colitis (UC) (right) patient. Bars = 500 μm; B: Lamina propria mononuclear cells (LPMCs) per tissue weight in the appendix of controls (n = 10) and UC (n = 10). Data are shown as mean ± SEM; C: Experimental design of single-cell RNAsequencing analysis. Appendiceal LPMCs were isolated, and EpCAM^- and 7-amino-actinomycin D^- cells were sorted using FACSAria II and subjected to single-cell RNAsequencing; D: Uniform manifold approximation and projection of appendiceal LPMCs UC (n = 4) and controls (n = 5); E: Expression of genes across cell types; F: Frequencies of cell types in UC (n = 4) and controls (n = 5). Scatter plots with a median line. ILC: Innate lymphoid cell; LPMCs: Lamina propria mononuclear cells; NK: Natural killer; scRNA-seq: Single-cell RNAsequencing; UC: Ulcerative colitis; UMAP: Uniform manifold approximation and projection; 7-AAD: 7-amino-actinomycin D.

Figure 3 Aberrant immunoglobulin G production in the appendices of patients with ulcerative colitis. A: Uniform manifold approximation and projection (UMAP); unsupervised clustering of appendiceal plasma cells; B: Dot plot; expression frequencies and signature gene levels across clusters; C: Immunoglobulin heavy constant alpha 1 expression on UMAP; D: Immunoglobulin heavy constant gamma 1 (left) and immunoglobulin heavy constant gamma 2 (right) expression on UMAP; E: Cell cycle scores (S score and G2M score) for clusters; F: Frequencies of plasma cell clusters in ulcerative colitis (n = 4), controls (n = 5); G: Frequencies of each Ig isotype in ulcerative colitis/controls. Scatter plots with a median line (F and G). ^aP < 0.05. Ig: Immunoglobulin; IGHA1: Immunoglobulin heavy constant alpha 1; IGHG1: Immunoglobulin heavy constant gamma 1; IGHG2: Immunoglobulin heavy constant gamma 2; UMAP: Uniform manifold approximation and projection; UC: Ulcerative colitis.

Figure 4 Inflammatory milieu influences germinal center reactions in the appendices of patients with ulcerative colitis. A: Uniform manifold approximation and projection; single-cell landscape of appendiceal B cells; B: Dot plot; expression levels of marker genes across B-cell subsets; C: Frequencies of B-cell subsets in ulcerative colitis/controls; D: Isotype distribution in germinal center B (GC B) cells; E: Immunoglobulin somatic mutation frequencies subsets. Box-and-whisker plot; median and interquartile range; F: Canonical pathway enhanced in GC B cells; G: Predicted upstream regulators enhanced or suppressed in GC B cells. Scatter plots with median line for each group. ulcerative colitis, n = 4; controls, n = 5 (C and D). GC B: Germinal center B; Ig: Immunoglobulin; UC: Ulcerative colitis; UMAP: Uniform manifold approximation and projection.

Figure 5 Increased frequencies of immunoglobulin G-positive cells in the appendices of patients with ulcerative colitis and their reduction in the rectum and colon after appendectomy. A: Immunohistochemistry of immunoglobulin (Ig) G-positive cells in a control appendix (upper left, bar = 500 μm) and a patient with ulcerative colitis (UC) (bottom left and right, bars = 500 μm and 50 μm, respectively); B: Flow cytometric analysis of IgG-positive and IgA-positive cells in appendiceal lamina propria mononuclear cells in controls and UC [IgG-positive cells: (1) UC (n = 12); and (2) Control (n = 11); IgA-positive cells: (1) UC (n = 8); and (2) Control (n = 10)]; C: Immunohistochemistry of IgG-positive cells in the appendix and colorectal regions of a patient with UC. Bars = 100 μm; D: IgG-positive cells per area of immunohistochemistry sections in the appendix and colorectal regions of patients with UC (n = 15); E: IgG-positive and IgA-positive cells in immunohistochemistry sections of ulcerative appendicitis (n = 15), acute appendicitis (n = 10); F: IgG-positive cells in UC colorectal regions with (n = 5) or without (n = 15) appendectomy. Bars = 100 μm. Data are shown as mean ± SEM (B and D-F). ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; and ^dP < 0.0001. Ig: Immunoglobulin; UC: Ulcerative colitis.

Figure 6 Immunoglobulin G-positive cells express C-C chemokine receptor 10 in the appendices and rectums of patients with ulcerative colitis. A: C-C chemokine receptor 10 (CCR10) expression on uniform manifold approximation and projection of single-cell RNA sequencing data; B: Flow cytometric gating plots; CCR10 and immunoglobulin (Ig) G expression in appendiceal plasma cells; C: Immunohistochemistry for CCR10, IgG, and IgA in ulcerative colitis (UC) appendix. Top panel, bars = 100 μm; bottom (enlarged view of square in top panel), bars = 50 μm; D: Immunohistochemistry for CCR10, IgG, and IgA in the UC rectum. Top panel, bars = 100 μm; bottom (enlarged view of square in top panel), bars = 50 μm; E: Correlation between IgG-positive and CCR10-positive cells per immunohistochemistry field of the UC appendix. Spearman’s rank correlation. Ig: Immunoglobulin; UC: Ulcerative colitis; UMAP: Uniform manifold approximation and projection.

Figure 7 Somatically mutated, arginine-glycine-aspartic acid-motif-containing immunoglobulin G-producing cells are the hallmark of appendiceal antibody response in ulcerative colitis. A: Immunoglobulin (Ig) somatic mutation frequencies of all isotypes expressed by appendiceal plasma cells. Cells colored by degree of mutations on uniform manifold approximation and projection plot (left). Box-and-whisker plots show mutation frequencies across the controls (blue) and ulcerative colitis groups (orange), and the median and interquartile range (right); B: Mutation frequencies of IgA; C: Mutation frequencies of IgG; D: Arginine-glycine-aspartic acid-motif-containing cells (arginine-glycine-aspartic acid⁺) in the total plasma cell fraction and IgG-expressing cells of patients with ulcerative colitis (n = 4) and controls (n = 5). The scatter plots with median line for each group. ^aP < 0.05. Ig: Immunoglobulin; RGD: Arginine-glycine-aspartic acid; UC: Ulcerative colitis; UMAP: Uniform manifold approximation and projection.