Fig 1: A C5aR1 antagonist, PMX205, impedes AOM/DSS-induced CRC tumorigenesis. (A, B) The effect of PMX205 treatment on AOM/DSS-induced CRC in mice. Images of the colorectum (A) and quantitative analysis of tumor number and mass (B). Scale bar, 1 cm. (C, D) Flow cytometry analysis of MDSCs (C) and CD8+ T cell (D) proportions in the indicated tissues of AOM/DSS-treated mice with or without PMX205 administration. (E) Representative immunohistochemistry staining of CD8+ T cell in colons. Left panel, mucosa-associated lymphoid tissue (MALT) (Scale bar, 100 µm, 50 µm, 20 µm); Right panel, tumor tissue (Scale bar, 50 µm, 20 µm). Quantification of the numbers of CD8+ T cell shown on the right. (F-N) Local levels of the proinflammatory cytokines TNF-a (F), IL-1a (G), IL-6 (H), IL-1ß (I) and IL-11 (J); the anti-inflammatory cytokine IL-10 (K); and the chemokines CCL2 (L), CXCL1 (M) and CXCL5 (N). Data are represented as mean ± SEM or Min to Max; n=5 in each group; ns, not significant; * P<0.05; ** P<0.01; *** P<0.001; and **** P<0.0001.
Fig 2: Schematic for the mechanisms by which C5a/C5aR1 signaling initiates colorectal tumorigenesis by modulating versatile immune responses. Complement C5a/C5aR1 signaling, independent of C3 activation, recruits MDSCs into the inflamed colorectal tissues to impair CD8+ T cells, and modulates the production of a variety of cytokines/chemokines (IL-1, IL-6, IL-11, IL-17A, TNF-α, IL-9, IL-10, IL-23, IL-27, CCL2, CCL17, CXCL1/5), thus fostering AOM/DSS-induced colorectal tumorigenesis. C5aR1 inhibition by PMX205 impedes CRC tumorigenesis. ACF, aberrant crypt foci.
Fig 3: BM transplantation reveals the sufficiency of C5aR1 expression in immune cells for initiating CRC. (A, B) The effect of mutual BM transplantation between WT and C5ar1-deficient mice on AOM/DSS-induced colorectal tumorigenesis. Images of the colorectum (A) and quantitative analysis of tumor number and mass (B). Scale bar, 1 cm. (C) MDSC proportions in the colon. (D-I) Local levels of the proinflammatory cytokines TNF-a (D), IL-1a (E) and IL-6 (F) and the chemokines CCL2 (G), CXCL1 (H) and CXCL5 (I). Data are represented as mean ± SEM.; n=8 in each group; ns, not significant; * P<0.05; ** P<0.01; *** P<0.001; and **** P<0.0001.
Fig 4: The profiles of multiple cytokines/chemokines in AOM/DSS-induced CRC mice. (A-F) The effect of C3, C5, and C5ar1 deficiency on the local levels of the proinflammatory cytokines TNF-a (A), IL-1a (B), IL-6 (C), IL-1ß (D), IL-17A (E) and IL-11 (F). (G-N) Local levels of the anti-inflammatory cytokines IL-23 (G), IL-9 (H), IL-27 (I) and IL-10 (J); and the chemokines CCL2 (K), CCL17 (L), CXCL1 (M) and CXCL5 (N) in the related mice upon AOM/DSS treatment. Data are represented as mean ± SEM; n=5 in each group; ns, not significant; * P<0.05; **P<0.01; *** P<0.001; and **** P<0.0001.
Fig 5: Mast cells produce chemokines upon stimulation, production of which is blocked by drugs used in treatment of cGVHD. Mast cells produce high levels of (A) CCL2, (B) CCL3, and (C) CCL4 upon stimulation with IgE + antigen or IgE + antigen + IL-33 (column 1 vs. columns 2 and 6). Production of these chemokines is inhibited by treatment with either ibrutinib or ruxolitinib in a dose-dependent manner. Results shown are representative of 2–4 independent assays. Error bars are the SD of technical replicates. Chemokine assays were performed using the LEGENDplex Inflammatory Chemokine Assay kit, which measures levels of 13 chemokines. Mast cells did not produce significant amounts of CCL5, CCL11, CCL17, CXCL1, CXCL9, CXCL10, CXCL13, CXCL5, or CCL22 (data not shown). *P = 0.01–0.05, **P = 0.001–0.01, ***P = 0.0001–0.001, ****P < 0.0001, NS, not significant.
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