Volume 137, Issue 3, Pages 1006-1018.e3 (September 2009) Expression of the Chemokine Binding Protein M3 Promotes Marked Changes in the Accumulation of Specific Leukocytes Subsets Within the Intestine  Limin Shang, Nanthakumar Thirunarayanan, Abel Viejo–Borbolla, Andrea P. Martin, Milena Bogunovic, Federica Marchesi, Jay C. Unkeless, Yin Ho, Glaucia C. Furtado, Antonio Alcami, Miriam Merad, Lloyd Mayer, Sergio A. Lira  Gastroenterology  Volume 137, Issue 3, Pages 1006-1018.e3 (September 2009) DOI: 10.1053/j.gastro.2009.05.055 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Expression pattern of chemokines in intestinal segments. (A) Sampling of intestinal segments for expression analysis is schematically depicted. Total RNA was isolated from 5–7 pieces of duodenum, jejunum, ileum, cecum, proximal colon, and distal colon for each mouse (n = 3). (B) Differential expression of chemokines in intestinal segments. The complementary DNA samples from 5–7 pieces of each intestinal segment were pooled for analysis of messenger RNA. The messenger RNA expression values were normalized to ubiquitin in each sample, and the data shown are the mean of samples isolated from 3 BDF1 mice. Gastroenterology 2009 137, 1006-1018.e3DOI: (10.1053/j.gastro.2009.05.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Expression of M3 in the intestine of V-M3 mice. (A) Diagram of the V-M3 transgene. p(A), SV40 poly A sequences. (B) M3 messenger RNA expression in different tissues of V-M3 mice. The values were normalized to ubiquitin in each sample. H, Heart; Lu, Lung; K, Kidney; Br, Brain; LI, Large intestine; SI, Small intestine. (C) Western blot analysis of M3 expression in different segments of intestine of V-M3 mice. (D and E) Representative immunostaining for M3 in small intestine of (D) WT and (E) V-M3 mice. Gastroenterology 2009 137, 1006-1018.e3DOI: (10.1053/j.gastro.2009.05.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 PP of V-M3 mice are smaller and have fewer cells than WT PP. (A and B) V-M3 mice had much smaller PP (B, arrows) than WT littermates (A). (C) The percentage of T and B cells in PP of WT and V-M3 mice. (D) Absolute numbers of B and T cells in PP of WT and V-M3 mice. For C and D: WT, n = 3; V-M3, n = 4; **P < .01. Gastroenterology 2009 137, 1006-1018.e3DOI: (10.1053/j.gastro.2009.05.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 The effects of M3 expression on lymphocyte subsets in the intestinal epithelium of V-M3 mice. (A) Representative FACS analysis of intraepithelial T-cell subsets in V-M3 mice. The upper panel was gated on CD3+ T cells, the middle panel was gated on CD8+ T cells, and the bottom panel was gated on CD8αα+ T cells. (B) Quantitative analysis of intraepithelial T-cell subsets in V-M3 mice. The upper panel represents the percentage of CD3+ cells for each T-cell subset, and the bottom panel shows the absolute cell number of each subset (n = 4 for each group, *P < .05, **P < .01). Gastroenterology 2009 137, 1006-1018.e3DOI: (10.1053/j.gastro.2009.05.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Reduction of CD8+ T cells in the small intestine but not large intestine LP of V-M3 mice. (A) FACS analysis of B and T cells in the small intestine LP of WT and V-M3 mice (n = 4 for each group). (B) FACS analysis of CD4 and CD8 T cells in the small intestine LP of WT and V-M3 mice. The analysis was gated on CD3+ cells (n = 4 for each group, *P < .05). (C) FACS analysis of B and T cells in the large intestine of WT and V-M3 mice (n = 4 for each group). (D) FACS analysis of CD4 and CD8 T cells in the large intestine of WT and V-M3 mice. The analysis was performed on CD3+ cells. Gastroenterology 2009 137, 1006-1018.e3DOI: (10.1053/j.gastro.2009.05.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Effect of M3 expression on myeloid cell subsets in the LP of WT and V-M3 mice. (A and B) FACS analysis of LPLs in the (A) small intestine and (B) large intestine of WT and V-M3 mice. The lower FACS panel was gated on the CD11b+/CD11clow subset of the upper panel. The R1, R2, and R3 populations in the right panel correspond to the gates in the lower FACS panel (n = 4 for each group, *P < .05, **P < .01, ***P < .001). (C) Cytospin morphology of cells from R1, R2, and R3 gates of CD11b+/CD11clow cells (as shown in A) from the LP of WT mice. (D) Analysis of CD11b+/CD11clow cells from LP of WT mice for eosinophil markers SIGLEC-F and CCR3. The analysis was performed on the CD11b+/CD11clow cells from LP of WT mice. Gastroenterology 2009 137, 1006-1018.e3DOI: (10.1053/j.gastro.2009.05.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Chemokine receptor profiles of CD8+ IEL T cells and of CD11b+/CD11clow LPL myeloid cells that were affected by M3 expression. (A and B) Quantitative PCR analysis of chemokine receptor profiles of the (A) CD8αβ/TCRαβ+ subset, CD4+/TCRαβ+ subset, and (B) CD8αα/TCRαβ+ subset of IELs in WT mice. (C and D) Chemokine receptor profiles of the (C) eosinophils and (D) CD11b+/CD11clow/MHCII+/Gr-1low macrophages from the LP of WT mice. Gastroenterology 2009 137, 1006-1018.e3DOI: (10.1053/j.gastro.2009.05.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 8 Effects of M3 expression on DSS-induced myeloid cell accumulation in the colon. (A and B) DSS treatment of C57BL/6 WT mice altered expression of many (A) CC, (B) CXC, and CX3C chemokines in the colon. Chemokines shown here were those significantly altered by DSS treatment (P < .05, n = 5 for WT and n = 3 for WT + DSS). (C) 6V-M3 transgenic mice (dotted line) lost less body weight than WT littermates (solid line) in response to DSS treatment (*P < .05). (D and E) M3 expression reduced DSS-induced (D) dendritic cell, (E) eosinophil, and macrophage accumulation in the colon of 6V-M3 mice (n = 4 per group, *P < .05, **P < .01, ***P < .001). Gastroenterology 2009 137, 1006-1018.e3DOI: (10.1053/j.gastro.2009.05.055) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 1 Chemokine expression profiles of intestinal segments. Many CC (A), CXC, CX3C, and XC (B) chemokines are expressed in different intestinal segments of BDF1 mice. The expression values were normalized to ubiquitin in each sample and the data shown are the mean of three independent samples (n = 3). Gastroenterology 2009 137, 1006-1018.e3DOI: (10.1053/j.gastro.2009.05.055) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 2 Interaction of M3 with chemokines expressed in the intestine. Individual sensorgrams showing the interaction of M3 with the murine chemokines CCL5, CCL6, CCL11, CCL22, CCL24, CCL25, and CCL28. No interaction was observed between M3 and murine CXCL12α or TNFα. The chemokines and TNFα were injected at a concentration of 100 nM. Gastroenterology 2009 137, 1006-1018.e3DOI: (10.1053/j.gastro.2009.05.055) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 3 M3 expression in the gut of V-M3 mice did not affect the lymphocyte homeostasis in spleen or LNs. (A–C) FACS analysis of the relative and absolute number of B and T cells in the spleen (A), inguinal LN (B), and MLN (C) of WT and V-M3 mice (n = 3 for each group). Gastroenterology 2009 137, 1006-1018.e3DOI: (10.1053/j.gastro.2009.05.055) Copyright © 2009 AGA Institute Terms and Conditions