Volume 120, Issue 4, Pages 900-913 (March 2001) Continuous stimulation by normal luminal bacteria is essential for the development and perpetuation of colitis in Tgϵ26 mice  Claudia Veltkamp, Susan L. Tonkonogy, Ype P. de Jong, Carol Albright, Wetonia B. Grenther, Edward Balish, Cox Terhorst, R.Balfour Sartor  Gastroenterology  Volume 120, Issue 4, Pages 900-913 (March 2001) DOI: 10.1053/gast.2001.22547 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Photomicrographs of the colons of SPF and GF Tgϵ26 mice after BM transplant or MLN cell transfer. Representative H&E-stained sections from the distal colon are shown (original magnification 66×). (A) Normal colon from a nontransplanted SPF Tgϵ26 mouse. (B) Moderate colitis in a SPF Tgϵ26 mouse 5 weeks after BM transplantation showing crypt hyperplasia, decreased goblet cells, and pronounced lamina propria lymphocytic infiltration. (C) Normal colon from a nontransplanted GF Tgϵ26 mouse. (D) No signs of colonic inflammation are seen in a BM⇒GF Tgϵ26 recipient 12 weeks after transplantation. (E) Severe colitis in an SPF Tgϵ26 mouse injected 2 weeks previously with MLN cells from a BM⇒SPF Tgϵ26 mouse with colitis. Extensive lamina propria cellular infiltration, crypt hyperplasia, and crypt abscesses are evident. (F) No signs of inflammation are seen in the colon of a GF Tgϵ26 mouse 8 weeks after transfer of the same source of MLN cells used in the SPF mouse shown in E. (G) Severe cecal inflammation in a BM⇒GF Tgϵ26 animal maintained in a GF environment for 8 weeks and then colonized with SPF intestinal bacteria for 4 weeks. (H) Active colitis in an SPF Tgϵ26 recipient 3 weeks after transfer of MLN cells obtained from a BM⇒GF Tgϵ26 mouse free of colitis 8 weeks after transplantation. Gastroenterology 2001 120, 900-913DOI: (10.1053/gast.2001.22547) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 (A) Blinded histologic score and (B) IL-12 (p40) secretion detected in supernatants of cultured fragments of the colon of BM⇒Tgϵ26 mice in the presence and absence of SPF bacteria. GF or SPF Tgϵ26 recipients were transplanted with BM from normal mice (n = 24 in both groups) and killed 4–6 weeks (SPF) or 8, 12, or 24 weeks (GF) later. Inflammation was scored on a scale of 0–4 (see Materials and Methods), and results are expressed as means ± SEM. Supernatants were collected after 18 hours of culture, and IL-12 (p40) was detected by ELISA. *P < 0.0001 vs. GF mice. Gastroenterology 2001 120, 900-913DOI: (10.1053/gast.2001.22547) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Cytokine production detected in supernatants of MLN cell cultures 3 days after stimulation with anti-CD3 antibody. (A) IFN-γ and (B) IL-4 were measured by ELISA. Values represent means ± SEM nanograms per milliliter (IFN-γ) or picograms per milliliter (IL-4) in supernatants of MLN cell cultures from SPF (n = 17) and GF (n = 16) Tgϵ26 mice after BM transplantation and from SPF normal mice (n = 18). Limits of detection are 5 pg/mL of IFN-γ and 1 pg/mL of IL-4. *P < 0.05 vs. GF Tgϵ26 and vs. normal mice; **P < 0.05 vs. SPF Tgϵ26 and normal mice. Gastroenterology 2001 120, 900-913DOI: (10.1053/gast.2001.22547) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Intracytoplasmic staining of (A and C) IFN-γ and (B and D) TNF. Dot plots for gated CD4+ MLN cells of representative GF and SPF Tgϵ26 mice after BM transplantation or an SPF control mouse analyzed by flow cytometry after overnight stimulation with immobilized anti-CD3 antibody. The summation of intracytoplasmic staining of (C) IFN-γ and (D) TNF is shown in gated CD4+ MLN cells from GF (n = 5) and SPF (n = 6) Tgϵ26 mice after BM transplantation and from normal mice (n = 3). The mean percentages ± SEM of cytokine-positive cells are shown. (C) *P < 0.01 vs. GF and vs. normal mice; **P < 0.001 vs. normal mice. (D) *P < 0.001 vs. GF and vs. normal mice. Gastroenterology 2001 120, 900-913DOI: (10.1053/gast.2001.22547) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 IFN-γ production by CD4+ T cells stimulated with APCs pulsed overnight with cecal extracts from SPF or GF Tgϵ26 mice, epithelial cells from GF Tgϵ26 mice, KLH, or no antigen. Supernatants collected on day 3 of culture were assayed for IFN-γ by ELISA. Values represent means ± SD of units per milliliter of IFN-γ in supernatants of pooled CD4+ MLN cells from 3 BM⇒SPF Tgϵ26 mice with colitis in duplicate or triplicate cultures. *P < 0.005 vs. all other groups. Gastroenterology 2001 120, 900-913DOI: (10.1053/gast.2001.22547) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) Blinded colonic histologic scores and (B) IL-12 (p40) secreted from cultured fragments of the colons of GF or SPF Tgϵ26 recipients that received MLN cells obtained from BM⇒SPF Tgϵ26 mice with colitis (n = 12, SPF group; n = 5, GF group) and were killed 2–4 weeks (SPF) or 8 weeks (GF) later. Histologic scores and IL-12 measurements were performed as described in Figure 2 and expressed as means ± SEM. *P < 0.001 vs. GF mice. Gastroenterology 2001 120, 900-913DOI: (10.1053/gast.2001.22547) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Comparison of infiltrating CD4+ T lymphocytes in the colonic mucosa of (A) GF and (B) SPF Tgϵ26 mice, both reconstituted with MLN cells from BM⇒SPF Tgϵ26 mice with colitis (original magnification 66×). CD4+ cells were identified by immunohistochemistry using the AEC substrate shown in brown, counterstained with H&E. Gastroenterology 2001 120, 900-913DOI: (10.1053/gast.2001.22547) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 8 Flow-cytometric analysis of the expression of activation/memory markers in CD4+ MLN cells in SPF and GF Tgϵ26 mice after MLN cell transfer from BM⇒SPF Tgϵ26 mice with colitis and in normal mice. (A) Dot plots showing representative proportions of CD4+ and CD8+ MLN cells in SPF and GF recipient Tgϵ26 mice after MLN cell transfer from SPF Tgϵ26 mice with colitis and proportions of CD4+ and CD8+ MLN cells in normal mice (top row). Representative histograms are shown for the expression of L-selectin, CD44, CD45RB, and CD69 on gated CD4+ MLN cells. (B) The percentages of CD4+ cells that are L-selectinneg, CD44hi, CD45RBlo, and CD69pos were determined for MLN cells from SPF (n = 6) or GF (n = 5) Tgϵ26 mice after MLN cell transfer from BM⇒SPF Tgϵ26 mice with colitis or from normal mice (n = 11). Data are the mean values ± SEM calculated for each group. *P < 0.0001 vs. normal mice; **P < 0.01 vs. normal mice. Gastroenterology 2001 120, 900-913DOI: (10.1053/gast.2001.22547) Copyright © 2001 American Gastroenterological Association Terms and Conditions