1. Volume 132, Issue 5, Pages 1778-1790 (May 2007)
Inducible Nitric Oxide Synthase From Bone Marrow-Derived Cells Plays a Critical Role in Regulating Colonic Inflammation 
Paul L. Beck, Yan Li, J. Wong, Chang–Wen Chen, Catherine M. Keenan, Keith A. Sharkey, Donna–Marie McCafferty 
Gastroenterology 
Volume 132, Issue 5, Pages (May 2007)
DOI: /j.gastro
Copyright © 2007 AGA Institute Terms and Conditions

2. Figure 1
(A–D) Expression of congenic Thy 1.1 and Thy 1.2 on thymocytes from chimeric lines at 6 weeks post-bone marrow transplantation, as determined by flow cytometry. A and B show expression of Thy 1.1 and Thy 1.2 on thymocytes from a Thy 1.1 wild-type (wt) congenic mouse (recipient) reconstituted with Thy 1.2 iNOS−/− (donor) BM. C and D show expression of Thy 1.1 and Thy 1.2 on thymocytes from a Thy 1.2 iNOS−/− mouse (recipient) reconstituted with Thy 1.1 wt congenic (donor) BM. In all of the chimeric lines, the vast majority of thymocytes were positive for the congenic marker expressed by the donor cells. (E and F) Y chromosome in situ hybridization at day 5 of DSS exposure in E shows a wt female mouse reconstituted with wt BM from a female donor and in F a wt female mouse reconstituted with wt BM from a male donor. Only the infiltrating inflammatory cells were positive for the Y chromosome (F), and none of the epithelial or endothelial cells were found to be of donor (male; Y chromosome +ve) origin. Bars, 50 μm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
(A) Weight loss expressed as the percentage basal body weight loss at day 7 of DSS exposure. The chimeric lines that were reconstituted with iNOS−/− BM (iNOS→iNOS or iNOS→wt) had less severe DSS-induced weight loss then the lines reconstituted with wt BM (wt→wt or wt→iNOS). Transplantation of iNOS−/− BM resulted in resistance to DSS-induced colitis in wt (donor) animals, and transplantation of wt (iNOS+/+) reversed the resistance of the iNOS−/− mice (recipient) to DSS-colitis. wt, wild type (iNOS+/+); iNOS, iNOS−/−. **P < .01, *P < .05 vs wt→wt (n = a minimum of 8 animals per group). (B) Hematocrit at day 7 of DSS exposure. There was no difference in the hematocrit in any of the untreated chimeric lines. All lines had evidence of fecal blood loss at day 7 of DSS. The chimeric lines that were reconstituted with iNOS−/− BM (iNOS→iNOS or iNOS→wt) had less fecal blood loss, as indicated by higher hematocrits, than the lines reconstituted with wt BM (wt→wt or wt→iNOS). Transplantation of iNOS−/− BM resulted in resistance to DSS-induced colitis in wt (donor) animals, and transplantation of wt (iNOS+/+) reversed the resistance of the iNOS−/− mice (recipient) to DSS-colitis. wt, wild type (iNOS +/+); iNOS, iNOS−/− **P <.01, *P < .05 vs wt→wt, ••P < .01 vs wt→iNOS (n = a minimum of 8 animals per group). (C) Histologic assessment at day 7 of DSS in the chimeric lines. The chimeric lines that were reconstituted with iNOS−/− BM (iNOS→iNOS or iNOS→wt) had lower levels of inflammation and less ulceration compared with the lines reconstituted with wt BM (wt→wt or wt→iNOS). Transplantation of iNOS−/− BM resulted in resistance to DSS-induced colitis in wt (donor) animals, and transplantation of wt (iNOS+/+) reversed the resistance of the iNOS−/− mice (recipient) to DSS-colitis. wt, wild type (iNOS +/+); iNOS, iNOS−/−. **P < .01, *P < .05 vs wt→wt, ••P < .01, •P < .05 vs wt→iNOS (n = a minimum of 8 animals per group). (D) Colonic tissue levels of myeloperoxidase (MPO) activity at day 7 of DSS. The chimeric lines that were reconstituted with iNOS−/− BM (iNOS→iNOS or iNOS→wt) had lower levels of inflammation as assessed by tissue MPO levels compared with the lines reconstituted with wt BM (wt→wt or wt→iNOS). Transplantation of iNOS−/− BM resulted in resistance to DSS-induced colitis in wt (donor) animals, and transplantation of wt (iNOS +/+) reversed the resistance of the iNOS−/− mice (recipient) to DSS-colitis. wt, wild type (iNOS +/+); iNOS, iNOS−/−. **P < .01, *P < .05 vs wt→wt, ••P < .01 vs wt→iNOS (n = a minimum of 8 animals per group).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
Serum nitrate/nitrite levels in the chimeric lines prior to DSS exposure (untreated) and at DSS day 7. An increase in serum nitrate/nitrite was noted in wt→wt at DSS day 7 compared with untreated wt→wt animals (■P < .05). The chimeric lines that were reconstituted with iNOS−/− BM (iNOS→iNOS or iNOS→wt) had lower serum nitrate/nitrite levels compared with the lines reconstituted with wt BM (wt→wt or wt→iNOS) in both prior DSS (untreated) exposure and at DSS day 7. wt, wild type (iNOS +/+); iNOS, iNOS−/−. ***P < .001, **P < .01, *P < .05 vs similarly treated wt→wt and ••P < .01 untreated vs similarly treated wt→iNOS animals (n = a minimum of 8 animals per group).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
Immunohistochemical staining of iNOS and neutrophils in wild-type animals at DSS day 7. Dual labeling of iNOS (FITC; green) and neutrophils (CY3; red) revealed that iNOS was expressed by epithelial cells and neutrophils. Of all the inflammatory cells that expressed iNOS (stained green), the majority were neutrophils (stained red and had classic histologic characteristics of neutrophils). (A) Shows an ulcerated area with submucosal infiltration of predominantly neutrophils, some of which are expressing iNOS. (B and C) Show similar staining (at higher magnification) of cells in the lamina propria and submucosa at DSS day 7. Bars, 50 μm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
Leukocyte adhesion in vessels of the jejunum and colon over a 100-μm length following IP administration of 50 μg/kg lipopolysaccharide (LPS). iNOS→iNOS chimeric lines had reduced LPS-induced leukocyte recruitment than wt→wt animals (*P < .05). The chimeric lines iNOS→wt and wt→iNOS had extremely variable results (data not shown, P > .05) (n = a minimum of 6 animals per group).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
(A) Chemokine expression in chimeric lines at DSS day 7 as assessed by multitemplate RNase protection assays. Chemokine expression was assessed from RNA extracted from full-thickness segments of colon. In general, the wt→wt animals had higher levels of chemokine expression than the other chimeric line, but the only significant difference (*P < .05) that was noted was in MIP-1α (vs the other chimeric lines). DSS exposure resulted in increased expression of all the chemokine assessed in all of the chimeric lines compared with DSS naïve animals (data not shown) (n = a minimum of 8 animals per group). (B) Keratinocyte growth factor (KGF) and tumor necrosis factor α (TNF-α) in chimeric lines at DSS day 7 as assessed by semiquantitative RT-PCR. RT-PCR was performed on RNA extracted from full-thickness segments of colon. All of the chimeric lines that had some form of iNOS deficiency (wt→iNOS, iNOS→iNOS, and iNOS→wt) had higher levels of KGF expression and lower levels of TNF-α expression. ***P < .001, **P < .01, *P < .05 vs wt→wt (n = a minimum of 8 animals per group).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

8. Figure 7
Colonic lamina propria leukocyte populations at DSS day 7 and intracellular cytokine expression by colonic lamina propria CD4+ve cells isolated at DSS day 7. (A) Lamina propria leukocyte populations isolated from colonic tissue and DSS day 7 and analyzed by flow cytometry gated for monocyte/lymphocyte populations. Compared with wt→wt animals, mice that were reconstituted with iNOS−/− BM (iNOS→iNOS or iNOS→wt) had higher numbers of CD3+ve cells and macrophages (F4/80 +ve cells). There was also a reduction in the number of B cells (B220 +ve) in the mice reconstituted with iNOS−/− BM. Interesting, the iNOS−/− mice that were reconstituted with wt BM (wt→iNOS) had lower CD3 +ve cells and increased B cells compared with wt→wt mice. No significant differences were noted in the NK 1.1 +ve cells. (B) Intracellular cytokine expression by colonic lamina propria CD4 +ve cells at DSS day 7, expressed as the percentage of cells staining for the cytokine in question. The mice reconstituted with iNOS−/− BM (iNOS→iNOS or iNOS→wt) had lower levels of expression of INF-γ, IL-2, and IL-10 compared with wt→wt There were also lower levels of IL-4 expression, but this failed to reach significance at P < .05. There was no difference in the wt→iNOS vs wt→wt. For each assay, a colonic segment from 3 to 4 animals were pooled together because of the small number of cells, and each experiment was done a minimum of 3 times. ***P < .001, **P < .01, *P < .05 vs similarly treated wt→wt; ••P < .01, •P < .05 vs wt→iNOS.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

9. Figure 8
iNOS-deficient BM confers resistance to TNBS-induced colitis. TNBS colitis model was induced in 8 weeks following BM transplantation. The wt→wt mice were more susceptible to TNBS colitis than the chimeric line with iNOS−/− BM. (A) Histology 3 days following induction of TNBS colitis. Note the severe transmural inflammation in the wt→wt mice. In wt→wt animals, there is a large ulcerated area with inflammatory cells extending through the muscularis and into the attached segment of serosa. An inflammatory cell infiltrate can be seen in the iNOS→wt animal that extends through the submucosa and into the muscularis. The mucosal architecture is disrupted in the iNOS→wt animals with an obvious crypt abscess (*), but there is no frank ulceration present (n = a minimum of 7 animals per group). Bars, 50 μm. (B) Histology 3 days following induction of TNBS colitis. The line that was reconstituted with iNOS−/− BM (iNOS→wt) had lower levels of inflammation and less ulceration compared with the line reconstituted with wt BM (wt→wt). Transplantation of iNOS−/− BM resulted in resistance to TNBS-induced colitis in wt animals (***P < .001, **P < .01 vs wt→wt) (n = a minimum of 7 animals per group). (C) Colonic tissue levels of myeloperoxidase (MPO) activity 3 days following induction of TNBS colitis. The line that was reconstituted with iNOS−/− BM (iNOS→wt) had lower levels of inflammation as assessed by tissue MPO levels compared with the lines reconstituted with wt BM (wt→wt) (*P < .05 vs wt→wt) (n = a minimum of 7 animals per group).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions