1. Volume 130, Issue 2, Pages 398-411 (February 2006)
Chitinase 3–Like-1 Exacerbates Intestinal Inflammation by Enhancing Bacterial Adhesion and Invasion in Colonic Epithelial Cells 
Emiko Mizoguchi 
Gastroenterology 
Volume 130, Issue 2, Pages (February 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Terms and Conditions

2. Figure 1
Increased CHI3L1 expression in murine models of colitis. (A) CHI3L1 mRNA expression by quantitative PCR in wild-type, DSS days 4 and 8, TCRα KO, B-cell–deficient TCRα KO (αμ), and IL-10 KO mice are shown as fold increased compared with wild-type mice. Average disease severity39 was 1.4 ± .5, 2.6 ± .5, and 2.0 ± .6 in TCRα KO, αμ KO, and IL-10 KO, respectively, and the score is shown in parenthesis. Each value represents the mean of 6 mice/group ± SEM. *P < .5, and **P < .01. (B–G) Immunohistochemical analysis of the cellular expression of CHI3L1 is shown. CHI3L1 is detected by staining with rabbit anti-mouse CHI3L1 Ab predominantly in the CECs on (D and E) DSS day 3 and LP cells on (F) DSS day 8 and (G) day 12, but no staining on (C) day 0. (B) Control rabbit serum in DSS day 0. (B, C, and E–G) Objective, 20×; (D) objective, 10×.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

3. Figure 2
Increased CHI3L1 expression in human IBD. (A–F) Immunohistochemical analysis of the expression of CHI3L1 is shown (A: objective, 20×; B–F: objective, 40×). Colonic tissue obtained from (A and B) normal individual, (C and D) CD, and (E and F) UC patients were stained with (A–C, E, and F) rabbit anti-human CHI3L1 or (D) normal rabbit serum. (G) Quantitative PCR analysis of human CHI3L1 expression in biopsy samples of patients with active UC (uc+, n = 4), inactive UC (uc−, n = 5), involved region of CD (cd+, n = 4), uninvolved region of CD (cd−, n = 5), diverticulitis (dd, n = 3), and normal individuals (n = 5) are shown as mean ± SEM. *P < .1, and **P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

4. Figure 3
Constitutive and inducible CHI3L1 expression in human colonic cell lines. (A) CHI3L1 mRNA were detected in macrophage cells (THP-1), T cells (Jurkat), and colonic epithelial cells (Caco-2, SW480, HT29, T84, and COLO205) by reverse-transcription PCR. (B) SW480 cells and (C) differentiated T84 cells were treated with recombinant human tumor necrosis factor α, IL-6, IL-1β, IL-4, IL-13, IL-10, or interferon γ for 5 hours or no stimulation (ns). CHI3L1 mRNA was detected by reverse-transcription PCR as described in the Materials and Methods section. The dose of human recombinant cytokines was 10 ng/mL unless specified.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

5. Figure 4
Effect of CHI3L1 on adhesion and invasion of S typhimurium in CECs. (A) Cell-associated and (B) intracellular bacteria were quantified after infection with S typhimurium for 1 hour. The number of invasive bacteria was determined after 100 μg/mL gentamicin treatment for 1 hour. Results are expressed as cell-associated (adherent + intracellular) or intracellular bacteria relative to the number of originally inoculated S typhimurium. The mean numbers of cell-associated and intracellular S typhimurium was 1.5 × 106 ± 1.1 × 106 and 1.1 × 105 ± 4.3 × 104 CFU/well, respectively. Each value is the mean ± SD of at least 6 separate experiments. *P < .1, **P < .05, and ***P < (C) Dose-dependent effect of pcDNA4 CHI3L1 vector (upper panel) and anti-CHI3L1 Ab (lower panel) in the number of intracellular bacteria were quantified after 2 hours of infection with S typhimurium. Results are expressed as the fold increase of the bacteria number compared with the control group (nontransfected CMT93 cells), which is taken as 1. Each value is the mean ± SEM of 3 separate experiments. *P < .1, **P < .05, and ***P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

6. Figure 5
Inhibitory effect of CHI3L1 expression by siRNA in CMT93 cells. (A) Real-time PCR analysis of β-actin mRNA expression (upper panel) and CHI3L1 (lower panel) in CMT93 cells untreated or treated by pcDNA4/CHI3L1 vector (.5 μg/well) that was cotransfected with or without combined pooled siRNA (1 μg of each siRNA/well). (B) Cell-associated bacteria (upper panel) and intracellular bacteria (lower panel) were quantified after a 1-hour infection period with S typhimurium in CMT93 cells untreated or treated by pcDNA4/CHI3L1 vector (.5 μg/well) that was cotransfected with or without combined pooled siRNA that was quantified in A. The number of invasive bacteria was determined after gentamicin treatment for 1 hour. Results are expressed as cell-associated bacteria, and intracellular bacteria compared with those obtained for mock (pcDNA4 vector) transfection control, which is taken as 1. Each value is the mean ± SD of at least 6 separate experiments. *P < .05, **P < .01, and ***P < .001.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

7. Figure 6
Effect of CHI3L1 on adhesion and invasion of different strains of E coli in CECs. Results are expressed as cell-associated (adherent + intracellular) or intracellular bacteria relative to the number of originally inoculated bacteria. (A) Cell-associated and intracellular bacteria were quantified after 1 hour of infection with AIEC (LF82 strain). The number of invasive bacteria was determined after gentamicin treatment for 1 hour. The mean number of cell-associated and intracellular LF82 bacteria was 5.8 × 105 ± 2.8 × 105 and 7.4 × 103 ± 3.1 × 103CFU/well, respectively. Each value is the mean ± SD of at least 6 separate experiments. *P < .1, **P < .05, and ***P < (B) Cell-associated (left panel) and intracellular bacteria (right panel) were quantified after a 2-hour infection period with EPEC JpNAR 15 strain. The mean number of cell associated and intracellular EPEC JpNAR 15 was 3.0 × 105 ± 2.6 × 105 and 4.2 × 103 ± 4.0 × 103 CFU/well, respectively. Each value is the mean ± SD of duplicated 3 separate experiments. *P < .1, **P < .05, and ***P < .01. (C) Cell-associated (left panel) and intracellular bacteria (right panel) were quantified after a 2-hour infection with E coli DH10B strain. The mean number of cell-associated and intracellular E coli DH10B was 3.2 × 104 ± 2.7 × 104 and 1.2 × 102 ± .6 × 102 CFU/well, respectively. Each value represents the mean ± SD of 3 duplicated individual experiments. Numbers on each bar indicate the P value compared with the nontransfected control group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

8. Figure 7
Role of CHI3L1 in S typhimurium infection in vivo. (A) Organ load with S typhimurium (2 × 108 CFU) 48 hours after infection was analyzed in C57BL/6 that was injected intraperitoneally with 1 mg anti-CHI3L1 Ab (▨) or purified rabbit IgG (■) on day −1. (B) Survival rate of C57BL/6 mice that was pretreated with 1 mg of anti-CHI3L1 Ab (□) or purified rabbit IgG (■) on days −3 to −1 and infected orally with 2 × 108 CFU of S typhimurium on day 0. n = 12 in each group of mice.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

9. Figure 8
CHI3L1 contributes to the exacerbation of acute colitis by the enhancement of bacterial invasion to the host. (A) Percent body weight change and (B) clinical scores of DSS colitis that was treated with anti-CHI3L1 (○) or purified rabbit IgG (▴) at days −1 to 2 (left) and days 5–8 (right) are shown. The values represent the mean ± SEM of 12 mice in each group. (C) Representative H&E and anti-BrdU staining of a distal part of the colon of DSS-treated mice (day 12) that received anti-CHI3L1 Ab or control rabbit IgG on days −1 to 2 are shown. (D) Histology score (upper panel) or number of BrdU incorporated cells per crypt unit of epithelium (lower panel) in anti-CHI3L1 Ab- (▨) or control rabbit IgG- (■) treated DSS mice on day 12 are shown. (E) Tissue invading gram-positive/negative (left panel) and gram-negative (right panel) in DSS colitis (day 12) that received anti-CHI3L1 Ab or rabbit IgG administered on days −1 to 2 are shown. Arrowheads indicate colonized bacteria in the surface tip of epithelium. (F) Number of invading bacteria in spleen, mesenteric lymph nodes, and liver was shown in C57BL/6 on DSS day 6 with 1 mg anti-CHI3L1 Ab (▨) or control rabbit IgG (■) on days −1 to 2.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions