Volume 138, Issue 4, Pages 1395-1405 (April 2010) In Vitro-Derived Alternatively Activated Macrophages Reduce Colonic Inflammation in Mice  Meaghan M. Hunter, Arthur Wang, Kuljit S. Parhar, Michael J.G. Johnston, Nico Van Rooijen, Paul L. Beck, Derek M. McKay  Gastroenterology  Volume 138, Issue 4, Pages 1395-1405 (April 2010) DOI: 10.1053/j.gastro.2009.12.041 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 RT-PCR reveals that infection with H diminuta increases markers indicative of alternatively activated macrophages in mouse colon. Panel A shows representative gels where β-actin served as a housekeeping gene. Panel B is the densitometry analysis of the mRNA products. Cotreatment of mice with a neutralizing anti-IL-10 AB inhibits the ability of infection with H diminuta (H.d) to increase expression of FIZZ1 and arginase-1 mRNA in the colon (mean ± SEM; n = 5 mice; *P < .05 compared with control). Gastroenterology 2010 138, 1395-1405DOI: (10.1053/j.gastro.2009.12.041) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 The ability of infection with H diminuta to protect mice from DNBS-induced colitis is lost by cotreatment with clodronate liposomes (clod). Mice were infected with H diminuta 8 days before DNBS, and liposomes (200 μL, IP) were given on days 3, 7, and 9 postinfection. Colitis was assessed by disease activity scores (A), colonic MPO levels (B), and histology damage scores (C). Panel D is representative H&E-stained sections of colon (control mice given PBS-liposomes; original magnification, 200×) (mean ± SEM; n = 3 to 4 mice from 1 representative experiment of 3 experiments; *P < .05 compared with control and #P < .05 compared with other DNBS groups; M, muscle; L, lumen). Gastroenterology 2010 138, 1395-1405DOI: (10.1053/j.gastro.2009.12.041) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 (A) Representative RT-PCR gel showing differential expression of arginase-1 and iNOS in IL-4+IL-13 and IFN-γ treated cells indicative of AAMs and CAMs, respectively (representative of 5 samples; MO, nontreated macrophages). Panel B shows representative immunoflorescent images of cryostat sections showing AAMs in the colon following IV transfer (n = 4 mice). Transfer of in vitro differentiated AAMs, but not CAMs, reduces colitis when given 2 days before DNBS as gauged by disease activity scores (C), colonic MPO activity (C), and histologic assessment (D). Panel E shows representative H&E-stained sections of murine colon (original magnification, 100×) (mean ± SEM; n = 21 mice from 5 experiments, except for CAMs were n = 8 mice from 2 experiments; *P < .05 compared with control, #P < .05 compared with all other groups). Panel F is representative images of colon (n = 4 mice) in an experiment in which CAMs or AAMs were administered IP 2 days before DNBS or 6 hours after (AAM (6h)) DNBS (arrows indicate a length of 7.5 cm). Gastroenterology 2010 138, 1395-1405DOI: (10.1053/j.gastro.2009.12.041) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 (A) Representative Mason's trichrome staining of collagen (prophylactic protocol) and (B) bar graph showing that levels of collagen in mice treated with DNBS (3 mg intrarectal) ± AAM or CAM (106 cells, IP, 6 hours after DNBS) are not statistically significantly different (mean ± SEM; n = 3 to 4; mice autopsied 72 hours after DNBS; M, muscle; L, lumen; arrow, epithelial erosion). Gastroenterology 2010 138, 1395-1405DOI: (10.1053/j.gastro.2009.12.041) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 (A) Five days post-AAM (106/mouse) administration (also 3 days post-DNBS (3 mg in 100 μL of 50% EtOH intrarectal) in the appropriate groups), arginase-1 mRNA is increased in colon and spleen (representative of 4 mice). (B) Splenocytes from mice treated with AAMs ± DNBS challenged in vitro with conA (2 μg for 48 hours) show increased IL-10 production (n = 8; *P < .05 compared with control and DNBS groups). (C) Disease activity and (D) histology damage scores in mice receiving AAMs + anti-IL-10 AB are intermediate between DNBS and AAM + DNBS-treated mice (mean ± SEM; n = 4 mice, letters indicate groups that are not statistically different; 1-way ANOVA followed by Tukey test). Gastroenterology 2010 138, 1395-1405DOI: (10.1053/j.gastro.2009.12.041) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Panel A shows increased mRNA expression of the mannose receptor (CD206) in biopsy specimens from patients with CD whose disease is inactive (n = 9). (B) Immunostaining revealed AAMs, as characterized by CD68+CD206+, are reduced in biopsy specimens from active disease (n = 12) but are increased in inactive (n = 8), noninflamed CD compared with control (n = 15) (panel C is representative images with double-positive cells identified by arrowheads (original magnification, 200×; omission of the primary AB resulted in no immunostaining; *lumen) (n ≥ 5 patients). (D) Human peripheral blood-derived monocytes can be differentiated into CAMs and AAMs by exposure to IFN-γ or IL-4 (20 ng/mL) (based on CD206 mRNA expression), and (E) the in vitro differentiated AAMs are hyporesponsive to LPS (1 μg/mL, 24 hours) (mean ± SEM; n = 10; *,#,†P < .05 compared to controls, CD ulcerated/inflamed, and CAMs, respectively). Gastroenterology 2010 138, 1395-1405DOI: (10.1053/j.gastro.2009.12.041) Copyright © 2010 AGA Institute Terms and Conditions