and water throughout the study. All experiments were performed in accordance with European guidelines and were reviewed by the local eth- ics committee on animal experiment (Région Midi-Pyrénees). A persistently hunched posture and labored respiration, a weight loss of more than 20% were considered as end-points where animals were euthanized. DSS-induced acute colitis. Wistar rat received drinking water supplemented with 5% (wt/vol) DSS (molecular weight 36,000– 50,000; MP Biomedicals) from day 0 to day 7. Controls were all time-matched and consisted of rats receiving normal tap water (n = 10 per group). Oral administrations of Nissle WT or Nissle ΔclbA were initiated 7 d before inflam- mation and prolonged during the DSS treat- ment. Each rat received a daily oral gavage of 109 cfu dissolved in PBS. Body weight, stool consistency and bleeding were monitored daily. Clinical scoring was based on the evaluation (0–4) from the last two parameters: 0, formed stool and absence of blood trace; 1, diarrhea; 2, diarrhea and presence of blood trace in stool; 3, diarrhea and rectal bleeding; 4, diarrhea and rectal bleeding with rectal prolapse. Animals were sacrificed on day 7 and distal segments of colon were stored at -80°C until assayed for measurement of myeloperoxydase (MPO) activity as previously described37 and ELISA. MPO is a granule associated enzyme present in neutrophils and other cells of myeloid ori- gin, and widely used as a marker of intestinal inflammation. MPO was expressed in units per gram of protein, where 1 U corresponds to the activity required to degrade 1 μmol of hydrogen peroxide in 1 min at room temperature. Protein concentrations were determined using the BC Assay Uptima kit (Interchim). For ELISA, tis- sue proteins were extracted with RIPA buffer (1% Igepal, 0.5% deoxycholic acid and 0.1% sodium dodecyl sulfate in Tris-buffered saline 1X; pH = 7.4) with protease inhibitor cocktail (Roche diagnotics). Protein concentrations were determined as above. Clear lysates were processed for ELISA: Interleukin-1β (IL-1β) and Interleukin-10 (IL-10) were estimated by Milliplex Rat cytokine immunoassay kit (Millipore). CD4+ CD45RBhigh T cell adoptive transfer colitis model. Eight-week-old SCID mice (mice with severe combined immu- nodeficiency, background C.B-17lcr-Prkdcscid/lcrCrl) were used as recipients of transferred cells.38,39 Conventional BalB/c AnNCrl were used as the donors of transferred cells. Single-cell suspensions of spleens from sex-matched mice were treated with red blood cell lysing buffer. CD4+ T cells were isolated using a negative www.landesbioscience.com Gut Microbes 507 Figure 5. Genetic complementation of the clbA mutant of Nissle 1917 restores its probiotic activity. c D4+ c D45RBhigh T cells were purified from spleens of BalB/c a nNc rl mice and 2 × 105 cells were injected (i.p.) into recipient sc ID mice. Groups of 9 mice fed with Nissle WT, Nissle ΔclbA or Nissle ΔclbA + clbA (2.5 × 109 cfu/mouse) every 3 d and sacrificed prema- turely 36 d after transfer due to global exacerbation of colitis severity. The extent of colitis damage was assessed blindly before tissue samples were collected. error bars represent the seM and the data shown result from one experiment. For related data, see Figure S2. (A) plotted data represent colitis scores of each individual mouse and medians are indicated. *p < 0.05, Kruskal-Wallis test, Dunn’s multiple comparison test. (B) Representative gross organ morphologies of the cecum and colon from each group of the recipient mice at day 36 are shown. (C) extracted colons were measured, rinsed with pBs and weighed. c olon length and mucosa thickening that are dependent of the severity of colitis are modulated according to the oral treatment. Mean ratios of colon weight/length are represented. *p < 0.05 and ***p < 0.001 by one-factor a NOVa analysis, Bonferroni’s multiple comparison test. (D) c olonic sections were stained with h &e to determine disease severity. c ompared with histological architecture observed in colonic sections of sc ID mice fed with Nissle ΔclbA after c D4+ c D45RBhigh T cells transfer, oral treatment of these mice with the complemented Nissle strain drives attenuated histological damage similar to that seen in mice fed with Nissle WT strain. Left, the scores for several parameters were summed for a total severity. Right, the images shown are representative h &e stained sections from mice colons before and after c D4+ c D45RBhigh T cells transfer followed by gavages. Bars = 500 μm. selection CD4+ isolation kit (Miltenyi Biotec). Cells were stained with 5 μg/ml anti-CD4-FITC and 2 μg/ml anti-CD45Rb-PE (eBioscience). CD4+ CD45RBhigh cells were isolated by fluores- cence activated cell sorting (FACS). Cells (2 × 105) were injected intraperitoneally into SCID mice (n = 9–11 per group, 2 inde- pendent experiments). Injected SCID mice were orally admin- istered Nissle WT or Nissle ΔclbA (2.5 109 cfu/mouse) or PBS every 3 d for 45 d. Controls consisted of non-injected SCID mice