Nuclear orphan receptor NR2F6 as a safeguard against experimental murine colitis

Objective Nuclear receptors are known to regulate both immune and barrier functions in the GI tract. The nuclear orphan receptor NR2F6 has been shown to suppress the expression of proinflammatory cytokines in T lymphocytes. NR2F6 gene expression is reduced in patients with IBS or UC, but its functional role and tissue dependency in healthy and inflamed gut have not yet been investigated. Design Intestinal inflammation was induced in wild-type, Nr2f6-deficient, Rag1-deficient or bone marrow-reconstituted mice by administration of chemical (dextran sodium sulfate (DSS)) and immunogenic (T cell transfer) triggers. Disease phenotypes were investigated by survival, body weight, colon length and analysis of immune cell infiltrates. Additionally, histology, intestinal permeability, tight junction proteins, bacterial fluorescence in situ hybridisation, apoptosis, cell proliferation and mucus production were investigated. Results Nr2f6-deficient mice were highly susceptible to DSS-induced colitis characterised by enhanced weight loss, increased colonic tissue destruction and immune cell infiltration together with enhanced intestinal permeability and reduced Muc2 expression. T cell transfer colitis and bone marrow reconstitution experiments demonstrated that disease susceptibility was not dependent on the expression of Nr2f6 in the immune compartment but on the protective role of NR2F6 in the intestinal epithelium. Mechanistically, we show that NR2F6 binds to a consensus sequence at −2 kb of the Muc2 promoter and transactivates Muc2 expression. Loss of NR2F6 alters intestinal permeability and results in spontaneous late-onset colitis in Nr2f6-deficient mice. Conclusion We have for the first time identified a fundamental and non-redundant role of NR2F6 in protecting gut barrier homeostasis.

immediately fixed in 4% buffered formalin or in Carnoy's fixative (60% methanol, 30% chloroform, and 10% acetic acid). Fixed tissues were further transferred into an automatic tissue processor (Leica, Wetzlar, Germany). Sections of 5 μm were stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS). All slides were scanned on a Pannoramic Scan digital slide scanner and investigated using Pannoramic Viewer software (both 3DHistech, Budapest, Hungary) in a blinded fashion. Sections from DSS-treated mice were assessed as described previously [46]; Briefly, distal colon was scored on a scale of 0-4 based on % of colon involvement by inflammation, % of crypt loss, presence of lymphoid follicles, edema, erosions, and density of inflammatory cells. The scores display the sum of the parameters for a total severity score. Numbers of goblet cells were assessed on PAS-stained slides and expressed as percentage per intestinal epithelial cells along the crypt axis. Numbers of goblet cells were assessed on PAS-stained slides and expressed as percentage per intestinal epithelial cells along the crypt axis.
Samples were viewed and imaged on a Zeiss AxioObserver.Z1 in combination with a LSM700 confocal laser scanning system and scored as described previously [33]. Only slides with clear and crisp hybridization signals were included into enumeration. Mean values and standard deviations were calculated for bacterial concentrations and measurements of mucus layer thickness.
Immunohistochemistry 4 µm sections were deparaffinized, rehydrated and antigens were retrieved by pressure cooking in antigen unmasking solution (Vector Laboratories, Burlingame, CA). Non−specific protein binding sites were blocked using 1% BSA. Slides were incubated with their respective primary and secondary (anti-rabbit, anti-rat IgG Alexa Fluor 488 ® or Alexa Fluor594) antibodies at RT for 1 hour (for immunofluorescence) or 4°C overnight (for immunohistochemistry). Slides were mounted with Dako fluorescent mounting medium (S3023), which includes DAPI staining. IHC analysis of colons was performed in triplicates of 4 biopsy specimens of Nr2f6 +/+ and Nr2f6 -/mice. Digital IHC images were acquired with an Axiovert 40 CFL (ZEISS) microscope.

In vivo BrdU labeling assay
For proliferation assays, animals were injected with 2.5 mg bromodeoxyuridine (BrdU, Becton Dickinson, Franklin Lakes, NJ) in 500 μl PBS. Colons were harvested after 2 or 24 hours. BrdU−positive nuclei were identified using a BrdU detection kit (Becton Dickinson).
Data are expressed as BrdU-positive nuclei per crypt axes normalized to total nuclei per crypt.

Apoptosis analysis by TdT−dTptosis dUTP nick end labeling (TUNEL)
TUNEL staining was performed using the ApopTag kit (Roche, Basel, Switzerland) according to the manufacturer's instructions. TUNEL-positive cells were counted in 5 randomly selected crypts per animal and expressed as the ratio to total intestinal epithelial cells (IEC).
Six mice from each group were studied.
Next, fragments were vortexed at maximum speed and the supernatants (containing IELs) were transferred into fresh tubes. The remaining fragments were digested in RPMI 20% FCS, 100U/ml type IV Collagenase and fresh 20U/ml DNAse II and rotated at 37°C for 60 min.
Thereafter, the remaining tissue fragments were re-suspended and sequentially passed through 100, 70, to 40 µm cell strainers. LPLs were pelleted by centrifugation, counted and stained for FACS analysis.

Flow cytometry
LPLs, lymph nodes and spleens were incubated with FcR Block (BD Biosciences, 553142) to prevent nonspecific antibody binding before staining with appropriate surface antibodies for   Gene expression was investigated in steady state and during DSS colitis (d7). Muc1 expression was different in steady state (p=0.019), but no altered expression of Muc3 , Muc4 and Muc5ac was detected in Nr2f6-deficient scrapings when compared with wild-type controls. Data are presented as mean ± SEM error bars and are representative of at least two independent experiments. Unpaired Student´s t test * p<0.05.