Neural Adhesion Molecule Close Homolog of L1 Deficiency Exacerbates DSS-Induced Colitis in Mice

Background: The cell adhesion molecule CHL1, which belongs to the immunoglobulin superfamily, functions in a variety of physiological and pathological processes including neural development, tissue injury and repair. Recently, we found CHL1 was co-localized with GFAP positive cells in mouse colon tissue. Methods: Here, Colon tissues were collected from CHL1+/+ , CHL1+/- and CHL1-/- mice after dextran sodium sulfate (DSS) induced to investigate the effects of CHL1 on the development of DSS-induced colitis. Results: The data showed that CHL1 expression was increased in distal colon in a time-dependent manner after DSS-treatment. CHL1 deficiency induced more pronounced colitis features, an exacerbation of inflammation and damage to colonic tissues in DSS-induced mouse than that of wild type mouse. Moreover, CHL1-/- mice showed a remarkable increase of neutrophil and macrophage infiltration into colonic tissues, and then result in more severe damage to the intestinal epithelial cells and FITC leakage in CHL1 deficiency mice than that in WT mouse. Conclusions: Our results revealed a distinct colonic role for CHL1 in regulating DSS-induced colitis, CHL1 deficiency exacerbates DSS-induced colitis in mice, indicating that CHL1 may be an attractive therapeutic target for IBD. et al. Our previous results have shown the colocalization of CHL1 with glial fibrillary acidic protein (GFAP) positive glial cells in mouse colon tissue (SFig1A). Here, we investigated the effects of CHL1 in the development of DSS-induced colitis. To study the correlation between CHL1 and colitis, we employed CHL1 +/+ , CHL1 +/− and CHL1 −/− mice in DSS-induced colitis model. We found that the level of CHL1 protein expression was increased in colon tissue of DSS induced mouse. CHL1 deficiency induced more pronounced colitis features, an exacerbation of inflammation and damage to colonic tissues in DSS-induced colitis than that of wild type mouse, CHL1 −/− mice showed a remarkable increase of neutrophil and macrophage infiltration into colonic tissues. This study provides a novel functional role of CHL1 in regulating colitis. the pro-inflammatory cytokines, the leakage of FITC in colon and serum. These results suggested that CHL1 could be involved in regulating the occurrence and development of IBD.

Introduction elevated protein synthesis and translocation of protein kinaseδ (PKCδ) from cytosol to the membrane fraction (Wu et al. 2010). Our previous results have shown the colocalization of CHL1 with glial fibrillary acidic protein (GFAP) positive glial cells in mouse colon tissue (SFig1A). Here, we investigated the effects of CHL1 in the development of DSS-induced colitis.
To study the correlation between CHL1 and colitis, we employed CHL1 +/+ , CHL1 +/− and CHL1 −/− mice in DSS-induced colitis model. We found that the level of CHL1 protein expression was increased in colon tissue of DSS induced mouse. CHL1 deficiency induced more pronounced colitis features, an exacerbation of inflammation and damage to colonic tissues in DSS-induced colitis than that of wild type mouse, CHL1 −/− mice showed a remarkable increase of neutrophil and macrophage infiltration into colonic tissues. This study provides a novel functional role of CHL1 in regulating colitis.

Mice Models
In all experiments, the ethics guidelines for investigations in conscious animals were followed and experiments were approved by the Animal Care and Use Committee of Institute of Basic Medical Sciences. Wild-type (CHL1 +/+ ) mice were purchased from Laboratory Animal Center of the Academy of Military Medical Sciences. Mice (CHL1 -/-) that lacked CHL1 was described previously (Montag-Sallaz et al. 2002). The background of CHL1 -/mice is the C57BL/6 strain. The heterozygous mice (CHL1 +/-) was propagated by breeding CHL1 -/mice with C57BL/6 mice; the offspring were genotyped at weaning age using an PCR assay. Male mice aged seven to eight-week-old were used in this study.
To explore the alternation of CHL1 level in response to colitis, CHL1 +/+ mice were fed with ad libitum access to drinking sterile water containing 2.2% dextran sulfate sodium (DSS, MP Biomedicals, US) for 7 days followed by normal drinking water for 2 days. Mice were euthanized on the 5 th , 7 th , 9 th days after the initiation of DSS treatment, respectively. Then the colon was removed and washed with PBS for the following assays.
To explore the effects of deficiency of CHL1 on the development of DSS-induced colitis, CHL1 +/+ , CHL1 +/and CHL1 -/mice were exposed to ad libitum access to sterile water containing 1.5% DSS, given that the CHL1 -/mice and CHL1 +/mice were more sensitive to the DSS-induced colitis than CHL1 +/+ mice. The mice were euthanized on 9 th days after DSS exposure, and then the colon was removed for the following assays.

Assessment of Colitis Symptoms and Disease Activity Index
To explore the time-dependent effects of DSS treatment in CHL1 +/+ mice, animal body weight was evaluated daily. To explore the effects of deficiency of CHL1 on the development of DSS-induced colitis, the animal body weight, stool consistency and the presence of rectal gross blood were individually evaluated daily. Each parameter was assigned a score according to the criteria previously proposed (Bibi et al. 2017;da Silva et al. 2016) and used to calculate on average daily the Disease Activity Index (DAI) (Supplementary Table 1).

Histological Staining
The mice were perfused with saline to wash out circulating blood cells under anesthesia.
Subsequently, the colon tissues were collected from mice and the length was measured in a relaxed position without stretching. Then the colon tissues were fixed in 10% buffered formaldehyde and embedded in paraffin. Colon tissue were cut and stained with H&E. H&E staining tissue sections were scanned by Nanozoomer-XR Scanner C12000 (Hamamatsu Inc, JP) or taken on an Olympus BX51 microscope (Olympus, JP) using the Spot insight image capture system CCD camera.

Histological Scoring
To explore the effects of deficiency of CHL1 on the development of DSS-induced colitis by histological scoring, over 100 fields were photographed for each group. Each field was evaluated for tissue damage and inflammatory cell infiltration by two independent pathologists in a blinded manner.
Tissue damage was assessed as follows: 1 (normal), no mucosal damage; 2 (mild), punctuate mucosal erosions; 3 (moderate), focal ulceration or surface mucosal erosion; 4 (severe), extensive mucosal damage and extension into deeper structures of the bowel wall. Inflammatory cell infiltration was assessed as follows: 1 (normal), occasional presence of inflammatory cells in the lamina propria; 2 (mild), increased presence of inflammatory cells in the lamina propria; 3 (moderate), confluence of inflammatory cells infiltrating into the submucosa; 4 (severe), transmural extension of the inflammatory cells. The data represent the percentage of fields for Normal, Mild, Moderate, and Severe in all fields for each group.

Intestinal Permeability Assay in Vivo
The intestinal permeability was measured by determining the amount of FITC-dextran in blood after it was orally administered as described previously (Xie et al. 2014). At the 5 th day, mice were gavaged with 0.6mg/g body weight of FITC-Dextran (Sigma-Aldrich, UK) for 4h, and the blood samples were taken from hepatic vein. The blood sample were firstly centrifuged (3,000rpm, 4℃, 30min), and serum was collected and added to a 96-well microplate. The concentration of FITC was tested by Fluoroskan Ascent Fc (Thermo Scientific, US) with excitation wave 480 nm and emanation wave 530nm using serially diluted samples of the marker as standard. Then the colon tissues were cleaned with ice-cold PBS. The distribution of FITC-dextran in sectioned colonic tissue was determined by Nikon Ti-A1 fluorescent inverted microscope (Nikon Corporation, JP).
in response to DSS-induced colitis by western-blot assay. DSS treatment evidently up-regulated CHL1 expression levels in mice colon tissue at 9 th day (Fig 1G and 1H). These data implied that CHL1 is related to the occurrence of colitis.

CHL1 Deficiency Augmented DSS-induced Colitis in Mice
To further assess the impact of CHL1 on the development of DSS-induced colitis, wild-type (CHL1 +/+ ), CHL1 heterozygous (CHL1 +/-) and CHL1 deficient (CHL1 -/-) mice were given access to 1.5% DSScontaining drinking water ad libitum (Fig 2A). At baseline and throughout the course of treatment, we tracked these features on daily basis. DSS-induced injury reproduces some clinical features of human colitis including weight loss, diarrhea, and bloody stools. All control (CHL1 +/+ , CHL1 +/and CHL1 -/-) mice were negative for weight loss, diarrhea and fecal blood. As expected, the body weights in CHL1 +/+ , CHL1 +/and CHL1 -/mice were decreased after DSS treatment. The weight of CHL1 +/+ was significantly higher than that of CHL1 +/and CHL1 -/mice at 8 th , 9 th days after DSS-treatment ( Fig   2B). The DAI (Disease activity index, DAI) score of CHL1 -/and CHL1 +/mice were significantly higher than that of CHL1 +/+ mice on day 7, day 8 and day 9 after DSS induced mouse (Fig 2C). These findings demonstrated that deficiency of CHL1 exacerbated DSS-induced colitis and implied a critical role of CHL1 in IBD.
Moreover, we observed that the length of colon tissue in DSS-induced CHL1 -/mice were significantly shorter than CHL1 +/+ mice (Fig 2D and 2E). Importantly, H&E staining presented more severe colitis symptom in DSS-induced CHL1 +/mice compared with DSS-induced CHL1 +/+ mice. Meanwhile, the colon tissue of CHL1 -/mice, compared with CHL1 +/and CHL1 +/+ mice, exhibited severe inflammation and crypt damage (Fig 2F). The pathological changes of colonic tissue, including inflammatory cell infiltration and tissue damage were scored. The histology scores in the mice of DSS groups were increased. DSS-induced CHL1 +/mice had a significantly higher inflammatory score and tissue damage than DSS-treated CHL1 +/+ group. Moreover, the colon tissue of CHL1 -/group had a Figure 1 The Level of CHL1 Expression was Increased in DSS-induced Colitis (A) DSS-induced acute colitis mice model. CHL1+/+ mice were exposed to ad libitum access to drinking sterile water containing 2.2% DSS for 7 days followed by normal drinking water for 2 days. Mice were euthanized on the 5th, 7th, 9th day after the initiation of DSS treatment, respectively.  model. CHL1+/+, CHL1+/-and CHL1-/-mice were exposed to ad libitum access to drinking sterile water containing 1.5% DSS for 7 days followed by normal drinking water for 2 days.

Figure 3
The Deficiency of CHL1 Aggravated Epithelial Barrier damage in IBD (A) High magnification histologic images of colonic tissue from CHL1+/+ and CHL1-/-mice with and without DSS treatment showed altered intestinal epithelial cellular shape in tissues from CHL1-/-animals.

Supplementary Files
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