Milk Fat Globule-EGF Factor 8 Is a Critical Protein for Healing of Dextran Sodium Sulfate-Induced Acute Colitis in Mice
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Milk fat globule-EGF factor 8 (MFG-E8) has been shown to play an important role in maintaining the integrity of the intestinal mucosa and to accelerate healing of the mucosa in septic mice. Herein, we (a) analyzed the expression of MFG-E8 in the gut of wild-type (WT) C57BL/6 (MFG-E8+/+) mice with and without dextran sulfate sodium (DSS)-induced colitis, (b) characterized the pathological changes in intestinal mucosa of MFG-E8+/+ and MFG-E8-/- mice with DSS-induced colitis and (c) examined the therapeutic role of MFG-E8 in inflammatory bowel disease by using DSS-induced colitis model. Our data documented that there was an increase in colonic and rectal MFG-E8 expression in MFG-E8+/+ mice during the development of DSS colitis. MFG-E8 levels in both tissues decreased to below baseline during the recovery phase in mice with colitis. Changes in MFG-E8 gene expression correlated to the levels of inflammatory response and crypt-epithelial injury in both colonic and rectal mucosa in MFG-E8+/+ mice. MFG-E8-/- mice developed more severe crypt-epithelial injury than MFG-E8+/+ mice during exposure to DSS with delayed healing of intestinal epithelium during the recovery phase of DSS colitis. Administration of MFG-E8 during the recovery phase ameliorated colitis and promoted mucosal repair in both MFG-E8-/- and MFG-E8+/+ mice, indicating that lack of MFG-E8 causes increased susceptibility to colitis and delayed mucosal healing. These data suggest that MGF-E8 is an essential protective factor for gut epithelial homeostasis, and exogenous administration of MFG-E8 may represent a novel therapeutic target in inflammatory bowel disease.
Inflammatory bowel disease (IBD) is a term used to describe inflammatory conditions of the large and small intestine including Crohn’s disease and ulcerative colitis. The etiology of IBD has not been elucidated completely as yet. The pathogenesis is being unraveled gradually and seems to be the result of a combination of genetic, environmental and immunological factors in which an uncontrolled exaggerated immune response within the intestinal lumen leads to inflammation in genetically predisposed individuals. IBD could be considered to be an imbalance between proinflammatory and antiinflammatory mediators in the bowel mucosa (1). Dysfunctions of the intestinal immune system and cross-reactivity against host epithelial cells have been implicated as major inflammatory mechanisms in IBD (2). A number of peptides expressed in the intestine including ghrelin, galanin, melatonin, vasoactive intestinal peptide and adrenomedullin have been shown to have an antiinflammatory effect in IBD research models (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13).
Milk fat globule-EGF factor 8 (MFG-E8) is a glycoprotein that corresponds to the human protein lactadherin (BA46), which is known to be a marker of breast carcinomas and a major component of milk fat globules (14). MFG-E8 is an important milk mucin-associated defense component that inhibits enteric pathogen binding and infectivity (15). MFG-E8 has been shown to be present in the gut and expressed in the murine intestinal lamina propria macrophages (16, 17, 18). MFG-E8 reduces levels of proinflammatory cytokines in the inflamed colonic mucosa of dextran sodium sulfate (DSS)-treated mice (19). MFG-E8-mediated antiinflammatory effects are generated by NF-kB inhibition via the modulation of αvβ3 integrin signaling (19). MFG-E8 plays an important role in maintaining the integrity of the intestinal mucosa and accelerates healing of the mucosa in septic mice (18).
In the present study, we further examined whether MFG-E8/lactadherin is a critical peptide in the pathogenesis of IBD and if it plays a protective role. We also explored the therapeutic role of MFG-E8 in experimental colitis.
Materials and Methods
C57BL/6 wild-type (WT) mice (male, 6–10-wks-old) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). MFG-E8/SED1-knockout mice were bred in our laboratory according to the methods described previously (18,20). Mice were backcrossed to C57BL/6 for 10 generations, so they were considered to be fully congenic with the C57BL/6 background. WT C57BL/6 mice were used as controls. Animals were housed in a specific pathogen-free animal facility at the Children’s Memorial Research Center (Chicago, IL, USA). All animal experiments were conducted in accordance with the National Institutes of Health (NIH) guidelines and were approved by the Institutional Animal Care and Use Committee of the Children’s Memorial Hospital.
Colitis Induction and Administration of MFG-E8 to Mice with Established Colitis
Colitis was induced in the WT C57BL/6 mice and the MFG-E8 knockout mice by using 3.5% DSS added to drinking bottles for 1 wk. Control mice were fed only regular drinking water. The acute colitis was followed by a recovery phase by changing the drinking water containing DSS to distilled water on day 7. Mice were euthanized with CO2 inhalation at appropriate time points designed. During experiments, the mice were weighed and stools were tested for occult blood every day. The clinical colitis score was calculated by using a modified scoring system by using weight loss, hemoccult status and presence of diarrhea. In some experiments, we administrated recombinant MFG-E8 (20 ng/kg, intraperitoneally [i.p.]) to mice twice a day for 9 d during the recovery phase of colitis. Mice in control groups were given saline instead.
Animals were euthanized by carbon dioxide inhalation on days 0, 3, 7, 10 and 16 counted from day of onset of DSS feeds. Intestinal tissues were harvested immediately and fixed in 10% buffered formalin overnight and processed for routine histology. Sections were stained with hematoxylin and eosin (H&E). The histological examination was performed in a blinded manner by using a scoring system modified from a method described by Krieglstein et al. (21). Briefly, three independent parameters were measured: severity of inflammation (0 to 3: none, slight, moderate, severe), depth of injury (0 to 3: none, mucosal, mucosal and submucosal, transmural), and crypt damage (0 to 4: none, basal one-third damaged, basal two-thirds damaged, only surface epithelium intact, entire crypt and epithelium lost). The score of each parameter was multiplied by a factor reflecting the percentage of tissue involvement (×1, 0% to 25%; ×2, 26% to 50%; ×3, 51% to 75%; ×4, 76% to 100%). The final scores of severity of inflammation and depth of injury were added to a sum which was defined as inflammatory injury score. In addition, the final score of crypt damage was defined as crypt-epithelial injury score. The maximum possible inflammatory injury score and crypt-epithelial injury score are 24 and 16, respectively.
Protein Extraction, Western Blot
We used our standard protocol for isolation of total protein from intestinal tissues and immunoblotting (22). Rabbit polyclonal antibody against murine MFG-E8 (1:500; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), was used to detect MFG-E8.
Total RNA from the colonic and rectal tissues was extracted by using the RNeasy kit (QIAGEN, Valencia, CA, USA) according to the protocol of the manufacturer. RNA concentration was determined by optical densitometry at 260 with Smart Spec plus spectropho-meter (Bio-Rad, Hercules, CA, USA).
cDNA Synthesis from Total RNA by Reverse Transcription
cDNA was synthesized by using iScript cDNA synthesis kit (Bio-Rad) according to the protocol provided by the manufacturer. Briefly, 0.7 μg of RNA from each tissue sample was added to 25 μL of reaction mixture containing dNTP mix, 1 × random hexamers primer, 1 × reaction buffer, 1 μL MMLV-erived iScript reverse transcriptase which was preblended with RNAse inhibitor. The reaction was run at 25°C for 5 min, 42°C for 30 min and stopped by incubation at 85°C for 5 min. The resulting cDNA was used for the following quantitative real-time polymerase chain reaction (PCR).
Quantitative Real-Time PCR
QuantiTect SYBR Green PCR kit (QIAGEN, Valencia, CA, USA) was used for the study. In brief, mastermix (73 μL) containing 0.4 nmol/L primers and 1 × SYBR Green PCR Universal Mastermix (QIAGEN) was added to 2 μL cDNA before aliquoting in triplicate to a 96-well microtiter plate (25 μL/well). The cDNA was amplified by using a Fast 7500 realtime PCR system (AB Applied Biosystems, Foster City, CA, USA) under the following conditions: 50°C for 5 min, 95°C for 10 min, and then 40 cycles of amplification (95°C for 15 sec and 60°C for 1 min). All PCR reactions were performed in 96-well plate by using a final volume of 25 μL. The cycle at which each sample crossed a fluorescence threshold, CT (at 0.1–0.2 fluorescence units), was determined. The triplicate values for each cDNA were averaged. Sequences for murine forward (F) and reverse (R) primers for real-time PCR were the following: for 18S rRNAF, 5′-TGCCCTATCA ACTTTCGATG-3′; for 18S rRNAR, 5′-GATGTGGTAGCCGTTTCTCA-3′; for MFG-E8F, 5′-ATCTACTGCCTCTGC CCTGA-3′; and for MFG-E8R, 5′-CCAGA CATTTGGCATCATTG-3′. Fold changes in expression levels of MFG-E8 mRNA in different tissues and different postnatal stages were calculated by using the 2-AACT method by using 18S rRNA as the internal reference (23). The AACT value is defined as the CT difference between the normalized amount of sample and the normalized amount of calibrator.
Data were expressed as means ± SEM analysis of variance and one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test were used to assess the significance of differences; P < 0.05 was considered significant.
All supplementary materials are available online at https://doi.org/www.molmed.org.
Clinical Course of DSS Colitis in WT and MFG-E8 Deficient mice
Histological Changes in Colonic and Rectal Mucosa Due to DSS-Induced Colitis in WT and MFG-E8 Deficient Mice
Furthermore, we analyzed histological alteration of colonic and rectal mucosa during the water recovery phase in DSS-treated WT and MFG-E8 knockout mice. It was revealed that mucosa of colon and rectum in DSS-treated WT and MFG-E8 knockout mice exhibited evidence of healing such as regenerating glands, decreased mucosal inflammation and ulcerations. In the colon, repair of crypt-epithelial injury but not recovery of inflammatory response was revealed to be delayed markedly in MFG-E8-null mice compared with WT mice (see Figure 2A, B; and Supplementary Figure 1). Similar to the colon, healing of mucosa in rectum was delayed in MFG-E8 knockout mice (see Figure 2C, D; and Supplementary Figure 1).
Altered Expression of MFG-E8 in DSS-Induced Acute Colitis
Administration of MFG-E8 during the Recovery Phase of Colitis Attenuates Inflammation and Enhances Epithelial Repair
MFG-E8 is being investigated currently for its cytoprotective role in many tissues including the intestinal mucosa. It has been shown that MFG-E8 is present in the gut and expressed in the murine intestinal lamina propria macrophages (16, 17, 18). Previously, we showed that MFG-E8 promotes migration of intestinal epithelial cells through a PKCɛ-dependent mechanism (18). It binds to phosphatidylserine and triggers reorientation of the actin cytoskeleton in intestinal epithelial cells at the wound edge. Aziz et al. demonstrated that pretreatment with MFG-E8 attenuates intestinal inflammation in experimental colitis by modulating osteopontindependent αvβ3 integrin signaling (19). However, the therapeutic efficacy of MFG-E8 in colitis remains unknown. In the present study, we further examined the role of MFG-E8 in DSS-induced colitis by using MFG-E8 deficient mice. We found that MFG-E8 knockout mice develop more severe crypt-epithelial injury during the onset of DSS-induced acute colitis. Interestingly, we did not reveal significant difference in intestinal inflammatory response between WT and MFG-E8 knockout mice in the colitic phase. During the recovery phase of DSS colitis, MFG-E8 knockout mice showed a delayed healing of damaged intestinal epithelium compared with WT mice. Rescue therapy with MFG-E8 during the recovery phase is effective in MFG-E8 knockout mice with DSS-induced colitis. In addition, we demonstrated that treatment of WT mice with recombinant MFG-E8 during the recovery phase also enhances repair of intestinal mucosa. Collectively, our data in conjunction with previous findings strongly suggest that MFG-E8 is a crucial protein not only for protection against colitis but also for healing of colitic mucosa.
Several lines of evidence support the role of inflammation in regulation of MFG-E8 gene expression in vivo. For example, MFG-E8 levels are revealed to decrease in numerous tissues during acute inflammation (24). We found that polymicrobial sepsis-induced intestinal injury is associated with downregulation of MFG-E8 gene expression (18). On the other hand, Atabai et al. recently reported that MFG-E8 expression is increased after pulmonary injury induced by bleomycin, a chemotherapeutic agent (25). In the present study, we examined whether intestinal MFG-E8 gene expression is altered during colitis by using a classic experimental colitis model. We found that MFG-E8 gene is expressed constitutively in both colon and rectum in mice. Increased expression of MFG-E8 in both colon and rectum is revealed during the induction of inflammation with DSS treatment. Higher levels of MFG-E8 are revealed to persist in the rectum as compared with the colon on day 7. The MFG-E8 levels in both tissues decrease to below baseline during the water recovery phase in mice with colitis. The alteration of MFG-E8 gene expression is correlated to the levels of inflammatory response and crypt-epithelial injury in both colonic and rectal mucosa in WT mice. Furthermore, administration of MFG-E8 during the recovery phase of colitis attenuates inflammation and improves epithelial repair. Taken together, our novel findings, in combination with previous reports, suggest that maintenance of MFG-E8 level in the intestinal mucosa is critical for tissue repair from colitis.
Aziz et al. previously reported that MFG-E8 is downregulated in the colon and rectum during the acute phase of colitis (corresponding to the DSS induction period), while it gradually became up-regulated during the healing phase when DSS was no longer added to the drinking water in BALB/c mice (19). Clearly, their findings differ from what we observed in C57BL/6J mice in the present study. By comparing data obtained from the inbred C57BL/6J mouse strain in our study to results from BALB/c mice reported by other investigators, we speculated that different inbred mouse strains exhibit differences in alteration of MFG-E8 gene expression during colitis. However, it remains to be determined how genetic background influences MFG-E8 gene expression during the inflammation.
In addition, we show here that MFG-E8 gene expression is increased in the first week of DSS treatment, which may play an important role in protecting the colonic and rectal mucosa from histological damage. Increased expression could be due to increased influx of macrophages into the tissue during DSS treatment. Decrease in the MFG-E8 levels corresponds to increased level of histological damage in the recovery phase. Therapeutic treatment with recombinant MFG-E8 during the recovery phase of DSS colitis promotes intestinal epithelial repair and attenuates inflammation in the intestinal mucosa. Given the established role of MFG-E8 in the maintenance of homeostasis of intestinal epithelium as demonstrated in the present study as well as previous reports (18,19), restoration of MFG-E8 may represent a novel therapeutic target in IBD.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported in part by the Grant R01DK064240 (to X-D Tan) from National Institutes of Health, the Excellence in Academic Medicine Award from Illinois Department of Public Aid (to X-D Tan) and Eloise and Warren Batts Investigator Chair (to X-D Tan). We thank Barry D Shur (Emory University School of Medicine) for MFG-E8 deficient mouse colony.
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