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Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

The immune system, including the innate and adaptive immune cells, plays an important role in the maintenance of tissue homeostasis and prevention of cancer. To date, the majority of immunotherapies have focused on stimulating T cell responses. However, macrophages and other myeloid immune cells also offer much promise as effectors of cancer immunotherapy. The CD47/SIRPα complex is a critical regulator of myeloid activation that controls the process of phagocytosis. Cancer cells display elevated levels of expression of CD47 which correlates with poor cancer prognosis. The mechanism by which cancer cells hijack the process of inhibition of macrophage phagocytosis through the CD47/SIRPα axis is still being elucidated, but it has been proposed that binding of the CD47 receptor on the cancer cell to a SIRPα receptor on the macrophage leads to phosphorylation of SIRPα and recruitment and activation of the protein tyrosine phosphatases SHP-1 and SHP-2, which in turn suppress function of cytoplasmic myosin IIA. This results in repression of phagocytosis. In recent years, blocking the CD47/SIRPα complex has become a leading immunotherapeutic approach for treating advanced solid tumours and hematologic cancers with many new CD47-blocking drugs entering clinical trial pipelines.

CD47 has been found to be ubiquitously expressed in numerous cell types in a range of tissues and it has been shown to regulate many cellular processes including inflammatory responses and tissue repair. It is foreseeable that blocking CD47 function can lead to potential unintended complications for cancer patients. Therefore, it is paramount to investigate the role of CD47 in specific tissues to inform us of the safety and side effects of the novel CD47-based immunotherapies.

It has been previously reported that CD47 plays a role in tissue repair with improved healing and survival in several in vivo models, including skin thermal injury and ischemia/reperfusion in organ transplant models of the liver, kidney and skin. In this report, authors have studied the function of CD47 in intestinal mucosal wound healing.

Using miniaturised endoscopic biopsy instruments, multiple wounds were inflicted in the intestines of Cd47 whole gene knockout mice (Cd47-/-). Wounds were observed and digitally quantified over two days and were compared with wild-type controls. Cd47-/- mice showed markedly inhibited wound closure. In addition, systemic and local administration of CD47-blocking antibodies into C57Bl/6 mice displayed a similar inhibitory effect on mucosal wound healing.

Mice with selective loss of CD47 in the intestinal epithelium (Cd47ΔIEC) were generated and characterised. Despite the loss of CD47 in intestinal cells, guts from Cd47ΔIEC mice exhibited no sign of intestinal inflammation or alteration in major tissue-resident immune cell populations, nor epithelial barrier dysfunction. Histological analysis revealed normal architecture of the intestinal mucosa. Similar results were observed in mice where loss of CD47 in the intestinal epithelium (Cd47ERΔIEC) could be temporarily induced by tamoxifen.

The difference was observed however, in rates of wound healing of the intestinal epithelium in incision wounds in all Cd47-/-, wild-type mice treated with anti-CD47 antibodies, Cd47ΔIEC and Cd47ERΔIEC in comparison with littermate controls. Mice lacking CD47 displayed less wound area closure than the control group over three days of wound observations. Although the number of proliferating (Ki67 positive) epithelial cells within the crypts were similar between groups, wounds depleted from CD47 were disorganised and lacked expression of brush border protein Villin, a marker of epithelial cell polarity.

In addition, when Cd47ΔIEC and Cd47ΔIEC mice were exposed to a cycle of mucosal injury-inducing assaults by DSS followed by periods of water-only recovery, both groups of mice developed escalating clinical colitis scores as determined by decreased weight loss, looser stool consistency and presence of blood in stools. Histological analysis revealed increased loss of crypts and mucosal ulceration particularly evident in the distal colon. The disease symptoms were more pronounced in Cd47ΔIEC mice where depletion of Cd47 was temporarily induced by tamoxifen. Interestingly, in contrast to chronic colonic mucosal injuries induced by cycles of DSS and water administration, when acute DSS treatment (without water-only recovery period) was applied, no significant differences in the manifestation of the disease were observed between groups.

Analysis of scratch wounds in 2D cultures of primary epithelial cells derived from murine (from gut of Cd47f/f and Cd47ERΔIEC strains) and human intestine (derived from human colonoids treated with selection of well-characterised CD47-blocking and CD47-non-blocking control antibodies), revealed that CD47 is required for epithelial cell migration in response to injury. The closure of the scratched wound was significantly reduced in cultures lacking CD47 or cultures depleted of CD47 by incubation with CD47-blocking antibodies.

The analyses of whole cell lysates derived from freshly isolated 2D epithelial cells, confirmed that CD47 regulates wound closure through B1 integrin-dependent signaling, Focal adhesion kinase (FAK), Src-protein-tyrosine kinase and Crk-associated substrate p130Cas. It appears that CD47 controls the formation of focal cell matrix adhesions, which in turn control epithelial cell motility, and thus contribute to wound closure. In addition, loss of CD47 negatively regulated the level of expression of its ligand thrombospondin-1 (TSP-1) and effector cytokine TGF-β1, both known to facilitate wound healing.

In summary, this study demonstrated an essential role of CD47 in promoting cell migration via focal cell matrix adhesion regulation in mucosal repair in the intestinal tissue. The blockage of CD47 leads to impaired cell migration in wound healing and makes the recipient more susceptible to IBD-inducing chronic assaults. The study raises the possibility of altered wound healing as a potential complication in clinical trials exploring the blockage of CD47 to enhance clearance of CD47-overexpressing tumour cells in humans. This should be taken into the account when assessing the score for benefit versus possible complications of new immunotherapy treatments for cancer patients.

Reference

Michelle Reed et al, Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo. Nature Communications 2019, 10:5004

https://doi.org/10.1038/s41467-019-12968-y

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Epistem offers pre-clinical models of wound healing to determine the effect of test items on specific processes and cell types involved in wound healing. These include in vivo excisional and incisional wounds, wounding of human skin engrafts, diabetic (db/db) and LPS-induced inflammation wound healing models. In vitro wound healing assays are also available to assess cell proliferation/migration, wound closure and angiogenesis. We work with many types of epithelial cells, including gut cells.

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