Osteopontin Mediates Citrobacter rodentium-Induced Colonic Epithelial Cell Hyperplasia and Attaching-Effacing Lesions

Although osteopontin (OPN) is up-regulated in inflammatory bowel diseases, its role in disease pathogenesis remains controversial. The objective of this study was to determine the role of OPN in host responses to a non-invasive bacterial pathogen, Citrobacter rodentium, which serves as a murine infectious model of colitis. OPN gene knockout and wild-type mice were infected orogastrically with either C. rodentium or Luria-Bertani (LB) broth. Mouse-derived OPN^+/+ and OPN^−/− fibroblasts were incubated with C. rodentium and attaching-effacing lesions were demonstrated using transmission electron microscopy and immunofluorescence. Colonic expression of OPN was increased by C. rodentium infection of wild-type mice. Furthermore, colonic epithelial cell hyperplasia, the hallmark of C. rodentium infection, was reduced in OPN^−/− mice, and spleen enlargement by infection was absent in OPN^−/− mice. Rectal administration of OPN to OPN^−/− mice restored these effects. There was an 8- to 17-fold reduction in bacterial colonization in OPN^−/− mice, compared with wild-type mice, which was accompanied by reduced attaching–effacing lesions, both in infected OPN^−/− mice and OPN^−/− mouse fibroblasts. Moreover, adhesion pedestals were restored in OPN^−/− cells complemented with human OPN. Therefore, lack of OPN results in decreased pedestal formation, colonization, and colonic epithelial cell hyperplasia responses to C. rodentium infection, indicating that OPN impacts disease pathogenesis through bacterial attachment and altered host immune responses.Osteopontin (OPN) is a multifunctional glycophosphoprotein involved in a variety of cellular functions, including stimulation of T helper(_H)1 cytokines and adhesion through binding to integrins and CD44 receptors on cell surfaces. This cytokine is highly expressed in chronic inflammatory and autoimmune diseases¹ and is localized in and around inflammatory cells.² OPN is believed to exacerbate inflammation in a variety of settings, including infectious diseases.¹ It is involved in recruiting inflammatory cells to sites of injury, as indicated by a decrease in macrophage infiltration in ischemic kidneys in OPN-deficient (OPN^−/−) mice.¹ OPN is also involved in controlling expression of inflammatory mediators, such as down-regulation of the anti-inflammatory cytokine interleukin (IL)-10 and an increase in levels of the T_H1 cytokine interferon (IFN)γ.³Accumulating evidence indicates that defects in the dynamic balance between organisms in the commensal intestinal microbiota and host innate defensive responses at the intestinal mucosal surface results in the induction of inflammatory bowel diseases (IBD).⁴ An association between luminal bacteria and IBD is further supported by animal models,⁵ including the use of Citrobacter rodentium infection. C. rodentium is the causative agent of transmissible murine colonic epithelial cell hyperplasia that harbors a locus of enterocyte effacement pathogenicity island, similar to enterohemorrhagic Escherichia coli (EHEC) O157:H7, and is capable of forming dense F-actin bacterial attachment pedestals, known as attaching–effacing (A/E) lesions, in mouse colon.⁶ The resulting T_H1 response and accompanying pathological changes represent findings observed in patients with IBD.⁷ As a result, C. rodentium serves as a relevant animal model to study potential infectious mechanisms of IBD.⁸An association between OPN and IBD was recently assessed using dextran sodium sulfate (DSS) to induce colitis in wild-type and OPN^−/− mice. However, the results arising were contradictory. One study suggested that lack of OPN has a protective effect,⁹ whereas others reported a detrimental outcome during the acute phase^10,11,12 and protection from chronic exposure to DSS.¹¹ This controversy prompted us to study an association between OPN and gut inflammation, using C. rodentium as a model of infectious colitis, to provide insight regarding the role of OPN in the pathogenesis of IBD.Herein, we describe the attenuation of C. rodentium-induced colonic epithelial cell hyperplasia and a reduction in bacterial colonization in OPN^−/− mice. In addition, we demonstrate dependence of adhesion pedestal formation in response to C. rodentium on the presence of OPN. Taken together, these findings contribute to an improved understanding of the role of OPN in response to intestinal infection.