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Osteopontin as two-sided mediator of intestinal inflammation

Osteopontin (OPN) is characterized as a major amplifier of Th1-immune responses. However, its role in intestinal inflammation is currently unknown. We found considerably raised OPN levels in blood of wild-type (WT) mice with dextran sodium sulfate (DSS)-induced colitis. To identify the role of this mediator in intestinal inflammation, we analysed experimental colitis in OPN-deficient (OPN−/−) mice. In the acute phase of colitis these mice showed more extensive colonic ulcerations and mucosal destruction than WT mice, which was abrogated by application of soluble OPN. Within the OPN−/– mice, infiltrating macrophages were not activated and showed impaired phagocytosis. Reduced mRNA expression of interleukin (IL)-1 β and matrix metalloproteinases was found in acute colitis of OPN−/– mice. This was associated with decreased blood levels of IL-22, a Th17 cytokine that may mediate epithelial regeneration. However, OPN–/– mice showed increased serum levels of tumour necrosis factor (TNF)-α, which could be due to systemically present lipopolysaccharide translocated to the gut. In contrast to acute colitis, during chronic DSS-colitis, which is driven by a Th1 response of the lamina propria infiltrates, OPN−/– mice were protected from mucosal inflammation and demonstrated lower serum levels of IL-12 than WT mice. Furthermore, neutralization of OPN in WT mice abrogated colitis. Lastly, we demonstrate that in patients with active Crohn’s disease OPN serum concentration correlated significantly with disease activity. Taken together, we postulate a dual function of OPN in intestinal inflammation: During acute inflammation OPN seems to activate innate immunity, reduces tissue damage and initiates mucosal repair whereas during chronic inflammation it promotes the Th1 response and strengthens inflammation.

proteins and by inducing the PI3K/Akt signalling cascade [7,8]. In its soluble form OPN is a strong chemoattractant and a proinflammatory cytokine that functionally activates dendritic cells and macrophages and induces their differentiation towards a Th1-polarizing phenotype [1]. OPN, produced by epithelial cells and mononuclear cells, enables IL-12 release through integrin engagement and dampens the IL-10 response through CD44 binding in macrophages [1]. It has been suggested that this binding of the same molecule to two different receptors on a cell differentially regulates the key cytokines towards type 1 immunity [1].
In inflammatory bowel disease (IBD), a dysregulated immune response to bacterial or food antigens plays a major role. Crohn's disease (CD) is characterized as a Th1 directed immune response with increased CD4 ϩ T-cell production of interferon (IFN)-␥ and activated macrophages that secret tumour necrosis factor (TNF)-␣ and IL-12 [9]. In contrast, ulcerative colitis (UC) is associated with an atypical Th2 response mediated by a distinct subset of NK T cells that produce IL-13 and are cytotoxic for epithelial cells [10]. Recently, it has been shown that IL-12 induction by OPN could only be detected in lamina propria mononuclear cells (LPMC) from CD patients, but not in LPMC from the control group or UC patients, which further implements the role of OPN in a Th1 immune response [11].
Consequently, in OPN -/mice activated mononuclear cells produce significantly more IL-10 and virtually no IL-12 [12]. In the mouse model deletion of OPN has been demonstrated to ameliorate the development of several autoimmune diseases including experimental autoimmune encephalomyelitis and collagen-induced arthritis, although contradictory reports have been published [2,[13][14][15][16].
To further clarify the pleiotropic function of OPN in immune responses our experiments were designed to assess the OPN function in the DSS colitis model in OPN -/mice and in patients with CD.

Materials and methods Patients
Healthy individuals, patients with non-IBD related colitis and patients with chronic IBD treated at the Charité University Medicine Berlin, were included in this study with approval from the institutional human research ethics committee. All patients with IBD had an established diagnosis of CD or UC based on standard clinical, endoscopic and histological criteria (Table 1). Disease duration, manifestation and therapy were evaluated in all patients. Disease activities were evaluated by the CD activity index (CDAI) and by the clinical activity index (CAI) for UC [17,18]. CDAI Ͼ 150 and CAI Ͼ 4, were both considered to define active disease. Peripheral blood samples and endoscopic biopsies from areas of active inflammation or normal appearing colon mucosa were taken. Informed consent was obtained from all patients with approval from the institutional human research ethics committee following the Helsinki Guidelines.

Mice
OPN-deficient (OPN -/-) mice, kindly provided by Drs. Susan Rittling and David T. Denhardt (Rutgers University, NJ, USA), were backcrossed with C57BL/6 mice for 10 generations. Intercrosses with C57BL/6 CD44v7 -/mice were performed to produce C57BL/6 OPN -/-CD44v7 -/double deficient mice [19]. Mice were bred and housed under specific pathogen-free conditions in the animal facility of the Forschungseinrichtung Experimentelle Medizin (Berlin, Germany). All experiments were performed with age-and sex-matched animals in accordance with institutional, state and federal guidelines.

Induction of acute and chronic DSS colitis in mice
Acute colitis in mice was established with 2.5% (w/v) DSS (MP Biomedicals, OH, USA) in tap water ad libitum for 7 days. Mice were killed 1 week after the last DSS feeding. In some experiments, recombinant OPN (10 g in 200 l phosphate buffered saline (PBS), R&D systems, Wiesbaden, Germany) was given i.p. daily from day 4 to day 7. In the chronic approach, the initial DSS application was followed by normal drinking water for 10 days and the treatment scheme was repeated four times. Control mice received tap water without DSS. Progression of colitis was evaluated by weight loss as well as loose and bloody stools. In some experiments an OPN antibody (antimouse-OPN-antibody, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was given i.p. (10 g in 200 l PBS) twice a week to wild-type (WT) mice. Mice were killed 2 weeks after the last DSS feeding. Post mortem the entire colon was excised and 1-cm segments each of the distal, transverse and proximal colon were fixed in 4% formaldehyde for histological analysis. Two-to 3-m serial sections were stained with haematoxylin and eosin. Histological scoring was performed $ Diverticulitis (6), lymphocyte colitis (2) and ischemic colitis (2); *CDAI > 150 and # CAI > 4.  (3) severe mucosal damage with extensive ulcerations extending into the bowel wall. The combined histological score (inflammatory score) ranged from 0 (no changes) to 6 (extensive cell infiltration and tissue damage). The extent of inflammation was given as the percentage of inflamed mucosa of the colon sections. The colitis index comprises the product of the inflammatory score and the extent of inflammation [20].

Immunohistochemistry
Sections of 4 m were cut, deparaffinized and subjected to a heat-induced epitope retrieval step before incubation with antibodies.

RNA analysis
Total RNA of LPMC from OPN -/and WT mice with acute and chronic inflammation versus healthy mice was isolated using the RNeasy kit from Qiagen (Hilden, Germany). Five hundred nanograms of total RNA were reverse transcribed as described elsewhere [23]. TLR2, 4 and 9 were detected using the primers as described [24]. NOD2 was amplified using the primers as described [25].   (Fig. 1C).

OPN deficient mice are protected from chronic experimental colitis
Analysing the chronic DSS colitis model, which more reflects the clinical situation of CD patients, we observed that OPN -/mice showed no chronic infiltrates compared to the WT mice, which is consistent with a suppression of the Th1 immune response (Fig. 2A). The inflammatory activity was reflected by a reduced colon length in WT mice ( (Fig. 2B). Furthermore, OPN -/mice developed a significant anti-DSS-antibody response in chronic colitis compared to WT mice (P Ͻ 0.05), which indicates a stronger adaptive immune response (Fig. 2C). An anti-OPN antibody was applied to WT mice during the chronic approach, which significantly impaired inflammation, as demonstrated in the colitis index (P Ͻ 0.007) (Fig. 2D) and led to a decrease of the serum OPN levels (48 ng/ml, Fig. 2E). Additionally, we observed an enormous increase (5000-fold) of OPN serum levels in WT mice with acute (93 ng/ml) and chronic colitis (105 ng/ml) and in WT mice reconstituted with recombinant OPN (134 ng/ml) compared to the healthy control (20 pg/ml) (Fig. 2E). These results allude to a dichotomic function of OPN: Low concentration of OPN is required for tissue repair, but higher concentrations enhance Th1-driven immune responses.

OPN absence impairs activation of lamina propria macrophages during acute colitis
We compared the expression of iNOS within the inflamed mucosa in OPN -/versus WT mice because OPN has been described to be involved in acute tissue repair [26]. iNOS, an indicator of inflammatory processes, is induced upon activation of antigen presenting cells [27]. Remarkably, whereas cell infiltrates were equally distributed in the inflamed lesions in WT and OPN -/animals, we observed a clear induction of iNOS protein in the WT mice, but almost complete absence in OPN -/mice (Fig. 3A). Furthermore, OPN -/phagocytes have a significantly impaired capacity to perform phagocytosis (Fig. 3B) (Fig. 3D). In these samples we found no differences in IL-22 expression between WT and OPN -/mice (Fig. 3D) Lastly, we questioned the further consequences of the reduced activation of macrophages in inflamed colons of OPN -/mice and analysed the expression of anti-bacterial proteins (Defcr5 and S100A9) and matrix metalloproteinases (MMPs) in the gut tissue. In doing so, we found diminished expression in particular of MMP2 but also of MMP10 and not MMP9 in the inflamed colons of 7-day DSS-treated OPN -/mice (acute colitis) in comparison to the colons of WT mice (Fig. 3E).

OPN deficiency causes different systemic cytokines responses
In the next step we searched for possible systemic differences between WT and OPN -/mice during acute and chronic DSS-colitis. First, we investigated the cytokine mRNA expression in MLNs. As demonstrated in Fig. 4A, (Fig. 4A). Correspondingly, the OPN -/mice showed reduced serum levels of IL-22 (Fig. 4B) at day 4 and IL-17A (Fig. 4C). Interestingly, the levels of TNF-␣ in these samples were elevated in OPN -/in comparison to WT mice during acute DSS colitis (Fig. 4B)

. The application of recombinant OPN increased IL-17 blood levels in OPN -/mice comparable to those of WT mice (Fig. 4C) (in these samples the levels of IL-22 and TNF-␣ were not evaluated). Lastly, the secretion of IFN-␥ and IL-10 was evaluated from MLN cells in WT and OPN -/mice in acute (day 7) and in chronic colitis (day 26).
In line with the mRNA results, no differences in the IFN-␥ levels were observed in acute colitis in OPN -/compared to WT mice. However, in the chronic DSS colitis, IFN-␥ levels were significantly lower in OPN -/mice (P Ͻ 0.01), whereas IL-10 secretion was increased (P Ͻ 0.001) compared to WT mice (Table 2). During the second, chronic, DSS treatment period at day 24, but not during the first DSS treatment period at day 0, we found a clear reduction of IL-12 mRNA expression in MLNs (Fig. 4D). (Fig. 5A). In patients with active CD the OPN serum levels were strongly higher (323 Ϯ 81 ng/ml) than in the healthy control group (27 Ϯ 11 ng/ml) and in patients with inactive CD (45 Ϯ 9 ng/ml) (P Ͻ 0.01). Consequently, a direct  (Fig. 5B). OPN levels in UC patients were slightly elevated in patients with acute symptoms (140 Ϯ 29 ng/ml) (P Ͻ 0.05) (Fig. 5A), compared to patients with inactive UC (126 Ϯ 20 ng/ml), but no correlation between CAI and OPN serum level could be seen (r 2 ϭ 0.007, P ϭ 0.73) (Fig. 5B) [30][31][32], but not by an RGD peptide that blocked the integrin receptor of OPN. The elimination of debris and pathogens by phagocytosis is essential in tissue repair and reepithelialization. A defective phagocytosis thus should result in the persistence of neutrophilic infiltrates and tissue destruction [33,34]. Our interesting observation that CD44v7 plays an important role in phagocytosis, and that neutralization or deletion of CD44v7 [31] alone protected mice from acute DSS colitis, but not double deletion of OPN plus its receptor CD44v7, suggests that OPN is essential for phagocytosis that is mediated via other receptors in the absence of CD44v7.

OPN exists as an intracellular and a secreted protein. The latter circulates as soluble molecule or can be specifically bound to the cell surface via the OPN receptors. Levels of soluble OPN were determined in the serum of CD and UC patients with different actual disease activities
Coming back to the cytokine analysis we clearly show a reduced expression of Th17 cytokines, in particular IL-22, during acute colitis in MLNs and serum of OPN -/compared to WT animals. IL-22 represents a novel type of immune mediator that regulates the function of tissue cells, e.g. in the skin, in the digestive and respiratory tract [35,36]. Additionally, we recently described a protective systemic role of IL-22 in acute DSS colitis and probably in CD [37]. We and other groups have already demonstrated that IL-22 induces MMPs, protects tissue from damage and may lead to regeneration of epithelial cells [38][39][40]. It is also well documented, that IL-1Њcan induce some MMPs in e.g. fibroblasts [41]. In our study, the reduction of MMP2 was approximately five times more pronounced than that of MMP10 in OPN -/mice in comparison to WT mice. Very interestingly, the group of Sitaraman demonstrated that selective deletion of MMP2 exacerbates both DSS and colitis induced by Salmonella enterica subsp. Serovar Typhimurium [42]. Additionally, the authors showed that mucosa-derived MMP2 was required for its protective effects Results represent median Ϯ S.E.M from four to six mice in each group. *Significant differences in cytokine secretion over all groups (P Ͻ 0.01).  [28]. It is well demonstrated that Th1 cells play an important pathogenic role in chronic DSS colitis. OPN expression is regulated by T-bet, a transcription factor that controls cell polarization in Th1-mediated diseases [46]. Thus, opposed to acute inflammation, OPN -/mice fully recovered between days 11 to 14. In chronic colitis we found clearly reduced IL-12 mRNA expression and IFN-␥ secretion of LPMC was not increased in OPN -/mice. Additionally, we demonstrate that the antibody response against DSS in WT mice with acute colitis was diminished significantly. In WT mice the shift towards a Th1 cytokine response might result in a decreased antibody secretion whereas OPN -/mice demonstrate a Th2 directed immune response in colitis [11,47]. The mouse data in our study very well match with the data in human CD, a Th1-mediated disease, where soluble OPN concentration in the serum strictly correlated with the CDAI in contrast to those of patients with colitis ulcerosa.
Our data now provide an interpretation of the conflicting data published on the impact of OPN in inflammation and autoimmune diseases [48][49][50]. The activities of OPN are not only confined to the adaptive immune responses, but OPN is part of an inherent protective mechanism, because recombinant OPN down-regulates acute inflammation in OPN -/mice.
Very recently, additional dichotomic functions of OPN have been shown in allergic airway inflammation. In this study, administration of OPN at primary antigenic contact provided protection from allergic disease. This was mainly mediated through a shift towards an anti-allergic Th1 response, as shown by increased levels of IL-12 and induction of regulatory dendritic cells whereas in the phase of antigen challenge OPN dampened the pro-allergic Th2 response [43]. Although most studies focused on the activity of secreted OPN as a Th1-directing cytokine, OPN is partially retained as intracellular OPN in the cytoplasm distinct from secretory vesicles also detected in the gut mucosa [51,52]. Shinohara and coworkers have shown that especially intracellular OPN in dendritic cells is required for TLR9-dependent expression of IFN-␣, which is supposed to be protective in inflamed mucosa [53]. Nevertheless, in our study OPN deletion has no significant effect on the expression of TLR2, 4 and 9.
However, the key question remains if targeting OPN is rather beneficial or deleterious in the treatment of IBD. Because the ongoing immune response is IL-12 driven and the epithelial damage is less significant in CD, neutralizing OPN might be a promising approach in this disease, but not in UC. In conclusion, the impact of OPN on intestinal homeostasis is reflected by two different functions. OPN -/mice are protected from chronic experimen-tal colitis by abrogating Th1 polarization, most probably as a consequence of abrogated receptor ligation on macrophages [1]. Furthermore, expression of OPN promotes macrophage activation via binding of CD44, induces a scavenger function and acts against acute tissue destruction. CD44v7 is the most plausible candidate as OPN receptor in colitis because CD44v7 is involved in Th1-driven intestinal inflammation, up-regulated on macrophages and necessary for macrophage maturation [54,55].