CD and UC are chronic relapsing disorders of the gastrointestinal tract which exert devastating personal and professional consequences on the approximately 1 million sufferers.14
Disease progression is relentless, with the eventual outcome being surgical intervention for up to 70% of patients.38
The era of the biologics has ushered in hope for improving therapeutic strategies; however, this approach is not without serious contraindications and adverse events.14
There remains an urgent need for novel therapeutics particularly to control the exaggerated inflammation within the intestine of patients with IBD. Recent studies have implicated the neuronal guidance molecule netrin-1 in mediating tissue inflammatory responses.2
Particularly, netrin-1 expression was shown to be induced under conditions of limited oxygen availability and to ameliorate hypoxia-driven inflammation.2
Based on these findings, we hypothesised that netrin-1 could represent an endogenous anti-inflammatory in the context of IBD—a condition that is also characterised by profound tissue hypoxia.19
Excitingly, our studies revealed that mice with partial netrin-1 deficiency (Ntn-1+/−
) demonstrated increased disease susceptibility in a model of DSS-colitis. This was associated with a robust neutrophil infiltration into the colonic LP and a significant increase in tissue cytokine levels in the Ntn-1+/−
mice compared to their wild-type controls. Conversely, supplementation with exogenous netrin-1 was associated with attenuated weight loss, improved tissue histology and diminished colonic inflammation. Moreover, mechanistic studies pointed towards a role for netrin-1 in preferentially dampening neutrophil tissue infiltration rather than improving intestinal barrier function or attenuating intestinal epithelial cell apoptosis. Studies using antibody blockade of the UNC5B receptor or gene-targeted mice for the Adora
2b point towards adenosine-receptor-mediated netrin-1 protection during DSS-colitis. Together, these studies demonstrate for the first time a role of netrin-1 in the suppression of intestinal inflammation as occurs in the context of IBD.
Consistent with our observations, previous studies have demonstrated that netrin-1 is induced by inflammatory signalling pathways in intestinal epithelia via NFκB (nuclear factor κ B) signalling.27
We demonstrate that netrin-1 expression is enhanced at the protein and mRNA level during the course of DSS-colitis. Despite netrin-1 mRNA upregulation by inflammatory stimulation of HMEC-1 endothelial cells, immunohistochemical analysis of the murine colon during DSS revealed limited, if any, detectable expression of netrin-1 at the level of the endothelium. However, netrin-1 protein was robustly expressed in the intestinal epithelium where it appears to be increased during DSS exposure. This correlates with enhanced netrin-1 expression observed in the intestinal epithelia of patients with CD and UC.15
Consistent with these studies, netrin-1 induction in IBD could involve the NFκB pathway.27
However, a recent study demonstrates that HIF-1α (hypoxia-inducible factor 1α), a transcription factor stabilised in intestinal epithelia in murine models of IBD,19
can induce netrin-1 expression in intestinal epithelia.2
Additionally, the Ets-1 transcription factor that is upregulated during IBD40
has been shown to induce netrin-1 expression in melanocytes.41
Therefore, multiple pathways can regulate netrin-1 expression in the context of inflammation.
Immunohistochemical analysis revealed consistent expression of netrin-1 in colonic myenteric neural units during DSS. Recent studies demonstrated a role for netrin-1 in the developing enteric nervous system where enteric neural-derived crest cells produce netrin-1.42
This intriguing observation led us to analyse the enteric neuronal anatomy of netrin-1-insufficient mice during DSS-colitis. Independent analysis revealed no difference in the enteric neuronal anatomy of netrin-1-insufficient mice in comparison with their wild-type controls either at baseline or during disease course. We conclude that netrin-1 insufficiency does not alter the neuronal anatomy in adult mice prior to or during DSS.
Supporting our observations, an anti-inflammatory effect of netrin-1 has been observed in models of acute inflammation.2
Originally, netrin-1 was demonstrated to attenuate LPS-induced sepsis through inhibition of leucocyte migration.6
Studies in kidney ischaemia–reperfusion injury,7
acute lung injury8
support this observation by demonstrating a tissue-protective role for netrin-1 through suppression of inflammation. Presently, a number of downstream receptor signalling pathways have been implicated in netrin-1-mediated tissue protection observed in these studies. The transmembrane receptors, deleted in colorectal cancer, uncoordinated receptor 5 (mouse UNC5A-D; human UNC5H1-4) and the A2B adenosine receptor (ADORA2B/Adora
2b) represent the known netrin-1 receptors.2
Independent studies have demonstrated that either UNC5B or Adora
2b expression on leucocytes is responsible for netrin-1 inhibition of tissue inflammation.2
Of additional relevance to the present study is the observation that netrin-1 receptors can mediate the apoptotic response of the intestinal epithelium.33
Enforced expression of netrin-1 in the intestinal epithelium resulted in decreased epithelial apoptosis and a modest increase in tumour formation in the presence of APC mutation.33
Furthermore, blockade of netrin-1 interaction with epithelial deleted in colorectal cancer receptor enhanced tumour cell death in a model of colorectal cancer progression.15
Intestinal epithelial cell apoptosis, along with alterations in epithelial tight junction formation, is central to the mucosal disruption observed in patients with IBD.46
Similarly, epithelial cell apoptosis and tight junction breakdown are key features of DSS-colitis, while inhibition of apoptosis in this model is barrier protective.34
Therefore, it is conceivable that antiapoptotic effects of netrin-1 signalling may be central to its beneficial effects during experimental colitis. In vitro studies of cytokine-induced tight junction rearrangement reveal no effect of netrin-1 on this aspect of epithelial permeability.49
Importantly, in vitro and in vivo studies indicate that netrin-1 does not influence DSS-induced epithelial barrier disruption and does not have a direct impact on intestinal epithelial cell apoptosis.
In addition to epithelial barrier breakdown, early and persistent infiltration of neutrophils into the LP forming crypt abscesses is a key feature of human disease and murine colitis.29–32
The full extent of the role that neutrophils play during DSS-colitis remains controversial. Studies using direct depletion of neutrophils with anti-Gr1 prior to DSS reveal modest effects of neutrophil depletion on early disease activity but no significant effect on disease outcome.50
However, the authors propose that macrophages are key regulators of DSS-colitis that mediate protective effects through limitation of neutrophil infiltration.50
Interestingly, one study identified neutrophil depletion leading to worsening disease during DSS-colitis.52
Conversely, therapeutic targeting of neutrophil recruitment to the colon in models of colitis using blockade of the neutrophil chemokine receptor (CXCR2) or ligand (CXCL5) results in diminished neutrophil recruitment during DSS and amelioration of disease.53–55
Taken together, these studies suggest that while complete blockade of neutrophil recruitment during colitis might not be desirable, limitation of the neutrophil influx into the LP would act to control disease severity. Present in crypt abscesses during human disease are other leucocyte populations including monocytes/macrophages and lymphocytes. Therapeutic strategies in human disease and mouse models to deplete granulocytic and monocytic tissue infiltration have met with some success.56
Therefore, treatment to attenuate tissue infiltration of granulocytes and monocytes may be of therapeutic benefit. In support of these studies, we demonstrate that netrin-1 directly limits trafficking of neutrophils in vitro and in vivo to ameliorate outcome in DSS-colitis. Further dissection of netrin-1 effects on leucocyte recruitment during DSS-colitis indicates that netrin-1 has no discernible effect on monocyte/macrophage recruitment to the colonic LP. Therefore, this study presents the preferential limitation of neutrophil recruitment by netrin-1 during DSS as a novel therapeutic strategy in an acute model of colitis.
Present findings place netrin-1 as an endogenous protective mediator in experimental colitis. Interestingly, supplementation with netrin-1 in DSS-colitis where its expression is induced is therapeutically effective. A similar phenomenon is observed in two independent studies where stabilisation of the locally produced HIF-1α transcription factor during experimental colitis is of therapeutic benefit.16
These studies support the potential benefit of enhancing in vivo protective responses for therapeutic intervention.
As previously mentioned, netrin-1 has an array of receptors through which it mediates tissue-protective effects. We focused our studies on receptors that have been implicated in mediating netrin-1-driven inhibition of leucocyte recruitment in models of acute inflammation, the UNC5B and the Adora2b. Lack of functional Adora2b abrogated the protective effect of netrin-1 on DSS-colitis. These studies implicate this purinergic signalling pathway in netrin-1-mediated protection in experimental colitis.
Consideration of netrin-1 as a therapeutic strategy in IBD must take into account the breadth of literature demonstrating the anti-inflammatory component as well as the antiapoptotic role of netrin-1 signalling. Studies indicating the involvement of netrin-1 in malignant transformation in the intestine,15
coupled with our observation that netrin-1 significantly attenuates intestinal leucocyte infiltration in experimental colitis, point to a tailored therapeutic strategy with netrin-1 administration perhaps being more suited to treat periods of disease flare rather than a maintenance treatment.
In summary, our present studies demonstrate for the first time that netrin-1 expression is an endogenous tissue-protective mechanism in experimental colitis. Therapeutic studies indicate that targeting netrin-1 may be useful in treatment of aberrant intestinal leucocyte accumulation, as occurs in colitis.