Neuronal guidance molecule netrin-1 attenuates inflammatory cell trafficking during acute experimental colitis

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1 Gut Online First, ublished on August 3, 2011 as /gutjnl < Additional materials are ublished online only. To view these files lease visit the journal online (htt://gut.bmj. com). 1 Mucosal Inflammation Program, Deartment of Anesthesiology, University of Colorado Denver, Aurora, Colorado, USA 2 Mucosal Inflammation Program, Deartment of Medicine, University of Colorado Denver, Aurora, Colorado, USA 3 Gastrointestinal Eosinohilic Diseases Program, Section of Pediatric Gastroenterology, Heatology and Nutrition, Digestive Health Institute, Children s Hosital Colorado, Aurora, Colorado, USA 4 Deartment of Cell and Develomental Biology, Rocky Mountain Taste and Smell Centre, University of Colorado, Denver, Aurora, Colorado, USA 5 Deartment of Pathology, University of Colorado, Denver, Aurora, Colorado, USA Corresondence to Dr Holger K Eltzschig, Mucosal Inflammation Program, Deartment of Anesthesiology, University of Colorado Denver, E 19th Avenue, Mailsto B112, Research Comlex 2, Room 7124, Aurora, CO 80045, USA; holger.eltzschig@ ucdenver.edu Original data related to this ublication are available uon request. Revised 15 June 2011 Acceted 20 June 2011 This aer is freely available online under the BMJ Journals unlocked scheme, see htt:// gut.bmj.com/site/about/ unlocked.xhtml Neuronal guidance molecule netrin-1 attenuates inflammatory cell trafficking during acute exerimental colitis Carol M Aherne, 1 Colm B Collins, 2 Joanne C Masterson, 2,3 Marco Tizzano, 4 Theresa A Boyle, 5 Joseh A Westrich, 1 Jason A Parnes, 4 Glenn T Furuta, 2,3 Jesús Rivera-Nieves, 2 Holger K Eltzschig 1 ABSTRACT Background Inflammatory bowel diseases, encomassing Crohn s disease and ulcerative colitis, are characterised by ersistent leucocyte tissue infiltration leading to eretuation of an inaroriate inflammatory cascade. The neuronal guidance molecule netrin-1 has recently been imlicated in the orchestration of leucocyte trafficking during acute inflammation. We therefore hyothesised that netrin-1 could modulate leucocyte infiltration and disease activity in a model of inflammatory bowel disease. Design DSS-colitis was erformed in mice with artial genetic netrin-1 deficiency (Ntn-1 +/ mice) or wild-tye mice treated with exogenous netrin-1 via osmotic um to examine the role of endogenous and theraeutically administered netrin-1. These studies were suorted by in vitro models of transeithelial migration and intestinal eithelial barrier function. Results Consistent with our hyothesis, we observed induction of netrin-1 during intestinal inflammation in vitro or in mice exosed to exerimental colitis. Moreover, mice with artial netrin-1 deficiency demonstrated an exacerbated course of DSS-colitis comared to littermate controls, with enhanced weight loss and colonic shortening. Conversely, mice treated with exogenous mouse netrin-1 exerienced attenuated disease severity. Imortantly, ermeability studies and quantitative assessment of aotosis reveal that netrin-1 signalling events do not alter mucosal ermeability or intestinal eithelial cell aotosis. In vivo studies of leucocyte transmigration demonstrate suression of neutrohil trafficking as a key function mediated by endogenous or exogenously administered netrin-1. Finally, genetic studies imlicate the A2B adenosine recetor in netrin-1-mediated rotection during DSS-colitis. Conclusions The resent study identifies a reviously unrecognised role for netrin-1 in attenuating exerimental colitis through limitation of neutrohil trafficking. INTRODUCTION Netrin-1 was originally identified as a diffusible factor released by neural floor late cells in the develoing sinal cord that regulates axonal outgrowth. 1 Studies have exanded to eriheral organs where netrin-1 modulates cell develoment, migration and inflammatory end oints to reduce tissue inflammation. 2e10 Inflammatory bowel diseases (IBDs), encasulating Crohn s disease (CD) and ulcerative colitis Significance of this study What is already known about this subject? < As a neuronal guidance molecule, netrin-1 has been imlicated in the develoing brain < Previous studies have found high exression levels of netrin-1 in the intestine, including in atients with inflammatory bowel disease (IBD) < Netrin-1 has been imlicated in immune functions What are the new findings? < At resent, the functional role of netrin-1 in IBD is unknown. As such, our studies rovide, for the first time, evidence for a rotective role of netrin-1 signalling in an exerimental model of IBD < Mice with a deficiency of netrin-1 exerience enhanced disease severity during exerimental colitis < Treatment with exogenous netrin-1 using osmotic um delivery attenuates exerimental colitis < Netrin-1 does not alter eithelial ermeability or aotosis in vivo but directly limits neutrohil trafficking to attenuate disease How might it imact on clinical ractice in the foreseeable future? < Continuous low-dose administration of netrin-1 as demonstrated here in an exerimental model of colitis may be exloited as a theraeutic agent directed towards the suression of neutrohil infiltration in atients with IBD (UC), are heterogeneous diseases affecting the gastrointestinal tract that occur at the cross section of genetics and environment Disrution of the intestinal eithelial barrier and aberrant accumulation of leucocytes within the intestinal lamina roria (LP) leading to continued mucosal damage characterise CD and UC. 13 While biologics offer more targeted treatment, the search for increased secificity in IBD theraeutics continues. 14 The role of netrin-1 in IBD remains undefined. Previous reorts demonstrate enhanced netrin-1 exression in the intestinal eithelium of atients with CD or UC. 15 Hyoxia-deendent signalling induces intestinal mucosal netrin-1 exression, 2 Aherne CM, Collins CB, Masterson JC, et al. Gut (2011). doi: /gutjnl of 11 Coyright Article author (or their emloyer) Produced by BMJ Publishing Grou Ltd (& BSG) under licence.

2 with recent reorts demonstrating a tissue-rotective role for the hyoxic resonse in IBD. 16e19 This notion couled with an inhibitory effect of netrin-1 on leucocyte transmigration 26 led us to hyothesise that netrin-1 may mediate tissue rotection in IBD. We established that netrin-1 exression was enhanced within the colonic mucosa during murine colitis. Deficiency of netrin-1 resulted in exacerbated murine colitis accomanied by exaggerated neutrohil infiltration into the colonic LP. This was attenuated by recombinant netrin-1. Mechanistic studies reveal that netrin-1 mediates its effects through interaction with migrating leucocytes without directly modulating intestinal eithelial barrier function or eithelial cell aotosis. Furthermore, we demonstrate a role for the urinergic signalling athway in netrin-1-mediated rotection. Our data demonstrate, for the first time, a rotective role for netrin-1 in exerimental colitis. MATERIALS AND METHODS Cell culture Caco-2, T84 and HMEC-1 cells were cultured as described. 20e22 Time course studies were erformed with recombinant human tumor necrosis factor (TNF) a, interleukin (IL) b and interferon (IFN) g. DSS-colitis Netrin-1 heterozygous mice (Ntn-1 +/ mice), A2B adenosine recetor-deficient (Adora2b / ) mice, matched littermate controls or C57BL/6 mice were used in DSS (dextran sulhate sodium) studies as described. 23e25 Recombinant netrin-1 was delivered with subcutaneous osmotic ums (1 mg/mouse/day). Anti-UNC5B antibody was administered intraeritoneally (800 mg/kg/mouse). Flow cytometry Flow cytometric analysis of LP leucocytes with anti-mouse GR-1, SiglecF, Ly-6G, Ly-6C, CD11b, CD45, LIVE/DEADÒ cell stain or isotye controls was erformed as described. 26 Immunohistochemistry Paraffin-embedded tissues were incubated with anti-mouse netrin-1 followed by DAB (3,3-diaminobenzidine substrate) develoment and methyl green counterstain. Immunofluorescence Frozen tissues were incubated with anti-pgp9.5 or anti-cgrp, followed by fluorescent detection and DAPI (4,6-diamidino-2- henylindole) counterstain. Western blotting Tissue immunoblotting was erformed as described. 2 Real-time reverse transcritase olymerase chain reaction (RT-PCR) Messenger RNA (mrna) analysis was erformed as described. 2 In vivo ermeability FITC-labelled (fluorescein isothiocyanate) dextran ermeability was erformed as described. 19 TUNEL assay TUNEL (deoxynucleotidyl transferase mediated deoxyuridine trihoshate) assay using araffin-embedded tissues was erformed as er manufacturer s instructions. In vitro ermeability Permeability studies in Caco-2 cells treated with DSS or cytokines were erformed as described. 20 In vitro transmigration Polymorhonuclear leucocytes (PMN) transmigration was erformed as described. 2 Statistical analysis Statistical comarisons were made with GrahPad Prism Analysis software using analysis of variance (ANOVA) or Student t test where aroriate and are exressed at mean6sem. Further details can be found in the sulementary material. RESULTS Induction of netrin-1 exression during intestinal inflammation Previous study suggests that inflammation regulates netrin-1 exression. 27 To examine the effects of inflammation on intestinal exression of netrin-1, we assessed netrin-1 mrna levels in human intestinal eithelial cell lines (T84 and Caco-2; figure 1A, B, resectively) and a human microvascular endothelial cell line (HMEC-1; figure 1C) following 2 h and 6 h stimulation with a combination of mediators closely associated with IBD athogenesis (cytomix; TNFa, IL-1b and IFNg). Netrin-1 mrna exression significantly increased in all cell lines following 6 h treatment. To examine this resonse and identify the cellular source of netrin-1 in vivo, netrin-1 exression was analysed in a DSS-colitis time course (figure 1DeG). Immunohistochemical analysis demonstrated robust exression of netrin-1 in colonic eithelial cells and in myenteric neural units (figure 1D; arrows identify staining of relevant cells). PCR analysis of HMEC-1 cells revealed induction of netrin-1 mrna uon inflammatory stimulation (figure 1C); however, immunohistochemistry failed to show netrin-1 exression in colonic endothelial cells during DSS. Netrin-1 exression remained secifically localised to the intestinal eithelium and the myenteric neural units of the colon throughout the DSS time course, with the aearance of increasing exression over time. mrna (figure 1E) and western blotting (figure 1F,G) confirmed a quantifiable increase in netrin- 1 exression in the whole colon (figure 1E,F) and in the mucosal lining layer (figure 1G) following 7 days of DSS. These observations indicate induction of netrin-1 exression during exerimental colitis with significant exression by the eithelium. Mice with artial netrin-1 deficiency demonstrate reduced netrin-1 exression and normal enteric neuronal anatomy in exerimental colitis Netrin-1-deficient mice do not survive beyond ostnatal day 3 due to imroer axonal develoment. 28 Mice with artial netrin- 1deficiency (Ntn-1 +/ ) develo normally comared to wild-tye controls (Ntn-1 +/+ ). 28 Having observed netrin-1 exression in the colon following exerimental colitis, we assessed netrin-1 exression in Ntn-1 +/- mice following DSS. Netrin-1 exression in Ntn-1 +/ mice remained below Ntn-1 +/+ at baseline and was similarly reduced following DSS (figure 2AeC). Therefore, we conclude that during DSS-colitis, endogenous netrin-1 levels are dramatically diminished in Ntn-1 +/ mice. Initial findings demonstrated netrin-1 exression in colonic myenteric neural units (figure 1D); therefore, we comared the colonic neuronal anatomy of Ntn-1 +/ and Ntn-1 +/+ mice at baseline and during DSS. Staining for eriheral nerve fibres (anti-pgp9.5) revealed no areciable difference in the neuronal anatomy of the colon of Ntn-1 +/ mice comared to Ntn-1 +/+ rior to or during DSS-colitis (figure 2D). Additionally, no difference in ain nerve fibres was observed between grous (anti-cgrp; data not shown). We therefore conclude that mice with netrin-1 insufficiency do not demonstrate altered neuronal atterning during DSS. 2 of 11 Aherne CM, Collins CB, Masterson JC, et al. Gut (2011). doi: /gutjnl

3 Figure 1 Netrin-1 exression in resonse to inflammation. T84 intestinal eithelial cells (A), Caco-2 intestinal eithelial cells (B) or HMEC-1 microvascular endothelial cells (C) were exosed to a combination of tumor necrosis factor (TNF) a, interleukin (IL) 1b and interferon (IFN) g (cytomix; all at 10 ng/ml) or vehicle (0.1% bovine serum albumin (BSA)/hoshatebuffered saline (PBS)) for 2 h or 6 h followed by total RNA isolation and analysis of netrin-1 transcrit levels (NTN-1). NTN-1 exression was calculated relative to b-actin and exressed as fold change relative to vehicle treatment6sem for each timeoint. Results are derived from indeendent exeriments at each individual time oint, erformed in trilicate. (D) Gender-, age- and weightmatched C57BL/6 mice exosed to water or DSS (4.5%) over a timecourse of 7 days were sacrificed at 2-day intervals ost DSS administration (days 0, 1, 3, 5 and 7), and whole colon was harvested by blunt dissection. Paraffinembedded sections of whole colon were dearaffinised and rehydrated for immunohistochemical staining with a chicken anti-netrin-1 secific antibody (1:100) followed by DAB develoment (brown staining) and counterstaining with methyl green (green staining). Primary antibody was omitted as negative control (control). Reresentative images were acquired at 10X using a Nikon Eclise Ti-S microscoe, DS-Fi1 1.0X camera. Bar reresents 100 mm. (E) Total RNA was isolated from whole colonic tissue isolated at different time intervals of DSS administration as described (D), and netrin-1 transcrit levels (Ntn-1) were determined by real-time reverse transcritase olymerase chain reaction (RT-PCR). Ntn-1 exression was calculated relative to b-actin and exressed as fold change relative to water-exosed mice6sem. (F) Whole colon was harvested following DSS exosure as described (D). Total rotein was isolated, and netrin-1 (Ntn-1) exression at 7 days ost DSS was determined by sodium dodecyl sulfate olyacrylamide gel electrohoresis (SDS-PAGE) with b-actin as loading control. (G) Colonic mucosal scraings were harvested ost DSS (described in D). Netrin-1 (Ntn-1) exression in the mucosal layer at 7 days ost DSS was determined by SDS-PAGE with b- actin as loading control. In vivo DSS time course data reresent five mice er time oint, and western blot data are reresentative of greater than three indeendent exeriments with six to eight mice er grou. Partial netrin-1 deficiency exacerbates disease activity in a model of exerimental colitis Recent study indicates that mice with artial genetic deficiency of netrin-1 (Ntn-1 +/ ) exerience an enhanced inflammatory resonse in models of acute inflammation, thus romting our investigation of these mice in exerimental colitis. Consistent with our hyothesis, we observed that Ntn-1 +/ mice exerienced increased disease severity comared to wild-tye controls (Ntn-1 +/+ ) as measured by weight loss, disease activity index, colonic shortening and histologic damage (figure 3AeE, resectively). Thus, we conclude that artial deficiency of netrin-1 is detrimental to the develoment of DSS-colitis. Partial netrin-1 deficiency results in heightened colonic inflammation in exerimental colitis Previous observations demonstrate that netrin-1 can inhibit leucocyte tissue infiltration, with studies demonstrating netrin-1 inhibition of neutrohil and monocyte recruitment in acute inflammation. 2 6e9 Inaroriate neutrohil accumulation within the LP is a key feature of early and active UC. 29e31 DSScolitis is characterised by raid neutrohil influx into the LP, closely modelling human disease. 32 Raid onset of weight loss in the Ntn-1 +/ mice romted investigation of early and late disease activity. Flow cytometric analysis of leucocytes isolated from the whole colon following 3 and 7 days of DSS revealed no significant difference in dendritic cell (CD11c + MHCII Hi ), Aherne CM, Collins CB, Masterson JC, et al. Gut (2011). doi: /gutjnl of 11

4 Figure 2 Netrin-1 exression and enteric neuronal atterning in mice with artial netrin-1 deficiency following DSS-colitis. Age-, weight-, and sexmatched mice with artial netrin-1 deficiency (Ntn-1 +/ ) and their wildtye controls (Ntn-1 +/+ ) were exosed to water or DSS (4.5%) for 7 days, followed by sacrifice and harvesting of the whole colon by blunt dissection. (A) Total RNA was extracted, and Ntn-1 transcrit levels determined by realtime reverse transcritase olymerase chain reaction (RT-PCR). Ntn-1 exression was calculated relative to b-actin and exressed as fold change relative to water-exosed Ntn-1 +/+ mice6sem. (B) Total rotein was extracted, and Ntn-1 levels were determined by sodium dodecyl sulfate olyacrylamide gel electrohoresis (SDS-PAGE) with b-actin as the loading control. (C) Densitometric analysis of netrin-1 exression in mucosal scraings as mean fold change relative to water-exosed Ntn-1 +/+ mice6sem. Results are reresentative of at least three indeendent exeriments with six to eight mice er grou. (D) Reresentative images of immunofluorescence staining erformed on frozen sections from whole colon of mice with artial netrin- 1 deficiency (Ntn-1 +/ ) or wild-tye controls (Ntn-1 +/+ ) exosed to water or DSS using an anti-pgp9.5 antibody (red) and counterstaining with DAPI (blue). Images were acquired with a QImaging Retiga-400RV at 10X. Bar reresents 100 mm. Results reresent analysis of mice at 3 and 7 days ost DSS stained as a whole mount and in sequential sections (n¼2 mice er grou). natural killer cell (NK1.1 + ), eosinohil (SiglecF + ), macrohage (F4/80 + ), B cell (B220 + ), CD4 T cell (CD4 + ), CD4 T-regulatory cell (CD4 + CD25 + FoxP3 + ), CD8 T cell (CD8 + ) or CD8 T- suressor cell (CD8 + CD103 + ) frequency or number between Ntn-1 +/ mice and Ntn-1 +/+ controls (data not shown). However, following 3 (figure 4A,B) and 7 days (figure 4C) of DSS, a dramatic and sustained increase was observed in the frequency and number of neutrohils (Gr-1 + cells) in the LP of Ntn-1 +/ mice comared with Ntn-1 +/+. Secificity of the netrin-1 effect on this granulocyte oulation is demonstrated by the dot lot revealing no change in eosinohil frequency (SiglecF + cells) between the two grous (figure 4A). Additionally, exression levels of the ro-inflammatory cytokines TNFa (figure 4D) and IL-1b (figure 4E) were significantly enhanced in Ntn-1 +/ mice following 7 days of DSS, confirming the exaggerated inflammatory resonse observed in these mice. Netrin-1 treatment attenuates disease severity during exerimental colitis The observation that netrin-1 deficiency exacerbates DSS-colitis directed us to investigate the consequences of sulementation with netrin-1 in this model. Mice that were treated with recombinant mouse netrin-1 for 7 days (1 mg/mouse/day) exerienced diminished disease severity as demonstrated by body weight loss, disease activity index, histologic damage and colonic shortening (figure 5AeD, resectively) comared with vehicle-treated controls. Reduced inflammation in these mice was indicated by decreased TNFa and IL-1b colonic exression following netrin-1 treatment (figure 5EeF). These observations indicate that treatment with exogenous netrin-1 exerts a rotective effect in exerimental colitis. Netrin-1 does not affect eithelial barrier function Eithelial barrier dysfunction is a key feature of human UC and a major comonent of DSS-colitis. 32 The rominent influx of neutrohils into the LP of Ntn-1 +/ mice comared to wild-tye controls (Ntn-1 +/+ ), articularly at early onset of DSS-colitis (day 3, figure 4B), led us to investigate netrin-1 effects on eithelial barrier function. Furthermore, netrin-1 signalling has been imlicated in antiaototic effects on intestinal eithelial cells 33 ; therefore, we could hyothesise that netrin-1 mediates rotection during DSS by inhibiting intestinal eithelial cell aotosis. For this urose, serum levels of FITC were determined following gavage with FITC-dextran (4 kda) in Ntn-1 +/ mice comared to wild-tye controls after 3 days of DSS. This day was chosen as eithelial barrier dysfunction occurs at early time oints ost DSS 32 and Ntn-1 +/ mice exerience significantly differential body weight loss by day 3 (figure 3A). No difference in eithelial ermeability was noted between the Ntn-1 +/ and wild-tye water controls. DSS exosure resulted in a significant increase in serum FITC in wild-tye mice comared to their water controls. Imortantly, we observed no additional increase in ermeability in Ntn-1 +/ mice following DSS comared to wild-tye counterarts (figure 6A). This indicates that loss of netrin-1 exression does not directly affect eithelial barrier function. This study was reeated in mice treated with exogenous netrin-1 (1 mg/mouse/day) at the same time oint 4 of 11 Aherne CM, Collins CB, Masterson JC, et al. Gut (2011). doi: /gutjnl

5 Figure 3 Disease activity in mice with artial netrin-1 deficiency during DSS-colitis. Gender-, age- and weightmatched mice with artial netrin-1 deficiency (Ntn-1 +/ ) and their wildtye controls (Ntn-1 +/+ ) were exosed to DSS (4.5%) for 7 days, followed by sacrifice and harvesting of the whole colon by blunt dissection. (A) Daily weight measurements were obtained for each grou of mice. <0.05 as measured by analysis of variance (ANOVA). (B) Daily disease activity measurements encomassing weight, stool consistency and resence of blood were assessed for each grou of mice. <0.05 as measured by ANOVA. (C) At harvest, colon length was measured for each mouse and is dislayed as the mean6sem. (D) Reresentative histological sections from whole colon of Ntn-1 +/+ or Ntn-1 +/ mice harvested following 7 days of DSS or water. Bar reresents 100 mm. Images acquired at 10X using Olymus BX51. (E) Blinded histological scoring of colonic tissue ost DSS. All results are reresentative of three indeendent exeriments with six to eight mice er grou and are dislayed as mean6sem. ost DSS. These mice exerience imroved disease outcome comared to vehicle-treated controls (figure 5). Mice exosed to DSS demonstrated significant increase in serum FITC comared to water controls as reviously noted 32 ; netrin-1 treatment had no effect on barrier function as measured by serum FITC levels (figure 6B). To more secifically analyse the effect of netrin-1 on intestinal eithelial cell aotosis, colonic sections from mice treated with vehicle or netrin-1 exosed to water or DSS for 6 days were TUNEL stained (figure 6C,D). Limited basal intestinal eithelial cell aotosis was identified in water controls which was unaltered by netrin-1 treatment (figure 6C,D; arrows indicate aototic nuclei). The ercentage of aototic colonic eithelial cells was dramatically enhanced in mice exosed to DSS, as reviously noted. 34 Netrin-1 treatment during DSS did not alter the ercentage of aototic eithelial cells (figure 6C,D). Taken together, these studies indicate that the rotection conveyed by endogenous or exogenous netrin-1 during exerimental colitis does not rimarily involve modulation of intestinal barrier function or eithelial cell aotosis. In vitro exeriments using Caco-2 intestinal eithelial cells as a model of eithelial barrier function confirmed in vivo findings. High-resistance Caco-2 monolayers were treated with DSS (4.5%; basolaterally and aically) for 6 h (figure 6E) or cytokine cocktail for 48 h (basolateral IFNg, TNFa, IL-1b; all at 10 ng/ml; figure 6F,G). Co-incubation with netrin-1 at the basolateral membrane or at both sides of the membrane (data not shown for cytomix) had no effect on barrier function as measured by transeithelial electrical resistance (TEER; figure 6E,F) or by FITCdextran (3 kda) flux erformed at 48 h ost cytomix treatment (figure 6G), suorting in vivo findings that netrin-1 does not directly modulate the intestinal eithelial barrier function. Netrin-1 limits colonic leucocyte infiltration to attenuate inflammation in exerimental colitis Our revious observation of increased neutrohil (Gr-1 + cells) accumulation within the LP of netrin-1-insufficient mice (figure 4AeD) couled with studies indicating that netrin-1 regulates leucocyte transmigration 2 6 romted investigation of the effects of exogenous netrin-1 on leucocyte migration. Transmigration of neutrohils across the eithelial barrier is a morhologic hallmark of colitis Initial investigation revealed that netrin-1 suressed chemo-attractant-induced (Formyl-Methinyl-Leucyl-Phenylalanine; fmlp) human neutrohil (PMN) transmigration across a Caco-2 intestinal eithelial barrier in a concentration-deendent manner (figure 7A), in suort of revious observations 2 6 and our conclusions from initial in vivo studies (figure 4A-C). Flow cytometric analysis revealed that the frequency and number of Gr-1 + neutrohils within the LP were significantly attenuated following netrin-1 treatment during DSS comared to vehicle-treated controls (figure 7B). Previous studies demonstrated that netrin-1 can Aherne CM, Collins CB, Masterson JC, et al. Gut (2011). doi: /gutjnl of 11

6 Figure 4 Colonic inflammatory infiltrate following DSS-colitis in Ntn-1 +/ + and Ntn-1 +/ mice. Gender-, age- and weight-matched mice with artial netrin-1 deficiency (Ntn-1 +/ ) and their wild-tye controls (Ntn- 1 +/+ ) were exosed to DSS (4.5%) for 3 or 7 days, followed by sacrifice and harvesting of the whole colon by blunt dissection. Colonic lamina roria leucocytes were isolated, and flow cytometric analysis of GR-1 + (neutrohils) and SiglecF + (eosinohils) granulocytes was erformed. (A) Reresentative dot lots showing GR-1 + granulocytes from the colonic lamina roria of Ntn-1 +/+ and Ntn-1 +/ mice ost DSS. (B) The number of GR-1 + granulocytes in the colonic lamina roria following 3 days of water or DSS in Ntn-1 +/+ and Ntn-1 +/ mice. (C) The number of GR-1 + granulocytes in the colonic lamina roria following 7 days of water or DSS in Ntn-1 +/+ and Ntn-1 +/ mice. (D, E) Following whole colon harvest, total RNA was extracted, and tumor necrosis factor (TNF) a (D) or interleukin (IL) 1b (E) transcrit levels were determined by real-time reverse transcritase olymerase chain reaction (RT-PCR). Gene exression was calculated relative to b-actin and exressed as fold change relative to water-exosed Ntn-1 +/+ mice6sem. Results reresent two indeendent exeriments with four mice er grou (flow cytometry) or six to eight mice er grou (RT-PCR). limit neutrohil and monocyte recruitment during instances of acute tissue injury Given that the anti-gr-1 antibody recognises Ly-6G and Ly-6C antigens, with Ly-6G reresenting a secific neutrohil surface marker and Ly-6C acting as a marker for inflammatory monocytes/macrohages, 37 we sought to dissect whether netrin-1 was limiting transmigration of both cell tyes to the LP during DSS-colitis. Flow cytometric analysis of leucocytes isolated from the LP of vehicle- and netrin-1- treated mice demonstrated that the frequency and number of Ly-6G Hi CD11b + neutrohils were considerably increased during DSS-colitis comared to water controls, with both arameters being significantly diminished by netrin-1 treatment (sulementary figure 1AeC). To negate neutrohilic contribution due to binding of Ly-6C to neutrohils, the frequency of monocyte oulations was assessed gating on Ly-6G negative cells. The frequency and number of Ly-6C high (Ly6G - Ly-6C Hi CD11b + ) and Ly-6C intermediate (Ly6G - Ly-6C Int CD11b + ) monocytes/ macrohages were augmented during DSS but were not significantly altered by netrin-1 (sulementary figure 1D,E). Therefore, detailed flow cytometric analysis using two distinct markers for neutrohil recognition demonstrates that netrin-1 can significantly attenuate neutrohil recruitment to the LP during DSS. Our analysis reveals no distinct effect of netrin-1 on monocyte/macrohage tissue infiltration during DSS. Therefore, we conclude that netrin-1 is art of an endogenous rotective athway within the colonic eithelium that damens DSS-colitis through referential limitation of neutrohil tissue infiltration. Purinergic signalling athways contribute to netrin-1-mediated rotection from exerimental colitis Having identified that netrin-1 mediates rotection during DSScolitis through limitation of leucocyte recruitment, we roceeded to investigate the secific netrin-1 recetor involved. Previous study has demonstrated significant levels of exression of the UNC5B (uncoordinated recetor 5B) recetor and Adora2b (A2B adenosine recetor) on leucocytes which have each been demonstrated to mediate netrin-1 rotective effects in models of acute inflammation Taking this into account, we initially erformed an antibody blocking study of the UNC5B recetor using a strategy shown to be effective in reversing netrin-1-mediated rotection in a model of renal ischaemia. 10 Anti-UNC5B blocking antibody or control immunoglobulin (Ig) G was administered (800 mg/kg/mouse) 1 day rior to commencement of netrin-1 delivery (1 mg/mouse/day) and 2 days rior to DSS with blocking antibody or control IgG administration at 2-day intervals until day 6 ost DSS (day 2, 0, 2, 4) when mice were sacrificed. Consistent with revious results (figure 5), netrin-1 significantly attenuated severity of DSS-colitis as measured by weight loss, colon shortening and histologic tissue injury (figure 8AeC, resectively). Co-administration of a UNC5B blocking antibody did not alter netrin-1- mediated rotection, with mice exhibiting no differences in weight loss, colon length or histologic injury to those treated with control IgG (figure 8AeC), thus indicating that netrin-1 was not acting through UNC5B to rotect in DSS. Subsequent studies were erformed with Adora2b-deficient mice (Adora2b / ; figure 8DeF). Adora2b / mice treated with netrin-1 (1 mg/mouse/day with subcutaneous um) demonstrated comarable disease activity as measured by weight loss, colon shortening or histologic injury (figure 8DeF) comared to vehicle-treated Adora2b / controls. Consistent with revious studies in acute inflammatory models, 2 8 we observed that netrin-1-mediated rotection during DSS-colitis involves adenosine recetors. DISCUSSION CD and UC are chronic relasing disorders of the gastrointestinal tract which exert devastating ersonal and rofessional consequences on the aroximately 1 million sufferers. 14 Disease rogression is relentless, with the eventual outcome being surgical intervention for u to 70% of atients The era of the biologics has ushered in hoe for imroving theraeutic strategies; however, this aroach is not without serious 6 of 11 Aherne CM, Collins CB, Masterson JC, et al. Gut (2011). doi: /gutjnl

7 Figure 5 Exogenous netrin-1 treatment in DSS-colitis. Gender-, ageand weight-matched C57BL/6 mice were treated with netrin-1 (1 mg/ mouse/day) or vehicle (1% bovine serum albumin (BSA)/hoshatebuffered saline (PBS)) using a subcutaneous osmotic um beginning 1 day rior to administration of (day 1) water or DSS (4.5%) for 6 days, followed by sacrifice and harvesting of the whole colon by blunt dissection. (A) Daily weight measurements were obtained for each grou of mice. <0.05 by analysis of variance (ANOVA). (B) Assessment of daily disease activity measurements, encomassing weight, stool consistency and resence of blood, was erformed for each grou of mice. <0.05 by ANOVA. (C) Reresentative histological images from vehicle- and netrin-1-treated mice exosed to water or 4.5% DSS. Bar reresents 100 mm. Images acquired at 10X using Olymus BX51. Bar grah of blinded histological scoring of colonic tissue ost treatment dislayed as the mean6sem. (D) Colon length measurements for each mouse were taken following harvest at day 6 ost DSS and are dislayed as mean6sem. (E, F) Following whole colon harvest, total RNA was extracted, and tumor necrosis factor (TNF) a (E) or interleukin (IL) 1b (F) transcrit levels were determined by real-time reverse transcritase olymerase chain reaction (RT-PCR). Gene exression was calculated relative to b-actin and exressed as fold change relative to water-exosed vehicle-treated mice6sem. Results reresent 3 indeendent exeriments with six to eight mice er grou. contraindications and adverse events. 14 There remains an urgent need for novel theraeutics articularly to control the exaggerated inflammation within the intestine of atients with IBD. Recent studies have imlicated the neuronal guidance molecule netrin-1 in mediating tissue inflammatory resonses. 2 6e9 Particularly, netrin-1 exression was shown to be induced under conditions of limited oxygen availability and to ameliorate hyoxia-driven inflammation. 2 Based on these findings, we hyothesised that netrin-1 could reresent an endogenous anti-inflammatory in the context of IBDda condition that is also characterised by rofound tissue hyoxia. 19 Excitingly, our studies revealed that mice with artial netrin-1 deficiency (Ntn- 1 +/ ) demonstrated increased disease suscetibility in a model of DSS-colitis. This was associated with a robust neutrohil infiltration into the colonic LP and a significant increase in tissue cytokine levels in the Ntn-1 +/ mice comared to their wild-tye controls. Conversely, sulementation with exogenous netrin-1 was associated with attenuated weight loss, imroved tissue histology and diminished colonic inflammation. Moreover, mechanistic studies ointed towards a role for netrin-1 in referentially damening neutrohil tissue infiltration rather than imroving intestinal barrier function or attenuating intestinal eithelial cell aotosis. Studies using antibody blockade of the UNC5B recetor or gene-targeted mice for the Adora2b oint towards adenosine-recetor-mediated netrin-1 rotection during DSS-colitis. Together, these studies demonstrate for the first time a role of netrin-1 in the suression of intestinal inflammation as occurs in the context of IBD. Consistent with our observations, revious studies have demonstrated that netrin-1 is induced by inflammatory signalling athways in intestinal eithelia via NFkB (nuclear factor k B) signalling. 27 We demonstrate that netrin-1 exression is enhanced at the rotein and mrna level during the course of DSS-colitis. Desite netrin-1 mrna uregulation by inflammatory Aherne CM, Collins CB, Masterson JC, et al. Gut (2011). doi: /gutjnl of 11

8 Figure 6 Netrin-1 effects on the intestinal eithelial barrier. (A) Mice with artial netrin-1 deficiency (Ntn-1 +/- ) and their wild-tye controls (Ntn-1 +/+ ) were exosed to DSS for 3 days (described in figure 3) rior to oral gavage with FITC-dextran (4 kda; 0.6 mg/g at 80 mg/ml). Fluorescence measurement (478 nm) was used to determine FITC in the serum 4 h later and is dislayed as mean FITC (mg/ ml)6sem. (B) Mice with vehicle or netrin-1 treatment (described in figure 5) were exosed to DSS for 3 days rior to oral gavage with FITC-dextran (4 kda 0.6 mg/g at 80 mg/ml), and serum FITC levels were determined and dislayed as described in A. (C) Reresentative images of aototic colonic eithelial cells identified by TUNEL staining of araffin-embedded colonic sections following treatment of mice with netrin-1 or vehicle and exosure to DSS for 6 days (described in figure 5). Images were acquired at 20X using a Nikon Eclise Ti-S microscoe, DS-Fi1 1.0X camera and reresent at least six mice er grou. (D) Quantification of the ercentage of aototic colonic eithelial cells in vehicle- or netrin-1-treated mice following 6 days of DSS exosure (described in figure 5). Scoring was carried out in a blinded fashion with counting of 600 cells over three randomly selected fields er section with six mice er grou. (E) Caco-2 intestinal eithelial monolayers cultured as monolayers on transwell ermeable suorts for at least 3 weeks rior were exosed to 4.5% DSS in the aical and basolateral chamber, with vehicle or human recombinant netrin-1 (500 ng/ ml) co-treatment. Transeithelial electrical resistance (TEER) measurements were taken at time intervals indicated (24 h not shown) and are dislayed as mean resistance measurements (Ohms.cm 2 )6SEM. (F) Caco-2 cells were cultured as described (E) rior to basolateral treatment with tumor necrosis factor (TNF) a, interleukin (IL) 1b and interferon (IFN) g (cytomix; all 10 ng/ml) lus vehicle or human recombinant netrin-1 (500 ng/ml) for 48 h. TEER measurements were taken at time intervals indicated. (G) FITC-dextran (3 kda; 250 mg/ml) was alied to the aical comartment of Caco-2 monolayers treated for 48 h as described in F. Aearance of FITC in the basolateral comartment was measured over a 3 h eriod and analysed as described in A. Results dislayed as the aarent ermeability of the monolayer (Pa; cm 2 /s). In vivo FITC measurements reresent five mice er grou. In vitro exeriments reresent four individual wells er treatment from two searate exeriments. stimulation of HMEC-1 endothelial cells, immunohistochemical analysis of the murine colon during DSS revealed limited, if any, detectable exression of netrin-1 at the level of the endothelium. However, netrin-1 rotein was robustly exressed in the intestinal eithelium where it aears to be increased during DSS exosure. This correlates with enhanced netrin-1 exression observed in the intestinal eithelia of atients with CD and UC. 15 Consistent with these studies, netrin-1 induction in IBD could involve the NFkB athway. 27 However, a recent study demonstrates that HIF-1a (hyoxia-inducible factor 1a), a transcrition factor stabilised in intestinal eithelia in murine models of IBD, 19 can induce netrin-1 exression in intestinal eithelia. 2 Additionally, the Ets-1 transcrition factor that is uregulated during IBD 40 has been shown to induce netrin-1 exression in melanocytes. 41 Therefore, multile athways can regulate netrin- 1 exression in the context of inflammation. Immunohistochemical analysis revealed consistent exression of netrin-1 in colonic myenteric neural units during DSS. Recent studies demonstrated a role for netrin-1 in the develoing enteric nervous system where enteric neural-derived crest cells roduce netrin This intriguing observation led us to analyse the enteric neuronal anatomy of netrin-1-insufficient mice during DSS-colitis. Indeendent analysis revealed no difference in the enteric neuronal anatomy of netrin-1-insufficient mice in comarison with their wild-tye controls either at baseline or during disease course. We conclude that netrin-1 insufficiency does not alter the neuronal anatomy in adult mice rior to or during DSS. 8 of 11 Aherne CM, Collins CB, Masterson JC, et al. Gut (2011). doi: /gutjnl

9 Figure 7 Netrin-1 effects on inflammatory infiltrate in vitro and in vivo. (A) Neutrohils (PMN) were harvested from whole human blood and alied at 1X10 6 PMN in the resence of vehicle or increasing concentrations of human recombinant netrin-1 (50e500 ng/ml) to the basolateral asect of Caco-2 intestinal eithelial cells grown to confluence on inverted ermeable suorts. fmlp (1 mm) was added to the aical asect of each well. Presence of transmigrated neutrohils in the aical comartment was measured by myeloeroxidase levels and is dislayed as the mean fold change in PMN numbers relative to vehicle6sem. Results reresent at least trilicate wells from three indeendent exeriments. (B) C57BL/6 mice were treated with netrin-1 (1 mg/mouse/day) or vehicle rior to exosure to water or DSS as described in figure 5. Following colon harvest at day 6 ost DSS, colonic lamina roria leucocytes were isolated, and flow cytometric analysis of GR-1 + (neutrohils) and SiglecF + (eosinohils) granulocytes was erformed. The number of GR-1 + granulocytes is dislayed as mean6sem for four mice. Suorting our observations, an anti-inflammatory effect of netrin-1 has been observed in models of acute inflammation. 2 6e9 Originally, netrin-1 was demonstrated to attenuate LPS-induced sesis through inhibition of leucocyte migration. 6 Studies in kidney ischaemiaereerfusion injury, 7 10 hyoxia-induced inflammation, 2 acute lung injury 843 and eritonitis 9 suort this observation by demonstrating a tissue-rotective role for netrin-1 through suression of inflammation. Presently, a number of downstream recetor signalling athways have been imlicated in netrin-1-mediated tissue rotection observed in these studies. The transmembrane recetors, deleted in colorectal cancer, uncoordinated recetor 5 (mouse UNC5A-D; human UNC5H1-4) and the A2B adenosine recetor (ADORA2B/Adora2b) reresent the known netrin-1 recetors e Indeendent studies have demonstrated that either UNC5B or Adora2b exression on leucocytes is resonsible for netrin-1 inhibition of tissue inflammation e10 Of additional relevance to the resent study is the observation that netrin-1 recetors can mediate the aototic resonse of the intestinal eithelium. 33 Enforced exression of netrin-1 in the intestinal eithelium resulted in decreased eithelial aotosis and a modest increase in tumour formation in the resence of APC mutation. 33 Furthermore, blockade of netrin-1 interaction with eithelial deleted in colorectal cancer recetor enhanced tumour cell death in a model of colorectal cancer rogression. 15 Intestinal eithelial cell aotosis, along with alterations in eithelial tight junction formation, is central to the mucosal disrution observed in atients with IBD Similarly, eithelial cell aotosis and tight junction breakdown are key features of DSS-colitis, while inhibition of aotosis in this model is barrier rotective. Therefore, it is conceivable that antiaototic effects of netrin-1 signalling may be central to its beneficial effects during exerimental colitis. In vitro studies of cytokine-induced tight junction rearrangement reveal no effect of netrin-1 on this asect of eithelial ermeability. 49 Imortantly, in vitro and in vivo studies indicate that netrin-1 does not influence DSS-induced eithelial barrier disrution and does not have a direct imact on intestinal eithelial cell aotosis. In addition to eithelial barrier breakdown, early and ersistent infiltration of neutrohils into the LP forming cryt abscesses is a key feature of human disease and murine colitis. 29e32 36 The full extent of the role that neutrohils lay during DSS-colitis remains controversial. Studies using direct deletion of neutrohils with anti-gr1 rior to DSS reveal modest effects of neutrohil deletion on early disease activity but no significant effect on disease outcome However, the authors roose that macrohages are key regulators of DSScolitis that mediate rotective effects through limitation of neutrohil infiltration. 50 Interestingly, one study identified neutrohil deletion leading to worsening disease during DSScolitis. 52 Conversely, theraeutic targeting of neutrohil recruitment to the colon in models of colitis using blockade of the neutrohil chemokine recetor (CXCR2) or ligand (CXCL5) results in diminished neutrohil recruitment during DSS and amelioration of disease. 53e55 Taken together, these studies suggest that while comlete blockade of neutrohil recruitment during colitis might not be desirable, limitation of the neutrohil influx into the LP would act to control disease severity. Present in cryt abscesses during human disease are other leucocyte oulations including monocytes/macrohages and lymhocytes. Theraeutic strategies in human disease and mouse models to delete granulocytic and monocytic tissue infiltration have met with some success Therefore, treatment to attenuate tissue infiltration of granulocytes and monocytes may be of theraeutic benefit. In suort of these studies, we demonstrate that netrin-1 directly limits trafficking of neutrohils 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/macrohage recruitment to the colonic LP. Therefore, this study resents the referential limitation of neutrohil recruitment by netrin-1 during DSS as a novel theraeutic strategy in an acute model of colitis. Present findings lace netrin-1 as an endogenous rotective mediator in exerimental colitis. Interestingly, sulementation with netrin-1 in DSS-colitis where its exression is induced is theraeutically effective. A similar henomenon is observed in two indeendent studies where stabilisation of the locally roduced HIF-1a transcrition factor during exerimental colitis Aherne CM, Collins CB, Masterson JC, et al. Gut (2011). doi: /gutjnl of 11

10 Figure 8 Secific blockade of netrin-1 recetors during exogenous netrin-1 treatment in DSS-colitis. (A) Gender-, age- and weight-matched C57BL/6 mice were treated with anti-unc5b antibody (800 mg/kg) or control goat immunoglobulin (Ig) G (800 mg/kg) intraeritoneally 2 days rior to administration of water or DSS (3.5%) followed by reeated administration of blocking antibody or control IgG at days 0, 2, and 4 of DSS exosure. Netrin-1 (1 mg/mouse/day) or vehicle (1% bovine serum albumin (BSA)/hoshatebuffered saline (PBS)) delivery using a subcutaneous osmotic um commenced 1 day rior to administration (day 1) of water or DSS for 6 days, followed by sacrifice and harvesting of the whole colon by blunt dissection. (A) Daily weight measurements were obtained for each grou of mice. <0.05 by analysis of variance (ANOVA). (B) Colon length measurements for each mouse were taken following harvest at day 6 ost DSS and are dislayed as mean6sem. (C) Reresentative histological images from (i) water controls, (ii) DSSexosed mice treated with control IgG and BSA/PBS vehicle, (iii) DSS-exosed mice treated with netrin-1 and control IgG and (iv) DSS-exosed mice treated with netrin-1 and anti-unc5b antibody. Bar reresents 100 mm. Images acquired at 10X using an Olymus BX51. (DeF) Gender-, age- and weightmatched A2B recetor-deficient mice (Adora2b / ) were treated with netrin- 1(1mg/mouse/day) or vehicle (1% BSA/PBS) using a subcutaneous osmotic um commencing 1 day rior to administration (day 1) of water or DSS (3.5%) for 6 days, followed by sacrifice and harvesting of the whole colon by blunt dissection. (D) Daily weight measurements were obtained for each grou of mice. (E) Colon length measurements for each mouse were taken following harvest at day 6 ost DSS and are dislayed as mean6sem. (F) Reresentative histological images from (i) water controls, (ii) DSS-exosed mice treated with BSA/PBS vehicle and (iii) DSS-exosed mice treated with netrin-1. Bar reresents 100 mm. Images acquired at 10X using an Olymus BX51. Exeriments are reresentative of six to eight mice er grou. is of theraeutic benefit These studies suort the otential benefit of enhancing in vivo rotective resonses for theraeutic intervention. As reviously mentioned, netrin-1 has an array of recetors through which it mediates tissue-rotective effects. We focused our studies on recetors that have been imlicated 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 rotective effect of netrin-1 on DSS-colitis. These studies imlicate this urinergic signalling athway in netrin-1-mediated rotection in exerimental colitis. Consideration of netrin-1 as a theraeutic strategy in IBD must take into account the breadth of literature demonstrating the anti-inflammatory comonent as well as the antiaototic role of netrin-1 signalling. Studies indicating the involvement of netrin-1 in malignant transformation in the intestine, 15 couled with our observation that netrin-1 significantly attenuates intestinal leucocyte infiltration in exerimental colitis, oint to a tailored theraeutic strategy with netrin-1 administration erhas being more suited to treat eriods of disease flare rather than a maintenance treatment. In summary, our resent studies demonstrate for the first time that netrin-1 exression is an endogenous tissue-rotective mechanism in exerimental colitis. Theraeutic studies indicate that targeting netrin-1 may be useful in treatment of aberrant intestinal leucocyte accumulation, as occurs in colitis. 10 of 11 Aherne CM, Collins CB, Masterson JC, et al. Gut (2011). doi: /gutjnl

11 Acknowledgements The authors would like to thank Marc Tessier-Lavigne for roviding the Ntn-1 mice for this roject. Thank you to Sean P. Colgan, Almut Grenz, Eric T. Clambey, Eoin N. McNamee, Kelley Brodsky, Julee Dalton, Timothy Lubbert and Douglas Ridyard for technical assistance as well as manuscrit discussion. Funding The resent manuscrit is suorted by United States National Institutes of Health grant R01-HL0921, R01-DK and R01HL to HKE, and Crohn s and Colitis Foundation Senior Research Award to HKE. Suorted by Crohn s and Colitis Foundation Fellowshi to CMA, CBC and JCM. Suorted in art by the Histology and Imaging Core of NIDCD Grant P30 DC04657 to D. Restreo and T. Finger Cometing interests None. Contributors All authors contributed to data generation, data analysis and interretation as well as writing of the manuscrit. Provenance and eer review Not commissioned; externally eer reviewed. REFERENCES 1. Serafini T, Kennedy TE, Galko MJ, et al. 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