Netrin-1 Signaling Dampens Inflammatory Peritonitis

Previous studies implicated the anti-inflammatory potential of the adenosine 2B receptor (A2BAR). A2BAR activation is achieved through adenosine, but this is limited by its very short t1/2. To further define alternative adenosine signaling, we examined the role of netrin-1 during acute inflammatory peritonitis. In this article, we report that animals with endogenous repression of netrin-1 (Ntn1+/−) demonstrated increased cell count, increased peritoneal cytokine concentration, and pronounced histological changes compared with controls in a model of zymosan A peritonitis. Exogenous netrin-1 significantly decreased i.p. inflammatory changes. This effect was not present in animals with deletion of A2BAR (A2BAR−/−). A2BAR−/− animals demonstrated no change in cell count, i.p. cytokine concentration, or histology in response to netrin-1 injection. These data strengthen the role of netrin-1 as an immunomodulatory protein exerting its function in dependence of the A2BAR and further define alternative adenosine receptor signaling.

T he activation of immune cells and the subsequent inflammatory response are critical steps during immune surveillance and host defense facilitating the elimination of foreign or infectious agents (1)(2)(3)(4). Leukocyte migration and the control of an inflammatory response are tightly controlled multistep processes and have been well characterized in recent years (4,5). Control mechanisms exist to prevent an exaggerated inflammatory response that might result in exaggerated tissue destruction and subsequent organ dysfunction. Only recently, neuronal-guidance proteins were identified to be part of these control mechanisms and, as such, to attenuate leukocyte infiltration and the associated cytokine release (6)(7)(8)(9)(10). The neuronal-guidance protein netrin-1 is involved in the control of local inflammation during tissue hypoxia and the early stages of acute lung injury, and its antiinflammatory function is dependent on the interaction of netrin-1 with the adenosine 2B receptor (A2BAR) (11)(12)(13). This opens the possibility of endogenous adenosine receptor activation independent of the short-lived nucleoside adenosine.
Several reports demonstrated a protective role for adenosine and reported the tissue-protective potential of A2BAR activation. As such, signaling through A2BAR dampens pulmonary inflammation and controls fluid exchange during acute lung injury (14,15). During ischemia-reperfusion injury, signaling through A2BAR reduces the associated reperfusion injury and possesses tissue-protective potential (16,17). The coordinated induction of A2BAR during cellular hypoxia and an acute inflammatory process has to be seen as an adaptation of the affected tissues to increase vascular barrier response and to induce tissue-protective signaling through the A2BAR (18)(19)(20)(21). However, a major limitation of adenosine signaling is that once adenosine is generated into the extracellular milieu, it is also rapidly cleared through passive or active uptake. This occurs through nucleoside transporters, termed equilibrative nucleoside transporters and concentrative nucleoside transporters, respectively. As a result, adenosine action is marked by its very short t 1/2 (22,23). Systemic administration of adenosine as an anti-inflammatory drug is not possible because this results in a variety of undesirable side effects, such as bradycardia or vasodilation (24). Therefore, mechanisms that are able to signal through the A2BAR are highly desirable and need further definition.
Given the role of netrin-1 during hypoxic inflammation, we aimed to define its role during acute exudative inflammation and corroborate a possible role for the A2BAR. In this article, we report that netrin-1 is expressed in intestinal organs, and this expression is reduced during acute peritoneal inflammation. Animals with endogenous repression of netrin-1 (Ntn1 +/2 ) demonstrated an increased inflammatory response following zymosan A (ZyA)-induced peritonitis compared with littermate controls. The injection of exogenous netrin-1 significantly dampened the extent of inflammatory peritonitis; this was dependent on the A2BAR. Therefore, these data corroborate the role of netrin-1 as an endogenous anti-inflammatory protein with significant influence on inflammatory control that is dependent on the A2BAR.

Transcriptional and protein analysis
Murine transcriptional analysis of Ntn1 mRNA was performed using sense primer 59-GAG CGG GGG AGT CTG TCT-39 and antisense primer 59-TGG TTT GAT TGC AGG TCT TG-39. Murine b-actin expression was evaluated using sense primer 59-CTC TCC CTC ACG CCA TCC TG-39 and antisense primer 59-TCA CGC ACG ATT TCC CTC TCA G-39. Semiquantitative analysis of human NTN1 mRNA expression was performed using primer sets containing sense primer 59-GAG TGC GTG GCC TGT AAC-39 and the antisense primer 59-AGG CAG ACA CCT CCG CTC TT-39. Samples were controlled for b-actin using the following primers: sense 59-GGT GGC TTT TAG GAT GGC AAG-39, antisense 59-ACT GGA ACG GTG AAG GTG ACA G-39. All PCR analyses were performed using real-time PCR (iCycler; Bio-Rad, Hercules, CA), as described previously (12).
For Western blot analysis, animal samples were homogenized, normalized for protein levels, and applied in nonreducing conditions to SDS containing polyacrylamide gels. Abs used for Western blotting included rabbit polyclonal anti-netrin-1 (sc 20786; Santa Cruz Biotechnology, Santa Cruz, CA) for murine Ntn1 analysis. Actin was stained using rabbit anti-Actin (Cell Signaling Technology, Beverly, MA). Blots were washed, and species-matched peroxidase-conjugated secondary Ab (Santa Cruz Biotechnology) was added. Labeled bands from washed blots were detected by ECL (Amersham Biosciences, Piscataway, NJ).

Immunofluorescent staining
For murine immunofluorescence, animals were sacrificed; colon and peritoneum were embedded in paraffin; and tissue sections were placed on slides, air-dried, and fixed in methanol followed by 4% acetone. Air-dried tissue sections were washed three times in PBS and blocked with 5% BSA (Sigma-Aldrich, St. Louis, MO). Rabbit polyclonal anti-netrin-1 (sc 20786; Santa Cruz Biotechnology), at a dilution of 1:1000, was used as the primary Ab. Texas Red 545-conjugated goat anti-rabbit (Invitrogen, Eugene, OR) was used as the secondary Ab. DAPI (Invitrogen) was used for nuclear counterstaining.

Histopathological evaluation
Following peritonitis, mice were killed, and tissues were fixed in 10% formaldehyde solution. Tissues were embedded in paraffin and stained with H&E. Fluorescence microscope LSM 510 Meta (Carl Zeiss, Jena, Germany) was used for imaging.

Peritonitis model
All animal experiments were in accordance with the regulations of the Regierungspräsidium Tübingen and the local ethical committee. Mice were injected i.p. with 1 ml ZyA (1 mg/ml; Sigma-Aldrich), and subsequently with vehicle (saline + 0.2% BSA) or 1 mg recombinant murine netrin-1 (R&D Systems, Minneapolis, MN) in 150 ml total volume. Recruited leukocytes were obtained at the described time points by peritoneal lavage with calcium-and magnesium-free ice-cold PBS solution containing 10 U/ml unfractionated heparin. Collected cells were washed, resuspended in 2 ml HBSS, and counted, and cytospin samples were prepared. All reagents used were endotoxin-free.

Measurement of peritoneal cAMP
After peritoneal lavage was obtained, cAMP concentrations were determined with a cAMP EIA Kit (Cayman Chemical, Ann Arbor, MI).

Data analysis
All data are presented as mean 6 SEM from 9-11 animals per condition. GraphPad 5.0 software (GraphPad, San Diego, CA) was used for statistical analysis. We performed statistical analysis using one-way ANOVA to determine group differences using post hoc analysis, followed by the unpaired Student t test (two sided, a , 0.05). A p value ,0.05 was considered statistically significant.

Netrin-1 is repressed during an acute inflammatory response
We initially addressed the question of whether netrin-1 is expressed outside the CNS in intestinal organs to validate the model subsequently used; we found a robust expression of netrin-1 within several murine organs (Fig. 1A, 1B). We then aimed to identify whether netrin-1 expression is affected during an acute inflammatory process FIGURE 1. Netrin-1 is expressed outside the CNS. A, Ntn1 mRNA expression in the brain (Br) and outside the CNS in lung (Lu), liver (Li), spleen (Sp), and intestine (In). B, Pooled Western blot analysis of four independent animals demonstrating Ntn1 expression in brain (Br), lung (Lu), liver (Li), spleen (Sp), and intestine (In). C, Immunofluorescent localization of Ntn1 expression (red) in colon of WT animals 4 h following i.p. injection of NaCl 0.9% or ZyA (original magnification 3400). D, Ntn1 protein analysis in WT animals 4 h following i.p. injection of NaCl 0.9% or ZyA. E, NTN1 mRNA expression in CaCo-2 cells exposed to increasing concentrations of TNF-a for 24 h. F, NTN1 mRNA expression in CaCo-2 cells exposed to increasing concentrations of IL-6 for 24 h. Data are mean 6 SEM (n = 4 per condition). **p , 0.01; ***p , 0.001. in the peritoneal cavity in vivo and found a significant reduction in netrin-1 in response to sterile peritonitis induced through ZyA (Fig.  1C, 1D). To corroborate this finding, we tested whether netrin-1 expression was changed following inflammatory stimulation in vitro and exposed the intestinal CaCo-2 cell line to TNF-a and IL-6 stimulation. This corroborated that netrin-1 expression is significantly reduced in intestinal epithelial cells in response to inflammatory cytokines (Fig. 1E, 1F).
To gain additional data about the anti-inflammatory role of netrin-1, we stimulated CaCo-2 cells with ZyA, which resulted in a significant induction of TNF-a and IL-6 transcripts within these cells. When adding netrin-1 to this, we found a significant reduction in the transcriptional response to ZyA. Following preincubation of CaCo-2 cells with the A2BAR antagonist 1-propyl-8-(p-sulfophenyl)xanthine, this effect was significantly attenuated but not completely abolished (Supplemental Fig. 6).

Discussion
Signaling through the A2BAR possesses anti-inflammatory potential and dampens inflammatory tissue damage. Endogenous activation of A2BAR occurs through adenosine, but recent reports FIGURE 6. Exogenous netrin-1 does not reduce inflammatory peritonitis in A2BAR 2/2 animals. A2BAR 2/2 animals were injected i.p. with NaCl 0.9% or 1 mg of ZyA, followed by i.v. injection with vehicle or recombinant netrin-1 (1 mg); samples were taken after 4 h. Cell count (A), MPO activity (B), protein content (C), and representative histological analysis of the peritoneum, the mesenterial fat tissue, and cytospin samples (D) in the peritoneal lavage. Sections were stained with H&E (original magnification 3400; insets 31000). Data are mean 6 SEM (n = 9 per group). **p , 0.01; ***p , 0.001. suggested a role for the neuronal repellent netrin-1 in A2BAR activation. Therefore, we assessed the role of exogenous and endogenous netrin-1 in a model of inflammatory peritonitis to further define its anti-inflammatory role, as well as its potential to exert its function in dependence of the A2BAR. We report in this article that netrin-1 possesses significant anti-inflammatory potential during exudative inflammation, and this effect is dependent on the A2BAR.
The role of netrin-1 was initially described in the context of axonal growth and migration. Floor plate cells at the ventral midline of the mammalian embryonic neural tube secrete netrin-1 and generate a circumferential gradient of netrin-1 protein in the neuroepithelium (25). This protein gradient possesses bifunctional potential, in that it attracts some axons to, as well as repels other axons from, the ventral midline. Subsequent studies demonstrated the expression of netrin-1 outside the nervous system within the vasculature, a reduction in netrin-1 expression during an acute inflammatory process, and that netrin-1 is a potent inhibitor of myeloid cell migration toward chemotactic stimuli in vitro (12,26). This anti-inflammatory potential of netrin-1 was corroborated by Wang et al. (27) during renal ischemia-reperfusion injury. In this study, the investigators demonstrated that the administration of netrin-1 resulted in reduced tissue injury and improved kidney function following ischemia-reperfusion injury. Consistent with these findings, we revealed a role for mucosal netrin-1 in the attenuation of hypoxic inflammation (13). Our current findings corroborate the anti-inflammatory potential and the tissueprotective role of netrin-1 during inflammatory peritonitis and indicate a role for A2BAR in mediating netrin-1 action during an acute inflammatory response.
Signaling through A2BAR was suggested to possess protective potential in a variety of organ systems and to be important during the orchestration of an acute inflammatory response (17,28,29). As such, A2BAR activation is involved in the inflammatory response of mast cells, epithelial cells, smooth muscle cells, and fibroblasts (30,31). A2BAR also limits an inflammatory response of the endothelium, determines its permeability, and suppresses macrophage activation, thereby preventing tissue injury after episodes of hypoxia and ischemia (32,33). Thus, A2BAR might be an important pharmacological target in treating conditions associated with barrier dysfunction and acute inflammation. Although investigations of pharmacological approaches for A2BR activation are underway, a possible endogenous activation of A2BAR through netrin-1 has not been well investigated. The present study demonstrated that netrin-1 does not exert antiinflammatory function in A2BAR 2/2 animals and, therefore, showed that its function is dependent on A2BAR.
For central nervous function of netrin-1, the deleted in colorectal cancer receptor and the UNC5 homologs UNC5A, -B, -C, and -D were initially reported (34). Corset et al. (11) subsequently demonstrated that netrin-1 also signals through A2BAR during axonal outgrowth. In this study, netrin-1 stimulation resulted in an intracellular cAMP increase that was dependent on A2BAR. However, other reports implicated that the repulsive function of netrin-1 during axonal growth is independent of A2BAR function; rather, it is mediated through the deleted in colon cancer receptor (35). In a study by McKenna et al. (36), the authors demonstrated that activation or overexpression of A2BAR reduced the level of the repellent UNC5A receptor on the surface of neuronal cells. This resulted in a conversion of repulsion to attraction of these cells. Ly et al. (26) demonstrated that the anti-inflammatory function of netrin-1 was dependent on a member of the UNC family, the UNC5b receptor in vitro through chemotactic migration studies, but they did not translate this finding into in vivo evidence. In recent studies, we demonstrated that netrin-1 dampens hypoxic inflammation through A2BAR and corroborated this during acute lung injury (12,13). In the present study, we investigated the role of netrin-1 during acute exudative inflammation and assessed its role during inflammatory peritonitis. We found that netrin-1 significantly reduced the number of inflammatory cells and dampened the histological changes of an acute inflammatory response in dependence of the A2BAR. Furthermore, netrin-1 significantly reduced the release of inflammatory cytokines; this was not observed in animals with genetargeted deletion of A2BAR. Given the findings by McKenna et al. (36), a role for A2BAR in the expression of other known netrin-1Rs on immunocompetent cells might be possible and explain our findings; however, further studies are needed. The results that we presented in this article corroborate an important role for A2BAR for the anti-inflammatory potential of netrin-1.
In summary, our results strengthen the role of netrin-1 as an endogenous modulator of an inflammatory response during an acute exudative inflammation. Exogenous netrin-1 exerts significant anti-inflammation that is dependent on the A2BAR. As such, these findings increase our knowledge about a possible role for adenosine receptor activation during netrin-1 anti-inflammation.

Disclosures
The authors have no financial conflicts of interest.