Immunomodulatory Functions of Neuronal Guidance Proteins

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Review

Immunomodulatory Functions of Neuronal Guidance Proteins Valbona Mirakaj1,* and Peter Rosenberger1,* Neuronal guidance proteins (NGPs) were originally identified for their role during the embryonic development of the nervous system. Recent years have seen the discovery of NGP functions during immune responses. In this context, NGPs were demonstrated to control leukocyte migration and the release of cytokines during conditions of acute inflammation, such as lung injury or sepsis. However, NGPs also display potent actions in the resolution of inflammation, chronic inflammatory conditions, the development of atherosclerosis, and during ischemia followed by reperfusion. Here, we provide an overview of the current state of knowledge about the role of NGPs in the immune system and describe their immunomodulatory function.

Trends NGPs are critical in the development of the nervous system, but recent studies have shown that they also have important immune roles. Netrin-1 is the best-described NGP in the immune system. It influences the switch from pro- to anti-inflammatory macrophages and induces the production of pro-resolving lipid mediators, promoting the resolution of inflammation. Semaphorins have diverse functions in the immune system. Members of this protein family can be proinflammatory (e.g., Sema7A), while other members have anti-inflammatory functions (e.g., Sema3E). Semaphorins can also interact with Toll-like receptors to enhance the inflammatory response (e.g., Sema3A).

Discovery of Neuronal Guidance Proteins Guidance of growing axons through NGPs was first described during the 1990s by Kolodkin and Serafini [1,2]. The authors described the influence of netrin-1 and the protein class of semaphorins on axonal growth. They showed that these proteins guide axons to their final location through a balance of chemoattractive and chemorepulsive signals, ultimately allowing neurons to form meaningful synapses within the 3D system of the developing brain. NGPs exert their function through guidance of the growth cone at the tip of the growing axon. Concentration gradients of NGPs mediate the role of attraction or repulsion. This can be done via not only direct cellular interactions, but also long-distance diffusion, which then results in fasciculation, turning, and eventual target innervations of the axon. Following this initial description of NGP function, other protein families were identified that display potent guidance activities during axonal growth, including the broader family of ephrins, which includes netrin-1, and the semaphorin receptor families of plexins and neuropilins. Recent years have seen the discovery of biological parallels between the nervous and the immune system and, within this context, discovery of the function of NGPs during important immunological processes. NGPs can not only be expressed on the cellular surface on a variety of cells, but also be cleaved and be present in a soluble form within serum. The first indication of an immunomodulatory function of NGPs was provided by Wu et al., who showed that Slit-2 not only repels axons during their growth, but also stops the migration of neutrophils in response to a chemotactic stimulus [3]. In both acute and chronic inflammatory conditions, immune cell migration to and within tissues is controlled in large part by proteins of the chemokine family. Yet, in contrast to chemokines that have critical roles in the attraction of leukocytes, NGPs can also act as repulsive molecules. In addition, NGPs hold several other functions in the immune system. Here, we present current evidence for the immunomodulatory function of NGPs.

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Atherosclerosis as a model of chronic inflammation is significantly influenced by NGPs. An important mechanism in this condition appears to be the interaction of NGPs with macrophages within atherosclerotic plaques.

1

Department of Anesthesia and Intensive Care Medicine, Tübingen University Hospital, Faculty of Medicine, Eberhard-Karls University Tübingen, Hoppe-Seyler-Straße 3, 72076 Tübingen, Germany

*Correspondence: [email protected] (V. Mirakaj) and [email protected] (P. Rosenberger).

http://dx.doi.org/10.1016/j.it.2017.03.007 © 2017 Elsevier Ltd. All rights reserved.

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Role of Neuronal Guidance Proteins during the Acute Inflammatory Response The initial phase of acute inflammation is marked by the extravasation and infiltration of inflammatory cells into the affected tissue areas. Of significant importance in this phase is the infiltration of neutrophil granulocytes with the aim to fight and/or clear the pathological insult. Initial evidence for the possible influence of NGPs on this phase of inflammation indicated that Slit-2 could inhibit chemotactic leukocyte migration [3]. The inhibition of chemokine-induced chemotaxis could be reconstructed in vitro by the co-expression of a chemokine receptor and Roundabout (Robo), a Slit receptor. Work on an additional guidance protein, netrin-1, demonstrated that this protein is expressed at the vascular barrier in endothelial cells. Netrin-1 expression is reduced in response to Staphylococcus aureus infection, which results in an increased infiltration of neutrophils into the extravascular space [4]. The capacity of netrin-1 to guide axonal outgrowth is mediated by the uncoordinated receptor 5 (UNC5b), the neogenin receptor, and the adenosine 2B receptor (A2BAR). The stop function of netrin-1 in the nervous system for axonal growth has translated into the potential to stop leukocyte migration at the endothelial interface (Figure 1). In this study [4], the function of netrin-1 was found to be mediated by UNC5b. There is close interplay between inflammation and hypoxia and inflamed lesions often become severely hypoxic; vice versa, tissue hypoxia can result in inflammation that is clinically relevant [5,6]. When looking at the role of netrin-1 during hypoxia, another study showed that netrin-1 is induced in areas of tissue hypoxia through hypoxia inducible factor 1alpha (HIF-1a). Although the anti-inflammatory role of netrin-1 was confirmed in this study, the authors also found that the netrin-1 function was dependent on A2BAR [7]. Relevant preclinical work in models of acute lung injury and inflammatory peritonitis corroborated the anti-inflammatory potential of netrin-1 [8–10]. Subsequently, models of intestinal and renal inflammation also showed significant anti-inflammatory and protective capacities of netrin-1 [11–13]. The anti-inflammatory effect of netrin-1 is mediated by suppression of COX-2 expression through regulation of nuclear factor (NF)-kB activation. This results in reduced PGE2 and thromboxane B2 production and the reduced apoptosis of renal epithelial cells. In addition, the reduced production of cytokines and chemokines resulted in a reduction in neutrophil infiltration after ischemia followed by reperfusion [14]. During acute inflammation, netrin-1 also appears to promote the differentiation of alternatively activated anti-inflammatory M2 macrophages. Overexpression of netrin-1 in transgenic mice resulted in a phenotypic switch in macrophage polarization toward M2 polarization. This was associated with an increase in arginase-1, interleukin (IL)-4, and IL-13 expression. In vitro studies confirmed that netrin-1 induced the expression of M2 markers and suppressed the interferon (IFN)-g-induced M1 polarization and production of inflammatory mediators [15]. In addition to these anti-inflammatory contributions, netrin-1 retains macrophages in atherosclerotic plaques and in adipose tissues during obesity, promoting disease development and inflammation in these conditions [16]. This is not surprising given the chemoattractive and chemorepulsive impacts of netrin-1 on axonal migration as well as leukocytes, and suggests that netrin-1 has dual roles in regulating inflammation depending on the organ and local microenvironment. The essential roles of NGPs in inflammation were extended during work showing that the repulsive guidance molecule A (RGM-A) attenuates chemotactic leukocyte migration during the early phases of acute inflammation [17]. Although RGM-A, a glycosylphosphatidylinositolanchored proline- and cysteine-rich chemorepulsive protein, is structurally not related to netrin-1, it also instructs the navigation of axonal growth. The function of RGM-A in the nervous system is mediated by the neogenin (Neo1) receptor and the binding of RGM-A to Neo1 has a repulsive function on axonal growth [18,19]. During the initial phase of inflammation, RGM-A binds to Neo1 and reduces the infiltration of granulocytes and the production of proinflammatory cytokines [17]. The role of neogenin during conditions of inflammation was also highlighted in the absence of specific RGM-A interactions. Neo1 induces the infiltration of neutrophils into

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Key: Netrin-1

UNC5b A2BAR

RGM-A Sema7A (A)

Neo1 PlexinC1 (B)

Intravascular

Inducon NF-κB HIF-1α

Inflammaon

Inflammaon Extravascular Figure 1. The Infiltration of Granulocytes Is Modulated by Neuronal Guidance Proteins (NGPs) during Acute Inflammation. [467_TD$IF](A) During acute inflammation, netrin-1 is expressed on the endothelial surface. Netrin-1 and repulsive guidance molecule A (RGM-A) reduce the infiltration of neutrophil granulocytes into the extravascular space and [468_TD$IF]attenuate the release of proinflammatory cytokines. ([469_TD$IF]B) Endothelially expressed semaphorin 7A (Sema7A) is induced during conditions of tissue inflammation [via nuclear factor (NF)-kB] or hypoxia [via hypoxia-inducible factor (HIF)-1a] and facilitates the recruitment of neutrophils into sites of inflammation. Sema7A also increases the release of proinflammatory cytokines into the circulation from immune cells. NGPs can not only be expressed on the cellular surface, but also be cleaved and present in soluble forms. Abbreviations: [470_TD$IF]A2BAR, adenosine 2B receptor; Neo1, neogenin; PlexinC1, plexin C1 receptor; UNC5b, uncoordinated receptor 5b.

sites of acute inflammation during the initial phase of inflammation. It also activates the expression of NF-kB and, thus, increases the production of cytokines and chemokines [20,21]. RGM-A not only activates protective cell type-specific programs, but also contributes to the invasion of inflammatory cells into the central nervous system (CNS) during autoimmune encephalomyelitis (EAE). In a model of EAE, RGM-A was able to bind to Neo1 and increase the attachment of CD4+ cells to ICAM-1, resulting in invasion into the CNS [22]. Subsequently, these CD4+ cells produced IFN-g, IL-2, IL-4, and IL-17 to induce inflammation. This reaction could be reduced using specific anti-RGM-A antibodies. These data demonstrate that the RGM-A–Neo1 interaction can have various functions in different cell types and disease modalities. Semaphorins are NGPs that also appear to have a multifaceted role in the regulation of inflammation. The semaphorins are classified into eight classes based on their structural homology, and their target receptors are the plexins and neuropilins (Table 1) [23]. Sema3A

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Table 1. NGPs Expressed in Endothelial Cells and Macrophagesa,b[472_TD$IF] NGPs [473_TD$IF]expressed in endothelial cells Ntn1 Ntn3 Ntn4 Ntn5

Slit2 Slit3

Efna1 Efna4 Efna5 Efnb1 Efnb2 Efnb3

Sema3(a,b,c,d,e,f,g) Sema4(a,b,c,d,g) Sema5a Sema6(a,b,c,d) Sema7a

PlexinA1 PlexinA2 PlexinA4 PlexinB1 PlexinB2 PlexinB3 PlexinC1 PlexinD1

Neo1 DCC

UNC5a UNC5b UNC5c UNC5d

Nrp1 Nrp2

Sema3(a,b,c,d,e,f,g) Sema4(a,b,c,d,g) Sema5(a,b) Sema6(a,b,c,d) Sema7a

PlexinA1 PlexinA2 PlexinA3 PlexinA4 PlexinB1 PlexinB2 PlexinB3 PlexinC1 PlexinD1

Neo1 DCC

UNC5a UNC5b UNC5c UNC5d

Nrp1 Nrp2

NGPs [47_TD$IF]expressed in macrophages Ntn1 Ntn3 Ntn4 Ntn5

Slit2 Slit3

Efna1 Efna2 Efna3 Efna4 Efna5 Efna6 Efna7 Efna8 Efna10 Efnb1 Efnb2 Efnb3

a

NGPs are expressed on the surface of these cells, but can also be cleaved from the surface and be present in soluble forms within the serum. b Information from [16,80,83,88].

markedly influences the inflammatory response during bacterial sepsis, particularly by exacerbating a Toll-like receptor (TLR)-dependent cytokine storm [24]. The binding of Sema3A to its receptor, Plexin A4, is required for TLR-induced activation of Rac1, which is accompanied by JNK and NF-kB activation. As a result, inflammatory cytokines are produced through the binding of NF-kB and AP-1 to the cytokine promoters. Of note, TLR engagement induces the expression of Sema3A, thus completing a proinflammatory autocrine loop. Sema3A in this context also influences the recirculation of leukocytes via their entry into the lymphatic system and inhibits tumor growth [25,26]. A proinflammatory function has also been highlighted for Sema3E and its target receptor PlexinD1, demonstrating a direct chemoattractive impact on the recruitment of classical macrophages into obese tissues that then leads to adipose tissue inflammation and metabolic disorders [27]. Thus, the disruption of PlexinD1 expression in these macrophages and of adipose tissue p53 expression led to downregulation of Sema3E with reduced adipose tissue inflammation. By contrast, Sema3E reduced the infiltration of neutrophils through PlexinD1 in a model of allergic airway inflammation [28]. Sema4A is an important regulator of Th2-driven lung pathophysiology, where it markedly reduces the severity of allergic airway inflammation. Genetic deletion of Sema4A resulted in an inflammatory response that was associated with a selective increase in bronchoalveolar lavage IL-13 content, augmented airway hyper-reactivity, and lower regulatory T cell (Treg) numbers following ovalbumin-induced inflammation [29]. Sema4A also activates mTOR signaling through Plexin B2 and helps to induce the differentiation of CD8+ cells. This has a significant impact on the processing on pathogen-specific CD8+ responses [30]. There are diverse additional ways in which semaphorins modulate an inflammatory response. During nephritis, Sema4D activates the recruitment of macrophages and other inflammatory cells through the PlexinB1 receptor [31]. Sema4D also induces tumor necrosis factor (TNF)-a and IL-6 production through CD14+ monocytes, and antibodies against Sema4D diminished this response in patients with rheumatoid arthritis [32]. Sema4D also impacts B cell activation and controls B cell receptor (BCR) signals in this cell population [33]. In this respect Sema4D is likely important for the fine-tuning of the B cell interactions, BCR signaling, and the production

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of autoantibodies [34]. Sema4D–CD72 interactions also account for the selection of highaffinity B cells by selecting survival signals from T cells [35]. Alongside the proinflammatory actions of Sema4D, Sema7A triggers a T cell-mediated inflammatory response through the a1b1 integrin receptor [36]. Suzuki et al. showed that Sema7A activates monocytes to induce the production of cytokines. However, cytokine-producing effector T cells were still able to develop and migrate into antigen-challenged sites in Sema7a–/–[476_TD$IF] mice, but Sema7a–/– T cells failed to induce contact hypersensitivity. Subsequent work showed that Sema7A is expressed on the surface of endothelial cells and increases the infiltration of neutrophils during tissue hypoxia [37]. This is likely mediated, at least in part, through the Plexin C1 receptor (Figure [478_TD$IF]1). Plexin C1 itself propagates the infiltration of immune cells into sites of sterile pulmonary inflammation and also during Zymosan A-induced inflammation [38,39]. By contrast, Sema7A was also found to be protective during dextran sulfate sodium (DSS)-induced colitis, where it increased the production of the anti-inflammatory cytokine IL-10 through the anb1 integrin receptor [40]. Therefore, the action of Sema7A is likely to be dependent on the expression of its target receptors within specific organ systems. The diversity in controlling inflammatory programs is also marked when semaphorins act via neuropilins as target receptors. Neuropilin-1 (NRP-1) is expressed by human dendritic cells (DCs) and resting T cells, with the initial contact between DCs and resting T cells resulting in NRP-1-induced polarization of T cells. Incubation of DCs or resting T cells with antibodies blocking NRP-1 inhibited DC-induced proliferation of resting T cells, suggesting that NRP-1 mediates interactions between DCs and T cells that are essential for initiation of the primary immune response [41]. NRP-1 was also expressed in alveolar epithelial cells to control cellular apoptosis and pulmonary emphysema following recurrent cigarette smoking-induced inflammation [42]. In a model of Bacillus Calmette-Guérin (BCG)-induced bladder inflammation, treatment with NRP1-neutralizing antibodies reduced polymorphonuclear cell and DC infiltration [43]. NRP-1 is generated at high levels on natural Treg cells controlled by transforming growth factor (TGF)-b. These cells highly express Foxp3 and their activity is essential for the normal response of the immune system, immune homeostasis, and self-tolerance [44]. This action of NRP-1 could be used in the future for the treatment of sepsis, since Gao et al. were able to show that the NRP-1 agonist tuftsin improved sepsis survival through a reduction of Treg-induced immunosuppression [45,46]. Neuropilin 2 (NRP-2) protects the vascular barrier and increases lymphatic drainage during acute inflammation through endogenous Sema3F [47]. This demonstrates the growing recognition of the role of neuropilins in the regulation of acute inflammatory conditions. A proinflammatory role has also been described for EphrinA2 (EphA2), whereby it augments the response to lipopolysaccharide (LPS) during pulmonary inflammation. EphA2 antagonism reduced the expression of phospho-p85, phosphoinositide 3-kinase 110gamma, phosphoAkt, NF-kB, and proinflammatory cytokines, and inhibited the phosphoinositide 3-kinase-Akt pathway [48]. Furthermore, a role for EphB2 was described during B cell activation in which EphB2 was involved in human naive B cell activation via the Src-p65 and Notch1 signaling pathways and could be regulated by miR-185. As a result, TNF-a secretion and immunoglobulin (Ig)-G production were depressed concordantly with downregulated EphB2 expression [49]. This shows that the function of NGPs within the nervous system is only partially conserved during acute inflammation. The function of Netrin-1 to stop axonal growth has translated into a stop function on immune cell migration with a reduction in cytokine production. This is also the case for Slit-2 and RGM-A, whose function as a repellent of axonal growth has translated into a potential repellent of chemotactic neutrophil migration. The translation of the function of the semaphorins is more complex. Although acting as a repellent during axonal growth, Sema3A was shown to have an activating function during acute

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(A)

Granulocyte Proinflammatory Ly6hi monocyte Netrin-1

An-inflammatory Ly6lo monocyte

–

–

Intravascular Netrin-1

+

+

Extravascular Iniaon

Resoluon

4h

12h Ly6Chi monocytes

PMN infiltraon Generaon of proresolving lipidmediators

24h Generaonof Ly6Clo monocytes

48h Return to homeostasis

Efferocytosis Organ regeneraon

(B)

+ Netrin-1 Edema

PMN

Monocytes/MF

Resoluon– phase

Time to resoluon

Edema

PMN

Monocytes/MF

Resoluonphase

Time to resoluon

Time enhanced recovery

Figure 2. Function of Guidance Protein Netrin-1 during the Resolution of Inflammation. Resolution is a critical terminal phase of an inflammatory response, ideally (Figure legend continued on the bottom of the next page.)

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inflammation, as well as being involved in TLR-dependent signaling. The function of Sema3E in axonal development is complex and reprograms neuronal function; similarly, its function within the immune system is also complex and not fully understood. Sema4D is known to induce growth cone collapse during axonal development, whereas it has an activating function on immune cell migration and cytokine production. In line with its enhancement of growth within the nervous system, Sema7A also activates immune cell migration and has a proinflammatory function. In the nervous system, NRP-1 and NRP-2 mediate complex functions during growth cone guidance and Sema3A signaling, and this also appears to translate into their function in the immune system. This shows that, although it was initially thought that NGPs might have conserved functions and/or signaling roles within the nervous and immune system, this does not appear to be the case for all NGPs. In particular, our understanding of the role of semaphorins in the immune system remains preliminary and requires further study.

Impact of Neuronal Guidance Proteins on the Resolution of Inflammation Following the initial phase of inflammation the active process of resolution initiates the return to tissue hemostasis [50]. Recent work has shown that NGPs actively influence this process. Netrin-1 stimulates the production of specialized proresolving lipid mediators, such as resolvins, protectins, or lipoxins, and increases non-inflammatory mononuclear cell recruitment and the capacity of macrophages to phagocytose apoptotic neutrophils following tissue injury [51]. As a result, netrin-1 significantly shortens the time required for resolution, which is a quantitative measure of resolution and tissue regeneration. Extension of this work showed that netrin-1 is an innate immune system resolvent that can induce tissue-specific regeneration programs and promote organ recovery [52] (Figure 2[479_TD$IF]). Non-resolving inflammation can result in tissue fibrosis or chronic and ongoing inflammation. Conditions that are marked by chronic and continued inflammation result in tissue destruction and functional impairment [53]. Examples of such conditions are colitis, asthma, and rheumatoid arthritis. Semaphorins have significant influence over the resolution of inflammation. Sema3E expression levels correlate with disease activity and the severity of histological neutrophil infiltration in rheumatoid arthritis [54]. It also suppresses the ability of CD4+ cells to abolish an allergic response [55]. Fibrosis is the end result of ongoing inflammation and the failed resolution of inflammation. Sema4A activates the Akt pathway in lung fibroblasts, a mechanism that was shown to increase pulmonary fibrosis via Plexin D1 [56]. Sema4A also promotes inflammation during rheumatoid arthritis, a condition marked by ongoing and nonresolved inflammation [57]. These findings suggest that Sema4A negatively influences the resolution of an inflammatory process. The resolution of inflammation is also dependent on appropriate T cell functions. As described above, Sema7A functions as an effector molecule in T cell-mediated inflammation, and also activates macrophages via a1b1integrin as part of the immunological synapse [36]. A study investigating the role of Sema7A during lung injury confirmed the capacity of Sema7A to serve as a negative feedback loop and to foster the production of proinflammatory cytokines [58]. Sema7A is also involved into the development of pulmonary fibrosis. TGF-b1 activated phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB)/AKT via a SEMA 7A-dependent mechanism that leads to pulmonary tissue fibrosis and

leading to a return of the tissue to homeostasis. (A) Netrin-1 has multiple functions during the resolution phase. It reduces the infiltration of neutrophil granulocytes and proinflammatory monocytes into the tissue site of inflammation, and then activates the switch from proinflammatory to anti-inflammatory monocytes, generally marked as a transition from a Ly6Chi to a Ly6Clo phenotype. Netrin-1 also promotes the phagocytosis of cellular debris and apoptotic cells (i.e., efferocytosis), thus promoting a return to tissue homeostasis. (B) Schematic drawing of complete resolution as the ideal outcome of inflammation. Resolution of inflammation involves a temporal series of edema formation, granulocyte infiltration, and subsequent monocyte infiltration with the differentiation of monocytes to macrophages (Ly6Chi[471_TD$IF] to a Ly6Clo). Netrin-1 dampens the infiltration of polymorphonuclear leukocytes into sites of inflammation, activates specialized proresolving lipid mediator production, and enhances the process of efferocytosis, reducing the time to complete the resolution of inflammation.

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impaired oxygenation [59]. It will be interesting to determine whether additional NGPs have the capacity to accelerate the resolution of inflammation and influence tissue regeneration.

Neuronal Guidance Protein Signaling during Conditions of Ischemia Followed by Reperfusion A large concern of modern healthcare is the care of patients experiencing organ ischemia followed by subsequent reperfusion (IR). IR not only affects the heart during myocardial infarction, but is also observed following major surgery or presents as part of sepsis [60]. Several studies have addressed the role of NGPs during IR ([480_TD$IF]Figure 3). Initial work showed that netrin-1 is protective during renal and myocardial IR injury in murine models [61,62]. Work on renal IR showed that netrin-1 and its target receptors are differentially expressed in the various tissues of the kidney [63]. The administration of netrin-1 reduced caspase-3 activity and, thus, dampened the apoptosis of tubular epithelial cells [64]. This effect influences the balance between T helper subtypes and induces an anti-inflammatory effect on Th1/Th2 and Th17dependent cytokine production via the UNC5b receptor [65], a reduction in granulocyte infiltration and the induction of the anti-inflammatory M2 macrophage phenotype [15,66]. The COX-2-dependent production of prostaglandin E2 is decreased by netrin-1, reducing the extent of IR-derived kidney injury [14]. Studies have also examined the role of netrin-1 during myocardial IR injury. Its protective effect following myocardial IR injury was reported to be mainly mediated by factors such as the induction of nitric oxide (NO) production and reduction of apoptosis-induced cardiomyocyte death [67] (Figure 3[481_TD$IF]). The netrin-1-induced NO production preserves mitochondrial function and simultaneously appears to reduce the activity of NADPH oxidase 4 (NOX4) [68,69]. This effect on NO induction and cardioprotection is mediated by the DCC receptor [70]. NGPs also have the capacity to promote tissue regeneration following IR injury. As mentioned above, Schlegel et al. demonstrated that netrin-1 increases the regeneration capacity following hepatic IR injury. This was associated with improved liver repair via the stimulation of proresolving lipid mediators (SPMs) and the generation of hepatic growth factors [52]. Several other guidance proteins and guidance receptors affect the extent of IR injury. The deletion of Neo1 and its functional inhibition resulted in the reduced infiltration of inflammatory cells and reduced tissue injury following hepatic IR injury [71]. This was also observed for the UNC5b receptor. In a model of hepatic and myocardial IR injury, partial deletion of UNC5b resulted in reduced myocardial as well as hepatic reperfusion tissue injury [72,73]. However, the role of UNC5b in this context needs to be further clarified, since work in the kidney has shown that mice with deletion of UNC5b in tubular epithelial cells (GGT-cre) exhibited significantly worse renal function and renal tissue damage, increased tubular epithelial cell death, enhanced p53 activation, and exacerbated inflammation compared with controls. An approach using small interfering (si)RNA-mediated suppression of UNC5B expression in cultured tubular epithelial cells exacerbated cisplatin-induced cell death. This demonstrates that UNC5B has a critical role in cell survival following kidney injury [74]. The role of semaphorins during conditions of IR injury is unclear. In the myocardium, a reduction in Sema3A is a protective strategy adopted by damaged myocardial cells to better resist the secretion of hypoxia-induced inflammatory factors, cell viability decline, cardiomyocyte apoptosis, and reactive oxygen species (ROS) release [75]. The genetic inactivation of Sema3A also resulted in protection against IR-induced acute kidney injury. Ranganathan et al. demonstrated not only a reduced neutrophil infiltration paired with attenuated epithelial cell apoptosis, but also increased chemokine excretion in urine. Pharmacological-based inhibition of Sema3A receptor binding likewise protected against IR-induced acute kidney injury (AKI). This effect was TLR-4 dependent in epithelial cells, macrophages, and DCs [76]. Evidence was reported for a target receptor of Sema7A, the Plexin C1 receptor. During hepatic IR injury, Plexin C1 knockout mice

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Specialized proresolving lipid mediators

Extravascular (A)

(B)

g Prostaglandin E2

(C)

M2

M1 Sw SSwitch witch

Apoptosis

Tissue hypoxia yp Mitochondrial driall p preservaon reservaon

Caspase p 3

COX2

Inducon

4 NOX4 HIF - 1α

NO NO

Intravascular

Key: Granulocyte

Macrophage

Netrin-1

Figure 3. Netrin-1 Function during Ischemia Followed by Reperfusion. During conditions of ischemia followed by reperfusion (A), Netrin-1 reduces intracellular caspase-3 activity and, as a result, reduces apoptosis. It also dampens cyclooxygenase 2 (COX-2) activity, decreases prostaglandin E2 production, and, via the increased generation of nitric oxide (NO) production, dampens NADPH oxidase 4 (NOX4) activity, preserving mitochondrial function as a result. (B) Netrin-1 is induced during tissue hypoxia via hypoxia-inducible factor 1-a (HIF1-a) and dampens the infiltration of neutrophil granulocytes and the release of proinflammatory cytokines. (C) Netrin-1 activates the switch of M1 macrophages to M2 macrophages and has anti-inflammatory and proresolving functions; it also furthers the release of proresolving specialized lipid mediators. NGPs can not only be expressed on the cellular surface, but also be cleaved and present in soluble form.

displayed reduced hepatic-specific injury enzymes, reduced polymorphonuclear leukocyte (PMN) infiltration, and significantly reduced proinflammatory cytokine levels. An approach using antibody-based inhibition of Plexin C1 confirmed these results [77]. Little evidence exists regarding the role of ephrins during IR injury. EphA2 was shown to be strongly upregulated through a Src kinase-dependent pathway that may provide critical cell contact-dependent, bidirectional cues for cytoskeletal repair during renal IR [78]. A recent study demonstrated that the inhibition of EphA4 signaling through the use of a EphA4-Fc fragment significantly reduced TNF-a production and infiltration of neutrophils into tissues, and reduced signs of vascular leakage [79].

Neuronal Guidance Proteins in Atherosclerosis Atherosclerosis is marked by the development of plaques in the vascular wall that ultimately lead to vessel occlusion. Plaque development is thought to be initiated by the presence of macrophages within the vascular wall. NGPs have a significant impact on macrophage biology during vascular inflammation and atherogenesis (Table 2). Netrin-1 is secreted by macrophages in human and mouse atheroma, where it inactivates the migration of macrophages out of plaques. Acting via UNC5b, netrin-1 inhibited the migration of macrophages directed by the chemokines CCL2 and CCL19, activation of GTPase Rac1, and actin polymerization. Deletion of netrin-1 in macrophages resulted in reduced atherosclerosis and promoted the emigration of

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Table 2. NPGs Known to [475_TD$IF]Be Involved in the Development of Atherosclerosis NGP

Function

Refs

Ntn1

Induced during tissue hypoxia, retains macrophages in atherosclerotic plaques through Unc5b, and promotes atherosclerosis

[4,7,16]

Sema3E

Promotes macrophage retention in atherosclerotic plaques and atherosclerotic progression

[83,88]

Sema4D

Protective against the development of atherosclerosis through the protection of platelets in dyslipidemia; mediates monocyte adhesion to endothelial cells via Plexin B1 and B2; might promote atherosclerosis

[82,85]

EphA2

Promotes endothelial cell inflammation and potentially increases atherosclerosis

[86]

EphB2/EphA4

Promotes the inflammatory activation of endothelial cells and increases monocyte adhesion

[87]

macrophages from plaques [80]. Netrin-1 is highly expressed in obese but not lean adipose tissue of humans and mice. A further study looking at the role of netrin-1 showed that induction of netrin-1 expression furthered the retention of macrophages within the adipose tissue. As a result, ongoing inflammation within adipose tissue resulted in decreased glucose tolerance in a glucose tolerance test, with the development of insulin resistance [16]. Importantly, using an adenovirus vector to deliver hNetrin-1 in vivo resulted in a significant reduction in plaque formation, as determined by larger aortic lumen size, thinner intima-media thickness, and reduced blood velocity, thus demonstrating that netrin-1 is a viable target in atherosclerosis [81]. Further studies have investigated the role of semaphorins during the development of atherosclerotic plaques. One target in this regard is Sema4D. Zhu et al. demonstrated that the loss of Sema4D expression reduced platelet hyperactivity otherwise found in dyslipidemia, and conferred protection against the development of atherosclerosis [82]. An overview of the expression patterns in atherosclerotic plaque of guidance cues showed that several candidates are induced during the development of atherosclerotic material [83]. Sema3E is upregulated in the macrophages of advanced plaques and acts as a negative regulator of macrophage migration, promoting macrophage retention and chronic inflammation [84]. This finding was corroborated by a study by Luque et al. that demonstrated that monocytes are retained on endothelial cells via the Sema4D–Plexin B1 axis [85]. In human samples of carotid artery plaques, the expression of Sema4D was enhanced, yet incubation with Sema4D reduced the formation of foam cells [85]. These studies point to a potential role of semaphorin–plexin interactions during the development of atherosclerotic lesions. Evidence for the involvement of ephrins during atherosclerosis showed that macrovascular endothelium expressed EphA1, EphA2, and EphA4 as the dominant isoforms. Endothelial activation with oxidized low-density lipoprotein and proinflammatory cytokines resulted in induction of EphA2 and EphA1 with sustained EphA2 activation. This resulted in proinflammatory gene expression and VCAM-1 and E-selectin induction, and identified these ephrins as potential regulators of atherosclerotic plaque development [86]. Van Gils et al. showed that EphB2 is upregulated under proatherosclerotic flow conditions and functions as a chemoattractant, increasing leukocyte migration in the absence of additional chemokines [83]. A proadhesive effect of monocytes on endothelial cells was shown for EphB2 via the Rho

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signaling pathway and actin filament polymerization. This effect was mediated via the receptor EphA4 and EphB2 was induced by TNF-a [87].

Concluding Remarks NGPs and their target receptors significantly influence multiple aspects of the immune response. During the initial phase of inflammation, netrin-1 and RGM-A stop the infiltration of neutrophils to sites of inflammation and dampen the production of proinflammatory cytokines, whereas semaphorins can have opposing functions. Netrin-1 has dual functions and shortens the resolution phase of inflammation; by contrast, semaphorins have significant influence on ongoing inflammatory processes and the development of tissue fibrosis. NGPs also affect the degree of atherosclerosis by influencing the balance of macrophage retention within the vascular wall and the inflammatory processes that drive the development of this disease. A better understanding of this field will broaden our insight into several other disease processes and pathological conditions. Future work will have to pay more attention to potential crosstalk between NGP signaling in the immune system and nervous system (see Outstanding Questions). Indeed, it remains unclear whether NGPs were simply co-opted by both the nervous and immune systems, where they serve different functional roles, or participate in the crosstalk between these two systems. Furthermore, the interplay between cytokines and NGPs is also of interest. If this crosstalk can be deciphered further, then future therapeutic developments will likely be based on our expanded knowledge of NGPs and their impact outside the nervous system.

Outstanding Questions What is the interplay between NGPs and cytokines and/or chemokines during the initial phase of an acute inflammatory response? What is the timeline of NGP expression during an inflammatory response and how does this influence the resolution of inflammation? What is the organ-specific expression of NGPs and how does this affect their function in the immune system. What is the net effect of NGPs during inflammation within a specific organ system? What is the interplay between the role of NGPs within the nervous system and their role within the immune system? Do NGPs have a concentrationdependent function in the immune system as they do within the nervous system?

Acknowledgments This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG): grants DFG-RO 3671/6-2, as well as DFG-RO 3671/8-1 to P.R. and DFG-MI 1506/4-1, as well as DFG-MI [482_TD$IF]1506/5-1 to V.M.

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