Neurokinin 1 receptor isoforms and the control of innate immunity

Review

Neurokinin 1 receptor isoforms and the control of innate immunity Florin Tuluc1,2, Jian Ping Lai1, Laurie E. Kilpatrick1,2, Dwight L. Evans3 and Steven D. Douglas1,2 1

Division of Allergy and Immunology, Joseph Stokes Jr. Research Institute, The Children’s Hospital of Philadelphia, USA Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, USA 3 Department of Psychiatry, University of Pennsylvania School of Medicine, Philadelphia, USA 2

Substance P is the prototype tachykinin peptide and triggers a variety of biological effects in both the nervous and immune system. Two naturally occurring variants of the neurokinin 1 receptor (NK1R) mediate the effects of SP: a ‘classic’ full-length receptor and a truncated (tail-less) form that lacks 96 amino acid residues at the C-terminus. Most research has focused on the full length receptor and the truncated NK1R has not been extensively explored. Recent data demonstrate that truncated NK1R has important functional roles, including modulation of responses triggered by cytokines, chemotaxis of macrophages and regulation of HIV replication. Targeting the truncated NK1R with pharmacologic agents might result in novel therapeutic approaches in diseases which affect the immune system, including HIV disease. Substance P and neurokinin-1 receptors The undecapeptide substance P (SP) is the prototype tachykinin and it has been identified in the central and peripheral nervous system, and in the immune system [1– 3]. The tachykinin family of neuropeptides initially included SP, neurokinin A (NKA), and neurokinin B (NKB), which are encoded by two preprotachykinin genes (TAC1, and TAC3) [4]. The gene encoding the polypeptide that includes the SP sequence was discovered first and it was termed TAC1. The name TAC2 was assigned to the gene encoding the precursor of NKA, but later it was discovered that TAC2 and TAC1 are identical. The TAC3 gene encodes the precursor of NKB. Each tachykinin is generated by proteolytic cleavage from precursor polypeptides. A fourth tachykinin gene (TAC4) was discovered later and subsequently several other tachykinin peptides were characterized; among them four peptides termed endokinins, hemokinin 1 (HK1) and its shorter derivative, HK1(4–11). Virokinin, a tachykinin-like peptide, is a part of the viral fusion protein of the bovine respiratory syncytial virus (RSV) and can represent a novel type of molecular mimicry used by the bovine RSV to modulate the inflammatory and immune responses in the host [4,5]. Virokinin, however, is absent in the human RSV. A detailed review of the extended family of tachykinins has been recently published [4]. Three human tachykinin receptors have been described (NK1R, NK2R, NK3R). They are G protein-coupled receptors (GPCR) encoded by three distinct genes (TACR1, Corresponding author: Douglas, S.D. ([email protected]).

TACR2, TACR3, respectively) [4,6–8]. SP, hemokinins and endokinins A and B bind with high affinity to NK1R. Here, we focus on the functional differences between the full length and truncated NK1R and on the roles of the NK1R in immune regulation and in host defense against viral infections. Furthermore, we describe how NK1R mediates important proinflammatory signals to cells in the immune system, in particular in monocytes and macrophages. The human NK1R gene is divided into five exons, allowing the generation of multiple splice variants. The only splice variant identified in human tissues at mRNA and protein level is the isoform which lacks 96 amino acid residues at the C-terminus [7,9,10] (Figure 1). This ‘tailless’ NK1R isoform is generated when the intron between exons 4 and 5 is not removed and, therefore, a premature stop codon is encountered before the start of exon 5 [4]. Functional differences between full length and truncated NK1R The most important functional studies and drug development efforts have investigated primarily the full length NK1R, but knowledge on the biology and functions of the truncated form is currently accumulating. The amino acid residues that are required for SP binding to full length NK1R have been mapped by extensive studies with numerous mutant receptors [11]. Many NK1R antagonists have been studied in clinical trials for a variety of potential applications, particularly for depressive disorder. To date, a single drug (aprepitant) is marketed in the USA for the prevention of nausea and vomiting in patients receiving chemotherapy for cancer and in patients undergoing general anesthesia. NK1R mediates a variety of cellular responses, which include pain transmission, exocrine and endocrine secretion, vasodilatation, modulation of cell proliferation, modulation of the immune and inflammatory responses [6,7,11,12]. NK1R is important in a variety of complex brain functions, such as neuronal sensory transmission associated with emesis, pain, depression, anxiety and central responses to stress [1,3,13–16]. Most of these functions are assumed to be mediated by the full length receptor and there are few systematic studies aimed to distinguish the functional responses mediated by the truncated NK1R. The two receptor isoforms have distinct intracellular signaling properties (Box 1).

1471-4906/$ – see front matter ß 2009 Published by Elsevier Ltd. doi:10.1016/j.it.2009.03.006 Available online 7 May 2009

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Box 1. Intracellular signaling mechanisms triggered by full length and truncated NK1R. Classically, the full length NK1R is regarded as a Gq/11-coupled receptor. Interaction of NK1R with agonists leads to activation of phospholipase C b, and results in a transient increase in intracellular inositol 1,4,5 triphosphate (IP3), diacyl-glycerol and increased cytosolic calcium concentration. Subsequently, a cascade of intracellular events occurs and leads to cellular responses that might differ from one cell type to another depending on the activated effectors. Several studies have shown that NK1R can also induce adenylyl cyclase activation and production of cAMP via the Gs protein [50], although the potency of the NK1R agonists in generating cAMP accumulation was lower compared to their ability to induce Ins(1,4,5)P3 formation and intracellular calcium increase [50]. NK1R was also linked to inhibition of adenylyl cyclase via the pertussis toxin-sensitive Gi protein in rat submandibular cells [51]. Most likely there are differences in signaling mechanisms triggered by full length NK1R depending on the cell type and possibly on the agonist that activates the receptor. The signaling properties of the truncated NK1R have been much less extensively studied, but they seem to be different from the full length receptor. The missing Cterminal domain seems to be essential for the coupling to Gq/11 proteins [9,32]. The truncated receptor can enhance CCR5-induced intracellular calcium increase [32] and mediates Erk1/2 phosphorylation [33].

Figure 1. Schematic representations of full length and truncated NK1R. (a) The sequence of full length NK1R contains 407 amino acid residues. A cysteine residue in position 323 is a putative palmitoylation site responsible for anchoring the Cterminus of the receptor to cell membrane. (b) The truncated NK1R includes in its sequence only 311 amino acid residues, lacking 96 amino acid residues corresponding to the C-terminus of the full length receptor. Two N-linked glycosylation sites, Asparagine-14 and Asparagine-18 have been described in full length NK1R, although they might not have an important role for receptor function [48]. A single polymorphism in the human neurokinin-1 receptor gene has been identified [49]; this is the Y192H variant, which displays affinity, receptor kinetics, functional calcium response and receptor internalization properties similar to the wild type variant [49].

Several different truncated forms of NK1R have been generated to identify the role of each part of the molecule in the interaction with ligands and with the downstream effectors and cell signaling regulators. An artificially truncated NK1R that lacks the last 82 amino acid residues in the C-tail mediates calcium mobilization in response to stimulation with agonists, suggesting that none of the residues between F325 and S407 is essential for the coupling of full length NK1R with downstream signals via Gq/11 proteins [17,18]. Cells which express the naturally occurring truncated NK1R (that lacks 96 amino acid residues of the Cterminal domain) does not couple to Gq/11. Furthermore, SP has relatively low binding affinity for the truncated receptor (10 fold lower than the full length receptor) [9]. Taken together, we therefore speculate that the amino acid residues between R311 and P324 in the C-terminal domain of full length NK1R are essential for coupling to Gq and, most likely, for the correct folding of the protein, thus ensuring the optimal conformation of the binding site for SP on the extracellular domains of the receptor. Agonist-induced endocytosis of full length NK1R is largely dependent on the phosphorylation of multiple 272

tyrosine residues in the C-terminal domain, with Y341 and Y349 being the most important [17]. Similarly, association with b-arrestins, Erk 1/2 activation and receptor desensitization upon SP stimulation is reduced in cells expressing truncated receptor mutants missing the C-terminal domain [19]. Despite the 311 amino acids sequence identity, the missing C-terminal domain in the truncated receptor is most likely essential for NK1R folding. We hypothesize that the conformation of the agonist binding pocket is altered in the truncated receptor, leading to lower binding affinity of SP [9]. The truncated NK1R might, therefore, the have capability for more prolonged responses after receptor-ligand interactions because its desensitization and internalization are impaired. The roles of NK1R in the immune system Tachykinins have well-established roles in the innate and adaptive immune system [2,15]. Our investigations [2,20– 26] have demonstrated the presence of NK1 receptors at mRNA and protein levels in cells of the human immune system and the nervous system, including human peripheral blood mononuclear cells (PBMC) [2], lymphocytes [20] and microglia [21]. SP triggers functional responses in many cells of the immune system, including monocytes and macrophages [2], lymphocytes [20], microglia [21], natural killer cells [27] and precursors of immune cells [15]. There are multiple lines of evidence supporting the role of NK1R in the development of immune cells. SP has synergistic effects with hematopoietic growth factors on stimulating the colony formation of both erythroid and myeloid lineages (see Ref. [15] for a detailed review). In vitro studies have shown that SP has proliferative effects on bone marrow cells in culture, most likely because of increased production of hematopoietic growth factors [28]. Stimulation with the inflammatory cytokines IFNg, IL-1b and TNFa decreases SP and HK-1 mRNA expression, in

Review addition to NK1R receptor expression in the monocyte and macrophage cell lines Wehi-3 and RAW264.7 [29]. These events coincide with monocytic differentiation of the cells. Incubation of primary bone marrow macrophages with a cytokine cocktail (IFN-g, IL-1b and TNF-a) also resulted in decreased mRNA levels of SP and NK1R, further suggesting that this receptor-ligand pair is controlled by cytokine networks and might serve a developmental role in the innate immune system [29]. SP can also function as a proliferative signal for myeloid progenitors, however, the proliferating effect occurs at relatively high concentrations of SP (10 7 M or above). Because SP triggers a variety of biological effects mediated by the full length receptor at concentrations significantly lower than 10 7 M and SP has a lower affinity for the truncated NK1R [9], it is more likely that the truncated isoform rather than the full length receptor mediates the effect of SP on myelopoiesis. NK1R in monocytes and macrophages The detection of the truncated NK1R poses difficulties which are related to the identity of its primary structure with the full length receptor. The only difference between the two polypeptide sequences in the two receptors is the missing 96 amino acids in the C-terminus of the truncated receptor [30]. Using reverse transcriptase-polymerase chain reaction (RT–PCR) assays it is possible to distinguish between the two receptor isoforms by using primers designed based on the non-coding part of the gene [30]. However, immunodetection by western blotting, immunohistochemistry or fluorescence microscopy of the truncated NK1R poses considerable challenges. The full length receptor can be detected in a specific manner using antibodies raised against amino acid sequences present in the C-terminal domain, the detection of the truncated receptor can be accomplished only in an indirect manner because it is very difficult to develop antibodies that bind to the truncated NK1R, but not to the full length receptor. Using one antibody against the amino acid sequences in the Cterminus of full length NK1R and one antibody against a common sequence in the N-terminus of both isoforms, we have demonstrated that human monocytes and non-differentiated THP-1 cells express only the truncated NK1R isoform [31,32]. We have detected the truncated NK1R in the non-differentiated monocytic THP-1 cell line using RT–PCR, whereas both full length and truncated forms were detected in the THP-1 cells differentiated into macrophages using phorbol myristate acetate (PMA) [32]. The undifferentiated THP-1 cells stained positively only with an antibody raised against a sequence of amino acids at the N-terminus of NK1R, whereas the differentiated THP-1 cells stained positively with both anti-N-terminus and anti-C-terminus antibodies. Treatment with SP did not trigger cytosolic calcium increase in undifferentiated THP1 cells. By contrast, addition of SP to PMA-differentiated THP-1 cells led to increased cytosolic calcium concentrations and this effect was inhibited by specific NK1R antagonists. Truncated NK1R, however, potentiated CCL5-induced calcium increases in THP-1 differentiated cells, which is mediated by the chemokine receptor CCR5. We have concluded that the truncated NK1R is functional in THP cells, and has a positive modulatory

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effect on CCL5-induced calcium increases and that the Cterminal domain of full length NK1R is essential for SPinduced calcium mobilization [32]. More recently, we have demonstrated that the truncated NK1R is also expressed in primary human monocytes [31]. Similar to non-differentiated THP-1 cells, SP enhanced CCR5-mediated intracellular calcium increase induced by the chemokine CCL5. More importantly, SP treatment also enhanced CCL5induced chemotaxis of monocytes, and this effect was inhibited by the selective NK1R antagonist aprepitant [31]. Thus, the tail-less receptor has well defined functional roles in monocytes, modulation of chemokine-induced chemotaxis in monocytes being the first response to be characterized. The truncated NK1R mediates selective phosphorylation of extracellular signal-regulated kinase 2 (Erk2) in human monocytes and it enhances CCL5-induced phosphorylation of both Erk1 and Erk2 [31]. Activation of the truncated NK1R in human monocytes also resulted in increased phosphorylation of serine residues in CCR5. This observation demonstrates the cross-talk between CCR5 and truncated NK1R. Other functional responses and intracellular signaling mechanisms are triggered by the activation of the truncated NK1R in HEK293 cells stably expressing the truncated receptor. Treatment with SP of HEK293 cells expressing the truncated receptor results in Erk1/2 phosphorylation that starts after 15 min of incubation with SP, whereas HEK293 cells stably expressing the full length NK1R respond with Erk1/2 phosphorylation in less that 1 min after SP application [33]. This finding, in addition to the observation that the full length receptor mediates robust intracellular calcium increase whereas the truncated receptor does not signal through calcium, clearly demonstrate the two NK1R isoforms are both active and completely different in terms of their intracellular signaling mechanisms. A newly identified mediator of the effects of SP in monocytes and macrophages is the peroxisome proliferator-activated receptor (PPAR) gamma. Peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors that mediate multiple biological effects. Among the three PPAR isoforms known so far (PPARa, PPARb/d and PPARg), the PPARg is best characterized due to its therapeutic application for treatment of diabetes type 2 [34] and it is recognized for its roles in inflammation [35,36]. The production of several inflammatory cytokines (such as TNFa, IL-1b and IL-6), PPARg inducible production of nitric oxide and the expression of matrix metalloproteinase 9 (MMP9) and macrophage scavenger receptor 1 are inhibited by PPARg-specific ligands in various cell types, including monocytes, macrophages and epithelial cells [37,38]. PPARg is constitutively present in human monocytes and its expression is upregulated along with differentiation to monocyte-derived macrophages (MDM) [39]. SP stimulates the expression of PPARg in monocytes and MDM in a NK1R-dependent manner [39]. Furthermore, the finding that the PPARg antagonist GW9662 enhances SP-induced TNFa release from monocytes further supports the possibility that NK1R-mediated PPARg regulation is a novel mechanism 273

Review of modulation of macrophage function, which is induced by SP and might contribute to a range of chronic inflammatory disorders [39]. NK1R and HIV In a recent ex vivo study on natural killer cells collected from HIV-seropositive depressed and non-depressed women, the effects of CP-96345, a selective NK1R antagonist, citalopram, a selective serotonin reuptake inhibitor (SSRI), and RU486, a glucocorticoid antagonist, were compared. Natural killer cell cytolytic activity was significantly increased by CP-96345 and by citalopram, whereas RU486 showed no effect [27]. Thus, NK1R seems to have an important contribution in modulating the function of natural killer cells; however it remains to be determined whether HIV-related impairment in natural killer cytolytic activity is improved by treatment with SP antagonists in vivo. The potential use of SP antagonists and serotonergic agents for improving the defense mechanisms mediated by natural killer cells, delaying HIV disease progression and extending survival of patients with HIV infection is a novel and promising approach for developing new therapies in HIV disease [27]. There is increasing evidence that SP and NK1R are involved in modulating responses to viral infections [40]. We have shown elevated SP levels in plasma from men [41] and from women [42] infected with HIV and this finding might partially explain the neuropathic pain in HIV disease. Neurokinin-1 antagonists inhibit HIV infection of both MDM [43] and PBMC [44]. We have recently demon-

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strated that aprepitant suppresses infection with HIV-1 in MDM [45]. Treatment of MDM with aprepitant inhibited infection with the AZT-resistant viruses and suppressed infection of macrophages with reverse transcriptase inhibitor-resistant viruses. Furthermore, treatment with aprepitant enhanced the anti-HIV activity of several antiretrovirals, including AZT, efavirenz and indinavir in vitro [45]. The mechanism of action of aprepitant as an antiviral is dependent on the HIV co-receptor CCR5. Aprepitant inhibited CCR5 expression on human macrophages in vitro. Heterogeneity of responses was observed with macrophages obtained from different donors and the factors that contribute to this heterogeneity could be related to the relative expression of the full length and truncated NK1R isoforms in macrophages [45]. Aprepitant and several other NK1R antagonists had antiretroviral activity against infection with various HIV-1 subtypes, including CXCR4- and CCR5-tropic primary isolates propagated in PBMC. Thus, the antiviral effect of NK1R antagonists observed in PBMCs could also be related to decreased expression of the CXCR4 co-receptor, although other mechanisms are possible (e.g. modulation of the HIV1 long terminal repeat promoter [43]). Synergistic effects were observed for aprepitant with ritonavir [44], an antiretroviral drug from the protease inhibitor family. This observation is particularly interesting because ritonavir is a strong inhibitor of cytochrome P450–3A4 (CYP3A4), the enzyme which is primarily responsible for aprepitant metabolism. Thus, the combination of ritonavir-aprepitant might be very effective in

Figure 2. Potential interactions between full length and truncated NK1R receptors and the HIV co-receptor CCR5 in macrophages. Binding of SP to full length NK1R triggers activation of proteins from the Gq/11 family, whereas the truncated NK1R initiate intracellular responses through G proteins that are yet to be identified (Gx). Blockade of NK1Rs in macrophages using NK1R antagonists results in decreased expression of CCR5, which is a Gi-coupled receptor that functions as a HIV co-receptor. NK1R antagonists inhibit HIV replication in MDM and this finding is associated with reduced expression of CCR5. We hypothesize that NK1R antagonists might interfere with the synthesis or recycling of CCR5. Signaling mechanisms initiated by NK1R also lead to changes in chemokine and cytokine synthesis and release that might impact on neighboring cells (paracrine effects) or on the macrophage itself (autocrine effects). Recent data demonstrate that the truncated receptor has an important modulatory effect on chemokine-induced chemotaxis in monocytes.

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Review vivo because ritonavir has antiretroviral properties and in addition it boosts plasma levels of aprepitant. The antiviral effect of aprepitant might be highly beneficial in combination therapies in selected patients. It is not clear which NK1R isoform is involved in modulating viral replication in macrophages. The full length NK1R couples to Gq/11 proteins and mediates inositol phosphate turnover and intracellular calcium increase. However, SP does not cause intracellular calcium increase in macrophages, thus it is likely that the antiviral effect induced by NK1R antagonists are mediated by the truncated form of NK1R. Data from other laboratories also support the involvement of SP in viral replication. The genome of herpes simplex virus type-1 (HSV-1) is transcriptionally silent during latency periods, with the exception of a single region encoding the latency-associated transcript (LAT). Transfection of primary cultures of rat trigeminal neurons with plasmids encoding LAT, resulted in an increased percentage of SP-immunoreactive neurons. This finding indicates that one of the mechanisms that promote HSV-1 persistence might be related to SP production [46]. HSV type 2 (HSV-2) can induce a increase of SP in the genital tract of mice. Lack of SP signaling in NK1R-deficient mice revealed an important role for the tachykinin system in the innate defense against HSV-2 [47]. NK1R-deficient mice had enhanced levels of HSV-2 in the genital tract and in the CNS after infection and an accelerated disease progression, which was associated with an impaired natural killer cell activity [47]. These findings indicate an important role for SP and NK1R in modulating the infection with herpes viruses and the mechanisms that mediate the effect of SP on HSV latency and replication involve not only the immune system but also the neurons. Concluding remarks Many of the studies that investigated the biological roles of NK1R assumed that the full length receptor exclusively mediates responses induced by SP. However, there is now direct evidence that SP-induced modulation of important functional responses in monocytes, such as chemokineinduced chemotaxis, is mediated by its truncated splice variant (Figure 2). The intracellular signaling mechanisms triggered by the activation of the truncated NK1R are obviously distinct from those initiated by its full length counterpart. Existing data strongly support the view that the presence of the C-terminal domain has an impact on the folding of NK1R, and the lack of the C-terminus confers to the truncated receptor distinct pharmacologic properties, despite the identity between the primary structure of the truncated receptor with the first 311 amino acids in the sequence of the full length receptor. The truncated form of NK1R most likely mediates prolonged cellular responsiveness after stimulation, and is resistant to homologous desensitization in comparison to the full length NK1R isoform because the missing C-terminal domain is essential for receptor desensitization, internalization and recycling. The wide distribution of the truncated receptor, including cells from the nervous and the immune system, makes it an excellent candidate for drug development. Based on the existing evidence, it is likely that the differ-

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ences between the 3D structures of the two receptor isoforms can be exploited for the development of selective ligands, although this could turn out to be a challenging task. We anticipate that in the near future more efforts will be made for the development of NK1R ligands able to selectively target the tail-less NK1R and important developments in this field are expected in the future. Acknowledgements Supported by NIH P01-MH076388 and R01-MH049981 (S.D.D.) and by the Joseph Stokes, Jr. Research Institute Foerderer-Murray research award (F.T).

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