Neuropeptide Y (NPY), a polypeptide consisting of 36 amino acid residues, belongs to the gut neurohormone NPY family, which also includes peptide YY (PYY) and pancreatic polypeptide (PP).¹⁾ NPY was first isolated from the porcine brain in 1982 and was identified as a sympathetic cotransmitter. It contains highly evolutionarily-conserved peptides ubiquitously present throughout the body.^2,3) NPY has a 92% sequence homology between cartilaginous fish and extant mammals, suggesting that this high level of conservation of NPY must serve important physiological roles within the human body.³⁾ Activation of NPY is known to play an essential role in various physiological functions. It is involved in the pathogenesis of human diseases such as obesity, hypertension, metabolic disorders, heart failure, atherosclerosis, and cancers.¹⁾

The NPY family is involved in several biological functions in humans, including food intake, stress modulation, energy homeostasis, circadian rhythm, and anxiolytic properties.⁴⁾ These actions are mediated by one of its six G-protein coupled receptors (GPCR), termed Y1, Y2, Y3, Y4, Y5 and Y6.⁵⁾ Among them, NPY primarily binds to and activates the Y1 receptor (Y1R) and Y2 receptor (Y2R) in order to mediate antisecretory responses in the body.⁶⁾ Y1Rs are involved in regulating absorption of ion and fluid across the epithelial cell layer by being localized in the basolateral domain of crypt epithelia and epithelial cells in the colon.⁶⁾ On the other hands, Y2R has a more discrete distribution pattern in the mammalian enteric neuron system (ENS) and intestine compared to that of Y1R. And Y2R plays a major role in the antisecretory/proabsorptive effect of NPY.⁷⁾

Emerging evidence supports the distinctive role of NPY in immunity in the gastrointestinal (GI) tract by allowing crucial enteric neuron-immune crosstalk in the gut.⁸⁾ In the ENS, NPY levels are altered by several inflammatory diseases. Furthermore, immune cell cytokines can modulate NPY expression, establishing a feedback loop between enteric immune and ENS.⁹⁾

In this review, we focused on the role of NPY as an immune mediator in the gut, particularly in inflammatory bowel disease (IBD) pathogenesis. First, we summarize the localization of NPY and its receptors in the gut, including ENS, sympathetic neurons, intestinal mucosa, and submucosa. Secondly, we briefly introduce the IBD. Third, we discuss the alteration of NPY expression in the colon, beyond the brain, during both mouse and human colitis. Finally, we identify the specific role of NPY in intestinal inflammation by summarizing the supporting evidence that demonstrates blocking of NPY and its receptor signaling.

NPY peptide is expressed and located within the submucosal and myenteric plexus neurons throughout the colon and small intestine, providing a dense network of immunoreactive fibers within the lamina propria.¹⁰⁾ The submucosal and myenteric plexus are two main networks of ENS and play a role in the secretory and motor function of the GI tract through multiple neurotransmitters, such as NPY⁹⁾ (Fig. 1). In addition to enteric neurons, NPY is abundantly produced in postganglionic sympathetic neurons and is co-released with norepinephrine and adenosine triphosphate, which induces colonic relaxation⁷⁾ (Fig. 1). The localization of NPY in major ganglionic networks provides extensive intrinsic innervation of smooth muscle layers and mucosal targets.⁶⁾ Therefore, NPY is abundantly produced by submucosal neurons and released within the lamina propria of both crypts and villi.⁹⁾

Fig. 1. (A) The organization of the nervous system. NPY is widely expressed in sympathetic neurons and the enteric nervous system (ENS), which are both part of the autonomic nervous system (ANS). The myenteric plexus and submucosal plexus are the two main networks of ENS that express NPY. (B) Expression of NPY in the nervous system. In case of sympathetic nerves (orange), NPY is abundantly produced postganglionic axons and is co-released with norepinephrine. In the colon, NPY is mainly produced in two parts of the ENS. The submucosal plexus (green) is located within the submucosa and the myenteric plexus (red) is found between circular muscle and longitudinal muscle layers.

Y1R and Y2R are also found in the colon and small intestine with NPY and are localized to submucosal cell bodies and both submucosal and myenteric nerve terminals.¹⁰⁾ In addition, Y1R is located in a basolateral distribution in both crypt and surface epithelium, and submucosal cell bodies.¹⁰⁾ Analysis of the localized patterns of Y1R and Y2R using autoradiography showed that NPY receptors are widely distributed in the myenteric and submucosal plexus, smooth muscle, vessels, mucosa, and muscularis mucosae, but not in the lymphoid follicles, in the human colon.¹¹⁾ NPY signaling plays a pivotal role in regulating appetite and food intake and is highly associated with intestinal biology, such as gastric emptying, gastric and pancreatic secretion, and absorption of water and electrolytes.^5,6)

NPY is now considered a critical neuroimmune transmitter linked to the nervous and immune systems.¹⁾ NPY nerve fibers are in close contact with immune cells in the ileum lamina propria.¹²⁾

NPY is produced from T cells, macrophages and dendritic cells during inflammation.⁹⁾ Early studies have demonstrated that NPY affects the mucosal immune system by improving the proliferation of human colonic lamina propria lymphocytes. This effect partially depends on the production of IL-1β in monocytes.¹³⁾ NPY is also a paracrine or autocrine immune mediator produced and released by immune cells.^1,7) NPY modulates inflammation by regulating immune cell functions like neutrophil chemotaxis, T helper cell differentiation, suppression of lymphocyte proliferation and activation of antigen-presenting cell (APC).⁹⁾

After inflammation, neuropeptide levels are richly innervated in the ENS of both epithelial and smooth muscle cells. These inflammation-induced neuronal remodeling can impact intestinal epithelial permeability, electrolyte and water secretion, intestinal motility, and vascular tone by NPY-Y1R activation.⁹⁾ NPY and its receptors regulate epithelial secretory functions and contribute to proabsorptive effects by changes in intestinal permeability, through stimulation of ion exchange activity and cytokine production.⁹⁾ Moreover, chronic impairment of colonic relaxation was observed after DSS treatment in WT mice, and genetic deficiency of NPY resulted in a reduced relaxation response. This suggests that NPY is a major determinant of nitric oxide-mediated colonic relaxation.¹⁴⁾

IBD, including Crohn’s disease (CD) and ulcerative colitis (UC), is marked by long-term repetitions of relapse and remission.^15,16) The main symptoms of CD and UC are similar, including severe diarrhea, rectal bleeding, abdominal pain, loss of appetite, and weight loss.¹⁷⁾ IBD has increased considerably and is currently a common chronic inflammatory disease worldwide.¹⁸⁾ During the past two decades, an epidemiological shift has appeared, and the incidence of IBD has continued to increase in previously low incidence areas such as Asia and Eastern Europe. This phenomenon is closely related to socioeconomic status, such as urbanization, modernization of lifestyle, and accompanying changes in environmental factors.^19,20) Currently, South Korea has the second highest incidence and prevalence of IBD in East Asia. The total number of patients with CD and UC in 2010 were 7,700 and 16,136, respectively, which in 2019 were 18,463 (2.37-fold increase) and 37,439 (2.32-fold increase), respectively.²¹⁾

Despite several scientific studies on the mechanism of IBD development, the etiology and actual causes of IBD are not well understood. However, several studies suggest that IBD pathogenesis involves the combined effects of genetic susceptibility, dysregulation of host immune response, alteration of intestinal microbiota, and environmental influences such as cigarette smoking, hygiene, and western diet.^22,23) The primary treatments for IBD include anti-inflammatory drugs such as mesalazine and corticosteroids, immunosuppressants such as azathioprine and 6-mercaptopurine, and biological agents using the anti-tumor necrosis factor (TNF)-α such as infliximab, adalimumab, and natalizumab.²⁴⁾ However, these treatments limit the host immune system and affect nonspecific cells in the body, resulting in undesirable side effects such as local irritation, lymphoproliferative disorder, and opportunistic infections.¹⁸⁾ Therefore, specific molecular targets must be developed to overcome these limitations.

NPY is widely expressed in sympathetic neurons, a part of the autonomic nervous system (ANS) (Fig. 1A). ANS dysfunction may contribute to the pathogenesis of IBD as significantly lower sympathetic activity was observed in patients with IBD during clinical remission compared with that in healthy individuals.²⁵⁾ This suggests that NPY contributes to the immune system during colitis.

According to the literature, the expression levels of NPY and its receptors were altered by intestinal inflammation (Table 1). In dextran sulfate sodium (DSS)-induced colitis mouse model, NPY gene expression was found to be increased in the enteric ganglia and hypothalamus.^14,26) Trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats showed an increase in hypothalamic NPYergic activity and release of NPY from the paraventricular nucleus of the hypothalamus.²⁷⁾ In a recent study, both hypothalamic NPY mRNA and circulating NPY were significantly increased in mice with DSS-induced colitis.²⁸⁾ An increased expression of NPY in the hypothalamus due to colitis may reflect a counterregulatory response to colitis-evoked anorexia, as NPY is one of the most potent orexigenic peptides.²⁸⁾ In the hippocampus, Y1R mRNA levels are increased in colitis, although these do not alter NPY expression.²⁶⁾

Table 1

A summary of NPY, Y1R, and Y2R expression levels in murine colitis models

Colitis model (Animal)	Detection	Alteration	Location	References
TNBS (rat)	NPY protein	Increased	Hypothalamus	27
DSS (mouse)	NPY protein	Increased	Enteric nerves from colon	32
DSS & S.T. infection (mouse)	NPY mRNA, protein	Increased	Enteric nerves from colon	14
DSS (rat)	NPY protein	Increased	Colon tissue	31
DSS (mouse)	NPY protein	Increased	Colon tissue	29, 30, 33
TNBS (mouse)	NPY protein	Increased	Colon tissue	29
DSS (mouse)	NPY mRNA	Increased	Hypothalamus	26, 28
DSS (mouse)	NPY protein	Increased	Blood plasma	28
DSS & T cell transfer (mouse)	NPY mRNA	Increased	Colon tissue	8
DSS (mouse)	Y1R mRNA, protein	Decreased	Colon tissue	38
DSS (mouse)	Y1R mRNA	Increased	Hippocampus	26
DSS (mouse)	Y1R and Y2R mRNA	Decreased	Colon tissue	8

In the colon tissues, beyond the brain, the concentration of NPY protein was significantly increased in both DSS- and TNBS-induced colitis mouse model.^14,29,30) A significant increase in NPY expression was also observed in rat colon tissues due to DSS-induced colitis.³¹⁾ NPY-positive nerve fibers in the myenteric and submucosa plexus increased at the site of damaged crypts in the inflamed mouse colon induced by DSS.³²⁾ The expression of NPY in enteric ganglion was increased in both DSS- and Salmonella typhimurium (S.T.)-induced colitis.¹⁴⁾ Dysfunction of enteroendocrine cells in the intestinal crypt showed increased susceptibility to DSS-induced colitis and is associated with reduced intestinal protein levels of NPY in the colon tissue.³³⁾

In human cases, the density of NPY immunoreactive nerve fibers was higher in the myenteric ganglia of the ileum from patients with CD.³⁴⁾ Another clinical study showed that plasma levels of NPY were significantly higher in patients with CD and UC compared to those in the healthy controls.^35,36) In contrast, another study indicated that NPY was slightly lower in plasma from patients with CD and UC than in controls, although the difference was not statistically significant.³⁷⁾ In addition, the levels of NPY in patients with IBD were similar in remission or relapse, irrespective of the disease activity.³⁵⁾ These results suggest that higher NPY expression during intestinal inflammation may have pro-inflammatory functions. However, there are limitations to these clinical studies as there is a lack of validation cohort due to the small number of patients. Further studies are needed to provide more precise information about the alterations and roles of NPY expression during IBD.

On the other hand, the expression of NPY receptors showed the opposite pattern. The loss of Y1R mRNA and protein has been observed in the colonic epithelium in DSS treatment.³⁸⁾ A more recent study also showed decreased expressions of both Y1R and Y2R DSS treatments.⁸⁾ This suggests an inherent compensatory mechanism of receptor desensitization in response to inflammation-induced NPY up-regulation from enteric neurons.⁸⁾

In summary, the NPY expression increased consistently in the brain, ENS, blood and colon tissues, while Y1R increased in the brain but decreased in colon tissues during colitis (Fig. 2).

Fig. 2. The alteration of the expression of NPY and its receptors during intestinal inflammation. In the brain, NPY expression increases in the hypothalamus and Y1R expression increases in the hippocampus during colitis. Plasma NPY levels from total blood also increa se in both animal and clinical studies. NPY expression also consistently increases in colon tissues and the ENS. In some clinical studies, Y1R and Y2R expression shows a decreasing pattern in the colon.

As mentioned in the previous section, there is an increase in NPY production during colitis. In this section, we discuss whether NPY signaling is dominant during the pro-inflammatory response in colitis (Fig. 3).

Fig. 3. A schematic representation of the immunomodulatory functions of NPY signaling. Since NPY has a pro-inflammatory role in colitis, blocking NPY and Y1R signaling has protective effects by reducing intestinal inflammation in experimental colitis mode ls.

First, genetic deletion and reduction of the NPY gene attenuate the severity of intestinal inflammation in mice. In DSS-induced colitis mouse model, body weight loss, disease activity, histological deterioration, and the colonic accumulation of myeloperoxidase (MPO) were alleviated in NPY knockout (KO) mice as compared to those in NPY wild type (WT) mice.^14,39) Similarly, NPY KO mice showed significantly reduced inflammation during S.T. infection.¹⁴⁾ In addition, administering NPY antisense oligodeoxy-nucleotides (ODNs) to rats with DSS-induced colitis showed significant improvements in disease activity index and MPO levels and the histological score.³¹⁾

Second, blocking of Y1R can also be used to treat colitis, as Y1R is widely expressed in immune cells and promotes inflammatory signaling.⁹⁾ The severity of DSS-induced colitis is also attenuated by the genetically deficient NPY Y1 receptor, as well as the antagonism of the Y1 receptor.^8,32) Localized intraluminal administration of the Y1R antagonist BIBP-3222 attenuates intestinal inflammation in the T-cell transfer colitis model.⁸⁾ Another Y1R antagonist, H409/22, reduces colonic ulcerations and the inflammatory response in DSS-induced mouse colitis.³²⁾ In another study that used DSS-induced mouse colitis, systemic NPY signaling inhibited Y1 or Y2 antagonists (BIBP 3226 and BIIE 0246, respectively) slightly but significantly and slowed weight loss progression. However, it did not show other pathological improvements, such as colon length, histology, and number of goblet cells.³⁰⁾ Blocking Y1R signaling by ex vivo treatment with BIBP-3222 significantly reduced TNF-a and interferon (IFN)-g expression in UC biopsy supernatants.⁸⁾

Increasing oxidative stress and inflammatory cytokine production subsequently exacerbate damage in colitis. The levels of plasma IL-6, IL-18, and TNF-α were significantly increased by DSS treatment.^26,28) Additionally, both serum and colonic IL-6 concentrations increased, and serum IL-10 concentration decreased after TNBS injection in mice.²⁹⁾ Colitis-induced NPY can lead to the production of these cytokines by activation of immune cells such as macrophages, neutrophils, and lymphocytes.⁹⁾ Early studies showed that exogenous NPY affects the mucosal immune system by enhancing the proliferation of lamina propria lymphocytes by promoting IL-1β production in monocytes.¹³⁾ In a recent study, exogenous NPY treatment induced nitric oxide production and TNF-α release in enteric neuronal cultures.¹⁴⁾ NPY1R inhibition showed protective effects in colitis by T cell transfer mouse model due to significant inhibition of IL-6, IL-12, and TNF-α.⁸⁾ Administering NPY antisense ODNs also reveals a reduction in TNF-α and phosphorylation of NF-κB and Akt in rats with DSS-induced colitis, without any alteration in the NPY levels.³¹⁾

Interestingly, NPY acts as an activator of APC function, and Y1R expression on APC is essential to produce Th1-promoting cytokines, such as IFN-γ, and present antigens to T cells.^7,40) Systemic deletion of Y1R shows significantly reduced IFN-γ levels in serum during DSS-induced inflammation.⁴¹⁾ However, in contrast, T cells isolated from Y1R KO mice are capable of differentiating into active IFN-γ-producing Th1 T cells and become hyperreactive to DSS, showing severe colitis upon transfer to lymphopenic mice.⁴¹⁾ These results showed a novel bimodal role for NPY and Y1R. Y1R signaling acts as a key activator of APC function and is important for T cell differentiation, while NPY signaling via Y1R also functions as a negative regulator of T cells. This suggests a negative feedback loop to prevent sustained or hyperactivation of the T cell response.40,⁴¹⁾

As a ubiquitous neurotransmitter in both the central nervous system as well as outside the nervous system, NPY exerts its effects on adjacent tissue and the regulatory system in our whole body.³⁾ Recently, NPY has been implicated as an immune regulator in various inflammatory diseases. In the case of intestinal inflammation, up-regulated expressions of NPY are consistently observed in various murine colitis models. Furthermore, inhibition of NPY signaling by directly targeting the NPY gene or inhibiting activation of the NPY receptor indirectly significantly reduces the development of acute colitis and attenuates the progression of symptoms. Consequently, we speculate that modulation of NPY signaling serves as a novel molecular target to treat IBD. However, the expression levels of NPY in clinical studies are uncertain, and the specific molecular mechanisms of the pro-inflammatory activities of NPY in IBD are poorly understood. More research is required to develop specific and effective therapies without adverse side effects.

This work was supported by a 2-Year Research Grant of Pusan National University.

All authors declare that they have no conflict of interest.