Neuro-immune-metabolism: The tripod system of homeostasis

Abstract

Homeostatic regulation of cellular and molecular processes is essential for the efficient physiological functioning of body organs. It requires an intricate balance of several networks throughout the body, most notable being the nervous, immune and metabolic systems. Several studies have reported the interactions between neuro-immune, immune-metabolic and neuro-metabolic pathways. Current review aims to integrate the information and show that neuro, immune and metabolic systems form the triumvirate of homeostasis. It focuses on the cellular and molecular interactions occurring in the extremities and intestine, which are innervated by the peripheral nervous system and for the intestine in particular the enteric nervous system. While the interdependence of neuro-immune-metabolic pathways provides a fallback mechanism in case of disruption of homeostasis, in chronic pathologies of continued disequilibrium, the collapse of one system spreads to the other interacting networks as well. Current review illustrates this domino-effect using diabetes as the main example. Together, this review attempts to provide a holistic picture of the integrated network of neuro-immune-metabolism and attempts to broaden the outlook when devising a scientific study or a treatment strategy.

Abbreviations

AGE

advanced glycated end-products

AhR

aryl hydrocarbon receptor

Akt/PKB

protein kinase B

ATP

adenosine triphosphate

BCR

B cell receptor

BMP2

bone morphogenic protein 2

CCR

CC chemokine receptor

CD

cluster of differentiation

CGRP

calcitonin gene related peptide

CNS

central nervous system

CSF1

colony stimulating factor 1

DC

dendritic cell

DRG

dorsal root ganglia

ENS

enteric nervous system

FFAR3

free fatty acid receptor 3

Gal

galanin

GLUT

glucose transporter

GLP-1

glucagon like peptide-1

Hbalc

Hemoglobin A1C

HFD

high fat diet

HIF1 α

hypoxia-inducible factor 1α

IBD

inflammatory bowel disease

IFN

interferon

ILC

innate lymphoid cell

IL

interleukin

LDL

low density lipoprotein

LOX1

oxidised low density lipoprotein (lectin-like) receptor 1

MC4R

melanocortin receptor 4

MOR

mu opioid receptor

mTOR

mechanistic target of rapamycin

NADPH

nicotinamide adenine dinucleotide phosphate

NF-kB

nuclear factor ‘kappa-light-chain-enhancer’ of activated B cells

NGF

nerve growth factor

NMU

neuromedin U

NMUR

neuromedin U receptor

nNOS

neuronal nitric oxide synthase

NK

natural killer

NK1R

neurokinin 1 receptor

NPY

neuropeptide Y

OxPhos

oxidative phosphorylation

PENK

proenkephalin

PI3K

phosphatidylinositol 3 kinase

PNS

peripheral nervous system

POMC

proopiomelanocortin

P2X3

P2X purinoceptor 3

RAGE

receptor for advanced glycated endproducts

RAMPI

receptor activity modifying protein 1

ROS

reactive oxygen species

SP

substance P

TCA

tricarboxylic acid pathway

TNF-α

umor necrosis factor a

TLR

toll like receptor

TrkA

tropomyosin receptor kinase A

TRPV1

transient receptor potential cation channel subfamily V member 1

VEGF

vascular endothelial growth factor

VIP

vasoactive intestinal peptide

VIPR

vasoactive intestinal peptide receptor

1. Introduction

Homeostasis is a state of being, wherein an evenness is maintained between multiple intercellular processes, necessary for the proper physiological functioning of body organs. This state of equilibrium is preserved despite constant environmental fluctuations. Maintaining homeostasis requires sensing of the environmental cues, dynamic adaptation of the cellular and molecular processes and effective communication between multiple organs. One such system, which can perform the above-mentioned functions, is the nervous system.

Broadly, the nervous system may be classified as the central nervous system (CNS; which includes the brain and spinal cord) and the peripheral nervous system (PNS; which includes the peripheral nerves that innervate the rest of the body) [1]. One of the main functions of the PNS is to integrate the sensory information from the peripheral organs and relay it to the CNS. Under normal physiological condition, there is a basal level of neuronal excitation occurring due to usual sensory triggers (e.g. walking on ground) and also a counteracting inhibitory signaling to terminate the excitatory signaling. Thus, the nervous system maintains a sensory homeostasis by thresholding the innocuous sensory triggers, so that they are not perceived as painful stimuli [2].

Apart from the sensory triggers, body is also continually challenged by pathogenic and non-pathogenic mircobes, especially at the mucosal surfaces and the intestinal barrier. Here, it is the immune system, which maintains the balance between mounting an appropriate response to eliminate the microbes without causing autoimmunity [[3],[4],[5]].

The efficient functioning of both neuronal and immune pathways demands energy input. The neurons, among all cell types in the body, are the highest consumers of energy [6]. The neurons consume 60% more energy (of basal energy expenditure of the body) to resolve a situation of acute pain and take precedence to claim energy resources of the body [7]. Similarly, the immune system requires energy for house-keeping functions and 25–30% more energy (of basal energy expenditure of the body) in acute infections, thereby ranking first in hierarchy for energy availability in such emergency situations [[8],[9]]. The nervous and immune systems are therefore annotated as ‘selfish systems’ when it comes to sharing energy resources to overcome external threats [10]. This indicates that it is extremely necessary for the metabolic pathways to function with utmost efficiency to be able to provide the required nutrients to the systems on one hand and not deprive other cells of the required metabolic substrates. This is achieved by maintaining a metabolic homeostasis.

The question then arises, whether these three above-mentioned systems can carry out their homeostatic functions individually. There is ample literature, which shows close interactions between neuroimmune, neuro-metabolic and immune-metabolic pathways. This review attempts to consolidate the information to present a holistic overview of the cellular interactions regulating neuro-immune-metabolic system. It further shows the functional interdependence of the individual components of this closely integrated system. It focuses on the molecular mechanisms occurring in the periphery i.e. outside the brain, since brain is comparatively an immunologically privileged site (Fig.1). Lastly, using diabetes as the main example, the review shows that collapse of one of the components spreads to the other two branches like a domino effect, creating disruption in nervous, immune and metabolic homeostasis (Fig.2).

Fig. 1. Neuro-immune-metabolic interactions during homeostasis.

The upper panel shows interactions in the intestine. Glucose is used as a metabolic substrate by all neurons and immune cells Th2 cells, Th17 cells, B cells and ILC3. The M2 macrophages, ILC2, naïve T cells and also B cells utilize fatty oxidation pathway to derive energy. The naïve T cells derive energy from amino acid metabolism as well. The immune cells secrete factors, which affect the enteric neuronal functions. For e.g., IL-10 and IL-4 bind to their cognate receptors on neurons to prevent inflammatory signaling. Neuronal function is also affected availability of metabolic substrates. They in turn regulate the glycolysis via the neuropeptides POMC, GLP-1 and Gal, whereas norepinephrine is required for efficient lipolysis via fatty acid oxidation. Together these interactions are required for physiological functions in the gut namely gut motility, mucosal barrier integrity and nutrient absorption. The lower panel depicts interactions occurring in the extremities. The legs, for e.g., are innervated by the sensory neurons broadly classified as CGRP⁺, NF200⁺ or IB4 binding. A sensory stimulus triggers cellular interactions aimed to restore homeostasis. The neurons release the neuropeptides CGRP and SP, which recruit macrophages, mast cells, neutrophils and DCs. The inflammatory factors such as TNF-α, histamines may cause proinflammatory signaling and hyperexcitability of neurons. However, antiinflammatory cytokines released by the immune cells (IL-10) and inhibitory neuropeptides released by the neurons (POMC and GABA) deactivate the immune cells and also prevent neuronal hyperexcitability and ensuing pain. The immune cells and neurons utilize glycolysis to perform these functions.

Fig. 2. Dysfunction neuro-immune-metabolic interactions during diabetes.

Upper panel shows dysfunction in the intestine. Hyperglycaemia, dyslipidemia and oxidative stress together causes increased proliferation and activation of M1 macrophages, B cells, Th1 cells, CD8⁺ T cells, DC and mast cell degranulation. The proinflammatory mediators released by these immune cells (IL-1ß, IL-6, TNF-α, IFN-γ) cause chronic neuronal excitation. Elevated glucose and fatty acid levels are also responsible for neuronal excitation. This leads to reduced levels of norepinephrine and increased levels of SP and CGRP. Dysbalance in neurotransmitters is unable to regulate metabolic pathways, further contributing metabolic distress. For instance, inefficient fatty acid oxidation and increased lipid storage can be attributed to decreased norepinephrine levels. Together this results in heightened inflammation, compromised mucosal barrier integrity, uncontrolled glucose levels and leads to abdominal pain and altered gut motility. Lower panel shows dysfunction in the extremities. Hyperglycaemia causes neuronal activation and chronic release of excitatory neurotransmitters (CGRP and SP). These recruit the immune cells, which also have a proinflammatory phenotype due the dysregulated metabolite levels. The immune cells contribute to inflammation and neuronal hyperexcitability by releasing proinflammatory mediators (IL-1ß, Il-6, TNF-α, IFN-γ). The decreased levels of inhibitory neuropeptides (POMC) is unable to terminate the excitatory transmission leading to excitotoxicity, hypersensitivity and eventual neuronal degeneration.

2. Immuno-metabolic regulation

2.1. Interdependence of immune responses and metabolism

Metabolism is the process by which, nutrients are made available to the cells of the body for growth and survival. The process includes the anabolic reactions, wherein nutrients are synthesized, and catabolic reactions, which break down the ingested food ingredients. The major metabolic pathways utilized by all mammalian cells are: aerobic glycolysis, tricarboxylic acid (TCA) cycle, anaerobic glycolysis (or the lactic acid pathway), pentose phosphate pathway, fatty acid oxidation and amino acid metabolism (which constitute the catabolic pathways) and glycogenic pathway, de novo fatty acid synthesis and de novo amino acid synthesis (which constitute the anabolic pathways).

In multicellular organisms, that metabolic pathways and immune responses can influence each other is now well accepted. However, the nature of their interaction has eluded scientists for many years. One of the reasons is that, inherently, the metabolic pathways function to conserve energy, whilst the immune responses against foreign invasions are energy demanding. Yet, the sharing of the energy resources among the immune and metabolic processes highlights the interdependence between the two systems [10].

This is particularly observed at the interfaces, such as the small intestine, which is constantly exposed to microbes including the commensal microbiota, as well as, the ones ingested via food, some of which may be pathogenic [11]. Therefore, the various immune cells deployed in the gut must exhibit diverse functions and work symbiotically at the same time. These immune cells show tolerance to the gut microbes to maintain immunological homeostasis, whilst mounting an immune response to the harmful pathogenic attack. Which immune cell will exhibit what function is governed not only by the type of microbe encountered, but also by the energy metabolism of that cell. This is evinced by the fact that the rate of aerobic glycolysis and TCA cycle are different in quiescent and activated immune cells [12].

The ingested nutrients or nutrient availability in tissues control the metabolic pathways within the body, which in turn regulate immune responses. Conversely, cellular stress driven by metabolic perturbation due to deficient or inappropriate nutrient intake may manifest as an inflammatory response, putatively aimed at restoring homeostasis by adjusting broader biological functions, including endocrine and metabolic processes [[13],[12]]. This nutrient-dependent interaction of metabolism and immune system, regulating immune functions is discussed below.

2.1.1. Metabolic regulation of immune cells

Diet modulates immune functions in multiple ways and determines the type of immune response. In the intestines, the nutrients acquired by the consumption of cruciferous vegetables and associated chemicals (indole-3-carbinol) get converted to aryl hydrocarbon receptor (AhR) ligands [14]. AhR ligands bind to the AhR receptors expressed highly by Th17 cells [15] the intestinal intraepithelial lymphocytes (IELs) [16], ILC2s [17] and ILC3s [[14],[18],[19]]. It has been shown that AhR ligands stimulate the production of IL-22 from Th17 cells IELs [[20],[15]] and ILC3s [19]. Unlike other cytokines, IL-22 is mainly acting on non-hematopoietic cells, such as epithelial cells in the intestine, where the IL-22 receptor is expressed. In epithelial cells, IL-22 induces production of antimicrobial peptides, such as Reg3β and Reg3γ and promotes barrier immunity against bacterial and viral infections, such as enteropathogenic Escherichia coli (Citrobacter rondentium) or Rotavirus infections and containment of the commensal microbiota [[21],[22], [5],[23]].

Another important metabolite regulating IL-22 production from ILC3s and also CD4⁺ T cells in the gut are the short-chain fatty acids (SCFA), metabolized by the gut microbiota [[24],[25]]. The SCFA induce IL-22 production by ILC3s and CD4⁺ T cells by inhibition of histone deacetylase and GPR41. IL-22 production is also upregulated via SCFA-mediated interaction between AhR and HIF1α [[26],[27]].

While promoting IL-22 production from ILC3 [[14],[18],[19]]., Ahr ligands were shown to inhibit ILC2 activation and might therefore regulate type 2 versus type 3 immunity [17]. Similar findings were reported for Vitamin A, which suppresses ILC2 and stimulates ILC3 both expressing Vitamin A receptor [28]. Further, Vitamin A was shown to promote the formation of secondary lymphoid organs [29]. Vice versa, SCFA suppressed ILC2s by modulating GATA-3 in the context of airway hyperreactivity [30]. Thus, availability of metabolites can influence immune response at the mucosal barrier.

The availability of nutrients acquired from diet dictates the intracellular metabolic pathway and therefore regulates the immune response at the mucosal barrier. In resting condition, the ILC3s rely on specific metabolic program controlled by mechanistic target of rapa-mycin (mTOR). mTOR, a member of the phosphatidylinositol-3 kinase (PI3K) related kinase family, is a serine/threonine kinase. mTOR constitutes the catalytic subunit of two distinct complexes, mTORC1 and mTORC2. mTORC1 is the key link between the availability of nutrients in the environment and the control of most anabolic and catabolic processes, whereas mTORC2 governs cytoskeletal behavior and activates several pro-survival pathways [31]. The ILC3s show an mTORC1-HIF1α (hypoxia-inducible factor 1α), [32], which then triggers glycolytic pathway and TCA cycle to synthesize ATP [33]. However, during an inflammatory challenge e.g. by bacteria, the ILC3s are activated and proliferate. While the anabolic mTOR-HIF1α pathway is required to sustain ILC3 numbers [34], the activated ILC3s also markedly increase mitochondrial respiration and generation of ROS, which together with aerobic glycolysis aides ILC3 proliferation and their effector functions to control Citrobacter rodentium infections [32]. Similar to ILC3s, ILC1s and NK cells trigger mTOR signaling, in addition to increased aerobic glyocolysis (ATP synthesis in the presence of oxygen) and the lactic acid pathway (wherein ATP is synthesized from the glycolytic end product, pyruvate, in absence of oxygen), to meet with the high-energy demand required during activation and proliferation. ILC2s, on the other hand, upon stimulation with IL-33, increase glycolysis and arginase catabolism to provide byproducts that further fuel glycolysis [[35],[36]]. Lack of arginine severely affects ILC2 proliferation and cytokine secretion. Fatty acid oxidation is also necessary for ILC2s for the production of IL-13 and IL-5 [[37],[38]]. Upon activation, the ILC2s increase lipid uptake and fatty acid oxidation, which promotes ILC2 proliferation. However, this lipid metabolism required by the ILC2s is governed by glucose availability and therefore, scarcity of glucose prevents ILC2 activation by impairing fatty acid metabolism [39]. Arginase catabolism is not thought to be required by ILC3s to fuel glycolysis when activated [36]. Thus, the ILCs utilize multiple metabolic pathways including aerobic and anaerobic glycolysis, fatty acid oxidation and amino acid catabolism to generate energy. Upon activation, ILC metabolism is adapted to the cellular needs and can trigger precise immune responses necessary to overcome external challenges such as pathogen invasion.

The naive T cells exhibit low metabolic activity and utilize fatty acid oxidation as an energy source [12]. Effector T cells (Th1, Th2, Th17) require increased glycolysis to rapidly produce ATP in order to be able to differentiate and proliferate [40]. This is achieved by upregulating GLUT1 transporter to increase glucose uptake and followed by activation of mTOR pathway, which further aides glycolysis via HIF1α [41]. mTOR also activates fatty acid oxidation and promotes T cell expansion and proliferation [42]. T cell proliferation also requires glutamine provided by the catabolism of proteins consumed. Glutamine elevates mitochondrial activity, ROS production by the activated T cells [43]. Another TCA cycle enzyme AAC1 mediates synthesis of fatty acids and is required for the development of Th17 cells [44]. The T cells also express receptors for vitamin D and retinoic acid. The catabolism of vitamin D produces 1, 25(OH)2D3 (active form of vitamin D), which then engages the two receptors to eventually regulate differentiation of the intestinal lymphocytes [45] and tight-junction stabilization [46]. The deficiency of vitamin D results in decreased cell numbers of the intestinal lymphocytes [47]. Similar to the T cells, B cells also demonstrate an increased glucose uptake and glycolysis when activated. Inhibition of glycolysis blocks B cells activation via B cell receptor (BCR) mediated antibody production [48]. B cells are also activated by IL-4, which mediates B cell survival by regulating glucose metabolism [49]. B cells proliferation and differentiation is enabled by fatty acid synthesis and production of acetyl-co-A from mitochondria-derived citrate [50]. ROS production by mitochondria also supports BCR-mediated proliferation and regulates B cell functions [51]. In summary, the T cells derive energy from intracellular fatty acid oxidation in resting state, whereas, activation of lymphocytes results in increased glucose, fatty acids and amino acids catabolism to provide for increased energy demand.

Alike the lymphocytes, myeloid cells adapt their metabolic pathways in response to pathogen invasion and also nutrient availability. The metabolic changes and requirements of macrophages have been well-characterized. The resting macrophages differentiate into proinflammatory M1 macrophages or anti-inflammatory M2 macrophages. Consumption of high saturated fatty acids triggers TLR4 signaling, similar to LPS stimulation, and induces differentiation of resting macrophages to the proinflammatory M1 phenotype [52]. The M1 macrophages exhibit elevated glycolysis and reduced mitochondrial oxidation pathway. The following TCA cycle is so modulated to produce high levels of citrate and succinate. Citrate promotes NO production via fatty acid synthesis pathways and succinate stimulates IL-1ß production [53]. The differentiation to M2 phenotype is triggered by omega-3-polyunsaturated lipids [54]. The M2 macrophages high levels of fatty acid oxidation and mitochondrial oxidation pathways [55]. M2 macrophages also require glutamine and therefore glutamine deprivation decreases M2 polarization [56]. Thus, inflammatory M1 macrophages mainly utilize glycolysis and anti-inflammatory M2 macrophages rely on fatty acid oxidation pathways.

Similar to the other immune cell types discussed above, the metabolic requirements of dendritic cells (DC) are different depending on cell stage and its activation status. GM-CSF and IL-4 stimulation results in diffrentiation of DCs from their progenitors [57]. This stimulation triggers increased mitochondrial biogenesis (increased ROS production and TCA cycle) [58], mTOR pathway [59] and fatty acid synthesis pathway [60], which are necessary for DC differentiation. Under normal physiological conditions, the differentiated DC stay quiescent (i.e. immature DCs) are less secretory and do not interact much with T cells. Immature DCs rely on fatty acid oxidation to fuel mitochondrial OxPhoS [61]. Following stimulation with TLR agonists, the DCs are activated and now use glycolysis are a primary energy-producing pathway [61]. To fulfill the excess ATP necessity, activated DCs also utilize anaerobic glycolysis [62]. The excess glycolytic intermediates are routed to the fatty acid synthesis pathways of the activated DCs [62]. Activated DCs can release IL-18 and IL-1ß [53]. At later stages after activation, DCs remain glycolytic, but turn off their OxPhos pathway for continued survival [63]. Furthermore, the DC metabolism and their activation status can be directly modulated by exogenous dietary metabolites. For instance, fatty acid rich food producing oxysterols can activated DCs and increase proinflammatory gene expression. On the other hand, excess exogenous oxidized free fatty acids induce tolerogenic DCs and decrease proin-flammatory gene expression. Tolerogenic DCs are also induced by Vitamin A and D. Lastly, the SCFA metabolized by the commensal microbiota are also sensed by the DCs. The SCFA are required to maintain the DCs in steady state (secreting IFN-I and IL-10) and decrease DC differentiation. The immature DCs are therefore programmed to a metabolic poised state, which allows them to rapidly respond to environmental triggers when required [64].

In summary, immune cells are poised to respond to metabolic milieu and availability of excess metabolites are associated with immune system activation. Hence, it follows that controlling diet may inhibit immune responses. This is exemplified by a study that showed that frequent water-only diet caused apoptosis of B cells in the Peyer’s patches. The B cell numbers could be restored by refeeding, however the cellular composition was drastically altered. Such fasting regime also led to failed antigen-induced IgA production and eventually exacerbated food antigen-induced diarrhea upon oral ovalbumin application [65]. Water only diet also reduced monocyte mobilization necessary during acute infection and wound healing [66]. Yet another study which applied a 50% calorie restriction instead of water-only fasting, an enhanced protection against infection and tumors was reported [67]. Calorie-restricted diet resulted in routing of the memory T cells to the bone marrow, promoted their survival in glucocorticoid-dependent pathway and enhanced their protective effects. Another study showed that short-term fasting in patients with multiple sclerosis (MS) or rheumatoid arthritis, showed significantly reduced numbers of circulating inflammatory monocytes derived from bone marrow. This mobilization of monocytes was not compromised in acute inflammatory conditions requiring monocyte mobilization, such as during a response to tissue injury [66]. Together these studies indicate that while complete lack of nutrients may cause some immune impairment, restricted diet holds the immune system in an energy-conserve mode and therefore retains the ability to launch an immune response when necessary. These data give valuable insight into how chronic pathologies involving autoimmunity or unchecked immune response can be manipulated simply by dietary interventions. This is underlined by the findings that periodic fasting in mouse models of MS, could halt the progression the disease and also resulted in regeneration of nerve axons, as well as their remyelination [68].

Taken together, nutritional signals are critical cues that guide immune cell numbers, their activation status and ultimately required for maintaining metabolic-immune homeostasis.

2.1.2. Immune regulation of metabolism

The way nutrient availability and metabolic pathways can regulate immune responses, there are also reports of immune participation to maintain metabolic homeostasis. Most studies on this topic have been conducted in adipose tissue, since it is a central hub for metabolic homeostasis, regulating insulin-dependent glucose uptake and long-term regulation of body weight via leptin secretion.

In steady state condition, Tregs function to keep inflammation in check. Depletion of Tregs not only leads to activation of Th17 and CD4⁺ T cells, but also increase in TNF-α [69]. TNF-α is known to antagonize insulin receptors and a key mediator in the development of insulin resistance [70]. Consequently, Treg ablation is shown to impair insulin receptor signaling and resultant increased insulin levels in adipose tissue [71]. This indicates the importance of Treg cells in maintaining glucose uptake and metabolic homeostasis even in the absence of environmental stressors. Moreover, it has also been shown that Foxp3⁺ Treg cells are enriched in the adipose tissue of lean, but not obese mice, and show a unique TCR repertoire [[71],[72]]. Metabolic homeostasis maintenance by Treg cells is influenced by Th17 and γδT cells, which regulate IL-33 secretion from stromal cells via IL-17 [73]. The cytokine IL-33 is produced by stromal cells in adipose tissue and is tightly linked to the regulation of metabolic hemostasis via different cell types including Tregs and ILC2s [[74],[75],[76],[77]]. IL-33 induces Treg proliferation, and therefore is indirectly involved in metabolic homeostasis regulation [75]. Th2 cell subset has also been shown to contribute to metabolic homeostasis in the adipose tissue. A study showed negative correlation between Th2 cell numbers and insulin resistance in humans [78], whereas another study demonstrated that after adoptive transfer into obese Rag1-null mice, CD4⁺ T cells gained a Th2 profile, which was associated with reversal of enhanced weight gain and insulin resistance [79]. Additionally, a recent study demonstrated that a direct role for intestinal T cell in nutrient sensing and modulating tissue metabolism. In response to increased dietary carbohydrate intake (i.e. high fat diet), intestinal γδ T cells showed alterations in their transcriptome, tissue localization, and behavior. The resulting inhibition IL-22 released by ILC3s regulated carbohydrate metabolism controlled by gut epithelium [80]. In addition to the ability of the T cells to influence glucose meta-bolism as described above, the study by Sullivan et al. underlines the role for T cells in mediating a balance between nutrient availability for immune cells and for smooth functioning of normal metabolic pathways in gut-immune homeostasis.

Cytokines produced by the T cells and ILC2s (IL-4, IL-5 and IL-13) play important role in M2 polarization of macrophages [[81],[82]]. The importance of M2 macrophages have been implicated in metabolic homeostasis, since depletion of CD8⁺ T cells followed by decrease in M1 macrophages improves insulin sensitivity in the adipose tissue. The M1 macrophages and proinflammatory cytokines secreted by them, namely TNF-α, aggravates insulin resistance [83].

The protective type-2 cytokines are also secreted by another immune cell subset in the adipose tissue-NKT cells. The NKT cells produce IL-4, IL-5 and IL-13 and lower levels of IFN-γ [84], thereby driving a M2 macrophage phenotype and contributing to metabolic homeostasis. Depletion of NKT cells in obese mice exacerbates weight gain, fatty liver, and insulin resistance, whereas activating NKT cells by lipid ligand α-galactocylceramidee (αGalCer) in obese wild-type mice reverses HFD-induced phenotypes [[85],[86]], indicating their role in glucose and lipid metabolism [87].

Several studies have demonstrated the role of ILCs in regulation of metabolic homeostasis. ILC2s are an important source of IL-5 and IL-13 in adipose tissue and pancreas. Pancreatic insulin secretion has been shown to be promoted by the IL-33-mediated activation of ILC2s [[88], [37]]. ILC2s promote accumulation of eosinophils via IL-4 signaling and alternatively activated macrophages, both of which have been implicated in metabolic homeostasis [[74],[89],[90],[37],[91]]. Conversely, ILC1s are enriched in inflamed tissues and are detrimental for metabolic equilibrium. ILC1s drive M1 macrophage polarization and induce adipose tissue fibrosis in an IFN-γ mediated pathway, whereas inhibiting ILC1 accumulation improves glycaemic tolerance in diabetic mice [[92], [93]].

Additionally, a role for the type 3 immune response via secretion IL-22 from ILC3s and CD4⁺ T cells is emerging. The dietary stimulation of ILC3s via AhR signaling produces IL-22, which also improves insulin sensitivity [94]. ILC3s can also influence lipid resorption via IL-22 signaling and are necessary to maintain lipid metabolism under steady state conditions [95], but IL-33 might regulate metabolic hemostasis in multiple tissues. [[96],[97],[94]]. However, more research is necessary to uncover the action and pathways regulated by IL-22 in multiple tissues.

Together, these data indicate that the presence of non-inflammatory conditions and type 2 immunity are critically important to maintain steady state metabolic homeostasis.

2.2. Immune-metabolic dysfunction

In order to maintain proper immune functions during homeostasis and pathogen attack, metabolic pathways must operate with highest efficiency. Conversely, for smooth functioning of metabolic pathways, immune system homeostasis or activation must be kept in check. The imbalance in one system can cause the collapse of its interacting partner system. An example of an unresolvable metabolic imbalance is diabetes. Type-1 diabetes is caused due to autoimmune-induced inflammation that destroys the insulin producing beta cells of the pancreas. Whereas, type-2 diabetes is characterized by obesity-mediated insulin resistance. Although the major causal factor underlying type-1 and type-2 diabetes (referred to as diabetes here onwards, unless stated otherwise) is different, the ensuing metabolic perturbations due to hyperglycaemia are similar in both conditions.

In diabetes, uncontrolled glucose uptake sets off a high rate of glycolysis, resulting in excess pyruvate systemically. If glycolysis is ineffective in disposing off the intracellular glucose, alternative metabolic pathways are triggered. For instance, aldose reductase converts glucose to sorbitol, generating osmotic stress in the cells. Excess glucose is also shunted to the hexose pathway, the end product of which is uridine diphosphate N acetylglucosamine. This molecule can modify serine and threonine residues on specific transcription factors (e.g. Sp1) implicated in hyperglycaemic inflammatory injury [98]. In diabetes, the oxidizing environment and high carbohydrate accumulation accelerate the formation of advanced glycation end products (AGE). These AGEs can directly bind to the receptor of AGE (RAGE) to trigger an NF-kB mediated inflammatory cascade [99]. This pathway results in accumulation of NADPH oxidase and further promotes an oxidative stress within cells [100].

Another feature underlying diabetes is dyslipidemia, i.e. increase in plasma lipids. Excess lipid catabolism generates an excess of free fatty acids. The free fatty acids can cause toxicity by lysosomal dysfunction and can send mitochondrial oxidative pathway into overdrive, leading to high oxidative stress [101]. In addition, freely floating LDLs get oxidized or glycated. Binding of these modified LDLs to their receptors, LOX1 and TLR4 results in activation of NADPH oxidase and releases free fatty acids [[102],[103]].

Thus, a continuous presence of excess free fatty acids, glucose and glycolytic byproducts, such as AGEs, is observed in diabetes. Together, they lead to mitochondrial hyperactivity generating superoxide radicals, which activate injurious inflammatory signaling pathways.

As mentioned above, nutrient availability dictates immune cell function. Naturally, excess of all the metabolites, along with increased oxidative stress, severely affects immune homeostasis during diabetes and obesity. Due to excess glucose and fatty acids in the surroundings, the T cells are in an over activated state. The percentage of IFN-γ–producing Th1 cells and CD8⁺ T is increased in the gut, whilst Tregs are decreased in a mouse model of diabetes [[72],[104]]. Depleting CD4⁺ T cells polarized to Th1 phentoype or IFN-γ in the diabetic mice, significantly improves insulin sensitivity, thereby tying the IFN-γ and T cell repertoire directly with metabolic dysbiosis [[105],[52]]. Several studies have also observed a reduction in Th17 cells and IL-22 secreted by them in different parts of the intestine of diabetic mice and patients, thereby compromising the integrity of the epithelial barrier [[107], [108],[109]]. Similar to T cells, circulating B cells in diabetic patients exhibit a proinflammatory phenotype with increased production of IL-6, IL-8 and TNF-α along with dampened IL-10 production [110]. A couple of studies using mouse model of diabetes have shown that B cell deficiency reduces TNF-α producing M1 macrophages and improves insulin sensitivity [[110],[111]]. The relative reductions in IL-10, coupled with increasing local TNF-α, may compromise mucin production and IgA class switching and affect gut hormone secretion [[112],[113],[114]]. This shows that adaptive immune system is constantly activated during diabetes.

With increased pro-inflammatory cytokines within the gut and in blood circulation, coupled with presence of excess metabolites, polarization of resting macrophages towards M1 phenotype occurs during diabetes. An increase in the number of CD68⁺ M1 macrophages, DCs and NK cells is reported in the intestine of insulin resistant patients [109]. Another study using rats revealed that a insulin deficiency resulted in a disruption in phagocytosis of macrophages [115], while NK cell dysfunction was also reported respect to CD159 expression, which was negatively correlated with HbA1c levels [116]. Beside altered macrophages, monocytes also play a key role in the progression of diabetes. Patients suffering from type-2 diabetes exhibited increased expression of monocyte activation markers (CD11b and CD36) [117]. Also, diabetic patients exhibited significant decrease in the number of neutrophils and eosinophils [91].

Finally, ILCs are a family of innate immune cells that respond to cytokines produced by surrounding macrophages, dendritic cells, and epithelial cells [118]. Although much is not known regarding the involvement of ILCs in diabetes, a study has shown that ILC1s driven M1 macrophage polarization and contribute to pro-inflammatory conditions and are directly implicated in obesity-induced insulin resistance [92]. The protective role of ILC2s is also implied in metabolic dysfunction during obesity-induced diabetes, since ILC2s and eosinophils play a prominent role in promoting M2-like macrophage development and ILC2 depletion greatly decreases eosinophil and M2 macrophage populations [[119],[37]].

Taken together, these data indicate a shift from the type 2 immunity, which is present in steady state to proinflammatory type 1 immunity owing to the presence of excess metabolites, metabolism-induced inflammation and ensuing oxidative stress. A systemic skewing of the immune system towards a proinflammatory phenotype is observed systemically and in individual tissues. Due to the inflammatory milieu, an infiltration of immune cells in observed in several tissues, including in the enteric and peripheral nervous system. The chronic immune activation affects physiological function of several tissues during diabetes, such as in the intestine causing gastric dysbiosis [120]. This in turn affects nutrient absorption and metabolic disequilibrium setting off a persistent and vicious circle of events. Metabolic perturbations and gastric dysfunction are integral component of disease pathophysiology, believed to be primarily caused due to immune dysfunction, such as, in inflammatory bowel disease (IBD), reviewed in detail elsewhere [121].

3. Neuro-immune regulation

The immune system is mainly activated due to reasons other than metabolic disturbances, namely, infections and tissue injuries. The immune responses are directed to counter the infection or to promote tissue repair, thereby resolving these acute conditions and limiting their spread. So long these immune response triggers are controlled and the inflammation is resolved, the immune activation remains brief and an immune-homeostasis condition can be reacquired. However, if the trigger is persistent, the immune system stays chronically activated. The dysbalance of the immune system can result in the loss of equilibrium in other systems, which closely interact with the immune system, notably, the nervous system.

3.1. Neuro-immune interactions

That the immune and nervous systems interact with each other, has been long since known. However, more and more reports are now emerging demonstrating the extent of co-dependance of the neuroimmune systems to counter both neuro- and immunopathies. Maximum interaction of the immune system with the nervous system occurs with the neural network outside of the CNS, which is immunologically privileged compared to the PNS [122]. The PNS innervating all peripheral organs can be further sub-classified as the somatosensory nervous system and the autonomous nervous system (ANS). The ANS comprises the sympathetic, the parasympathetic and the enteric nervous system (ENS) [1].

The PNS innervates the peripheral tissues including limbs, skin, lymphoid organs and mucosal barriers. The PNS comprises the dorsal root ganglia (DRG) located just outside the spinal cord. Soma of the peripheral neurons lie in the DRG, whereas the axons constitute the nerves emerging from the DRG and the nerve endings of these neurons are located in the extremities. The somatosensory system innervates the skeletal muscles and the sensory component senses i.e. nociceptive stimuli by the nerve endings and electric signals are transmitted to the neuronal soma in the DRG via the axons. The DRG can integrate the sensory information and release neurotransmitter in response, as well as, communicate the information to the CNS via spinal cord. Since the PNS innervates peripheral tissues, it is proximal to a variety of cells of the tissue and immune cells.

In addition to the PNS innervation, the gut is extrinsically innervated by the sympathetic and parasympathetic nerve endings, which release the neurotransmitters norepinephrine and acetylcholine, respectively. The gut is also intrinsically innervated by the ENS, which is an extensive network of neurons located along the gastrointestinal tract. ENS is organized in two main ganglionated plexuses: the submucosal plexus (also called Meissner’s plexus) within the connective tissue of the submucosa of the intestinal wall and the myenteric plexus (also called Auerbach’s plexus) located between the circular and longitudinal muscle layers [123]. The ENS neurons can be broadly divided into nNOS⁺ neurons comprising secretomotor/vasodilatator motor neurons and inhibitory motor neurons. Excitatory motor neurons, interneurons and sensory are characterized by expression of choline acetyltransferase (Chat) [124]. The ENS interacts with the local immune system to control immune activation due to gut microbiota and maintain tolerance. Notably, immune cells express receptors for many neuronal factors, which describe the neurochemical signature of ENS neurons, such as VIP, acetylcholine, NMU, CGRP, SP, serotonin and NPY [1]. Furthermore, the ENS is emerging as a source of cytokines, such as IL-6 and IL-18 [[125],[126],[127]].

3.1.1. Immune regulation of neurons

The PNS and ENS can interact with the immune system, because these neurons have been shown to express receptors for several cytokines and immune-modulatory factors released during inflammation. In the context of skin inflammation, expression of receptors for the type 2 cytokines (TSLP, IL-4 and IL-31) were shown to trigger a signal pathway including JAK1 in neurons promoting itch behavior. Interestingly, inhibitory action of JAK1 have been proven successful in clinical treatment of itch symptoms illustrating the therapeutic importance of neuroimmune crosstalk [[128],[129]].

DRG neurons express receptors for canonical proinflammatory cytokines released by macrophages, neutrophils and mast cells, such as, TNF-α [130], IL-1ß [131] IFN-γ [220] and IL-6 [132]. These receptors are also expressed by the enteric neurons [[133],[134]]. Under inflammatory conditions, mast cell degranulation releases IL-5, histamines and nerve growth factor (NGF), all of which can bind to their respective cognate receptors (IL-5R, histamine receptor 2 and TrkA, respectively) on the neurons and stimulate them [135]. Neurons also express prostaglandin (PGE2) receptors 1–4 [[136], [137]] and purinergic receptors (P2X3) [138], which can be activated by the PGE2 released by macrophages and other innate immune cells. The DRG and enteric neurons express IL-17RA ([75], p. 17; [[139],[140]]) to respond to the IL-17A released by the Th17 and γδT cells. They also express receptors for type I and type II interferons [[141],[220]]. Moreover, the DRG and enteric neurons could potentially play a role in directly sensing microbes since they also express pattern-recognition receptors. Although the expression the pattern recognition receptors TLR 3, 4, 7, 8 and 9 was reported from neurons the physiological importance remains elusive [[142],[143]]. For TLR4, a function distinct from LPS sensing was also described [144]. For a more detailed discussion on sensing abilities of neurons with respect to allergens, toxins and metabolites the reader is kindly referred to a recent review on this topic [1].

Together, this shows that the neurons of PNS and ENS are thoroughly equipped to respond to the immune-mediated cues and serve as sentinels, should there occur any environmental challenge.

3.1.2. Neuronal regulation of immune system

Upon ‘sensing’ an inflammatory milieu, the neurons get activated and in turn release neuropeptides to regulate immune activation, as well as, uncontrolled activation of neuronal activation using a negative feedback loop. The immune cells have also been shown to express not just receptors to neuropeptides and neurotransmitters, but also release certain neurotransmitters, such as acetylcholine, CGRP and serotonin themselves [[145],[146],[147],[148],[149],[150],[151]].

Neuronal regulation of immune function is of particular significance in the intestine, where the diversity and complexity of neuronal and immune cell types is required to sense, react and adapt to ever changing conditions [127]. In the intestine, mast cells have been shown to express of receptors for the neuropeptides CGRP (CALCRL) and SP (Mrgprb2) [152]. Stimulation of mast cells by these neuropeptides has been shown to induce degranulation and release of histamines and cytokines such as TNF-α, GM-CSF and PAR-2 in human mast cells [[153],[154]], all of which can cause hyperexcitability of sensory enteric neurons [[155], [156]]. The muscularis of the intestine also contains a specialized subset of macrophages. These macrophages express the receptors for norepinephrine (ß2-adrenergic receptor) and CSF1 (CSF1R). Macrophages also respond to BMP2 released by the enteric neurons. Macrophage-neuron inter-signaling via the above-mentioned factors, which influence gut motility and peristalsis [157], indicates that gut macrophages communicate closely with the motor neurons in the intestine. ILCs are a population of immune cells, abundant at the mucosal barrier [123]. ILC2s express receptors for the neuropeptides norepinephrine (β2 adrenergic receptor) [158] neuromedin U (NMUR1) [[159],[160],[161]], acetylcholine [[162],[163],[164]] VIP [[165],[166]] and CGRP (Ramp1/-CALCRL) [[150],[167],[168]]. Activation of ILC2s leads to the production of type 2 cytokines, including IL-5 and IL-13, which promote an anti-helminthic immunity as part of the intestinal barrier defense system or trigger allergic lung inflammation. Some of these receptors are expressed by other subsets of ILCs, for example the β2 adrenergic receptor is upregulated upon NK cells activation is regulates proper expansion and control of cytamegalovirus infection [169]. Whereas, VIP-VIPR2 signaling in ILC3s has been demonstrated to regulate intestinal inflammation and lipid absorption IL-22 dependent manner [[170],[95]]. Similarly, glial cell derived signals, such as GDNF regulate IL-22 production in ILC3 via the Ret receptor [171]. Other immune subsets found in the gut mucosa have also been shown to be affected indirectly by neuropeptides, however the complexity of these interactions is massive and is reviewed in detail elsewhere [123].

The somatosensory nervous system, too, is a fertile soil for neuroimmune interaction, albeit somewhat less complex than the ENS. The sensory response is activated upon receiving noxious/pain stimulus. Release of neuropeptides alters the function of tissue resident immune cells in the skin, nerve or the tissue where the pain stimulus was generated. Macrophages and DCs express the receptor for CGRP (RAMP1), and upon being stimulated by CGRP release IL-10 to provoke anti-inflammatory, anti-nociceptive responses in the adjacent neurons. RAMP1 stimulation in macrophages also regulates VEGF production and plays a crucial role in wound healing [172]. CGRP activates RAMP1 to inhibit dendritic cell production of IL-12, IL-6, and TNF-α and expression of CCR7, which in turn regulates neuronal hypersensitivity [[173], [174]]. CGRP also stimulates dermal DCs, which modulates Th17 and γδ T cell function in the periphery [175]. In the context of type 2 inflammation nociceptors did respond to different allergens, with the production of SP. SP elicited migration of DC carrying MRGPRA1 and stimulation of T helper 2 cells in secondary lymphoid organs.

The macrophages, DCs, T and B lymphocytes also express different isotypes of the receptor for the neuropeptide NPY [[176],[177],[178]] and have been shown to respond to NPY stimulation. NPY can have immunoregulatory effects namely, inhibition of chemotaxis and TNF-α inhibition in macrophages [[179],[180]] and promote Th2 polarization [[181],[182]]. However, NPY has also been shown to promote M1 phenotype in macrophages (TNF-α secretion) [[183],[184],[185]], activation of DCs (IL-6 secretion) and CD4⁺ T cell activation (Th1 proliferation, IFN-γ secretion) [[176],[186],[187]]. This double-action is suggested to depend on NPY concentration and involvement of different NPY receptors on cells [[186],[188],[187]].

Apart from releasing neuropeptides, neurons may themselves be source of cytokines and chemokines, although cytokine secretion within the nervous systems is often associated with the glial cells. Cultured sympathetic neurons have been shown to secrete IL-1β and IL-6 [[189], [190]]. Stimulation of the purinergic receptor is written as P2X7 on cultured rat ganglion neurons resulted in secretion of IL-3, TNF-α, CXCL9, VEGF, L-selectin, IL-4, GM-CSF, IL-10, IL-1Rα, MIP and CCL20 [191]. P2X7 is also expressed by the enteric [192] and DRG neurons [193]. The DRG neurons could release type I IFNs upon stimulation STING agonists [194].

The importance of cytokines secreted by the ENS was highlighted in two recent studies. In steady state, the IL-6 released by the ENS controlled proportions of Fopx3⁺ and RORγt⁺ CD4⁺ cells by integrating signals from the commensal microbiota [126]. During Salmonella enterica infection, IL-18 production from the ENS was essential for pathogen control by induction of antimicrobial peptides from goblet cells [125].

Altogether, these studies indicate just as the immune cells have adopted neuropeptides and receptor expression, neurons utilize cytokine and cytokine receptor signaling pathways as well.

3.2. Function and dysfunction in neuro-immune crosstalk

3.2.1. Neuro-immune co-ordination

The shared molecular repertoire shows that the neuro- and immune axes comprise one integrated system, which is constantly in bidirectional communication, in order to restore any temporary imbalance caused in either neuronal or immune function by environmental triggers. For instance, an acute and inflammatory trigger caused by tissue injury activates the tissue resident immune cells. Injury initiates release of proinflammatory mediators (TNF-α and interleukins) by the macrophages [195] and induces mast cell degranulation [156]. Vice versa, the vagus nerve is a very potent suppressor of macrophages TNF production via release of acetylcholine and the nicotinic α7 acetylcholine receptor on macrophages during sepsis, known as the cholinergic anti-inflammatory pathway [196]. Notably, vagus nerve stimulation by an electronic device has been proven effective in patients suffering from rheumatoid arthritis and unresponsive to glucocorticoid treatment [197]. These findings indicate that the nervous system is an underappreciated regulator of acute and chronic inflammation.

In the context of type 2 inflammation mast cells degranulation triggers the rapid secretion of histamines, serotonin, nerve growth factor, cytokines, and leukotrienes. These immune-released factors bind to their respective receptors and stimulate the proximal nerve endings. The neurons are now hyperexcitable and result in heightened pain sensitivity [202]. The whole scenario is classic example of an acute shift in immune and neural equilibrium states.

However, neuro-immune axes also work in symbiotically to contain the pain and inflammation. The stimulation of the sensory neurons results in the release of neuropeptides such as CGRP and SP [199]. CGRP and SP results in vasodilation allowing recruitment of immune cells at the site of injury/inflammation [[200],[201]]. CGRP binds to the RAMP1 receptor on macrophages, DCs and neutrophils to initiate anti-inflammatory processes. RAMP1 stimulation causes inhibition on TNF-α production by the macrophages and induces IL-10 release. Macrophages exhibit functions in mediating phagocytosis and tissue repair. CGRP also inhibits mast cell degranulation and release of IL-12, IL-6, and TNF-α by DCs. Both macrophages and neutrophils inhibit nociceptive effects by releasing opioid peptides at injury sites [202]. A negative feedback loop is triggered in the sensory neurons due to the concerted action of anti-inflammatory cytokines and opioid peptides now released by the CGRP-stimulated immune cells and the binding of CGRP to the TRPV1 channels on the neurons. The inhibitory GABA-ergic and opioi-dergic pathways are activated within the neurons. The release of the neuropeptide GABA terminates the neuronal excitatory signaling upon binding to the GABA receptors on neurons [[203]]. The anti-inflammatory cytokines IL-4 and IL-10 binds to IL-4R and IL-10R on neurons to inhibit the inflammatory pathways activated within the neurons and release of opioid peptides by the neurons [[128],[204]]. The opioid peptides, namely ß-endorphin, binds to mu-opioid receptor (MOR) on neurons to initiate downstream analgesic signaling [205]. ß-endorphin can also bind to MOR is also expressed by macrophages, granulocytes and lymphocytes and trigger anti-inflammatory responses [[206],[207],[208]].

The integrated response of neuro and immune systems can efficiently resolve painful and inflammatory triggers due to injury or infection. Thus, the immune and nervous system can complement each other perfectly to maintain homeostasis. However, if one system collapses, the domino effect disturbs the other system too. This is observed during chronic inflammatory pathologies.

3.2.2. Neuro-immune discord

Inability of the immune or the nervous system to resolve the triggers causing disequilibrium, result in chronic pathologies. As mentioned in Section 2.2, immune system is in overdrive during type-1 and type-2 diabetes. A major complication of diabetes is also peripheral neuropathy, affecting nearly 50% of the diabetic patients [209]. In diabetic peripheral neuropathy (DPN), patients suffer from somatosensory dysfunction, resulting in heightened pain sensitivity in early stages followed by loss of pain sensation later due to degeneration of nerves in the extremities [210]. A chronic neurogenic inflammation is detected in the peripheral nerves during diabetes, shown by activated NF-kB signaling pathways [99]. The peripheral nerves of patients and animal models of diabetes were shown to have increased numbers of infiltrating macrophages, neutrophils and T cells [[211],[212],[213]]. These immune cells are responsible for higher levels of TNF-α, IL-6 and IL-1ß within the nerves, which also activates the glial cells and nerve-resident macrophages, all of which together contributes to chronic neurogenic inflammation. This inflammatory milieu has a direct consequence on neuronal function. The constant stimulation of the neurons by the inflammatory cytokines and loss of inhibitory/neuroprotective neuronal signaling pathways causes hyperactivation of ion channels in the nerves [[214],[131]]. The levels of the excitatory neurotransmitter, glutamate, were significantly higher in the patients with diabetes [242]. This eventually leads to excitotoxicity and degeneration of nerves [[215], [216]]. Thus, a continuous hyperactive immune response and ensuing inflammation can lead to neuronal dysfunction and degeneration.

Excitoxicity is not solely because of increased excitatory neurotransmitters, but also due to loss of inhibitory neuronal pathways. One such example is the impairment of analgesic POMC – MOR pathway in mice and patients with diabetes [205]. POMC promoter was shown to be suppressed, as a direct consequence of increased NF-kB activation within the neurons. This shows that tissue inflammation can directly control neuropeptide expression and release. POMC-derived peptides apart from having an analgesic function in neurons, also have an anti-inflammatory and immunosuppressive effect on immune cells expressing POMC receptors, such as the monocytes, macrophages, neutrophils, T and B cells [[207],[208]]. Since NF-kB activation is a common occurrence during inflammation, loss of such anti-inflammatory neuronal mediators can have far-reaching consequences in inflammatory pathologies beyond diabetes.

As the neuro-immune crosstalk fails at the mucosal barrier in the gut during IBD, similar neuroinflammatory pathology is observed. Neurological manifestations in IBD patients are more common than previously estimated, with over 40% patients reporting symptoms of peripheral neuropathy [[217],[218],[219]]. Symptoms involving CNS (migraine, cranial nerve palsy, optic neuropathy among many) have also been associated with IBD [[220],[217]]. As stated earlier (section 1.2.2), in IBD, there is an ongoing inflammation at the intestinal barrier. A surge of infiltrating eosinophils, neutrophils, mast cells and T cells is observed in enteric ganglia in experimental colitis [221]. Higher levels of both local and circulating pro-inflammatory cytokines (e.g., IL-1β, TNF-α, IL-6) are found in patients with IBD. These cytokines, causes hyperexcitability of enteric neurons, which regulate gut motility and immune response towards gut microbiota [222]. For example, hyperexcitation of the serotonergic neurons results in increased secretion of serotonin in experimental colitis, known to regulate peristalsis [[223],[224]]. Continuous excitation of cholinergic neurons results in elevated levels of the SP, which acts in a proinflammatory manner, in IBD patients [225]. Increased levels of IL-1β and IL-6 modulate the enteric secretomotor neurons via the suppression of norepinephrine at sympathetic synapses on the submucosal neurons [226]. Norepinephrine released by the sympathetic neurons in the ENS has been shown to suppress pathogenic T cell response [[227],[319]]. Whether this occurs directly by binding to adrenergic receptors on T cells or is an indirect effect by other immune cells expressing adrenergic receptors is not clearly proven [229]. Another recent study has reported contradictory results [230]. Admittedly, while there is discrepancy in understanding the effect of norepinephrine on T cells within the context of IBD, studies have indicated increased number of pro-inflammatory T cells and increased B cell reactivity in the gut of IBD patients [231]. In vivo, impaired neurotransmitter function in both inflamed and non-inflamed regions of the bowel during IBD [232], and altered neuronal signaling can persist up to 6 months after the initiation of colitis [233]. This implies that the inflammation may cause a prolonged change in the intrinsic properties of the neurons. The inflammation and neuron dysfunction although commences in the ENS [[234],[235]], structural nerve damage, neuronal dysfunction and eventual neuronal loss is also observed in PNS and CNS, suggesting that neuroinflammation traverses the interconnecting nervous systems and can have far-reaching consequences [[236],[219]].

This indicates the existence of a damaging neuro-immune vicious circle in chronic pathologies in not just diabetes or IBD, but also other diseases. The reader is kindly referred to other reviews extensively covering the topic of neuro-immune dysregulation in diseases such as intestinal disorders [237] multiple sclerosis [[238],[239]], Alzheimers disease [240], charcot marie tooth disease [241], obesity [242] and chronic infections resulting in neuropathies [243] to name a few.

4. Neuro-metabolic regulation

4.1. Neuro-metabolic interdependence

The neurons are the highest energy consuming cells of the body, with 20% of the total energy being consumed by the brain alone in steady-state condition [6]. This high-energy demand of the neurons is because of the continuous action potentials triggered within the neurons by constant sensory signals and resulting effector functions, cognition, imagination and other neurobehavioral aspects. The energy is also spent by the neurons to support the metabolic needs of the surrounding glial cells [244]. To meet this high-energy demand, neurons rely heavily on efficiently operating metabolic pathways. Conversely, dysfunction in the nervous system changes the way metabolites are uptaken causing a nutrient imbalance and skewing of the homeostatic metabolic status.

4.2. Metabolic regulation of neurons

Adult mammalian neurons rely mainly on glycolysis as an energy source. Under certain instances, however, neurons can use other substrates to derive energy too. For example, ketone bodies are utilized during fasting and lactate during high physical activity. Given the high-energy demands and negligible intrinsic energy stores, the nervous system is dependent on continuous supply of energy sources from circulation.

Blood glucose is made available to the neurons by the GLUT1 transporters in the blood brain barrier of the CNS and blood nerve barrier of the PNS [245]. The neurons of PNS and ENS also express GLUT3 transporters predominantly and GLUT 4, 8 to a lesser extent, of which GLUT1 and 3 are insulin-independent. These transporters allow unlimited uptake of glucose by the neurons to meet the high-energy demands, and the deficiency of which leads to an underdeveloped or degenerate nervous system in mice [246] and patients [[247],[248]]. This demonstrates how heavily the neurons depend on glucose to perform neuronal functions. The glucose taken up by the GLUT transporters enters either glycolytic pathway directly or indirectly and is followed by the TCA cycle and OxPhos pathway for producing ATP [249]. The ATP is then utilized for constantly generating and inhibiting action potentials via ion channel regulation, neurotransmitter production and release.

While glucose serves as a metabolic substrate for all neurons, there are certain neurons, which are especially sensitive to extracellular glucose concentrations and modulate their function in response to glucose fluctuations. In addition to GLUT3, these neurons take up glucose directly via the glucose-sensitive ion channels called SGLTs, and are present in the CNS [250] as well as PNS, especially the ENS [[251], [252]]. These neurons sense glucose in the euglycaemic range i.e. 5–20 mM glucose and help maintain homeostasis between neuronal excitation and inhibition [253]. In the ENS, glucose infusion results in the release of the neuropeptides calbindin and calrexin [254]. In contrast, glucose insufficiency results in decreased secretion of acetylcholine from the excitatory motor neurons of the ENS, thereby exerting an inhibitory effect [255]. Since, all three neuropeptides (calbindin, calrexin and acetylcholine) are required for gut motility, this indicates that glucose availability directly regulates physiological functioning of the gut.

Another substrate taken up by the enteric neurons is the amino acid, glutamine. Glutamine (source of nitrogen) is required for the activity of inhibitory nitrergic motor neurons expressing nNOS and VIP and also for the sensory neurons expressing CGRP [256]. Glutamine activates the inhibitory neurons and regulates hypercontractility observed during diseases, such as diabetes [[257],[258]]. Glutathione, which is synthesized from glutamine via the amino acid metabolic pathway, has more potent effect compared to glutamine [259].

Enteric neurons can also take up free fatty acids via the FFAR3 receptors. These receptors are mainly expressed by the cholinergic neurons. Binding of the free fatty acids to their receptor inhibits the release of acetylcholine and hence prevents motility of the gut [260].

In short, this shows that metabolite availability and absorption dictates which neurons will be activated or inhibited, subsequent neurotransmitter release and eventual physiology of the organ.

4.3. Neuronal regulation of metabolism

Neuronal regulation of metabolic pathways is a feedback regulatory mechanism to maintain metabolic homeostasis. This is necessary to maintain an uninterrupted supply of energy substrates to the neurons for the proper functioning of the central and peripheral nervous system.

The intestine is the first tissue to come in contact with the nutrients released from the catabolism of the ingested food. Many cells of the intestine are involved in detection of nutrients and regulation of metabolism, such as the enterocytes, brush cells and enteroendocrine cells. One of the main responders to the metabolites present in the intestine, are the neurons of the ENS [255]. The enteric neurons can sense the changes in metabolic substrates and release neuropeptides, which contribute to maintain metabolic homeostasis. For instance, the widely studied neuropeptide in maintaining systemic glucose homeostasis is POMC. POMC neurons are excited upon directly sensing extracellular glucose [261] or can also be activated hormonal ‘food-cues’ such as insulin and leptin [262]. In a fed state, i.e. under elevated blood glucose levels, POMC neurons are excitated and release the neuropeptides ß-endorphins and melanocortin, both of which signal to stop feeding [263]. This prevents hyperphagia and ensuing hyperglycaemia. Thus, POMC neurons can directly regulate feeding behavior and glucose metabolic pathways in the body. Although this pathway is widely studied in the CNS, ENS neurons have also been shown to express the MOR (receptor for the neuropeptide ß-endorphin derived from POMC) [264] and MC4R (receptor for the neuropeptide melanocortin derived from POMC) is expressed by the enteroendocrine cells of the intestine [265]. It has been shown that intragastric administration of MOR agonist, decreased food consumption and motor activity in food-deprived rats [266]. Furthermore, another study showed that MC4R deficient mice develop high triglyceride levels in the intestine and that stimulation of CNS neurons did not affect this POMC - MC4R mediated lipid metabolism in the gut [267]. These studies indicate the importance of the local POMC signaling in the gut in maintenance of glucose homeostasis. POMC, MOR and MC4R expression is also found on neurons of PNS [[205],[268]], indicating glucose absorption may also be controlled locally in DRG neurons.

Feeding and glucose metabolism is also controlled by neuropeptide Y (NPY), which is known to inhibit the POMC neurons [106]. This indicates that NPY positively regulates glucose metabolism by increasing glucose levels in the body. Unsurprisingly, insulin negatively regulates NPY and ghrelin, a gut hormone secreted during hunger [[269],[270]]. Ghrelin, in turn, is known to positively regulate NPY [[271],[272]]. NPY and its receptor is also expressed by neurons of ENS [273] and PNS [[274],[275]], indicating that NPY may be involved in local glucose homeostasis in the PNS. POMC and NPY expressing neurons also express the receptor for glucagon-like-peptide-1 (GLP-1) in ENS [276] and PNS [277]. GLP-1 is a peptide hormone known to be secreted by the enteroendocrine cells and has the ability to decrease blood glucose levels. This provides direct evidence how the NPY and POMC expressing neurons can sense changes in blood glucose levels. GLP-1R stimulation has been shown to inhibit cholinergic/excitatory and stimulate nitrergic/inhibitory neurons [278]. This fits well since, GLP-1 is known to suppress NPY neurons (excitatory) and stimulate POMC neurons (inhibitory neurons) and therefore plays a role in promoting feeding [271]. Together, the neuropeptides POMC, NPY and the hormones insulin, leptin, GLP-1 regulate carbohydrate intake.

Lipid metabolism is regulated by the neuropeptides galanin, enkephalin and cannabinoids [279]. Many studies have been carried out on the orexigenic peptide galanin/Gal with respect to fat intake. These studies show that Gal deficient animals are unable to increase their body weight despite being fed high-fat diet (HFD) [280], whereas high Gal levels are observed in obese individuals [281], suggesting a critical role for Gal in energy homeostasis. Increased Gal signaling stimulates feeding with a preference to fat intake [[282],[283]]. However another study argues that obese phenotype observed in Gal deficient mice is attributed to insulin intolerance and reduced energy expenditure [284]. Gal is expressed by those ENS and PNS neurons, which also express SP [124]. Gal receptors expression is also present in the neurons of ENS [285] and PNS [[286],[287],[288],[124]]. In the ENS, Gal triggers the inhibitory nitrergic signaling upon binding to the Gal-1R receptors expressed by nNOS⁺ neurons [258]. Gal was shown to reduce intestinal contractility and improve systemic glucose tolerance via ENS in diabetic mice [257].

Another peptide increased in CNS with high fat intake is enkephalin, which also is expressed by the ENS [289] and PNS [290], however, its direct involvement in the peripheral lipid metabolism is not well-established. On the other hand, there is more information regarding how the neurotransmitters of the sympathetic and parasympathetic nervous system can regulate peripheral lipid metabolism, since these neuron fibers innervate the adipose tissue. Secretion of noreinephrine results conversion of the stored lipid droplets to free fatty acids in the adipocytes (expressing β3-adrenergic receptor which binds norepinephrine) i.e. lipolysis is triggered to mobilize extra energy resources in emergency situations such as external threat [291]. Conversely, acetylcholine, the major neurotransmitter of the parasympathetic nervous system upregulates glucose uptake and fatty acid synthesis/lipogenesis i.e. energy accumulation and storage [[292],[293]]. Naturally, excess of blood glucose (or insulin secreted in response to increased glucose) inhibits sympathetic activity [[294],[295]]. Upregulation of the genes POMC and CGRP have been noted in the sensory innervation of adipose tissue when priming it for efficient fatty acid oxidation (i.e. beiging/browning), which help the process of thermogenesis in cold conditions [[296],[297]].

In summary, the metabolic changes in the periphery can be locally sensed be the PNS neurons and the neuropeptides released in response, can modulate the carbohydrate and lipid metabolic pathways.

4.4. Neuro-metabolic dysfunction

With so many players belonging to the class of neuropeptides and the division of the metabolic pathways, it is highly necessary to maintain a tight rope walk to maintain neurometabolic homeostasis on the playground of the enteric and peripheral nervous system. Not surprisingly, when one arm is disturbed, the other one is too with dire consequences.

Diabetes, characterized by a complete meltdown of metabolic pathways, not only affects PNS due to inflammatory injury, but also alters ENS activity leading to enteric neuropathy. In terms of neurometabolism, nutrient imbalance in the gut of animal models fed with HFD have been shown to incur structural and functional alterations in the enteric neurons. Diabetes is associated with degeneration of enteric axons [298] and loss of neurons in DRGs and myenteric plexus [[299], [300]]. The combined deleterious effect of inflammatory signaling along with gluco and lipotoxicity, oxidative stress and reduction of neurotrophic factors manifests as selective loss of inhibitory nitrergic signaling associated with intestinal hypercontractility [[257],[255]]. The loss of neurons occurs due to enhanced apoptotic neuron death in the ENS [301]. The loss of nitrergic function is due to the loss of inhibitory neuropeptides in the intestines during diabetes. For instance, a loss of VIP⁺ neurons, nNOS⁺ neurons and decreased level of Gal is observed in during of diabetes [[302],[303],[304]]. A reduction is also reported in the levels of the sensory peptides CGRP and SP [[305], [306]]. A defective MOR signaling pathway is also implicated in the pathogenesis of diabetic enteric neuropathy [307], suggesting altered levels of gut POMC peptides. Another inhibitory neuropeptide demonstrated to be decreased in the gut of diabetic patients in PENK [307]. In addition to loss of inhibitory and sensory peptides, an increase in the excitatory neurons expressing acetylcholine is noted in diabetic animal models [198]. These changes together are responsible for symptoms such as abdominal pain, intestinal hypercontractility, nausea, diarrhea etc. [308].

Due to hyperglycaemia, constant activation of glucose-sensing neurons drives them presumably towards an insensitivity or tolerance, leading to imbalanced neurotransmitter repertoire in gut. Since nutrient sensing is compromised due the maladaptation of gut neurons, downstream signaling to directly or indirectly control glucose levels, lipid metabolism is dysfunctional, further adding to the metabolic havoc. Several studies have shown supplementation with neuropeptides can improve metabolic status. For instance, Gal treatment improved glucose tolerance in diabetic mice [258], glutamine (required by nitrergic neurons) prevents enteric neuronal loss [299], MOR stimulation improved glucose metabolism [307], SP injection elevated insulin levels and lowered glucose levels [305]. Conversely, loss of nitrergic function in the stomach of diabetic mouse model could be restored by insulin therapy [309].

Similar disruption of metabolic pathways linked with dysregulated neuronal activity is also reported in other diseases affect the gastrointestinal tract [[310],[311],[312]] and PNS [[313],[314],[315]] alike.

5. Conclusion

Taken together, the neuro-immune-metabolic interactions form a closed loop in health and disease. It seems clear that whilst the neuroimmune-metabolic networks support each other to maintain homeostasis and physiological functioning of a body organ, they can also deregulate one another. Below are some synopsized examples from the above-mentioned studies to support this concept.

A study by Seillet et al. beautifully demonstrated the interdependence of the neuro-immune-metabolic network in steady state conditions with respect to lipid metabolism [170]. This study showed that food consumption activated the VIP neurons in the ENS. VIP released from these neurons bind to VIPR2 expressed by the ILC3s in close proximity in the gut. This stimulation induced IL-22 release from ILC3s. IL-22 released by the ILC3s is shown to improve insulin sensitivity and can also regulate lipid metabolism in liver and adipose tissue [94]. In diabetes, however, a loss of VIP⁺ neurons and nNOS inhibitory neurons is reported, leading to reduced VIP and Gal levels in the intestine [[302], [303],[304]]. A reduced VIP-VIPR2 interaction leads to decreased IL-22 secretion by the ILC3s, as is shown in fasting animals secreting less VIP [170]. Reduced IL-22 levels are directly correlated to the development of metabolic disorders. This was shown using mice deficient in IL-22 fed, which when with HFD, developed hyperglycaemia and insulin resistance [94]. Another study showed that HFD caused alterations in the intestinal γδ T cells subset, resulting in inhibition IL-22 released by ILC3s [80]. Supporting these findings, yet another study showed that IL-22 secretion by ILC3 negatively correlates with fat accumulation [97]. Furthermore, the reduced Gal levels during diabetes can be correlated with increased accumulation of fat leading [[282],[284]] to an obese phenotype in type-2 diabetes. Loss of inhibitory neuropeptides (VIP and Gal) may, in turn, contribute to the increased muscle contractions, gastric pain and dysbiosis, which are the symptoms of enteric neuropathy observed in diabetes [308]. However, it must be noted that there have been studies reporting contradictory findings of inverse relationship between IL-22 and lipid resorption [[96],[166]]. This suggests that although there is a thread of events linking the neurotransmitters VIP and Gal with ILC3s and fat accumulation, the understanding of ILC3s and fat metabolism is at its nascent stage and more studies in future are necessary to unravel them fully.

Another example of the complex cross interaction between neuro-immune-metabolic pathways is during the obese phenotype that exemplifies type-2 diabetes. Under normal physiological condition, feeding induces leptin release by the adipocytes, which can be sensed locally by the POMC neurons expressing leptin receptors (LepR) in the white adipose tissue [316]. The neuropeptide melanocortin cleaved off from POMC decreases food intake and stimulates sympathetic nervous system in the adipose tissue. GLP-1 is another neurohormone released post-prandially which induces the sympathetic activity in the adipose tissue [317] and the release of insulin [318]. Interaction of norepinephrine released by the sympathetic nerves with its receptor (adrenergic receptor ß3; ß3-AR) induces lipolysis generating free fatty acids, as well as inhibition of insulin-driven release of leptin. However, diabetes characterized by leptin and insulin resistance, results in POMC down-regulation [205]. The loss of POMC peptides known to mediate anti-inflammatory signaling in immune cells, may be the link between the observed increase in macrophages in obese adipose tissue [228]. The macrophages enriched in the obese adipose tissue can degrade norepinephrine [320]. Following decreased POMC level and increased degradation by the macrophages, norepinephrine is reduced. Decreased sympathetic activity promotes fat accumulation, which further contributes to insulin intolerance.

A third instance outlining the importance of homeostatic neuro-immune-metabolic cross communication is given below. Excess availability of glucose during diabetes causes proliferation and activation the pro-inflammatory M1 macrophages, ILC1, T and B cells. These cells are responsible for the increased levels of pro-inflammatory cytokines. The M1 macrophages release TNF-α and IL-1ß, IL-6 [[321],[322]]. Activated B cells release TNF-α and IL-6 during diabetes [323]. The CD8⁺ T cells, Th1, Th17 cells release are responsible for increases in IFN-γ and Th17 [[324],[325],[326],[327]]. The ILC1s also contribute to higher levels of IFN-γ during diabetes [[328],[93]]. The systemically elevated proinflammatory cytokines bind to their cognate receptors on neurons causing sustained neuronal excitation and consequent loss of neurons [329]. Loss of neurons means lower levels of certain neuropeptides, such as POMC, CGRP and SP [[205],[305],[306]], which can influence not only glucose levels, but also immune responses. The affliction of diabetes, however, is the chicken-and-the-egg problem. It is not possible to pinpoint the perturbation first started in the neuro, immune or metabolic system.

Such alterations in all three networks of neuro-immune-metabolism are seen in diseases either thought of primarily as metabolic disorders, namely diabetes [[330],[331]], inborn errors of metabolism [[332], [333]], porphyria [[334],[335]] or considered as typical neurological conditions, namely, charcot marie tooth [[336],[337],[338]], spinal muscular atrophy [[339],[340]] or conventionally regarded as immune-related pathologies HIV infection [[341],[342]], Crohn’s disease [[343],[344],[345]]. It is therefore important to think of them as a single integrated system. Although, this may appear complicated to fathom, understanding a holistic picture of the cellular interactions is necessary to pin down causative pathogenic mechanisms underlying a disease, to appreciate the consequences of a scientific finding and expand the scope of a scientific study. Such studies will prove most useful while devising a treatment strategy