Essential Neuroscience in Immunology

Reflexes maintain health in organ systems

A neural reflex is defined by three components: first a sensory receptor capable of responding to a change in the environment; second a sensory or afferent arc that transmits action potentials into the nervous system; third a motor or efferent arc that sends action potentials from the nervous system back to the periphery to modulate the environment. Charles Sherrington, one of the fathers of neuroscience, proposed that this basic unit of reflex action provides a fundamental building block upon which an entire nervous system can be assembled (1, 2). Reflexes function at the speed of action potentials, which is sufficient to control the output of organ systems nearly instantaneously. The cardiovascular, gastrointestinal, respiratory, hepatobiliary, and renal organ systems are all regulated by reflexes that respond to changes in the internal milieu and send corrective and compensatory signals to maintain homeostasis. Reflex integration of physiological output has evolutionarily ancient origins, dating back to C. elegans, a nematode with 302 neurons and a rudimentary immune system (3). Reflexes in mammals and vertebrates evolved to exquisitely maintain organ homeostasis despite a wide range of environmental variability and threat.

One example that perhaps best illustrates the function of a simple reflex controlling the output of a complex system can be found in the neural control of heart rate. The principle sensory nerve to the visceral organs, including the heart and reticuloendothelial system, is the vagus nerve. It is a paired structure, arising in the brain stem that descends through the thorax and abdomen to innervate the visceral organs. Comprised of 80,000–100,000 fibers in larger mammals, 80% of these neurons traveling in the vagus nerve are sensory. During exercise or physical exertion, heart rate increases, and this increase in resting heart rate is sensed by the afferent fibers traveling in the vagus nerve. Activation of this sensory arc in the brain stem activates motor fibers traveling in the efferent arc that descends to the cardiac sinoatrial node. Arrival of action potentials at the heart prolongs the time to subsequent heartbeats, thereby slowing heart rate. Simple reflexes, as in the regulation of heart rate, are activated when the output of the organ system deviates from a set point.

Within the immune system, sensory and motor nerves, the fundamental components of a simple reflex, were first identified in organs of the reticuloendothelial system more than 50 years ago. The thymus, lymph nodes, liver and spleen are all richly innervated by neurons capable of transmitting information to and from the nervous system in order to regulate the activity of hematopoetic cells during antigen exposure, infection, or sterile injury. Early studies of fever, inflammation, anorexia, and sickness behavior focused on products of inflammatory and immune responses modulating neuronal signals in the brain stem. For example, interleukin-1 (IL-1) was extensively studied as a mediator of fever capable of interacting with specific receptors expressed on neurons in the thermal regulatory centers of the brain stem hypothalamus (4–7). Activation of IL-1 receptor dependent signaling in the brain also mediates the development of anorexia and behavioral withdrawal, termed “sickness syndrome” (8, 9). These and other studies with other cytokine mediators established the dogma that cytokines produced in the peripheral tissues that gain access to the brain, or cytokines that are produced locally in the brain by astrocytes and microglial cells, are capable of mediating sickness behaviors and changing the neurological status of the host. Seminal studies by Linda Watkins soon presented a significant challenge to this simple dogma (10).

The Inflammatory Reflex

In the early 1990’s, when Watkins and her colleagues administered IL-1 to rats via the intraperitoneal route they observed, as expected, the onset of hyperthermia (10, 11). When they repeated these experiments in animals subjected to vagotomy, in which the connections in the vagus nerve to the brain were cut, they found that administration of IL-1 failed to produce a fever. This gave direct evidence that sensory signals traveling in the vagus nerve participates in the onset of the fever response (10). Independent work by Niijima demonstrated that the presence of IL-1 in the liver activates ascending signals in the vagus nerve that are transmitted to the brainstem, then relayed as efferent signals descending in the vagus nerve to the spleen, thymus and other organs (12–14). Together, these findings suggested that cytokines can activate functional afferent neural signals to the brain stem. An independent study and an unexpected experimental result in our laboratory led to the discovery of the inflammatory reflex.

We had developed a cytokine blocking molecule (CNI-1493) (15–18) and were studying its effects in the brain of animals with stroke, when we made the striking observation that production of TNF and other cytokines in the reticuloendothelial system was inhibited by the presence of CNI-1493 in the brain (16). Accordingly, we postulated that signals descending from the brain in the vagus nerve comprise a previously unknown motor arc in an “inflammatory reflex”, completing a sensory arc activated by the presence of IL-1 and other cytokines (19). We repeated the experiments after cutting the vagus nerve, and observed that CNI-1493 in the brain no longer inhibited cytokine production in the reticuloendothelial system (16). The inescapable conclusion from these experiments was that signals descending from the brain stem to the immune system in the vagus nerve regulated cytokine production.

We then began to directly explore the molecular and neurophysiological mechanisms using nerve stimulators positioned on the cervical vagus nerve in anesthetized animals during endotoxemia. The results defined the “inflammatory reflex,” a neural reflex circuit of action potentials traveling in the sensory and motor vagus nerve that regulate cytokine production in the spleen (19–22). The splenic nerve is an adrenergic nerve, producing norepinephrine in the vicinity of lymphocytes in the spleen (23). A discrete subset of lymphocytes regulated by the splenic nerve signals express choline acetyltransferase, the rate-limiting enzyme in the biosynthesis of acetylcholine (23, 24). Acetylcholine released by lymphocytes under the control of adrenergic splenic nerve signals inhibits macrophage cytokine release, and shifts them towards an M2 anti-inflammatory, tissue protective phenotype. Acetylcholine-induced signal transduction in monocytes/macrophages is mediated by alpha-7 nicotinic acetylcholine receptors (25) which inhibits the nuclear translocation of NFkB (26), activates JAK2/STAT3 pathway (27) and stabilizes mitochondrial membranes and downregulates the activity of the inflammasome (28) (Figure 1). The net effect of vagus nerve mediated signals within the spleen is the inhibition of cytokine release by red pulp and marginal zone macrophages, that together normally account for 90% of the TNF and IL-1 produced during acute endotoxemia (23, 29).

These mechanistic insights have enabled studies of selective nerve stimulation of the inflammatory reflex to block innate immunity and inflammation in animal models of sepsis, endotoxemia, collagen induced arthritis, hemorrhagic shock, ischemia reperfusion injury, autoimmune myocarditis, ileus, and colitis (30–40). A comprehensive and unabridged review of these studies is beyond the scope of this review, but this body of work enabled the development of a clinical trial for humans with rheumatoid arthritis. As reported quite recently, stimulation of the inflammatory reflex in rheumatoid arthritis patients significantly inhibited cytokine production and provided therapeutic benefit in two cohorts of patients, those resistant to methotrexate therapy, and those resistant to combined therapy with methotrexate and biological inhibitors of cytokines (41). This first in man study validated the experiments from the basic science laboratories and applied these concepts to patients.

What can be learned from the principles of the inflammatory reflex, and how can these be applied to other questions in immunology? In essence, these studies indicate that neurons have a highly conserved role in modulating the development of immunity. By approaching fundamental questions in immunology from the perspective of neuroscience, a wealth of hypotheses become testable. By focusing experimental design around principles of basic reflex circuits it becomes plausible to address the role of immunological mechanisms in activating sensory neurons, in modulating neurological signaling, and in responding to input from motor neurons. As reviewed in brief here, including the neuron as an active participant in the development of innate and adaptive immunity provides new understanding and new opportunities for therapy (Table 1, Figure 2).

Splenic neural circuits modulating antibody production

Lymphocytes and monocytes express alpha-adrenergic (α-ARs) and beta 2-adrenergic receptors (β2AR), and binding of norepinephrine to its receptor modulates cell homing, proliferation, and immune cell function. Catecholamines have been implicated in diverse responses to B cell activation and antibody production. Exposure of naïve B cells to norepinephrine can either increase (42) or decrease (43) Th2 (T helper 2) cell-dependent antibody production. Norepinephrine-mediated modulation of B cell antibody production requires signaling via β-ARs, but not α-ARs (44). Maturity states of B cells further govern the effects of norepinephrine on antibody production: activation of β2AR in early stages of B cell activation may increase the number of antibody-secreting cells or the level of antibody produced by antibody secreting cells, whereas B cell differentiation and function is inhibited by β2AR stimulation in more mature cells (45, 46). Animals lacking β2AR expression have normal lymphoid organ cell number/phenotype/histology, contact sensitivity responses, and T cell-dependent antibody responses as compared to wild type animals. B cells devoid of β2AR fail to respond to norepinephrine, indicating that other adrenergic receptors do not compensate for the absence in β2AR expression (47). Together, these data suggest that in the absence of β2AR, other non-adrenergic mechanisms can maintain immune homeostasis.

Reflex neural networks in lymphoid organs maintain the integrity of antibody responses. During pneumococcal invasion, neutrophils, monocytes and dendritic cells capture and transport the bacteria to the spleen, where resident antigen-specific marginal zone B cells and B1 cells are activated to differentiate into antibody-secreting cells. Marginal zone B cells migrate along splenic nerves, which secrete norepinephrine as the neurotransmitter, arriving at the red pulp venous sinuses where they become antibody-secreting cells, releasing antibodies into the blood (48). Adrenergic splenic neurotransmitter release is enhanced by stimulation of the vagus or splenic nerves, providing a critical regulatory step in splenic B-cell activation and migration that is key to the efficient secretion of antibodies (48).

As noted earlier, the adrenergic splenic nerve also modulates the activity of choline acetyltransferase expressing T cells that produce acetylcholine in spleen, and are necessary to suppress cytokine production by splenic macrophages under the control of the inflammatory reflex (24). Activation of the inflammatory reflex by direct electrical stimulation of the vagus nerve or administration of nicotine arrests neutrophil, dendritic cell and B-cell migration, and decreases antibody secretion. Chemical ablation of the adrenergic splenic nerves decreases antibody titers significantly, indicating that adrenergic signals modulate B cell activation and antibody responses. Importantly, ablation of splenic nerves suppresses the inhibitory effect of nicotine on antigen production, indicating that both cholinergic and adrenergic signals modulate B cell antibody responses. Detailed analysis of the neural regulation of antibody responses indicates that cholinergic stimulation induces accumulation of antigen specific activated B cells in the marginal zone (48). The implications of these results extend well beyond antibody production to other adaptive immune responses, because neural signals also control egress of immune cells from the bone marrow (49).

Vagus nerve-adrenal reflex

Torres-Rosas and colleagues (50) mapped a discreet immunoregulatory neural reflex that utilizes cholinergic and dopaminergic neurons. By subjecting animals to either adrenalectomy, cervical or subdiaphragmatic vagotomy, they mapped a sciatic to vagus neural reflex circuit that regulates inflammatory responses. Electrical stimulation of the sciatic nerve sends sensory signals to the brainstem that un turn activate efferent vagus nerve firing to the adrenal medulla. Vagus nerve mediated activation of the adrenal medulla increased production of dopamine that suppresses systemic inflammation to improve survival in animals with lethal sepsis by signaling through dopaminergic type 1 (D1) receptors (50).

Strikingly similar findings were obtained in other models of inflammatory diseases. Electrical stimulation of the sciatic nerve significantly reduced circulating levels of cytokine in a murine endotoxemia model in a vagus nerve dependent manner. Blocking of vagus nerve activity with either pharmacological antagonists or surgical ablation suppressed the inhibitory effects of electrical stimulation (51–53). In a collagen-induced arthritis model, electrical stimulation of the sciatic nerve significantly reduced arthritis incidence, attenuated inflammatory cytokine levels, and prevented joint destruction (54). More recently, activation of the sciatic nerve using low-power laser therapy significantly reduced localized inflammation in a murine paw edema model (55). The precise route by which the activation of the sciatic nerve transmits the signals to the brainstem is not known, but may involve sensory neurons in the sciatic nerve, the lumbosacral plexus, the vagus nerve, and a terminal dopaminigeric neurotransmitter. It is interesting to consider the therapeutic implications of a sciatic-vagus-neural circuit because it may be possible to stimulate afferent neural responses in the sciatic nerve to induce anti-inflammatory efferent vagus nerve signaling that will suppress subsequent inflammatory responses.

Enteric neural reflexes

Macrophages are anatomically stratified in the layers of the intestinal wall, and play a vital role in maintaining tissue homeostasis through scavenging bacteria and foreign antigens (56). In the gastrointestinal tract, which is continuously exposed to microbial products and dietary antigens, macrophage activation is tightly controlled to prevent chronic inflammation and tissue damage. Three recent studies provide unexpected evidence that enteric neurons reflexively modulate functional phenotypes of the intestinal macrophages under normal physiological conditions and in bacterial infection. In a murine model of postoperative ileus, electrical stimulation of the vagus nerve attenuated inflammation and improved postoperative intestinal transit. Anterograde tracing of vagus nerve to the gut demonstrated a dense network of cholinergic vagus fibers in close proximity to muscularis macrophages. Acetylcholine released by cholinergic myenteric neurons directly modulated inflammatory responses by muscularis macrophages, and activation of alpha 7 nicotinic receptors suppressed inflammatory responses in the muscle layer (57). Enteric neurons regulate homeostasis of muscularis macrophages through the production of colony stimulating factor-1, a growth factor necessary for macrophage survival (58). Muscularis macrophages, in turn, secrete bone morphogenetic protein (BMP)-2, which activates the BMP receptor (BMPR) expressed by enteric neurons. BMPR-mediated activation of enteric neurons is essential for maintaining proper tissue function. Selective ablation of the muscularis macrophage population is correlated with reduced neuronal activation of SMAD1, SMAD5 and SMAD8, the downstream signaling mediators of BMP receptors, resulting in impaired contractility. Interestingly, antibiotic-treated mice expressed significantly less Bmp2 as compared to control; indicating that luminal microbiota regulate the neuronal interaction with macrophages.

Recent studies identified a novel reflex mechanism in the gut activated by the gut microbiome that forces macrophages into a tissue protective phenotype (59). Intestinal resident macrophages exhibit distinct cell dynamics, morphological features and functional phenotypes depending on their proximity to the gut lumen. Lamina propria macrophages reside closer to the gut mucosa, and preferentially express a proinflammatory M1 phenotype. In contrast, muscularis macrophages are located more distal from luminal surface in the submucosal region between the circular and longitudinal muscle layers, and primarily express a M2 tissue protective gene profile. Muscularis macrophages share a niche with a dense network of neurons. Luminal bacterial infection results in activation of extrinsic adrenergic neurons innervating the intestine. Norepinephrine released by these neurons in turn activates development of a tissue protective phenotype in muscularis macrophages by signaling through β2 adrenergic receptors. Earlier studies using electron microscopy and immune staining have suggested that muscularis macrophages form direct synapses with enteric neurons (60, 61). Thus, the bacterial antigen mediated tissue protective program in muscularis macrophages is regulated by adrenergic neural circuits.

Sensory axon-axon reflex

Bacterial-derived N-formylated peptides or toxins interact with receptors expressed on nociceptor neurons (62). Immune cell activation is not necessary for bacteria-induced hyperalgesia during infection. Moreover, direct binding of bacterial products to nociceptor neurons induces an axonal-axonal reflex mechanism that modulates innate immune responses. Activated nociceptor neurons release neuropeptides, including galanin, somatostatin and calcitonin gene-related peptide (CGRP) that directly suppress innate immune activation in the region of infection (62). Accordingly, ablation of nociceptor neurons increases immune cell influx and lymphadenopathy. It is plausible that pathogenic bacteria utilize this neural mechanism to enhance virulence. Gram-negative bacteria induce hyperalgesia by directly activating TRPA1 expressing nociceptors. Lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria, activates and depolarizes TRPA1 expressing nociceptors in a TLR4-independent manner, and induces hyperalgesia (63). LPS-induced hyperalgesia may also play a major role in chronic pain and neuropathy observed in diabetic patients. Pattern recognition receptors, including TLR4 and CD14, which are normally expressed on immune cells, are also expressed on sensory neurons (64–66). Lipopolysaccharide binds directly to sensory neurons in a TLR4 dependent manner, and induces production of nociceptin (67), an opioid-related peptide that is upregulated during inflammation and associated with hyperalgesia (67–69). LPS binding also triggers activation of sensory neurons to enhance production of the vasodilator CGRP from dorsal root ganglion (70, 71). Together, these studies suggest that direct activation of nociceptors by bacterial products participate in the molecular mechanisms of pain and stimulate reflex regulation of innate immunity.

Nociceptive reflexes in lung

The lung is innervated by a rich network of sensory neurons that express nociceptive receptors (72–74). Activation of nociceptors enhances the release of neuropeptides, including CGRP and substance P. These mediators increase vascular permeability and vasodilation, and activate innate and adaptive immune cells. Recent evidence indicates that by-products of allergic inflammatory response activate lung nociceptors to modulate allergic lung inflammatory responses (75). Specifically, IL-5 released by activated immune cells induces the release of vasoactive peptide VIP by Nav1.8 positive nociceptors. VIP in turn activates CD4+ T cells and Type 2 innate lymphoid cells in a VIP receptor (VPAC2) dependent manner to produce elevated levels of IL-5, IL-13 and other cytokines. VIP signaling through VPAC2 activates and recruits immune cells to amplify the inflammatory responses in the lung. Silencing of nociceptors by either genetic ablation or chemical blocking significantly inhibits cytokine production, and increases resolution of allergic lung inflammation. These studies delineate neural circuits that drive a feed-forward amplification loop to exacerbate allergic lung inflammation opening new avenues for developing therapeutic strategies.

A gateway reflex

The blood brain barrier strictly limits the flow of proteins and cells from the blood stream to the central nervous system (CNS). In a mouse model of multiple sclerosis, Arima and colleagues (76) identified a specific neural circuit that modulates trafficking of pathogenic T cells into the CNS. In this adoptive transfer model of experimental allergic encephalomyelitis, myelinated nerve specific T cells were introduced into naïve recipient mice to maintain CNS quiescence. At the earliest phase, autoreactive T cells preferentially accumulated at specific sites in the spinal cord near the fifth lumbar level, rather than in the brain or at other levels of the spinal cord. In an attempt to elucidate the mechanism for this specificity of the “entry gateway”, they discovered that the expression of CCL20, a chemokine important for T cell accumulation, is increased in the dorsal blood vessels of the fifth lumbar cord. As the fifth lumbar vertebrae lies close to the dorsal root ganglia of sensory neurons that innervate the soleus and other leg muscles, they reasoned that weight bearing stimulation of the soleus muscles might contribute to the sensory nerve activation resulting in induction of high CCL20 expression in the blood vessels of the fifth lumbar cord. They studied this possibility by silencing the muscle sensitive neuronal pathway by suspending mice from their tail, so that forelimbs, and not the hind limbs, were subjected to weight bearing. This significantly decreased CCL20 expression and suppressed pathogenic T cell accumulation in the fifth lumbar cord level. Instead, these pathogenic T cells accumulated at the cervical cord, indicating that stimulation of the forelimbs opened a new gateway for the pathogenic cells. Moreover, pharmacological inhibition of adrenergic receptors significantly suppressed accumulation of pathogenic T cells, and improved clinical signs of the disease. Together, these studies reveal a reflex circuit that controls a critical step in the initiation and progression of autoimmune disease by regulating blood brain barrier permeability to pathogenic T cells.

Neural reflexes in cancer

Neural circuits play a critical role in both the development and spread of tumors. In an experimental model of prostate cancer, adrenergic nerves promote tumor growth by producing norepinephrine, which binds to and stimulates adrenergic receptors (β2 and β3) on the stromal cells in the tumor microenvironment. In addition, cholinergic nerves within the tumor tissue enhance tumor metastasis by releasing acetylcholine, which activates the stromal type 1 muscarinic receptor, and enhances prostate cancer metastasis to distant lymph nodes (77). Recent observations in patients with prostate cancer support the preclinical data, and strongly suggest that neural circuits are critical for tumor development and metastasis (77). Higher neuronal density is observed within tumors and in normal prostate tissue surrounding tumors in patients with aggressive prostate cancers as compared to patients who had less aggressive tumors. Specifically, normal prostate tissue surrounding the tumor is densely innervated with TH+ adrenergic fibers, whereas VAChT+ cholinergic fibers are restricted inside the tumor. High neural densities of adrenergic fibers are associated with a higher tumor proliferative index, shorter recurrence time, and tumor metastasis, whereas cholinergic fiber density is significantly associated with extra-prostatic extension. These observations correlate with the epidemiological data showing that beta-blockers, which lower blood pressure by blocking β-adrenergic receptors, is associated with improved survival in prostate cancer (78, 79).

Neural reflex circuits have also been implicated in development and progression of other cancers. Denervation of the stomach markedly reduced tumor incidence and progression in preclinical models of gastric cancer (80). In rodent models of melanoma, lung cancer and ovarian cancer, sustained adrenergic activation promotes cancer growth and metastasis (81–84). In addition to their effect on tumor cells, norepinephrine and epinephrine also stimulate endothelial cells, fibroblasts and modulate functions of immune cells, leaving open a broader potential impact of neurotransmitters on the tumor microenvironment (85). Several retrospective clinical studies have revealed that beta-blockers may have antitumor activity, reducing metastasis, tumor recurrence, cancer-specific mortality and increasing survival time for patients with melanoma, breast cancer, lung cancer, ovarian cancer and prostate cancer (78, 86–90).

Conclusions

The discovery that neurons operating reflexively are functional components of the immune system represents a fundamentally new insight into understanding of immunity. Cells of hematopoetic origin share the frontline of host defense with neurons. Monocytes and neurons both detect changes in the environment. Macrophages can respond by producing cytokines and other mediators, and neurons respond by producing action potentials. Neurons and macrophages indeed share the responsibility for mobilizing host responses to infection and injury, and share responsibilities for producing a coordinated and balanced immune response. Highly conserved neural circuits act reflexively to maintain homeostasis during the development of immunity, and inflammation. This discovery has raised many more questions that future students of immunology will likely approach, expanding their research focus from cells of hematopoetic origin to include neurons. These new studies of immunology that exploit essentials from neuroscience will continue to enlighten our understanding of basic principles governing immunity.