The Neuro-Immune Axis in Skin Sensation, Inflammation, and Immunity

Abstract

While connections between the immune and nervous systems have long been recognized, the precise mechanisms that underlie this reciprocal relationship are just starting to be elucidated. Advances in sensory biology have unveiled novel mechanisms by which inflammatory cytokines promote itch and pain sensations in order to coordinate host-protective behavioral responses. Conversely, new evidence has emphasized the critical ability of sensory neurons to regulate immune cell function. By focusing on the nascent field of itch biology and how it has been informed by the more established conceptual framework of pain, we highlight recent interdisciplinary studies that demonstrate how novel neuroimmune interactions underlie a diversity of sensory, inflammatory, and infectious diseases.

Introduction

The skin is one of the first lines of defense against chemical, mechanical, microbial, and thermal insults. While the immune system is an essential component of cutaneous immunity, it is increasingly evident that the sensory nervous system also plays a critical role in host defense. By evoking sensations such as pain and itch, the organism can immediately sense danger and rapidly initiate a protective behavioral response. Additionally, emerging evidence suggests sensory neurons further aid the body’s response to potentially harmful agents by directly modulating immune cell function through the release of mediators such as neuropeptides. In this review, we highlight recent advances in our understanding of how the sensory nervous system responds to and, in turn, regulates the immune system in the setting of cutaneous inflammation and immunity. Specifically, we will focus on itch biology and the studies in pain that helped to inform this burgeoning field of neuroimmunology (for more comprehensive reviews on pain and itch see (1-5)).

Neurophysiology of itch

The primary somatosensory neurons that innervate the skin are pseudounipolar, which means that their axon consists of two branches: one that synapses at the dorsal horn of the spinal cord (or brainstem) and another that innervates the peripheral tissue. Its cell body is housed in a peripheral ganglion, either the trigeminal ganglion (TG) if the nerve innervates the face/oral cavity or the dorsal root ganglion (DRG) if it innervates elsewhere in the body. Itch is sensed following the activation of receptors at peripheral sensory nerve endings in the skin, which ultimately triggers the opening of nonspecific cation channels such as transient receptor potential A1 (TRPA1) or TRPV1 (Figure 1) (6). The resulting membrane depolarization, if sufficient, results in the opening of voltage-gated sodium channels, such as Na_V1.7 and Na_V1.8, which initiate and propagate the action potential (Figure 1) (7). These signals are then rapidly transmitted by the peripheral sensory neuron to the spinal cord, where neurons of the central nervous system (CNS) relay the signal to the brain in order to evoke sensory perception. Yet while sensory neurons primarily transmit afferent information from the skin to the CNS, they can also release mediators (e.g. neuropeptides) in an efferent manner (such as through an axonal reflex) in order to communicate with other cell types in the peripheral tissue.

Figure 1. Neuronal Cytokine Receptors Exhibit Specialized Functions for Pain and Itch.

While some ligands such as histamine demonstrate significant overlap in terms of their ability to evoke pain and itch, to date, there appears to be distinct differences between what cytokines drive pain or itch. Cytokines like IL-1β, IL-6, IL-17A, and TNF-α are predominantly associated with pain-sensory responses, while cytokines like IL-4, IL-13, IL-31, IL-33, and thymic stromal lymphopoietin (TSLP), are predominantly associated with itch. Following the activation of specific nociceptive or pruriceptive pathways, a number of overlapping cation channels are activated at the neuronal membrane including TRPA1, TRPV1, Nav1.7, and Nav1.8. IL-1R, IL-1 receptor binds IL-1β; IL-17RA, IL-17 receptor A binds IL-17A; gp130, combines with soluble IL-6 receptor to bind IL-6; TNFR1, tumor necrosis factor receptor 1 binds TNF-α; IL-31RA, IL-31 receptor A binds IL-31; IL-4Rα, IL-4 receptor alpha binds IL-4 and IL-13; ST2, IL-33 receptor binds IL-33; TSLPR, thymic stromal lymphpopoietin receptor binds TSLP; TRPA1, transient receptor potential ankyrin 1; TRPV1, transient receptor potential vanilloid 1; Na_V1.7 and Na_V1.8, voltage-gated sodium channel 1.7 and 1.8.

Delineating the precise identity of skin-innervating neurons involved in itch transmission is a highly active area of investigation as somatosensory neurons are remarkably diverse. Traditionally, somatosensory neurons are classified based their size, conduction velocity, and degree of myelination. Itch is believed to be largely transmitted by small, unmyelinated, slow conducting c-fibers, although thinly myelinated, Aδ fibers may also play a role (8). Both of these fibers also transmit pain. Itch was originally believed to be mediated by the same neurons that signaled pain, with the intensity of neuronal firing coding which signal is transmitted (9). Indeed, both itch- and pain-sensory neurons employ many of the same ion channels to transmit their signals including Na_V1.7, Na_V1.8, TRPA1, and TRPV1 (10). However, through the discovery of gastrin-releasing peptide receptor (GRPR), Mas-related G protein-coupled receptor (Mrgpr), and the neuropeptide Nppb, it is now recognized that there are specialized pathways that can distinctly mediate itch in the periphery (4, 5, 11-14). Currently, the expression of these hallmark receptors and neuropeptides is used to classify itch-sensory neurons. Recent studies employing single-cell RNA-sequencing of DRG neurons have begun to unveil a more comprehensive classification of itch-sensory neurons, proposing the existence of a number of different subsets (15, 16). However, further work is needed to assess the in vivo functionality of these subsets and what modalities are distinct, shared, or synergistic.

Cytokines and itch

The immune response is organized into specialized effector modules that are tailored to combat different types of pathogens. Type 1 immunity is broadly utilized to combat infections involving intracellular bacteria and viruses, and is characterized by the production of the effector cytokines IFN-γ and/or TNF-α. The production of IL-17A and/or IL-22 is a hallmark of type 3 immunity, which is specialized for extracellular bacterial and antifungal defense. Finally, parasitic infections, along with noxious environmental substances, result in the generation of a type 2 immune response driven by the production of IL-4, IL-5, and IL-13 (17). While cytokines are central in coordinating these specialized immune responses, several of these mediators also modulate sensory perception and behavior, another key aspect of host defense (18, 19). The early discovery that the canonical proinflammatory cytokine IL-1β can induce pain led to a significant paradigm shift in our understanding of how the immune system participates in sensation and behavior (20). Subsequently, over the past decade, a number of cytokines spanning these specialized immune responses have been discovered to elicit either pain or itch by directly binding to their receptors expressed on sensory neurons. This provokes the hypothesis that the specific sensory response that is evoked depends on the type of pathologic insult and the resulting immune response that is generated.

Building on the initial discovery that IL-1β can induce pain in vivo, a number of studies have identified additional cytokines that can also elicit pain by modulating sensory neuronal signaling (1). For example, in a rodent model of bone cancer-induced pain, IL-6 was found to critically regulate neuronal hyperexcitability as well as increased sensitivity to pain (hyperalgesia) (21). TNF-α was also found to be important in mediating hyperalgesia, specifically in the setting of nerve injury (22). Finally, in another study, injection of IL-17A was found to induce pain in the joints of rats in a TNF-α- and IL-6-independent manner and treatment with an anti-IL17 antibody (Ab) reduced pain symptoms in a model of arthritis independently of effects on joint swelling (23). Taken together, a number of cytokines associated with type 1 and type 3 immune responses including IL-1β, IL-6, TNF-α, and IL-17A have been found to modulate neuronal signaling in various models of pain behavior (Figure 1) (1). However, it is important to note that much of the early literature on neuroimmune regulation of pain is derived from the study of other tissues than the skin such as the bone, joint, and nerve.

In contrast to pain which typically involves deeper structures, itch is predominantly confined to the skin. Similar to pain, a number of different cytokines have been found to mediate itch, however in contrast, the itch-associated cytokines thus far identified are all associated with a type 2 immune response. Poised to respond to environmental insults, keratinocytes are key initiators of a host-protective immune response through the production of alarmins or epithelial cell-derived cytokines such as IL-33 and thymic stromal lymphopoietin (TSLP). These cytokines potently activate a variety of both innate and adaptive immune cell populations, which results in a robust type 2 immune response characterized by the production of IL-4 and IL-13. However, in addition to driving type 2 immunity, it was recently shown that IL-33 and TSLP can directly activate sensory neurons to evoke itch (24, 25). However, whether these cytokines are the key mediators of itch in type 2 inflammatory skin disorders such as atopic dermatitis (AD) remains to be clearly defined. Thus, epithelial cell-derived cytokines, in response to epidermal stress or disruption, have the capacity to simultaneously and rapidly activate both innate immune responses and scratching behavior.

Downstream of IL-33 and TSLP, a number of cell populations are elicited to produce IL-4 and/or IL-13 including basophils, eosinophils, group 2 innate lymphoid cells (ILC2s), mast cells, and T helper type 2 (Th2) cells (26-29). These effector cytokines, in addition to their well-known role in promoting barrier inflammation, were recently shown to modulate itch responses in mice (30). IL-31, which is predominantly produced by Th2 cells, also is an important mediator of itch in vivo (31, 32). However, in contrast to IL-4 and IL-13, IL-31 may not play a prominent role in driving cutaneous inflammation. While IL-31-deficient mice have reduced scratching behavior compared to controls, they appear to have similar levels of skin inflammation in a mouse model of contact hypersensitivity (33). In support of this concept, anti-IL-31RA monoclonal Ab (mAb) treatment (nemolizumab) appeared to preferentially target symptoms of itch rather than inflammation in AD patients in a recent phase 2 clinical trial (34). In contrast, inhibition of the shared receptor subunit for IL-4 and IL-13 (anti-IL-4Rα mAb, dupilumab) resulted in a dramatic reduction in both overall disease severity (i.e., cutaneous inflammation) as well as itch in phase 3 clinical trials for AD (35). In light of the complex network of cytokines involved in promoting type 2 skin inflammation and itch, futures studies will be required to determine how these cytokines come together to specifically modulate itch in the setting of different inflammatory skin disorders.

Broadly, cytokines that underlie type 1 and/or type 3 immune responses such as IL-1β, IL-6, TNF-α, and IL-17A (1, 21-23) have been associated with pain, while those associated with a type 2 immune response such as IL-4, IL-13, IL-31, IL-33, and TSLP involve itch. Additionally, many diseases associated with type 2 inflammatory features are highly pruritic, such as AD, acute and chronic urticaria, and prurigo nodularis (36). Although some skin conditions associated with type 1 and/or type 3 immune responses are pruritic, such as allergic contact dermatitis (ACD), psoriasis, and superficial fungal infections, whether effector cytokines specifically associated with these types of immune responses can act as pruritogens remains poorly defined and is an exciting area of inquiry. However, based on the current body of work in sensory neuroimmunology, we speculate that specialized immune responses specifically evoke the protective behavioral response of either pain or itch depending on the environmental stimulus. Pain responses appear to be more commonly associated with bacteria where aversion to movement may be needed to minimize the spread of infection (e.g., sepsis) and promote healing, while the scratching response to itch sensation may aid in the expulsion of larger ectoparasites and noxious environmental substances.

Acute versus chronic itch

While acute itch is likely a protective behavioral response, chronic itch is a highly debilitating medical disorder (37). A current focus of the itch field is identifying pruritogens, molecules that directly activate sensory neurons to induce itch. A standard technique used to identify such molecules is the injection of a putative pruritogen intradermally into the skin. Potential pruritogens are often injected into the cheek skin in order to allow researchers to distinguish itch from pain behavior, which are defined as hind limb scratching and front paw wiping, respectively (38, 39). While this technique has been extremely valuable in identifying key pruritogens, it is important to note that it is an acute itch model. Scratching bouts are evoked within several minutes of introducing the stimulant into the skin and typically last for under an hour. Thus, although it is a very powerful and efficient technique, it may have potential limitations in defining important mediators of chronic itch, where spontaneous scratching commences independently of acute stimuli. These include both genetic and chemically-induced models of chronic itch conditions such as allergic contact dermatitis, dry skin, and AD where itch that can last days to weeks (30, 31, 40-42).

A classic example where a potent mediator of acute itch may not play a key role in driving chronic itch is histamine. Although a canonical pruritogen that was used to validate the intradermal cheek model (38), antihistamines are generally poorly efficacious in many chronic itch disorders such as AD (43). Conversely, IL-4 and IL-13 are poor acute pruritogens and yet, they are critical drivers of chronic itch in the setting of AD-like disease in mice through their direct activity on sensory neurons. This appears to be due to the ability of these cytokines to sensitize neurons to other pruritogens like histamine, IL-31, and TSLP (30). Cytokines thus may have additional direct roles in modulating itch beyond the immediate induction of itch signaling. Collectively, these studies demonstrate that understanding how various models of acute and chronic itch function can bring novel insight into the biology and clinical implications of the pathways being studied.

Cytokine signaling in neurons: Janus kinases (JAKs)

The intracellular signaling pathways downstream of cytokine-receptor binding on sensory neurons and how they mediate specific sensations is an exciting area of research that is just starting to be elucidated. One signaling pathway that was found to alter neuronal excitability downstream of IL-1β and TNFα is phosphorylation of Na_V1.8 via p38 MAPK (44-46). However, in contrast to the IL-1 and TNF families, it is well known that in immune cells many of cytokine receptors utilize JAKs to activate STAT transcription factors. Similarly, it appears that neurons utilize JAKs. For example, JAK phosphorylation has been found to be elevated in neurons following spinal cord injury in mice (47). However, the functional significance of JAK signaling in sensory neurons was not entirely clear until more recent studies in itch biology.

Oetjen et al. demonstrated that neuronal JAK1 is a critical mediator of chronic AD-associated itch in vivo (30). However, the signaling pathways that are activated in neurons by JAKs remain poorly defined. In contrast to classical JAK-STAT signaling in lymphocytes, it is likely that cytokine-mediated stimulation of sensory neurons results in STAT-independent effects given that cytokine stimulation of isolated DRG neurons by IL-4 and IL-13 results in rapid neuronal activation as indicated by a calcium influx within seconds to minutes following cytokine application (30). Given the dependence of this calcium response on TRP channels, we speculate that JAKs may either directly or indirectly influence calcium influx into the cell by modulation of these channels. How this occurs is an exciting area of inquiry. JAKs additionally appear to have long term effects on the excitability of neurons such as by altering the expression of TRPA1 and membrane trafficking of TRPV1 (21, 48). These JAK-dependent alterations in trafficking and transcription may be additional ways by which pain and itch sensitization occurs in the periphery and symptoms such as allodynia and alloknesis can develop. Thus, we hypothesize that, in addition to rapidly altering ion channel function through STAT-independent phosphorylation events, there may also be STAT-dependent transcriptional events that underlie the chronicity of pain and itch. We anticipate that neuronal JAK-induced signaling pathways may unveil novel biochemistry with regard to JAK signaling and have broad implications for the treatment of both chronic pain and itch disorders.

The significance of neuronal JAK1 signaling in itch is corroborated by clinical trials where topical JAK inhibitors have demonstrated rapid anti-itch effects within 24–48 hours of initial treatment in AD patients, prior to any detectable effect on clinical skin inflammation (49, 50). Additionally, in a small proof-of-concept study, we found that even in a condition where there is an absence of noticeable skin inflammation, chronic idiopathic pruritus (CIP) or generalized pruritus of unknown origin (GPUO) (51), patients experienced a rapid reduction in itch symptoms in response to a JAK inhibitor (tofacitinib) (30). The importance of neuronal JAK1 in mediating itch is further reinforced by the finding that patients with germline JAK1 gain-of-function (GOF) mutations develop chronic pruritus that is selectively responsive to treatment with a JAK inhibitor (ruxolitinib) over broader anti-inflammatory agents (i.e., systemic steroids) (52). Collectively, these studies show that investigating cytokine-neuronal interactions can led to novel therapeutic insights that can be exploited for treatment of itch, pain, and possibly other sensory disorders.

The role of peripheral nerves in driving inflammatory skin disorders

In contrast to other barrier surfaces, somatosensory neurons are the primary source of neuropeptides in the skin. Historically associated with signaling in neural tissues, it is now recognized that these small peptides can also act on other cell types such as keratinocytes and immune cells, provoking the hypothesis that sensory neurons directly and critically regulate skin inflammation (53). This hypothesis is supported by a number of observational studies which documented that patients with inflammatory skin disorders such as AD and psoriasis experienced disease resolution in body parts that had a loss of innervation (54-57). Similarly, in murine models of psoriasis, surgical denervation as well a chemical ablation of Na_V1.8⁺ TRPV1⁺ sensory nerve fibers also resulted in the improvement of disease severity (58-60). This improvement may be due to, at least in part, the loss of IL-23 production from dermal dendritic cells (dDCs) (60). Identifying which neuropeptides critically regulate these inflammatory processes is currently a highly active field of neuroimmunology.

Thus far, the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) have been strongly implicated in skin inflammation. Psoriasis-like inflammation in denervated skin can be restored by intradermal delivery of SP and CGRP (58). Specifically, SP appears to critically regulate immune cell recruitment while CGRP promotes epidermal hyperplasia (acanthosis) in this setting (58). Similarly, in the context of AD, sensory neurons have been shown to regulate human keratinocyte proliferation in a CGRP-dependent manner (61). While thus far CGRP and SP have been the primary neuropeptides linked to skin inflammation, additional neuropeptides have been found to critically modulate inflammation at other barrier surfaces although their role in the skin has yet to be clearly demonstrated. For example, recent studies have shown that ILC2s are regulated by neuromedin U (NMU) released from cholinergic neurons in the gut (62, 63) and somatosensory afferents in the lung (64). Vasoactive intestinal peptide (VIP) is another neuropeptide found to activate lung ILC2s upon its release from cholinergic neurons of the vagal nodose ganglia, which along with somatosensory neurons, also innervate the lung (65). Given that both NMU and VIP are expressed by somatosensory neurons, whether these neuropeptides also play an important role in regulating cutaneous inflammation is an exciting area of future inquiry (64, 66, 67). One study suggests that VIP can alter the capacity of Langerhans cells to present antigens and thus inhibit the generation of Th1 cell responses (68). Finally, while the primary focus of research in the skin thus far has concentrated on the role of neuropeptides, there is emerging interest in the ability of small molecule neurotransmitters in mediating cutaneous inflammation.

Pathogen-neuron interactions

Recent studies have found that sensory neurons, like immune cells, are able to directly sense and respond to microbes. One example is lipopolysaccharide (LPS), a major component of gram-negative bacteria and a key endotoxin that binds TLR4. Along with being immunostimulatory, LPS also directly activates sensory neurons to modulate both pain and itch signaling. While LPS injection is known to induce pain but not itch (69), one study found that TLR4 signaling promotes histamine-mediated itch by potentiating TRPV1 activity (70). Although studies have shown that sensory neurons detect LPS directly through TLR4 (71-73), others have found that LPS can directly stimulate sensory neurons in an TLR4-independent manner through mechanical perturbation of the neuronal membrane, resulting in the activation of TRPA1 (74, 75). Collectively, these studies demonstrate how bacterial endotoxins can directly manipulate the peripheral nervous system (PNS) to modulate sensation.

Pore-forming toxins (PFTs) are another class of virulence factors which, in contrast to classical endotoxins, are also produced by gram-positive bacteria that commonly cause bacterial skin infections such as cellulitis and necrotizing fasciitis. Notably, these infections are strongly associated with disproportionate levels of pain. Recent studies have shown that these PFTs can penetrate and activate sensory neurons directly to evoke pain (76, 77) (Figure 2). Specifically, α-toxin/hemolysin (HIa), phenol-soluble modulins (PSMs), and leukocidin HlgAB from Staphylococcus aureus (S. aureus) (76) and streptolysin S (SLS) from Streptococcus pyogenes (S. pyogenes) (77) were shown critically mediate bacterial infection-associated pain in the skin through direct neuronal activation. Thus, it appears that, in addition to stimulating the host inflammatory response, the bacterium can directly influence pain sensation and behavior through a variety of toxins, likely independently of pain-associated cytokines such as IL-1β, IL-6, IL-17A, and TNF-α. Future studies may reveal many more mechanisms by which bacteria and other microorganisms are sensed by sensory neurons and if these signals can modulate itch in addition to pain.

Figure 2. Pathogens Can Directly Activate Sensory Neurons to Release Neuropeptides in Skin Immunity.

Candida albicans (C. albicans) and pore-forming toxins (PFTs) released from bacteria like Staphylococcus aureus (S. aureus) and Streptococcus pyogenes (S. pyogenes) can activate sensory neurons. Stimulation of these sensory neurons results in the release of neuropeptides such as calcitonin gene-related peptide (CGRP) which can directly influence cutaneous immunity by acting on immune cells.

Sensory neurons mediate immunity

Similar to their contribution to inflammatory skin diseases, neurons also play an important role in modulating protective immunity. Recent studies have demonstrated that intact peripheral sensory innervation is critical for optimal production of IL-23 in order to drive antifungal immunity to Candida albicans (C. albicans). Administration of CGRP was sufficient to overcome the effects of denervation in this context (78). A study by Maruyama et al. suggests neurons detect C. albicans through the binding of neuronal Dectin-1 to β-glucan and this may stimulate the release of CGRP (Figure 2) (79). Whether neurons can sense other fungi and modulate immunity in a similar fashion remains an intriguing question. Similar to C. albicans, CGRP is also released by neurons upon S. pyogenes infection (Figure 2). However, while CGRP appears to be protective in the setting of C. albicans, it plays a detrimental role in S. pyogenes infection by suppressing neutrophil recruitment and antibacterial immunity (77). Ultimately, these studies bring forth a paradigm in which the sensory nervous system is both capable of sensing microbes directly and in turn shaping host immunity.

Conclusions

The skin harbors a vast neuro-immune network that is poised to provide a rapid, coordinated response to a variety of environmental insults. Recent advances covered in this review suggest that specialized protective immune modules may also encode highly specific sensory and behavioral responses. In the setting of pain, the withdrawal reflex (acute pain) and/or aversion to movement (prolonged pain) may help to promote wound healing, prevent the spread of infection, and conserve host metabolic resources. In contrast to pain, the scratching reflex may help to promote the expulsion of macro-parasites, toxins, and environmental irritants (80). Thus, scratching the skin in response to itch may parallel the “weep and sweep” responses promoted by type 2 inflammation in the intestine and airway. Finally, not only does the cytokine milieu dictate the sensation and behavioral response generated, sensory neurons in turn shape the immune response through the release of various neuropeptides. Collectively, advances in neuroimmunology demonstrate that the nervous system is an integral part of the overall immune response in both health and disease.

Three broad areas of investigation remain open to major advancement. First, the specific cellular sources of neuromodulatory cytokines during the course of an immune response and how these cells home towards and interact with specific sensory neurons remains poorly defined. Second, which chronic inflammatory and sensory disorders can be modulated by targeting specific cytokines and/or neuropeptides in human disease is an exciting area of therapeutic inquiry. Third, what specific mediators and signaling pathways drive the release of neuropeptides from neurons in the skin as well as mucosal surfaces remains largely unknown. Ultimately additional insights into these processes will likely open new avenues of therapeutic intervention for sensory, inflammatory, and infectious disorders of the skin and beyond.