The importance of the neuro‐immuno‐cutaneous system on human skin equivalent design

Abstract The skin is a highly complex organ, responsible for sensation, protection against the environment (pollutants, foreign proteins, infection) and thereby linked to the immune and sensory systems in the neuro‐immuno‐cutaneous (NIC) system. Cutaneous innervation is a key part of the peripheral nervous system; therefore, the skin should be considered a sensory organ and an important part of the central nervous system, an ‘active interface’ and the first connection of the body to the outside world. Peripheral nerves are a complex class of neurons within these systems, subsets of functions are conducted, including mechanoreception, nociception and thermoception. Epidermal and dermal cells produce signalling factors (such as cytokines or growth factors), neurites influence skin cells (such as via neuropeptides), and peripheral nerves have a role in both early and late stages of the inflammatory response. One way this is achieved, specifically in the cutaneous system, is through neuropeptide release and signalling, especially via substance P (SP), neuropeptide Y (NPY) and nerve growth factor (NGF). Cutaneous, neuronal and immune cells play a central role in many conditions, including psoriasis, atopic dermatitis, vitiligo, UV‐induced immunosuppression, herpes and lymphomas. Therefore, it is critical to understand the connections and interplay between the peripheral nervous system and the skin and immune systems, the NIC system. Relevant in vitro tissue models based on human skin equivalents can be used to gain insight and to address impact across research and clinical needs.


| INTRODUC TI ON
The skin is the largest organ in the body and one of the most complex, as a multi-layered, multiple cell type, multifunctional organ that serves as a key interface to the outside world. 1 The skin is composed of three main layers: epidermis, dermis and hypodermis (subcutis).
Each layer is composed of multiple cell types with unique and complementary functions to support homeostasis ( Figure 1, Table 1). The epidermis contains keratinocytes, melanocytes, Langerhans cells and Merkel cells. The dermis contains dermal fibroblasts, mast cells, vascular smooth muscle cells, specialized muscle cells, endothelial and immune cells. The hypodermis is composed of adipocytes, nerves and fibroblasts. The complex functional components of these layers include sweat glands, hair follicles, blood vessels and peripheral nerve endings (Aβ, Aδ, and C nerve fibres). [1][2][3][4][5][6][7][8][9][10][11] Efforts in skin research are typically divided into three areas of importance: clinical models, commercial in vitro testing and exploratory research ( Figure 2). Clinical research on skin focuses on the development of reliable human skin equivalents (HSEs) that can be used as dermal grafts, skin replacements, or wound coverings in acute cases, or for chronic cases that include diabetic ulcers or non-healing wounds. 12, 13 The general constraints for these models include that they must be biocompatible, integrate with the existing tissue beneath (ie subcutis/hypodermis), and interface with the surrounding tissue along the perimeter of the replacement, and they must be approved by the FDA, so material and regulatory concerns are met.
Clinical models have significantly advanced in the past decades along with the advent of tissue engineering, and currently, there are numerous options for clinicians to choose from, diverse in delivery format, in composition of cells or tissue, and in the choice of matrix material (Tables 2 and 3). The clinical research field is growing and expected to be valued at $24.3 billion USD by 2019. 3 However, as the demand continues to grow, there are various problems which are still pertinent to the clinical field, including rejection, scarring, size constraints and lack of integration with functional components of the skin (ie sensation may not return due to lack of innervation, hair follicles or pores may not develop). 12,13 Beyond clinical application, HSEs are also used for commercial applications (in vitro testing/diagnostics) for testing permeability, sensitization or toxicity studies ( Figure 2). 10 Typically, these HSEs only contain 2 or 3 cell types, keratinocytes and fibroblasts, and sometimes melanocytes. 10 Challenges with these systems include their differences from skin biology which impacts permeability and barrier functions, difficulty in recapitulating disease conditions or non-intact skin, and issues with biomaterial choices for the dermis; collagen hydrogels undergo contraction, deterioration, and can have homogeneity and reproducibility issues. 10,[14][15][16] Alternatives to in vitro HSEs include human explant tissue or animal models, however, there have been major efforts to develop relevant in vitro systems that circumvent ethical concerns, biological differences (animal vs human) and donor variation from explant tissue. 17,18 In vitro systems also provide opportunities to develop controlled experimental conditions or patient-specific/genetically engineered models. 19 Human skin equivalents as research tools are diverse in terms of applications, with wound healing as an example of a dominant focus in skin research. As tissue engineering has advanced, the capabilities of in vitro models have progressed with many formats including skin-on-chip (or as part of a multi-organ-chip), multi-compartment 2D or 3D devices, and monolayer or full-thickness models (Table 3).
However, most of these in vitro models still focus on only 2 or 3 cell types, generally keratinocytes and fibroblasts, with or without an additional cell type of interest (melanocytes, neurons, etc). While much of this work has been instrumental to the field, most in vitro tissue models do not address the NIC system because they lack the representative components. Ultimately, to discern the effects of cell types or components on the skin system, more complete in vitro tissue models are needed.

| NEURO -IMMUNO -CUTANEOUS (NIC) COMP ONENTS IN S K IN AND IN VITRO S K IN MODEL RE S E ARCH
The NIC system is a relatively new concept for inclusion in in vitro skin model research, although the connection from the brain, skin and host response has been studied with great interest across many fields (psychology, biology, engineering) for many decades. 20,21 The skin is a key organ to study the connection between the mind, nervous system and the host immune response, as the window to the outside world has tangible links between physical and mental health. 20 The interconnectedness of the NIC system, and the neuro-immuno-cutaneous-endocrine (NICE) systems, is founded by complex, and constant communication between neuropeptides, cytokines, neurotransmitters, small molecules and less defined processes like psychological stress, to maintain homeostasis in the skin ( Figure 3). 21 70 ; terminally differentiated keratinocytes of the outer epidermis play a role in immune modulation and also still communicate with stem cells, other skin cells and immune cells in the epidermal basal layer 71 • Keratinocytes of the stratum corneum produce lipids to serve, in part, as protective barrier layer but the microenvironment of the skin (ie lipid concentration, bacteria population/microbiota, moisturization) will be distinct with respect to location on the body 70,72 • Keratinocytes deposit keratins, proteins responsible for numerous processes distinct through differentiation stages of the keratinocyte that also add mechanical strength to the skin 10 • Keratinocytes have neurotransmitter receptors, respond to neuropeptide activity in the skin, and re-epithelialization can be stimulated via neuronal-keratinocyte signalling 31 Evidence of the interconnectedness and importance of a complex understanding of the skin has been reflected in several recent skin models or HSEs which address some components of the NIC or NICE system (Table 4). However, only one model system has addressed all components simultaneously. 16

| PERIPHER AL NERVE ANATOMY IN S KIN
Peripheral nerves consist of 2 types: afferent nerves (directed towards the central nervous system) and motor nerves (towards the peripheral nervous system). There are 3 main types of nociceptive nerve fibres: Aβ, Aδ and C fibres. 27 Nerve fibres refer to the axon of a nerve, which are responsible for conducting electrical impulses.
The thickness of the myelin sheath will differ, which in turn changes the rate of impulse travel. Thicker myelin sheaths relate to faster impulses. Cutaneous sensory nerves are characterized by their cell body size, axon diameters, degree of myelination and conduction velocity. 9

| Aβ nerve fibres
Aβ nerve fibre receptors are located in several areas: Meissner's cor- Merkel's discs (skin, hair follicles) and Ruffini's corpuscles (skin). 9 These fibres generally have a low threshold for static and dynamic touch, vibration, and skin stretching, as well as having fast conduction speeds due to heavy myelination. 9

| Aδ nerve fibres
Aδ nerve fibres consist of two subtypes: Type I and Type II. 27 Type I are high-threshold mechanical nociceptors, with a high-heat threshold.
Type I fibres respond to mechanical and chemical stimuli, and when injured, their heat threshold lowers, referred to as sensitization. Type II fibres have a high-mechanical threshold and low-heat threshold and mainly respond to intense mechanical stimuli. In general, Aδ fibres are thinly myelinated and found in all regions of the skin. Aδ fibres are most associated with localized pain and light touch. 27

| C nerve fibres
C nerve fibres are the smallest and most abundant subtype of nerve, unmyelinated and located in all regions of the skin. 27 C fibres are free nerve endings, generally referred to as nociceptors that respond to noxious mechanical or hot/cold stimuli 9 Impulses travel slowly in C fibres and are associated with poorly localized, slower pain. 27

| Neurons interaction with keratinocytes, dermal fibroblasts
The epidermis is populated with fine, unmyelinated nerve endings, and free, branched nerve endings in the dermis. 28

| Acetylcholine (Ach)
Acetylcholine (Ach) is synthesized in nerve terminals from acetyl coenzyme A and choline, it is an excitatory neurotransmitter. [33][34][35] Acetylcholine acts as an immune cytokine, which inhibits macrophages through cholinergic (receptors which respond to acetylcholine) anti-inflammatory pathways. 34 Several nicotinic Ach receptor (nAChR) agonists have been developed to treat subcutaneous inflammation due to the relationship of various immune cells (such as monocytes and macrophages) with nAChRs. Macrophage nAChRs can modulate functional activity of cholinergic anti-inflammatory pathways which regulate innate immune function and inflammation. 34 Keratinocytes have been shown to produce non-neuronal acetylcholine. 36 Additional evidence, such as sensory receptors located on keratinocytes, suggests that they are also sensory cells. 6,37 are all essential for growth, proliferation and maintenance of nerves.

| Neurotrophins in the skin
Neurotrophin 3 is responsible for the development of cutaneous nerves and promotes the survival of cutaneous sensory nerves. 6,8 Common neurotrophins include nerve growth factor (NGF), brainderived neurotrophic factor (BDNF) and neurotrophins 3, 4, 5. 38 In general, neurotrophins are key molecules in neuro-immuno-endocrine signalling. 4

| Neuropeptides
Neuropeptides are secreted by cutaneous nerves and can interact with many cutaneous cell types including keratinocytes, Langerhans and endothelial cells. 39 Sensory neurons secrete at least 17-20 different neuropeptides, including substance P (SP), neuropeptide Y (NPY) and nerve growth factor (NGF). 39,40

| Substance P
Substance P is secreted by sensory C fibres, 41 dorsal root ganglion, 38 can bind to keratinocytes, mast cells, 28 or induce interleukin (and other cytokine) release. 39 There are 3 main peripheral actions of SP: vasodilation or vascular permeability, local inflammation or immune system effects, and increased cellular proliferation (keratinocytes, fibroblasts, endothelial cells and immune cells). 41 SP has also been implicated in psoriasis. 4

| Neuropeptide Y (NPY)
Neuropeptide Y is a neuronal signalling molecule 42 which is a part of the NIC system that can act locally (ie inflammation) or act on entire systems (via endocrine or neuro-endocrine pathways). 6 NPY has several functions, including activating mast cells, induction of phagocytosis, stimulating antibodies and cytokines, and inducing vascular permeability. 4

| DYS REG UL ATI ON OF NEURO -IMMUNO -ENDO CRINE SYS TEMS IN S KIN PATHOLOG IE S
The immune system of the cutaneous system is extremely important as the first line of defence of the body against the environment and is composed of numerous cell types that are distributed throughout the skin in key locations for their function. 49 There has been significant advancement into the understanding of the complexity of the immune cells and dendritic cells which populate the skin. [50][51][52] While there is much information known, there is still exciting work to be done in discovery of the immune and neural systems of the skin to fully integrate of our understanding of the NIC/NICE systems with various skin pathologies ( Table 5).
Dysregulation of components of the NIC/NICE systems has been implicated in numerous skin pathologies.
Based on research into the effect of neuromediators, it has been suggested that they do not simply play a pro-inflammatory role in the skin but also can and do participate in the entire inflammatory response process. 40 Cutaneous innervation or immune components are targets for many treatments of disease; as a result, promising results have been gained from developments in therapeutics from a neuro-immuno-endocrine approach: neuropeptides like NGF, 53,54 hormonal (vitamin D), 55 anti-cytokine 56 and capsaicin to target sensory neurons of the skin. [57][58][59] Continuing emphasis on the NIC/NICE systems for multi-pronged treatments of these pathologies could be an important consideration for drug development and reveal further intricacies of the skin. In summary, the most important considerations for the design of an optimal skin biomaterial would be the following: mechanically robust and/or flexible to allow for skin movement, biodegradable at controlled rate to optimize integration with cells or the local tissue (for implants), bio-inertness of the scaffold, conductivity to aid neural integration and/or reintegration, and additional factors to promote healing to enhance neural regrowth and/or wound healing. 24,61 A crucial avenue of skin research, and of relevance to the innervation field, is wound healing. Neuromediators are involved in all stages of the wound-healing process, and it is crucial to consider the potential loss of sensation, innervation and re-innervation following trauma such as a burn, a common consequence following injury. 30,61,62 Future work could address the lack of medical treatment options for re-innervation of skin following burn injuries. 61 One way to expand research in this area would be to investigate 'bio-active' materials for skin tissue replacements or in vitro skin tissue models which enhance healing, regrowth of neural cells, and also include or reintegrate vasculature. 24 [117][118][119][120] • Hormonal or stressor-related 118,121 advances in skin bioprinting, such materials have yet to be developed for skin. 61,63,64 Recent research demonstrated that cutaneous cell types have complex communication that reaches across systems, that is keratinocytes (cutaneous) interact with neurons (neuronal system) and vice versa. 1,47,65,66 Several skin pathologies have known interactions between skin cells like keratinocytes, with neuronal and/or immune cells including psoriasis, atopic dermatitis and vitiligo. 67,68 The understanding of the impact of the NIC/NICE system on skin pathology is just at the beginning. Through developments in in vitro HSE design, by inclusion of NIC/NICE components, it would be possible to gain insights into human pathologies in a manner that avoids animal testing and is more translatable to human biology. Complete, complex in vitro HSEs could become fully viable alternatives to animal testing, increase accuracy of in vitro testing models, and serve as sensory and immunocompetent disease models. 5,69

ACK N OWLED G EM ENTS
Research for this paper was conducted with grant support:

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.