The Skin Microbiota and Itch: Is There a Link?

Itch is an unpleasant sensation that emanates primarily from the skin. The chemical mediators that drive neuronal activity originate from a complex interaction between keratinocytes, inflammatory cells, nerve endings and the skin microbiota, relaying itch signals to the brain. Stress also exacerbates itch via the skin-brain axis. Recently, the microbiota has surfaced as a major player to regulate this axis, notably during stress settings aroused by actual or perceived homeostatic challenge. The routes of communication between the microbiota and brain are slowly being unraveled and involve neurochemicals (i.e., acetylcholine, histamine, catecholamines, corticotropin) that originate from the microbiota itself. By focusing on itch biology and by referring to the more established field of pain research, this review examines the possible means by which the skin microbiota contributes to itch.


Introduction
Bacteria, viruses, fungi, archaea, helminths, and protozoa that inhabit our body are a prospering dynamic community shaping a symbiotic superorganism. Roughly 10 14 microbiota populate the entire body, with their number approximating that of human cells [1,2]. Evidence suggests that microbiota take part in maintaining human health [3,4].
As a crucial barrier to the exterior world, skin is one of the body's largest organ [5]. A square centimeter of human skin holds around 10 6 of microbiota [6][7][8]. The symbionts defend against illness by regulating the skin barrier and host immune response [9,10]. On the other hand, microbial imbalance (dysbiosis) has been noted to exacerbate skin lesions and delay wound healing [11,12]. Recently, the emerging role of the skin microbiota in itch has received attention [13].
Large-scale changes of the skin microbiota have been noted in itchy skin diseases. Staphylococcus aureus (S. aureus) participates in atopic dermatitis (AD) flare-up; its colonization correlates with disease severity and itch [14][15][16].
In the present review, we offer an integrative perspective on the skin microbiota and itch. The first section describes the interplay of the cutaneous microbiota with the epidermal barrier, the local immune system, and the sensory nerve, proposing the microbiota's peripheral mechanism of itch. The second section concentrates on the concept of microbial endocrinology and addresses the microbiota-skin-brain axis. Moreover, the interaction between the skin microbiota and the amygdala is discussed to explain the microbiota's central mechanism of itch. Overall, this article describes the putative role of the skin microbiota in itch.  Various microbiota (bacteria, fungi and viruses) cover the exterior of a healthy skin where the barrier is intact. In the event of dysbiosis, pathogens release proteases, which may disrupt the epidermal barrier. Delta-toxin causes mast cell degranulation, which prompt inflammation and itching. AMP: antimicrobial peptides; DRG: dorsal root ganglia; IL: interleukin; LTB4: leukotriene B4; PAMP: pathogen associated molecular pattern; PGE2: prostaglandin E2; TLR: Toll-like receptor; TRPA1: transient receptor potential antigen 1; TSLP: thymic stromal lymphopoietin.

The Skin Microbiota, The Immune System, and Itch
Skin is flushed with a wide scope of cells of the innate and adaptive immune system. The skin microbiota keeps immune homeostasis [19] by modulating the expression of diverse innate factors, including AMPs, interleukin 1a (IL-1a) [48], and complement [49].

Bacteria
Interactions with TLRs
Finally, S. epidermidis can finely tune the response of resident T cells and promote selective immunity against skin pathogens [57].
Alteration in the normal makeup of the skin microbiota induces inflammation. Moreover, the constitution of the cutaneous microbiota shifts dramatically in the course of inflammation [14]. For example, AD flares are tied with an overall decrease in microbial diversity with an expansion of staphylococcal species [14]. The resulting bacterial and viral infection can cause itch.
One possible mechanism of itch from S. aureus infection is mast cell-mediated pruriceptor stimulation. Nunez et al. discovered that S. aureus releases delta-toxin, an amphipathic peptide that stimulates chemical release from mast cells and mediates skin pathology in AD [58]. Serine protease from S. aureus is also involved in type-2 inflammation and itch [16,59].
Mast cells (MCs) are also an essential element of innate immunity. MCs recognize pathogens via pathogen-associated molecular pattern (PAMP) receptors (i.e., TLR) on their surface [66]. Once they detect pathogens, inflammatory mediators are released to attract other immune cells [67,68].
Th2 immunity is dominant in scabies and is complemented by a heavy inflow of IL-31(+) M2 macrophages [73]. Proteases from scabies mite stir epidermal KCs to express TSLP. TSLP activates Th2 cells and induces M2 macrophages to produce IL-31, causing severe itch [74]. The antigens of S. aureus have also been reported to induce IL-31 in individuals with AD [75].

The Skin Microbiota, The Sensory Nerve, and Itch
Skin is one of the first lines of defense against microbial threats. Though the immune system is an essential component of cutaneous immunity, it is evident that the sensory nervous system also plays an important part in host defense. By evoking the sensation of itch, the host can immediately sense danger and rapidly initiate a protective behavioral response [69].
A network of high-and low-threshold sensory nerves innervates the skin and is frequently exposed to bacterial pathogens ( Figure 2). J. Clin. Med. 2020, 9, x FOR PEER REVIEW 5 of 18 plays an important part in host defense. By evoking the sensation of itch, the host can immediately sense danger and rapidly initiate a protective behavioral response [69]. A network of high-and low-threshold sensory nerves innervates the skin and is frequently exposed to bacterial pathogens ( Figure 2). Pruriceptor neurons express cytokine receptors and G protein-coupled receptors that recognize immune mediators [76]. While we understand that microbial inflammation propagates itch, how the skin microbiota directly triggers sensory nerves is a new area of inquiry.
The latest studies suggest that sensory neurons, like immune cells, are able to detect microbiota [13,69,76,77]. Ji and colleagues reported TLR7 on pruriceptors and noted synthetic TLR7 ligands (i.e., imiquimod) causing itch behavior in mice [78]. TLR3 is also displayed by pruriceptors, where Polyl:C, a TLR3 ligand, stimulates neuronal activity and itch [79]. Viral single-stranded RNA and doublestranded RNA are known pathogen-derived ligands for TLR7 and TLR3, respectively, and there is a possibility that these viral ligands cause itch by directly interacting with pruriceptor neurons [76].
Lipopolysaccharide (LPS), an important component of the Gram-negative bacteria outer membrane, binds to TLR4 [80]. Although LPS has only been reported with pain [81], it can also modulate itch since TLR4 promotes histamine-mediated itch [82]. Interestingly, LPS has also been found to stimulate sensory neurons in an TLR4-independent manner, via the activation of TRPA1 [83,84]. Pruriceptor neurons express cytokine receptors and G protein-coupled receptors that recognize immune mediators [76]. While we understand that microbial inflammation propagates itch, how the skin microbiota directly triggers sensory nerves is a new area of inquiry.
The latest studies suggest that sensory neurons, like immune cells, are able to detect microbiota [13,69,76,77]. Ji and colleagues reported TLR7 on pruriceptors and noted synthetic TLR7 ligands (i.e., imiquimod) causing itch behavior in mice [78]. TLR3 is also displayed by pruriceptors, where Polyl:C, a TLR3 ligand, stimulates neuronal activity and itch [79]. Viral single-stranded RNA and double-stranded RNA are known pathogen-derived ligands for TLR7 and TLR3, respectively, and there is a possibility that these viral ligands cause itch by directly interacting with pruriceptor neurons [76]. Lipopolysaccharide (LPS), an important component of the Gram-negative bacteria outer membrane, binds to TLR4 [80]. Although LPS has only been reported with pain [81], it can also modulate itch since TLR4 promotes histamine-mediated itch [82]. Interestingly, LPS has also been found to stimulate sensory neurons in an TLR4-independent manner, via the activation of TRPA1 [83,84].
Besides TLR ligands, sensory neurons notice pathogens through various molecular means. Specifically, zymosan from Candida albicans [85], N-formylated peptides and α-hemolysin from S. aureus [86], and streptolysin S from S. pyogens [87] were shown to mediate pain through direct neuronal stimulation. It remains to be discovered whether pruriceptors recognize these pathogens in a matching manner to elicit itch.
Itch is bothersome in patients with cholestatic liver disease [88]. Recently, alteration of the skin microbiota was identified in cirrhosis patients where specified microbial taxa correlated with itch severity and serum autotaxin (ATX) level [89]. Lysophosphatidic acid (LPA), a powerful neuronal activator, and ATX (ectonucleotide pyrophosphatease/ phosphodiesterase 2), the enzyme that creates LPA, are pruritogens in cholestasis [90,91]. It is suggested that LPA directly activates TRPV1 on peripheral sensory neurons to mediate itch [92].

Microbial Endocrinology
Microbial endocrinology is a crossing of two supposedly distinct areas, microbiology and neurobiology, and is based on the shared presence of neurochemicals in the host and the microbiota [66].
The ability of the microbiota to not only respond to but also create the very same neurochemicals of mammalian systems, tells that host interplay with the microbiota is much more interactive than it was thought before.
Hence, microbial endocrinology could be applied beyond infectious disease to other conditions such as brain health through the microbiota-skin-brain axis.
Microbiota has multiple transmission pathways to access the brain: the neural signals carried by the afferent neurons, endocrine messages transmitted by neurochemicals and the immune messages transferred by cytokines [112,113].
The skin is one important platform for microbial communication with the brain. In an evolutionary standpoint, it is reasonable for the skin to support the cutaneous microbiota, which in turn assists skin barrier function and local immune system and helps the skin communicate with other organ systems, including the brain (microbiota-skin-brain axis) [114].

Stress, The Skin Microbiota, and Itch
Stress is a complex dynamic condition where homeostasis, or the stability of an organism is altered, promoting the adaptation of the host. Stress aggravates itch [115][116][117], which proves that the brain is engaged in the final common stage of itch processing [118,119].
Stress acts by the central nervous system (CNS) and alters the microbiota via the release of neurochemicals [120,121]. Glucocorticoids, an essential component of the stress response, repress AMP release/localization in the epidermis, weaken the barrier, and raise host susceptibility to infection [122][123][124].
Substance P is released in sweat during stress and increases the virulence of Gram-positive skin bacteria, namely S. aureus and S. epidermidis [95,96].
Substance P is released in sweat during stress and increases the virulence of Gram-positive skin bacteria, namely S. aureus and S. epidermidis [95,96].
Thus, the effect of stress on the skin microbiota may be twofold: dampening the host defense to infection and adjusting the microenvironment ideal for pathogens [124]. The resultant dysbiosis can exacerbate itch ("stress aggravated itch") ( Table 2). Table 2. Effects of stress mediators on the skin microbiota.

Staphylococcus epidermidis
Glucocorticoids decrease the effects of super antigen activated T cells and inhibit staphylococcal exotoxin-induced T cell proliferation, cytokine secretion [137].

Pseudomonas aeruginosa
Norepinephrine increases expression of the attachment factor PA-1 of P. aeruginosa and increase biofilm formation [135,138].

Staphylococcus aureus
Acetylcholine augments susceptibility to infection by S. aureus [124]. Norepinephrine increases S. aureus' ability to remove iron from host and therefore facilitates the bacteria to form biofilms [138,140].
Catecholamines enhance Group A Streptococcus growth likely by increasing iron availability [138,142].

The Skin Microbiota, The Amygdala, and Itch
Itch encompasses sensory-discriminative and affective-motivational aspects and undergoes extensive processing in the higher brain centers [119,125].
The Amygdala is involved in pain, especially in the emotional-affective aspects of pain perception [144]. The central nucleus of the amygdala (CeA) is commonly called the "nociceptive amygdala" [145] and receives peripheral pain signals via the parabrachial nucleus [146].
The role of amygdala in itch is also shown in animal studies [147]. A recent study noted that scratching was suppressed after blocking itch-mediating spinal neurons connected to the spinoparabrachial pathway [148]. Additionally, an animal functional MRI (fMRI) study presented amygdala activation during itch stimuli [149]. The findings hint that itch signals are delivered by both the spinothalamic pathway and the spinoparabrachial-CeA path. It was claimed that the injection of muscimol (γ-aminobutyric acid agonist) to the amygdala minimized scratching elicited by the injection of serotonin to the cheek, implying a modulatory role of the amygdala in itch processing [150].
Chronic stress brings functional and configurational changes in the amygdala (central sensitization) (Figure 3) [151]. This change may influence itch processing in the brain, which explains why stress worsens itch in individuals with chronic itch [152,153].
Studies suggest that the amygdala itself is susceptible to microbial influences [154]. Most convincingly, data from germ-free (GF) mice imply that the amygdala transcriptome becomes hyperactive in the absence of microbiota [155,156]. This hyperactive state is in line with the altered pain sensitivity [157] and stress response in GF mice [158,159].
We do not know how microbial signals navigate through the skin-brain axis to reach the amygdala specifically yet; however, there are some strong candidate paths, including the blood stream (circulation) and the spinal cord [112,154,160].

Conclusions and Future Perspectives
With increased recognition of the presence and functionality of the microbiota, the human body is not what we perceive. Evidence suggests that our microbiota occupies a prominent role in the human body than formerly thought.
Cutaneous microbiota delivers a diverse and far-reaching influence on our physiology by calling upon the host nervous system. Bacteria make metabolites, toxins, and structural components that are recognized by peripheral and central neurons via matching receptors. Microbiota also indirectly affects neural function by causing endocrine (i.e., keratinocytes) and immune cells to transmit signals (i.e., cytokines, proteases). Itch is a prototypic sensory neural function, and the microbiota propels the itch-scratch cycle.
Some descriptive studies have differentiated the microbiota found in itchy skin versus those of healthy skin. While dysbiosis is found in various pathologies, these raise a "chicken-or-the-egg" type question, as we are not sure if dysbiosis leads to disease, or whether the underlying conditions cause microbial imbalance.
To differentiate cause and effect, a deeper and more mechanistic (functional) understanding of the skin microbiota's role in itch is required. Increased grasp of this area will help find microbiological markers in itchy conditions and develop alternative therapeutics which utilize host-microbiota relationship.
The gut and skin are uniquely related in function, and numerous studies link gut microbiota to skin homeostasis (skin-gut axis or skin-gut-brain axis) [35,[161][162][163][164]. Commonalities have also been found between itch transition in the skin and neural signaling in the lower intestinal tract, which raises the possibility of intestinal microbiota playing a role in itching [165,166].
Various types of microbiota-based therapy may be applied in the upcoming years: (1) Whole microbiota transplant, a process that offers microbiota from healthy donors to patients with significant skin dysbiosis, such as AD, to correct the dysbiosis. The favorable effects of microbial transplant can be studied for other itchy conditions as well. (2) Topical probiotics can be used to introduce known advantageous microbiota to a patient (especially at a critical age for immune and limbic brain wiring).
(3) Topical prebiotics (nutrients that stimulate beneficial skin micrbiota, or biomass or dead extracts of non-pathogenic bacteria which antagonize substance P) can be adopted. (4) Host-microbiota interplay can be studied by analyzing microbial metabolites, re-imposing commensal microbial activity by offering signaling molecules (Figure 4). J. Clin. Med. 2020, 9, x FOR PEER REVIEW 9 of 18 Some descriptive studies have differentiated the microbiota found in itchy skin versus those of healthy skin. While dysbiosis is found in various pathologies, these raise a "chicken-or-the-egg" type question, as we are not sure if dysbiosis leads to disease, or whether the underlying conditions cause microbial imbalance.
To differentiate cause and effect, a deeper and more mechanistic (functional) understanding of the skin microbiota's role in itch is required. Increased grasp of this area will help find microbiological markers in itchy conditions and develop alternative therapeutics which utilize host-microbiota relationship.
The gut and skin are uniquely related in function, and numerous studies link gut microbiota to skin homeostasis (skin-gut axis or skin-gut-brain axis) [35,[161][162][163][164]. Commonalities have also been found between itch transition in the skin and neural signaling in the lower intestinal tract, which raises the possibility of intestinal microbiota playing a role in itching [165,166].
Various types of microbiota-based therapy may be applied in the upcoming years: (1) Whole microbiota transplant, a process that offers microbiota from healthy donors to patients with significant skin dysbiosis, such as AD, to correct the dysbiosis. The favorable effects of microbial transplant can be studied for other itchy conditions as well. (2) Topical probiotics can be used to introduce known advantageous microbiota to a patient (especially at a critical age for immune and limbic brain wiring). (3) Topical prebiotics (nutrients that stimulate beneficial skin micrbiota, or biomass or dead extracts of non-pathogenic bacteria which antagonize substance P) can be adopted. (4) Host-microbiota interplay can be studied by analyzing microbial metabolites, re-imposing commensal microbial activity by offering signaling molecules (Figure 4).
In conclusion, the interplay between the skin microbiota and itch is an emerging area to explore. In future, cosmetics/transdermal drugs with a concept of 'topical microbiota modulator' could have the potential to claim that they do not only make you look good but also that make you feel good. . Two main approaches of controlling the human skin microbiota for the itch control. Topical pre-and probiotics target to increase the number of advantageous bacteria (green) and reduce pathogens (red). Skin microbial transplant is a new approach that transfers beneficial microbiota from healthy skin to itchy and dysbiotic skin [167]. . Two main approaches of controlling the human skin microbiota for the itch control. Topical pre-and probiotics target to increase the number of advantageous bacteria (green) and reduce pathogens (red). Skin microbial transplant is a new approach that transfers beneficial microbiota from healthy skin to itchy and dysbiotic skin [167].
In conclusion, the interplay between the skin microbiota and itch is an emerging area to explore. In future, cosmetics/transdermal drugs with a concept of 'topical microbiota modulator' could have the potential to claim that they do not only make you look good but also that make you feel good.

Conflicts of Interest:
Yosipovitch reports serving on the scientific board and being a consultant for Trevi, Sanofi Regeneron, Galderma, Pfizer, Novartis, Kiniksa, Eli Lilly, Bellus, LEO and is supported by Sun Pharma, Pfizer, Novartis, Kiniksa, Leo Pierre Fabre. None of these involvements had influence on the content of the presented paper.

Glossary Term Definition
16S rRNA gene sequencing Genomic analysis of 16S ribosomal RNA phylotypes from DNA that is extracted directly from bacterial communities in clinical or environmental samples, a process that circumvents culturing [29].
Skin microbiota Total of microbes in/on our skin [168].

Microbiota
The group of microbes found in/on a specific environment or living host [169].
Microbial diversity Degree of variability of the microbiota. α-diversity describes within-sample variability, while β-diversity signifies variability between samples [169].

Probiotics
Live microorganisms that have a favorable impact on host health when given in proper amounts [169].

Antibiotics
Antibiotics block the growth of or destroy bacteria and other microbes [168].