Skin immunity and its dysregulation in atopic dermatitis, hidradenitis suppurativa and vitiligo

ABSTRACT

While the epidermis is the frontline defense against infections and indeed, it is a peripheral lymphoid organ, the same immunological mechanisms may initiate and sustain pathological conditions. Indeed, a deregulated action against exogenous pathogens could activate a T cell response in atopic dermatitis, hidradenitis suppurativa and vitiligo. Atopic dermatitis (AD) is a chronic inflammatory skin condition with a complex pathophysiology. Although T helper 2 immunity dysregulation is thought to be the main cause of AD etiopathogenesis, the triggering mechanism is not well understood, and the treatment is often difficult. As the AD, hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a dramatic impact on the quality of life of the affected patients. The exact pathophysiology of HS is still unclear, but many evidences report a follicular obstruction and subsequent inflammation with TNF-α, interleukin (IL)-1β, IL-10, and IL-17 involvement. Vitiligo is an autoimmune epidermal disorder which consists of melanocytes destruction and skin depigmentation. Melanocytes destruction is mainly due to their increased oxidative-stress sensitivity with a consequent activation of innate first and adaptative immunity (CD8⁺ T cells) later. The understanding of the triggering mechanisms of AD, HS and Vitiligo is pivotal to outline novel therapies aimed at regaining the physiological immune homeostasis of healthy skin. The aim of this review is to provide new insight on the pathogenesis of these skin diseases and to highlight on the new therapeutic approaches adopted in the treatment of AD, HS and Vitiligo.

Introduction

Skin is a complex organ which provides a strong barrier against external insults and acts as an amphitheater for a broad variety of inflammatory processes, including immunity against infections, tumor immunity, autoimmunity, and allergy [1]. Therefore, it is considered both as a mechanical barrier, limiting water loss and preventing the entry of potentially dangerous environmental elements and micro-organisms, and as an active barrier providing the first line of immunological defense against infections.

Moreover, the various microbial populations colonizing the skin surface, regularly interact with the host’s epithelial and immune cells, influencing local and systemic immunity [2].

Skin immune homeostasis is based on a finely regulated equilibrium between different cellular and microbial components. Dysregulation of this balance contributes to the pathogenesis of inflammatory skin diseases such as atopic dermatitis, vitiligo and hidradenitis [3,4]. From the concept of skin immunity and skin-associated lymphoid tissue firstly introduced by Streilein nowadays skin is considered a peripheral lymphoid organ where stromal cells (keratinocytes, fibroblasts, endothelial cells, and adipocytes) interact with bone marrow-derived cells (dendritic cells, macrophages, natural killer cells, mast cells, T cells, and others) [5–7]. The cells derived from the bone marrow, which are found in the skin, can be divided into resident cells that migrate to the skin where they differentiate and reside mainly and in recirculating cells that play a surveillance role. The latter can be recruited to fight short-term infection and stored as memory cells to protect against future re-invasion.

Bone marrow–derived cells can be further subdivided into innate and adaptive immune populations. While innate cells give a nonspecific response to infection, adaptive immune populations have a pathogen-specific response to infection through specialized and unique antigen-specific receptors produced via genetic rearrangement [4]. Between adaptative immune cells, T lymphocytes are the central effector lymphocytes in skin immunity with different functions [8]. Particularly, CD4 + T helper (Th) cells are considered the principal responsible in maintaining skin immunity homeostasis. They can be subdivided into four major subtypes with divergent molecular and functional features. These subgroups embrace Th1, Th2, Th17 and regulatory Th cells (Tregs). The prevalence of a subtypes over another can lead to the development of a skin disease rather than another. The differentiation from naïve Th lymphocytes begins with the stimulation of their receptor (TCR), together with the presence of co-stimulatory factors and lineage-driving cytokines. These subgroups embrace Th1, Th2, Th17 and regulatory Th cells (Tregs). Thus, innate immune system and APCs are responsible of the Th cells activation [9] and especially cytokines have crucial functions in skin immunity, allowing the development and regulation of the immune response [10].

Indeed, while Interleukin(IL)-12 has a central role in Th1 differentiation, IL-4 promotes the development of Th2 cells [11]. Therefore, a complex interchange between the immune cells and cytokines mediates and regulates immunity and inflammation. On this basis, the definition of Th subsets determined by their cytokine profiles could be useful to understand their role in autoimmune and inflammatory diseases.

Interferon (IFN)-γ is the central Th1 cytokine; however, Th1 cells also secrete Tumor Necrosis Factor (TNF)-a and IL-2 [12]. The latter one promotes T cell spread and CD8+ cytotoxic T cells stimulation. IFN-γ, instead, triggers macrophages to produce IL-12, TNF-a, and nitric oxide (NO) to act against intracellular pathogens.

Regarding the Th2 subset, it produces IL-4, IL-5, and IL-13. IL-4 is the key cytokine in the development of Th2 cells from naïve T cells, it downregulates Th1 responses and supports the IgE class switch in B lymphocytes, whereas IL-5, is involved in eosinophilic activation [11].

Among CD4+ cells, Tregs (FoxP3+) play a crucial role in controlling Th1 and Th17 cell-dependent inflammatory responses within the skin, by secreting immunosuppressive cytokines such as IL-10 and Transforming Growth Factor- β (TGF-β), and therefore maintaining immune self-tolerance [11]. Particularly, IL-10 plays a key role in controlling dendritic cell (DC) function during the effector phase of inflammatory skin reaction and also inhibiting macrophage functions [13]. Thus, Tregs are crucial in the mechanisms of immune tolerance interplaying with DCs, which represent the major link between the innate and adaptive immune response. They are characterized by a functional plasticity and play a key role in immunological reactions and tolerance. Particularly, tolerogenic subsets of DCs (tDCs) are involved in central and peripheral tolerance, through negative selection of autoreactive T cells and regulatory T cells production in the thymus. TDCs also promote Tregs in the periphery [14]. Any alteration of DC/Treg function contributes to autoimmune disease. Although the mechanisms by which TDC regulate the homeostasis of IL-10–producing cells are not completely understood, it is possible to assume that TDC con mediate their tolerogenic properties at least in part via IL-10–producing CD4 + T cells.

Beyond the typical Th1/Th2 opposition, other specific Th cells subsets are found in the skin, such as Th17 and Th22 subtypes. Th 17 are categorized by the expression of IL-17A, IL-17F, IL-21, IL-22, and TNF-a and have a central role in the defense against extracellular pathogens, as well as in the development of autoimmune inflammatory disorders [15].

Th22 subset, firstly described by Duhen et al., mainly produces the lineage-defining cytokine IL-22 (Th22), and it is implicated in the pathogenesis of inflammatory and autoimmune disorders [16].

Recently, an IL-9-producing Th (Th9) subset has been described by Schlapbach et al. IL-9 is a pleiotropic cytokine, responsible for immunity activation and tissue inflammation by enhancing inflammatory cytokine production by other Th cell subset [17]. However, the homeostatic control of skin immunity also depends on the cutaneous microbiota [18]. Indeed, the physiological, as well as the pathological, the microbiome can interplay with genetic factors, infections, metabolic status, especially in the presence of specific inflammatory conditions to affect the immunological landscape and significantly affect the progression of skin diseases. This complex interaction also affects the delicate balance of skin formation and homeostasis [19–21]. Indeed, skin commensals modulate the function of local T cells through their ability to modify the local innate immune setting and in particular IL-1 production. This action results in an increased production of cytokines such as IL-17-A and IFN-y involved in both host defense and inflammatory diseases, i.e. psoriasis, atopic dermatitis, and acne that have all been associated with dysbiosis of the skin flora [22].

In this manuscript, we would like to synthetize recent advances on the immunity of skin disorders, and we will discuss the immunological implications in Atopic Dermatitis, Hidradenitis Suppurativa and Vitiligo. The analysis of these diseases shows how the involvement of T lymphocytes, and the secrete cytokines and lymphokines can trigger and sustain the pathology. Its understanding offers a novel venue of pharmacological intervention.

The role of the immune system in atopic dermatitis

Atopic dermatitis (AD) is a common chronic or recurrent inflammatory skin disease, affecting 15–20% of children and 1–3% of adults worldwide. It is characterized by acute flare-ups of eczematous pruritic lesions over dry skin. AD is considered the result of an immune system impairment mixed with an impaired skin barrier genetically determined [23–25]. These defects of skin barrier function facilitate the penetration of allergens and microbial pathogens into the skin which induces the Th2 polarization [26]. Indeed, a predominant Th2 phenotype is considered the main cause in the etiopathogenesis of AD [27]. Th2 cells can, in fact, induce isotype class switching to IgE production, and favor the eosinophils proliferation [28]. The levels of Th2 cytokines (IL-4, IL-5 and IL-13) are higher in lesional and perilesional skin during the active phase of AD [29]. Particularly, there is a correlation between high levels of IL-13 and IL-4 in cord blood and the future development of AD [30], while IL-13 is also capable of inducing tissue remodeling at sites of Th2 inflammation.

Since IL-13 and IL-4 share a lot of functions in AD development, several studies demonstrated that it could be due to a common receptor subunit, IL-4Rα, which binds both. The ability of IL-4 to reduce the expression of numerous genes involved in the production of extracellular matrix, in fact, is also shown by IL-13 [30,31]. Both IL-4 and Il-13 reduce the expression of other late cornified envelope molecules horner in, involucrin and loricrin and the production of lamellar bodies, responsible for the secretion of lipids and protease inhibitors, in the upper keratinocyte layers. IL- 4/IL-13 can induce kallikrein 7 production by keratinocytes which promotes skin desquamation by degrading corneodesmosomal proteins, worsening micro epidermal fissuring and water loss [32]. On the other side, IL-5 is associated with the increase of eosinophil survival, explaining why eosinophilia is a marker for the active phase of AD [33], while IL-31, a novel Th2 cytokine, seems to be linked to pruritus, justifying its increase in pruritic skin conditions such as AD [34].

Even if the Th2 activation is predominant, the precise mechanisms underlying the immune response in AD remain unclear. Several evidences suggest that it could be related to impaired APCs, innate deficiency of T cells and deregulated cytokine expression. The defective function of the skin barrier modifying the epidermal microenvironment could induce IgE-switching and finally cause Th2-mediated allergic skin inflammation (Figure 1) [35,36].

Figure 1.

Immunopathogenesis of AD. The genetic impairment of skin barrier increases allergens and microbial pathogens penetration into the skin. The allergens induce IgE production by B cells, and the complex IgE-allergens activates the DCs and mast cells. The activation of DCs induces the Th2 polarization. The main Th2 cytokines, IL-4, IL-5, and IL-13 may downregulate the expression of TLRs and AMPs in keratinocytes, exacerbating the disease. Th22 lymphocytes producing Il-22 and Th1 and Th17 mainly via IFN-γ and IL-17 play a role in skin inflammation especially in the chronic stage of AD. Pruritus can be caused by mast cells producing histamine, tryptase, and other inflammatory mediators. Mast cells can exacerbate AD-producing IL-4 and IL-13. IL-5 activates eosinophils. Pruritus may induce scratching which also damages the skin barrier and increases TEWL, which induces skin dryness exacerbating AD.

Despite the link between Th2 immune response and the acute phase of AD, the chronic phase of AD is Th1-predominant [41] with tissue remodeling and fibrosis.

Even Th17 cells, responsible for the production of IL-17A and IL-17F [37], seem to play a role in AD through their interface with eosinophils and the establishment of a Th2 immune response. However, although some studies proved that the rate of Th17 cells was higher in peripheral blood of AD patients, others reported controversial results [38–41]. This may be elucidated by the fact that the numbers of Th17 cells and the levels of IL-17 varies during the natural history of the disease. In active AD, IL-17 levels are high, while in chronic AD, are low, or even undetectable [42].

As in psoriasis, IL-22 is increased in AD [43]. It seems to block the formation of new keratinocytes, and helps their migration [44]. IL-22 might also reduce the expression of filaggrin and profilaggrin processing enzyme, causing an increase of the epidermal barrier impairment in AD [45].

The role of the immune system in hidradenitis suppurativa

Hidradenitis suppurativa (HS) is a chronic recurrent inflammatory disease of apocrine-bearing skin areas. Comedones, profound relapsing nodules and abscesses forming tortuous draining fistulas are the main clinical features of this disease [46].

This disease has a tremendous impact on the quality of life of affected patients, causing pain, secretion of malodorous fluids and impairing the social life. Several risk factors have already been associated with HS, i.e. female sex, smoking, obesity and family history [47].

Even if the pathogenesis of this disease is still unclear, a lot of evidence suggests a role of the immune system in HS.

Particularly, few studies have been described as a correlation between the expression of TNF-α in terms of mRNA and protein levels in the HS skin [48,49]. Moreover, TNF-α levels also are positively linked to disease severity measured trough the Hurley classification [49].

Even high levels of IL-1b have been described in HS lesions by van der Zee et al. [49]. This cytokine is a central mediator of the inflammatory response, promoting the development of Th17 cells [50], already known for their role in psoriasis and AD. High levels of IL-17 and IL-23 have been reported in HS-lesional skin suggesting that the activation of IL-1b/IL-23/TH17/axis is also involved in the pathogenesis of HS [51,52].

Additionally, increased levels of serum IL-17 have been correlated with severe disease. In recent studies, an imbalance between Th17 and Tregs, in favor of Th17 functions, has been demonstrated in the HS-lesional skin, especially for familial-HS related to mutation of γ-secretase. Interestingly, as for psoriasis, an increase of anti-microbial peptide LL-37 has also been implicated in the pathogenesis of this disease (Figure 2).

Figure 2.

Immunopathogenesis of HS. In a background of smoking habitus and obesity, the interaction between γ-secretase mutation, bacteria and follicular obstruction can allow the inflammasome activation mediated by Th1/Th17 axis and their cytokines.

Few studies have focused the attention also on IL-12, while oddly, IFN-γ, signature mediator of Th1 response, was not consistently found [53]. IL-12 secreted by macrophages, along with IL-23, has also been implicated in maintaining the Th17 inflammatory loop.

Furthermore, elevated levels of the anti-inflammatory cytokine IL-10 have been documented and may signify a negative feedback signal to the inflammation. IL-10 is produced by activated T cells, macrophages, mast cells, and B cells, of whom HS-lesional skin is rich [49,54,55].

The role of the immune system in vitiligo

Vitiligo is the most common skin depigmenting disorder with an incidence rate of between 0.1% and 2% worldwide. It is characterized by acquired, idiopathic, progressive, circumscribed hypomelanosis of the skin and hair, with a total absence of melanocytes microscopically [56–58]. Indeed, an intrinsic defect in melanocytes is, in fact, suggested by abnormalities in their regeneration and proliferation and in their defective defense against oxidative stress. Though, a major role of auto-inflammation and auto-immunity is evident, especially during the active phase of the disease [59–63].

The hypothesis of an increased vulnerability to pro-oxidant agents, which leads to melanocytes destruction, is supported by the presence of elevated levels of reactive oxygen species (ROS) in lesional and non-lesional vitiligo skin, as a result of ultraviolet radiation exposure or chemical damage [64]. Hence, the immune system could be activated by the ROS-generated local inflammation [65]. Another candidate which seems to stimulate the immune system in vitiligo is calreticulin (CRT), an omnipresent protein responsible of intracellular Ca²⁺ homeostasis. This protein is localized over the surface of immune cells during the immune response, controlling complement activation, antigen presentation and removal of apoptotic cells. Its role is to allow the production of pro-inflammatory cytokines (IL-6 and TNF-α), and the enhancement of melanocytes immunogenicity [66,67].

Vitiligo skin abounds also in NK cells activated by the local inflammation. These cells show cytotoxic functions, but can also trigger the adaptive immunity producing pro-inflammatory cytokines [12].

However, T cells and their related cytokines (mainly IFNγ and TNFα) are involved in the development of vitiligo [68,69], although the participation of autoreactive Th cells in melanocyte destruction has not been yet elucidated. Especially Th17 cells seem to act an important role in vitiligo, as IL-17 levels are increased in these patients and the higher the levels are, the more extensive the depigmentation is, even if this correlation has not been yet widely studied [70]. Several studies hypothesized a proper role of IL-17 in the melanogenesis, this cytokine, in fact, seems to enhance the effects of TNFα, IL-1β, and IL-6 on melanocytes, leading to their survival or destruction [71,72]. Furthermore, in melanoma, both TNFα and IL-17 induce the production of CXCL1 and IL-8 in melanocytes, suppressing the pigmentation pathway [72].

Moreover, Richmond et al. suggested that autoreactive T-lymphcytes are recruited to the skin, encounter IL-15 presented to them by keratinocytes, up-regulate IFN-y, and depend on IL-15 for survival once they become resident in the epidermis [73]. Finally, the infiltration of CD8 T cells into perilesional vitiligo skin is very frequent in patients with active disease, showing their major role in melanocyte destruction (Figure 3) [74]. This activation of CD8 T cells is linked to a defective role of Tregs, that are not able to maintain the immune tolerance in vitiligo [75] and are decreased in the periphery as well as in the lesional and non-lesional skin of vitiligo patients [76].

Figure 3.

Immunopathogenesis of vitiligo. In predisposed patients (due to exogenous triggers and genetic predisposition), abnormal melanocytes induce the release of inflammatory cytokines which contribute to activation of the innate immune response and subsequently to adaptive T cell responses, producing TNFα and IFNγ involved in melanocyte loss and development of white patches.

Therapeutic implications

Due to their heterogenicity, multiple therapeutic approaches are adopted in the treatment of Atopic dermatitis, Hidradenitis suppurativa and Vitiligo (Table 1). Because Atopic Dermatitis is mostly characterized by increased levels of Th2 cells and their cytokines production (IL-4, IL-5 and IL-13), targeting Th2 immune response entails disease improvement. Recently, the US Food and Drug Administration has approved Dupilumab, a specific IL-4 receptor monoclonal Antibody (mAb) able to block the effect of IL-4 and IL-13 on immune response [77–80]. Other Th2 antagonists’ molecules are also developed and their efficacy is yet to be determined (e.g. anti–IL-13/tralokinumab and lebrikizumab/NCT02347176 NCT02340234 and anti-thymic stromal lymphopoietin/tezepelumab/NCT00757042). Table 1 shows additional current Clinical Trials in Atopic Dermatitis.

Table 1.

Current clinical trials on atopic dermatitis, hidradenitis suppurativa and vitiligo.

Title	ClinicalTrials.gov Identifier	Location
Atopic Dermatitis
Safety and Efficacy of FURESTEM-AD Inj. in Patients With Moderate to Severe Chronic Atopic Dermatitis(AD)	NCT03269773	Pusan National University Hospital, Busan, Korea. Kang Stem Biotech Co., Ltd.
Pilot Study of Ustekinumab for Subjects With Chronic Atopic Dermatitis	NCT01806662	Rockefeller University, New York. USA
Evaluation of Upadacitinib in Adolescent and Adult Patients With Moderate to Severe Atopic Dermatitis (Eczema)- Measure Up 1	NCT03569293	Alliance Dermatology and MOHs/ID# 200,375. Phoenix, Arizona, USA
A Study of Baricitinib (LY3009104) in Adult Participants With Moderate to Severe Atopic Dermatitis	NCT03435081	University of Alabama at Birmingham, USA. Eli Lilly; Incyte Co.
A Study to Evaluate Upadacitinib in Combination With Topical Corticosteroids in Adolescent and Adult Participants With Moderate to Severe Atopic Dermatitis	NCT03568318	University of Alabama at Birmingham, USA. Abbvie
Hidradenitis Suppurativa
A Study of Bermekimab in Patients With Hidradenitis Suppurativa	NCT03512275	Tennessee Clinical Research Center Nashville, Tennessee, USA. XBiotech, Inc.
Study of Efficacy and Safety of Investigational Treatments in Patients With Moderate to Severe Hidradenitis Suppurativa	NCT03827798	Novartis Pharmaceuticals
A Placebo-Controlled Study of the Safety of INCB054707 in Participants With Hidradenitis Suppurativa	NCT03607487	Wiseman Dermatology Research Winnipeg, Manitoba, Canada. Incyte Corporation
Study of Efficacy and Safety of Two Secukinumab Dose Regimens in Subjects With Moderate to Severe Hidradenitis Suppurativa (HS) (SUNRISE)	NCT03713632	Novartis Pharmaceuticals
A Study to Evaluate the Efficacy, Safety, and Tolerability of Guselkumab for the Treatment of Participants With Moderate to Severe Hidradenitis Suppurativa (HS)	NCT03628924	University of Alabama at Birmingham, USA. Janssen Research & Development, LLC
Vitiligo
Combined Therapy With Narrow-Band Ultraviolet B Phototherapy and Apremilast for the Treatment of Vitiligo	NCT03123016	Icahn School of Medicine at Mount Sinai New York, New York, United States
Efficacy and Safety of Total Glucosides of Paeony Combined With NB-UVB on Treating Vitiligo	NCT03608917	Xijing Hospital Xi’an, Shaanxi, China
Human Beta-defensin 1 in Vitiligo	NCT03832751	Cairo University
NB-UVB and PUVA Vitiligo Study	NCT01732965	Henry Ford Dermatology Detroit, Michigan, USA
Prostaglandin F2-alpha (PGF2α) in Vitiligo	NCT03755830	Cairo University
Evaluation of Scanoskin for the Assessment of Vitiligo	NCT03133871	Chelsea and Westminster Hospital, London, UK

Hidradenitis suppurativa is usually treated for a short period with antibiotic therapies; however, biologic therapies can be given to patients for a prolonged period who failed antibiotic treatment. At the moment, Adalimumab is the only biologic molecule approved by the U.S. Food and Drug Administration and the European Medicines Agency for the treatment of HS. Adalimumab is a human IgG1 monoclonal antibody that specifically binds to tumor necrosis factor (TNF)-alpha neutralizing its activity and inducing apoptosis of TNF-expressing mononuclear cells [55,81]. In addition to this, other drugs and biologics are under investigation (Table 1). For example, M923 (an adalimumab biosimilar) and Humira are currently under clinical trial to evaluate their efficacy, safety, and immunogenicity (Table 1). Also, a phase II clinical trial (NCT02421172) using a fully human IgG1 anti-IL-17 monoclonal antibody (CJM112) was completed in November 2016 and a pilot study (NCT03099980) on the effect of secukinumab, an anti-IL17A antibody, and a phase II study (NCT03248531) with bimekizuma (anti-IL-17A and IL-17F) for moderate-to-severe HS have been initiated [53].

Current therapeutic approaches for Vitiligo are based on the use of topical corticosteroids (TCS) and calcineurin inhibitors (TCIs). They result in re-pigmentation of sun-exposed areas in 75% of cases. Melanocytes regeneration and skin re-pigmentation can be reached also through phototherapy. It has been demonstrated that phototherapy (UVB 311 nm, excimer laser or lamp UVB 308 nm) causes re-pigmentation through various mechanisms, including migration of melanocytes from perilesional skin and hair follicles, formation and melanization of melanosomes, and transfer of melanosomes into keratinocytes [82]. Other current clinical trials for the treatment of vitiligo are listed in table 1.

Conclusion

The epidermis responds to exogenous stimuli, protecting our body from external danger, activating distinct repair mechanisms [83]. On one side, these require the protection form wanton death by a fine-tuning of the Bcl-2 protein family [84–86] able to inhibit [87,88] or activate [89] the mitochondrial depolarization [90] to regulate cell fate [91–93]. On the other side, the transcription factor p53 [94–96] is able to activate a complex regulatory mechanism [72,97–100] and metabolism [101] able to kill the damaged cell [102,103] and thus prevent unwanted tumor formation [104,105]. Interestingly p53 is the main component of the p53 family including p73 and p63 [106], but while p73 does not seems to be involved in skin formation [107–111], p63 is a crucial element for epidermal development and homeostasis [21,112–114]. In addition to these drastic, life-threatening mechanisms, the skin is a battle ground for a fine immune-regulatory protective response that, when altered contribute to autoimmune disease. As also highlighted for psoriasis [115], the Th1/Th17 balance is involved in a classic inflammatory loop that is at the center of current biotechnological therapies based on anti-TNFs and anti-ILs. Herein, we discussed the unbalance of effector T (Th1/Th2 and Th17) and regulatory (Treg) cells, illustrating how IL-10 produced by Tregs may unbalance the Th1/Th2 versus Th17 equilibrium. Conversely, these mechanisms are currently exploited for innovative, highly effective biological therapies, able to adjust the correct cytokine balance in the treatment of the inflammatory/autoimmune skin disorders such as atopic dermatitis, hidradenitis suppurativa and vitiligo.

Funding Statement

This work has been supported by the Medical Research Council (to GM), Associazione Italiana per la Ricerca contro il Cancro (AIRC) to [GM IG#20473] (2018-2022), Ministry of Health IDI-IRCCS (RC 1.3 e 2.2 to GM, MM, MAP), Ministry of Health Italy–China cooperation grant . Work has been also supported by Regione Lazio through Lazio Innova Progetto Gruppo di Ricerca n 85-2017-14986.

Disclosure statement

No potential conflict of interest was reported by the authors.