The role of cytokines in skin aging

DCLI A 802303 indd CLIMACTERIC 2013;16 1–8 REVIEW Received 30 01 2013 © 2013 International Menopause Society Revised 24 04 2013 DOI 10 310913697137 2013 802303 Accepted 01 05 2013 Correspondence Dr J.

Correspondence: Dr J Calleja-Agius, Department of Obstetrics and Gynaecology, Mater Dei Hospital, B’Kara, Malta

The role of cytokines in skin aging

M Borg * , S Brincat * , G Camilleri * , P Schembri-Wismayer * , M Brincat † and J Calleja-Agius * , †

* Department of Anatomy, Faculty of Medicine and Surgery, University of Malta; † Department of Obstetrics and Gynaecology, Mater Dei Hospital, Malta

Key words: CUTANEOUS AGING , MENOPAUSE , CYTOKINES , TNF- α , COLLAGEN , INTERLEUKINS , INTERFERONS

ABSTRACT

Cutaneous aging is one of the major noticeable menopausal complications that most women want to fi ght

in their quest for an eternally youthful skin appearance It may contribute to some maladies that occur in aging which, despite not being life-threatening, affect the well-being, psychological state and quality of life

of aged women Skin aging is mainly affected by three factors: chronological aging, decreased levels of estrogen after menopause, and environmental factors Aged skin is characterized by a decrease in collagen content and skin thickness which result in dry, wrinkled skin that is easily bruised and takes a longer time

to heal Cytokines play a crucial role in the manifestation of these features of old skin The pro-infl ammatory cytokine tumor necrosis factor-alpha inhibits collagen synthesis and enhances collagen degradation by increasing the production of MMP-9 It also lowers the skin immunity and thus increases the risk of cutane-ous infections in old age Deranged levels of several interleukins and interferons also affect the aging process The high level of CCN1 protein in aged skin gives dermal fi broblasts an ‘ age-associated secretory phenotype ’ that causes abnormal homeostasis of skin collagen and leads to the loss of the function and integrity of skin Further research is required especially to establish the role of cytokines in the treatment of cutaneous aging

INTRODUCTION

It is estimated that within the next 50 years, one-third of all

women will be in their menopausal phase 1 Although many

people consider skin aging as an inevitable part of the aging

process, wrinkled and sagging skin is a disease which can be

fought 2

Several studies suggest that the imbalanced levels of various

cytokines, such as interleukins and tumor necrosis factor-alpha

(TNF- α ), during menopause contribute to the menopausal

com-plications 3 The role of cytokines in osteoporosis and

cardiovas-cular disease in menopause has been well studied 3 – 5 ; however,

their role in skin aging still needs to be explored further

SKIN AGING

Three important factors affect skin aging These are the

natu-ral process of aging (known as chronological aging) 6 , decreased

estrogen levels (which typically happen in women after

menopause) 7 , and harmful environmental factors like

ultra-violet (UV) radiation 8 and smoking 9

There are two major processes through which skin ages – the intrinsic and extrinsic processes Intrinsic aging varies among individuals and depends on their genetic make-up 8 This type of aging cannot be avoided In contrast, extrinsic skin aging can be modulated and depends on the individual ’ s lifestyle Extrinsic cutaneous aging is a type of premature skin aging It occurs by exposing the skin to harmful envi-ronmental factors such as poor nutrition, smoking, the sun and large alcohol intake 8

CHANGES IN THE SKIN AFTER MENOPAUSE

Skin which has undergone intrinsic aging sometimes looks nearly as smooth and fl awless as normal skin except for a few exaggerated expression lines 8 However, several changes occur

to the skin after menopause and the appearance of skin may change quite signifi cantly With the onset of menopause, the skin becomes dry because the functions of the sebaceous glands and sweat glands decline and with time they become unresponsive to stimuli This is associated with decreased estrogen levels 10

Menopause brings with it changes in collagen type, quality,

amount, metabolism and turnover 11 The skin becomes

rap-idly thinner after menopause at a rate similar to the decrease

in bone mass 10 The reduction in thickness cannot be

attrib-uted to age alone 10 The thickness of the skin decreases by

1.13% every year in the initial postmenopausal period 12 ,

while the collagen content decreases by 2.1% every

post-menopausal year 13 The decline in skin collagen and skin

thickness is refl ected in dry, fl aky, wrinkled and easily bruised

skin 11 The postmenopausal period is marked by low amounts

of soluble collagen, a slower turnover and collagen synthesis

This manifests itself in decreased skin resilience and

pliabil-ity 14 It is the decrease in collagen content and change in skin

elasticity that contribute to the appearance of old skin 15

There are both estrogen and androgen receptors in skin

fi broblasts 10,16 Skin is rich in estrogen receptors and estrogens

bind actively and are metabolized by the skin 14 Menopause

brings with it a decrease in the density of hair follicles in the

scalp and body 14 Changes in estrogen levels with the onset

of menopause alter the skin vascularization 17 and the

connec-tive tissue in the dermis 11 Changes in the dermal connective

tissue occur as a result of increased hydroxyproline turnover

and mucopolysaccharide incorporation 11 When estrogen

therapy is administered for 6 weeks, the number of capillaries

increases, atrophy of the epidermis disappears, and collagen

fi bers appear less fragmented 18 Both estriol succinate and

estradiol valerate have been shown to thicken the epidermis

of castrated women after 3 months from the start of

treatment 19

As a result of intrinsic aging, the epidermis and dermis

atro-phy, the amount of fi broblasts decreases and the epidermal

rete ridges become fl at 20 There is an increase in the ratio of

type III to type I collagen 8,21

Aging brings with it a decline in procollagen types I and

III mRNA and protein expression 22 As well as the

impair-ment in collagen synthesis, an increase in the production of

various matrix metalloproteinases (MMPs) also occurs

with aging, especially of MMP-1, MMP-2, MMP-3, and

MMP-9 22,23 The production of all of these MMPs except

gelatinase A (MMP-2) is governed by activator protein-1

(AP-1) 22 AP-1 is a transcription factor that inhibits the

expression of procollagen gene in fi broblasts as well as

stimu-lating MMP gene transcription in both fi broblasts and

kera-tinocytes 24 The expression of AP-1 is higher than normal in

aged fi broblasts taken from skin samples in vitro 22,25 and also

higher than in younger skin in vivo 22,23 Oxidative damage

to cells accumulates with years and the free radicals and

reactive oxygen species (ROS) from aerobic metabolism

initiate the aging process 26 Furthermore, the anti-oxidant

defenses decline with aging 27 The increased ROS participate

in various mitogen-activated protein (MAP) kinase pathways

that activate mitogen-activated protein kinase (MAPK)

MAPK induces AP-1 which consequently increases the

expression of MMPs These mechanisms are responsible

for the decreased amount of collagen in old skin 22

The main theory that is believed to contribute to the aging

process of skin is the accumulation of ROS 26 This oxidative

stress occurring in cells damages the DNA, causing mutations, and oxidizes proteins which then lose their function Further-more, it oxidizes lipids found in membranes which lose their transport capacity and have distorted transmembrane signalling 24

THE MICRO-INFLAMMATORY MODEL

OF SKIN AGING

External stimuli such as cigarette smoke and UV radiation, together with internal stimuli like hormone changes that accompany menopause, trigger the production of adhesion molecules, which are responsible for the fi rst step in the infl ammatory process 28 External stimuli also induce nerve endings to release various neuropeptides into the skin 29 These neuropeptides increase the synthesis of adhesion molecules Adhesion molecules enable circulating monocytes and granulocytes to roll over, adhere and diapedese through the endothelial wall of blood vessels and migrate into the der-mis 28 These white blood cells produce and secrete proteases and ROS that in turn change the turnover of the proteins of the dermis and also damage cells in the skin The damaged cells are stimulated to release leukotrienes and prostaglan-dins 28 These chemicals act as stimuli on the mast cells and induce the secretion of histamine and the cytokine TNF- α , which in turn stimulate endothelial cells to produce intracel-lular adhesion molecule-1 (ICAM-1) and also help liberate P-selectins 28

A second set of immune cells is stimulated to migrate, caus-ing more infl ammation, and the process goes on and on The

fi nal result would be an imbalance in the degradation and synthesis of elastin and collagen fi bers Since the fi broblasts

in old age are unable to synthesize these fi bers in oriented arrays, this infl ammatory process causes aging of the skin with a change in the composition of the dermis and epidermis, skin thickness and elastic properties of the skin 28

CYTOKINES

Cytokines play an important role in skin aging These cell-signalling proteins serve as an intercellular communication link They are produced by cells of the immune system such

as Langerhans cells and other cells in the skin, including kera-tinocytes and epithelial cells 30 Several cytokines that act on the skin target cells have pleiotropic and redundant effects 30 This is due to similarities in their amino acid sequence which enable some cytokines, especially the interleukins, to bind to the same cell receptors Usually, receptors have a very high affi nity to the cytokines; therefore, the concentration of cyto-kines in the body would be low, typically femtomolar or nano-molar However, this concentration increases with trauma or infection The action of cytokines is usually brought about in

a cascade involving several cytokines acting simultaneously or after each other Their action is brought to an end by other inhibitory cytokines or by the receptors themselves 31

TNF- a AND SKIN AGING

In the skin, TNF- α is produced by fi broblasts 32 , macrophages,

monocytes and keratinocytes 30 TNF- α is initially

membrane-bound but it is then cleaved by TNF- α -converting enzyme to

a soluble protein These two forms of TNF- α are both

biologi-cally active on forming non-covalently linked homotrimers 33

TNF- α brings about its effects by binding to the receptors

TNFR1 or TNFR2 which are both located on cell

mem-branes 33 Ultraviolet B (UVB) radiation promotes the

production of TNF- α by dermal fi broblasts and epidermal

keratinocytes and this increases infl ammation 34

Macrophages that reside in the skin secrete TNF- α 35 , a

cytokine which triggers infl ammatory responses 36 However,

macrophages in the skin of old people secrete only a very small

amount of TNF- α This decrease in cutaneous macrophage

TNF- α secretion with age results in defective activation of the

blood vessels of the dermis and hence a decreased recruitment

of antigen-specifi c CD4 ⫹ T cells from the blood to areas of

the skin where antigens would have entered 35 This leads to

an increased occurrence of cutaneous infections 37 while

immu-nity decreases with age 38 This is, in fact, shown by decreased

skin delayed-type hypersensitivity (DTH) responses to

previ-ously sensitized antigens 35

The DTH response of old skin to particular recall antigens

introduced via intradermal injections was investigated 35

Antigens used were the bacterium tuberculin purifi ed

pro-tein derivative, varicella zoster virus and Candida albicans

(fungus) For all the three different types of microorganisms,

skin biopsy samples showed low TNF- α levels and DTH

response The macrophages were studied to determine the

cause of the low TNF- α When Toll-like receptor (TLR)

ligands were added, the macrophages secreted a

considerable amount of TNF- α This showed that the

mac-rophages residing in old skin are not defective but are

inactivated CD4 ⫹ Foxp3 ⫹ regulatory T cells, which are

present in large amounts in the skin of elderly people, inhibit

macrophages from secreting TNF- α 35,39 These T regulatory

cells inhibit the process of macrophage activation and the

secretion of TNF- α 40

The DTH response of the skin studied in vitro manifests

the responsiveness of memory T cells in vivo As the skin

reactivity to antigens decreases with aging, old people are

more susceptible to skin infections and malignancy 37 TNF- α

stimulates the expression of adhesion molecules on

endothe-lial cells, mainly E-selectin, ICAM-1, and VCAM-1, and

hence helps leukocytes to migrate through the blood vessels

into the dermis 35 When anti-TNF- α is given as a treatment

for rheumatoid arthritis, patients often suffer from skin

infections due to the decreased leukocyte migration 41

In elderly people, the function of TLR1 and TLR2 on

cutaneous macrophages may be defective after they bind to

their ligands 42 This defect may be responsible for the

decreased secretion of TNF- α by macrophages when the

recall antigen C albicans was used to infect skin , as this

species binds to TLR1 and TLR2 35 A defect with TLR4

expression and function of macrophages in old skin was also

observed by other investigators 43 – 45 although not shown

by others 46 When these three receptors were stimulated

in vitro , cutaneous macrophages and monocytes from

peripheral blood secreted TNF- α and this confi rmed that the defect was reversible 35

Besides signals from TLRs to become activated, mac-rophages need cytokines like interferon gamma (IFN- γ ) in order to achieve their maximum functional capacity 35 How-ever, skin samples lacked this cytokine 35 and this could

be the reason for the low amount of T cells that migrated to the site of antigen challenge IFN- γ is required for the expression of some genes of the macrophage, including the MHC class II genes The expression of MHC class II by the macrophage is necessary for antigen presentation to the T cells 47 If this does not happen (as occurs in old people), the immune response cannot occur due to a block in the cascade 35

With old age, the decrease in macrophage activation leads to reduced memory T-cell immunosurveillence and chronic non-specifi c infl ammatory responses 35 This results in

an accumulation of debris in skin tissue and blood vessels which can act as a predisposing factor for infection and malignancy in aged skin and other complications like crystal arthritis and macular degeneration due to lipofuscin pigment accumulation 35,48

With menopause, levels of the cytokine TNF- α in the cir-culation are elevated 3,49,50 At high concentrations, TNF- α increases the synthesis of collagenase as well as inhibiting collagen synthesis 51

TNF- α has a key role in infl ammatory responses that occur in the skin It is able to modulate the expression of the MMP gene and is responsible for inducing the production of MMP-9, an enzyme that causes skin aging by causing skin damage and does not allow its repair 52 When the cells of the epidermis are exposed to persistent TNF- α , the production

of MMP-9 is disturbed and the epidermis can be damaged irreversibly 52

Infl ammatory signals upregulate MMP-9 53 The transcrip-tion of MMP genes is regulated by the transcriptranscrip-tion factors AP-1 54 and nuclear factor kappa B (NF- κ B) 52 TNF- α increases the binding activity of these transcription factors to the MMP-9 DNA sequence and hence increases the production of MMP-9 protein 52

The effect of 3-deoxysappanchalcone, a fl avonoid having anti-infl ammatory and antioxidant properties 55 , was investi-gated in combination with MMP-9 52 The 3-deoxysappanchal-cone decreased infl ammation by reducing the expression

of MMP-9 52 3-Deoxysappanchalcone also has anti-allergic properties 56 , is able to cause apoptosis 57 , and works against the infl uenza virus 58 The 3-deoxysappanchalcone inhibited the DNA binding activity of AP-1 52 The higher the concentra-tion of 3-deoxysappanchalcone used, the larger the reducconcentra-tion

in the expression and activity of AP-1 protein which decreased the expression of MMP-9 protein 52 The 3-deoxysappanchal-cone also inhibited directly TNF- α -induced NF- κ B 52 Further investigations were carried out to determine which part of the translocation process of NF- κ B 3-deoxysappanchalcone

affected Western blot showed that the degradation and

phos-phorylation of I κ Ba, a protein that inhibits NF- κ B, was not

affected by this anti-infl ammatory agent This suggests that

3-deoxysappanchalcone directly inhibited the activity of

NF- κ B 52

3-Deoxysappanchalcone can inhibit the expression of

MMP-9 at both the mRNA and protein levels in human

kera-tinocytes by blocking the activation of both AP-1 and NF- κ B

transcription factors 52 Cosmetic and pharmacological

products that inhibit the transcription factors of MMP-9 can

help skin renewal 52

TNF- a AND THE CELL CYCLE OF

KERATINOCYTES

TNF- α may help the skin to remove damaged cells and

mediates UVB-induced apoptosis When pre-malignant

keratinocytes are irradiated with UVB, their TNF- α

secre-tion in the epidermis increases rapidly This increased TNF- α

eliminates the G2/M checkpoint of the cell cycle of

kerati-nocytes and inhibits the repair of damaged DNA although

it increases apoptosis 33 The cells escape this checkpoint and

accumulate mutations, leading to the development of

tumors TNF- α activates protein kinase B (Akt) and

regu-lates the transcription factor FoxO3a, the pro-apoptotic

protein Bad, the protein kinase mTOR and the atypical

protein kinase C (aPKC) The reduction in the repair of

damaged DNA is caused by the aPKC – Akt axis However,

when these keratinocytes were treated with infl iximab (an

anti-TNF- α monoclonal antibody) therapy, DNA repair

was inhibited despite the enhancement of the G2/M cell

cycle checkpoint and apoptosis 33

TNF- α mediates skin aging by inhibiting collagen synthesis

and by increasing the production of MMP-9 which causes

collagen degradation 51,52 The decrease in cutaneous

mac-rophage TNF- α secretion with age decreases the skin

immu-nity, with a resultant increase in cutaneous infections in old

skin 37,38

INTERLEUKIN-1

Active interleukin-1 (IL-1) is found in the stratum corneum

of the epidermis 59 The stratum corneum is the outermost

layer of the epidermis and it is mostly composed of dead

cells 60,61 IL-1 α and interleukin-1 receptor antagonist (IL-1ra)

are both produced by keratinocytes in the skin IL-1ra

operates through competitive inhibition by binding to the

common receptors on several target cells 59 although other

investigators suggest that it only binds to IL-1 receptor

type I 62 Several stimuli such as UVB radiation stimulate

keratinocytes in the epidermis to release IL-1 α which

induces cytokines and adhesion molecules to bring about

skin infl ammation In turn, IL-1ra inhibits the activities of

IL-1 α and the balance between these two cytokines helps

preserve homeostasis of the skin 59

The effect of aging and gender (and hence also menopause indirectly) on the content of IL-1 α and IL-1ra in the stratum corneum has been investigated 59 The variables of skin exposed to UVB (the face) and skin not exposed (inside of upper arm) were also taken into account The IL-1 levels mea-sured in the facial skin layer did not change signifi cantly with age However, in the layer of the inside of the upper arm, IL-1 α levels were higher in aged skin while IL-1ra decreased with age There was no difference between the genders and hence menopause does not alter the concentration of IL-1 cytokines in the stratum corneum 59 However, other research-ers have reported an increase in the expression of IL-1ra

in cultured human keratinocytes with aging, while the expression decreased in photo-aged skin keratinocytes 63

INTERLEUKIN-18

IL-18, formerly known as interferon-gamma inducing factor

is an immunoregulatory cytokine produced by epithelial cells, dendritic cells and macrophages It is a proinfl ammatory cytokine that increases the expression of cell adhesion molecules, contributes to atherosclerosis and brings about the aging process 62 Therapeutic strategies aimed at reducing the level of IL-18 may slow the aging process 62

IL-18 also plays a role in type 2 T-helper cell polarization When it is overexpressed, it worsens both allergic and non-allergic skin infl ammation 64 It is also involved in autoimmune diseases 62

However, IL-18 protects against skin damage caused from exposure to UVB radiation and hence decreases UV-induced apoptosis 65 It also helps prevent UV-induced immunosuppression 65

INTERLEUKIN-6

IL-6 functions as a pro-infl ammatory and an anti-infl ammatory cytokine via different signalling mechanisms 66

It participates in various infl ammatory and immune responses 67 IL-6 synthesis is induced by transforming growth factor-alpha (TGF- α ) in human keratinocytes 68 IL-6 in turn helps the keratinocytes to proliferate 69 However, other investigators have not found an increase in keratinocyte pro-liferation with keratinocyte-directed IL-6 expression in trans-genic mice 67 IL-6 expression in transgenic mice causes the stratum corneum to become thicker; however, it does not enhance epidermal proliferation or cause leukocyte infi ltra-tion, suggesting that IL-6 does not have a direct proinfl amma-tory activity in the skin despite being elevated in infl ammaamma-tory diseases like rheumatoid arthritis 67

The decline in estrogen level that occurs in menopause is accompanied by an increase in IL-6 production 3 However, this increase is very small when compared to the elevated levels that occur in infection or tissue injury 70

Skin keratinocytes are stimulated to increase the production

of IL-6 after exposure to TNF- α , IFN- γ or IL-4 71 IL-6 is

involved in the aging of skin and in the formation of skin

wrinkles IL-6 levels are further upregulated on exposure to

ultraviolet radiation 72

INTERFERONS

Interferons have several antiproliferative and antiviral

activities Fibroblasts, the major type of cells in the dermis,

undergo senescence after exposure to interferon beta (IFN- β )

through a DNA damage signalling pathway 73 Fibroblasts

produce collagen, mainly types I and III 18 On the other hand,

continuous exposure to IFN- α decreases population doublings

of dermal microvascular endothelial cells and induces their

senescent phenotype 74

IFN- γ belongs to the macrophage-activating factor family

of cytokines It is produced by T lymphocytes, including

those that home in the skin and have the cutaneous

lympho-cyte antigen (CLA) It participates in both adaptive and

innate immunity There seems to be no change in the number

of IFN- γ -producing CLA ⫹ T cells in old age 75

T-lymphocyte-dependent immune reactions are stimulated

by CD1a ⫹ (Langerhans) cells Evidence shows that these cells

decrease linearly with age and, as a consequence, aged skin

has decreased immunosurveillance Studies on the effect of

IFN- α on Langerhans cells on biopsies of preauricular skin

showed that, after application of alpha-interferon cream, the

skin samples taken from the group aged between 57 and 75

years had increased numbers of cutaneous CD1a ⫹ cells 76

EFFECT OF CCN ON THE SKIN CONNECTIVE

TISSUE

The connective tissue of the skin consists primarily of collagen

that is produced by fi broblasts located in the dermal layer of

the skin These fi broblasts are also responsible for the

degra-dation of collagen in the extracellular matrix With old age,

dermal fi broblasts express an ‘ age-associated secretory

pheno-type ’ which is believed to be caused by a high level of

cysteine-rich protein 61 (CCN1) in aged skin 77 The end result is an

abnormal homeostasis of skin collagen type I whose

degrada-tion is upregulated and its producdegrada-tion reduced 78

CCN1 or CRY61 belongs to the protein family of CCNs, a

family made up of six matricellular proteins that inter-relate

with the extracellular matrix 77,79 This protein changes the

way some cytokines work, for example it causes TNF- α to

express cytotoxic properties without inhibiting NF- κ B

activ-ity It also supports the adhesion of murine macrophages and

enhances the expression of a pro-infl ammatory genetic profi le

that is typical of activated M1 macrophages that take part in

T1 helper cell responses Furthermore, CCN1 increases the

levels of the cytokines IL-1 α , IL-1 β , IL-6, IL-12b, and TNF- α

while it downregulates the expression of anti-infl ammatory

factor TGF- β 80 This change in genetic profi le is regulated via

two mechanisms, either by a direct activation of NF- κ B by

CCN1, known as the immediate-early response, or by a delayed response that is brought about through CCN1-induced TNF- α 80

CCN1 elevates the amount of ROS in the cell by interacting with integrins 81 The high level of ROS activates NF- κ B and MAPK signalling which then upregulate the production of IL-1 β and IL-6 82 The expression of MMPs in aged dermal

fi broblasts is increased by the cytokines IL-1 β and IL-6, which also decrease the production of collagen, further imbal-ancing collagen homeostasis 82,83 IL-1 β reduces the TGF- β type II receptor and hence impairs TGF- β signalling in aged dermal fi broblasts, causing aberrant collagen homeostasis 78 The secretory phenotype of dermal fi broblasts in old age results in aging of the skin connective tissue and brings about the loss of function and integrity of old skin 77

CONCLUSION

Cutaneous aging is one of the major noticeable menopausal complications that most women want to fi ght in their quest for an eternally youthful skin appearance Skin aging is not

a uniform biological event and varies between individuals 84

It is mainly affected by three factors: chronological aging 6 , decreased levels of estrogen after menopause 7 , and environ-mental factors 8 Aged skin is characterized by a decrease in collagen content and skin thickness, which result in dry, wrinkled skin that is easily bruised and takes a long time to heal 84 Cytokines play a central role in the manifestation of these features of old skin TNF- α inhibits collagen synthesis and enhances collagen degradation by increasing the produc-tion of MMP-9 51,52 It also lowers the skin immunity and thus increases the risk of cutaneous infections in old age 37,38 IL-1 α and IL-18 levels are higher in aged skin and promote skin infl ammation, while IL-1ra decreases with age 59 IL-18 also contributes to atherosclerosis 62 The level of IL-6 is slightly increased after menopause and this helps keratinocyte proliferation 69 and increases the stratum corneum thickness

in transgenic mice 67 The amount of IFN- γ -producing CLA ⫹

T cells is not altered in old skin IFN- γ participates in both adaptive and innate immunity 75 On the other hand, applica-tion of alpha-interferon cream on aged skin increases the number of cutaneous Langerhans cells that are responsible for skin immunosurveillance 76 Aged skin expresses a high level of CCN1 protein which gives dermal fi broblasts an ‘ age-associated secretory phenotype ’ , resulting in an abnormal homeostasis of skin collagen and causing old skin to lose its function and integrity 77,78 Further research is required espe-cially to establish the role of cytokines in the treatment of cutaneous aging

Confl ict of interest The authors report no confl ict of

interest The authors alone are responsible for the content and writing of this paper

Source of funding Nil

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