Tryptophan, an important link in regulating the complex network of skin immunology response in atopic dermatitis

Atopic dermatitis (AD) is a common chronic relapsing inflammatory skin disease, of which the pathogenesis is a complex interplay between genetics and environment. Although the exact mechanisms of the disease pathogenesis remain unclear, the immune dysregulation primarily involving the Th2 inflammatory pathway and accompanied with an imbalance of multiple immune cells is considered as one of the critical etiologies of AD. Tryptophan metabolism has long been firmly established as a key regulator of immune cells and then affect the occurrence and development of many immune and inflammatory diseases. But the relationship between tryptophan metabolism and the pathogenesis of AD has not been profoundly discussed throughout the literatures. Therefore, this review is conducted to discuss the relationship between tryptophan metabolism and the complex network of skin inflammatory response in AD, which is important to elucidate its complex pathophysiological mechanisms, and then lead to the development of new therapeutic strategies and drugs for the treatment of this frequently relapsing disease.

. At present, researchers believe that immune disorders play a crucial role in the pathogenesis of AD.T helper type 2 (Th2) and Th17-mediated immune disorders dominates the acute stage and chronic stages of AD respectively (5), both of them "interweave" each other to form a complex network of skin inflammation.Among them, the Th2 type immune response has attracted widespread attention and exploration.In relevant studies on the pathogenesis of AD, the pathogenic role of keratinocyte derived cytokines such as thymic stromal lymphopoietin (TSLP), interleukin-33 (IL-33) and IL-25 in inducing skin inflammation has been emphasized (6).These cytokines can promote the production of Th2 type cytokines such as IL-4, IL-5, and IL-13.Notably, IL-4 and IL-13 have previously been reported to damage the integrity of the skin barrier by inhibiting the production of key proteins such as filaggrin (Flg) and disrupting the stability of tight junctions (7,8), leading to increased penetration of allergens and pathogens.The downstream signal transduction of both also blocks the expression of innate immune response genes such as bdefensins (7,9), increasing the risk of skin infection with Staphylococcus aureus in AD patients.Meanwhile, IL-4 and IL-13 can drive the regeneration of eosinophils and mast cells, as well as stimulate the secretion of key cytokines IL-31 in pruritus stimulation (10).The repeated scratching behavior leads to further physical damage to the skin barrier, thereby forming a "vicious cycle" that exacerbates AD skin lesions.In addition, congenital skin barrier dysfunction and disruption of the microbiota in the skin and intestines are also considered the main contributors in driving the development of AD (11).However, the exact mechanism of AD pathogenesis mediated by intestinal microbiota is still not fully elucidated, which is presumed to be associated with tryptophan (Trp) metabolism.
Long-term itching symptoms and recurrent episodes of disease could severely affect the life quality of AD patients, and are closely related to the occurrence of negative psychology such as anxiety and depression (12).But, unfortunately, treating AD is challenging due to the high heterogeneity of the disease (13) and the limited therapeutic drugs (14).In traditional treatment regimens, topical corticosteroids are a classic first-line medication, although they could produce anti-inflammatory and immunosuppressive effects by inhibiting various inflammatory cells and cytokines, long term use may be limited by the side effects such as skin atrophy (15).The highly individualized selection of immunosuppressive drugs reduces the scope of application of the drugs and patient compliance.As an emerging treatment for AD, biological agents seem to be effective alternatives to traditional treatment.In 2017, dupilumab, a fully monoclonal antibody targeting IL-4R, became the first biological agent approved for use in adult patients in the United States due to its significant improvement in AD clinical manifestations.However, a higher incidence of herpes simplex virus infection and conjunctivitis was found in patients receiving dupilumab.Among other newly developed monoclonal antibodies, including Lebrikizumab and Tralokinumab combined with IL-13, Nemolizumab inhibiting IL-31, and Fezakinumab blocking the action of IL-22, various degrees of herpes infection, conjunctivitis, viral upper respiratory tract infection, peripheral edema, and elevated creatinine kinase levels have been found after treatment (16).Recent studies have shown that the activation of the JAK (Janus kinase)-STAT (signal transducer and activator of transcription) signaling pathway is essential for mediating downstream inflammatory cytokines in AD patients, including IL-4, IL-5, IL-13, IL-31, IL-22, and TSLP (17).These cytokines bind to immune cells, keratinocytes, and peripheral sensory neurons, leading to the spread of inflammation and itching symptoms in AD patients.Therefore, JAK inhibitors have become a new treatment strategy for AD because of immunosuppressive effects.Currently, commonly used JAK inhibitors include oral Baricinib targeting JAK1 and 2, and Upadacitinib targeting JAK1, as well as topical Tofacitinib targeting JAK1 and 3, Ruxolitinib targeting JAK1 and 2, and Delgocitinib targeting the entire JAK pathway.However, it is worth noting that, although JAK inhibitors could improve the severity of the condition and itching symptoms in AD patients, they also have the risk of increasing blood creatine phosphokinase and inducing headaches and nasopharyngitis (18).The long-term effectiveness and safety of these developing biological therapies are not yet fully understood, and with the continuous emergence of new therapies for AD, the comparison of drugs is crucial for patients to choose safe and effective treatment plans.Hence, in-depth study of the mechanism and looking for new potential treatment of AD is still valuable.Trp metabolism has recently been found to have a potential connection with the regulation of AD immune system, and greatly affect the development of other immune diseases such as metabolic syndrome, neuropsychiatric disorders and inflammatory bowel disease (19).However, throughout the literatures, the role of tryptophan metabolism in the pathogenesis of AD has not been well discussed.This article reviews the potential role of tryptophan metabolism in regulating the complex network of skin inflammatory response in AD, in order to enhance understanding of the occurrence and development of AD and seek treatment opportunities targeting tryptophan metabolism.

Tryptophan metabolism and its' role on regulating skin diseases by aryl hydrocarbon receptor
Tryptophan is an essential amino acid for the human body that is mainly produced by high-protein foods such as milk, seafood, grains and peanuts, which is a biosynthetic precursor of a large number of metabolites (19).As an important intestinal metabolite, Trp metabolism in the intestine follows three main pathways (20) under homeostatic conditions: (i) the Kynurenine (Kyn) pathway (KP) via indoleamine 2,3-dioxygenase 1 (IDO1) in immune cells (mainly macrophages) and intestinal epithelial cells (IEC); (ii) the serotonin pathway via Trp hydroxylase 1 (TpH1) in enterochromaffin cells.Above two pathways mainly occur in host cells, that are predominantly but not exclusively used by the host.(iii) The direct conversion of Trp into indole, indole derivatives, and tryptamine by the intestinal microbiota via the enzyme tryptophanase (21), which produces metabolites such as indole acetic acid (IAA), indole-3-acetaldehyde (IAAld), indole-3-aldehyde (IAld), indole propionic acid (IPA) and indoleacrylic acid (IA) (Figure 1).Trp metabolites mainly exists in human feces and have been shown to maintain the integrity of the skin and intestinal barrier and immune cell homeostasis by activating aryl hydrocarbon receptor (AhR) (22,23), thus are considered as the active biomarkers (24).
AhR is a ligand dependent transcription factor that could be observed in all skin cells.Although the expression levels vary in specific cell types (25), AhR signaling plays a major role in skin diseases due to its involvement in many important physiology processes such as regulating immune cells, maintaining redox balance in cells and epidermal barrier function (25)(26)(27).As a hybrid receptor, AhR could be activated by a variety of exogenous and endogenous ligands (28-31), among them, the mild and transient AhR activation caused by antioxidant phytochemicals or some tryptophan derivatives could effectively maintain healthy and complete skin barrier (32,33).When tryptophan derivative binds to the AhR ligand to form a complex, AhR dissociates from the cytoplasm and translocates to the nucleus.The AhR nuclear translocation protein (ARNT) dimerizes with the exposed nuclear translocation site of AhR, binding to be the upregulated target gene transcription response element (34).This signaling pathway initiates the activation of the OVO-like 1 (OVOL 1) transcription factor, and subsequently enhances the expression of Flg and olein (LOR), these kind of terminal differentiation proteins have specificity for fully differentiated keratinocytes (35), helping to accelerate the final differentiation of the epidermis and the formation of the epidermal barrier (36, 37).Notably, increasing evidences suggest that the binding of tryptophan derivatives with AhR play a role in the pathogenesis or treatment of many skin diseases (38,39), including inflammatory diseases, skin pigmentation diseases, and cancer (40,41).As expected, tryptophan metabolism has been reported to be closely related to the occurrence and development of AD (42), the tryptophan metabolite in the AD skin lesions is significantly reduced (43) that may be associated with the weakened tryptophan metabolism in the skin microbiota by Th2 type immune response (43).Due to AhR lacking physiological ligands in the Th2-deviated environment of AD, the skin barrier damage and colonization of pathogenic microorganisms such as Staphylococcus aureus would increase, thereby exacerbating Th2 type inflammation in the lesion of AD (44,45).Therefore, we speculate that tryptophan and its derivatives  Tryptophan has recently been also implicated in the crosstalk between gut microbiota (GM) and host in healthy and diseased states (19,46), and its metabolic impairment may affect the occurrence and development of many diseases, such as metabolic syndrome, neuropsychiatric disorders and inflammatory bowel disease (19).Research findings showed that, compared with the healthy control, inflammatory bowel disease (IBD) patients experienced intestinal microbiota homeostasis imbalance, while their serum tryptophan levels also significantly decreased.Interestingly, the composition of GM is significantly correlated with serum level of tryptophan, dietary supplementation with Trp can normalize the disordered GM in the IBD mouse model (47), and the AhR ligands produced subsequently by GM could alleviate the inflammatory response (48).Wilck et al. found that, Lactobacillus murinus and Lactobacillus reuteri could convert tryptophan into IAld and indole-3-lactic acid (ILA) by aromatic amino acid aminotransferase and indole lactic acid dehydrogenase (49,50), these generator of AhR would improve the severity of colitis in mice (23).This type of approach can even be expanded to other inflammatory diseases (51), suggesting that the biological effects of Trp metabolites may be related to the interactions between gut microbiota and may become potential therapeutic targets for certain diseases.Additionally, Metghalchi et al. found that plasma level of Trp predicts the likelihood of adverse cardiovascular events in patients with acute myocardial infarction (52).Based on the characteristics of Trp metabolism influenced by pathological conditions, the use of Trp and its metabolites as biomarkers to support diagnosis and prognosis and to guide treatment options is attractive.Therefore, this article will focus on exploring the potential diagnostic and therapeutic role of tryptophan metabolism in the complex pathogenesis of AD.

The potential role of tryptophan metabolism in atopic diseases
AD typically begins in early childhood and is usually the first manifestation of atopic progression.While clinical symptoms of some AD children would disappear with age, some children may experience food allergy during the course of the disease, even half of AD children may develop allergic asthma and two-thirds of AD children are at risk of developing allergic rhinitis in the future.This kind of disease progression is known as the "Atopic March" (53,54).Exploring the relationship between these atopic diseases and tryptophan can help us further understand its role in AD.

Food allergy
Although people often refer to any adverse reaction to food as an allergy, strictly speaking, food allergy (FA) is an adverse reaction to the food itself that is mediated by specific IgE antibody.Increased levels of tryptophan and indole metabolic pathway products were found in serum samples from FA children, while levels of metabolites from Kynurenine and 5-hydroxytryptamine pathways decreased with disease burden (55,56).However, these tryptophan metabolites can downregulate the T cells activity or induce various regulatory T cell (Treg) cells to regulate immune responses at the mucosal barrier (55,57).The reduction of tryptophan metabolites with potential anti-inflammatory effects would create a suitable environment for Th2 tilted immune responses, thereby increasing the occurrence risk of Th2 related diseases such as AD.Another study on peanut allergic mice suggests that the above mechanism is related to the activation of AhR (58,59), but to our knowledge, there are currently no clinical studies evaluating the functional role of AhR ligands in FA.

Allergic rhinitis and asthma
Atopic diseases are highly prevalent in children worldwide, with the prevalence rates of allergic rhinitis (60, 61) and asthma (62) being approximately 14% and 15%, respectively.Consistent with food allergy, levels of tryptophan are significantly elevated in patients with allergic rhinitis and asthma (55,(63)(64)(65)(66). Serum tryptophan levels could not only be used to distinguish between stable asthma children and healthy children, but also to distinguish between controlled and uncontrolled asthma patients (64).This kind of influence could even be traced back to the early stages of life, the tryptophan metabolite 5-hydroxyindolepyruvate in maternal amniotic fluid can determine whether the baby has wheezing attacks in the first year of life (65).The above evidences suggest the important value of tryptophan in atopic related diseases.Some researchers believe that, the high level of tryptophan in the serum of patients with atopic diseases may be related to the low activity of IDO-1 enzyme or the inhibition of IDO-2 enzyme activity (66,67).Among them, IDO-1 is widely expressed in tumor cells and inflammatory/antigen presenting cells (APCs), induces the production of Kyn by catabolizing tryptophan, the ratio of serum Kyn to tryptophan reflects IDO-1 activity to some extent (68,69).Kyn could up-regulate the expression of foxp3 transcription factor, increase the differentiation of Treg cells and decrease the expansion of Th17 type cells (67,(70)(71)(72).For instance, Th17 and Th1 type airway inflammation were observed in IDO deficient mice infected with the virus, characterized by significant infiltration of neutrophil, high production of IL-17 and interferon-g (IFN-g) and obvious airway responsiveness (73).In the asthma mouse model, activation of toll-like receptors (TLR)-9 through bacterial DNA motifs could induce the expression of IDO (74) and aromatic receptor (75,76) to reduce the inflammatory airway hyperresponsiveness.Therefore, although the specific role of tryptophan metabolism in the pathogenesis of atopic diseases such as asthma and allergic rhinitis is still not fully understood, it cannot be denied that tryptophan metabolism and its intermediate products are the important links in regulating immune responses (19).According to research reports, administration of D-tryptophan before experimental asthma induction in mice observed an increased number of regulatory T cells in the lungs, a decreased Th2 type immune response, and an improvement in allergic airway inflammation and airway hyperresponsiveness (77).Another tryptophan metabolite, 3-Hydroxy-Anthranilic acid (3-HAA), could directly target the phosphorylation of 3-phosphoinositol dependent kinase 1 (PDK 1) to inhibit NF-kB triggered by T cell antigen receptors, leads to dysfunction and cell death of activated TH2 cells in the body, which is sufficient to suppress experimental asthma induced by TH2 immune response in mice (78).In addition, PapaMichael et al. (79) also reported a positive correlation between the tryptophan metabolite 5-hydroxyindoleacetic acid (5-HIAA) and the asthma FEV 1/FVC (forced expiratory volume in the first second/forced vital capacity) ratio, as well as a negative correlation with fractional exhaled nitric oxide (FeNO).However, further research is needed to demonstrate the therapeutic potential of tryptophan and its metabolites in patients.
In summary, the atopic diseases such as food allergy, asthma, and allergic rhinitis all have varying degrees of tryptophan metabolism abnormalities, which may even affect early immune homeostasis.The use of tryptophan metabolites as biomarkers to support the diagnosis and prognosis of atopic diseases and guide treatment choices is an attractive choice.However, despite the extensive research supporting the important role of tryptophan metabolism in the occurrence and development of atopic related diseases, the mechanism connections between these diseases are still unclear (80), the specific mechanisms of action and therapeutic potential still need to be further explored.4 Trp metabolism as a bridge between atopic dermatitis and gut microbiota 4.1 Gut microbiota and its metabolites contribute to the development and prognosis of AD GM is an enormous and complex ecosystem.The number of bacterial cells within the human gut outnumber the host's cells by 10 times and the genes encoded by these bacteria even exceeds their host's genes by more than 100 times (81).Consequently, extensive research has been conducted on the gut microbiota and its role in the various diseases and health states.It is reported that the imbalance in the diversity and composition of GM could lead to negative changes in intestinal microbial metabolism and immune response, such as affecting the host's intestinal immune environment and barrier function, disrupting mucosal immune tolerance, and increasing the vulnerability of the host (82, 83).Actually, the connection between GM and skin inflammation was discovered as early as the 1930s (84).Up to now, numerous studies have revealed that the development of allergic diseases such as asthma, allergic rhinitis and AD is closely associated with GM disturbance (85)(86)(87)(88)(89)(90)(91).Although there are some conflicting results (92-95), lots of clinical trials have claimed that AD patients exhibit poor gut microbial diversity compared to healthy individuals (96-99), as well as a structural disorder in the gut microbiota, which includes the increased abundance of microorganisms related to inflammation and epithelial damage in the intestinal flora, such as Clostridium difficile, Coliform, pathogenic Escherichia coli, and Staphylococcus aureus (93, 97, 100-104), and the significantly decreased abundance of SCFA (short chain fatty acid) producers, such as Bifidobacteria, Fecal cocci and Fecal bacilli (92, 95, 96, 103, 105-108).Notably, SCFAs, including acetic acid, propionic acid, and butyric acid, have been proven to help maintain the balance of GM, affect immune cells, and are closely related to the remission of clinical manifestations of AD (96,109,110).SCFAs can bind G-Protein-Coupled Receptors (GPCRs, including GPR43, GPR41, and GPR109a) expressed on intestinal epithelial and immune cells to inhibit histone deacetylase (HDAC) (111,112), leading to acetylation of the Foxp3 coding region in T cells, driving Treg differentiation (113,114), downregulating expression of inflammatory cytokines (IL-6, IL-8, and tumor necrosis factor -a (TNF-a)) (115,116), and stimulating the production of antiinflammatory cytokines such as IL-10 by peripheral blood mononuclear cells (PBMCs) (117) which could suppresses proinflammatory types of Th17 and Th2 cells in turn.Therefore, intervention targeted GM and its metabolites may be an alternative method for controlling the inflammationary responses and improving the clinical symptoms of AD.

Probiotics alleviate the inflammatory response in AD by up-regulating tryptophan metabolism
In addition to SCFA, another prominent example of how microbiota affects host tissue level immune maturation is the probiotic metabolic pathway of tryptophan.As the important regulator of GM, probiotics could improve the clinical severity of patients by the reduction of pro-inflammatory cytokines such as IL-13 and IL-5 in AD skin lesions (118).It has been revealed that IL-13 is a key driver of activating Th2 type immune responses, and IL-15 is a key cytokine inducing development and survival of eosinophil (119).Thus, probiotics is expected to become an effective alternative strategy for the treatment of skin diseases based on the enormous potential in regulating immune function (120,121).It is worth mentioning that, current research has also confirmed a close link between probiotics and tryptophan metabolism.For instance, the application of Lactobacillus reuteri (122), Lactobacillus salivary (123), and Bifidobacterium (124) significantly increased the level of tryptophan metabolites in the serum, and even accompanied by the decreased pro-inflammatory response.In fact, tryptophan, as an important regulator of mammalian inflammatory response (125), has been proven to have an immunomodulatory effect in experimental colitis or IBD patients (126).The GM obtained from IBD patients showed poor ability to produce AhR ligands (23), and in addition, sterile mice susceptible to colitis observed significant improvement in intestinal inflammation after supplementation with AhR agonists and Lactobacillus strains capable of metabolizing tryptophan (22).Similarly, administration of Bifidobacterium in AD model mice showed reduced scratching behavior and an increase in the level of Kyn (127).Therefore, considering the role of the beneficial microbiomes in tryptophan metabolism, we speculate that increasing intestinal probiotics may medicate the immune inflammatory responses and alleviate clinical symptoms of AD by producing tryptophan derivatives.

Tryptophan metabolites promote the regression of skin inflammation in AD by repairing the intestinal barrier
It has been reported that, Trp metabolism and its derivatives have many beneficial effects on intestinal epithelial barrier function mediated by GM.The intestinal barrier is made up of epithelial cells, mucous layers, T cells, IgA, and dendritic cells, collectively forming the "mucosal firewall" (128).The imbalance of GM, decreased production of SCFA, and loss of immune tolerance in the intestines of AD results in the occurrence of inflammatory reactions, increased pathogenic microorganisms and damaged intestinal barrier (92).This kind of "leaky gut" paves the way for the occurrence of AD skin inflammation by releasing toxins, food residues and pathogenic microorganisms from the damaged intestinal epithelium into the systemic circulation, inducing the release of pro-inflammatory cytokines to propel Th2 type immune responses ultimately (129)(130)(131).Nevertheless, Trp metabolites such as indole can reinforce intestinal epithelial barrier function by enhancing the expression of genes involved in preserving epithelial cell structure and function in vivo and in vitro (132,133), IA (134) and IPA (134, 135) can enhance intestinal epithelial barrier function by reducing the expression of inflammatory factors in intestinal epithelial cells via activating the AhR and PXR (pregnenolone X receptor) receptors, respectively, to promote intestinal goblet cells differentiation and mucus production.Thus, it is supposed that the supplementation of tryptophan metabolites will play a beneficial role in AD through repairing the intestinal epithelial barrier function and consequently controlling "leaky gut" induced skin inflammation.

Tryptophan metabolites regulate the complex immune response in AD
Although the exact mechanisms of the disease pathogenesis remain unclear, accumulating evidence from experimental, genetic, and clinical studies indicates that impaired skin barrier and the immune dysregulation are the critical etiologies of AD (11).Although AD has long been considered to be Th2-dominated inflammation, it is also evident that its pathology is accompanied by an imbalance in immunity involving both innate immune cells such as keratinocytes, macrophages, dendritic cells, and adaptive immune cells such as Th1, Th2, Th17, Th22 and Treg cells, which interact and eventually intertwine into a complex inflammatory network.On the other hand, tryptophan metabolism has been firmly established as a key regulator of both innate and adaptive immune cells (68,136), and its derivative receptor, AhR, is a key component of the immune response at the barrier site.Alterations in AhR activity or AhR deficiency may disrupt the immune response or impair the development and function of the epidermal barrier (137).Next, we will delve into the innate and adaptive immune mechanisms mediated by the tryptophan metabolites in the pathological process of AD.

Keratinocytes
Keratinocytes represent the first line of the host defense system by sensing pathogens via innate immune receptors, initiating antimicrobial responses and producing various cytokines and antimicrobial peptides.Among the dysregulation of immune responses in AD, keratinocytes initiate cross-talk between innate and adaptive immune responses by regulating the release of several key molecules including eotaxin/CC chemokine ligand (CCL) 11, eotaxin-3/CCL26, monocyte chemotactic protein (MCP)-4/CCL13, regulated on activation, normal T cell-expressed and secreted (RANTES)/CCL5, and thymus and activation-regulated chemokine (TARC)/CCL17, that trigger inflammatory reactions and immune responses (138).It was found that Th2 type cytokine IL-4/IL-13 could stimulate the production of IL-24 in Keratinocyte by inducing the activity of STAT 6 (139), and then reduce the expression of Flg, ultimately leading to epidermal terminal differentiation damage and barrier dysfunction in AD patients.Interestingly, AhR ligands, such as Coal tar, Glyteer and 6-formylindolo[3,2-b]carbazole (FICZ), could block the activation of STAT 6 mediated by IL-4/IL-13 and promote the expression of Flg via mild and transient activation of AhR/ARNT pathway, thus restoring barrier dysfunction (140,141).Further experiments have proved that the defect of AhR in Keratinocyte may be the cause of worsening inflammation (142), while intense and sustained AhR activation would lead to excessive keratinization of Keratinocyte and sebocytes (43).In conclusion, the lack of AhR or the change of its activity is related to the imbalance of Keratinocyte 'response to inflammatory stimuli, which may damage the immune response or the normal function of the epidermal barrier.

Macrophages
Macrophages (Mj), as a natural immune cell, differentiate into two phenotypes, including the M1 phenotype activated by TLR ligands to secrete proinflammatory cytokines such as TNF-a and IL-1b, or the M2 phenotype activated by interferon or lipopolysaccharide (LPS) to secrete the anti-inflammatory cytokine such as IL-10 (143), and respond to immune responses in the AD microenvironment under different activation states.Remarkably, M2 macrophages exert immunosuppressive effects by expressing IDO to depresses T cell proliferation and stimulate regulatory T cells, which are cells with immune suppressive function (144,145).Tryptophan metabolism has been found to affect the polarization and immune function regulation of macrophages.5-methoxyltryptophan (5-MTP), a new endothelial factor produced by L-tryptophan metabolism that has recently been identified as a functional feature with protective and repair barriers (146), could inhibit the LPS-induced p38-MAPK pathway by interfering the binding of phosphor-p38 to peroxiredoxins (Prdx), thereby blocking the activation of macrophages and preventing the occurrence of systemic inflammation.Similarly, for other tryptophan metabolites, I3A could reduce the expression of inflammatory cytokine IL-1qwb, TNF-a and CXCL-1 in macrophages (146); IA could stimulate IL-10 production in macrophages, thereby reducing the secretion of TNF and IL-6 (115); Kyna was later confirmed to reduce the inflammatory response induced by LPS stimulation in monocytes and Mj through its interaction with GPR35 (147).Therefore, these findings support the idea that tryptophan metabolism could effectively control macrophage activation and "cytokine storm" in vivo, but the specific mechanism of action in AD needs further research.

Dendritic cells
As early as the 1970s, dendriticcells (DCs) has been known to coordinate immune responses by building a "bridge" between innate and adaptive immunity, while the normal physiological function of DCs could be severely affected under pathological condition, and results in the occurrence of abnormal immune responses subsequently.For instance, there are various inflammatory signals in the AD microenvironment, exposure of DCs to this immune stimulation environment would upregulate the expression of inflammatory mediators during antigen acquisition, and activate effector T cells via treated antigen peptides simultaneously (148).Despite not being the case for all DCs (149), exposure to stimuli such as IFN, TLR-4, TLR-9, TNF, and IL-1 could induce functional IDO expression in DCs, promoting tolerance in vivo and contributing to their ability to present antigens and stimulate T cells (150) either through effects of IDO on DCs or through direct action on T cells mediated by tryptophan depletion or tryptophan metabolites (151,152).A previous study proposed initially that CD4+naïve treated T cells could transformed into FoxP 3+functional regulatory T cells upon exposure to LT/ Kynurenine or IDO+ DCs (153).Additionally, the tryptophan metabolite IPA promotes the accumulation of anti-inflammatory DCs in the mesenteric lymph nodes, which was abolished by AhR antagonist (154).IPYA inhibits colon inflammation by increasing IL-10 production, reducing Th1 cell differentiation in the lamina propria of the colon, and altering the composition of DCs in the mesenteric lymph nodes (154).Therefore, we believe that the tryptophan metabolism mediated by DCs has a positive inflammatory regulatory potential in AD.

Th2 cells
The strong activation of adaptive immunity driven by Th2 cells seems to be the dominant mechanism in the acute phase of AD (155), mainly characterized by the secretion of cytokines such as IL-4, IL-5, IL-13 and IL-31, which are involved in the occurrence of keratinocyte apoptosis, inflammation and itching symptoms, to confirm the influence of T cells in the pathogenesis of AD.Previous studies have shown that tryptophan and its derivative ligand, AhR, are involved in regulating T cell populations, mediating immunosuppression, and maintaining the balance between Treg and T cells.A recent study has demonstrated that Th2-deviated environment is related to the reduction of the endogenous AhR ligand such as indole-3-aldehyde (IAId) produced by the skin symbiotic microbiota (43).IAId could promote the interaction of activated AhR receptors with TSLP, thereby inhibit the production of TSLP, and ultimately reduce the occurrence of AD-like dermatitis (43).Notably, TSLP, mainly produced by epidermal keratinocytes and fibroblasts, could stimulate Th2 differentiation by promoting the migration of DCs to the epidermis ( 156), playing a central role in initiating Th2 type adaptive immune responses in AD skin inflammation.In addition to IAId, significant alleviation in clinical manifestations and reversal of Th2 biased immune response were also observed after supplementation with D-tryptophan in a mice with allergic airway inflammation (77).D-tryptophan is a metabolite of Bifidobacteria, Lactobacillus and Lactococcus, strongly induces the production of anti-inflammatory factor IL-10 and reduces the secretion of IFN-g,IL-12, and IL-5 in LPS-induced KM-H2 (a human Hodgkin's disease cell line) cultures, and suppress the expression of the crucial chemokine CCL17 responsible for recruitment of Th2 cells in AD skin lesions (77,141).A newlyidentified role of D-tryptophan consists of slowing down the production of biofilm formation in Staphylococcus aureus and Pseudomonas aeruginosa (157,158).As is known, the skin of AD patients is more susceptible to the colonization and overgrowth of Staphylococcus aureus (159), which has been linked to the increased IgE responses and severity of AD disease (160).In addition, Yu et al. found that appropriate activation of the AhR/ARNT/Flg axis may be beneficial in treating AD (43,161).Th2-deviated environment could significantly reduce the expression of filaments and other barrier related molecules, while the activation of the AhR/ARNT/ Flg signaling pathway by rapidly metabolized AhR ligands (such as IAId or FICZ) and dioxins (2,162) would initiate the activation of OVOL 1 transcription factors, enhance the expression of Flg and LOR, thereby contributing to accelerating the final differentiation of the epidermis and the formation of the epidermal barrier (25,35).It should be mentioned that, FICZ, as an endogenous UVB photoproduct (28), has been found to be closely related to human skin physiology (163) that could limit the production of IL-17 and IL-22 in mouse dermatitis models by activating the AhR receptor (142,161), this barrier-protecting effect may partially explain why UVB phototherapy is effective in treating AD and psoriasis (164, 165).Therefore, the reduction of filaments and other barrier related molecules in the Th2-deviated environment, as well as the deficiency of AhR ligands, may underlie the skin lesions in AD (117), which may compensate for the up-regulation of AhR/ARNT signal transduction pathways to weaken the occurrence of Th2 type response mediated skin inflammation.However, the excessive activation of AhR may also induce the occurrence of pruritus dermatitis (2, 166, 167).

Th22 type immune response
IL-22, the effector cytokine of Th22 type immune, has been reported to play a leading role in the pathogenesis of AD.IL-22 could inhibit epidermal differentiation and promote inflammation in AD skin lesions by inducing the secretion of IL-6, and downregulate the expression of keratinocyte fibril aggregation proteins in keratinocytes, especially, to increase the degree of epidermal damage (168), which is closely related to the severity of AD.However, the role of IL-22 in AD pathogenesis may be bidirectional, as it has been reported to have protective effects in acute viral infection of the intestinal tract but is pathogenic in the chronic inflammatory environment of AD and rheumatoid arthritis (169)(170)(171)(172), this contradictory characteristic can even be derived into tryptophan metabolism.For instance, topical application of the tryptophan metabolic derivative FICZ could reduce the gene expression of IL-22 in a murine mite-induced dermatitis model by activating AhR receptors (161).On the other hand, IAld and I3A could actively stimulate IL-22 secretion via AhR, and the STAT3 phosphorylation is subsequently induced to accelerate proliferation of enterocytes for restoring damaged intestinal mucosa (173).Although there is evidence linking tryptophan metabolism to IL-22 secretion, the specific role of Th22 type immune response in the pathological development of AD or its connection with tryptophan require further relevant experimental confirmation.

Th17 type immune response
IL-17 is an important pro-inflammatory cytokine secreted mainly by Th17 type cells (174), could results in inflammatory reactions by inducing the production of pro-inflammatory cytokines and chemokines in keratinocytes, neutrophils and endothelial cells (175,176).Besides that, IL-17 would reduce the expression of Flg in keratinocytes (177), thereby promoting the colonization of Staphylococcus aureus in damaged epidermal barrier of AD.The superantigen Staphylococcin B secreted by Staphylococcus aureus reversely promotes the secretion of IL-17, leading to a "vicious cycle" further destroys the skin barrier function of AD (178).As was expected, tryptophan metabolites and their AhR receptors have recently been shown to control inflammation by reducing the proliferation of Th17 lymphocytes (49,179,180).In mice exposed to Colitogenic DSS, the tryptophan derivative dioxin TCDD triggered AhR to inhibit proliferation of Th17 and induce differentiation of Treg (181).Similarly, the AhR ligand 3,3'-diindolyl methane (DIM) alleviated oxazolone induced experimental colitis by reducing Th2/ Th17 cells and increasing Tregs (182).Increased expression of IL-17A in CD4+T cells was also observed in AhR -/-mice (183).The above evidences suggest that, the tryptophan metabolite, as an AhR ligand, is crucial for stimulating or inhibiting host immune responses and may be involved in the innate and adaptive immune response regulation of AD, although the further research about direct relationship between probiotics/tryptophan metabolites and AD is urgently needed.

Kyn-IDO pathway plays a crucial role in the development of AD
The Kyn -IDO pathway involved in the degradation of tryptophan was initially considered to have tolerance and immunomodulatory effects, moreover, there is mounting evidence that the Kyn-IDO pathway plays a crucial role in the development of atopy and allergy Kyn and its metabolites cover more than 90% of tryptophan metabolism, have been reported to activate AhR receptors with anti-inflammatory activity (184-187), thereupon then suppressing the activity of natural killer cells (NKT) (188) and APC such as dendritic cells (DC), monocytes, and macrophages (189,190) in mice.Therefore, the Kyn-AhR axis has been postulated to constitute one of the factors affecting the progression of chronic inflammation (184).Further research has shown that the treatment of ovalbumin (OVA) induced asthma mice with Kyn metabolite HAA could inhibit Th 2 lung inflammation through moderately inducing apoptosis of Th 2 cells (78).In addition, IDO 1-Kyn-AhR signaling may reveal feedback loops related to inflammation and reactive oxygen species (ROS) production (191,192).In the Kyn pathway, proinflammatory cytokines induce the IDO1 to create more Kyn, which in turn stimulates the expression of IDO1 by activating AhR.For example, when IFN-g acts on intestinal epithelial cells, IDO could be induced to interfere with the expression of IL-10 receptor.Subsequently, Kyn restores the upregulation of IL-10 receptor expression by activating AhR, which significantly reduces the occurrence of intestinal inflammation (193).The above evidences indicates that the anti-inflammatory properties of the Kyn pathway is achieved through a negative feedback regulatory mechanism.
IDO is widely expressed in various types of cells, including most tumor cells, dendritic cells, macrophages, microglia, eosinophils, fibroblasts, and endothelial cells (142,(191)(192)(193)(194)(195)(196)(197), the most significant and effective inducers of IDO expression mainly are cytokines (such as IFN-g, IFN-a, IFN-b and IL-10), as well as signaling through TLRs (198)(199)(200).IDO has long been considered to contribute significantly to the control of systemic inflammation (201), including its key role in reducing Th1 cell proliferation and inducing Treg cell differentiation (151, 189, 202), which could maintain normal immunological tolerance and limit the occurrence of tissue damage in the body.In support, enhanced CD4+ Th17 and Th1 responses were observed in airway of IDOablated mice following attacking by OVA and infection of human rhinovirus (hRV), characterized by significant infiltration of Th 17 and Th1 type neutrophils, high production of IL-17A, and IFN-g, and increased collagen deposition and epithelial proliferation (73).Additionally, the direct role of IDO in inhibiting target cells by activation of Treg cells has been confirmed from Munn's recent study (203,204), specifically, the enhanced infiltration of effector T cells in the lung cells of IDO−/− mice coincided with a sharp decrease in CD4+CD25+FoxP3+Treg cells, which could be attributed to decreased AhR activity and impaired Kyn production (185,205).Similarly, when co cultured with IDO +AML (Acute Myeloid Leukemia) cells, naïve T cells would convert to FoxP 3+Treg cells, and yet this conversion was completely eliminated by IDO inhibitor (206).The transformed Treg cells would subsequently suppress antigen-induced T cell responses in a "time delayed manner", so then offsetting the overactive immune response and bringing the immune system closer to physiological equilibrium.Compared to the inhibitory effect of IDO on Th 1 cells differentiation, the effect on Th 2 cells is more complex with both inhibitory and stimulatory actions reported (207).According to the report, a asthma mouse model sensitized with ovalbumin (OVA) observed that expression of IDO inhibited Th2 type airway inflammation in the lungs (74), while the IDO-expressing and Kynurenine-producing eosinophils co cultured with Th1 or Th2 cells, a preferential decrease in Th1 response and subsequent increase in Th2 cytokine production were observed (208), indicating that IDO-expressing eosinophils may create cytotoxic metabolites that maintain an imbalance between Th1 and Th2 cell populations, and the consistent results were obtained in the study of Molano et al. (209).Notably, Kositz et al. reported that the higher tryptophan levels in atopic patients may be result from lower IDO-1 activity (66).In contrast, Th2 cytokines such as IL-4 and IL-13 inhibit the expression of IDO (210, 211), which creates favorable conditions for a Th2 tilted immune environment, thereby increasing the risk of Th2 related diseases.Although the above evidences suggest that IDO and/or its metabolites have a protective effect in atopic inflammation, there are also studies demonstrating the contradictory results that expression of IDO on eosinophils may contribute to the development of allergic inflammation (195) and was up-regulated in the AD skin (212).Under these circumstances, we suspect that the upregulation of IDO expression is a result of efforts to prevent ongoing allergic inflammation.In a few words, whether the induction of IDO can inhibit the development of allergic reactions or induce immune tolerance of allergic inflammation has begun requires further research.

Discussion
Previous studies have confirmed the beneficial role of tryptophan derivatives and AhR ligand in the pathogenesis or treatment of many Decreased tryptophan metabolites mediated by gut microbiota disorder induce atopic dermatitis inflammation.AD patients are accompanied by intestinal microbiota disorders, including a downregulation of tryptophan producing probiotics and an increase in lipopolysaccharide producing pathogenic bacteria.The LPS could enter the bloodstream through increased gut permeability and cause systemic inflammation subsequently.On the contrary, downregulated tryptophan and its derivatives in the circulatory system have a decreased positive regulatory ability on the immune system, which could further exacerbate skin inflammation in AD patients.Frontiers in Immunology frontiersin.orgskin diseases, including inflammatory diseases, skin pigmentation disorders and cancer (38)(39)(40)(41)213). Therefore, we speculate that exogenous supplementation of tryptophan derivatives (such as FICZ, IAId), or targeted intervention of GM (such as Lactobacillus reuteri, Lactobacillus salivary, Bifidobacterium and Lactobacillus), or targeted induction of the Kyn-IDO pathway to accelerate endogenous tryptophan metabolism and produce AhR ligands, may ultimately alleviate clinical symptoms of AD by suppressing abnormal immune responses.In addition to being a potential therapeutic option, tryptophan metabolites such as 5-hydroxyindolepyruvate and 5hydroxyindoleacetic acid may become the new biomarkers supporting the diagnosis and prognosis of atopic diseases.Furthermore, supplementing tryptophan derivatives may alleviate some side effects such as herpes infections and headaches (214,215) during the treatment of AD with biological agents.However, the biological functions involved in tryptophan metabolism are complex.The outcome of AhR activation depends on the type of cell and ligand (216, 217), stimulation or inhibition of AhR in the skin results in different immune responses (218), such as inducing overexpression of proinflammatory cytokines and ROS production to arise the development of inflammatory diseases or carcinogenesis (219), or affecting the differentiation of Treg cells, thereby promoting the immune tolerance (220, 221).Moreover, there is still insufficient evidence to support the clinical application of tryptophan and its derivatives, and the long-term efficacy and safety of their therapeutic potential are not fully understood.In summary, while the causal relationship among tryptophan metabolism, AD and GM, as well as the specific molecular mechanisms remain to be determined, we have preliminarily provided evidences that tryptophan plays an antiinflammatory role in AD, laying the foundation for the exciting connection between tryptophan metabolism and AD immune regulation (Figure 2).We hope that this review will lead the way to further understand AD, and provide new insights into the pathogenesis and treatment direction of this disease.