Manipulating Microbiota to Treat Atopic Dermatitis: Functions and Therapies

Atopic dermatitis (AD) is a globally prevalent skin inflammation with a particular impact on children. Current therapies for AD are challenged by the limited armamentarium and the high heterogeneity of the disease. A novel promising therapeutic target for AD is the microbiota. Numerous studies have highlighted the involvement of the skin and gut microbiota in the pathogenesis of AD. The resident microbiota at these two epithelial tissues can modulate skin barrier functions and host immune responses, thus regulating AD progression. For example, the pathogenic roles of Staphylococcus aureus in the skin are well-established, making this bacterium an attractive target for AD treatment. Targeting the gut microbiota is another therapeutic strategy for AD. Multiple oral supplements with prebiotics, probiotics, postbiotics, and synbiotics have demonstrated promising efficacy in both AD prevention and treatment. In this review, we summarize the association of microbiota dysbiosis in both the skin and gut with AD, and the current knowledge of the functions of commensal microbiota in AD pathogenesis. Furthermore, we discuss the existing therapies in manipulating both the skin and gut commensal microbiota to prevent or treat AD. We also propose potential novel therapies based on the cutting-edge progress in this area.


Introduction
Atopic dermatitis (AD), also known as eczema, is a skin inflammation that exhibits chronic, persistent, pruritic lesions, and is often associated elevated levels of IgE [1,2]. AD affects 10-20% of the population during their lifetime in developed countries, with a particularly high prevalence among children [2,3]. Its prevalence is also rapidly increasing in developing nations [4,5]. The established pathogenesis of AD involves the initiation of barrier disruption, followed by the activation of type 2 (T H 2) immune responses [2,6]. Variants in the filaggrin (FLG), the gene encoding an important skin barrier protein, represent a significant risk factor for AD [7][8][9]. AD is often associated with the development of asthma and food allergies, which is known as "atopic march" [10]. Physical therapies that moisturize the skin, preventing water loss, controlling xerosis, and relieving barrier disruptions, are recommended for AD patients [11]. While corticosteroids remain the standard anti-inflammatory treatment against AD, the efficacy of blocking T H 2 responses is recognized by various clinical trials [12][13][14][15][16][17][18][19]. Unfortunately, the limited armamentarium [20] Pathogens 2022, 11, 642 2 of 34 and the high heterogeneity of the disease [21] make the management of AD challenging. Therefore, novel therapeutic strategies needed for AD treatment.
The past two decades have highlighted the role of commensal microbiota in health homeostasis and disease [22]. As the largest and outmost organ of the body, the human skin has been estimated to host about 1 billion bacteria per 1 cm 2 area [23]. Conversely, the gastrointestinal tract harbors the largest microbiota population in our body, exceeding 10 14 bacterial cells [24,25]. AD is probably the most well-characterized disease in which skin dysbiosis plays a causal role [6]. In addition, AD is also associated with gut dysbiosis [26]. The investigations from the pre-clinical animal studies and the emerging human 3D skin models furthered our understanding on the complex interplay between the microbiota and the AD context [27,28]. In this review, we comprehensively summarize the roles of both the skin and gut microbiota in AD and the current approaches aimed at manipulating commensal microbiota to prevent or treat AD. We also provide insights for future investigations to improve the efficacy of current agents and establish novel therapeutic strategies that leverage the microbiota.

The Skin Microbiota Alternation in AD Patients: A Particular Focus on Staphylococcus aureus
It is now well established that changes in the normal skin microbiota composition, a condition known as dysbiosis, contribute to the disruption of cutaneous immune homeostasis and promotes the development of skin diseases, including AD [29,30]. Studies that have demonstrated the association of AD with skin dysbiosis are summarized in Table 1. AD is often accompanied with dysbiosis, featured by increased colonization of staphylococcal species and decreased richness and diversity of other bacterial communities [31]. S. aureus, a dominant species among the family of Staphylococcae, can be 100 times more abundant in AD skin compared to normal healthy skin [32]. On the skin surface, S. aureus secretes virulence factors, including phenol-soluble modulins (PSMs) and proteases, thus disrupting normal skin barrier functions, which alter the epidermal environment favouring the development of AD [33][34][35][36][37]. We will discuss the mechanisms involved later in this review.
The increased abundance of S. aureus colonization in AD is associated with a depletion in the coagulase-negative staphylococcal species (CoNS), such as S. epidermidis, S. hominis, and other skin commensal bacterial communities, including Streptococcus salivarius, Propionibacterium, Streptococcus, Acinetobacter, Corynebacterium, Prevotella and Proteobacteria [31,[38][39][40]. In contrast, these skin commensal microbiota produce antimicrobial substances that inhibit the growth of pathogenic S. aureus and its biofilm formation [30,41,42]. In the non-lesional skin samples of AD patients, CoNS are the dominant bacterial communities [38]. However, their abundances were lower on the skin of healthy individuals compared with non-lesional AD skin [38,43]. This suggests the existence of complex interactions between the different skin microbial communities, which modulate the host's susceptibility to AD.

S. aureus in AD Pathogenesis
Probably the best understood association between skin microbiota and AD is the involvement of S. aureus. As previously discussed, S. aureus is commonly identified in the skin of AD patients [34,55,56]. S. aureus colonization precedes the onset of AD in infancy [57], and topical application of S. aureus isolates from AD skin suffices to induce AD-like skin inflammation in mice [27]. This evidence supports the causal role of S. aureus in AD pathogenesis. S. aureus adheres to AD skin biopsy specimens more efficiently compared to healthy ones because of the changes to the composition of the stratum corneum and

Other AD Pathogeneses Regulated by the Skin Microbiota
In addition to the pathogenic role of S. aureus, homeostasis of skin microbiota is vital for normal skin barrier function, the cutaneous immune balance, and the elimination of possible pathogens, thus modulating AD pathogenesis [86]. Germ-free mice exhibited a significantly different skin transcriptome [87]. Germ-free mice displayed impaired skin barrier function, a direct consequence of the abnormal epithelial development and differentiation [88]. Treating a 3D skin tissue model with a mix of selected members of normal skin microbiome profoundly improved the barrier integrity of the tissue [89]. In this section, we discuss how the skin microbiota regulates two major aspects: (1) AMPs, and (2) the tryptophan metabolites-aryl hydrocarbon receptor (AHR) axis. Together with the pathogenic roles of S. aureus in AD, these mechanisms are summarized in Figure 1.
significantly different skin transcriptome [87]. Germ-free mice displayed impaired skin barrier function, a direct consequence of the abnormal epithelial development and differentiation [88]. Treating a 3D skin tissue model with a mix of selected members of normal skin microbiome profoundly improved the barrier integrity of the tissue [89]. In this section, we discuss how the skin microbiota regulates two major aspects: (1) AMPs, and (2) the tryptophan metabolites-aryl hydrocarbon receptor (AHR) axis. Together with the pathogenic roles of S. aureus in AD, these mechanisms are summarized in Figure 1.

Antimicrobial Peptides
Using antimicrobial mechanisms [90], the skin barrier and its associated microbiota protect against pathogenic microorganisms such as S. aureus in AD. For example, the production of AMPs such as cathelicidin and β-defensin by the host and the microbiota inhibit certain microorganisms. It appears that normal commensal bacteria are more resistant to host AMPs [91], and host production of AMPs is regulated by commensal bacteria in poorly understood mechanisms [92][93][94]. Not only can commensal bacteria Figure 1. Mechanisms of how skin microbiota regulates AD pathogenesis. Excessive S. aureus colonization on the skin leads to the formation of biofilms and the secretion of virulence factors. These virulence factors facilitate mast cell degranulation, enhance inflammatory cytokine productions and the release of histamine, and increase IgE levels. In addition, S. aureus directly stimulates keratinocytes (KC) and Langerhans cells (LC) to release proinflammatory cytokines, including TH2 cytokines, TSLP, IL-8, and IL-1β, thus inducing TH2 differentiation and inflammation. Together, excessive cutaneous S. aureus colonization promotes AD pathogenesis. Conversely, commensal Coagulase-negative staphylococci (CoNS) inhibit the colonization of S. aureus by producing lantibiotics, PSMγ, and PSMδ. They also inhibit S. aureus biofilm formation by producing the serine protease glutamyl endopeptidase (Esp). Tryptophan metabolites derived from skin microbiota can activate AHR, thus inhibiting TSLP production by KCs and improving the epidermal barrier of the skin. AHR, aryl hydrocarbon receptor; AHRE, AHR element; AMPs, antimicrobial peptides; CoNS, Coagulase-negative staphylococci; IAId, indole-3-aldehyde; IL, interleukin; ILC3, lymphoid cells type 3; KC, keratinocyte; LC, Langerhans cell; PSM, phenol-soluble modulin, TSLP, thymic stromal lymphopoietin; SE, Staphylococcal enterotoxin; TSST-1, toxic shock syndrome toxin-1. Created with BioRender.com (accessed on 4 May 2022).

Antimicrobial Peptides
Using antimicrobial mechanisms [90], the skin barrier and its associated microbiota protect against pathogenic microorganisms such as S. aureus in AD. For example, the production of AMPs such as cathelicidin and β-defensin by the host and the microbiota inhibit certain microorganisms. It appears that normal commensal bacteria are more resistant to host AMPs [91], and host production of AMPs is regulated by commensal bacteria in poorly understood mechanisms [92][93][94]. Not only can commensal bacteria control the production of AMPs by the host, they also produce some antimicrobial agents able to suppress pathogenic competitors, thereby providing an additional antimicrobial barrier on the skin. For instance, S. epidermidis produces the peptides PSMγ and PSMδ, which inhibit the growth of pathogenic bacteria on the skin [95,96]. Similarly, S. hominis is another CoNS that produces lantibiotics, a class of cyclic AMPs containing lanthionine and methyllanthionine [30]. Skin dysbiosis likely leads to the dysregulation of the antimicrobial function against pathogenic bacteria. Impaired production of cathelicidin and β-defensins is responsible for the dominance of S. aureus in AD skin [62]. Therefore, the skin microbiota has an interaction with the host AMPs production and contributes to the control of AD.

Tryptophan Metabolites-AHR Axis
AHR is a ligand-activated transcription factor that modulates tissue homeostasis and immune responses [97][98][99]. AHR agonists range from xenobiotic chemicals to endogenous indole by-products derived from tryptophan metabolism [100][101][102]. The activation of AHR is mostly anti-inflammatory through the regulation of cytokine production and other immune-related transcription factors, such as NF-κB [97][98][99]. AHR is expressed by keratinocytes, epidermal Langerhans cells, dermal and epidermal innate and adaptive immune cells [103]. Although T H 2 cells exhibited negligible AHR expression [104], activation of AHR in dendritic cells (DCs) suppressed T H 2 differentiation [105,106]. The topical application of coal tar, which is a traditional therapy against AD, works by activating AHR [107]. In the skin epidermis, dead keratinocytes and broken keratin are used as substrates in the tryptophan metabolism of skin microbiota which leads to AHR agonists production [108]. The barrier dysfunction observed in germ-free mice skin is attributed to the attenuated AHR pathway [88]. Activation of AHR upregulates the expression of barrier-related molecules like filaggrin, loricrin and involucrin, by keratinocytes, thus maintaining the healthy epidermal barrier [109][110][111]. This can be achieved by the tryptophan metabolites derived from the skin microbiota [111][112][113][114]. For example, Indole-3-aldehyde (IAld), a skin microbiota-derived AHR agonist, suppresses TSLP expression and protects mice against MC903-induced AD. In humans, the skin of AD patients displays a lower level of IAld compared to that of their healthy counterparts [113]. AHR signalling is essential for AMPs production and can shape the skin microbiome [53], as demonstrated with AHR-deficient mice, which display a more variable and complex skin microbiota compared to WT controls [115].

Therapeutic Manipulation of the Skin Microbiota in the Management of AD
Reducing the abundance of pathogenic bacteria and restoring the normal microbial balance in the skin may be of clinical benefit for AD management. In animal models, antibiotic treatment established the causal role of skin dysbiosis in AD. Although a randomized clinical trial (RCT) has demonstrated that antibiotics suppressing the growth of S. aureus significantly attenuated AD [116], the clinical efficacy of antibiotics in this pathology remains questionable [117]. Skin bacteria abundantly reside in hair follicles, eccrine glands or beneath the epidermal barrier, where antibiotics are difficult to reach [63,118]. In addition, most antibiotics can (1) disrupt normal microbiota due to their lack of specificity and (2) favour antibiotic-resistant strains of S. aureus. Nevertheless, a phase III trial (NCT02840955) investigates the therapeutic potential of a bacteriophage endolysin (Staphefekt) in AD adult patients. Staphefekt is a specific bactericidal agent against S. aureus with unlikeliness of resistance and is effective against the methicillin-resistant S. aureus (MRSA) strains that have already acquired resistance to conventional antibiotics. Because bacterial killing by this endolysin is independent of the involvement of the bacterial metabolism [119][120][121].
The reintroduction of normal commensal bacteria represents a promising strategy for AD treatment. Topical application of antimicrobial CoNS strains able to produce specific AMPs against S. aureus, decreased colonization of this bacteria and effectively attenuated AD severity [30,122]. Another angle in manipulating skin microbiota is by reducing the skin surface pH. The acidic epidermal surface is essential for maintaining the skin barrier [123]. Acidity favours commensal bacteria and suppresses pathogenic bacteria, including S. aureus [124]. In healthy individuals, it has been observed that higher skin pH was associated with increased S. aureus skin colonization [125]. It is well known that AD lesions display higher pH values than healthy skin [126,127], and topical application of acids improved AD in murine models [128,129].
Current attempts to manipulate skin microbiota to control AD are limited and lack RCTs. However, as more causal roles of the skin microbiota in AD are being revealed, therapeutic manipulation of skin microbiota emerges as a promising way to improve the management of AD.

The Gut Microbiota Profiles in AD Patients
Aside from the skin microbiota, the gut microbiota, known to have a systemic impact on the host immune responses, is also closely associated with atopic diseases such as AD. A hypothesis of the "gut-skin" axis has been proposed and has opened new paths related to the prevention and treatment of AD [26]. Various studies demonstrated that AD is associated with gut dysbiosis, especially during early life ( Table 2). AD patients display poor gut microbial diversity in several clinical trials [130][131][132][133][134][135][136][137][138], while contradictory results exist [139][140][141][142]. Similarly, to the skin, AD patients exhibit abundant S. aureus in their gut microbiota [143][144][145][146]. Other microbes associated with inflammation and epithelial damage, such as Clostridiodes difficile, and coliforms, including pathogenic Escherichia coli, are increased in the gut microbiota of AD patients [136,140,[146][147][148][149][150][151][152]. Metagenomic analysis has shown that the gut microbes in AD patients carry extra genes associated with inflammatory responses and the breakdown of gut epithelial layers [139,141]. Breastfed infants have a lower risk of atopic diseases [153][154][155][156][157][158], likely because of the abundance of Bifidobacteria in their gut [159][160][161][162], a genus that is reduced in the gut microbiota of AD patients [151,[163][164][165][166]. However, it has been reported that AD development is also associated with the higher abundance of certain species of Bifidobacteria, such as Bifidobacterium catenulatum, B. bifidum and B. pseudocatenulatum [167,168]. Short-chain fatty acids (SCFAs) are microbial metabolites well known for their anti-inflammatory effects. High fecal levels of SCFAs are significantly associated with a lower risk of AD [139,169]. The SCFA-producing bacteria Coprococcus eutactus is less abundant in the gut microbiota of AD patients [135]. Subspecies of Faecalibacterium prausnitzii are enriched in AD fecal samples. Although F. prausnitzii is a major SCFA producer in healthy subjects, this species is an inefficient SCFA producer in AD patients [139]. Furthermore, genes encoding carbohydrate active enzymes (CAZymes), which break down resistant starch into SCFAs, are deficient in the gut microbiota of AD patients [142]. While infantile gut dysbiosis may predict the development of AD [131,132,134,[147][148][149]152], childhood AD history had prolonged imprints in gut microbiota that neonates with childhood AD history had a lower abundance of Bifidobacteria, Akkermansia, and Faecalibacteria compared to their healthy counterparts [170].  Despite these reports linking gut dysbiosis with AD, the actual roles of gut microbiota in AD development are still largely unknown. Establishing a causative role for the gut microbiota in the development of diseases remains challenging. An invaluable tool for this type of investigation is the use of germ-free animals. Germ-free mice showed similar disease severity in the oxazolone-induced ear atopic dermatitis model as conventional mice [171]. However, germ-free mice have higher levels of serum IgE and inflammatory cytokines, such as TNF and IL-6 in ear tissues, suggesting exacerbated skin inflammation [171]. Furthermore, the sensitivity to oxazolone is transferable to germ-free mice with fecal microbiota transplant (FMT) [171]. These findings suggest a causative role of gut microbiota in the development of AD. However, the direct link between gut microbiota and type 2 inflammatory responses in AD is still unknown.

The Gut Microbiota Regulates AD-Related Immune Responses and the Underlying Mechanisms
There is firm evidence supporting the role of gut microbiota-regulated immune responses in atopic diseases and AD [172]. For example, early colonization of Bifidobacteria subtly regulates the T H 1/T H 2 immune balance, reducing the risk of AD [173]. Most of the knowledge regarding this arises from the use of probiotics in different clinical trials, as discussed below. In this section, we focus on three aspects: (1) SCFAs-anti-inflammation axis, (2) tryptophan metabolites-AHR axis, and (3) toll-like receptor signalling (Figure 2). These pathways that can be modulated by gut microbiota are well characterized and highly relevant to AD and other allergic immune responses.
inflammatory cytokines, such as TNF and IL-6 in ear tissues, suggesting exacerbated skin inflammation [171]. Furthermore, the sensitivity to oxazolone is transferable to germ-free mice with fecal microbiota transplant (FMT) [171]. These findings suggest a causative role of gut microbiota in the development of AD. However, the direct link between gut microbiota and type 2 inflammatory responses in AD is still unknown.

The Gut Microbiota Regulates AD-Related Immune Responses and the Underlying Mechanisms
There is firm evidence supporting the role of gut microbiota-regulated immune responses in atopic diseases and AD [172]. For example, early colonization of Bifidobacteria subtly regulates the TH1/TH2 immune balance, reducing the risk of AD [173]. Most of the knowledge regarding this arises from the use of probiotics in different clinical trials, as discussed below. In this section, we focus on three aspects: (1) SCFAs-anti-inflammation axis, (2) tryptophan metabolites-AHR axis, and (3) toll-like receptor signalling (Figure 2). These pathways that can be modulated by gut microbiota are well characterized and highly relevant to AD and other allergic immune responses.

Figure 2.
Mechanisms of how gut microbiota regulate AD pathogenesis. Short-chain fatty acids (SCFAs) produced by the gut microbiota are able to activate SCFA-sensing G-protein coupled receptors (GPCRs) and/or inhibit histone deacetylases (HDACs), thus activating downstream signalling cascades that suppress inflammatory responses and restoring TH1/TH2 balance. Microbial metabolite, D-tryptophan can also restore TH1/TH2 balance. Bifidobacteria, a genus of bacteria to which many probiotics belong, are an important source of these metabolites. Microbial tryptophan metabolites can activate the aryl hydrocarbon receptor (AHR), which inhibits inflammatory responses and improve the epidermal barrier of skin. Pathogen-associated molecular patterns produced by the gut microbiota can activate toll-like receptors (TLRs) thus restoring Figure 2. Mechanisms of how gut microbiota regulate AD pathogenesis. Short-chain fatty acids (SC-FAs) produced by the gut microbiota are able to activate SCFA-sensing G-protein coupled receptors (GPCRs) and/or inhibit histone deacetylases (HDACs), thus activating downstream signalling cascades that suppress inflammatory responses and restoring TH1/TH2 balance. Microbial metabolite, D-tryptophan can also restore TH1/TH2 balance. Bifidobacteria, a genus of bacteria to which many probiotics belong, are an important source of these metabolites. Microbial tryptophan metabolites can activate the aryl hydrocarbon receptor (AHR), which inhibits inflammatory responses and improve the epidermal barrier of skin. Pathogen-associated molecular patterns produced by the gut microbiota can activate toll-like receptors (TLRs) thus restoring TH1/TH2 balance. All these mechanisms benefit AD. IAld, indole-3-aldehyde; IAA, indole-3-acetic acid; IPA, indole-3-propionic acid; TA, tryptamine; ARNT, aryl hydrocarbon receptor nuclear translocator. Created with BioRender.com (accessed on 4 May 2022).

Short-Chain Fatty Acids and Their Anti-Inflammatory Effects
As briefly discussed above, the gut microbiota of AD patients contains less SCFAproducers or is defective in generating SCFAs [135,139,142]. SCFAs are microbial metabolites derived from dietary fibre [174]. The most abundant SCFAs found in the host are acetate (two carbons), propionate (three carbons), and butyrate (four carbons) [175]. Recent studies established a potent anti-inflammatory role of SCFAs against numerous inflammatory diseases, including atopic diseases [169,172,[176][177][178]. In atopic models, the anti-inflammatory effects of SCFAs are promoted through the regulation of DCs leading to the inhibition of T H 2 [179] and the activation of regulatory T cell (Treg) differentiation [180]. Subcutaneous injection and/or topical application of sodium butyrate attenuate the hapten-induced AD in mice by recruiting Tregs and inducing the production of the anti-inflammatory cytokine, IL-10 [181]. Still, the mechanisms underlying the protective effect of SCFAs in AD are poorly understood and insights from other atopic diseases would be invaluable.
The immune regulations by SCFAs are largely mediated by two key mechanisms: (1) the activation of certain G-protein coupled receptors (GPCRs) and (2) the inhibition of histone deacetylases (HDACs) [176].

SCFA-Sensing GPCRs
There are three GPCRs activated by SCFAs. GPR41 and GPR43 are exclusively activated by all three abundant SCFAs, while GPR109A can only be activated by butyrate [182,183]. The role of GPR41 and GPR109A in the immune cells remains elusive, while more information is available about GPR43, which is more widely expressed by the immune cells compared to the other two receptors [182,184]. Activation of GPR41 and GPR43 inhibits the TNF-induced or LPS-induced production of the pro-inflammatory cytokines IL-6 and IL-8 in human endothelial cells [185]. GPR43 was the first SCFA-sensing receptor to be deorphanized. It plays an anti-inflammatory role in various disease models, including atopic diseases [180,184,[186][187][188][189]. A report demonstrated that T cells recruited to the skin after butyrate administration showed higher expression of GPR43 [181]. GPR43 is essential for the expansion of Tregs and the regulatory effects of Tregs induced by SC-FAs [190,191]. Activation of GPR43 promotes the production of the anti-inflammatory cytokine IL-10 by CD4 + T cells [192]. Regulation of neutrophil biology, which is involved in AD [193][194][195][196][197], by SCFAs is also dependent on GPR43 activation [184,198].
Conversely, SCFAs significantly reduce the luminal pH in the colon [204,205], thus inhibiting the survival of potential pathogens [206] and activating proton-sensing GPCRs of the host [172,182]. We recently reported that GPR65, a proton sensor highly expressed in various leukocyte subsets, plays a protective role in AD by inhibiting inflammatory cytokines production and immune cell migrations [207]. The polymorphisms resident in or near the human gene GPR65, rs3742704, and rs8005161 were significantly associated with the risk of AD and asthma [207,208].

HDAC Inhibition
HDACs are enzymes that remove acetyl groups from an ε-N-acetyl lysine amino acid on histones [209]. The histone deacetylation facilitates high-affinity binding between histone proteins and DNA, leading to DNA compaction [209], results in lower chromatin accessibility for transcription factors and prevents gene expression [209]. HDACs play a critical role in many pathologies, including atopic diseases, making HDAC inhibitors promising drug candidates against atopic diseases and AD [210]. High HDAC activity impairs tight junction function [211]. HDAC inhibitors such as trichostatin A (TSA) inhibit 2,4-dinitrofluorobenzene-induced dermatitis in mice [212,213]. A recent study demonstrates that belinostat, a pan HDAC inhibitor, can rescue the defective skin barrier in AD through restoring epidermal miR-335 expression [214]. miR-335 directly represses SOX6, which impairs epidermal differentiation [214]. However, a report also demonstrated that HDAC inhibited IL-4 production by human T cells, and TSA promoted IL-4 production [215].
SCFAs, particularly butyrate, are potent inhibitors of HDACs [216][217][218][219][220][221]. SCFAs are able to inhibit HDACs directly by entering the cells [222] and this inhibition is closely associated with GPCR signalling. The activation of GPR41 can suppress histone acetylation in Chinese hamster ovary cell lines, possibly through inhibiting HDACs [223]. HDAC inhibition in colonic Tregs by SCFAs may be dependent on GPR43 [190]. SCFAs restrict pro-inflammatory cytokine production through inhibiting HDAC activity. Acetate reduced HDAC activity in human monocytes in vitro, which correlated with lower production of IL-6, IL-8, and TNF [224]. SCFAs showed a similar effect as TSA in suppressing NF-κB activation and TNF production in human peripheral mononuclear cells, but HDAC activity was not tested [225]. Similarly, HDAC inhibition can partially mediate SCFAs' inhibition of pro-inflammatory cytokine production by human endothelial cells [185]. Whether SCFAs regulate T H 2 cytokine production through HDAC inhibition remains unknown.

Tryptophan, Its Metabolites and Aryl Hydrocarbon Receptor Signalling
Tryptophan is an essential amino acid in human. While L-tryptophan represents the proteinogenic enantiomer, its D-enantiomer, D-tryptophan, is a bacterial metabolite from Bifidobacterium, Lactobacillus and Lactococcus, which are known probiotics [231]. A recent study demonstrated that D-tryptophan expands the Treg population in the colon [231], and suppresses the expression of T H 2-associated CCL17 by the human Hodgkin lymphoma T-cell line KM-H2 and the inflammatory activity of human DCs [231].
Meanwhile, by breaking down dietary L-tryptophan into indole, indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), tryptamine (TA), and 3-methyl indole (skatole) [232,233], the gut microbiota also serves as a vital source of AHR agonists. A landmark study demonstrated that the AHR agonist IAld produced from the tryptophan metabolism of Lactobacillus reuteri activates AhR, induces IL-22 secretion, and promotes gut homeostasis [234]. This suggests that AHR serves as the bridge in the crosstalk between commensal bacteria and host health. Knowing that AHR agonists, such as IAld, have potent protection against AD, we hypothesize that AHR agonists derived from gut microbiota have distal protection for AD in the skin.

Toll-like Receptor Signalling
In addition to skin microbiota, gut microbiota also produces PAMPs recognized by TLRs, thus contributing to systemic immune homeostasis [235], including AD-related immune responses. As mentioned earlier, TLR2 and TLR4 play essential roles in maintaining the balance between T H 1 and T H 2 immune responses, thus regulating AD symptoms [236][237][238][239]. Gut microbiota composition is associated with TLR signalling in atopic diseases. Infants with eczema have lower fecal Ruminococcaceae, which is negatively associated with TLR2induced IL-6 and TNF production [240]. Moreover, TLR4 SNP rs10759932 and fecal E. coli had a significant multiplicative interaction regarding allergic sensitization [241]. To the best of our knowledge, few studies have looked at the crosstalk between gut microbiota and TLRs in AD development.

Therapeutic Manipulation of Gut Microbiota in the Prevention and Treatment of AD
Since gut microbiota is likely to regulate AD pathogenesis, both gut microbiota and microbial metabolites are promising tools in controlling AD prevalence. The major therapeutic methods relevant to gut microbiota include FMT, the use of prebiotics, probiotics, synbiotics, and postbiotics. These methods all aim to restore the balance of gut ecology in order to modulate allergic immune responses. While abundant trials attempted to use these as therapies to prevent and treat AD, at this stage, their efficacies remain inconclusive due to inconsistent results and potential risks.

Fecal Microbiota Transplantation
The most direct method to re-establish the balance of the gut microbiota is FMT. FMT has been successfully used to treat various diseases, including Clostridium difficile infection [242]. A recent study reported that FMT using feces from healthy BALB/c donors successfully attenuated ovalbumin-induced AD in BALB/c mice [243], hinting at the promise of FMT in AD management. However, caution should be taken when applying FMT. FMT has resulted in several deaths in the recent decade through the spread of multidrug-resistant bacteria, aspiration, and toxic megacolon [244][245][246][247] and has elicited an important safety alert from United States Food and Drug Administration in 2019 [244]. Due to the limited knowledge on the gut microbiota, the actual operative agents in FMT treatments are mostly unknown, and the uncertain risks are not well known. Since patients with gut dysbiosis tend to have compromised gut barrier, they are more susceptible to the risks associated with FMT. Furthermore, regulatory benchmarks and standardized protocols for FMT are still absent or at least limited in many jurisdictions [248]. The alternative microbiota-based therapies with rationally selected microorganisms or microbial metabolites may be preferable for future AD treatments.

Prebiotics in Preventing and Treating AD
Prebiotics are non-viable substances that are selectively utilized by host microorganisms conferring health benefits [249]. Prebiotics are naturally rich in human milk and vegetarian food, including cereal, fruits, and vegetables. Modern industry also produces some prebiotics. Therefore, prebiotics can be supplemented either directly or by modifying food intake.
Investigations into the health effects of breastfeeding on infants led to the first applications of prebiotics to benefit health. Breastfed infants have different fecal bacterial composition compared to non-breastfed infants, with enrichment of Bifidobacteria, and such difference correlates with the health status of the infants [159][160][161][162]. Recent studies confirmed that intake of breast milk is the most significant factor shaping the early life gut microbiota [250,251]. Breastfed infants have a reduced risk of childhood AD compared to non-breastfed infants [153][154][155][156][157][158]. The beneficial effect of breast milk has been attributed to the prebiotics, human milk oligosaccharides (HMOs) [252][253][254]. HMOs selectively expand Bifidobacteria, particularly B. bifidum and B. longus, which, together with B. breve are the most abundant bacteria in the gut microbiota of breastfed infants [255]. Supplementing HMO effectively modulates the infant gut microbiota [256]. However, whether supplementing breast milk-derived prebiotics prevents AD is still questionable. Supplementing HMOs, mostly galacto-oligosaccharide (GOS) and fructo-oligosaccharide (FOS), effectively reduced the incidence of AD during infancy but exhibited poor long-term protection in several RCTs [257][258][259][260][261][262][263][264]. The results from other trials were not sufficiently in favour of supplementing HMOs in treating AD [265][266][267][268][269][270][271]. Of note, no adverse side effects were noted in all these trials and prebiotic HMOs have been added to infant milk formula to mimic the bifidogenic effect of breast milk [258,272,273].
Fermentable dietary fibres are probably the most popular prebiotics in recent biomedical studies [249]. They can be fermented by the commensal gut microbiota into SCFAs, whose anti-inflammatory functions have been discussed above. However, whether supple-menting fermentable dietary fibre would benefit AD still requires more investigations in both pre-clinical and clinical studies.
Due to the lack of solid scientific evidence, the use of prebiotics is not recommended by World Allergy Organization (WAO) as a preventive or therapeutic measure against AD [270]. Future studies may demonstrate the efficacy of using non-digestible carbohydrates in preventing or even treating AD.

Probiotics in Preventing and Treating AD
Probiotics are living microorganisms that confer a health benefit on the host when administered in an adequate amount [274]. The benefits of probiotics in AD are studied more extendedly and thus have more solid conclusions. The most commonly used probiotics in RCTs against AD belong to Lactobacilli and Bifidobacteria genus, which are enriched in dairy products. These bacteria are able to modulate immune cells to restore the T H 1/T H 2 immune balance, enhance the production of the regulatory cytokine IL-10 and expand the population of Tregs [275], all of which benefit AD management. In addition, they compete with pathogenic bacteria, including S. aureus associated with AD, for nutrition and binding mucin [276]. Several RCTs attempted to test the efficacy of probiotics in preventing the development of AD ( Table 3). Application of a single strain of probiotic demonstrates potent and enduring prevention against AD. Supplementation with L. rhamnosus GG (LGG) successfully reduced the incidence of AD in the first year of life by~50% compared to placebo treatment in an RCT performed in Finland [277], and such protection extended to 4 years of age [278]. Another large-scale RCT in New Zealand suggested that early exposure to L. rhamnosus (HN001) had enduring protection against AD, at least in the first decade of life [279][280][281][282]. The further investigation suggested that the prevention against AD provided by HN001 may overcome the risk of AD caused by certain genetic deficiencies, particularly in genes encoding TLRs [283,284]. Nevertheless, supplements of HN001 did not have significant impacts on the infant gut microbiota [285]. A combination of various probiotics appears to be effective in preventing AD as well. An RCT performed in Norway demonstrated that maternal uptake of a combination of three probiotics, LGG, L. acidophilus La-5 and B. lactis Bb-12, had a potent preventive effect against the development of AD up to 6 years, which is accompanied with a reduction in T H 22 [286][287][288]. A probiotic mix comprised of B. bifidum BGN4, B. animalis subsp. lactis AD011 and L. acidophilus AD031 also significantly reduced the incidence of AD in the first year of life in a Korean RCT [289]. Supplement of B. breve and B. longum together exhibited similar benefits and reduced fecal Proteobacteria [290]. However, probiotic treatments, particularly prenatal treatments alone, failed to prevent the development of AD in early life in multiple other RCTs without comprehensive follow-ups [291][292][293][294][295]. Such variations may be due to the differences in the participants enrolled, experimental designs, cultures of the probiotics and the dose administered. A follow-up study from the Norwegian RCT [286][287][288] suggested that the efficacy of probiotics may depend on the intrinsic microbiota, that high levels of fecal B. dentium is associated with weaker protection against AD by the probiotics treatment [296]. Overall, systematic reviews and meta-analyses based on the literature above suggest that probiotics have a long-term preventive effect against AD [297][298][299][300][301][302][303][304][305][306][307][308][309][310][311]. Therefore, although contradictory results exist, WAO recommends using probiotics during pregnancy, lactation and infancy to prevent the development of allergy [312].
Supplementation with probiotics has also been used to treat AD in RCTs (Table 3). Many RCTs selected L. rhamnosus, particularly the strain LGG [277,278]. The first RCT using probiotics to manage AD achieved improvement in AD infants by orally applying LGG [313].
LGG alleviated infantile AD with no major impact on the gut microbiota in another RCT [314]. Nevertheless, LGG supplement failed to rescue infantile AD in multiple more recent RCTs [315][316][317]. Another strain of L. rhamnosus, MP108, showed a therapeutic effect on AD [318]. More recently, a mixture of three strains of L. rhamnosus (ŁOCK 0900, ŁOCK 0908, ŁOCK 0918) significantly benefitted infants with AD [319]. Supplement of other Lactobacilli appeared effective in alleviating AD symptoms as well, including L. salivarius [320,321], L. fermentum [322,323], L. sakei [324], L. plantarum [325][326][327], and L. paracasei [323], although contradictory results exist for L. paracasei [328]. Combination treatment of different Lactobacilli also potently attenuate AD [323,329,330]. Bifidobacteria are also used in RCTs against AD, particularly B. lactis. B. lactis Bb-12 was effective in improving AD in the first trial using probiotics to manage AD [313]. Supplementing B. lactis LKM512 expanded Lactobacilli in gut microbiota and alleviated AD, likely through the increased production of kynurenic acids, a product of tryptophan metabolism [331]. However, B. lactis CNCM I-3446 demonstrated no benefit in the treatment of AD [328]. Using the mixtures of Lactobacilli and Bifidobacteria appropriately may be a promising therapy against AD. Supplements of L. salivarius and B. breve combination conferred a significant improvement in adult AD patients and a potent regulation on the T H 1/T H 2 immune balance [332]. A mixture of B. lactis, B. longum and L. casei accelerated the recovery of moderate AD with steroid treatment [333]. Another combination of probiotics, L. salivarius and S. thermophilus is also beneficial for AD management [334]. Probiotic treatments selectively attenuated IgE-sensitized AD [335,336] and food-sensitized AD infants [337], suggesting probiotics may not be a wide-spectral therapy against AD. Aligned with the mixed results, systematic reviews and meta-analyses cannot conclude the efficacy of probiotics in treating AD [299,311,[338][339][340][341][342][343].
While future RCTs may continue to identify novel species or strains of probiotics that may be effective in controlling and treating AD, more effort is needed to standardize the current probiotics applications in the battle against AD and to set up regulations in the use of probiotics.

Synbiotics and Postbiotics in Treating AD
There are attempts to combat AD by combining the administration of prebiotics and probiotics. A new term "synbiotic" was defined in 2019 as "a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefit on the host" [344]. RCTs against AD using synbiotics with limited scales demonstrated mixed results [264,[345][346][347][348][349][350][351]. Nevertheless, well-designed synbiotics are likely to lead to novel, effective therapeutics against AD.
Postbiotics are inanimate microorganisms and/or their components with beneficial effects on host health [352], including the lysates of microorganisms, heat-inactivated microorganisms, and microbial metabolites. The trials using lysates and heat-inactivated microorganisms are highly contradictory. Early attempts using heat-inactivated LGG failed to benefit AD and even resulted in adverse gastrointestinal symptoms, including diarrhea [314]. Similarly, heat-treated L. paracei GM-080 did not accelerate the recovery of infantile AD with topical corticosteroid treatment [353]. On the contrary, trials using the postbiotics from the different strains of the same species were potent in improving AD. Tyndallized L. rhamnosus IDCC3201 (RHT3201) had therapeutic improvement in children with moderate AD and decreased eosinophil cationic protein and IL-31 in blood [354]. Heat-killed L. paracasei K71 alleviated adult AD and reduced the use of topical corticosteroids [355]. Heat-killed L. acidophilus strain L-92 attenuated AD symptoms in both children and adults [356,357], decreased eosinophil count, and increased serum TGF-β [357]. In addition, lysate of Vitreoscilla filiformis significantly improved AD and reduced fecal S. aureus [358]. The application of lysates and heat-inactivated microorganisms faces similar challenges as the application of probiotics in lacking standard and consistency.
Microbial metabolites have advantages in standardization, but current investigations are still limited to the pre-clinical stage [359]. With sufficient knowledge from pre-clinical studies, more trials should utilize microbial metabolites, including SCFAs, D-tryptophan, and IAld to test their clinical efficacy against AD.

Concluding Remarks
The recent discoveries on how the skin and gut microbiota modulate AD pathogenesis point out a bright potential in manipulating commensal bacteria to prevent and manage AD. Current therapies that leverage the skin microbiota, however, are still limited. Further investigations are needed to improve the specificity of the therapies against pathogenic bacteria, such as S. aureus, while encouraging the growth of normal bacteria at the same time. Clinical trials have been conducted to manipulate the gut microbiota for the prevention and treatment against AD. Although the efficacies of certain treatments are still controversial, the oral application of probiotics during pregnancy, lactation, and infancy is recommended for the prevention of allergic diseases, including AD [312]. More efforts are needed to standardize gut microbiota-based interventions against AD. In addition, microbial metabolites may aid in AD management, but they need to be tested in future trials. Furthermore, using these microbiota-based treatments in conjunction with the traditional treatments may achieve improved clinical outcomes. The role of commensal bacteria in AD is still a relatively new area. Increased understanding in this area would improve the management of AD, an increasingly prevalent atopic disease.