Staphylococcus aureus: The Bug Behind the Itch in Atopic Dermatitis

Pruritus or itch is a defining symptom of atopic dermatitis (AD). The origins of itch are complex, and it is considered both a defense mechanism and a cause of disease that leads to inflammation and psychological stress. Considerable progress has been made in understanding the processes that trigger itch, particularly the pruritoceptive origins that are generated in the skin. This perspective review discusses the implications of a recent observation that the V8 protease expressed by Staphylococcus aureus can directly trigger sensory neurons in the skin through activation of protease-activated receptor 1. This may be a key to understanding why itch is so common in AD because S. aureus commonly overgrows in this disease owing to deficient antimicrobial defense from both the epidermis and the cutaneous microbiome. Increased understanding of the role of microbes in AD provides increased opportunities for safely improving the treatment of this disorder. Pruritus or itch is a defining symptom of atopic dermatitis (AD). The origins of itch are complex, and it is considered both a defense mechanism and a cause of disease that leads to inflammation and psychological stress. Considerable progress has been made in understanding the processes that trigger itch, particularly the pruritoceptive origins that are generated in the skin. This perspective review discusses the implications of a recent observation that the V8 protease expressed by Staphylococcus aureus can directly trigger sensory neurons in the skin through activation of protease-activated receptor 1. This may be a key to understanding why itch is so common in AD because S. aureus commonly overgrows in this disease owing to deficient antimicrobial defense from both the epidermis and the cutaneous microbiome. Increased understanding of the role of microbes in AD provides increased opportunities for safely improving the treatment of this disorder. Atopic dermatitis (AD) is a common disease that typically occurs during infancy and childhood, affecting up to 20% of children and 5% of adults in many industrialized countries (Weidinger et al., 2018Weidinger S. Beck L.A. Bieber T. Kabashima K. Irvine A.D. Atopic dermatitis.Nat Rev Dis Primers. 2018; 4: 1Google Scholar). Chronic itch is a defining symptom of AD and is associated with impaired QOL and avoidance of social interaction (Yosipovitch et al., 2023Yosipovitch G. Kim B. Luger T. Lerner E. Metz M. Adiri R. et al.Similarities and differences in peripheral itch and pain pathways in atopic dermatitis [epub ahead of print].J Allergy Clin Immunol. 2023; https://doi.org/10.1016/j.jaci.2023.10.034Google Scholar). This disorder has been referred to as the itch that rashes, a phrase that reflects the observation that the itch symptom in many patients may be observed prior to any apparent signs of skin inflammation. Itch is an important element in the progression of disease. Scratching behavior triggered by itch promotes further barrier disruption and skin inflammation. There is also abundant indirect evidence for a critical role of itch in AD. In infants, lesions appear predominantly on dorsal extremities and face and then shift to ventral surfaces and trunk as children age. This pattern follows the physical ability of the maturing infant to scratch these areas of the body. In addition, patients report that symptoms of itch improve rapidly after initiation of anti–IL-4Ra therapy, occurring as rapidly as 2 days after the start of therapy and preceding the improvement in skin lesions by several weeks (Silverberg et al., 2020Silverberg J.I. Yosipovitch G. Simpson E.L. Kim B.S. Wu J.J. Eckert L. et al.Dupilumab treatment results in early and sustained improvements in itch in adolescents and adults with moderate to severe atopic dermatitis: analysis of the randomized phase 3 studies SOLO 1 and SOLO 2, AD ADOL, and CHRONOS.J Am Acad Dermatol. 2020; 82: 1328-1336Google Scholar). Understanding and treating itch is therefore an important goal in the care of patients with AD and an active and ongoing area of investigation. The pathogenesis of AD is complex and remains incompletely understood. Host genetic abnormalities such as sequence variants in FLG and/or other genes that contribute to the epidermal barrier are major risk factors but not causative by themselves for the disease. Defects in the host immune system, including activating allelic variants in the IL-4 receptor (Hershey et al., 1997Hershey G.K. Friedrich M.F. Esswein L.A. Thomas M.L. Chatila T.A. The association of atopy with a gain-of-function mutation in the alpha subunit of the interleukin-4 receptor.N Engl J Med. 1997; 337: 1720-1725Google Scholar), can also promote the disease but are rare. Several large-scale GWASs have identified multiple risk loci associated with AD, but these account for only a minority of patients afflicted by the disease (Brown, 2021Brown S.J. What have we learned from GWAS for atopic dermatitis?.J Invest Dermatol. 2021; 141: 19-22Google Scholar). AD is now best understood from the perspective of the holobiome, which recognizes that the function of the skin depends not only on the behavior of cells from the host but also on the community of bacteria and other microbes that live on and within the body (Burger and Gallo, 2023Burger E. Gallo R.L. Host-microbiome interactions in the holobiome of atopic dermatitis.J Allergy Clin Immunol. 2023; 151: 1236-1238Google Scholar). AD is not universally caused by genetic abnormalities, evidence now suggests that it is driven by how the host and its microbiome interact. AD is not an infectious disease as defined by Koch's postulates, but common to almost all patients with AD is a change in the number and strains of some of the bacteria that populate the skin. In particular, Staphylococcus aureus and S. epidermidis overgrow on the skin of most patients with AD and produce multiple gene products that damage the epidermal barrier, trigger a type 2 inflammatory response, or influence the composition of the microbiome (Saheb Kashaf et al., 2023Saheb Kashaf S. Harkins C.P. Deming C. Joglekar P. Conlan S. Holmes C.J. et al.Staphylococcal diversity in atopic dermatitis from an individual to a global scale.Cell Host Microbe. 2023; 31: 578-592.e6Google Scholar). These species overgrow owing to a defect in the ability of AD skin to release adequate amounts of antimicrobial peptides such as cathelicidins and β-defensins and a lack of other species of bacteria that normally would fight against the survival of pathogenic strains of S. aureus (Nakatsuji et al., 2023Nakatsuji T. Brinton S.L. Cavagnero K.J. O'Neill A.M. Chen Y. Dokoshi T. et al.Competition between skin antimicrobial peptides and commensal bacteria in type 2 inflammation enables survival of S. aureus.Cell Rep. 2023; 42112494Google Scholar). Itch is important to the pathogenesis of AD and is not just a symptom of the disorder. The sensation of itch has been attributed at least in part to the activation of nonpeptidergic neurons in the skin. These sensory neurons exist as several subgroups that possess multiple different receptors that can trigger itch in response to specific chemical entities (puritogens). One group of receptors is unique G protein-coupled receptors (GPCRs) that respond to puritogens such as IL-31 and brain natriuretic peptide (Cevikbas et al., 2014Cevikbas F. Wang X. Akiyama T. Kempkes C. Savinko T. Antal A. et al.A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: involvement of TRPV1 and TRPA1.J Allergy Clin Immunol. 2014; 133: 448-460Google Scholar). Other GPCRs include histamine receptor H1/4R and protease-activated receptors (PARs) such as PAR2. PAR2 has been thought to be activated in AD by proteases released from keratinocytes such as the kallikreins or proteases released from mast cells such as tryptase (Ui et al., 2006Ui H. Andoh T. Lee J.B. Nojima H. Kuraishi Y. Potent pruritogenic action of tryptase mediated by PAR-2 receptor and its involvement in anti-pruritic effect of nafamostat mesilate in mice.Eur J Pharmacol. 2006; 530: 172-178Google Scholar). Type 2 cytokines have also been associated with direct activation of itch (Oetjen et al., 2017Oetjen L.K. Mack M.R. Feng J. Whelan T.M. Niu H. Guo C.J. et al.Sensory neurons co-opt classical immune signaling pathways to mediate chronic itch.Cell. 2017; 171: 217-228.e13Google Scholar), and IL-13 has been recently shown to act directly to enhance itch signals in a human dorsal root ganglia (DRG) model (Miron et al., 2022Miron Y. Miller P.E. Hughes C. Indersmitten T. Lerner E.A. Cevikbas F. Mechanistic insights into the antipruritic effects of lebrikizumab, an anti-IL-13 mAb.J Allergy Clin Immunol. 2022; 150: 690-700Google Scholar). Understanding the pathogenesis of AD is further complicated by observations that some neurons in the skin can also trigger inflammation (Cohen et al., 2019Cohen J.A. Edwards T.N. Liu A.W. Hirai T. Jones M.R. Wu J. et al.Cutaneous TRPV1+ neurons trigger protective innate Type 17 anticipatory immunity.Cell. 2019; 178: 919-932.e14Google Scholar). The prevailing paradigm in AD had been that skin inflammation could be a cause or result of itch. Inflammation can trigger neurons to promote itch due to the release of IL-31, which can act directly and indirectly (Datsi et al., 2021Datsi A. Steinhoff M. Ahmad F. Alam M. Buddenkotte J. Interleukin-31: the "itchy" cytokine in inflammation and therapy.Allergy. 2021; 76: 2982-2997Google Scholar), and histamine or endogenous proteases. Alternatively, neural stimulation might occur first, which would then trigger inflammation as a consequence of scratching or neural stimulation of inflammatory cytokines. Thus, evidence supports that inflammation can be either the cause or result of itch. From the perspective of the AD holobiome, it had been unclear what role microbes might play in the itch–scratch–inflammation cycle. A recent study by Deng et al., 2023Deng L. Costa F. Blake K.J. Choi S. Chandrabalan A. Yousuf M.S. et al.aureus drives itch and scratch-induced skin damage through a V8 protease-PAR1 axis.Cell. 2023; 186: 5375-5393.e25Google Scholar has answered this question and provided a more complete understanding of why patients with AD suffer from pruritus (Figure 1). Knowing the high correlation of S. aureus with AD lesional sites, Deng et al., 2023Deng L. Costa F. Blake K.J. Choi S. Chandrabalan A. Yousuf M.S. et al.aureus drives itch and scratch-induced skin damage through a V8 protease-PAR1 axis.Cell. 2023; 186: 5375-5393.e25Google Scholar tested the possibility that S. aureus can induce itch after epicutaneous application of the bacteria to mice. To assess itch, scratching behavior was studied using an iBOB (infrared behavior observation box) that could track scratching bouts and enable monitoring of spontaneous itch or alloknesis (touch-induced itch). Deng et al., 2023Deng L. Costa F. Blake K.J. Choi S. Chandrabalan A. Yousuf M.S. et al.aureus drives itch and scratch-induced skin damage through a V8 protease-PAR1 axis.Cell. 2023; 186: 5375-5393.e25Google Scholar demonstrated that topical application of S. aureus to the back skin of mice induced robust scratching, and the itch-induced scratching exacerbated skin damage similar to that seen in patients with AD. In contrast, subcutaneous infection by S. aureus did not trigger spontaneous itch or alloknesis, thus demonstrating the importance of the location of the bacterial in the skin and illustrating the difference between excessive colonization on the surface, a characteristic of AD, and deep tissue infection, which occurs more frequently in AD but is rarer and more often results in pain. Unexpectedly, S. aureus did not cause itch through previously known mechanisms. Mast cells have a role in itch (Dong and Dong, 2018Dong X. Dong X. Peripheral and central mechanisms of itch.Neuron. 2018; 98: 482-494Google Scholar), but mice lacking mast cells were still itchy after S. aureus exposure. IL-36 signaling through Myd88 and a role for basophils were also ruled out with knockout mice studies. IL-4, IL-13, and IL-31, all cytokines relevant to type 2 inflammation and associated with itch (Garcovich et al., 2021Garcovich S. Maurelli M. Gisondi P. Peris K. Yosipovitch G. Girolomoni G. Pruritus as a distinctive feature of type 2 inflammation.Vaccines (Basel). 2021; 9: 303Google Scholar), were not involved in S. aureus–induced itch. Because the triggers for itch that arise endogenously within the skin did not alter the itch response to S. aureus, factors produced directly by the bacteria were investigated for their contribution to spontaneous itch and alloknesis. They started with mutations that blocked S. aureus agr quorum sensing. The agr system is a global S. aureus virulence regulator that responds to an extracellular autoinducing peptide signal when a quorum of cells is reached, leading to enhanced expression of many important toxins and exoenzymes (Jenul and Horswill, 2019Jenul C. Horswill A.R. Regulation of Staphylococcus aureus virulence.Microbiol Spectr. 2019; 7https://doi.org/10.1128/microbiolspec.GPP3-0031-2018Google Scholar). This is clinically relevant because the quorum necessary for activation of the agr system is achieved in AD owing to the absolute abundance of S. aureus being high in many patients (Williams et al., 2019Williams M.R. Costa S.K. Zaramela L.S. Khalil S. Todd D.A. Winter H.L. et al.Quorum sensing between bacterial species on the skin protects against epidermal injury in atopic dermatitis.Sci Transl Med. 2019; 11eaat8329Google Scholar). Activation of the agr system increases alpha-toxin production, and sensory neurons directly respond to S. aureus alpha-toxin and phenol soluble modulin (PSM) peptides and produce pain during subcutaneous infections (Blake et al., 2018Blake K.J. Baral P. Voisin T. Lubkin A. Pinho-Ribeiro F.A. Adams K.L. et al.Staphylococcus aureus produces pain through pore-forming toxins and neuronal TRPV1 that is silenced by QX-314.Nat Commun. 2018; 9: 37Google Scholar; Chiu et al., 2013Chiu I.M. Heesters B.A. Ghasemlou N. Von Hehn C.A. Zhao F. Tran J. et al.Bacteria activate sensory neurons that modulate pain and inflammation.Nature. 2013; 501: 52-57Google Scholar). However, inactivation of alpha-toxin and PSMs had no role in itch. Considering that it had been demonstrated that S. aureus proteases also contribute to skin pathology (Williams et al., 2019Williams M.R. Costa S.K. Zaramela L.S. Khalil S. Todd D.A. Winter H.L. et al.Quorum sensing between bacterial species on the skin protects against epidermal injury in atopic dermatitis.Sci Transl Med. 2019; 11eaat8329Google Scholar) and that the success of outbreak methicillin-resistant S. aureus strains is partially attributed to these extracellular proteases (Ramirez et al., 2020Ramirez A.M. Beenken K.E. Byrum S.D. Tackett A.J. Shaw L.N. Gimza B.D. et al.SarA plays a predominant role in controlling the production of extracellular proteases in the diverse clinical isolates of Staphylococcus aureus LAC and UAMS-1.Virulence. 2020; 11: 1738-1762Google Scholar; Rom et al., 2017Rom J.S. Atwood D.N. Beenken K.E. Meeker D.G. Loughran A.J. Spencer H.J. et al.Impact of Staphylococcus aureus regulatory mutations that modulate biofilm formation in the USA300 strain LAC on virulence in a murine bacteremia model.Virulence. 2017; 8: 1776-1790Google Scholar; Zielinska et al., 2011Zielinska A.K. Beenken K.E. Joo H.S. Mrak L.N. Griffin L.M. Luong T.T. et al.Defining the strain-dependent impact of the Staphylococcal accessory regulator (sarA) on the alpha-toxin phenotype of Staphylococcus aureus.J Bacteriol. 2011; 193: 2948-2958Google Scholar), Deng et al., 2023Deng L. Costa F. Blake K.J. Choi S. Chandrabalan A. Yousuf M.S. et al.aureus drives itch and scratch-induced skin damage through a V8 protease-PAR1 axis.Cell. 2023; 186: 5375-5393.e25Google Scholar evaluated the role of these enzymes in itch. Most S. aureus isolates secrete at least 10 different proteases that mediate important functions, including nutrient acquisition, barrier penetration, and immune evasion (Nakatsuji et al., 2016Nakatsuji T. Chen T.H. Two A.M. Chun K.A. Narala S. Geha R.S. et al.Staphylococcus aureus exploits epidermal barrier defects in atopic dermatitis to trigger cytokine expression.J Invest Dermatol. 2016; 136: 2192-2200Google Scholar; Tam and Torres, 2019Tam K. Torres V.J. Staphylococcus aureus secreted toxins and extracellular enzymes.Microbiol Spectr. 2019; 7https://doi.org/10.1128/microbiolspec.GPP3-0039-2018Google Scholar). These include V8 (serine protease sspa), a metalloprotease (aureolysin [aur]), 2 cysteine proteases (staphopain A and staphopain B), and 6 serine protease-like proteins that are all positively regulated by agr quorum sensing (Jenul and Horswill, 2019Jenul C. Horswill A.R. Regulation of Staphylococcus aureus virulence.Microbiol Spectr. 2019; 7https://doi.org/10.1128/microbiolspec.GPP3-0031-2018Google Scholar). Through systematic single and combination mutants, the itch phenotype was attributed to just a dependence on V8 protease. The gene encoding V8 (sspa) was found to be upregulated on mouse skin in time frames of itch occurrences, and sspa was also upregulated in human AD skin lesional samples. Notably, direct V8 injection into an intradermal cheek model induced robust itch, and V8 intradermal back injections on mice drove skin damage, collectively demonstrating that V8 was solely sufficient to induce the itch and skin damage phenotypes. Mast cell tryptases can induce itch by activation of PARs, so Deng et al., 2023Deng L. Costa F. Blake K.J. Choi S. Chandrabalan A. Yousuf M.S. et al.aureus drives itch and scratch-induced skin damage through a V8 protease-PAR1 axis.Cell. 2023; 186: 5375-5393.e25Google Scholar next hypothesized that these receptors may sense or respond to V8 protease. They demonstrated that V8 binds and activates the host receptor PAR1, driving neural activation and itch. Specific cleavage assays and mass spectrometry confirmed the activation of PAR1, and additional assays ruled out PAR2 and PAR4. V8 was also found to activate human and mouse DRGs, which are known to express PAR1. Mice lacking PAR1 (F2r−/−) failed to respond to V8 protease through neural activation. The serine protease inhibitor TLCK and PAR1 antagonist vorapaxar prevented V8-induced calcium influx in neurons. Small interfering RNA targeting of PAR1 also eliminated the neuroimmune signaling in the presence of S. aureus–induced itch. Finally, vorapaxar inhibition of PAR1 reduced S. aureus–induced itch and skin damage phenotypes. The narrowing of all these phenotypes induced by S. aureus to the V8 protease was somewhat surprising. V8 was identified over 50 years ago (Drapeau et al., 1972Drapeau G.R. Boily Y. Houmard J. Purification and properties of an extracellular protease of Staphylococcus aureus.J Biol Chem. 1972; 247: 6720-6726Google Scholar) and is an endopeptidase that has been used extensively in the protein structure field owing to its ability to specifically cleave after glutamate residues. Although V8 has been identified as a virulence factor (Frey et al., 2021Frey A.M. Chaput D. Shaw L.N. Insight into the human pathodegradome of the V8 protease from Staphylococcus aureus.Cell Rep. 2021; 35108930Google Scholar; Shaw et al., 2004Shaw L. Golonka E. Potempa J. Foster S.J. The role and regulation of the extracellular proteases of Staphylococcus aureus.Microbiology (Reading). 2004; 150: 217-228Google Scholar), it was unclear exactly how V8 protease induced pathology. V8 was shown to have multiple actions, including the ability to self-cleave S. aureus surface proteins (McGavin et al., 1997McGavin M.J. Zahradka C. Rice K. Scott J.E. Modification of the Staphylococcus aureus fibronectin binding phenotype by V8 protease.Infect Immun. 1997; 65: 2621-2628Google Scholar) and cleave host targets (Frey et al., 2021Frey A.M. Chaput D. Shaw L.N. Insight into the human pathodegradome of the V8 protease from Staphylococcus aureus.Cell Rep. 2021; 35108930Google Scholar), and the purified enzyme can cause epidermal barrier dysfunction when applied directly to the skin of mice, inducing IgE responses (Hirasawa et al., 2010Hirasawa Y. Takai T. Nakamura T. Mitsuishi K. Gunawan H. Suto H. et al.Staphylococcus aureus extracellular protease causes epidermal barrier dysfunction.J Invest Dermatol. 2010; 130: 614-617Google Scholar). In a study examining the proteolytic activity of S. aureus strains isolated from AD lesions, both Aur and V8 were found in high abundance (Miedzobrodzki et al., 2002Miedzobrodzki J. Kaszycki P. Bialecka A. Kasprowicz A. Proteolytic activity of Staphylococcus aureus strains isolated from the colonized skin of patients with acute-phase atopic dermatitis.Eur J Clin Microbiol Infect Dis. 2002; 21: 269-276Google Scholar). Interestingly, V8 is secreted as a zymogen and is normally activated by the S. aureus metalloprotease aur (Drapeau, 1978Drapeau G.R. Role of metalloprotease in activation of the precursor of staphylococcal protease.J Bacteriol. 1978; 136: 607-613Google Scholar), but Deng et al., 2023Deng L. Costa F. Blake K.J. Choi S. Chandrabalan A. Yousuf M.S. et al.aureus drives itch and scratch-induced skin damage through a V8 protease-PAR1 axis.Cell. 2023; 186: 5375-5393.e25Google Scholar demonstrated that aur mutants had no phenotype in the itch model, suggesting activation of V8 in the skin by unknown factors, potentially relying on host metalloproteases. These findings open an important new area for study in itch and the overall understanding of the AD holobiome. How microbes on the skin influence cutaneous sensory responses requires further study. Not all patients with itch are known to be heavily colonized by S. aureus, so it is possible that other microbes may also contribute in similar ways. Could targeting the V8–PAR-1 initiation of itch be used to treat AD? Indeed, the therapeutic use of protease inhibitors has been an area of investigation in dermatology, and the focus has been on the human proteases such as kallikreins that play a role in diseases such as Rosacea (Two et al., 2014Two A.M. Hata T.R. Nakatsuji T. Coda A.B. Kotol P.F. Wu W. et al.Reduction in serine protease activity correlates with improved rosacea severity in a small, randomized pilot study of a topical serine protease inhibitor.J Invest Dermatol. 2014; 134: 1143-1145Google Scholar) or Netherton syndrome (Liddle et al., 2021Liddle J. Beneton V. Benson M. Bingham R. Bouillot A. Boullay A.B. et al.A potent and selective Kallikrein-5 inhibitor delivers high pharmacological activity in skin from patients with Netherton syndrome.J Invest Dermatol. 2021; 141: 2272-2279Google Scholar). In infectious diseases, the focus of protease inhibitors has been on viral pathogens such as HIV, but the current findings suggest that specific targeting of bacterial proteases may have benefits. Finally, current treatments of AD such as anti–-IL-4Ra therapy have a rapid effect on itch and a similarly rapid effect on bacterial colonization (Callewaert et al., 2020Callewaert C. Nakatsuji T. Knight R. Kosciolek T. Vrbanac A. Kotol P. et al.IL-4Ralpha blockade by dupilumab decreases Staphylococcus aureus colonization and increases microbial diversity in atopic dermatitis.J Invest Dermatol. 2020; 140: 191-202.e7Google Scholar). Perhaps the improvement in itch in these patients is due to the action on S. aureus or other microbes. We are only beginning to understand the full extent of the influence of the microbiome on human health. Richard L. Gallo: http://orcid.org/0000-0002-6187-2991 Alexander R. Horswill: http://orcid.org/0000-0002-5568-0096 RLG is a cofounder, is a consultant, receives income, and is an equity holder of MatriSys Bioscience. The remaining author states no conflict of interest. Grants from the NIHU01AI52038 (RG), UM1AI151958 (RG), R01AI153185 (AH AND RG), R01AR07082 (RG) Conceptualization: RLG, ARH; Writing – Original Draft Preparation: RLG, ARH; Writing – Review and Editing: RLG, ARH