Human Skin Explants Recapitulate Key Features of HSV-1 Infections

COMMENTARY instigating anti-dermatophyte responses was not addressed in this study, but would be an intriguing avenue of investigation. In summary, this work represents a notable advancement in the field of fungal immunology, illuminating host defenses to a superficial fungal infection that impacts a large portion of the world’s population. The study reinforces the idea that host mucosal and barrier surfaces are equipped with sophisticated immune defense networks that work in a synchronized manner to counter microbial pathogens, such as T. benhamiae, and limit spread to other distal sites. These mechanistic insights into the workings of the dermal immune system are foundations that will be needed in the pursuit of antifungal vaccines, none of which exist to date. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS The authors thank G. Trevejo-Nun˜ez for helpful suggestions. SLG was supported by National Institutes of Health grant DE022550. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at www.jidonline.org, and at https://doi.org/10.1016/j.jid.2018.10.022.

REFERENCES Acosta-Rodriguez EV, Rivino L, Geginat J, Jarrossay D, Gattorno M, Lanzavecchia A, et al. Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat Immunol 2007;8:639e46. Blanco JL, Garcia ME. Immune response to fungal infections. Vet Immunol Immunopathol 2008;125:47e70. Brown GD. How fungi have shaped our understanding of mammalian immunology. Cell Host Microbe 2010;7:9e11. Burstein VL, Guasconi L, Beccacece I, Theumer MG, Mena C, Prinz I, et al. IL-17mediated immunity controls skin infection and T helper 1 response during experimental Microsporum canis dermatophytosis. J Invest Dermatol 2018;138:1744e53. Casadevall A, Pirofski LA. Immunoglobulins in defense, pathogenesis, and therapy of fungal diseases. Cell Host Microbe 2012;11:447e56. Conti HR, Gaffen SL. IL-17-mediated immunity to the opportunistic fungal pathogen Candida albicans. J Immunol 2015;195:780e8. Conti H, Shen F, Nayyar N, Stocum E, JN S, Lindemann M, et al. Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis. J Exp Med 2009;206:299e311. Conti H, Peterson A, Huppler A, Brane L, Herna´ndez-Santos N, Whibley N, et al. Oralresident ‘natural’ Th17 cells and gd-T cells control opportunistic Candida albicans infections. J Exp Med 2014;211:2075e84.

Farah C, Hu Y, Riminton S, Ashman R. Distinct roles for interleukin-12p40 and tumour necrosis factor in resistance to oral candidiasis defined by gene targeting. Oral Microbiol Immunol 2006;21:252e5. Gladiator A, Wangler N, Trautwein-Weidner K, Leibundgut-Landmann S. Cutting edge: IL-17secreting innate lymphoid cells are essential for host defense against fungal infection. J Immunol 2013;190:521e5.

Park CO, Fu X, Jiang X, Pan Y, Teague JE, Collins N, et al. Staged development of long-lived T-cell receptor alphabeta TH17 resident memory Tcell population to Candida albicans after skin infection. J Allergy Clin Immunol 2018;142: 647e62. Puel A, Cypowji S, Bustamante J, Wright J, Liu L, Lim H, et al. Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin17 immunity. Science 2011;332:65e8.

Hernandez-Santos N, Wiesner DL, Fites JS, McDermott AJ, Warner T, Wuthrich M, et al. Lung epithelial cells coordinate innate lymphocytes and immunity against pulmonary fungal infection. Cell Host Microbe 2018;23:511e22 e5.

Quintin J, Saeed S, Martens JH, GiamarellosBourboulis EJ, Ifrim DC, Logie C, et al. Candida albicans infection affords protection against reinfection via functional reprogramming of monocytes. Cell Host Microbe 2012;12:223e32.

Kashem SW, Igyarto BZ, Gerami-Nejad M, Kumamoto Y, Mohammed J, Jarrett E, et al. Candida albicans morphology and dendritic cell subsets determine T helper cell differentiation. Immunity 2015a;42:356e66.

Santus W, Barresi S, Mingozzi F, Broggi A, Orlandi I, Stamerra G, et al. Skin infections are eliminated by cooperation of the fibrinolytic and innate immune systems. Sci Immunol 2017;2(15).

Kashem S, Riedl M, Yao C, Honda C, LVulchanova, Kaplan D. Nociceptive sensory fibers drive interleukin-23 production from CD301bþ dermal dendritic cells and drive protective cutaneous immunity. Immunity 2015b;43:515e26. Heinen M-P, Cambier L, Antoine N, Gabriel A, Gillet L, Bureau F, et al. Th1 and Th17 immune responses act complementarily to optimally control superficial dermatophytosis. J Invest Derm 2019;139:626e37. Naik S, Bouladoux N, Linehan JL, Han SJ, Harrison OJ, Wilhelm C, et al. Commensaldendritic-cell interaction specifies a unique protective skin immune signature. Nature 2015;520(7545):104e8. Naik S, Larsen SB, Gomez NC, Alaverdyan K, Sendoel A, Yuan S, et al. Inflammatory memory sensitizes skin epithelial stem cells to tissue damage. Nature 2017;550(7677):475e80.

St Leger AJ, Desai JV, Drummond RA, Kugadas A, Almaghrabi F, Silver P, et al. An ocular commensal protects against corneal infection by driving an interleukin-17 response from mucosal gammadelta T cells. Immunity 2017;47:148e58 e5. Sparber F, Dolowschiak T, Mertens S, Lauener L, Clausen BE, Joller N, et al. Langerinþ DCs regulate innate IL-17 production in the oral mucosa during Candida albicans-mediated infection. PLoS Pathog 2018;14(5):e1007069. Verma A, Richardson J, Zhou C, Coleman BM, Moyes D, Ho J, et al. Oral epithelial cells orchestrate innate Type 17 responses to Candida albicans through the virulence factor Candidalysin. Sci Immunol 2017;2:eeam8834. Whibley N, Tritto E, Traggiai E, Kolbinger F, Moulin P, Brees D, et al. Antibody blockade of IL-17-family cytokines in immunity to acute murine oral mucosal candidiasis. J Leukoc Biol 2016;99:1153e64.

See related article on pg 673

Human Skin Explants Recapitulate Key Features of HSV-1 Infections Megan H. Orzalli1 The development of novel antiviral compounds is hindered by the lack of model systems that recapitulate the pathophysiology of human infections. Tajpara et al. developed an ex vivo human abdominal skin model of HSV-1 infection to examine host-pathogen interactions and test the efficacy of antiviral compounds. This approach provides a platform for future development and testing of antiviral drugs. Journal of Investigative Dermatology (2019) 139, 519e521. doi:10.1016/j.jid.2018.09.015

1

Division of Gastroenterology, Hepatology and Nutrition, Boston Children’s Hospital, Boston, Massachusetts, USA Correspondence: Megan H. Orzalli, Boston Children’s Hospital, Enders 650, 300 Longwood Avenue, Boston, Massachusetts 02115, USA. E-mail: [email protected]

ª 2018 The Author. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.

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COMMENTARY

Clinical Implications  Discarded human abdominal skin can be infected with HSV-1 after perturbation of the outermost layers of the epidermis.  A clinical isolate of HSV-1 induces a classic antiviral immune response in human abdominal skin.  The antiviral drugs acyclovir and pritelivir inhibit HSV-1 replication in human abdominal skin.

Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) are ubiquitous pathogens that establish lifelong infections in their human hosts. After an initial round of lytic replication in epithelial cells at mucosal surfaces, HSV establishes latency in sensory neurons that innervate the primary site of infection. Periodic reactivation of the virus results in the recurrent development of oral and genital lesions that are the hallmark of infections with HSV-1 and HSV-2. In immunocompetent hosts, clinical manifestations of primary infection and reactivation are usually self-limiting. By contrast, HSV infections in immunocompromised patients can result in severe manifestations, such as herpes simplex encephalitis, which is associated with high mortality rates and neurological sequalae. No vaccines against HSV-1 or HSV-2 are available, and current treatment options are limited. The most commonly used therapies for HSV infections are nucleoside triphosphate analogues, such as the guanosine analogues acyclovir and penciclovir. After an initial monophosphorylation event by the viral thymidine kinase, guanosine analogues are converted into a

triphosphorylated molecule by cellular kinases. This active form of the molecule is incorporated into the viral genome, effectively terminating DNA

The use of bona fide human tissues to examine virus-cell interactions after infection is a powerful step toward understanding and countering the pathogenesis of HSV infections in humans. replication and inhibiting virus spread (Birkmann and Zimmermann, 2016). Mutations in viral thymidine kinase arise frequently in immunocompromised individuals, resulting in nucleoside-resistant infections (Piret and Boivin, 2011). In such cases, viral DNA polymerase inhibitors, including foscarnet and cidofovir, can be used to treat HSV infections. However, administration of these drugs is associated

with severe adverse effects (Birkmann and Zimmermann, 2016). Thus, there is an unmet need for additional therapeutic options, particularly those that are safe and effective in immunocompromised populations. Although new classes of antiviral molecules that target HSV-1 and HSV-2 are currently in development and undergoing clinical trials (Birkmann and Zimmermann, 2016), antiviral drug discovery on the whole has been limited by a lack of models that recapitulate the pathophysiology of human infections. Current models to study herpes simplex viruses rely predominantly on in vitro cell culture systems and in vivo infections of small animals, including mice. However, these systems do not always predict the clinical success of antiviral compounds accurately (Everts et al., 2017). Cell culture systems are often limited by their inability to mimic the threedimensional architecture of host tissues and the complexity of cell-to-cell and cell-to-matrix interactions that occur after infection. Similarly, in vivo small animal models of infection can be poor predictors of clinical outcomes because of differences in host physiology. Comparative studies in mouse and human skin have unveiled striking differences in the morphology and cellular composition of these two systems (Zomer and Trentin, 2018). Human skin has a thicker epidermis than mouse skin, resulting in increased barrier function. In addition, differences in immune cell composition in the epidermis of these two organisms are apparent. Although Langerhans cells and CD8þ T cells are observed in

HSV-1 Microneedle Roller

HSV-1 replication

Acyclovir/Pritelivir

Nectin 1 expression Nuclear NF- B Nuclear IRF3

Figure 1. HSV-1 productively infects abdominal skin after perturbation with a microneedle roller. HSV-1 infection results in decreased nectin 1 expression and increased accumulation of nuclear NF-kB and IRF3. Virus replication in the skin is inhibited by antiviral drugs. HSV-1, herpes simplex virus type 1.

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COMMENTARY human and mouse skin, mice have a prominent population of dendritic epidermal T cells that are not found in humans. Specific differences in the human and mouse molecular responses to HSV1 infection can also limit the ability to extrapolate findings from mice to humans. Mice restrict HSV replication by initiating an inflammatory cell death pathway known as necroptosis in infected cells (Orzalli and Kagan, 2017), which may have profound effects on the pathogenesis of HSV in mice. By contrast, HSV blocks necroptosis in human cells. Thus, differences between mice and humans at the molecular, cellular, and tissue levels must be considered when using mice as preclinical models of HSV infection. These differences in murine and human physiology underscore the need for models of infection that faithfully mimic pathogenesis in humans. Tajpara et al. (2019) describe an ex vivo infection model in human skin that can be used to study HSV pathogenesis and test the efficacy of antiviral compounds. Discarded human abdominal skin samples obtained from plastic surgery procedures were subjected to microneedle treatment to overcome the mechanical barrier function of the stratum corneum (Tajpara et al., 2018). This approach allowed for reproducible and productive infections of these tissues with a clinical isolate of HSV-1. The authors show that their model recapitulates the morphological features of HSV infections observed in lesional biopsy samples, and several cellular responses that have been observed in vitro, including down-regulation of the viral entry factor nectin 1 and nuclear accumulation of the innate immune signaling component NF-kB (Patel et al., 1998; Stiles and Krummenacher, 2010) (Figure 1). The authors also observe strong nuclear accumulation of the antiviral signaling factor IRF3 in infected epidermal keratinocytes, which has been reported to be blocked by HSV-1 in cell culture systems of infection (Kurt-Jones et al., 2017). This observation potentially highlights differences in the cellular

responses to clinical isolates of HSV from those used in laboratory settings. Alternatively, these differences may reflect a host response that cannot be captured in in vitro infection models. Future follow-up studies will likely provide insight into these differences. After the establishment and characterization of their abdominal tissue infection model, Tajpara et al. (2018) examined the utility of this model as a platform for testing the efficacy and toxicity of antiviral drugs. As a proof-ofprinciple experiment, the authors used this ex vivo system to compare the antiviral activities of acyclovir and pritelivir, a helicase-primase small molecule inhibitor that recently completed phase 2 clinical trials (Birkmann and Zimmermann, 2016). The authors observed that both compounds significantly reduced HSV-1 replication in tissues, consistent with their described antiviral activity in humans. These results suggest that this model can recapitulate the antiviral activities of specific drugs observed in clinical practice. In addition to providing a model that recapitulates the pathophysiology of HSV infections in humans, the approach established by Tajpara et al. (2018) provides an opportunity to examine potential sex-based differences in acute responses to HSV infection and antiviral compounds. A majority of dermatology-based research studies use cells and tissues from male donors, likely a result of the availability of foreskin samples for cell isolation and ex vivo tissue studies (Kong et al., 2016). However, research examining host responses to viral infection and putative antiviral compounds should include analysis of both sexes. The use of abdominal skin as a model for HSV infection allows for comparisons to be made between tissues isolated from male and female donors. It remains to be determined if this ex vivo tissue model of infection can be used in a high-throughput manner to screen novel antiviral compounds or whether it represents a pipeline step to test previously identified candidate antivirals for their toxicity and potency in

human tissues. Nevertheless, the use of bona fide human tissues to examine virus-cell interactions after infection is a powerful step forward to understanding and countering the pathogenesis of HSV infections in humans. Furthermore, the use of these tissues as an experimental model may be expanded to other viruses that are introduced via the skin or other mucocutaneus surfaces. CONFLICT OF INTEREST The author states no conflict of interest.

ACKNOWLEDGMENTS The author would like to thank Dr. Jonathan Kagan for his helpful feedback on this commentary. MHO is supported by National Institutes of Health grant K99AI130258.

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