Characterizing the Immune Infiltrates in Secondary Syphilis: Implications for Transmission and Pathology

Irène Gallais Sérézal ^1,2 Joseph Kirma ³ Mrinal K. Sarkar ³ Christopher Cole ³ Xianying Xing ³ Rachael Wasikowski ³ Jennifer Fox ³ Anthony Coon ³ Kelsey R vanStraalen ⁴ Linda H Xu ⁵ Craig Dobry ³ J. Michelle Kahlenberg ⁶ Paul Harms ⁷ Allison Chelsa Billi ³ Alex Tsoi ⁸ Lorenzo Giacani ⁵ Johann Eli Gudjonsson ^3*

• ¹ Centre Hospitalier Universitaire de Besançon, Besancon, France
• ² INSERM U1098 Interactions Hôte-Greffon-Tumeur & Ingénierie Cellulaire et Génique, Besançon, Franche-Comté, France
• ³ Department of Dermatology, University of Michigan, Ann Arbor MI48109 USA, Ann Arbor, United States
• ⁴ Laboratory for Experimental Immunodermatology, Department of Dermatology, Erasmus University Medical Center, Rotterdam, The Netherlands, rotterdam, Netherlands
• ⁵ Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA 98104, USA, Seattle, United States
• ⁶ Division of Rheumatology, Department of Internal Medicine, School of Medicine, Michigan Medicine, University of Michigan, Ann Arbor, Michigan, United States
• ⁷ Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA, Ann Arbor, United States
• ⁸ Department of Computational Medicine and Bioinformatics, School of Medicine, Michigan Medicine, University of Michigan, Ann Arbor, Michigan, United States

Background: Syphilis is a complex disease with variable clinical presentation where symptomatic and potentially infectious stages alternate with periods of latency, representing a fascinating model to study immune evasion and host immune responses. Methods: Immunohistochemistry (IHC), bulk, and single-cell RNA sequencing were performed on formalinfixed paraffin-embedded skin biopsies collected from subjects with secondary syphilis. Additionally, PBMCs from healthy individuals and either primary or MyD88 knock-out keratinocytes were exposed to live Treponema pallidum cells to define initial skin responses to the bacteria. Results: Immunohistochemistry of secondary syphilis skin lesions showed a polymorphous immune infiltrate with colocalization of T cells, B cells and antigen-presenting cells. Single-cell analysis revealed distinct cellular contributions to the immune response, with prominent immune-stromal crosstalk accompanied by altered keratinocyte differentiation and decreased intraepidermal communication. Notably, prominent inflammatory signals were countered by concomitant regulatory responses, particularly in infiltrating myeloid cells. Exposure of PBMCs to live T. pallidum inhibited immune responses, while exposure to sonicated cells triggered CXCL1 and CXCL3 upregulation. Keratinocytes responded to both intact and sonicated T. pallidum with upregulation of type-I interferon responses that, however, were abolished in MYD88-deficient but not in STING-deficient keratinocytes. Conclusion: Our data provide novel insights into the contribution of epidermal TLR sensing through MYD88 to the host response to syphilis infection, highlighting mechanisms by which T. pallidum evades immune responses in skin that may facilitate transmission of this pathogen through the skin.