Structural modeling and functional characterization of a novel gain-of-function TLR8 variant causing severe inflammatory syndrome
Nikolaos T. Skenteris, Elisa Luttermann, Sanjana Nair, Ioannis Evangelakos, Maria Pujantell, Marie Eggers, Fabian Hausmann, Marleen Bérouti, Benedetta Padoan, Felix J. Flomm, Janna M. Claussen, Benjamin Grünhagel, Anika Salfelder, Brigitte Beifuss, Saskia Biskup, Patrick Blümke, Katrin Rading, Heike Hildebrandt, Urte Matschl, Silke Giesemann-Jansen, Jana Hennesen, Viacheslav O. Nikolaev, Michael Kutsche, Christian Kubisch, Friedrich Koch-Nolte, Nicola M. Tomas, Eva Tolosa, Marc Lütgehetmann, Felix R. Stahl, Veit Hornung, Madeleine J. Bunders, Christian Schlein, Maya Topf, Ina Kötter, Marcus Altfeld
Nikolaos T. Skenteris, Elisa Luttermann, Sanjana Nair, Ioannis Evangelakos, Maria Pujantell, Marie Eggers, Fabian Hausmann, Marleen Bérouti, Benedetta Padoan, Felix J. Flomm, Janna M. Claussen, Benjamin Grünhagel, Anika Salfelder, Brigitte Beifuss, Saskia Biskup, Patrick Blümke, Katrin Rading, Heike Hildebrandt, Urte Matschl, Silke Giesemann-Jansen, Jana Hennesen, Viacheslav O. Nikolaev, Michael Kutsche, Christian Kubisch, Friedrich Koch-Nolte, Nicola M. Tomas, Eva Tolosa, Marc Lütgehetmann, Felix R. Stahl, Veit Hornung, Madeleine J. Bunders, Christian Schlein, Maya Topf, Ina Kötter, Marcus Altfeld
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Research Article Genetics Immunology Infectious disease

Structural modeling and functional characterization of a novel gain-of-function TLR8 variant causing severe inflammatory syndrome

Abstract

With the increasing use of genetic sequencing to investigate inborn errors of immunity, rare variants are frequently identified, yet their clinical relevance often remains uncertain. Establishing pathogenicity requires a multidisciplinary approach that integrates genetic, structural, functional, and clinical data. Here, we used such a strategy to investigate a previously unreported hemizygous missense variant — alanine (A) to threonine (T) at residue 518 — in Toll-like receptor 8 (TLR8), identified in 2 male siblings with recurrent infections and systemic inflammation, characterized by a proinflammatory immune signature and B cell dysregulation. Functional studies showed that the TLR8 A518T variant enhanced NF-κB activation and increased secretion of proinflammatory cytokines compared with WT TLR8 upon stimulation, consistent with a gain-of-function effect. Protein degradation and turnover assays revealed reduced abundance of the mutant TLR8 protein due to faster turnover and increased proteasomal degradation. Computational modeling predicted enhanced structural stabilization of the active TLR8 homodimer interface via additional water-mediated hydrogen bonds introduced by the A518T substitution. Together, these findings integrating structural modeling with functional assays identify a novel TLR8 ligand-specific gain-of-function mutation resulting in complex immunopathology in 2 siblings.

Authors

Nikolaos T. Skenteris, Elisa Luttermann, Sanjana Nair, Ioannis Evangelakos, Maria Pujantell, Marie Eggers, Fabian Hausmann, Marleen Bérouti, Benedetta Padoan, Felix J. Flomm, Janna M. Claussen, Benjamin Grünhagel, Anika Salfelder, Brigitte Beifuss, Saskia Biskup, Patrick Blümke, Katrin Rading, Heike Hildebrandt, Urte Matschl, Silke Giesemann-Jansen, Jana Hennesen, Viacheslav O. Nikolaev, Michael Kutsche, Christian Kubisch, Friedrich Koch-Nolte, Nicola M. Tomas, Eva Tolosa, Marc Lütgehetmann, Felix R. Stahl, Veit Hornung, Madeleine J. Bunders, Christian Schlein, Maya Topf, Ina Kötter, Marcus Altfeld

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Figure 8

Stimulation of TLR8 A518T variant induces p–NF-κB and cytokine release.

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Stimulation of TLR8 A518T variant induces p–NF-κB and cytokine release. ...
(A) Western blot against TLR8, p–NF-κB, total NF-κB, and γ-tubulin (γ-TUB) in BlaER1 TLR8–/– cells with the insertion of the TLR8 WT protein or the p.A518T and the previously published p.D543A (loss-of-function) TLR8 variants. Cells were stimulated with 100 ng/mL TL8-506 agonist for 15 minutes (left), and the ratio of p–NF-κB to total NF-κB to TLR8 levels, normalized to TLR8 levels, is shown (right). Statistical significance between groups was assessed using a 2-way ANOVA multiple-comparison test. Data represent mean ± SEM of n = 4 experiments. RQ, relative quantification. (B) IL-6 quantification of BlaER1 TLR8–/– cells with the insertion of the TLR8 WT protein or the p.A518T and p.D543A (loss-of-function) variants. Cells were stimulated with 1 μg/mL TL8-506 and 1 μg/mL ORN 06 agonists. Data are presented after the subtraction of the unstimulated measurements. Statistical significance between groups was assessed using a 2-way ANOVA multiple-comparison test. Data represent mean ± SEM of n = 3–4 experiments. Differences between groups were considered significant at P values less than 0.05.