Autoinhibitory regulation of S100A8/S100A9 alarmin activity locally restricts sterile inflammation
Thomas Vogl, … , Thomas Pap, Johannes Roth
Thomas Vogl, … , Thomas Pap, Johannes Roth
Published April 3, 2018
Citation Information: J Clin Invest. 2018;128(5):1852-1866. https://doi.org/10.1172/JCI89867.
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Research Article Autoimmunity Inflammation

Autoinhibitory regulation of S100A8/S100A9 alarmin activity locally restricts sterile inflammation

Abstract

Autoimmune diseases, such as psoriasis and arthritis, show a patchy distribution of inflammation despite systemic dysregulation of adaptive immunity. Thus, additional tissue-derived signals, such as danger-associated molecular patterns (DAMPs), are indispensable for manifestation of local inflammation. S100A8/S100A9 complexes are the most abundant DAMPs in many autoimmune diseases. However, regulatory mechanisms locally restricting DAMP activities are barely understood. We now unravel for the first time, to our knowledge, a mechanism of autoinhibition in mice and humans restricting S100-DAMP activity to local sites of inflammation. Combining protease degradation, pull-down assays, mass spectrometry, and targeted mutations, we identified specific peptide sequences within the second calcium-binding EF-hands triggering TLR4/MD2-dependent inflammation. These binding sites are free when S100A8/S100A9 heterodimers are released at sites of inflammation. Subsequently, S100A8/S100A9 activities are locally restricted by calcium-induced (S100A8/S100A9)2 tetramer formation hiding the TLR4/MD2-binding site within the tetramer interphase, thus preventing undesirable systemic effects. Loss of this autoinhibitory mechanism in vivo results in TNF-α–driven fatal inflammation, as shown by lack of tetramer formation in crossing S100A9–/– mice with 2 independent TNF-α–transgene mouse strains. Since S100A8/S100A9 is the most abundant DAMP in many inflammatory diseases, specifically blocking the TLR4-binding site of active S100 dimers may represent a promising approach for local suppression of inflammatory diseases, avoiding systemic side effects.

Authors

Thomas Vogl, Athanasios Stratis, Viktor Wixler, Tom Völler, Sumita Thurainayagam, Selina K. Jorch, Stefanie Zenker, Alena Dreiling, Deblina Chakraborty, Mareike Fröhling, Peter Paruzel, Corinna Wehmeyer, Sven Hermann, Olympia Papantonopoulou, Christiane Geyer, Karin Loser, Michael Schäfers, Stephan Ludwig, Monika Stoll, Tomas Leanderson, Joachim L. Schultze, Simone König, Thomas Pap, Johannes Roth

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Figure 5

Identification of the S100A9-binding site on TLR4/MD2.

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Identification of the S100A9-binding site on TLR4/MD2. (A–C) The PDB fil...
(A–C) The PDB files of the S100A8/S100A9 tetramer (1XK4, A) and S100A9 dimer (1IRJ, C) were retrieved from the RSCB PDB website. (B) 1XK4 was modified containing only the E (S100A8, gray) and G (S100A9, green) chains resembling 1 heterodimer. Masked aa in the tetramer were analyzed by Cluspro. (B, yellow- or cyan-labeled aa for S100A8 or S100A9). Labeled S100A9 aa were chosen for mutation studies (C). (D) Single mutated S100A9 was analyzed for TLR4/MD2 binding compared with WT S100A9. All S100A9 mutants showed reduced binding to TLR4/MD2. (E) Double-mutated S100A9 was analyzed for binding to TLR4/MD2 as shown in D and showed significantly reduced binding properties compared with WT S100A9. Data represent mean ± SD of 5 independent experiments. *P < 0.05; **P < 0.01, 1-way ANOVA. (F) Monocytes were stimulated for 4 hours with intact S100A9 (0 h = no trypsinization) or the fragment mixture, and TNF-α levels were quantified by ELISA. Western blot shows remaining intact S100A9 (insert). One representative of 3 independent experiments is shown. (G) Tryptic fragments of hS100A9 (F) were analyzed for TLR4/MD2 binding, and peptide 15 corresponding to aa 73–85 showed specific interaction with TLR4/MD2, as detected by nanoUPLC/ESI-Q-TOF MS/MS (m/z 1614). Peptide 6 (NIETIINTFHQYSVK) was also detected in the control setting without TLR4/MD2 and reflects unspecific binding. (H) Peptides comprising aa 63–79 of S100A9 (MEDLDTNADKQLSFEEF, MW: 2032 g/mol), control peptides 63–79 5A (MAALDTNADAALSFAEF, MW: 1758 g/mol) or a scrambled peptide (DSLEMTEENLADQFKDF, MW: 2032 g/mol) were investigated for TLR4/MD2 binding as shown in G. Unspecific binding was analyzed by using peptides 63–79 without TLR4/MD2. Only S100A9 peptides 63–79 showed binding to TLR4/MD2 (3 independent experiments). Asterisks indicate polyethylene glycol impurities.