The alarmin protein S100B, acting through the danger receptor RAGE (receptor for advanced glycation end-products), was shown in a mouse model of pulmonary aspergillosis to restrain Toll-like receptor (TLR)-driven inflammation through two unexplained limbs: extracellular nanomolar S100B induces a physical RAGE-TLR2 association that switches off TLR2 signalling in recruited neutrophils, and intracellular S100B binds pathogen- and host-derived nucleic acids so that TLR9 signalling converges on the adaptor TRIF instead of the canonical MyD88 route, terminating S100B production. The molecular mechanisms underlying both limbs remain unknown: nothing has been established about how S100B-bound DNA diverts TLR9 to TRIF, how RAGE-TLR2 association prevents TLR2 signalling, whether S100B redirects intracellular nucleic-acid trafficking (with a predicted attendant risk of autoimmunity). This problem asks for the causal molecular mechanisms, validated against the original calcium-, dose- and adaptor-dependence observations.

Background

Innate immunity operates two partially coupled sensing systems. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) — microbial and fungal components including nucleic acids — and relay the signal intracellularly through two Toll/interleukin-1 receptor domain adaptors: MyD88, used by all surface and most endosomal TLRs, and TRIF (TIR-domain-containing adaptor inducing interferon-beta), used canonically only by TLR3 and TLR4 (O'Neill, 2006). TLR9, the endosomal sensor of unmethylated CpG DNA, is in the standard framework a strictly MyD88-dependent receptor; where in the endosomal system a TLR9 ligand arrives and how it is structured can, however, change the quality of the response (Guiducci et al., 2006; Honda et al., 2005). A second system senses danger-associated molecular patterns (DAMPs), endogenous molecules released or induced upon tissue stress, through receptors such as RAGE, a multi-ligand receptor of the immunoglobulin superfamily. S100B is a small EF-hand calcium-binding protein of the S100 family with a dual life: an intracellular regulator of target proteins and, when released, an extracellular DAMP acting on RAGE (Donato et al., 2009). Nanomolar S100B is trophic/immunoregulatory whereas micromolar S100B multimers cross-link and activate RAGE by binding its V and C1 immunoglobulin domains (Leclerc et al., 2007; Ostendorp et al., 2007). DAMP-nucleic acid complexes can already shift TLR9 output in the neighbouring HMGB1 (high-mobility group box 1 protein) system: HMGB1-DNA complexes acting through RAGE potentiate TLR9 activation by DNA-containing immune complexes (Tian et al., 2007), while in other settings HMGB1 suppresses the plasmacytoid dendritic cell response to TLR9 agonists (Popovic et al., 2006). Against this backdrop, Sorci et al. (2011) delineated an integrated S100B circuit in pulmonary Aspergillus fumigatus infection. Alveolar and bronchiolar epithelial cells proved to be the source of infection-induced S100B. Extracellularly, nanomolar (but not micromolar) S100B caused physical association of RAGE with TLR2 (shown by co-immunoprecipitation and in situ proximity ligation) and inhibited TLR2-driven ERK (extracellular signal-regulated kinase) and NF-kappaB (nuclear factor kappaB) signalling and survival in recruited neutrophils — a paracrine epithelium-to-neutrophil braking circuit whose loss, in RAGE-deficient mice, produced exaggerated and persistent lung inflammation. S100B was also found to bind, in a calcium-dependent manner, class B CpG oligodeoxynucleotides, mammalian DNA, and fungal RNA and DNA; intracellularly this engaged TLR3 and TLR9 through a p38/TRIF-dependent route that activated noncanonical NF-kappaB (the p100-to-p52 processing branch) and transcriptionally shut off s100b expression — so that pathogen-derived nucleic-acid sensing terminates danger signalling. Two mechanistic anomalies in this circuit were explicitly left open by the authors. First, TLR9 signalling that converges on TRIF rather than MyD88 contradicts the canonical adaptor-assignment framework; the only independent precedent is the later finding that high doses of CpG oligodeoxynucleotides stimulate a tolerogenic TLR9-TRIF-TRAF6 pathway — activating the same noncanonical NF-kappaB branch — in plasmacytoid dendritic cells (Volpi et al., 2013), where the molecular basis of adaptor choice likewise remained unresolved. In the epithelial S100B-DNA context the molecular basis was stated to be under investigation; the authors proposed, by analogy with dendritic-cell work showing that DNA ligand structure controls subcellular localization and downstream adaptor usage, that S100B might change the intracellular compartmentalization of the bound DNA. Second, how the S100B-induced physical RAGE-TLR2 association actually prevents TLR2 signalling is unknown; the authors sketched, without evidence, a dynamic model in which S100B changes the orientation or conformation of TLR2 ligands (whose positioning on the TLR2 surface is known to control signalling; Kajava and Vasselon, 2010) or allosterically interferes with assembly of the signal-inducing ternary receptor complex. They further predicted an inherent autoimmunity risk from S100B chaperoning of self nucleic acids, and suggested that RAGE splice isoforms might underlie individual differences in the clinical course of invasive aspergillosis.

Problem Statement

Determine the causal molecular mechanisms by which the alarmin S100B rewires Toll-like receptor signalling through RAGE, as observed by Sorci et al. (2011) in epithelial cells and neutrophils during pulmonary Aspergillus fumigatus infection. Concretely, an answer must establish: (1) Adaptor choice by TLR9 — identify the molecular determinants that make TLR9 signalling on S100B-bound DNA in epithelial cells converge on the TRIF adaptor and noncanonical NF-kappaB instead of the canonical MyD88 route (candidate determinants include the structure or conformation of the S100B-DNA complex, the composition of the receptor-ligand assembly, and the intracellular compartment or trafficking route of the complex), and demonstrate causality by perturbing the determinant(s) demonstrated to be causal; (2) Inhibition of TLR2 by RAGE — identify the biophysical mechanism by which S100B-driven physical association of RAGE with TLR2 prevents TLR2 signalling, distinguishing among candidate mechanisms such as altered ligand orientation or conformation, altered receptor conformation, and prevention of assembly of the signal-inducing ternary receptor complex, and demonstrate causality by structure-guided or mutational perturbation; (3) Nucleic-acid trafficking and its immunological consequence — determine whether S100B binding changes the intracellular compartmentalization and sorting of pathogen- and host-derived nucleic acids in epithelial cells in a way that redirects TLR (TLR9 and TLR3) adaptor recruitment, and test the authors' predicted consequence that S100B-mediated chaperoning of self nucleic acids carries an inherent risk of autoimmunity, assessed by a defined readout of self-nucleic-acid-driven TLR9/RAGE activation or of loss of self-tolerance. In addition to the mandatory requirements (1)-(3), a complete answer must account for the load-bearing original observations, each of which underpins the requirement(s) noted: calcium-dependent binding of S100B to class B CpG oligodeoxynucleotides, mammalian DNA, and fungal RNA/DNA (underpins (1) and (3)); inhibition of TLR2 signalling at nanomolar but not micromolar S100B (underpins (2)); RAGE-dependence of the TLR2 inhibition in neutrophils (underpins (2)); and TRIF/noncanonical-NF-kappaB-dependence of the transcriptional shut-off of s100b expression (underpins (1)). The problem does not presuppose that the original mechanistic interpretation is correct: a systematic, controlled demonstration that one or more of the load-bearing observations named above does not replicate, with assay-competence controls, is also an admissible answer, discharging the requirement(s) listed against each contested observation and nothing else, while every requirement not so discharged stays mandatory.

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