Introduction
Systemic lupus erythematosus (SLE) describes a heterogeneous set of clinical phenotypes associated with type I interferon (IFN) signaling upregulation and the presence of autoantibodies targeting nuclear autoantigens [
1]. Familial aggregation and higher concordance rates between monozygotic versus dizygotic twins suggest a major hereditary component, with rare monogenic forms of SLE providing important insights into disease pathogenesis [
2,
3]. Expressed in sentinel cells such as macrophages and dendritic cells, Toll-like receptors (TLRs) are a family of single-pass membrane-spanning proteins that engage structurally conserved microbial features as part of a coordinated innate and adaptive immune response to pathogens. Endosomal TLR3, TLR7/8, and TLR9 traffic from the ER to the endosomal compartment where they sense nucleic acids, respectively, dsRNA, ssRNA, and CpG DNA. Inherited TLR3 deficiency underlies herpes simplex encephalitis [
4] and severe influenza or COVID-19 pneumonia [
5], and inherited TLR7 deficiency predisposes to critical COVID-19 pneumonia [
6]. Importantly, TLR7/8 and TLR9 can signal upon sensing both viral- and self-derived nucleic acid [
7]. Reflective of the latter situation, disease in murine models of SLE is attenuated in animals deficient for TLR7 [
8], while SLE-like pathology is driven by TLR7 overexpression [
9]. Further, gain-of-function (GOF) mutations in human TLR7 were recently identified to cause severe autoimmune phenotypes, specifically, SLE and neuromyelitis optica [
10]. These observations indicate the importance of endosomal TLR signaling in immunological homeostasis. Here, we describe two novel mutations in TLR7, causing a spectrum of systemic and neuro-inflammatory diseases.
Primers Used for Site-Directed Mutagenesis of TLR7
F507S | TAG TAT ATT TTC TGT CAA GTC CTC | TTT TTA CTT AGA TCC AAG GTC |
F507L | AGT ATA TTT TTG GTC AAG TCC TC | ATT TTT ACT TAG ATC CAA GGT C |
L528I | TGT CAG GAA ATA TCA TTA GCC AAA C | GAT TCA GGC ATT TGA GGA AAG |
Luciferase Reporter Assay
For each condition, 2.104 HEK293T cells per well were plated in a 96-well plate in duplicate and co-transfected with a plasmid containing the Firefly luciferase gene under the control of the human NF-κB promoter (pGL4.32, Promega), a plasmid constitutively expressing the Renilla luciferase gene for normalization (pRL-SV40, Promega), as well as a plasmid encoding WT, empty vector (EV) or variant TLR7 and a plasmid encoding UNC93B1, using the TransIT®-293 Transfection Reagent (Mirus, # MIR2700) according to the manufacturer’s instructions. After incubation for 24 h, cells were left either unstimulated or stimulated with the TLR7 agonist R848 0.01, 0.1, and 1 µg/mL for 24 h. Cells were then lysed, and luciferase levels were measured with the Dual-Luciferase® Reporter assay system (Promega, #E1980) according to the manufacturer’s protocol. Luminescence intensity was acquired on an Infinite® F200 Pro microplate reader (TECAN). Firefly luciferase activity values were normalized against Renilla luciferase activity before further data processing (see figure legend).
Discussion
TLRs are type I transmembrane proteins with an extracellular LRR domain, a single transmembrane helix, and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain [
18]. On recognition of ligand by the LRR domain, inactive monomeric or preformed dimeric TLRs are induced to form a face-to-face activated dimer [
19]. In this way, TIR domains are brought into proximity with each other, thereby recruiting downstream adaptor proteins to initiate transcriptionally-driven immune responses. TLR7 contains two distinct ligand-binding sites, recognizing ssRNAs in the form of degradation products, nucleosides, and oligoribonucleotides [
20]. Site 1, which is highly conserved between TLR7 and TLR8, recognizes both nucleosides (guanosine for TLR7, uridine for TLR8) and nucleoside analogs and is essential for receptor dimerization. In contrast, site 2 is not conserved, is spatially distinct between TLR7 and TLR8, and appears to play an auxiliary role in receptor dimerization by enhancing the binding affinities of site 1 ligands.
Here, we describe two novel mutations in TLR7, F507S and L528I. While the L528I mutation is predicted to impact intermolecular contacts at the TLR7 dimer interface, this does not necessarily mean that dimerization will be completely disrupted. Notably, there is considerable variability in the precise orientation of the TLR7 dimer in the available experimental structures, suggesting that it might be robust to a single amino acid change. Comparable to the F507L mutation described by Brown et al. [
10], the F507S substitution is predicted to have a minimal effect on monomeric and dimeric TLR7 structures. Neither of the L528 and F507 residues are in direct contact with the guanosine (or other RNA ligand) binding site in any structure. Brown et al. described a Y264H substitution to map to ligand-binding site 1 [
10], suggesting an enhanced affinity of the Y264H for guanosine. However, since Y264 lies at the TLR7 dimer interface, it remains possible that this substitution might also play a role in homo-dimerization. We highlight this point by noting that four (F507L, Y264H, F507S, L528I) of the five mutations in human TLR7 described to date, not including R28G (possibly a signal peptide-related variant: Brown et al. [
10]), occur directly at the homodimer interface in multiple structures. Of possible further interest, although TLR7-TLR7 molecules are in contact with one another, no noticeable interactions (hydrogen bond, intimate contact, etc.), that might be important in dimer stabilization, were observed in the structure reported by Ishida et al. [
17], indicating that dimerization is loosely achieved (and perhaps partly explaining why the isolated endosomal domain of TLR7 mainly exists as a monomer in solution [
21]). All told, these observations suggest the importance of the TLR7 interface in maintaining immune homeostasis, where we predict that altered homo-dimerization enhances TLR7 signaling. It will be interesting to map the position of further mutations in human TLR7 as they are reported.
While GOF mutations in TLR7 can result in a phenotype consistent with SLE, our data emphasize a broader spectrum of disease, including significant neurological involvement. Thus, the child with a de novo Y264H mutation described by Brown et al. [
10] demonstrated recurrent hemichorea requiring treatment with haloperidol (Table
S2), and in a second case reported by the same authors, a child with a maternally inherited F507L mutation, the phenotype was exclusively neurological, consistent with neuromyelitis optica and positive for AQP4 autoantibodies (a phenomenon recognized in the context of enhanced type I interferon signaling [
22]). In our series of four affected patients, we observed cerebral vasculitis and cerebral calcification in the female proband in family AGS571 and developmental delay, epilepsy, and marked intracranial calcification in her brother. Further, white matter disease, atrophy, and calcification were seen in the proband in family AGS3740, which was progressive despite an apparently excellent response of life-threatening immune cytopenias to HSCT.
Finally, given that the TLR7 gene is situated on the X chromosome, the observation of a severely affected male in our series is notable, with all other cases so far described being female (and where variable levels of X-inactivation might affect the clinical phenotype).
Acknowledgements
Y.J.C. would like to thank Maggie MacDonald for assistance with referencing. C.D. is supported by the Fondation pour la Recherche Médicale (Grant: FDM202106013329). M.Z. acknowledges the Italian Ministry of Heath: Ricerca Corrente 08045818. Y.J.C. acknowledges the European Research Council (786142 E-T1IFNs), a UK Medical Research Council Human Genetics Unit core grant (MC_UU_00035/11), a state subsidy from the Agence Nationale de la Recherche (France) under the ‘Investissements d’avenir’ program bearing the reference ANR-10-IAHU-01, and the ANR T1-UNC project grant (ANR-23-CE15-0015-02). A.I. wishes to acknowledge work generated within the European Reference Network for immunodeficiencies, autoinflammatory and autoimmune diseases (ERN-RITA). S.O. thanks Dr Riccardo Castagnoli for valuable clinical discussions relating to AGS3740.
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