Decoding the Double-Edged Sword: Novel TLR7 Mutations Unveil a Spectrum of Autoimmune and Neurological Disorders

Toll-like receptor 7 (TLR7) protein plays a crucial role in the immune system, particularly in the innate immune response. TLR7 is part of the Toll-like receptor family, which is known for recognizing structurally conserved molecules derived from microbes. These receptors are essential for the early detection of invading pathogens and for initiating an immune response. It is particularly sensitive to single-stranded RNA (ssRNA) from viruses, allowing the immune system to detect viral infections. Upon recognizing ssRNA, TLR7 activates signaling pathways that lead to the production of type I interferons and other pro-inflammatory cytokines. These molecules help to establish an antiviral state in the cells and alert the immune system to the presence of a viral invader. It is expressed in various immune cells, including dendritic cells, macrophages, and B cells. Its activation and subsequent signaling pathways play a critical role in antiviral defense, the modulation of immune responses, and the development of adaptive immunity. Due to its involvement in immune responses, TLR7 is also a subject of research for its potential roles in autoimmune diseases, where the immune system mistakenly targets the body’s own cells, and in the development of therapeutics aimed at modulating immune responses for various conditions.

The recent study published in the Journal of Clinical Immunology by Professors Yanick Crow and Joe Marsh, alongside a comprehensive team of researchers from the University of Edinburgh, investigated the role of TLR7 in the pathogenesis of systemic and neuro-inflammatory diseases. The authors identified two novel mutations in TLR7, underscores the importance of innate immune signaling pathways in maintaining immunological homeostasis and their implications in the development of autoimmune and inflammatory disorders. They conducted a series of comprehensive experiments to investigate the functional implications of two novel mutations in TLR7 identified as F507S and L528I. These mutations were explored in the context of their roles in systemic lupus erythematosus (SLE)-like diseases and neurological involvement. The research team obtained blood samples from probands and their parents with informed consent. DNA was extracted using standard protocols. Two different families (AGS571 and AGS3740) were studied using high-throughput sequencing techniques: For family AGS571, a custom panel encompassing around 8000 disease-causing genes was used for library preparation, followed by sequencing on a NovaSeq6000 platform. For family AGS3740, exome sequencing was performed using a SureSelect Human All Exon Kit for targeted enrichment and sequencing on an Illumina HiSeq2000 platform.

The authors analyzed the identified variants using in silico tools such as SIFT and PolyPhen2, with allele frequencies checked against the gnomAD database. Sanger sequencing confirmed the TLR7 variants. The researchers looked for TLR7 structures with >90% homology over at least 50 amino acids in the Protein Data Bank. Using FoldX 5.0, they estimated the change in Gibbs free energy (ΔΔG) due to the mutations, indicating their impact on protein stability and dimerization. Structures were visualized using PyMOL to understand the mutations’ placement and potential impact on TLR7 function.

The team cultured human embryonic kidney (HEK) 293 T cells and used for transfection experiments due to their lack of endogenous TLRs. They cloned wild-type TLR7   into a vector, and mutant TLR7 plasmids (F507S and L528I) were generated via site-directed mutagenesis. A vector encoding human UNC93B1, crucial for TLR7 function, was also used. Cells were co-transfected with NF-κB promoter-driven Firefly luciferase plasmid, Renilla luciferase for normalization, and either wild-type or mutant TLR7 plasmids. Post-transfection, cells were stimulated with the TLR7 agonist R848, and luciferase activity was measured to assess NF-κB activation indicative of TLR7 signaling.

The researchers found the F507S mutation in three related individuals from family AGS571, and the L528I mutation was identified as a de novo mutation in the proband from family AGS3740. Both F507S and L528I mutations were characterized as gain-of-function, enhancing TLR7 signaling in response to agonist stimulation compared to wild-type TLR7. This was evident from the increased NF-κB-driven luciferase activity in the cell culture experiments. Protein modeling suggested that these mutations, particularly L528I, could disrupt TLR7 dimerization, a critical step for its activation. This is consistent with the gain-of-function behavior observed in the luciferase assays.

The study’s findings highlight the critical role of TLR7 dimerization in immune homeostasis and suggest that alterations in this process can lead to autoimmune and neuro-inflammatory diseases. The experiments successfully linked the novel TLR7 mutations to enhanced receptor signaling, providing a molecular basis for the observed clinical phenotypes and expanding the understanding of TLR7’s role in disease pathogenesis.  The clinical manifestations observed in the study’s subjects, ranging from severe neurological deficits to systemic autoimmune responses, underscore the diverse impact of TLR7 dysregulation. The cases presented in the study, including a severely affected male, challenge the previously held notion that TLR7-associated diseases predominantly affect females, given the gene’s location on the X chromosome. This research advances our understanding of the genetic and molecular mechanisms underlying autoimmune and inflammatory diseases, highlighting the critical role of TLR7 and the innate immune system in disease pathogenesis. The identification of novel TLR7 mutations and their functional characterization provide valuable insights into the complex interplay between genetic factors and immune responses, paving the way for the development of targeted therapeutic interventions. In conclusion, the study by Professors Crow, Marsh, and their colleagues represents a significant contribution to the field of clinical immunology, offering new perspectives on the role of TLR7 in autoimmunity and inflammation. The findings emphasize the need for ongoing research into the genetic underpinnings of immune system dysregulation, with the ultimate goal of improving the diagnosis, management, and treatment of autoimmune and inflammatory disorders.