Background/Purpose: Endosomal nucleic acid-sensing toll-like receptors 3, 7 and 9 are key innate immune sensors of dsRNA, ssRNA and ssDNA, respectively, whose activities must be tightly regulated to prevent systemic autoimmune or autoinflammatory disease (TLR7, TLR9) or virus-associated immunopathology (TLR3). The 12-pass transmembrane protein UNC93B1 chaperones nascent TLR3, TLR7 and TLR9 molecules from the ER to endosomes that support ligand recognition and signaling. Release from UNC93B1 is required for TLR3 and TLR9 signaling, whereas a persistent association between UNC93B1 and TLR7 is important for limiting TLR7 responses to endogenous nucleic acids.

Methods: Here we undertook a systematic scanning mutagenesis screen of all cytosolic and endosomal loops of UNC93B1 in murine RAW macrophages, totaling 93 mutated single or triplet amino acid residues.

Results: We identified both negative and positive regulatory regions – in particular, 21, 31 and 17 of these mutations yielded a 2-fold or higher enhancement of TNFa production downstream of TLR3, TLR7 or TLR9, respectively. The number and effect size of negative regulatory UNC93B1 mutations was noted to be highest for TLR7 responses.

We were subsequently contacted by several groups of clinicians who had identified patients with coding mutations in UNC93B1, including: (i) a family of 3 adolescent siblings and with pediatric onset isolated cutaneous tumid lupus, harboring a heterozygous T93I mutation; (ii) an adolescent girl with pediatric onset extended oligoarticular JIA, chillblain and tumid lupus rashes and an ataxic/dystonic movement disorder with CNS basal ganglia calcifications reminiscent of Aicardis-Goutieres syndrome, harboring a heterozygous R336C mutation not present in either of her parents; and (iii) a young man diagnosed at age 10 with SLE complicated by aggressive class IV nephritis, harboring a homozygous G590E mutation. When introduced in to human THP-1 monocytes and murine RAW macrophages, these mutations enhanced cytokine responses to TLR7 (expressed in mouse and human cells), TLR8 (human cells only), and to a lesser extent TLR3 (mouse cells only) stimulation, phenocopying mutants in the same UNC93B1 regions from our original screen. Knock-in mice have been generated for all three human variant alleles, and to date analysis of Unc93b1^(R336C) mice has revealed a 30% reduction in body weight, splenomegaly, expansion of Age-Associated B cells and plasma cells, monocytes and neutrophils, and titers of serum ANA IgG comparable to TLR7.1 transgenic animals.

Conclusion: Altogether our results implicate the UNC93B1-TLR7/8 axis in human monogenic autoimmune disease and provide a functional resource and analysis pipeline to assess the impact of yet-to-be-reported UNC93B1 mutations.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mutational-analysis-of-unc93b1-identifies-regulatory-regions-mutated-in-human-sle/