Rare variants in Toll-like receptor 7 results in functional impairment and downregulation of cytokine-mediated signaling in COVID-19 patients

Abstract

Toll-like receptors (TLR) are crucial components in the initiation of innate immune responses to a variety of pathogens, triggering the production of pro-inflammatory cytokines and type I and II interferons, which are responsible for innate antiviral responses. Among the different TLRs, TLR7 recognizes several single-stranded RNA viruses including SARS-CoV-2. We and others identified rare loss-of-function variants in X-chromosomal TLR7 in young men with severe COVID-19 and with no prior history of major chronic diseases, that were associated with impaired TLR7 signaling as well as type I and II IFN responses. Here, we performed RNA sequencing to investigate transcriptome variations following imiquimod stimulation of peripheral blood mononuclear cells isolated from patients carrying previously identified hypomorphic, hypofunctional, and loss-of-function TLR7 variants. Our investigation revealed a profound impairment of the TLR7 pathway in patients carrying loss-of-function variants. Of note, a failure in IFNγ upregulation following stimulation was also observed in cells harboring the hypofunctional and hypomorphic variants. We also identified new TLR7 variants in severely affected male patients for which a functional characterization of the TLR7 pathway was performed demonstrating a decrease in mRNA levels in the IFNα, IFNγ, RSAD2, ACOD1, IFIT2, and CXCL10 genes.

Fig. 1. DEGs in PBMC from HDs and patients carrying TLR7 variants stimulated with IMQ.

A–C Volcano plots showing DEGs. Red dots show upregulated genes (log2FC ≥ 1.5 with adjusted p value ≤ 0.05) and blue dots represent downregulated genes (log2FC ≤ −1,5; adjusted p value ≤ 0.05). The DEGs with the top 50 absolute FC are reported. A Three healthy donors. B Patients (n = 3) carrying the Ala288Val, Ala488Val, and Val219 Ile variants. C Patients carrying Ala1032Thr (n = 1) and Ser301Pro (n = 1) variants. Gene Ontology biological process terms significantly overrepresented among the genes increased by IMQ are shown in the lower panel in (A, B, D). Heatmap of logCPM values for the top DEGs in HDs and patients carrying TLR7 variants after IMQ stimulation. IMQ imiquimod, NS non stimulated.

Fig. 2. Gene expression analysis in PBMC of patients carrying TLR7 variants stimulated with IMQ.

A Pedigree of P6 (A upper panel) and P10 (A lower panel) shows the segregation of the variant within the family. Squares represent male family members; circles, females. Black symbols indicate individuals harboring the TLR7 variant. Individuals infected by SARS-CoV-2 are indicated by a virus symbol () close to the individual symbol. B–G PBMC from COVID-19 patients and four unaffected male controls (HDs) were stimulated with IMQ at 5 μg/mL or cell culture medium. A quantitative PCR assay was performed and 2^−ΔΔCt was calculated using HPRT1 as the housekeeping gene. Fold change in mRNA expression of genes induced by IMQ with respect to cell culture medium was calculated: *p < 0.05; **p < 0.01; ***p < 0.001.