Species-Specific Minimal Sequence Motif for Oligodeoxyribonucleotides Activating Mouse TLR9

Abstract

Synthetic oligodeoxyribonucleotides (ODNs) containing unmethylated CpG recapitulate the activation of TLR9 by microbial DNA. ODNs are potent stimulators of the immune response in cells expressing TLR9. Despite extensive use of mice as experimental animals in basic and applied immunological research, the key sequence determinants that govern the activation of mouse TLR9 by ODNs have not been well defined. We performed a systematic investigation of the sequence motif of B class phosphodiester ODNs to identify the sequence properties that govern mouse TLR9 activation. In contrast to ODNs activating human TLR9, where the minimal sequence motif for the receptor activation comprises a pair of closely positioned CpGs we found that the mouse TLR9 requires a single CpG positioned 4–6 nt from the 5′-end. Activation is augmented by a 5′TCC sequence one to three nucleotides from the CG. The distance of the CG dinucleotide of four to six nucleotides from the 5′-end and the ODN’s length fine-tunes activation of mouse macrophages. Length of the ODN <23 and >29 nt decreases activation of dendritic cells. The ODNs with minimal sequence induce Th1-type cytokine synthesis in dendritic cells and confirm the expression of cell surface markers in B cells. Identification of the minimal sequence provides an insight into the sequence selectivity of mouse TLR9 and points to the differences in the receptor selectivity between species probably as a result of differences in the receptor binding sites.

Structural basis for species‐specific activation of mouse Toll‐like receptor 9

“…(B) NF ‐κB‐dependent luciferase reporter assay for mouse TLR 9 mutants in HEK 293T cells. Cells were stimulated with the CpG oligonucleotide TCGTTTCGTTTTTTTTTTTTTTT . Error bars represent the standard deviation of three independent wells.…”

Protective microbiomes can limit the evolution of host pathogen defense

“…We capture the impacts of pathogen and commensal microbiota on fitness through a simple mapping from their abundance to host survival (rather than developing a complete dynamical description of the interactions between pathogens, commensal microbiota, and host immunity; e.g., as in Leung and Weitz 2019). To capture the discrimination problem faced by innate immunity, we assume that pathogens and commensal microbiota can be reflected as being distributed along a single axis, representing a range from a "small" to a "large" signal detectable by the immune system, which might reflect aspects such as the number of CpG repeats (detectable by Toll-like-receptor 9; Pohar et al 2015), immunogenically variable aspects of flagellin (Felix et al 1999;Trdá et al 2014), or a combined signal integrating presence of highly conserved microbe-associated molecules with functional characteristics indicative of pathogen's presence (e.g., via NBS-LRR proteins in plants, DeYoung and Innes 2006; and signatures of "stress" or deviation from homeostasis more broadly, Chovatiya and Medzhitov 2014). From this, we develop a model framework constructed around core principles from epidemiology to better understand the drivers of the evolution of host defense in the context of pathogen-microbiome interactions.…”

“…To capture the discrimination problem faced by innate immunity, we assume that pathogens and commensal microbiota can be reflected as being distributed along a single axis, representing a range from a “small” to a “large” signal detectable by the immune system, which might reflect aspects such as the number of CpG repeats (detectable by Toll‐like‐receptor 9; Pohar et al. ), immunogenically variable aspects of flagellin (Felix et al. ; Trdá et al.…”

The Role of Nucleic Acid Sensing in Controlling Microbial and Autoimmune Disorders

“…Additionally, TLR9 detects ssDNA of at least 21 nucleotides, and methylated ssDNA or dsDNA only weakly activate TLR9 (Pohar et al, 2015a,b). However, the addition of oligonucleotides as short as two nucleotides augments TLR activation (Pohar et al, 2017).…”