A non-coding single-stranded oligonucleotide (ssON), 35 nucleotides in length, has demonstrated promising immunomodulatory effects in vivo, including in the skin of non-human primates, as previously reported by Järver et al. Mechanistically, extracellularly added ssON inhibits the endocytic uptake of Toll-like receptor (TLR) ligands in monocyte-derived dendritic cells (moDC), thereby transiently suppressing endosomal TLR3/4/7 signalling. However, a comprehensive understanding of its mechanism of action remained incomplete, particularly regarding the cellular mediators involved in ssON-induced immune regulation in the skin.

To address this, we first investigated the immune cell populations recruited following local injection of the 35-nucleotide long ssON in mice. In Paper I, repeated injections in mice resulted in a significant local influx of CD11b⁺ Ly6C⁺ monocytic cells to the site of injection. Transcriptomic and flow cytometric analyses revealed that ssON treatment induced the expression of checkpoint proteins PD-L1 and ILT3 on dermal CD11b⁺ subsets. Functional assays and cytokine profiling further showed that ssON promoted the development of monocytic-myeloid derived suppressor-like cells (M-MDSC), which suppressed Th1, Th2, and Th9 responses in vitro and attenuated Th17-driven inflammation in an imiquimod-induced psoriasis model in vivo, while also supporting Treg induction in vitro.

To evaluate relevance in human systems, Paper II examined the effects of ssON on human moDCs. ssON exposure during differentiation altered the process and resulted in upregulation of both CD1a and PD-L1. Furthermore, these ssON-conditioned moDCs exhibited impaired maturation, characterized by reduced expression of MHC II and CD86 and diminished pro-inflammatory cytokine production upon LPS stimulation. Transcriptomic profiling indicated a tolerogenic phenotype dependent on activation of the transcription factor PPARγ. These findings are consistent with the murine data, where ssON-induced skin-infiltrating cells similarly displayed tolerogenic properties.

Given that monocytes are key precursors of dermal macrophages, Paper III assessed whether ssON priming influences macrophage polarization. Transcriptomic, phenotypic, and cytokine analyses revealed that ssON priming altered both M1- and M2-like macrophage profiles, notably increasing expression of the anti-inflammatory cytokine IL1RN. Following IL-4-induced polarization, ssON-primed macrophages showed enhanced production of IL-1RA and IL-10, along with reduced expression of HLA-DR and CD86.

Finally, in Paper IV, we investigated the effects of ssON on keratinocytes (KCs), the predominant cell type in the epidermis. ssON treatment attenuated poly I:C-induced inflammatory transcriptional and cytokine responses in KCs. Moreover, Staphylococcus enterotoxin B (SEB) loaded KCs stimulated with poly I:C in the presence of ssON, exhibited reduced T cell stimulatory capacity compared to poly I:C stimulated KCs.

In summary, this thesis demonstrates that ssON exerts immunomodulatory effects in the skin, promoting tolerogenic immune responses across multiple cell types in vitro and in vivo.