Is There a Role for Immunoregulatory and Antiviral Oligonucleotides Acting in the Extracellular Space? A Review and Hypothesis.

Here, we link approved and emerging nucleic acid-based therapies with the expanding universe of small non-coding RNAs (sncRNAs) and the innate immune responses that sense oligonucleotides taken up into endosomes. The Toll-like receptors (TLRs) 3, 7, 8, and 9 are located in endosomes and can detect nucleic acids taken up through endocytic routes. These receptors are key triggers in the defense against viruses and/or bacterial infections, yet they also constitute an Achilles heel towards the discrimination between self- and pathogenic nucleic acids. The compartmentalization of nucleic acids and the activity of nucleases are key components in avoiding autoimmune reactions against nucleic acids, but we still lack knowledge on the plethora of nucleic acids that might be released into the extracellular space upon infections, inflammation, and other stress responses involving increased cell death. We review recent findings that a set of single-stranded oligonucleotides (length of 25-40 nucleotides (nt)) can temporarily block ligands destined for endosomes expressing TLRs in human monocyte-derived dendritic cells. We discuss knowledge gaps and highlight the existence of a pool of RNA with an approximate length of 30-40 nt that may still have unappreciated regulatory functions in physiology and in the defense against viruses as gatekeepers of endosomal uptake through certain routes.

A Non-Coding Oligonucleotide Recruits Cutaneous CD11b+ Cells that Inhibit Thelper Responses and Promote Tregs.

Skin-resident antigen-presenting cells (APC) play an important role in maintaining peripheral tolerance via immune checkpoint proteins and induction of T regulatory cells (Tregs). However, there is a lack of knowledge on how to expand or recruit immunoregulatory cutaneous cells without causing inflammation. Here, it is shown that administration of a non-coding single-stranded oligonucleotide (ssON) leads to CCR2-dependent accumulation of CD45+CD11b+Ly6C+ cells in the skin that express substantial levels of PD-L1 and ILT3. Transcriptomic analyses of skin biopsies reveal the upregulation of key immunosuppressive genes after ssON administration. Functionally, the cutaneous CD11b+ cells inhibit Th1/2/9 responses and promote the induction of CD4+FoxP3+ T-cells. In addition, ssON treatment of imiquimod-induced inflammation results in significantly reduced Th17 responses. It is also shown that induction of IL-10 production in the presence of cutaneous CD11b+ cells isolated after ssON administrations is partly PD-L1 dependent. Altogether, an immunomodulatory ssON is identified that can be used therapeutically to recruit cutaneous CD11b+ cells with the capacity to dampen Th cells.

Amelioration of Compound 48/80-Mediated Itch and LL-37-Induced Inflammation by a Single-Stranded Oligonucleotide.

Numerous inflammatory skin disorders display a high prevalence of itch. The Mas-related G protein coupled receptor X2 (MRGPRX2) has been shown to modulate itch by inducing non-IgE-mediated mast cell degranulation and the release of endogenous inducers of pruritus. Various substances collectively known as basic secretagogues, which include inflammatory peptides and certain drugs, can trigger MRGPRX2 and thereby induce pseudo-allergic reactions characterized by histamine and protease release as well as inflammation. Here, we investigated the capacity of an immunomodulatory single-stranded oligonucleotide (ssON) to modulate IgE-independent mast cell degranulation and, more specifically, its ability to inhibit the basic secretagogues compound 48/80 (C48/80)-and LL-37 in vitro and in vivo. We examined the effect of ssON on MRGPRX2 activation in vitro by measuring degranulation in a human mast cell line (LAD2) and calcium influx in MRGPRX2-transfected HEK293 cells. To determine the effect of ssON on itch, we performed behavioral studies in established mouse models and collected skin biopsies for histological analysis. Additionally, with the use of a rosacea mouse model and RT-qPCR, we investigated the effect on ssON on LL-37-induced inflammation. We reveal that both mast cell degranulation and calcium influx in MRGPRX2 transfected HEK293 cells, induced by the antimicrobial peptide LL-37 and the basic secretagogue C48/80, are effectively inhibited by ssON in a dose-dependent manner. Further, ssON demonstrates a capability to inhibit LL-37 and C48/80 activation in vivo in two mouse models. We show that intradermal injection of ssON in mice is able to block itch induced via C48/80 in a dose-dependent manner. Histological staining revealed that ssON inhibits acute mast cell degranulation in murine skin treated with C48/80. Lastly, we show that ssON treatment ameliorates LL-37-induced inflammation in a rosacea mouse model. Since there is a need for new therapeutics targeting non-IgE-mediated activation of mast cells, ssON could be used as a prospective drug candidate to resolve itch and inflammation in certain dermatoses.

Single-Stranded Oligonucleotide-Mediated Inhibition of Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children. Currently, there is no RSV vaccine or universally accessible antiviral treatment available. Addressing the urgent need for new antiviral agents, we have investigated the capacity of a non-coding single-stranded oligonucleotide (ssON) to inhibit RSV infection. By utilizing a GFP-expressing RSV, we demonstrate that the ssON significantly reduced the proportion of RSV infected A549 cells (lung epithelial cells). Furthermore, we show that ssON's antiviral activity was length dependent and that both RNA and DNA of this class of oligonucleotides have antiviral activity. We reveal that ssON inhibited RSV infection by competing with the virus for binding to the cellular receptor nucleolin in vitro. Additionally, using a recombinant RSV that expresses luciferase we show that ssON effectively blocked RSV infection in mice. Treatment with ssON in vivo resulted in the upregulation of RSV-induced interferon stimulated genes (ISGs) such as Stat1, Stat2, Cxcl10, and Ccl2. This study highlights the possibility of using oligonucleotides as therapeutic agents against RSV infection. We demonstrate that the mechanism of action of ssON is the inhibition of viral entry in vitro, likely through the binding of the receptor, nucleolin and that ssON treatment against RSV infection in vivo additionally results in the upregulation of ISGs.

A Single-Stranded Oligonucleotide Inhibits Toll-Like Receptor 3 Activation and Reduces Influenza A (H1N1) Infection.

The initiation of an immune response is dependent on the activation and maturation of dendritic cells after sensing pathogen associated molecular patterns by pattern recognition receptors. However, the response needs to be balanced as excessive pro-inflammatory cytokine production in response to viral or stress-induced pattern recognition receptor signaling has been associated with severe influenza A virus (IAV) infection. Here, we use an inhibitor of Toll-like receptor (TLR)3, a single-stranded oligonucleotide (ssON) with the capacity to inhibit certain endocytic routes, or a TLR3 agonist (synthetic double-stranded RNA PolyI:C), to evaluate modulation of innate responses during H1N1 IAV infection. Since IAV utilizes cellular endocytic machinery for viral entry, we also assessed ssON's capacity to affect IAV infection. We first show that IAV infected human monocyte-derived dendritic cells (MoDC) were unable to up-regulate the co-stimulatory molecules CD80 and CD86 required for T cell activation. Exogenous TLR3 stimulation did not overcome the IAV-mediated inhibition of co-stimulatory molecule expression in MoDC. However, TLR3 stimulation using PolyI:C led to an augmented pro-inflammatory cytokine response. We reveal that ssON effectively inhibited PolyI:C-mediated pro-inflammatory cytokine production in MoDC, notably, ssON treatment maintained an interferon response induced by IAV infection. Accordingly, RNAseq analyses revealed robust up-regulation of interferon-stimulated genes in IAV cultures treated with ssON. We next measured reduced IAV production in MoDC treated with ssON and found a length requirement for its anti-viral activity, which overlapped with its capacity to inhibit uptake of PolyI:C. Hence, in cases wherein an overreacting TLR3 activation contributes to IAV pathogenesis, ssON can reduce this signaling pathway. Furthermore, concomitant treatment with ssON and IAV infection in mice resulted in maintained weight and reduced viral load in the lungs. Therefore, extracellular ssON provides a mechanism for immune regulation of TLR3-mediated responses and suppression of IAV infection in vitro and in vivo in mice.

The viral protein corona directs viral pathogenesis and amyloid aggregation.

Artificial nanoparticles accumulate a protein corona layer in biological fluids, which significantly influences their bioactivity. As nanosized obligate intracellular parasites, viruses share many biophysical properties with artificial nanoparticles in extracellular environments and here we show that respiratory syncytial virus (RSV) and herpes simplex virus type 1 (HSV-1) accumulate a rich and distinctive protein corona in different biological fluids. Moreover, we show that corona pre-coating differentially affects viral infectivity and immune cell activation. In addition, we demonstrate that viruses bind amyloidogenic peptides in their corona and catalyze amyloid formation via surface-assisted heterogeneous nucleation. Importantly, we show that HSV-1 catalyzes the aggregation of the amyloid ß-peptide (Aß42), a major constituent of amyloid plaques in Alzheimer's disease, in vitro and in animal models. Our results highlight the viral protein corona as an acquired structural layer that is critical for viral-host interactions and illustrate a mechanistic convergence between viral and amyloid pathologies.

Single-Stranded Nucleic Acids Regulate TLR3/4/7 Activation through Interference with Clathrin-Mediated Endocytosis.

Recognition of nucleic acids by endosomal Toll-like receptors (TLR) is essential to combat pathogens, but requires strict control to limit inflammatory responses. The mechanisms governing this tight regulation are unclear. We found that single-stranded oligonucleotides (ssON) inhibit endocytic pathways used by cargo destined for TLR3/4/7 signaling endosomes. Both ssDNA and ssRNA conferred the endocytic inhibition, it was concentration dependent, and required a certain ssON length. The ssON-mediated inhibition modulated signaling downstream of TLRs that localized within the affected endosomal pathway. We further show that injection of ssON dampens dsRNA-mediated inflammatory responses in the skin of non-human primates. These studies reveal a regulatory role for extracellular ssON in the endocytic uptake of TLR ligands and provide a mechanistic explanation of their immunomodulation. The identified ssON-mediated interference of endocytosis (SOMIE) is a regulatory process that temporarily dampens TLR3/4/7 signaling, thereby averting excessive immune responses.