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

Sandra Pålsson,Joseph Bergenstråhle,Vanessa Contreras,Roger Legrand,Aleksandra Dondalska,Joakim Lundeberg,Peter Järver,Candice Poux,John S Tregoning,Anna-Lena Spetz,Claudia Arasa,Jan Albert,David C Busse

doi:10.3389/fimmu.2019.02161

Abstract

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.

Highlights

According to new estimates by the United States Centers for Disease Control and Prevention (US-CDC), the World Health Organization and global health partners, seasonal influenza infections annually cause up to 650.000 deaths worldwide
As expected, based on the viability results, we predominantly found influenza A H1N1 (IAV) IAV nucleoprotein (NP) staining in live Monocyte-derived dendritic cells (DCs) (MoDC) at 24 h p.i. (Figure 1F), while there was an accumulation of IAV NP+ cells among dead cells 48 h p.i. (Figure 1G)
As we recently showed that single-stranded oligonucleotides (ssON) with the length of 25–35 bases inhibit both clathrin mediated endocytosis (CME) and Caveolin-Dependent Endocytosis (CDE) in MoDC, we here sought to investigate ssON’s capacity to limit IAV infection

Summary

Introduction

According to new estimates by the United States Centers for Disease Control and Prevention (US-CDC), the World Health Organization and global health partners (www.who.int), seasonal influenza infections annually cause up to 650.000 deaths worldwide. The annual influenza vaccines incorporate variants of influenza A(H1N1), A(H3N2), B/Yamagata and B/Victoria, which WHO predicts will dominate the following season. Influenza viruses pose a constant threat of human pandemics due to the risk of transmission of new variants from animals for which we currently lack appropriate vaccines [1, 2]. There is a pressing need to develop treatments targeting key steps in the life cycle utilized by many influenza viruses, thereby increasing the likelihood to achieve broad anti-viral activity [3]. The pivotal step of viral entry into cells, may be an Achilles’ heel accessible for novel broad antiviral compounds as it is shared by many viruses after binding to cellular receptors. Due to the notion that IAV utilizes cellular endocytic machinery for viral entry, we here assessed ssON’s capacity to affect IAV infection

Methods

Results

Conclusion