Background Acute respiratory viral infections cause significant morbidity, especially in vulnerable individuals and is a topic of immense significance during the current COVID-19 global pandemic. Respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD) involve inflammation of the airways and viruses are a major cause of exacerbations. Whilst new biological drugs targeting allergic and eosinophilic immune pathways have been developed for allergic asthma, therapeutic options for treating asthma exacerbations remain limited and rely on decades-old blunt tools such as oral corticosteroids. Development of new therapies and vaccines to combat viral respiratory tract infections is slow, partly because of the limited understanding of innate immune responses at the respiratory mucosal site of disease. Detailed characterization of such responses can facilitate biomarker definition for respiratory diseases, providing mechanistic insights and a platform for the testing of novel therapeutics.
Our understanding of the role of innate immunity to infection has recently undergone a paradigm shift as it is now recognized that innate cells (and not just adaptive immune cells) have the capacity to undergo "training" after infection via metabolic changes and epigenetic reprogramming. Pathogens can therefore modulate chromatin structure and the transcriptional programme of innate immune cells, which can result in altered but non-specific responses upon reinfection either with the same or heterologous stimuli. These epigenetic mechanisms may be particularly relevant in understanding how viral triggers (e.g. influenza) can lead to increased susceptibility to bacterial infections and cause pneumonia (e.g. due to streptococcus spp.).
Most studies have focused on peripheral immune cells such as macrophages, but the concept of innate immune training has been extended to long-lived non-immune skin epithelial stem cells, which have the capacity for "inflammatory memory". Allergic inflammatory memory has also recently been established in respiratory progenitor cells. Murine studies have highlighted the potential for structural cells (such as epithelial cells) to have the potential to mount immune responses. However, mechanisms of innate immune "memory" in human respiratory mucosal epithelial and immune cells in response to microbes and pathogen associated molecular patterns (PAMPs) are not well understood despite the respiratory tract being under a constant barrage of infectious stimuli.
Toll-like receptors (TLRs) play a critical role in the initial detection of microbes and PAMPs. TLR7/8 specifically detects RNA viruses such as influenza, rhinovirus, and coronavirus. Airway epithelial and immune cells express TLR7/8 and are therefore vital in mounting the innate immune response to viral infections, as well as helping shape adaptive immunity. Resiquimod (R848) is a single-stranded RNA viral analogue that binds to TLR7/8. When given systemically to treat human hepatitis C infection, it causes cytokine release and a flu-like syndrome. However, R848 has been successfully employed topically as a skin cream for actinic keratosis. TLR7 agonists have been developed for therapeutic use by repeated dose intranasal administration to target allergic inflammation in healthy volunteers and those with allergic rhinitis.
Rationale for current study Intranasal R848 administration has now been established in a human challenge model in healthy volunteers and in those with allergic rhinitis (AR) and allergic rhinitis with asthma (ARwA) by the chief investigator. In total, 44 volunteers have received intranasal R848 at a range of doses; 9 healthy volunteers received 10µg per nostril (n=9) with one person receiving 100µg per nostril (n=1); subsequently 35 volunteers received between 1-2µg per nostril based on weight (0.02µg/kg, healthy n=12, AR n=12, ARwA n=11). This dose was well tolerated by all volunteers with minimal nasal symptoms and no significant change in airflow obstruction (FEV1) in volunteers with asthma. Intranasal R848 was well tolerated systemically with no change in physiological measures or serum inflammatory mediators compared to saline challenge. The study demonstrated that individuals with AR and ARwA have increased nasal mucosal interferon and chemokine responses compared to healthy volunteers. This is coupled with the observation that there is a small and transient reduction in peripheral eosinophil counts at 4 hours and in lymphocyte counts at 24 hours, a finding limited to volunteers with AR and ARwA. This suggests that leukocyte trafficking may be occurring from the peripheral blood to the nasal mucosa. These findings highlight that dysregulated mucosal innate immune responses are likely to be important in determining the clinical outcome of viral triggers.
R848 nasal challenge (up to 2µg per nostril) is therefore a practical, tolerable, and non-invasive method to stimulate and investigate TLR-driven respiratory mucosal innate immune responses in both health and disease.
Defining the cellular basis for TLR-driven innate immunity will help provide mechanistic insights into how these responses differ between individuals, its dysregulation in disease (such as during an asthma exacerbation) and susceptibility to secondary infections. Understanding the epigenetic mechanisms which regulate interferon and chemokine production may help identify the molecular drivers of excessive or dampened inflammation, helping to refine potential therapeutic targets. Given the current COVID-19 pandemic, the clinical translational model utilised in this study allows for a practical and straightforward method to assess the host factors which determine respiratory mucosal innate immunity.
The goal of this project is to utilize an established tolerable human nasal TLR agonist challenge model combined with single-cell transcriptomic and epigenetic techniques to precisely characterize innate immune responses and mechanisms of innate immune training in airway epithelial and immune cells. Samples will be collected from the nasal mucosa and blood before and after saline and R848 challenge. Cell cultures from airway epithelium and blood will also be established to assess the functional impact of R848 on cellular response to repeat stimulation with viral and bacterial ligands. Study populations will include healthy participants and those with allergic rhinitis with/without asthma in order to investigate how in-vivo innate immunity is altered in these populations. Some participants will also undergo repeat nasal challenge to assess mechanisms of innate immune memory in the respiratory tract.
