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Summary The intracellular innate immune receptor NOD1 detects Gram-negative bacterial peptidoglycan (PG) to induce autophagy and inflammatory responses in host cells. To date, the intracellular compartment in which PG is detected by NOD1 and whether NOD1 directly interacts with PG are two questions that remain to be resolved. To address this, we used outer membrane vesicles (OMVs) from pathogenic bacteria as a physiological mechanism to deliver PG into the host cell cytosol. We report that OMVs induced autophagosome formation and inflammatory IL-8 responses in epithelial cells in a NOD1- and RIP2-dependent manner. PG contained within OMVs colocalized with both NOD1 and RIP2 in EEA1-positive early endosomes. Further, we provide evidence for direct interactions between NOD1 and PG. Collectively, these findings demonstrate that NOD1 detects PG within early endosomes, thereby promoting RIP2-dependent autophagy and inflammatory signaling in response to bacterial infection.

Aims Viral upper respiratory tract infections caused by rhinovirus (RV) and influenza A virus (IAV) are associated with 50–80% of asthma exacerbations. Inhaled glucocorticosteroids (GCS) are routinely used to relieve asthma symptoms, and while it has been widely reported that GCS reduce inflammation associated with viral infection, the effects of GCS on viral replication has not been comprehensively examined. Methods Using primary human airway cells and a mouse model of IAV infection we examined the effect of GCS on the production of inflammatory mediators, anti-viral responses and viral replication following RV and/or IAV infection. Results GCS pre-treatment of epithelial cells significantly reduced IL-8, IL-6 and IP-10 production following RV and IAV infection, compared to untreated controls. The expression of anti-viral genes, including ISG56, ISG15, viperin and MxA, were significantly reduced in epithelial cells, fibroblasts and macrophages pre-treated with GCS prior to infection with RV or IAV. The impairment of anti-viral responses observed following GCS pre-treatment was associated with enhanced RV and IAV replication in both epithelial cells and fibroblasts. GCS pre-treatment of mice prior to IAV infection was associated with significant weight loss and severe disease progression compared with untreated mice. Viral loads were significantly elevated in both the lungs and nasal tissues of the GCS treated mice, and there were significantly elevated levels of IL-6, MCP-1 and KC in BAL fluid. Interferon and anti-viral ISG expression were significantly reduced in GCS treated mice. Treatment with recombinant interferon after infection delayed the onset of disease and improved survival. Conclusions Our data indicate that GCS at therapeutic doses enhanced virus replication through the suppression of innate anti-viral responses. Interferon treatment improved disease severity in the mouse model, suggesting this may be a viable therapeutic option to counteract the adverse effects of GCS treatment on viral replication and innate immune suppression.

Aims Nucleotide oligomerization domain 1 (NOD1) is an intracellular host receptor that senses microbial pathogens by detecting a conserved structure of Gram negative bacterial peptidoglycan (PG). Detection of peptidoglycan by NOD1 ultimately results in a pro-inflammatory cytokine response. However, to date, the intracellular location and the mechanisms whereby NOD1 detects peptidoglycan resulting in the development of pro-inflammatory cytokine responses and autophagy are unknown. Methods We used peptidoglycan-containing bacterial outer membrane vesicles (PG-OMVs) as a tool to elucidate the intracellular location of NOD1 and the mechanisms of NOD1-dependent responses that result in cytokine production and autophagy. Results Upon entry into host epithelial cells, PG-OMVs from mucosal pathogens induced NOD1-dependent autophagy and IL-8 responses. Fluorescent labelling of peptidoglycan contained within bacterial OMVs revealed that upon entry into host cells, peptidoglycan migrated to early endosomes where it interacted with NOD1 and the NOD1-adaptor protein RIP-2, facilitating the development of an inflammatory response from this location. We showed that migration of PG-OMVs to early endosomes occurred in a NOD1 dependent manner, identifying a previously unknown role for NOD1 in the intracellular migration of peptidoglycan. Most importantly, using fluorescent lifetime imaging microscopy (FLIM)-fluorescence energy transfer (FRET), we were able to show for the first time the direct interaction between bacterial peptidoglycan and NOD1 within host cells. Finally, we found that the NOD1 adaptor protein RIP-2 is essential for the development of NOD1-dependent autophagy and IL-8 production in response to PG-OMVs [1] . Conclusions This study reveals for the first time the intracellular location and the early recognition events required for the detection of Gram negative bacterial pathogens by NOD1. Moreover, this study is the first to visualise a direct interaction between bacterial peptidoglycan and NOD1. These findings will significantly expand our limited knowledge of the contribution of NOD1 in Gram negative bacterial pathogenesis, innate immunity and inflammatory disorders.