Protection from Influenza A Virus Infection by Modulating Nucleotide‐Binding Oligomerization Domain Containing 2 (NOD2) Signaling

Rationale Seasonal influenza A virus (IAV) infection is a highly contagious respiratory illness that results in over 200,000 hospitalizations per year in the United States. Nucleotide‐binding oligomerization domain containing 2 (NOD2) is an important component of the innate immune response, the body's first line of defense against viral infection. Its activation results in the production of type I interferons which promote pro‐inflammatory cytokines and restriction of viral replication. NOD2 deficient mice are more susceptible to IAV infection, but interestingly amplified expression of NOD2 has also been associated with increased lethality. This suggests that tight regulation of NOD2 signaling is required to adequately control infection without producing immunopathology. We hypothesize that modulation of the NOD2 signaling pathway will result in improved outcomes following IAV infection. Methods A549 cells were infected with PR8 influenza and NOD2 mRNA expression was measured via quantitative real‐time PCR (qRT‐PCR). NOD2 knockdown (NOD2 KD ) of human THP‐1 macrophages was accomplished via anti‐NOD siRNA. These cells were subsequently infected with IAV or treated with 5′pppRNA, a NOD2 agonist, and IFN‐β levels were assessed with an ELISA. In vivo, forty plaque‐forming units of WSN Influenza A virus was instilled intratracheally in 8–10 week‐old wild type (WT), NOD2 knock‐out (NOD2 −/− ), and NOD2 heterozygous knock‐out (NOD2 +/− ) mice and a death curve was obtained. In a separate experiment, the animals were sacrificed at set time points post‐infection, total cell number, protein concentration and cytokines (IFN‐α, IFN‐β, IL‐18) in bronchoalveolar lavage fluid (BALF) were measured. Lung tissue was collected and used for viral plaque assay and western blot analysis. Results Induction of NOD2 signaling following IAV infection was confirmed via qRT‐PCR in vitro. NOD2 KD cells infected with IAV or 5′pppRNA failed to stimulate the production of IFN‐β. In vivo, WT and NOD2 −/− mice infected with IAV had 100% mortality at 10 days post‐infection. In contrast 50% of the NOD2 +/− were alive at this time point. Complete knockdown of NOD2 resulted in the absence of the downstream type I interferon products in BALF. The NOD2 +/− mice demonstrated a diminished BALF interferon response when compared to WT. Conclusions Type I interferons are a critical component to influenza A recovery, but in excess can contribute to immunopathology and increased lethality. Modulation of type I interferon production via a heterozygous knock‐out mouse model improved IAV mortality. These data suggest that regulating the activity of NOD2 may allow the advantageous inflammatory aspects of its signaling to be preserved while limiting the likelihood of immunopathology. Support or Funding Information Kristin M. Wiese is supported in part by NIH/NHLBI training grant 2T32HL076139‐12 and also through funding from HL071643