Damage sensing through TLR9 Promotes Viral Clearance and Recovery During Influenza Infection

Toll-like receptor-9 is a pattern-recognition receptor that binds to the unmethylated CpG sequences in DNA. Its role in influenza infection is not well understood. Using mice deficient in TLR9, we demonstrated that these animals mounted a reduced inflammatory response during early infection on day 7, driven by the myeloid cells and associated cytokine responses. However, despite this diminished inflammation, TLR9-/- mice had increased viral burden on day 7 post-infection. To understand the cellular specificity and underlying mechanism of increased infection susceptibility in TLR9-/- mice, we performed single-cell RNA sequencing of lung tissues. scRNA sequencing data indicate that TLR9-/- immune cells, especially those of myeloid origin are highly susceptible to influenza infection. This susceptibility of TLR9-/- cells appeared to be independent of type I interferon signaling as the type I interferon pathway appeared the most upregulated pathway in TLR9-/- immune cells. We confirmed increased susceptibility of TLR9-/- myeloid cells using ex vivo murine alveolar macrophage and human peripheral blood monocyte cells. Despite the initial amelioration of the lung injury, the widespread infection of immune cells in TLR9-/- lungs upregulated both influenza and inflammatory responses in myeloid cells. This persistent inflammatory response manifested as the accumulation of both myeloid and lymphoid cells in the airways and increased injury markers in TLR9-/- on day 14. Elevated injury on day 14 delayed the weight loss recovery in TLR9-/- mice. Finally, we demonstrate elevated levels of TLR9 ligands during influenza infection in humans. These data demonstrate a key role of TLR9 in regulating host response to influenza infection.