This study aimed to characterize the immune response mediated by TLR7 in the URT and LRT using a moderately pathogenic model of IAV. TLR7 KO mice exhibited increased levels of type II/III IFNs and proinflammatory cytokines in the URT, while showing a decrease in overall inflammatory responses in the LRT. This pattern was associated with significant alterations in both innate and adaptive immune cell populations in the airways and lungs of TLR7 KO mice. Despite these immune alterations, the responses of macrophages, NKT cells, eosinophils, and T cells remained largely intact in the nasal tissue. These findings suggest that TLR7 plays a crucial role in driving immune responses in the LRT, but its role in the URT is more limited. Additionally, the expression and stimulatory capacity of TLR7 were highest in cells of the LRT, reinforcing its predominant function in driving LRT inflammation. The reduced body weight loss observed in TLR7 KO mice highlights TLR7 as a key driver of LRT disease during IAV infection.

The role of TLR7 in the URT and LRT was clearly distinct in terms of immune response. Our study mapped TLR7 expression in various regions of the URT and LRT, providing valuable insight into the spatial distribution of immune responses during IAV infection. The findings reveal that TLR7-driven inflammatory responses are predominantly localized to the LRT, where macrophages and monocytes are the primary cell types expressing TLR7. Hyperactivation of macrophages and monocytes, in particular, in the LRT has been strongly associated with poor clinical outcomes during IAV infection, highlighting the potentially detrimental role of TLR7 signalling in exacerbating lung pathology^16,29,30. In contrast, while TLR7 was also detected in the nasal tissue, its expression was markedly lower compared to the LRT. Notably, TLR7 expression in the nasal tissue was largely confined to immune cells, such as macrophages and NK cells. These data suggest that the contribution of TLR7 to the URT inflammatory response has been underestimated in current literature, likely due to its relatively low expression in epithelial cells²⁶, which are commonly used to model viral infection. Our study, therefore, offers a more comprehensive view of TLR7 responses to IAV infection in the nasal compartment by also considering both resident and infiltrating immune cells.

This investigation into TLR7 expression in nasal tissues highlights the importance of the immune microenvironment in the URT, including specialised structures like the nasal-associated lymphoid tissue (NALT), which plays a crucial role in initiating mucosal immune responses to pathogens. The nasal immune microenvironment, comprising macrophages, DCs, and NK cells, functions to survey the local area and initiate mucosal immune responses to combat invading pathogens³¹. Many of the TLR7-expressing cells we observed are contained within the NALT and this specialized lymphoid structure is rich in B cells, T cells, macrophages, and DCs^32,33. NK cells, which are recruited to the NALT following intranasal immunization, have also been shown to be activated by TLR7 during IAV infection^34,35. Moreover, the NALT is known to play a pivotal role in the generation of CD8 + T cell memory responses following IAV infection, with the priming of naïve CD8 + T cells occurring at the draining cervical lymph nodes³⁶. This may help explain why we did not detect IAV-specific T cells in the nasal tissue, as these cells are likely primed and recruited to the lung, where the bulk of the immune response and viral clearance occur. Future experiments examining later timepoints will be needed to validate this hypothesis.

After mapping TLR7 expression and immune cell contributions in the URT, it was essential to explore how TLR7 deficiency affected the immune responses in the airways and lungs, especially during IAV infection. The effects of TLR7 deficiency were most pronounced in the airways and lungs, where both innate and adaptive immune responses were significantly altered. This resulted in a marked reduction in proinflammatory gene expression in the lungs of TLR7 KO mice compared to WT mice, suggesting that TLR7 is important for driving robust inflammatory responses in the LRT. In contrast, TLR7 deficiency in the nasal tissue impaired responses from monocytes, pDCs, and B cells, while macrophage, NK cell, eosinophil, and T cell responses remained largely intact. Interestingly, despite these immune deficiencies, TLR7 KO mice exhibited higher expression of proinflammatory markers, as well as elevated levels of TLRs and RLRs, in the nasal tissue compared to WT mice. This suggests a compensatory upregulation of alternative immune pathways that might help mitigate the effects of TLR7 deficiency in the absence of overt pathology, thereby reducing disease severity that may be driven by TLR7-mediated inflammation. Indeed, intact antiviral and some T cell responses to IAV infection can occur via IL-1R or MAVS signalling in the absence of TLR7^37,38,39. These compensatory responses may be more pronounced in the URT, given the higher expression of those components in this region, possibly resulting in the observed intact macrophage, NKT and T cell responses in the nasal tissue. Additionally, increased levels of IFNG, which likely reflects the heightened presence of NK cells in the nasal tissue, were observed. Anti-inflammatory IL10 expression mirrored the levels of proinflammatory cytokines, both in the URT and LRT, potentially indicating the host’s attempt to resolve inflammation and limit tissue damage⁴⁰. Overall, TLR7 KO mice showed a higher degree of inflammation in the URT and reduced inflammation in the LRT compared to WT mice. Despite these differences in inflammatory profiles, viral load in both nasal and lung tissues was similar between TLR7 KO and WT mice, suggesting that TLR7 does not significantly affect viral replication at these sites, at least at the time point studied. This finding highlights the complex role of TLR7 in modulating the immune response, rather than directly influencing viral load.

This duality in immune responses suggests that TLR7 acts pivotally in the inflammatory response to IAV infection, particularly by regulating the transition of inflammation from the URT to the LRT. In the absence of TLR7, inflammation remains sustained in the URT, potentially preventing excessive inflammatory damage in the lungs. This hypothesis is further supported by our observation that TLR7 KO mice exhibited less body weight loss during infection, suggesting a less severe disease course compared to WT mice. Disease severity is often attributed to changes in lung histopathology. Further studies that comprehensively assess histological alterations across multiple regions of the respiratory tract, and examine how these changes correlate with inflammatory markers in these tissues, will provide additional insight into how TLR7-mediated inflammation contributes to disease progression. It is important to recognize, however, that impaired immune memory to IAV infection could be a long-term consequence of TLR7 deficiency^39,41. Although previous research has linked enhanced neutrophilic and monocytic inflammatory signatures in nasal samples to poorer IAV outcomes—reflecting the extensive inflammation observed in the LRT^6,17—our data indicate that the higher inflammatory responses in the URT at day 6 post-infection might actually be protective, especially when accompanied by reduced inflammation in the LRT. In addition, localized inflammation in the URT, such as that caused by bacterial colonization in the nasal mucosa, has been shown to mitigate LRT disease during subsequent IAV infections^42,43, further suggesting that localized inflammation in the URT can be protective. Furthermore, we found that the monocyte response, which can drive lung damage during IAV infection^16,29, was impaired in both the URT and LRT of TLR7 KO mice, suggesting that these mice may be protected from monocyte-driven pathology. Further work is needed to fully understand the role of TLR7 in monocyte-mediated IAV disease and its broader implications for immune regulation.

A potential mechanism underlying the TLR7-dependent propagation of inflammation from the URT to the LRT may involve the differential contributions of type I and type III IFN signalling during IAV infection, specifically in the URT. Galani et al.⁹ demonstrated that, upon IAV infection, IFN-λ is initially produced at the epithelial barrier, followed by the production of type I IFN, which drives proinflammatory cytokine production and immune cell recruitment. Importantly, a higher viral inoculum was found to trigger a more rapid type I IFN response, leading to earlier inflammation and more severe disease outcomes. Moreover, Davidson et al.⁴⁴ showed that therapeutic administration of IFN-λ during active IAV infection reduced viral replication in the airway epithelia and conferred protection, while IFNα stimulated inflammation in the lungs and exacerbated LRT disease. Similar protective effects of intranasal IFN-λ have also been observed in mice infected with SARS-CoV-2⁴⁵. These findings underscore that type III IFNs exert less immunostimulatory effect on immune cells compared to type I IFNs during infection, suggesting that an early dominance of type I IFN responses may drive more extensive inflammation in the LRT. This could be due to the broader expression of the IFNAR1 across many cell types, whereas the IFN-λ receptor (IFNLR1) is primarily expressed on epithelial cells. As a result, type III IFNs exert their effects more specifically at mucosal surfaces, providing antiviral protection without driving excessive proinflammatory responses⁴⁶. In terms of the IFN response in the lungs, we found lower type I and type III IFNs in TLR7 KO mice following IAV infection. We hypothesize that this reduced response specifically in the lungs may be protective, as sustained type I IFN signalling in the LRT can impair lung epithelial regeneration and repair during the later stages of infection, contributing to chronic inflammation and fibrosis⁴⁷.

In contrast, in the URT, TLR7 deficiency had a more specific effect, primarily reducing IFNB1 production while enhancing IFNL3 levels. This shift resulted in a more balanced type I/III IFN response in the nasal tissue of TLR7 KO mice, compared to a dominant type I IFN response in WT mice. We hypothesize that this altered IFN response in TLR7 KO mice leads to reduced recruitment and activation of inflammatory cells to the LRT without altering viral load, thus protecting these mice from severe LRT disease. Although further work is needed to mechanistically validate this hypothesis, we propose that TLR7-dependent production of type I and III IFNs by specific cell types, and its temporal dynamics, plays a crucial role in this process. Type III IFNs are primarily produced by the respiratory epithelium during IAV infection, with pDCs also contributing to this production^9,48. We speculate the primary source of IFN-λ in the URT of TLR7-deficient mice to derive primarily from epithelial cells, possibly through RIG-I/MDA5 recognition⁴⁹ since TLR7 expression is minimal in these cells and RLR expression was found to be elevated in the nasal tissue after infection. In contrast, pDCs, which typically express TLR7, would have a diminished capacity to produce type I and III IFNs to IAV infection in these mice⁵⁰. This would lead to an antiviral response predominately regulated by epithelial-derived IFN-λ, without the additional proinflammatory effects of type I IFN. We therefore propose that TLR7 exacerbates IAV disease by skewing the immune response in favour of type I IFN, potentially sustaining IFN production for longer than necessary, which in turn promotes sustained downstream LRT inflammation. Supporting this, PBMCs treated with the TLR7/8 agonist R848 secreted more IFN-α than IFN-λ, highlighting the capacity of TLR7 to bias the immune response toward type I IFN⁵¹. Furthermore, SARS-CoV-2 infection of pDCs promoted type I IFN production via TLR7, and this led to transcriptional and epigenetic changes in lung macrophages that enhanced their proinflammatory phenotype²². Thus, these TLR7-dependent events in the URT, along with the production of IFNs, may influence the inflammatory status of cells in the LRT, contributing to the overall immune response and potentially exacerbating disease outcomes.

In summary, this study highlights the pivotal role of TLR7 in shaping the immune response during IAV infection, providing evidence that TLR7 mediates the dissemination of the inflammatory response from the URT to the LRT. We utilized an acute IAV model of Hk-X31 infection thereby enabling us to assess concordant immune responses in both the URT and LRT. Future work assessing the temporal progression of immune alterations in across multiple compartments of the respiratory tract will provide a more comprehensive understanding of the immune dynamics driven by TLR7 during IAV infection. Furthermore, characterising the dissemination of inflammation from the URT to LRT following infection with more pathogenic strains of IAV will provide important insights into the broad role of TLR7. Previous studies have shown that either activating TLR7 via IMQ prior to IAV infection or inhibiting it with IRS661 during established infection reduces LRT pathology^21,52. These treatments were administered intranasally, implying that TLR7 within cells of both the URT and LRT were targeted, although the immune responses in the URT were not specifically addressed. Building on these findings, our results imply that early intranasal administration of TLR7 antagonists could be a more effective approach in IAV treatment. Such a strategy may have two key mechanistic outcomes to limit LRT inflammation: (1) it could reduce the immunostimulatory effects of type I IFNs, thereby suppressing the indirect activation and recruitment of downstream inflammatory cells, and (2) it could dampen the direct activation of TLR7-expressing immune cells. By limiting excessive inflammation in the LRT, this approach could alleviate IAV disease severity. Therefore, targeting TLR7 may be a therapeutic strategy to improve outcomes in severe IAV infections by modulating pathogenic responses in both the upper and lower respiratory tracts.