Dysregulated Immune Responses via Toll-Like Receptor 7 Drive Maternal and Fetal Disease Pathogenesis During Gestational Influenza A Virus Infection

Whilst influenza A virus (IAV) infections are typically considered self-limiting, certain demographics, such as pregnant women suffer disproportionate morbidity and mortality in response to IAV infection. Gestational IAV infection increases the risk of hospitalisation and death in the pregnant mother and is evidenced to drive adverse pregnancy and fetal outcomes, including miscarriage, fetal growth restriction, congenital birth defects and most strikingly, altered neurodevelopment, which presents as the emergence of neuropsychiatric conditions, such as schizophrenia later in life. Fetal outcomes from gestational IAV infection arise despite a lack of vertical transmission of the virus in-utero. The mechanisms of increased disease severity during gestational IAV infection remain unknown. However, recent data has revealed that gestational IAV infection is associated with the development of an inflammatory “vascular storm” in the maternal aorta. This “vascular storm” is characterised by immune cell infiltration, inflammation and vascular dysfunction. This “vascular storm” was evidenced to be a pregnancy-specific pathology. The identification of the “vascular storm” has prompted a new understanding that the exacerbated pathology associated with gestational IAV infection may be immune-mediated. One of the key findings in the “vascular storm” was an elevation in the expression of the pattern recognition receptor, Toll-like receptor 7 (TLR7) in the aorta, in particular. Accordingly, we sought to elucidate the mechanisms by which TLR7 may contribute to the immunopathology of gestational IAV infection. Using an established pre-clinical mouse model of gestational IAV infection, which recapitulates clinical characteristics of IAV pathology during pregnancy we investigated how gestational IAV immunopathology was modified in the absence of TLR7, utilising wild-type (WT) mice and TLR7-knockout (TLR7-/-) to discern differences in immune pathology. Here, we demonstrate that in the absence of TLR7 the pathology of gestational IAV infection is less severe. TLR7-/- mice display normal bodyweight gain across their pregnancy following infection, comparatively WT mice experienced blunted weight gain following infection. Additionally, this more mild disease phenotype was also observed within the pulmonary system. During typical disease progression, gestational IAV infection drives a pulmonary pathology, that is characteristic of acute respiratory distress syndrome (ARDS). TLR7-/- mice infected with IAV do not display this ARDS-like pathology. Moreover, the immune response to IAV infection within the lungs of infected mice differs between WT and TLR7-/- mice. WT mice display a robust type-I interferon (IFN-I) response to infection along with elevated levels of the cytokines, interleukin-10 (IL-10), and oncostatin M (OSM). Previous research indicates that OSM disrupts epithelial and endothelial barrier integrity and leads to the development of ARDS during IAV infection. Moreover, the production of OSM occurs in an IFN-I-dependent manner. As TLR7 activation is a primary driver of IFN-I production, we therefore have proposed a new mechanism of gestational IAV infection, which is mediated by a TLR7/IFN-I/OSM axis. In contrast to WT mice, TLR7-/- mice display an immune response characterised by IFN-II anti-viral responses, and more surprisingly, this reduced immune pathology occurred despite higher viral loads within the lungs, compared to infected WT mice. Upon establishing a proposed TLR7-dependant mechanism for the increased pulmonary disease severity exhibited by pregnant women in response to IAV infection, we next sought to understand how TLR7 may contribute to the pathology of the “vascular storm”. Significantly, we found that gestational IAV infection drives the development of bradycardia and systolic hypotension in WT mice, but these cardiovascular manifestations of IAV infection are absent in TLR7-/- mice. Furthermore, we found that in the absence of TLR7, viral dissemination to the maternal cardiovascular system was prevented and dissemination of the virus in WT mice was strongly correlated with the levels of OSM in the lungs. Therefore, we have provided evidence of a TLR7-dependent mechanism that facilitates viral dissemination during gestational IAV infection. Consistent with previous findings, we find that gestational IAV infection induces an endothelial-dependant vascular dysfunction in WT mice, which was associated with an increase in the infiltration of CD4+ T cells into the aorta. Again, in the absence of TLR7, we found no evidence of increased vascular dysfunction, along with a reduction in the numbers of infiltrating CD4+ T cells. Using selective depletion, we further show that CD4+ T cells mediate vascular dysfunction during gestational IAV infection. Together, these results provide novel insight into the cardiovascular pathologies associated with gestational IAV infection. This restoration of maternal cardiovascular function led us to investigate if, in the absence of severe systemic disease, fetal development is restored. Indeed, we find that in the absence of cardiovascular pathology, fetal growth restriction is prevented. Offspring from IAV-infected WT mice had significantly smaller offspring following infection. Comparatively, offspring from TLR7-/- mice did not display any alterations in size in response to infection. This restored growth pattern appeared to be related to the differences in immune profiles at the maternofetal interface of the placenta. In response to infection, the placentae of WT mice display an immune phenotype characteristic of type-1 T helper cell phenotype, characterised by increased placental IFN-γ. Conversely, IAV infection TLR7-/- mice resulted in a more pronounced anti-inflammatory type-2 T helper cell phenotype, with elevated expression of IL-4. Finally, we sought to understand if the protective effects of TLR7-/- extended to the neurodevelopment of offspring. In response to maternal IAV infection, WT offspring displayed elevated markers of neuroinflammation within the hippocampus. IAV infection caused increased expression, of proinflammatory cytokines, complement system components and markers of cell death. In TLR7-/- offspring, maternal infection did not cause any elevation in inflammatory gene expression, indicating that in the absence of TLR7, fetal development is protected during gestational IAV infection. The findings from these studies provide novel, mechanistic insight into the pathology of gestational IAV infection in relation to immune dysfunction and provide a hitherto unappreciated hypothesis for the viral origins of cardiovascular and neurodevelopment disease. Our results have provided invaluable insight into the mechanisms by which TLR7 mediates immunopathology during gestational IAV infection and present TLR7 as a potential therapeutic target with application for gestational IAV infection. By targeting TLR7, it may be possible to ameliorate, if not prevent the pathologies that drive increased maternal and fetal morbidity and mortality.