Early-Life Respiratory Syncytial Virus (RSV) Infection Triggers Immunological Changes in Gut-Associated Lymphoid Tissues in a Sex-Dependent Manner in Adulthood.

Severe respiratory syncytial virus (RSV) infection during early life has been linked to gut dysbiosis, which correlates with increased disease severity and a higher risk of developing asthma later in life. However, the impact of such early-life RSV infections on intestinal immunity in adulthood remains unclear. Herein, we show that RSV infection in 3-week-old mice induced persistent differential natural killer (NK) and T cell profiles within the lungs and gastrointestinal (GI) lymphoid tissues (GALT) in adulthood. Notably, male mice exhibited more pronounced RSV-induced changes in immune cell populations in both the lungs and GALT, while female mice displayed greater resilience. Importantly, early-life RSV infection was associated with the chronic downregulation of CD69-expressing T lymphocytes, particularly T regulatory cells in Peyer's patches, which could have a significant impact on T cell functionality and immune tolerance. We propose that RSV infection in early life is a trigger for the breakdown in immune tolerance at mucosal surfaces, with potential implications for airways allergic disease, food allergies, and other GI inflammatory diseases.

Gestational influenza A virus infection elicits nonresolving vascular dysfunction and T-cell accumulation in the aorta of mice.

T-cell accumulation within the aorta promotes endothelial dysfunction and the genesis of cardiovascular disease, including hypertension and atherosclerosis. Viral infection during pregnancy is also known to mediate marked acute endothelial dysfunction, but it is not clear whether T cells are recruited to the aorta and whether the dysfunction persists postpartum. Here, we demonstrate that influenza A virus (IAV) infection during pregnancy in a murine model resulted in endothelial dysfunction of the aorta, which persisted for up to 60 days postinfection and was associated with higher levels of IFN-γ mRNA expression within the tissue. In the absence of infection, low numbers of naïve CD4+ and CD8+ T cells, central memory T cells, and effector memory T cells were observed in the aorta. However, with IAV infection, these T-cell subsets were significantly increased with a notable accumulation of IAV-specific CD8+ effector memory T cells. Critically, this increase was maintained out to at least 60 days. In contrast, IAV infection in nonpregnant female mice resulted in modest endothelial dysfunction with no accumulation of T cells within the aorta. These data, therefore, demonstrate that the aorta is a site of T-cell recruitment and retention after IAV infection during pregnancy. Although IAV-specific memory T cells could theoretically confer protection against future influenza infection, nonspecific memory T-cell activation and IFN-γ production in the aorta could also contribute to future endothelial dysfunction and cardiovascular disease.NEW & NOTEWORTHY Pregnancy is a risk factor for cardiovascular complications to influenza A virus (IAV) infection. We demonstrate that gestational IAV infection caused endothelial dysfunction of the maternal aorta, which persisted for 60 days postinfection in mice. Various T cells accumulated within the aorta at 60 days because of the infection, and this was associated with elevated levels of the proinflammatory cytokine, IFN-γ. Our study demonstrates a novel "long influenza" cardiovascular phenotype in female mice.

Brain region-specific alterations in gene expression trajectories in the offspring born from influenza A virus infected mice.

Influenza A virus (IAV) infection during pregnancy can increase the risk for neurodevelopmental disorders in the offspring, however, the underlying neurobiological mechanisms are largely unknown. To recapitulate viral infection, preclinical studies have traditionally focused on using synthetic viral mimetics, rather than live IAV, to examine consequences of maternal immune activation (MIA)-dependent processes on offspring. In contrast, few studies have used live IAV to assess effects on global gene expression, and none to date have addressed whether moderate IAV, mimicking seasonal influenza disease, alters normal gene expression trajectories in different brain regions across different stages of development. Herein, we show that moderate IAV infection during pregnancy, which causes mild maternal disease and no overt foetal complications in utero, induces lasting effects on the offspring into adulthood. We observed behavioural changes in adult offspring, including disrupted prepulse inhibition, dopaminergic hyper-responsiveness, and spatial recognition memory deficits. Gene profiling in the offspring brain from neonate to adolescence revealed persistent alterations to normal gene expression trajectories in the prefronal cortex, hippocampus, hypothalamus and cerebellum. Alterations were found in genes involved in inflammation and neurogenesis, which were predominately dysregulated in neonatal and early adolescent offspring. Notably, late adolescent offspring born from IAV infected mice displayed altered microglial morphology in the hippocampus. In conclusion, we show that moderate IAV during pregnancy perturbs neurodevelopmental trajectories in the offspring, including alterations in the neuroinflammatory gene expression profile and microglial number and morphology in the hippocampus, resulting in behavioural changes in adult offspring. Such early perturbations may underlie the vulnerability in human offspring for the later development of neurodevelopmental disorders, including schizophrenia. Our work highlights the importance of using live IAV in developing novel preclinical models that better recapitulate the real-world impact of inflammatory insults during pregnancy on offspring neurodevelopmental trajectories and disease susceptibility later in life.

Low dose aspirin prevents endothelial dysfunction in the aorta and foetal loss in pregnant mice infected with influenza A virus.

Influenza A virus (IAV) infection in pregnancy resembles a preeclamptic phenotype characterised by vascular dysfunction and foetal growth retardation. Given that low dose aspirin (ASA) is safe in pregnancy and is used to prevent preeclampsia, we investigated whether ASA or NO-conjugated aspirin, NCX4016, resolve vascular inflammation and function to improve offspring outcomes following IAV infection in pregnant mice. Pregnant mice were intranasally infected with a mouse adapted IAV strain (Hkx31; 104 plaque forming units) and received daily treatments with either 200µg/kg ASA or NCX4016 via oral gavage. Mice were then culled and the maternal lungs and aortas collected for qPCR analysis, and wire myography was performed on aortic rings to assess endothelial and vascular smooth muscle functionality. Pup and placentas were weighed and pup growth rates and survival assessed. IAV infected mice had an impaired endothelial dependent relaxation response to ACh in the aorta, which was prevented by ASA and NCX4016 treatment. ASA and NCX4016 treatment prevented IAV dissemination and inflammation of the aorta as well as improving the pup placental ratios in utero, survival and growth rates at post-natal day 5. Low dose ASA is safe to use during pregnancy for preeclampsia and this study demonstrates that ASA may prove a promising treatment for averting the significant vascular complications associated with influenza infection during pregnancy.

TLR9 Monotherapy in Immune-Competent Mice Suppresses Orthotopic Prostate Tumor Development.

Prostate cancer is ranked second in the world for cancer-related deaths in men, highlighting the lack of effective therapies for advanced-stage disease. Toll-like receptors (TLRs) and immunity have a direct role in prostate cancer pathogenesis, but TLR9 has been reported to contribute to both the progression and inhibition of prostate tumorigenesis. To further understand this apparent disparity, we have investigated the effect of TLR9 stimulation on prostate cancer progression in an immune-competent, syngeneic orthotopic mouse model of prostate cancer. Here, we utilized the class B synthetic agonist CPG-1668 to provoke a TLR9-mediated systemic immune response and demonstrate a significant impairment of prostate tumorigenesis. Untreated tumors contained a high abundance of immune-cell infiltrates. However, pharmacological activation of TLR9 resulted in smaller tumors containing significantly fewer M1 macrophages and T cells. TLR9 stimulation of tumor cells in vitro had no effect on cell viability or its downstream transcriptional targets, whereas stimulation in macrophages suppressed cancer cell growth via type I IFN. This suggests that the antitumorigenic effects of CPG-1668 were predominantly mediated by an antitumor immune response. This study demonstrated that systemic TLR9 stimulation negatively regulates prostate cancer tumorigenesis and highlights TLR9 agonists as a useful therapeutic for the treatment of prostate cancer.

TLR7 promotes chronic airway disease in RSV-infected mice.

Respiratory syncytial virus (RSV) commonly infects the upper respiratory tract (URT) of humans, manifesting with mild cold or flu-like symptoms. However, in infants and the elderly, severe disease of the lower respiratory tract (LRT) often occurs and can develop into chronic airway disease. A better understanding of how an acute RSV infection transitions to a LRT chronic inflammatory disease is critically important to improve patient care and long-term health outcomes. To model acute and chronic phases of the disease, we infected wild-type C57BL/6 and toll-like receptor 7 knockout (TLR7 KO) mice with RSV and temporally assessed nasal, airway and lung inflammation for up to 42 days post-infection. We show that TLR7 reduced viral titers in the URT during acute infection but promoted pronounced pathogenic and chronic airway inflammation and hyperreactivity in the LRT. This study defines a hitherto unappreciated molecular mechanism of lower respiratory pathogenesis to RSV, highlighting the potential of TLR7 modulation to constrain RSV pathology to the URT.

Influenza A virus infection during pregnancy causes immunological changes in gut-associated lymphoid tissues of offspring mice.

Maternal influenza A virus (IAV) infection during pregnancy can affect offspring immune programming and development. Offspring born from influenza-infected mothers are at increased risk of neurodevelopmental disorders and have impaired respiratory mucosal immunity against pathogens. The gut-associated lymphoid tissue (GALT) represents a large proportion of the immune system in the body and plays an important role in gastrointestinal (GI) homeostasis. This includes immune modulation to antigens derived from food or microbes, gut microbiota composition, and gut-brain axis signaling. Therefore, in this study, we investigated the effect of maternal IAV infection on mucosal immunity of the GI tract in the offspring. There were no major anatomical changes to the gastrointestinal tract of offspring born to influenza-infected dams. In contrast, maternal IAV did affect the mucosal immunity of offspring, showing regional differences in immune cell profiles within distinct GALT. Neutrophils, monocytes/macrophages, CD4+ and CD8+ T cells infiltration was increased in the cecal patch offspring from IAV-infected dams. In the Peyer's patches, only activated CD4+ T cells were increased in IAV offspring. IL-6 gene expression was also elevated in the cecal patch but not in the Peyer's patches of IAV offspring. These findings suggest that maternal IAV infection perturbs homeostatic mucosal immunity in the offspring gastrointestinal tract. This could have profound ramifications on the gut-brain axis and mucosal immunity in the lungs leading to increased susceptibility to respiratory infections and neurological disorders in the offspring later in life.NEW & NOTEWORTHY Influenza A virus (IAV) infection during pregnancy is associated with changes in gut-associated lymphoid tissue (GALT) in the offspring in a region-dependent manner. Neutrophils and monocytes/macrophages were elevated in the cecal patch of offspring from infected dams. This increase in innate immune cell infiltration was not observed in the Peyer's patches. T cells were also elevated in the cecal patch but not in the Peyer's patches.

Glycolysis and the Pentose Phosphate Pathway Promote LPS-Induced NOX2 Oxidase- and IFN-ß-Dependent Inflammation in Macrophages.

Macrophages undergo a metabolic switch from oxidative phosphorylation to glycolysis when exposed to gram-negative bacterial lipopolysaccharide (LPS), which modulates antibacterial host defence mechanisms. Here, we show that LPS treatment of macrophages increased the classical oxidative burst response via the NADPH oxidase (NOX) 2 enzyme, which was blocked by 2-deoxyglucose (2-DG) inhibition of glycolysis. The inhibition of the pentose phosphate pathway with 6-aminonicotinamide (6-AN) also suppressed the LPS-induced increase in NOX2 activity and was associated with a significant reduction in the mRNA expression of NOX2 and its organizer protein p47phox. Notably, the LPS-dependent enhancement in NOX2 oxidase activity was independent of both succinate and mitochondrial reactive oxygen species (ROS) production. LPS also increased type I IFN-ß expression, which was suppressed by 2-DG and 6-AN and, therefore, is dependent on glycolysis and the pentose phosphate pathway. The type I IFN-ß response to LPS was also inhibited by apocynin pre-treatment, suggesting that NOX2-derived ROS promotes the TLR4-induced response to LPS. Moreover, recombinant IFN-ß increased NOX2 oxidase-dependent ROS production, as well as NOX2 and p47phox expression. Our findings identify a previously undescribed molecular mechanism where both glycolysis and the pentose phosphate pathway are required to promote LPS-induced inflammation in macrophages.

Influenza A virus elicits peri-vascular adipose tissue inflammation and vascular dysfunction of the aorta in pregnant mice.

Influenza A virus (IAV) infection during pregnancy initiates significant aortic endothelial and vascular smooth muscle dysfunction, with inflammation and T cell activation, but the details of the mechanism are yet to be clearly defined. Here we demonstrate that IAV disseminates preferentially into the perivascular adipose tissue (PVAT) of the aorta in mice. IAV mRNA levels in the PVAT increased at 1-3 days post infection (d.p.i) with the levels being ~4-8 fold higher compared with the vessel wall. IAV infection also increased Ly6Clow patrolling monocytes and Ly6Chigh pro-inflammatory monocytes in the vessel wall at 3 d.p.i., which was then followed by a greater homing of these monocytes into the PVAT at 6 d.p.i. The vascular immune phenotype was characteristic of a "vascular storm"- like response, with increases in neutrophils, pro-inflammatory cytokines and oxidative stress markers in the PVAT and arterial wall, which was associated with an impairment in endothelium-dependent relaxation to acetylcholine. IAV also triggered a PVAT compartmentalised elevation in CD4+ and CD8+ activated T cells. In conclusion, the PVAT of the aorta is a niche that supports IAV dissemination and a site for perpetuating a profound innate inflammatory and adaptive T cell response. The manifestation of this inflammatory response in the PVAT following IAV infection may be central to the genesis of cardiovascular complications arising during pregnancy.

Endothelial NOX4 Oxidase Negatively Regulates Inflammation and Improves Morbidity During Influenza A Virus Lung Infection in Mice.

Endosomal NOX2 oxidase-dependent ROS production promotes influenza pathogenicity, but the role of NOX4 oxidase, which is highly expressed in the lung endothelium, is largely unknown. The aim of this study was to determine if endothelial NOX4 expression can influence viral pathology in vivo, using a mouse model of influenza infection. WT and transgenic endothelial NOX4 overexpressing mice (NOX4 TG) were infected intranasally with the Hong Kong H3N2 X-31 influenza A virus (104 PFU; HK x-31) or PBS control. Mice were culled at either 3 or 7 days post-infection to analyse: airway inflammation by bronchoalveolar lavage fluid (BALF) cell counts; NOX4, as well as inflammatory cytokine and chemokine gene expression by QPCR; and ROS production by an L-012-enhanced chemiluminescence assay. Influenza A virus infection of WT mice resulted in a significant reduction in lung NOX4 mRNA at day 3, which persisted until day 7, when compared to uninfected mice. Influenza A virus infection of NOX4 TG mice resulted in significantly less weight loss than that of WT mice at 3-days post infection. Viral titres were decreased in infected NOX4 TG mice compared to the infected WT mice, at both 3- and 7-days post infection and there was significantly less lung alveolitis, peri-bronchial inflammation and neutrophil infiltration. The oxidative burst from BALF inflammatory cells extracted from infected NOX4 TG mice was significantly less than that in the WT mice. Expression of macrophage and neutrophil chemoattractants CXCL10, CCL3, CXCL1 and CXCL2 in the lung tissue were significantly lower in NOX4 TG mice compared to the WT mice at 3-days post infection. We conclude that endothelial NOX4 oxidase is protective against influenza morbidity and is a potential target for limiting influenza A virus-induced lung inflammation.

Therapeutic Targeting of Endosome and Mitochondrial Reactive Oxygen Species Protects Mice From Influenza Virus Morbidity.

There is an urgent need to develop effective therapeutic strategies including immunomodulators to combat influenza A virus (IAV) infection. Influenza A viruses increase ROS production, which suppress anti-viral responses and contribute to pathological inflammation and morbidity. Two major cellular sites of ROS production are endosomes via the NOX2-oxidase enzyme and the electron transport chain in mitochondria. Here we examined the effect of administration of Cgp91ds-TAT, an endosome-targeted NOX2 oxidase inhibitor, in combination with mitoTEMPO, a mitochondrial ROS scavenger and compared it to monotherapy treatment during an established IAV infection. Mice were infected with IAV (Hkx31 strain; 104PFU/mouse) and 24 h post infection were treated with Cgp91ds-TAT (0.2 mg/kg), mitoTEMPO (100 µg) or with a combination of these inhibitors [Cgp91ds-TAT (0.2 mg/kg)/mitoTEMPO (100 µg)] intranasally every day for up to 2 days post infection (pi). Mice were euthanized on Days 3 or 6 post infection for analyses of disease severity. A combination of Cgp91ds-TAT and mitoTEMPO treatment was more effective than the ROS inhibitors alone at reducing airway and neutrophilic inflammation, bodyweight loss, lung oedema and improved the lung pathology with a reduction in alveolitis following IAV infection. Dual ROS inhibition also caused a significant elevation in Type I IFN expression at the early phase of infection (day 3 pi), however, this response was suppressed at the later phase of infection (day 6 pi). Furthermore, combined treatment with Cgp91ds-TAT and mitoTEMPO resulted in an increase in IAV-specific CD8+ T cells in the lungs. In conclusion, this study demonstrates that the reduction of ROS production in two major subcellular sites, i.e. endosomes and mitochondria, by intranasal delivery of a combination of Cgp91ds-TAT and mitoTEMPO, suppresses the severity of influenza infection and highlights a novel immunomodulatory approach for IAV disease management.

Influenza A virus causes maternal and fetal pathology via innate and adaptive vascular inflammation in mice.

Influenza A virus (IAV) infection during pregnancy causes severe maternal and perinatal complications, despite a lack of vertical transmission of IAV across the placenta. Here, we demonstrate a significant alteration in the maternal vascular landscape that underpins the maternal and downstream fetal pathology to IAV infection in mice. In IAV infection of nonpregnant mice, the local lung inflammatory response was contained to the lungs and was self-resolving, whereas in pregnant mice, virus dissemination to major maternal blood vessels, including the aorta, resulted in a peripheral "vascular storm," with elevated proinflammatory and antiviral mediators and the influx of Ly6Clow and Ly6Chigh monocytes, plus neutrophils and T cells. This vascular storm was associated with elevated levels of the adhesion molecules ICAM and VCAM and the pattern-recognition receptors TLR7 and TLR9 in the vascular wall, resulting in profound vascular dysfunction. The sequalae of this IAV-driven vascular storm included placental growth retardation and intrauterine growth restriction, evidence of placental and fetal brain hypoxia, and increased circulating cell free fetal DNA and soluble Flt1. In contrast, IAV infection in nonpregnant mice caused no obvious alterations in endothelial function or vascular inflammation. Therefore, IAV infection during pregnancy drives a significant systemic vascular alteration in pregnant dams, which likely suppresses critical blood flow to the placenta and fetus. This study in mice provides a fundamental mechanistic insight and a paradigm into how an immune response to a respiratory virus, such as IAV, is likely to specifically drive maternal and fetal pathologies during pregnancy.

Mitochondrial Reactive Oxygen Species Contribute to Pathological Inflammation During Influenza A Virus Infection in Mice.

Aims: Reactive oxygen species (ROS) are highly reactive molecules generated in different subcellular sites or compartments, including endosomes via the NOX2-containing nicotinamide adenine dinucleotide phosphate oxidase during an immune response and in mitochondria during cellular respiration. However, while endosomal NOX2 oxidase promotes innate inflammation to influenza A virus (IAV) infection, the role of mitochondrial ROS (mtROS) has not been comprehensively investigated in the context of viral infections in vivo. Results: In this study, we show that pharmacological inhibition of mtROS, with intranasal delivery of MitoTEMPO, resulted in a reduction in airway/lung inflammation, neutrophil infiltration, viral titers, as well as overall morbidity and mortality in mice infected with IAV (Hkx31, H3N2). MitoTEMPO treatment also attenuated apoptotic and necrotic neutrophils and macrophages in airway and lung tissue. At an early phase of influenza infection, that is, day 3 there were significantly lower amounts of IL-1ß protein in the airways, but substantially higher amounts of type I IFN-ß following MitoTEMPO treatment. Importantly, blocking mtROS did not appear to alter the initiation of an adaptive immune response by lung dendritic cells, nor did it affect lung B and T cell populations that participate in humoral and cellular immunity. Innovation/Conclusion: Influenza virus infection promotes mtROS production, which drives innate immune inflammation and this exacerbates viral pathogenesis. This pathogenic cascade highlights the therapeutic potential of local mtROS antioxidant delivery to alleviate influenza virus pathology.

Intranasal and epicutaneous administration of Toll-like receptor 7 (TLR7) agonists provides protection against influenza A virus-induced morbidity in mice.

Toll-like receptor 7 (TLR7) is a pattern recognition receptor that recognizes viral RNA following endocytosis of the virus and initiates a powerful immune response characterized by Type I IFN production and pro-inflammatory cytokine production. Despite this immune response, the virus causes very significant pathology, which may be inflammation-dependent. In the present study, we examined the effect of intranasal delivery of the TLR7 agonist, imiquimod or its topical formulation Aldara, on the inflammation and pathogenesis caused by IAV infection. In mice, daily intranasal delivery of imiquimod prevented peak viral replication, bodyweight loss, airway and pulmonary inflammation, and lung neutrophils. Imiquimod treatment also resulted in a significant reduction in pro-inflammatory neutrophil chemotactic cytokines and prevented the increase in viral-induced lung dysfunction. Various antibody isotypes (IgG1, IgG2a, total IgG, IgE and IgM), which were increased in the BALF following influenza A virus infection, were further increased with imiquimod. While epicutaneous application of Aldara had a significant effect on body weight, it did not reduce neutrophil and eosinophil airway infiltration; indicating less effective drug delivery for this formulation. We concluded that intranasal imiquimod facilitates a more effective immune response, which can limit the pathology associated with influenza A virus infection.