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-β 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-β 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-β 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.

Severe lower respiratory tract disease following influenza A virus (IAV) infection is characterized by excessive inflammation and lung tissue damage, and this can impair lung function. The effect of toll-like receptor 7 (TLR7), which detects viral RNA to initiate antiviral and proinflammatory responses to IAV, on lung function during peak infection and in the resolution phase is not fully understood. Using wild-type (WT) C57BL/6 and TLR7 knockout (TLR7 KO) mice, we found that IAV infection induced airway dysfunction in both genotypes, although in TLR7 KO mice, this dysfunction manifested later, did not affect lung tissue elastance and damping, and was associated with a different immune phenotype. A positive correlation was found between lung dysfunction and the infiltration of neutrophils and Ly6C^lo patrolling monocytes at day 7 post-infection. Conversely, in TLR7 KO mice, eosinophil and CD8+ cytotoxic T cells were associated with airway hyperactivity at day 14. IL-5 expression was higher in the airways of IAV-infected TLR7 KO mice, suggesting an enhanced Th2 response due to TLR7 deficiency. This study highlights an underappreciated duality of TLR7 in IAV disease: promoting inflammation-driven lung dysfunction during the acute infection but suppressing eosinophilic and CD8+ T cell-dependent hyperresponsiveness during disease resolution.

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.

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 plays a key role in recognizing viral RNA to initiate an immune response. Sex-based differences in the severity of RSV respiratory infections have been noted, and this may be related to higher expression of X-linked toll-like receptor 7 (TLR7) in female immune cells. Indeed, TLR7 has been shown to influence sex differences in responses to other respiratory viruses; however, its role in RSV infection remains underexplored. We infected adult C57Bl/6 or TLR7 knockout mice with RSV and compared the specific lung immune responses between different sexes. Gene expression analysis revealed that infected female mice had elevated levels of type I and II interferons, proinflammatory cytokines, chemokines, and viral transcripts in their lungs compared to males. Additionally, females exhibited increased numbers of macrophages and higher antibody responses in the airways. Deletion of TLR7 diminished the sex differences in certain cytokine and antibody responses. Furthermore, ex vivo infection of male alveolar macrophages with RSV resulted in greater production of proinflammatory cytokines and viral transcripts than in female macrophages, suggesting inherent sex differences in macrophage responses. These findings provide new insights into the mechanisms underlying sex differences in RSV pathophysiology and suggest that TLR7 contributes to an enhanced inflammatory response in females.

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.