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.

Measurement invariance testing using confirmatory factor analysis and alignment optimization: A tutorial for transparent analysis planning and reporting.

Measurement invariance-the notion that the measurement properties of a scale are equal across groups, contexts, or time-is an important assumption underlying much of psychology research. The traditional approach for evaluating measurement invariance is to fit a series of nested measurement models using multiple-group confirmatory factor analyses. However, traditional approaches are strict, vary across the field in implementation, and present multiplicity challenges, even in the simplest case of two groups under study. The alignment method was recently proposed as an alternative approach. This method is more automated, requires fewer decisions from researchers, and accommodates two or more groups. However, it has different assumptions, estimation techniques, and limitations from traditional approaches. To address the lack of accessible resources that explain the methodological differences and complexities between the two approaches, we introduce and illustrate both, comparing them side by side. First, we overview the concepts, assumptions, advantages, and limitations of each approach. Based on this overview, we propose a list of four key considerations to help researchers decide which approach to choose and how to document their analytical decisions in a preregistration or analysis plan. We then demonstrate our key considerations on an illustrative research question using an open dataset and provide an example of a completed preregistration. Our illustrative example is accompanied by an annotated analysis report that shows readers, step-by-step, how to conduct measurement invariance tests using R and Mplus. Finally, we provide recommendations for how to decide between and use each approach and next steps for methodological research. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

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.

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.

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.

Addressing a crisis of generalizability with large-scale construct validation.

Because of the misspecification of models and specificity of operationalizations, many studies produce claims of limited utility. We suggest a path forward that requires taking a few steps back. Researchers can retool large-scale replications to conduct the descriptive research which assesses the generalizability of constructs. Large-scale construct validation is feasible and a necessary next step in addressing the generalizability crisis.

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.

Novel endosomal NOX2 oxidase inhibitor ameliorates pandemic influenza A virus-induced lung inflammation in mice.

BACKGROUND AND OBJECTIVE: Influenza A viruses (IAV) cause respiratory tract infections that can be fatal when the virus spreads to the alveolar space (i.e. alveolitis), and this is mainly observed with highly pathogenic strains. Reactive oxygen species (ROS) production by the NOX2 NADPH oxidase in endosomes has been directly implicated in IAV pathology. Recently, we demonstrated that treatment with a novel endosome-targeted NOX2 oxidase inhibitor, cholestanol-conjugated gp91dsTAT (Cgp91ds-TAT), attenuated airway inflammation and viral replication to infection with a low pathogenic influenza A viral strain. Here, we determined whether suppression of endosome NOX2 oxidase prevents the lung inflammation following infection with a highly pathogenic IAV strain. METHODS: C57Bl/6 mice were intranasally treated with either DMSO vehicle (2%) or Cgp91ds-TAT (0.2 mg/kg/day) 1 day prior to infection with the high pathogenicity PR8 IAV strain (500 PFU/mouse). At Day 3 post-infection, mice were culled for the evaluation of airway and lung inflammation, viral titres and ROS generation. RESULTS: PR8 infection resulted in a marked degree of airway inflammation, epithelial denudation, alveolitis and inflammatory cell ROS production. Cgp91ds-TAT treatment significantly attenuated airway inflammation, including neutrophil influx, the degree of alveolitis and inflammatory cell ROS generation. Importantly, the anti-inflammatory phenotype affected by Cgp91ds-TAT significantly enhanced the clearance of lung viral mRNA following PR8 infection. CONCLUSION: Endosomal NOX2 oxidase promotes pathogenic lung inflammation to IAV infection. The localized delivery of endosomal NOX2 oxidase inhibitors is a novel therapeutic strategy against IAV, which has the potential to limit the pathogenesis caused during epidemics and pandemics.

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.

NOX2 oxidase expressed in endosomes promotes cell proliferation and prostate tumour development.

Reactive oxygen species (ROS) promote growth factor signalling including for VEGF-A and have potent angiogenic and tumourigenic properties. However, the precise enzymatic source of ROS generation, the subcellular localization of ROS production and cellular targets in vivo that influence tumour-promoting processes, are largely undefined. Here, using mRNA microarrays, we show increased gene expression for NOX2, the catalytic subunit of the ROS-generating NADPH oxidase enzyme, in human primary prostate cancer compared to non-malignant tissue. In addition, NOX4 gene expression was markedly elevated in human metastatic prostate cancers, but not in primary prostate tumours. Using a syngeneic, orthotopic mouse model of prostate cancer the genetic deletion of NOX2 (i.e. NOX2 -/y mouse) resulted in reduced angiogenesis and an almost complete failure in tumour development. Furthermore, pharmacological inhibition of NOX2 oxidase suppressed established prostate tumours in mice. In isolated endothelial cells, and in human normal and prostate cancer cells, NOX2 co-located to varying degrees with early endosome markers including EEA1, Appl1 and Rab5A and the late endosome marker Rab7A, and this correlated with significant VEGF-A-dependent ROS production within acidified endosomal compartments and endothelial cell proliferation that was NOX2 oxidase- and hydrogen peroxide dependent. We concluded that NOX2 oxidase expression and endosomal ROS production were important for prostate cancer growth and that this was required to positively regulate the VEGF pathway. The research provides a paradigm for limiting tumour growth through a better understanding of NOX2 oxidase's effect on VEGF signalling and how controlling the development of tumour vasculature can limit prostate tumour development and metastasis.

Endosomal NOX2 oxidase exacerbates virus pathogenicity and is a target for antiviral therapy.

The imminent threat of viral epidemics and pandemics dictates a need for therapeutic approaches that target viral pathology irrespective of the infecting strain. Reactive oxygen species are ancient processes that protect plants, fungi and animals against invading pathogens including bacteria. However, in mammals reactive oxygen species production paradoxically promotes virus pathogenicity by mechanisms not yet defined. Here we identify that the primary enzymatic source of reactive oxygen species, NOX2 oxidase, is activated by single stranded RNA and DNA viruses in endocytic compartments resulting in endosomal hydrogen peroxide generation, which suppresses antiviral and humoral signaling networks via modification of a unique, highly conserved cysteine residue (Cys98) on Toll-like receptor-7. Accordingly, targeted inhibition of endosomal reactive oxygen species production abrogates influenza A virus pathogenicity. We conclude that endosomal reactive oxygen species promote fundamental molecular mechanisms of viral pathogenicity, and the specific targeting of this pathogenic process with endosomal-targeted reactive oxygen species inhibitors has implications for the treatment of viral disease.Production of reactive oxygen species is an ancient antimicrobial mechanism, but its role in antiviral defense in mammals is unclear. Here, To et al. show that virus infection activates endosomal NOX2 oxidase and restricts TLR7 signaling, and that an endosomal NOX2 inhibitor decreases viral pathogenicity.