Tolerance to Haemophilus influenzae infection in human epithelial cells: Insights from a primary cell-based model.

Haemophilus influenzae is a human respiratory pathogen and inhabits the human respiratory tract as its only niche. Despite this, the molecular mechanisms that allow H. influenzae to establish persistent infections of human epithelia are not well understood. Here, we have investigated how H. influenzae adapts to the host environment and triggers the host immune response using a human primary cell-based infection model that closely resembles human nasal epithelia (NHNE). Physiological assays combined with dualRNAseq revealed that NHNE from five healthy donors all responded to H. influenzae infection with an initial, 'unproductive' inflammatory response that included a strong hypoxia signature but did not produce pro-inflammatory cytokines. Subsequently, an apparent tolerance to large extracellular and intraepithelial burdens of H. influenzae developed, with NHNE transcriptional profiles resembling the pre-infection state. This occurred in parallel with the development of intraepithelial bacterial populations, and appears to involve interruption of NFκB signalling. This is the first time that large-scale, persistence-promoting immunomodulatory effects of H. influenzae during infection have been observed, and we were able to demonstrate that only infections with live, but not heat-killed H. influenzae led to immunomodulation and reduced expression of NFκB-controlled cytokines such as IL-1ß, IL-36γ and TNFα. Interestingly, NHNE were able to re-activate pro-inflammatory responses towards the end of the 14-day infection, resulting in release of IL-8 and TNFα. In addition to providing first molecular insights into mechanisms enabling persistence of H. influenzae in the host, our data further indicate the presence of infection stage-specific gene expression modules, highlighting fundamental similarities between immune responses in NHNE and canonical immune cells, which merit further investigation.

Development of a novel angiotensin converting enzyme 2 stimulator with broad implications in SARS-CoV2 and type 1 diabetes.

Angiotensin-converting enzyme 2 (ACE2) is protective in cardiovascular disease, lung injury and diabetes yet paradoxically underlies our susceptibility to SARs-CoV2 infection and the fatal heart and lung disease it can induce. Furthermore, diabetic patients have chronic, systemic inflammation and altered ACE2 expression resulting in increased risk of severe COVID-19 and the associated mortality. A drug that could increase ACE2 activity and inhibit cellular uptake of severe acute respiratory syndrome coronavirus 2 (SARs-CoV2), thus decrease infection, would be of high relevance to cardiovascular disease, diabetes and SARs-CoV2 infection. While the need for such a drug lead was highlighted over a decade ago receiving over 600 citations,1 to date, no such drugs are available.2 Here, we report the development of a novel ACE2 stimulator, designated '2A'(international PCT filed), which is a 10 amino acid peptide derived from a snake venom, and demonstrate its in vitro and in vivo efficacy against SARs-CoV2 infection and associated lung inflammation. Peptide 2A also provides remarkable protection against glycaemic dysregulation, weight loss and disease severity in a mouse model of type 1 diabetes. No untoward effects of 2A were observed in these pre-clinical models suggesting its strong clinical translation potential.

Environmentally persistent free radicals enhance SARS-CoV-2 replication in respiratory epithelium.

Epidemiological evidence links lower air quality with increased incidence and severity of COVID-19; however, mechanistic data have yet to be published. We hypothesized air pollution-induced oxidative stress in the nasal epithelium increased viral replication and inflammation. Nasal epithelial cells (NECs), collected from healthy adults, were grown into a fully differentiated epithelium. NECs were infected with the ancestral strain of SARS-CoV-2. An oxidant combustion by-product found in air pollution, the environmentally persistent free radical (EPFR) DCB230, was used to mimic pollution exposure four hours prior to infection. Some wells were pretreated with antioxidant, astaxanthin, for 24 hours prior to EPFR-DCB230 exposure and/or SARS-CoV-2 infection. Outcomes included viral replication, epithelial integrity, surface receptor expression (ACE2, TMPRSS2), cytokine mRNA expression (TNF-α, IFN-ß), intracellular signaling pathways, and oxidative defense enzymes. SARS-CoV-2 infection induced a mild phenotype in NECs, with some cell death, upregulation of the antiviral cytokine IFN-ß, but had little effect on intracellular pathways or oxidative defense enzymes. Prior exposure to EPFR-DCB230 increased SARS-CoV-2 replication, upregulated TMPRSS2 expression, increased secretion of the proinflammatory cytokine TNF-α, inhibited expression of the mucus producing MUC5AC gene, upregulated expression of p21 (apoptosis pathway), PINK1 (mitophagy pathway), and reduced levels of antioxidant enzymes. Pretreatment with astaxanthin reduced SARS-CoV-2 replication, downregulated ACE2 expression, and prevented most, but not all EPFR-DCB230 effects. Our data suggest that oxidant damage to the respiratory epithelium may underly the link between poor air quality and increased COVID-19. The apparent protection by antioxidants warrants further research.

Environmental Impacts on COVID-19: Mechanisms of Increased Susceptibility.

Background: Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in >554M cases and >6.3M deaths worldwide. The disease caused by SARS-CoV-2, COVID-19, has resulted in a broad range of clinical symptoms differing in severity. Initially, the elderly were identified as particularly susceptible to severe COVID-19, with children experiencing less severe disease. However, as new variants arise, the epidemiology of SARS-CoV-2 infection is changing, and the disease severity in children is increasing. While environmental impacts on COVID-19 have been described, the underlying mechanisms are poorly described. Objective: The Pacific Basin Consortium for Environment and Health (PBC) held meeting on September 16, 2021, to explore environmental impacts on infectious diseases, including COVID-19. Methods: The PBC is an international group of environmental scientists and those interested in health outcomes. The PBC met to present preliminary data and discuss the role of exposures to airborne pollutants in enhancing susceptibility to and severity of respiratory tract viral infections, including COVID-19. Findings: Analysis of the literature and data presented identified age as an important factor in vulnerability to air pollution and enhanced COVID-19 susceptibility and severity. Mechanisms involved in increasing severity of COVID-19 were discussed, and gaps in knowledge were identified. Conclusions: Exposure to particulate matter (PM) pollution enhanced morbidity and mortality to COVID-19 in a pediatric population associated with induction of oxidative stress. In addition, free radicals present on PM can induce rapid changes in the viral genome that can lead to vaccine escape, altered host susceptibility, and viral pathogenicity. Nutritional antioxidant supplements have been shown to reduce the severity of viral infections, inhibit the inflammatory cytokine storm, and boost host immunity and may be of benefit in combating COVID-19.

Ancestral SARS-CoV-2, but not Omicron, replicates less efficiently in primary pediatric nasal epithelial cells.

Children typically experience more mild symptoms of Coronavirus Disease 2019 (COVID-19) when compared to adults. There is a strong body of evidence that children are also less susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remain to be determined. Here, we use primary nasal epithelial cells (NECs) from children and adults, differentiated at an air-liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the NECs of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the NECs of children. Importantly, the Delta variant also replicated to significantly lower titers in the NECs of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves.

Resveratrol and Astaxanthin Protect Primary Human Nasal Epithelial Cells Cultured at an Air-liquid Interface from an Acute Oxidant Exposure.

Oxidative stress (OS) in the airway epithelium is associated with cell damage, inflammation, and mitochondrial dysfunction that may initiate or worsen respiratory disease. However, it is unclear whether exogenous antioxidants can provide protection to the airway epithelium from OS. Resveratrol and astaxanthin are nutritional compounds that have shown diverse benefits including protection against OS and inflammation in various situations. The aim of this study was to examine the utility of pre-treatment with resveratrol and astaxanthin to prevent the negative effects of oxidant exposure and restore redox homeostasis in a well-differentiated epithelium grown from primary human nasal epithelial cells (NECs) at the air-liquid interface. Fully differentiated NECs were pretreated with the antioxidants for 24 hours and the cultured epithelia was subsequently exposed to hydrogen peroxide (H2O2) for 1 hour to induce an acute OS. Responses measured included mitochondrial reactive oxygen species (mtROS) generation, redox status (GSH/GSSG ratio), cellular ATP, and signaling pathways (SIRT1, FOXO3, p21, PINK1, PARKIN, NRF2). Following H2O2 exposure, mtROS production increased by 4-fold compared with control (p<0.01) and pre-treatment with resveratrol or astaxanthin reduced this by 50% (p<0.05). H2O2 exposure reduced GSH/GSSG ratio and this decline was prevented by antioxidants pre-treatment. H2O2 exposure caused 2.5-fold increase in p21 mRNA expression compared with control (p<0.05), while a slight decrease in p21 mRNA expression was observed when cells were pre-treated with resveratrol or astaxanthin. Our results demonstrate that antioxidants, resveratrol, and astaxanthin were able to protect cells from an acute OS. These agents show promise that encourages further research.

Redox Homeostasis in Well-differentiated Primary Human Nasal Epithelial Cells.

Oxidative stress (OS) in the airway epithelium is associated with inflammation, cell damage, and mitochondrial dysfunction that may initiate or worsen respiratory disease. Redox regulation maintains the equilibrium of pro-oxidant/antioxidant reactions but can be disturbed by environmental exposures. The mechanism(s) underlying the induction and impact of OS on airway epithelium and how these influences on respiratory disease is poorly understood. The aim of this study was to develop a stress response model in primary human nasal epithelial cells (NECs) grown at the air-liquid interface (ALI) into a well-differentiated epithelium and to use this model to investigate the mechanisms underlying OS. Hydrogen peroxide (H2O2) was used to induce acute OS and the responses were measured with trans epithelial electrical resistance (TEER), membrane permeability, cell death (LDH release), mitochondrial reactive oxygen species (mtROS) generation, redox status (GSH/GSSG ratio), cellular ATP, and signaling pathways (SIRT1, FOXO3, p53, p21, PINK1, PARKIN, NRF2). Following 25 mM (sensitive) or 50mM (resistant) H2O2 exposure, cell integrity decreased (p<0.05), GSH/GSSG ratio reduced (p<0.05), and ATP production declined by 83% (p<0.05) in the sensitive and 55% (p<0.05) in the resistant group; mtROS production increased 3.4-fold (p<0.001). Significant inter-individual differences between healthy humans with regards to susceptibility to OS, and differential activation of various pathways (FOXO3, PARKIN) were observed. These intra-individual differences in susceptibility to OS may be attributed to resistant individuals having more mitochondria or greater mitochondrial function.

CXC chemokine ligand-13 promotes metastasis via CXCR5-dependent signaling pathway in non-small cell lung cancer.

The CXC chemokine ligand-13 (CXCL13) is a chemoattractant of B cells and has been implicated in the progression of many cancers. So far, CXCL13 and its related receptor CXCR5 have been proved to regulate cancer cell migration as well as tumour metastasis. However, the role of CXCL13-CXCR5 axis in metastasis of lung cancer is still poorly understood. In this study, we found that CXCL13 and CXCR5 were commonly up-regulated in lung cancer specimens compared with normal tissues among different cohorts. Our evidence showed that CXCL13 obviously promoted migration of lung cancer cells, and this effect was mediated by vascular cell adhesion molecule-1 (VCAM-1) expression. We also confirmed that CXCR5, the major receptor responsible for CXCL13 function, was required for CXCL13-promoted cell migration. We also test the candidate components which are activated after CXCL13 treatment and found that phospholipase C-ß (PLCß), protein kinase C-α (PKCα) and c-Src signalling pathways were involved in CXCL13-promoted cell migration and VCAM-1 expression in lung cancer cells. Finally, CXCL13 stimulated NF-κB transcription factor in lung cancer cells, contributing to VCAM-1 expression in translational level. These evidences propose a novel insight into lung cancer metastasis which is regulated by CXCL13.

Involvement of matrix metalloproteinase-3 in CCL5/CCR5 pathway of chondrosarcomas metastasis.

CCL5 (previously called RANTES) was originally recognized as a product of activated T cells, and plays a crucial role in the migration and metastasis of human cancer cells. It has been reported that the effect of CCL5 is mediated via CCR receptors. We found that human chondrosarcoma tissues had significant expression of the CCL5 and CCR5, which was higher than that in normal cartilage. We also found CCL5 increased the migration and matrix metalloproteinases-3 (MMP)-3 expression in human chondrosarcoma cells (JJ012 cells). In addition, MMP-3 small interfering RNA and inhibitor inhibited the CCL5-induced cell migration. Activations of phosphatidylinositol 3-kinase (PI3K), Akt and NF-kappaB pathways after CCL5 treatment was demonstrated, and CCL5-induced expression of MMP-3 and migration activity was inhibited by the specific inhibitor of PI3K, Akt and NF-kappaB cascades. Taken together, these results indicate that CCL5 and CCR5 interaction enhanced migration of chondrosarcoma cells through the increase of MMP-3 production.