Lung Inflammatory Response Research Articles

Exposure to Particulate Matter in the Broiler House Causes Dyslipidemia and Exacerbates It by Damaging Lung Tissue in Broilers

The high concentration of particulate matter (PM) in broiler houses seriously endangers the biological safety of broilers and causes low growth performance, deserving more attention. This study aimed to investigate the effects of PM collected from a broiler house on the lung and systemic inflammatory responses and liver lipid anabolic process in broilers. Broilers were systemically exposed to fresh air (control) and 4 mg·m-3 and 8 mg·m-3 total suspended particles (TSP). Lung, liver, and serum were sampled after 7 (E7) and 14 (E14) days of PM exposure and 7 days after self-recovery (R 7). Corresponding kits were used to assay the inflammatory cytokines and serum biochemical indicators. The expression levels of genes related to lipid metabolism were detected by real-time polymerase chain reaction (RT-PCR) assay. The results showed a significant decrease in the average daily gain in broilers for 7 days of PM exposure (p < 0.05) and clear lung and liver inflammations in PM groups. In addition, upregulation of lung interleukin (IL)-1β and IL-8 and serum low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) occurred after 7 days of PM exposure (p < 0.05), and upregulation of lung serum tumor necrosis factor (TNF)-α and cholesterol (CHOL) occurred after 14 days of PM exposure (p < 0.05). A decrease in serum total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-px) levels was found after 14 days of PM exposure (p < 0.05), and the GSH-px level was maintained until 7 days after cessation of exposure (p < 0.05). Seven days after cessation of exposure, the expression levels of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) and fatty acid synthase (Fas) genes significantly increased (p < 0.05) and decreased (p < 0.05), respectively. These results demonstrate that exposure to PM in broiler houses can induce systemic inflammation and dyslipidemia through local pulmonary inflammation and also exert toxic effects on the liver by disturbing the expression of genes involved in the hepatic lipid anabolic process.

C57Bl/6N mice have an attenuated lung inflammatory response to dsRNA compared to C57Bl/6J and BALB/c mice

BackgroundLower respiratory infections caused by ssRNA viruses are a major health burden globally. Translational mouse models are a valuable tool for medical research, including research on respiratory viral infections. In in vivo mouse models, synthetic dsRNA can be used as a surrogate for ssRNA virus replication. However, studies investigating how genetic background of mice impacts the murine lung inflammatory response to dsRNA is lacking. Hence, we have compared lung immunological responses of BALB/c, C57Bl/6N and C57Bl/6J mice to synthetic dsRNA.MethodsdsRNA was administered intranasally to BALB/c, C57Bl/6N and C57Bl/6J mice once/day for three consecutive days. Lactate dehydrogenase (LDH) activity, inflammatory cells, and total protein concentration were analyzed in bronchoalveolar lavage fluid (BALF). Pattern recognition receptors levels (TLR3, MDA5 and RIG-I) were measured in lung homogenates using RT-qPCR and western blot. Gene expression of IFN-β, TNF-α, IL-1β and CXCL1 was assessed in lung homogenates by RT-qPCR. ELISA was used to analyze protein concentrations of CXCL1 and IL-1β in BALF and lung homogenates.ResultsBALB/c and C57Bl/6J mice showed infiltration of neutrophils to the lung, and an increase in total protein concentration and LDH activity in response to dsRNA administration. Only modest increases in these parameters were observed for C57Bl/6N mice. Similarly, dsRNA administration evoked an upregulation of MDA5 and RIG-I gene and protein expression in BALB/c and C57Bl/6J, but not C57Bl/6N, mice. Further, dsRNA provoked an increase in gene expression of TNF-α in BALB/c and C57Bl/6J mice, IL-1β only in C57Bl/6N mice and CXCL1 exclusively in BALB/c mice. BALF levels of CXCL1 and IL-1β were increased in BALB/c and C57Bl/6J mice in response to dsRNA, whereas the response of C57Bl/6N was blunt. Overall, inter-strain comparisons of the lung reactivity to dsRNA revealed that BALB/c, followed by C57Bl/6J, had the most pronounced respiratory inflammatory responses, while the responses of C57Bl/6N mice were attenuated.ConclusionsWe report clear differences of the lung innate inflammatory response to dsRNA between BALB/c, C57Bl/6J and C57Bl/6N mice. Of particular note, the highlighted differences in the inflammatory response of C57Bl/6J and C57Bl/6N substrains underscore the value of strain selection in mouse models of respiratory viral infections.

SARS-CoV-2-Induced TSLP Is Associated with Duration of Hospital Stay in COVID-19 Patients.

Thymic stromal lymphopoietin (TSLP) is an epithelium-derived pro-inflammatory cytokine involved in lung inflammatory responses. Previous studies show conflicting observations in blood TSLP in COVID-19, while none report SARS-CoV-2 inducing TSLP expression in bronchial epithelial cells. Our objective in this study was to determine whether TSLP levels increase in COVID-19 patients and if SARS-CoV-2 induces TSLP expression in bronchial epithelial cells. Plasma cytokine levels were measured in patients hospitalized with confirmed COVID-19 and age- and sex-matched healthy controls. Demographic and clinical information from COVID-19 patients was collected. We determined associations between plasma TSLP and clinical parameters using Poisson regression. Cultured human nasal (HNEpC) and bronchial epithelial cells (NHBEs), Caco-2 cells, and patient-derived bronchial epithelial cells (HBECs) obtained from elective bronchoscopy were infected in vitro with SARS-CoV-2, and secretion as well as intracellular expression of TSLP was detected by immunofluorescence. Increased TSLP levels were detected in the plasma of hospitalized COVID-19 patients (603.4 ± 75.4 vs 997.6 ± 241.4 fg/mL, mean ± SEM), the levels of which correlated with duration of stay in hospital (β: 0.11; 95% confidence interval (CI): 0.01-0.21). In cultured NHBE and HBECs but not HNEpCs or Caco-2 cells, TSLP levels were significantly elevated after 24 h post-infection with SARS-CoV-2 (p < 0.001) in a dose-dependent manner. Plasma TSLP in COVID-19 patients significantly correlated with duration of hospitalization, while SARS-CoV-2 induced TSLP secretion from bronchial epithelial cells in vitro. Based on our findings, TSLP may be considered an important therapeutic target for COVID-19 treatment.

Bacterial Outer Membrane Vesicles Promote Lung Inflammatory Responses and Macrophage Activation via Multi-Signaling Pathways.

Emerging evidence suggests that Gram-negative bacteria release bacterial outer membrane vesicles (OMVs) and that these play an important role in the pathogenesis of bacterial infection-mediated inflammatory responses and organ damage. Despite the fact that scattered reports have shown that OMVs released from Gram-negative bacteria may function via the TLR2/4-signaling pathway or induce pyroptosis in macrophages, our study reveals a more complex role of OMVs in the development of inflammatory lung responses and macrophage pro-inflammatory activation. We first confirmed that various types of Gram-negative bacteria release similar OMVs which prompt pro-inflammatory activation in both bone marrow-derived macrophages and lung alveolar macrophages. We further demonstrated that mice treated with OMVs via intratracheal instillation developed significant inflammatory lung responses. Using mouse inflammation and autoimmune arrays, we identified multiple altered cytokine/chemokines in both bone marrow-derived macrophages and alveolar macrophages, suggesting that OMVs have a broader spectrum of function compared to LPS. Using TLR4 knock-out cells, we found that OMVs exert more robust effects on activating macrophages compared to LPS. We next examined multiple signaling pathways, including not only cell surface antigens, but also intracellular receptors. Our results confirmed that bacterial OMVs trigger both surface protein-mediated signaling and intracellular signaling pathways, such as the S100-A8 protein-mediated pathway. In summary, our studies confirm that bacterial OMVs strongly induced macrophage pro-inflammatory activation and inflammatory lung responses via multi-signaling pathways. Bacterial OMVs should be viewed as a repertoire of pathogen-associated molecular patterns (PAMPs), exerting more robust effects than Gram-negative bacteria-derived LPS.

Issues in the inhalation toxicity testing and hazard assessment for low density particulate materials such as synthetic amorphous silica (SAS)

Inhalation toxicity testing of particulate materials is mandated for classification. According to CLP, particulate materials should be tested as marketed and many particulate materials are marketed as non-respirable particles. However, OECD TG 413 requires exposure to particle sizes that are respirable and reach the alveoli. The requirement for exposure of rats to respirable particles is thus in contrast to CLP and requires the application of high shear forces. The exposure to artificially small particles causes a number of issues that hamper the interpretation of the results of the testing. These issues are aerosol altering in the exposure system, assessment of the adversity of the inflammatory lung responses, inclusion of recovery groups, and extrapolation of the results to humans exposed under occupational condition. In addition, effects of many particulate materials after testing according to OECD 413 are not intrinsic properties, but a general reaction of the lung to the deposited material, show very similar NOAECs for chemical diverse materials, and often are completely reversible.

GPR109A controls neutrophil extracellular traps formation and improve early sepsis by regulating ROS/PAD4/Cit-H3 signal axis

BackgroundNeutrophil extracellular traps (NETs) is the key means for neutrophils to resist bacterial invasion. Sepsis is a systemic inflammatory response syndrome caused by infection.MethodsIn our study, qRT-PCR was used to detect the gene expression in neutrophils, Western blot was used to detect the protein expression in mouse tissues and neutrophils, flow cytometry was used to detect the purity of neutrophils in the whole blood and immunofluorescence was used to detect the NETs formation.ResultsIn this study, we analyzed the NETs formation in the blood of patients with sepsis. The results showed that a large number of NETs appeared. And the expression of GPR109A in neutrophils of patients with sepsis was significantly up regulated. Then we collected neutrophils from WT mice and GPR109A−/− mice and found that GPR109A knockout could significantly inhibit the early NETs formation of neutrophils. The results also showed that knockout of GPR109A or inhibition of the NETs formation could increase the inflammatory response of liver, spleen, lung and kidney in mice, thus affecting the disease process of sepsis. Then we observed the death of mice in 16 days. The results showed that inhibiting the NETs formation could significantly affect the early mortality of mice, while knocking out GPR109A could directly affect the mortality of the whole period.ConclusionsThis study confirmed the regulatory effect of GPR109A on early NETs formation for the first time, and provided a new target for the treatment of sepsis.

Effect and mechanism of apelin on lipopolysaccharide induced acute pulmonary vascular endothelial barrier dysfunction

Vascular endothelial barrier dysfunction is the most prominent manifestation and important cause of mortality in infectious acute lung injury (ALI). Exogenous apelin is effective in ameliorating lipopolysaccharide (LPS)-induced inflammatory response in ALI lungs, reducing exudation of lung tissue and decreasing mortality. This study set out to investigate the association between apelin and Friend leukemia integration-1 (Fli-1) in the prevention and treatment of ALI, and to elucidate the molecular mechanism by which apelin protects the permeability of the vascular endothelial barrier. At the vivo functional level, lung wet/dry weight ratio was used to detect whole lung permeability, evans blue assay and dual fluorescent protein tracking assay were used to detect lung vascular endothelial permeability, HE staining to observe the inflammatory status of lung tissue, and immunofluorescence staining for VE-cadherin expression levels in blood vessels. The changes in inflammatory factors in bronchoalveolar lavage fluid (BALF) were detected by ELASA. Western blot was used to detect the expression level of proteins. qRT-PCR was performed to detect changes in mRNA expression of Fli-1 and adherent junction-related proteins. The correlation analysis of Fli-1 with vascular endothelial permeability and SRC showed that Fli-1 participated in the process of ALI. After preventive and therapeutic treatment of ALI mice with exogenous apelin, Fli-1, APJ, VE-cadherin, phosphorylated-VE-cadherin (p-VE-cadherin) and β-catenin were up-regulated, while SRC, phosphorylated-SRC (p-SRC), VEGF and VEGF-R were down-regulated, which indicated that the stability of vascular endothelial barrier was enhanced. With the use of Fli-1 inhibitor irinotecan, the protective effect of apelin was weakened in various functional indexes, genes and proteins. The lung was maintained at the level of the injury. Our research shows that Fli-1 is involved in the LPS-induced ALI process. The molecular mechanism for apelin in preventing endothelial barrier dysfunction in ALI is through up-regulating Fli-1, thus regulating adherens junction-related proteins, and finally recovering the endothelial barrier function.

Toll-like receptor 2 mediates Acanthamoeba-induced allergic airway inflammatory response in mice.

Repeated intranasal exposure to Acanthamoeba has been revealed to induce allergic airway inflammatory responses in mice. Based on the role of toll-like receptors (TLRs) in the pathogenesis of allergic asthma, TLRs form a link between innate and adaptive immune responses, and play an important role in the activation of various cells in the innate immune system. To determine the TLRs that are related to these immune responses, we assessed the expression levels of inflammation-related genes in mouse lung epithelial (MLE)-12 cells treated with excretory-secretory proteins (ES-P) of the Acanthamoeba strain (KA/E2) with or without the TLR antagonists. The expression levels of inflammation-related genes, such as eotaxin, TARC, macrophage-derived chemokine (MDC), and TSLP, in the TLR2 and TLR9 antagonist treatment groups were decreased, compared to those in the ES-P alone or other TLR antagonist treatment groups. In particular, a greater decrease in the relevant gene expression levels was found in the TLR2 antagonist treatment group than in the TLR9 antagonist treatment group. Allergic airway inflammation was evaluated in the wild-type (WT) and TLR2 knockout (KO) groups following KA/E2 exposure. Based on the results, allergic airway inflammatory responses (airway resistance value, inflammatory cell infiltration, Th2-related cytokine expression, mucin production, and metaplasia of lung epithelial cells and goblet cells) by KA/E2 were reduced in the TLR2 KO groups. In addition, TLR2 knockout BMDCs displayed lower activation of surface markers owing to ES-P stimulation than normal BMDCs, and KA/E2 ES-P-treated TLR2-depleted BMDCs produced fewer Th2 cytokine-expressing cells from naïve T cells than WT BMDCs. When ES-P was administered after primary lung cells were obtained from WT and TLR2 KO mice, the expression levels of inflammation-related genes were found to be significantly decreased in TLR2 KO cells compared to those in WT cells. These results suggest that TLR2 is involved in lung inflammatory response activation in KA/E2 intranasal infection, especially in airway tissue.

Therapeutic Targeting of Inflammation and Virus Simultaneously Ameliorates Influenza Pneumonia and Protects from Morbidity and Mortality.

Influenza pneumonia is a severe complication caused by inflammation of the lungs following infection with seasonal and pandemic strains of influenza A virus (IAV), that can result in lung pathology, respiratory failure, and death. There is currently no treatment for severe disease and pneumonia caused by IAV. Antivirals are available but are only effective if treatment is initiated within 48 h of onset of symptoms. Influenza complications and mortality are often associated with high viral load and an excessive lung inflammatory cytokine response. Therefore, we simultaneously targeted the virus and inflammation. We used the antiviral oseltamivir and the anti-inflammatory drug etanercept to dampen TNF signaling after the onset of clinical signs to treat pneumonia in a mouse model of respiratory IAV infection. The combined treatment down-regulated the inflammatory cytokines TNF, IL-1β, IL-6, and IL-12p40, and the chemokines CCL2, CCL5, and CXCL10. Consequently, combined treatment with oseltamivir and a signal transducer and activator of transcription 3 (STAT3) inhibitor effectively reduced clinical disease and lung pathology. Combined treatment using etanercept or STAT3 inhibitor and oseltamivir dampened an overlapping set of cytokines. Thus, combined therapy targeting a specific cytokine or cytokine signaling pathway and an antiviral drug provide an effective treatment strategy for ameliorating IAV pneumonia. This approach might apply to treating pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Pulmonary inflammation, oxidative stress, and fibrosis in a mouse model of cholestasis: the potential protective properties of the dipeptide carnosine.

Cholestasis is a clinical complication that primarily influences the liver. However, it is well known that many other organs could be affected by cholestasis. Lung tissue is a major organ influenced during cholestasis. Cholestasis-induced lung injury could induce severe complications such as respiratory distress, serious pulmonary infections, and tissue fibrosis. Unfortunately, there is no specific pharmacological intervention against this complication. Several studies revealed that oxidative stress and inflammatory response play a role in cholestasis-induced lung injury. Carnosine (CARN) is a dipeptide found at high concentrations in different tissues of humans. CARN's antioxidant and antiinflammatory properties are repeatedly mentioned in various experimental models. This study aimed to assess the role of CARN on cholestasis-induced lung injury. Rats underwent bile duct ligation (BDL) to induce cholestasis. Broncho-alveolar lavage fluid (BALF) levels of inflammatory cells, pro-inflammatory cytokines, and immunoglobulin were monitored at scheduled intervals (7, 14, and 28days after BDL). Moreover, lung tissue histopathological alterations and biomarkers of oxidative stress were evaluated. A significant increase in BALF inflammatory cells, TNF-α, IL-1β, IL-6, and immunoglobulin-G (IgG) was detected in the BALF of BDL rats. Moreover, lung tissue histopathological changes, collagen deposition, increased TGF-β, and elevated levels of oxidative stress biomarkers were evident in cholestatic animals. It was found that CARN (100 and 500mg/kg, i.p.) significantly alleviated lung oxidative stress biomarkers, inflammatory response, tissue fibrosis, and histopathological alterations. These data indicate the potential protective properties of CARN in the management of cholestasis-induced pulmonary damage. The effects of CARN on inflammatory response and oxidative stress biomarkers seems to play a crucial role in its protective properties in the lung of cholestatic animals.

ITGB4 deficiency in airway epithelia enhances HDM-induced airway inflammation through hyperactivation of TLR4 signaling pathway.

Airway epithelial cells (AECs) are the first cell barrier of the respiratory system against external stimuli that play a critical role in the development of asthma. It is known that AECs play a key role in asthma susceptibility and severity. ITGB4 is a downregulated adhesion molecule in the airway epithelia of asthma patients, which was involved in the exaggerated lung inflammation after allergy stimulation. Toll-like receptor 4 (TLR4) in AECs has also been shown to play a crucial role in the development of lung inflammation in asthma patients. However, the specific intrinsic regulatory mechanism of TLR4 in AECs are still obscure. In this article, we demonstrated that ITGB4 deficiency in AECs enhances HDM-induced airway inflammation through hyperactivation of the TLR4 signaling pathway, which is mediated by inhibition of FYN phosphorylation. Moreover, TLR4-antagonist treatment or blockade of FYN can inhibit or exaggerate lung inflammation in HDM-stressed ITGB4-deficient mice, separately. Together, these results demonstrated that ITGB4 deficiency in AECs enhances HDM-induced lung inflammatory response through the ITGB4-FYN-TLR4 axis, which may provide new therapeutic approaches for the management of lung inflammation in asthma.

Role of Myeloid Cell-Specific TLR9 in Mitochondrial DNA-Induced Lung Inflammation in Mice.

Mitochondrial dysfunction is common in various pathological conditions including obesity. Release of mitochondrial DNA (mtDNA) during mitochondrial dysfunction has been shown to play a role in driving the pro-inflammatory response in leukocytes including macrophages. However, the mechanisms by which mtDNA induces leukocyte inflammatory responses in vivo are still unclear. Moreover, how mtDNA is released in an obese setting has not been well understood. By using a mouse model of TLR9 deficiency in myeloid cells (e.g., macrophages), we found that TLR9 signaling in myeloid cells was critical to mtDNA-mediated pro-inflammatory responses such as neutrophil influx and chemokine production. mtDNA release by lung macrophages was enhanced by exposure to palmitic acid (PA), a major saturated fatty acid related to obesity. Moreover, TLR9 contributed to PA-mediated mtDNA release and inflammatory responses. Pathway analysis of RNA-sequencing data in TLR9-sufficient lung macrophages revealed the up-regulation of axon guidance molecule genes and down-regulation of metabolic pathway genes by PA. However, in TLR9-deficient lung macrophages, PA down-regulated axon guidance molecule genes, but up-regulated metabolic pathway genes. Our results suggest that mtDNA utilizes TLR9 signaling in leukocytes to promote lung inflammatory responses in hosts with increased PA. Moreover, TLR9 signaling is involved in the regulation of axon guidance and metabolic pathways in lung macrophages exposed to PA.

Bacterial expression of a designed single‐chain IL‐10 prevents severe lung inflammation

Interleukin‐10 (IL‐10) is an anti‐inflammatory cytokine that is active as a swapped domain dimer and is used in bacterial therapy of gut inflammation. IL‐10 can be used as treatment of a wide range of pulmonary diseases. Here we have developed a non‐pathogenic chassis (CV8) of the human lung bacterium Mycoplasma pneumoniae (MPN) to treat lung diseases. We find that IL‐10 expression by MPN has a limited impact on the lung inflammatory response in mice. To solve these issues, we rationally designed a single‐chain IL‐10 (SC‐IL10) with or without surface mutations, using our protein design software (ModelX and FoldX). As compared to the IL‐10 WT, the designed SC‐IL10 molecules increase the effective expression in MPN four‐fold, and the activity in mouse and human cell lines between 10 and 60 times, depending on the cell line. The SC‐IL10 molecules expressed in the mouse lung by CV8 in vivo have a powerful anti‐inflammatory effect on Pseudomonas aeruginosa lung infection. This rational design strategy could be used to other molecules with immunomodulatory properties used in bacterial therapy.

STV-Na attenuates lipopolysaccharide-induced lung injury in mice via the TLR4/NF-kB pathway.

Acute lung injury (ALI) is a potentially fatal disorder that is largely caused by inflammation. Sodium isostevanol (STV-Na) is a terpenoid produced from stevioside, which possesses anti-inflammatory and antioxidative stress characteristics. nevertheless, it is still unclear how STV-Na affects ALI. Therefore, we investigated the possible STV-Na therapeutic impacts on lipopolysaccharide (LPS)-induced (ALI). We employed hematoxylin-eosin staining to observe the impact of STV-Na on lung histopathological alterations and used kits to detect the oxidative stress status of lung tissues, such as superoxide dismutase, malondialdehyde, and glutathione. The reactive oxygen speciesand myeloperoxidaseexpression in the tissues of lung was assessed by immunofluorescence and immunohistochemistry. Additionally, we detected the impact of STV-Na on inflammatory cell infiltration in lung tissue using Wright-Giemsa staining solution and immunohistochemistry, which was found to reduce inflammation in lung tissue by enzyme-linked immunosorbent assay. Finally, using WB, we examined the impact of STV-Na on the TLR4/NF-kB pathway. We observed that STV-Na attenuated lung histopathological alterations in LPS-induced lung damage in mice, reduced infiltration of inflammatory cell and oxidative stress in the tissue of lung, and via the TLR4/NF-kB pathway, there is a reduction in the inflammatory responses in mouse lung tissue. These outcomes indicate that the response of inflammatory cells to LPS-induced ALI in mice was attenuated by STV-Na.

Sleep quality in patients of chronic obstructive pulmonary disease

Background: Chronic obstructive pulmonary disease (COPD) is a common respiratory ailment with persistent airflow limitation associated with an enhanced chronic inflammatory response in airways and lungs to noxious particles or gases. This study aims to observe the sleep quality in these cases as it is known that most of them suffer from disturbed sleep. Aims and Objectives: The aims of this study were to assess the sleep quality in stable patients of COPD and compare with healthy controls. Material and Methods: This cross-sectional observational study was approved by ICMR and IEC and conducted in the department of Physiology and the Department of TB and Chest Medicine, Lady Hardinge Medical College, New Delhi. In this study, 33 clinically stable COPD patients and 33 normal subjects as controls were included in the study. Then, a detailed history including the demographic characteristics was recorded. Then, Pittsburg Sleep Quality Index (PSQI) Questionnaire (Hindi version was obtained from MAPI organization) was recorded by direct interview before lung functions were assessed by Spirometry. Results: In his study, COPD was found predominantly in male subjects compared to females. Poor sleep was found in majority of COPD cases. The median global PSQI scores were much higher in COPD cases compared to controls and the difference between them was statistically highly significant (P &lt; 0.001). Conclusion: This study shows that the prevalence of poor sleep quality among COPD patients of urban Delhi is very high. Limitations are small sample size and hospital-based subjects and lack of polysomnography.

In vitro immunogenic profile of recombinant SARS-CoV2 S1-RBD peptide in murine macrophage and microglial cells.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can infect common mice inducing significant pathological lung lesions and inflammatory responses. This substantially mimics coronavirus disease 19 (COVID-19) infection and pathogenesis in humans. To characterise the effects of recombinant SARS-CoV-2 S1 receptor-binding domain (RBD) peptide in murine macrophage and microglial cells' immune activation compared with classical PAMPs in vitro. Murine RAW 264.7 macrophages and BV2 microglial cells were exposed to increasing concentrations of the RBD peptide (0.01, 0.05, and 0.1 µg/mL), Lipopolysaccharide (LPS) and Poly(I:C) and evaluated after two and 24 h for significant markers of macrophage activation. We determined the effects of RBD peptide on cell viability, cleaved caspase 3 expressions, and nuclear morphometry analysis. In RAW cells, RBD peptide was cytotoxic, but not for BV2 cells. RAW cells presented increased arginase activity and IL-10 production; however, BV2 cells expressed iNOS and IL-6 after RBD peptide exposure. In addition, RAW cells increased cleaved-caspase-3, apoptosis, and mitotic catastrophe after RBD peptide stimulation but not BV2 cells. RBD peptide exposure has different effects depending on the cell line, exposure time, and concentration. This study brings new evidence about the immunogenic profile of RBD in macrophage and microglial cells, advancing the understanding of SARS-Cov2 immuno- and neuropathology.

Citrus peel extract protects against diesel exhaust particle-induced chronic obstructive pulmonary disease-like lung lesions and oxidative stress.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide and characterized by emphysema, small airway remodeling and mucus hypersecretion. Citrus peels have been widely used as food spices and in traditional Chinese medicine for chronic lung disease. Given that citrus peels are known for containing antioxidants and anti-inflammatory compounds, we hypothesize that citrus peel intake can suppress oxidative stress and inflammatory response to air pollution exposure, thereby alleviating COPD-like pathologies. This study aimed to investigate the efficacy of citrus peel extract, namely Guang Chenpi (GC), in preventing the development of COPD induced by diesel exhaust particles (DEPs) and its potential mechanism. DEP-induced COPD-like lung pathologies, inflammatory responses and oxidative stress with or without GC treatment were examined in vivo and in vitro. Our in vivo study showed that GC was effective in decreasing inflammatory cell counts and inflammatory mediator (IL-17A and TNF-α) concentrations in bronchoalveolar lavage fluid (BALF). Pretreatment with GC extract also significantly decreased oxidative stress in the serum and lung tissue of DEP-induced COPD rats. Furthermore, GC pretreatment effectively reduced goblet cell hyperplasia (PAS positive cells) and fibrosis of the small airways, decreased macrophage infiltration as well as carbon loading in the peripheral lungs, and facilitated the resolution of emphysema and small airway remodeling in DEP-induced COPD rats. An in vitro free radical scavenging assay revealed robust antioxidant potential of GC in scavenging DPPH free radicals. Moreover, GC demonstrated potent capacities in reducing ROS production and enhancing SOD activity in BEAS-2B cells stimulated by DEPs. GC treatment significantly attenuated the increased level of IL-8 and MUC5AC from DEP-treated BEAS-2B cells. Mechanistically, GC treatment upregulated the protein level of Nrf-2 and could function via MAPK/NF-κB signaling pathways by suppressing the phosphorylation of p38, JNK and p65. Citrus peel extract is effective in decreasing oxidative stress and inflammatory responses of the peripheral lungs to DEP exposure. These protective effects further contributed to the resolution of COPD-like pathologies.

Effects of chronic secondhand smoke exposure on cardiovascular regulation and the role of soluble epoxide hydrolase in mice.

Background: Secondhand smoke (SHS) is a significant risk factor for cardiovascular morbidity and mortality with an estimated 80% of SHS-related deaths attributed to cardiovascular causes. Public health measures and smoking bans have been successful both in reducing SHS exposure and improving cardiovascular outcomes in non-smokers. Soluble epoxide hydrolase (sEH) inhibitors have been shown to attenuate tobacco exposure-induced lung inflammatory responses, making them a promising target for mitigating SHS exposure-induced cardiovascular outcomes. Objectives: The objectives of this study were to determine 1) effects of environmentally relevant SHS exposure on cardiac autonomic function and blood pressure (BP) regulation and 2) whether prophylactic administration of an sEH inhibitor (TPPU) can reduce the adverse cardiovascular effects of SHS exposure. Methods: Male C57BL/6J mice (11weeks old) implanted with BP/electrocardiogram (ECG) telemetry devices were exposed to filtered air or 3mg/m3 of SHS (6hr/d, 5d/wk) for 12weeks, followed by 4weeks of recovery in filtered air. Some mice received TPPU in drinking water (15mg/L) throughout SHS exposure. BP, heart rate (HR), HR variability (HRV), baroreflex sensitivity (BRS), and BP variability were determined monthly. Results: SHS exposure significantly decreased 1) short-term HRV by ∼20% (p < 0.05) within 4weeks; 2) overall HRV with maximum effect at 12weeks (-15%, p < 0.05); 3) pulse pressure (-8%, p < 0.05) as early as week 4; and 4) BRS with maximum effect at 12weeks (-11%, p < 0.05). Four weeks of recovery following 12weeks of SHS ameliorated all SHS-induced cardiovascular detriments. Importantly, mice exposed to TPPU in drinking water during SHS-related exposure were protected from SHS cardiovascular consequences. Discussion: The data suggest that 1) environmental relevant SHS exposure significantly alters cardiac autonomic function and BP regulation; 2) cardiovascular consequences from SHS can be reversed by discontinuing SHS exposure; and 3) inhibiting sEH can prevent SHS-induced cardiovascular consequences.

Highly efficacious and safe neutralizing DNA aptamer of SARS-CoV-2 as an emerging therapy for COVID-19 disease

BackgroundThe paucity of SARS-CoV-2-specific virulence factors has greatly hampered the therapeutic management of patients with COVID-19 disease. Although available vaccines and approved therapies have shown tremendous benefits, the continuous emergence of new variants of SARS-CoV-2 and side effects of existing treatments continue to challenge therapy, necessitating the development of a novel effective therapy. We have previously shown that our developed novel single-stranded DNA aptamers not only target the trimer S protein of SARS-CoV-2, but also block the interaction between ACE2 receptors and trimer S protein of Wuhan origin, Delta, Delta plus, Alpha, Lambda, Mu, and Omicron variants of SARS-CoV-2. We herein performed in vivo experiments that administer the aptamer to the lungs by intubation as well as in vitro studies utilizing PBMCs to prove the efficacy and safety of our most effective aptamer, AYA2012004_L.MethodsIn vivo studies were conducted in transgenic mice expressing human ACE2 (K18hACE2), C57BL/6J, and Balb/cJ. Flow cytometry was used to check S-protein expressing pseudo-virus-like particles (VLP) uptake by the lung cells and test the immuogenicity of AYA2012004_L. Ames test was used to assess mutagenicity of AYA2012004_L. RT-PCR and histopathology were used to determine the biodistribution and toxicity of AYA2012004_L in vital organs of mice.ResultsWe measured the in vivo uptake of VLPs by lung cells by detecting GFP signal using flow cytometry. AYA2012004_L specifically neutralized VLP uptake and also showed no inflammatory response in mice lungs. In addition, AYA2012004_L did not induce inflammatory response in the lungs of Th1 and Th2 mouse models as well as human PBMCs. AYA2012004_L was detectable in mice lungs and noticeable in insignificant amounts in other vital organs. Accumulation of AYA2012004_L in organs decreased over time. AYA2012004_L did not induce degenerative signs in tissues as seen by histopathology and did not cause changes in the body weight of mice. Ames test also certified that AYA2012004_L is non-mutagenic and proved it to be safe for in vivo studies.ConclusionsOur aptamer is safe, effective, and can neutralize the uptake of VLPs by lung cells when administered locally suggesting that it can be used as a potential therapeutic agent for COVID-19 management.

Lung inflammation induced by silica particles triggers hippocampal inflammation, synapse damage and memory impairment in mice

BackgroundConsiderable evidence indicates that a signaling crosstalk between the brain and periphery plays important roles in neurological disorders, and that both acute and chronic peripheral inflammation can produce brain changes leading to cognitive impairments. Recent clinical and epidemiological studies have revealed an increased risk of cognitive impairment and dementia in individuals with impaired pulmonary function. However, the mechanistic underpinnings of this association remain unknown. Exposure to SiO2 (silica) particles triggers lung inflammation, including infiltration by peripheral immune cells and upregulation of pro-inflammatory cytokines. We here utilized a mouse model of lung silicosis to investigate the crosstalk between lung inflammation and memory.MethodsSilicosis was induced by intratracheal administration of a single dose of 2.5 mg SiO2/kg in mice. Molecular and behavioral measurements were conducted 24 h and 15 days after silica administration. Lung and hippocampal inflammation were investigated by histological analysis and by determination of pro-inflammatory cytokines. Hippocampal synapse damage, amyloid-β (Aβ) peptide content and phosphorylation of Akt, a proxy of hippocampal insulin signaling, were investigated by Western blotting and ELISA. Memory was assessed using the open field and novel object recognition tests.ResultsAdministration of silica induced alveolar collapse, lung infiltration by polymorphonuclear (PMN) cells, and increased lung pro-inflammatory cytokines. Lung inflammation was followed by upregulation of hippocampal pro-inflammatory cytokines, synapse damage, accumulation of the Aβ peptide, and memory impairment in mice.ConclusionThe current study identified a crosstalk between lung and brain inflammatory responses leading to hippocampal synapse damage and memory impairment after exposure to a single low dose of silica in mice.