American ginseng with different processing methods ameliorate immunosuppression induced by cyclophosphamide in mice via the MAPK signaling pathways.

This study aimed to clarify the effects of two processed forms of American ginseng (Panax quinquefolius L.) on immunosuppression caused by cyclophosphamide (CTX) in mice. In the CTX-induced immunosuppressive model, mice were given either steamed American ginseng (American ginseng red, AGR) or raw American ginseng (American ginseng soft branch, AGS) by intragastric administration. Serum and spleen tissues were collected, and the pathological changes in mice spleens were observed by conventional HE staining. The expression levels of cytokines were detected by ELISA, and the apoptosis of splenic cells was determined by western blotting. The results showed that AGR and AGS could relieve CTX-induced immunosuppression through the enhanced immune organ index, improved cell-mediated immune response, increased serum levels of cytokines (TNF-α, IFN-γ, and IL-2) and immunoglobulins (IgG, IgA, and IgM), as well as macrophage activities including carbon clearance and phagocytic index. AGR and AGS downregulated the expression of BAX and elevated the expression of Bcl-2, p-P38, p-JNK, and p-ERK in the spleens of CTX-injected animals. Compared to AGS, AGR significantly improved the number of CD4+CD8-T lymphocytes, the spleen index, and serum levels of IgA, IgG, TNF-α, and IFN-γ. The expression of the ERK/MAPK pathway was markedly increased. These findings support the hypothesis that AGR and AGS are effective immunomodulatory agents capable of preventing immune system hypofunction. Future research may investigate the exact mechanism to rule out any unforeseen effects of AGR and AGS.

Calcitriol modifies tight junctions, improves barrier function, and reduces TNF-α-induced barrier leak in the human lung-derived epithelial cell culture model, 16HBE 14o.

Using the 16HBE 14o- human airway epithelial cell culture model, calcitriol (Vitamin D) was shown to improve barrier function by two independent metrics - increased transepithelial electrical resistance (TER) and reduced transepithelial diffusion of 14 C-D-mannitol (Jm ). Both effects were concentration dependent and active out to 168 h post-treatment. Barrier improvement associated with changes in the abundance of specific tight junctional (TJ) proteins in detergent-soluble fractions, most notably decreased claudin-2. TNF-α-induced compromise of barrier function could be attenuated by calcitriol with a concentration dependence similar to that observed for improvement of control barrier function. TNF-α-induced increases in claudin-2 were partially reversed by calcitriol. The ERK 1,2 inhibitor, U0126, itself improved 16HBE barrier function indicating MAPK pathway regulation of 16HBE barrier function. Calcitriol's action was additive to the effect of U0126 in reducing TNF- α -induced barrier compromise, suggesting that calcitriol may be acting through a non-ERK pathway in its blunting of TNF- α - induced barrier compromise. This was supported by calcitriol being without effect on pERK levels elevated by the action of TNF-α. Lack of effect of TNF- α on the death marker, caspase-3, and the inability of calcitriol to decrease the elevated LC3B II level caused by TNF-α, suggest that calcitriol's barrier improvement does not involve a cell death pathway. Calcitriol's improvement of control barrier function was not additive to barrier improvement induced by retinoic acid (Vitamin A). Calcitriol improvement and protection of airway barrier function could in part explain Vitamin D's reported clinical efficacy in COVID-19 and other airway diseases.

The multiphasic TNF-α-induced compromise of Calu-3 airway epithelial barrier function.

Purpose: Airway epithelial barrier leak and the involvement of proinflammatory cytokines play a key role in a variety of diseases. This study evaluates barrier compromise by the inflammatory mediator Tumor Necrosis Factor-α (TNF-α) in the human airway epithelial Calu-3 model. Methods: We examined the effects of TNF-α on barrier function in Calu-3 cell layers using Transepithelial Electrical Resistance (TER) and transepithelial diffusion of radiolabeled probe molecules. Western immunoblot analyses of tight junctional (TJ) proteins in detergent soluble fractions were performed. Results: TNF-α dramatically reduced TER and increased paracellular permeability of both 14C-D-mannitol and the larger 5 kDa probe, 14C-inulin. A time course of the effects shows two separate actions on barrier function. An initial compromise of barrier function occurs 2-4 hours after TNF-α exposure, followed by complete recovery of barrier function by 24 hrs. Beginning 48 hrs. post-exposure, a second more sustained barrier compromise ensues, in which leakiness persists through 144 hrs. There were no changes in TJ proteins observed at 3 hrs. post exposure, but significant increases in claudins-2, -3, -4, and -5, as well as a decrease in occludin were seen at 72 hrs. post TNF-α exposure. Both the 2-4 hr. and the 72 hr. TNF-α induced leaks are shown to be mediated by the ERK signaling pathway. Conclusion: TNF-α induced a multiphasic transepithelial leak in Calu-3 cell layers that was shown to be ERK mediated, as well as involve changes in the TJ complex. The micronutrients, retinoic acid and calcitriol, were effective at reducing this barrier compromise caused by TNF-α. The significance of these results for airway disease and for COVID-19 specifically are discussed.

Synergism of TNF-α and IFN-ß triggers human airway epithelial cells death by apoptosis and pyroptosis.

Cytokine release syndrome, also called cytokine storm, could cause lung tissue damage, acute respiratory distress syndrome (ARDS) and even death during SARS-CoV-2 infection. However, the underlying mechanisms of cytokine storm still remain unknown. Among these cytokines, the function of TNF-α and type I IFNs especially deserved further investigation. Here, we first found that TNF-α and IFN-ß synergistically induced human airway epithelial cells BEAS-2B death. Mechanistically, the combination of TNF-α and IFN-ß led to the activation of caspase-8 and caspase-3, which initiated BEAS-2B apoptosis. The activated caspase-8 and caspase-3 could further induce the cleavage and activation of gasdermin D (GSDMD) and gasdermin E (GSDME), which finally resulted in pro-inflammatory pyroptosis. The knock-down of caspase-8 and caspase-3 could effectively block the activation of GSDMD and GSDME, and then the death of BEAS-2B induced by TNF-α and IFN-ß. In addition, pan-caspase inhibitor Z-VAD-FMK (ZVAD) and necrosulfonamide (NSA) could inhibit BEAS-2B death induced by TNF-α and IFN-ß. Overall, our work revealed one possible mechanism that cytokine storm causes airway epithelial cells (AECs) damage and ARDS. These results indicated that blocking TNF-α and IFN-ß-mediated AECs death may be a potential target to treat related viral infectious diseases, such as COVID-19.

The Expression Levels of SARS-CoV-2 Infection-Mediating Molecules Promoted by Interferon-γ and Tumor Necrosis Factor-α Are Downregulated by Hydrogen Sulfide.

Autoimmune thyroid diseases (AITDs), which include Hashimoto's thyroiditis (HT) and Graves' disease (GD), have a higher prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the literature. The effects of AITD-associated cytokines on SARS-CoV-2 infection-mediating molecule levels might be involved in the pathogenesis of susceptibility. We speculated that hydrogen sulfide (H2S) might attenuate this process since H2S has antiviral effects. Using immunohistochemistry, we found that angiotensin-converting enzyme-II (ACE2) expression was higher in the HT group and neuropilin 1 (NRP1) expression was higher in HT and GD groups than in the normal group, while transmembrane protease serine type 2 (TMPRSS2) expression was lower in HT and GD groups. When culturing primary thyrocytes with cytokines or sodium hydrosulfide (NaHS) plus cytokines, we found that ACE2 and NRP1 mRNA levels were upregulated while TMPRSS2 levels were downregulated by interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). After pretreatment with NaHS in thyrocytes, ACE2 and NRP1 expression were downregulated compared to IFN-γ or TNF-α treatment, and NaHS had no effect on TMPRSS2 expression. Our findings suggested that IFN-γ and TNF-α, which are elevated in AITDs, promoted ACE2 and NRP1 expression and inhibited TMPRSS2 expression. H2S might protect against SARS-CoV-2 infection by downregulating ACE2 and NRP1 levels.

Effects of tocilizumab and dexamethasone on the downregulation of proinflammatory cytokines and upregulation of antioxidants in the lungs in oleic acid-induced ARDS.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a life-threatening disease caused by the induction of inflammatory cytokines and chemokines in the lungs. There is a dearth of drug applications that can be used to prevent cytokine storms in ARDS treatment. This study was designed to investigate the effects of tocilizumab and dexamethasone on oxidative stress, antioxidant parameters, and cytokine storms in acute lung injury caused by oleic acid in rats. METHODS: Adult male rats were divided into five groups: the CN (healthy rats, n = 6), OA (oleic acid administration, n = 6), OA + TCZ-2 (oleic acid and tocilizumab at 2 mg/kg, n = 6), OA + TCZ-4 (oleic acid and tocilizumab at 4 mg/kg, n = 6), and OA + DEX-10 (oleic acid and dexamethasone at 10 mg/kg, n = 6) groups. All animals were euthanized after treatment for histopathological, immunohistochemical, biochemical, PCR, and SEM analyses. RESULTS: Expressions of TNF-α, IL-1ß, IL-6, and IL-8 cytokines in rats with acute lung injury induced by oleic acid were downregulated in the TCZ and DEX groups compared to the OA group (P < 0.05). The MDA level in lung tissues was statistically lower in the OA + TCZ-4 group compared to the OA group. It was further determined that SOD, GSH, and CAT levels were decreased in the OA group and increased in the TCZ and DEX groups (P < 0.05). Histopathological findings such as thickening of the alveoli, hyperemia, and peribronchial cell infiltration were found to be similar when lung tissues of the TCZ and DEX groups were compared to the control group. With SEM imaging of the lung tissues, it was found that the alveolar lining layer had become indistinct in the OA, OA + TCZ-2, and OA + TCZ-4 groups. CONCLUSIONS: In this model of acute lung injury caused by oleic acid, tocilizumab and dexamethasone were effective in preventing cytokine storms by downregulating the expression of proinflammatory cytokines including TNF-α, IL-1ß, IL-6, and IL-8. Against the downregulation of antioxidant parameters such as SOD and GSH in the lung tissues caused by oleic acid, tocilizumab and dexamethasone upregulated them and showed protective effects against cell damage.

Flavonoids as Potential Anti-Inflammatory Molecules: A Review.

Hydroxylated polyphenols, also called flavonoids, are richly present in vegetables, fruits, cereals, nuts, herbs, seeds, stems, and flowers of numerous plants. They possess numerous medicinal properties such as antioxidant, anti-cancer, anti-microbial, neuroprotective, and anti-inflammation. Studies show that flavonoids activate antioxidant pathways that render an anti-inflammatory effect. They inhibit the secretions of enzymes such as lysozymes and ß-glucuronidase and inhibit the secretion of arachidonic acid, which reduces inflammatory reactions. Flavonoids such as quercetin, genistein, apigenin, kaempferol, and epigallocatechin 3-gallate modulate the expression and activation of a cytokine such as interleukin-1beta (IL-1ß), Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8); regulate the gene expression of many pro-inflammatory molecules such s nuclear factor kappa-light chain enhancer of activated B cells (NF-κB), activator protein-1 (AP-1), intercellular adhesion molecule-1 (ICAM), vascular cell adhesion molecule-1 (VCAM), and E-selectins; and also inhibits inducible nitric oxide (NO) synthase, cyclooxygenase-2, and lipoxygenase, which are pro-inflammatory enzymes. Understanding the anti-inflammatory action of flavonoids provides better treatment options, including coronavirus disease 2019 (COVID-19)-induced inflammation, inflammatory bowel disease, obstructive pulmonary disorder, arthritis, Alzheimer's disease, cardiovascular disease, atherosclerosis, and cancer. This review highlights the sources, biochemical activities, and role of flavonoids in enhancing human health.

Circulating Microvesicle-Associated Inducible Nitric Oxide Synthase Is a Novel Therapeutic Target to Treat Sepsis: Current Status and Future Considerations.

To determine whether mitigating the harmful effects of circulating microvesicle-associated inducible nitric oxide (MV-A iNOS) in vivo increases the survival of challenged mice in three different mouse models of sepsis, the ability of anti-MV-A iNOS monoclonal antibodies (mAbs) to rescue challenged mice was assessed using three different mouse models of sepsis. The vivarium of a research laboratory Balb/c mice were challenged with an LD80 dose of either lipopolysaccharide (LPS/endotoxin), TNFα, or MV-A iNOS and then treated at various times after the challenge with saline as control or with an anti-MV-A iNOS mAb as a potential immunotherapeutic to treat sepsis. Each group of mice was checked daily for survivors, and Kaplan-Meier survival curves were constructed. Five different murine anti-MV-A iNOS mAbs from our panel of 24 murine anti-MV-A iNOS mAbs were found to rescue some of the challenged mice. All five murine mAbs were used to genetically engineer humanized anti-MV-A iNOS mAbs by inserting the murine complementarity-determining regions (CDRs) into a human IgG1,kappa scaffold and expressing the humanized mAbs in CHO cells. Three humanized anti-MV-A iNOS mAbs were effective at rescuing mice from sepsis in three different animal models of sepsis. The effectiveness of the treatment was both time- and dose-dependent. Humanized anti-MV-A iNOS rHJ mAb could rescue up to 80% of the challenged animals if administered early and at a high dose. Our conclusions are that MV-A iNOS is a novel therapeutic target to treat sepsis; anti-MV-A iNOS mAbs can mitigate the harmful effects of MV-A iNOS; the neutralizing mAb's efficacy is both time- and dose-dependent; and a specifically targeted immunotherapeutic for MV-A iNOS could potentially save tens of thousands of lives annually and could result in improved antibiotic stewardship.

SARS-CoV-2 Spike Protein S1-Mediated Endothelial Injury and Pro-Inflammatory State Is Amplified by Dihydrotestosterone and Prevented by Mineralocorticoid Antagonism.

Men are disproportionately affected by the coronavirus disease-2019 (COVID-19), and face higher odds of severe illness and death compared to women. The vascular effects of androgen signaling and inflammatory cytokines in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mediated endothelial injury are not defined. We determined the effects of SARS-CoV-2 spike protein-mediated endothelial injury under conditions of exposure to androgen dihydrotestosterone (DHT) and tumor necrosis factor-a (TNF-α) and tested potentially therapeutic effects of mineralocorticoid receptor antagonism by spironolactone. Circulating endothelial injury markers VCAM-1 and E-selectin were measured in men and women diagnosed with COVID-19. Exposure of endothelial cells (ECs) in vitro to DHT exacerbated spike protein S1-mediated endothelial injury transcripts for the cell adhesion molecules E-selectin, VCAM-1 and ICAM-1 and anti-fibrinolytic PAI-1 (p < 0.05), and increased THP-1 monocyte adhesion to ECs (p = 0.032). Spironolactone dramatically reduced DHT+S1-induced endothelial activation. TNF-α exacerbated S1-induced EC activation, which was abrogated by pretreatment with spironolactone. Analysis from patients hospitalized with COVID-19 showed concordant higher circulating VCAM-1 and E-Selectin levels in men, compared to women. A beneficial effect of the FDA-approved drug spironolactone was observed on endothelial cells in vitro, supporting a rationale for further evaluation of mineralocorticoid antagonism as an adjunct treatment in COVID-19.

Development of an Image-Based HCS-Compatible Method for Endothelial Barrier Function Assessment.

The recent renascence of phenotypic drug discovery (PDD) is catalyzed by its ability to identify first-in-class drugs and deliver results when the exact molecular mechanism is partially obscure. Acute respiratory distress syndrome (ARDS) is a severe, life-threatening condition with a high mortality rate that has increased in frequency due to the COVID-19 pandemic. Despite decades of laboratory and clinical study, no efficient pharmacological therapy for ARDS has been found. An increase in endothelial permeability is the primary event in ARDS onset, causing the development of pulmonary edema that leads to respiratory failure. Currently, the detailed molecular mechanisms regulating endothelial permeability are poorly understood. Therefore, the use of the PDD approach in the search for efficient ARDS treatment can be more productive than classic target-based drug discovery (TDD), but its use requires a new cell-based assay compatible with high-throughput (HTS) and high-content (HCS) screening. Here we report the development of a new plate-based image cytometry method to measure endothelial barrier function. The incorporation of image cytometry in combination with digital image analysis substantially decreases assay variability and increases the signal window. This new method simultaneously allows for rapid measurement of cell monolayer permeability and cytological analysis. The time-course of permeability increase in human pulmonary artery endothelial cells (HPAECs) in response to the thrombin and tumor necrosis factor α treatment correlates with previously published data obtained by transendothelial resistance (TER) measurements. Furthermore, the proposed image cytometry method can be easily adapted for HTS/HCS applications.

ACE2 Expression Is Upregulated in Inflammatory Corneal Epithelial Cells and Attenuated by Resveratrol.

Purpose: The ocular surface is considered an important route for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. The expression level of the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is vital for viral infection. However, the regulation of ACE2 expression on the ocular surface is still unknown. We aimed to determine the change in ACE2 expression in inflamed corneal epithelium and explore potential drugs to reduce the expression of ACE2 on the ocular surface. Methods: The expression of the SARS-CoV-2 receptors ACE2 and TMPRSS2 in human corneal epithelial cells (HCECs) was examined by qPCR and Western blotting. The altered expression of ACE2 in inflammatory corneal epithelium was evaluated in TNFα- and IL-1ß-stimulated HCECs and inflamed mouse corneal epithelium, and the effect of resveratrol on ACE2 expression in HCECs was detected by immunofluorescence and Western blot analysis. Results: ACE2 and TMPRSS2 are expressed on the human corneal epithelial cells. ACE2 expression is upregulated in HCECs by stimulation with TNFα and IL-1ß and inflamed mouse corneas, including dry eye and alkali-burned corneas. In addition, resveratrol attenuates the increased expression of ACE2 induced by TNFα in HCECs. Conclusions: This study demonstrates that ACE2 is highly expressed in HCECs and can be upregulated by stimulation with inflammatory cytokines and inflamed mouse corneal epithelium. Resveratrol may be able to reduce the increased expression of ACE2 on the inflammatory ocular surface. Our work suggests that patients with an inflammatory ocular surface may display higher ACE2 expression, which increases the risk of SARS-CoV-2 infection.

Epidemiology and the Impact of Therapies on the Outcome of COVID-19 in Patients With Inflammatory Bowel Disease.

INTRODUCTION: It has been hypothesized that people suffering from inflammatory bowel disease (IBD) have an increased risk of coronavirus disease (COVID-19). However, it is not known whether immunosuppressive therapies exacerbate the COVID-19 outcome. METHODS: We reviewed data on the prevalence and clinical outcomes of COVID-19 in patients with IBD. RESULTS: COVID-19 prevalence in patients with IBD was comparable with that in the general population. Therapies using antitumor necrosis factor-α agents have been associated with better clinical outcomes. DISCUSSION: Management and treatments provided by gastroenterologists were effective in reducing COVID-19 risk. Antitumor necrosis factor-α agents seem to mitigate the course of COVID-19.