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1.
BMC Immunol ; 23(1): 25, 2022 May 23.
Article in English | MEDLINE | ID: covidwho-1857991

ABSTRACT

BACKGROUND: Signal transducer and activator of transcription 6 (STAT6) is an intracelluar transcriotion factor and NLRP3 (Nod-like receptor containing a pyrin domain 3) is a component of NLRP3 inflammasome in pyroptotic cells. There was increased activation of STAT6 and expression of NLRP3 in mice with murine acute lung injury (ALI). However, it is unknown their roles in the development of murine ALI. We in this study, investigated the effects of STAT6 signaling on murine ALI and pyroptosis in STAT6 knock-out (KO) mice and macrophages. RESULTS: STAT6 was activated in the lung tissues of mice 2 days after intratracheal treatmemt with 5 mg/kg LPS. Lack of STAT6 expression in KO mice induced more severe lung inflammation, associated with elevated neutrophil influx and expression of TNF-alpha, IL-6 and IL-1beta in the inflamed lung tissues. In addition, the expression of NLRP3, ASC (apoptosis-associated speck-like protein containing a CARD), p-p38 MAPK (p38 mitogen-activated protein kinase) and ratio of LC3-II/I (microtubule-associated protein-1 light chain-3) was increased, accompanied with the increased polarization of Siglec-F(-) subtype macrophages in KO mice with ALI. Further studies in bone marrow-derived macrophages (BMDMs) revealed that lack of STAT6 increased the expression of NLRP3 and p-p38 MAPK, in association with elevated expression of TNF-alpha, IL-1beta and Calreticulin in LPS-treated KO BMDMs. CONCLUSIONS: Lack of STAT6 exacerbated murine ALI through improving the expression of NLRP3 and activation of p38 MAPK in macrophages. STAT6 has an immune suppressive role in the development of ALI and would be a promising therapeutic target in the treatment of ALI and possibly among patients with acute respiratory distress syndrome (ARDS).


Subject(s)
Acute Lung Injury , NLR Family, Pyrin Domain-Containing 3 Protein , Acute Lung Injury/drug therapy , Acute Lung Injury/metabolism , Animals , Humans , Inflammasomes/metabolism , Lipopolysaccharides/pharmacology , Macrophages , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , STAT6 Transcription Factor/genetics , STAT6 Transcription Factor/metabolism , STAT6 Transcription Factor/pharmacology , Signal Transduction , Tumor Necrosis Factor-alpha/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism
2.
Int Immunopharmacol ; 108: 108773, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1796626

ABSTRACT

Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is one of the leading pulmonary inflammatory disorders causing significant morbidity and mortality. Vincamine is a novel phytochemical with promising anti-inflammatory properties. In the current work, the protective effect of vincamine was studied in vitro (Raw 264.7 macrophages) and in vivo against lipopolysaccharide (LPS) induced ALI in Swiss albino mice. Vincamine significantly reduced nitrite and TNF-α release from the LPS stimulated macrophages and increased the levels of IL-10, indicating potent anti-inflammatory effects. It was observed that vincamine at the dose of 40 mg/kg, significantly reduced LPS induced inflammatory cell count in blood and in bronchoalveolar lavage (BAL) fluid. Further, vincamine exerted potent suppression of inflammation by reducing the expression of proinflammatory cytokines, while significantly increased (p < 0.001) the expression of anti-inflammatory cytokine (IL-10 and IL-22). Interestingly, histological changes were reversed in vincamine treated groups in a dose-dependent manner. Immunohistochemical analysis revealed significantly enhanced expression of NF-κB, TNF-α and COX-2 while reduced expression of Nrf-2 in disease control group, which were significantly (p < 0.001) ameliorated by vincamine. We, to the best of our knowledge, report for the first time that vincamine possesses protective potential against LPS induced inflammation and oxidative stress, possibly by inhibiting the NF-κB cascade, while positively regulating the Nrf-2 pathway. These findings are of potential relevance for COVID-19 management concerning the fact that lung injury and ARDS are its critical features.


Subject(s)
Acute Lung Injury , COVID-19 , Catharanthus , Respiratory Distress Syndrome , Vincamine , Acute Lung Injury/chemically induced , Acute Lung Injury/drug therapy , Acute Lung Injury/metabolism , Animals , Anti-Inflammatory Agents/pharmacology , Catharanthus/metabolism , Cytokines/metabolism , Inflammation/drug therapy , Interleukin-10/metabolism , Lipopolysaccharides/pharmacology , Lung/pathology , Mice , NF-kappa B/metabolism , Tumor Necrosis Factor-alpha/metabolism , Vincamine/metabolism , Vincamine/pharmacology , Vincamine/therapeutic use
3.
Cells ; 11(9)2022 04 20.
Article in English | MEDLINE | ID: covidwho-1792800

ABSTRACT

Cannabinoids, mainly cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), are the most studied group of compounds obtained from Cannabis sativa because of their several pharmaceutical properties. Current evidence suggests a crucial role of cannabinoids as potent anti-inflammatory agents for the treatment of chronic inflammatory diseases; however, the mechanisms remain largely unclear. Cytokine storm, a dysregulated severe inflammatory response by our immune system, is involved in the pathogenesis of numerous chronic inflammatory disorders, including coronavirus disease 2019 (COVID-19), which results in the accumulation of pro-inflammatory cytokines. Therefore, we hypothesized that CBD and THC reduce the levels of pro-inflammatory cytokines by inhibiting key inflammatory signaling pathways. The nucleotide-binding and oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome signaling has been implicated in a variety of chronic inflammatory diseases, which results in the release of pyroptotic cytokines, interleukin-1ß (IL-1ß) and IL-18. Likewise, the activation of the signal transducer and activator of transcription-3 (STAT3) causes increased expression of pro-inflammatory cytokines. We studied the effects of CBD and THC on lipopolysaccharide (LPS)-induced inflammatory response in human THP-1 macrophages and primary human bronchial epithelial cells (HBECs). Our results revealed that CBD and, for the first time, THC significantly inhibited NLRP3 inflammasome activation following LPS + ATP stimulation, leading to a reduction in the levels of IL-1ß in THP-1 macrophages and HBECs. CBD attenuated the phosphorylation of nuclear factor-κB (NF-κB), and both cannabinoids inhibited the generation of oxidative stress post-LPS. Our multiplex ELISA data revealed that CBD and THC significantly diminished the levels of IL-6, IL-8, and tumor necrosis factor-α (TNF-α) after LPS treatment in THP-1 macrophages and HBECs. In addition, the phosphorylation of STAT3 was significantly downregulated by CBD and THC in THP-1 macrophages and HBECs, which was in turn attributed to the reduced phosphorylation of tyrosine kinase-2 (TYK2) by CBD and THC after LPS stimulation in these cells. Overall, CBD and THC were found to be effective in alleviating the LPS-induced cytokine storm in human macrophages and primary HBECs, at least via modulation of NLRP3 inflammasome and STAT3 signaling pathways. The encouraging results from this study warrant further investigation of these cannabinoids in vivo.


Subject(s)
COVID-19 , Cannabidiol , Cannabinoids , Cannabidiol/pharmacology , Cannabinoids/pharmacology , Cytokine Release Syndrome , Cytokines/metabolism , Dronabinol/pharmacology , Humans , Inflammasomes/metabolism , Lipopolysaccharides/pharmacology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction , TYK2 Kinase/metabolism , TYK2 Kinase/pharmacology
4.
Physiol Rep ; 10(8): e15271, 2022 04.
Article in English | MEDLINE | ID: covidwho-1791646

ABSTRACT

Acute respiratory distress syndrome (ARDS) is a major clinical problem without available therapies. Known risks for ARDS include severe sepsis, SARS-CoV-2, gram-negative bacteria, trauma, pancreatitis, and blood transfusion. During ARDS, blood fluids and inflammatory cells enter the alveoli, preventing oxygen exchange from air into blood vessels. Reduced pulmonary endothelial barrier function, resulting in leakage of plasma from blood vessels, is one of the major determinants in ARDS. It is, however, unknown why systemic inflammation particularly targets the pulmonary endothelium, as endothelial cells (ECs) line all vessels in the vascular system of the body. In this study, we examined ECs of pulmonary, umbilical, renal, pancreatic, and cardiac origin for upregulation of adhesion molecules, ability to facilitate neutrophil (PMN) trans-endothelial migration (TEM) and for endothelial barrier function, in response to the gram-negative bacterial endotoxin LPS. Interestingly, we found that upon LPS stimulation, pulmonary ECs showed increased levels of adhesion molecules, facilitated more PMN-TEM and significantly perturbed the endothelial barrier, compared to other types of ECs. These observations could partly be explained by a higher expression of the adhesion molecule ICAM-1 on the pulmonary endothelial surface compared to other ECs. Moreover, we identified an increased expression of Cadherin-13 in pulmonary ECs, for which we demonstrated that it aids PMN-TEM in pulmonary ECs stimulated with LPS. We conclude that pulmonary ECs are uniquely sensitive to LPS, and intrinsically different, compared to ECs from other vascular beds. This may add to our understanding of the development of ARDS upon systemic inflammation.


Subject(s)
COVID-19 , Respiratory Distress Syndrome , Cell Adhesion Molecules/metabolism , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Humans , Inflammation/metabolism , Lipopolysaccharides/metabolism , Lipopolysaccharides/pharmacology , SARS-CoV-2
5.
Int J Mol Sci ; 23(6)2022 Mar 18.
Article in English | MEDLINE | ID: covidwho-1780037

ABSTRACT

BACKGROUND: Local anesthetics (LAs) have potent anti-inflammatory properties. Inflammatory down-regulation is crucial in diseases with overactive immune reactions, such as acute respiratory distress syndrome (ARDS) and chronic inflammation. We investigated the influence of four LAs, procaine, lidocaine, mepivacaine, and bupivacaine, on the reduction of tumor necrosis factor-alpha (TNF-α) secretion in lipopolysaccharide (LPS)-activated human leucocytes. METHODS: Blood samples of 28 individuals were stimulated with LPS. The reduction of TNF-α production by each of the four LAs added (0.5 mg/mL) was measured and correlated with biometric variables. A response was defined as reduction to <85% of initial levels. RESULTS: All four LAs down-regulated the TNF-α secretion in 44-61%: Bupivacaine (44.4%), lidocaine (61.5%), mepivacaine (44.4%), and procaine (50% of the individuals, "responders"). The TNF-α secretion was reduced to 67.4, 68.0, 63.6, and 67.1% of the initial values in responders. The effects in both patients and healthy persons were the same. Interindividual responses to LAs were not correlated with the duration or type of complaints, basal TNF-α serum level, sex, BMI, or age of responders. CONCLUSIONS: Four clinically relevant LAs (amid-LA and ester-LA) attenuate the inflammatory response provoked by LPS. They are potential candidates for drug repositioning in treating overactive immune reactions and chronic inflammation.


Subject(s)
Lipopolysaccharides , Tumor Necrosis Factor-alpha , Anesthetics, Local/pharmacology , Anti-Inflammatory Agents/pharmacology , Bupivacaine/pharmacology , Humans , Inflammation , Lidocaine/pharmacology , Lipopolysaccharides/pharmacology , Mepivacaine , Procaine/pharmacology
6.
Biomolecules ; 12(4)2022 03 31.
Article in English | MEDLINE | ID: covidwho-1776123

ABSTRACT

Intercellular communication between monocytes/macrophages and cells involved in tissue regeneration, such as mesenchymal stromal cells (MSCs) and primary tissue cells, is essential for tissue regeneration and recovery of homeostasis. Typically, in the final phase of the inflammation-resolving process, this intercellular communication drives an anti-inflammatory immunomodulatory response. To obtain a safe and effective treatment to counteract the cytokine storm associated with a disproportionate immune response to severe infections, including that associated with COVID-19, by means of naturally balanced immunomodulation, our group has standardized the production under GMP-like conditions of a secretome by coculture of macrophages and MSCs. To characterize this proteome, we determined the expression of molecules related to cellular immune response and tissue regeneration, as well as its possible toxicity and anti-inflammatory potency. The results show a specific molecular pattern of interaction between the two cell types studied, with an anti-inflammatory and regenerative profile. In addition, the secretome is not toxic by itself on human PBMC or on THP-1 monocytes and prevents lipopolysaccharide (LPS)-induced growth effects on those cell types. Finally, PRS CK STORM prevents LPS-induced TNF-A and IL-1Β secretion from PBMC and from THP-1 cells at the same level as hydrocortisone, demonstrating its anti-inflammatory potency.


Subject(s)
COVID-19 , Mesenchymal Stem Cells , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacology , Coculture Techniques , Culture Media, Conditioned/pharmacology , Humans , Leukocytes, Mononuclear , Lipopolysaccharides/pharmacology , Monocytes
7.
Cells ; 11(7)2022 03 24.
Article in English | MEDLINE | ID: covidwho-1771141

ABSTRACT

Due to limited data on the link between gut barrier defects (leaky gut) and neutrophil extracellular traps (NETs) in coronavirus disease 2019 (COVID-19), blood samples of COVID-19 cases-mild (upper respiratory tract symptoms without pneumonia; n = 27), moderate (pneumonia without hypoxia; n = 28), and severe (pneumonia with hypoxia; n = 20)-versus healthy control (n = 15) were evaluated, together with in vitro experiments. Accordingly, neutrophil counts, serum cytokines (IL-6 and IL-8), lipopolysaccharide (LPS), bacteria-free DNA, and NETs parameters (fluorescent-stained nuclear morphology, dsDNA, neutrophil elastase, histone-DNA complex, and myeloperoxidase-DNA complex) were found to differentiate COVID-19 severity, whereas serum (1→3)-ß-D-glucan (BG) was different between the control and COVID-19 cases. Despite non-detectable bacteria-free DNA in the blood of healthy volunteers, using blood bacteriome analysis, proteobacterial DNA was similarly predominant in both control and COVID-19 cases (all severities). In parallel, only COVID-19 samples from moderate and severe cases, but not mild cases, were activated in vitro NETs, as determined by supernatant dsDNA, Peptidyl Arginine Deiminase 4, and nuclear morphology. With neutrophil experiments, LPS plus BG (LPS + BG) more prominently induced NETs, cytokines, NFκB, and reactive oxygen species, when compared with the activation by each molecule alone. In conclusion, pathogen molecules (LPS and BG) from gut translocation along with neutrophilia and cytokinemia in COVID-19-activated, NETs-induced hyperinflammation.


Subject(s)
COVID-19 , Extracellular Traps , Pneumonia , beta-Glucans , Cytokines , Humans , Hypoxia , Lipopolysaccharides/pharmacology , SARS-CoV-2
8.
J Control Release ; 346: 421-433, 2022 Jun.
Article in English | MEDLINE | ID: covidwho-1763813

ABSTRACT

Acute Respiratory Distress Syndrome (ARDS), associated with Covid-19 infections, is characterized by diffuse lung damage, inflammation and alveolar collapse that impairs gas exchange, leading to hypoxemia and patient' mortality rates above 40%. Here, we describe the development and assessment of 100-nm liposomes that are tailored for pulmonary delivery for treating ARDS, as a model for lung diseases. The liposomal lipid composition (primarily DPPC) was optimized to mimic the lung surfactant composition, and the drug loading process of both methylprednisolone (MPS), a steroid, and N-acetyl cysteine (NAC), a mucolytic agent, reached an encapsulation efficiency of 98% and 92%, respectively. In vitro, treating lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages with the liposomes decreased TNFα and nitric oxide (NO) secretion, while NAC increased the penetration of nanoparticles through the mucus. In vivo, we used LPS-induced lung inflammation model to assess the accumulation and therapeutic efficacy of the liposomes in C57BL/6 mice, either by intravenous (IV), endotracheal (ET) or IV plus ET nanoparticles administrations. Using both administration methods, liposomes exhibited an increased accumulation profile in the inflamed lungs over 48 h. Interestingly, while IV-administrated liposomes distributed widely throughout the lung, ET liposomes were present in lungs parenchyma but were not detected at some distal regions of the lungs, possibly due to imperfect airflow regimes. Twenty hours after the different treatments, lungs were assessed for markers of inflammation. We found that the nanoparticle treatment had a superior therapeutic effect compared to free drugs in treating ARDS, reducing inflammation and TNFα, IL-6 and IL-1ß cytokine secretion in bronchoalveolar lavage (BAL), and that the combined treatment, delivering nanoparticles IV and ET simultaneously, had the best outcome of all treatments. Interestingly, also the DPPC lipid component alone played a therapeutic role in reducing inflammatory markers in the lungs. Collectively, we show that therapeutic nanoparticles accumulate in inflamed lungs holding potential for treating lung disorders. SIGNIFICANCE: In this study we compare intravenous versus intratracheal delivery of nanoparticles for treating lung disorders, specifically, acute respiratory distress syndrome (ARDS). By co-loading two medications into lipid nanoparticles, we were able to reduce both inflammation and mucus secretion in the inflamed lungs. Both modes of delivery resulted in high nanoparticle accumulation in the lungs, intravenously administered nanoparticles reached lung endothelial while endotracheal delivery reached lung epithelial. Combining both delivery approaches simultaneously provided the best ARDS treatment outcome.


Subject(s)
COVID-19 , Lung Diseases , Respiratory Distress Syndrome , Acetylcysteine/pharmacology , Animals , Humans , Inflammation/drug therapy , Lipopolysaccharides/pharmacology , Liposomes/therapeutic use , Lung , Mice , Mice, Inbred C57BL , Nanoparticles , Respiratory Distress Syndrome/drug therapy , Tumor Necrosis Factor-alpha
9.
J Leukoc Biol ; 111(5): 1107-1121, 2022 05.
Article in English | MEDLINE | ID: covidwho-1756612

ABSTRACT

Infection by SARS-CoV-2 may elicit uncontrolled and damaging inflammatory responses. Thus, it is critical to identify compounds able to inhibit virus replication and thwart the inflammatory reaction. Here, we show that the plasma levels of the immunoregulatory neuropeptide VIP are elevated in patients with severe COVID-19, correlating with reduced inflammatory mediators and with survival on those patients. In vitro, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), highly similar neuropeptides, decreased the SARS-CoV-2 RNA content in human monocytes and viral production in lung epithelial cells, also reducing cell death. Both neuropeptides inhibited the production of proinflammatory mediators in lung epithelial cells and in monocytes. VIP and PACAP prevented in monocytes the SARS-CoV-2-induced activation of NF-kB and SREBP1 and SREBP2, transcriptions factors involved in proinflammatory reactions and lipid metabolism, respectively. They also promoted CREB activation, a transcription factor with antiapoptotic activity and negative regulator of NF-kB. Specific inhibition of NF-kB and SREBP1/2 reproduced the anti-inflammatory, antiviral, and cell death protection effects of VIP and PACAP. Our results support further clinical investigations of these neuropeptides against COVID-19.


Subject(s)
COVID-19 , Vasoactive Intestinal Peptide , Humans , Lipopolysaccharides/pharmacology , NF-kappa B/metabolism , Pituitary Adenylate Cyclase-Activating Polypeptide/pharmacology , RNA, Viral , Receptors, Vasoactive Intestinal Polypeptide, Type I , SARS-CoV-2 , Transcription Factors/metabolism , Vasoactive Intestinal Peptide/pharmacology
10.
Bull Exp Biol Med ; 172(4): 423-429, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1696762

ABSTRACT

We studied the lung-protective effect and mechanisms of the anti-inflammatory and antioxidant effects of ultra-short-wave diathermy (USWD) in a rat model of LPS-induced acute lung injury. Histological examination of the lung tissues was performed and the levels of oxidative stress-related factors and inflammatory cytokines were measured. It was shown that the lung injury score, the lung wet-to-dry weight ratio (W/D), oxidative stress-related factors malondialdehyde and acyl-CoA synthetase long-chain family member 4 (ACSL4), and inflammatory cytokines were increased after LPS administration, while USWD treatment reduced these parameters. In addition, superoxide dismutase and glutathione peroxidase 4 were decreased in rats with LPS-induced acute lung injury, while USWD therapy up-regulated the expression of these enzymes. Thus, USWD could antagonize lung injury by inhibiting oxidative stress and inflammatory response in rats with acute lung injury. USWD can be a promising adjunctive treatment to counter oxidative stress and inflammation and a potential therapeutic candidate for the treatment of patients with this pathology.


Subject(s)
Acute Lung Injury , Diathermy , Acute Lung Injury/drug therapy , Acute Lung Injury/therapy , Animals , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Antioxidants/metabolism , Humans , Lipopolysaccharides/pharmacology , Lung , Oxidative Stress , Radio Waves , Rats
11.
Commun Biol ; 5(1): 102, 2022 01 28.
Article in English | MEDLINE | ID: covidwho-1655635

ABSTRACT

Emerging studies suggest that monocytes can be trained by bacterial endotoxin to adopt distinct memory states ranging from low-grade inflammation to immune exhaustion. While low-grade inflammation may contribute to the pathogenesis of chronic diseases, exhausted monocytes with pathogenic and immune-suppressive characteristics may underlie the pathogenesis of polymicrobial sepsis including COVID-19. However, detailed processes by which the dynamic adaption of monocytes occur remain poorly understood. Here we exposed murine bone-marrow derived monocytes to chronic lipopolysaccharide (LPS) stimulation at low-dose or high-dose, as well as a PBS control. The cells were profiled for genome-wide H3K27ac modification and gene expression. The gene expression of TRAM-deficient and IRAK-M-deficient monocytes with LPS exposure was also analyzed. We discover that low-grade inflammation preferentially utilizes the TRAM-dependent pathway of TLR4 signaling, and induces the expression of interferon response genes. In contrast, high dose LPS uniquely upregulates exhaustion signatures with metabolic and proliferative pathways. The extensive differences in the epigenomic landscape between low-dose and high-dose conditions suggest the importance of epigenetic regulations in driving differential responses. Our data provide potential targets for future mechanistic or therapeutic studies.


Subject(s)
Epigenomics , Inflammation/genetics , Lipopolysaccharides/pharmacology , Monocytes/drug effects , Transcriptome , Animals , COVID-19/virology , Dose-Response Relationship, Drug , Inflammation/immunology , Lipopolysaccharides/administration & dosage , Mice , SARS-CoV-2/isolation & purification
12.
Int J Mol Sci ; 22(24)2021 Dec 13.
Article in English | MEDLINE | ID: covidwho-1599176

ABSTRACT

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.


Subject(s)
Cell-Derived Microparticles/metabolism , Nitric Oxide Synthase Type II/metabolism , Sepsis/therapy , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Monoclonal, Humanized/pharmacology , Cell-Derived Microparticles/immunology , Disease Models, Animal , Humans , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred BALB C , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide Synthase Type II/immunology , Tumor Necrosis Factor-alpha/pharmacology
13.
Sci Rep ; 11(1): 24432, 2021 12 24.
Article in English | MEDLINE | ID: covidwho-1585772

ABSTRACT

Despite the initial success of some drugs and vaccines targeting COVID-19, understanding the mechanism underlying SARS-CoV-2 disease pathogenesis remains crucial for the development of further approaches to treatment. Some patients with severe Covid-19 experience a cytokine storm and display evidence of inflammasome activation leading to increased levels of IL-1ß and IL-18; however, other reports have suggested reduced inflammatory responses to Sars-Cov-2. In this study we have examined the effects of the Sars-Cov-2 envelope (E) protein, a virulence factor in coronaviruses, on inflammasome activation and pulmonary inflammation. In cultured macrophages the E protein suppressed inflammasome priming and NLRP3 inflammasome activation. Similarly, in mice transfected with E protein and treated with poly(I:C) to simulate the effects of viral RNA, the E protein, in an NLRP3-dependent fashion, reduced expression of pro-IL-1ß, levels of IL-1ß and IL-18 in broncho-alveolar lavage fluid, and macrophage infiltration in the lung. To simulate the effects of more advanced infection, macrophages were treated with both LPS and poly(I:C). In this setting the E protein increased NLRP3 inflammasome activation in both murine and human macrophages. Thus, the Sars-Cov-2 E protein may initially suppress the host NLRP3 inflammasome response to viral RNA while potentially increasing NLRP3 inflammasome responses in the later stages of infection. Targeting the Sars-Cov-2 E protein especially in the early stages of infection may represent a novel approach to Covid-19 therapy.


Subject(s)
Coronavirus Envelope Proteins/metabolism , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , SARS-CoV-2/metabolism , Animals , Bronchoalveolar Lavage Fluid/chemistry , COVID-19/pathology , COVID-19/virology , Coronavirus Envelope Proteins/genetics , Down-Regulation/drug effects , Endoplasmic Reticulum Stress , Humans , Inflammasomes/drug effects , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Janus Kinases/genetics , Janus Kinases/metabolism , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/deficiency , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Poly I-C/pharmacology , RNA, Viral/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/isolation & purification
14.
Phytomedicine ; 95: 153784, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1521466

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a pandemic that has caused a high number of deaths worldwide. Inflammatory factors may play important roles in COVID-19 progression. Yindan Jiedu granules (YDJDG) can inhibit the progression of COVID-19, but the associated mechanism is unclear. PURPOSE: To evaluate the therapeutic effects of YDJDG on COVID-19 and explore its underlying mechanism. METHODS: We recruited 262 participants and randomly assigned 97 patients each to the YDJDG and control groups using one-to-one propensity score matching (PSM). Clinical effects were observed and serum inflammatory and immune indicators were measured. The target network model of YDJDG was established by predicting and determining the targets of identified compounds. The main constituents of the YDJDG extracts were identified and evaluated using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and molecular docking. Besides, the anti-inflammatory effects of YDJDG and its specific biological mechanism of action were studied. RESULTS: After PSM, the results showed that compared with the control group, the YDJDG group had a shorter time of dissipation of acute pulmonary exudative lesions (p < 0.0001), shorter time to negative conversion of viral nucleic acid (p < 0.01), more rapid decrease in serum amyloid A level and erythrocyte sedimentation rate (p < 0.0001), and a higher rate of increase in CD4+T cell count (p = 0.0155). By overlapping the genes of YDJDG and COVID-19, 213 co-targeted genes were identified. Metascape enrichment analysis showed that 25 genes were significantly enriched in the NF-κB pathway, which were mainly targets of luteolin, quercetin, and kaempferol as confirmed by MS analysis. Molecular docking revealed that the ligands of three compounds had strong interaction with NF-κB p65 and IκBα. In vivo, YDJDG significantly protected animals from lipopolysaccharide (LPS)-induced acute lung injury (ALI), decreasing the lung wet/dry weight ratio, ALI score, and lung histological damage. In LPS-treated RAW264.7 cells, YDJDG suppressed nuclear translocation of NF-κB p65. In vivo and in vitro, YDJDG exerted anti-inflammatory effects by inhibiting the production of inflammatory cytokines (IL-6, IL-1ß, and TNF-α). These effects were accompanied by the inhibition of NF-ĸB activation and IκBα phosphorylation. CONCLUSION: YDJDG may shorten the COVID-19 course and delay its progression by suppressing inflammation via targeting the NF-κB pathway.


Subject(s)
COVID-19 , NF-kappa B , Animals , Anti-Inflammatory Agents/pharmacology , Cytokines , Humans , Lipopolysaccharides/pharmacology , Molecular Docking Simulation , NF-kappa B/metabolism , SARS-CoV-2 , Signal Transduction , Tandem Mass Spectrometry
15.
Elife ; 102021 04 27.
Article in English | MEDLINE | ID: covidwho-1513055

ABSTRACT

Dendritic cells (DCs) regulate processes ranging from antitumor and antiviral immunity to host-microbe communication at mucosal surfaces. It remains difficult, however, to genetically manipulate human DCs, limiting our ability to probe how DCs elicit specific immune responses. Here, we develop a CRISPR-Cas9 genome editing method for human monocyte-derived DCs (moDCs) that mediates knockouts with a median efficiency of >94% across >300 genes. Using this method, we perform genetic screens in moDCs, identifying mechanisms by which DCs tune responses to lipopolysaccharides from the human microbiome. In addition, we reveal donor-specific responses to lipopolysaccharides, underscoring the importance of assessing immune phenotypes in donor-derived cells, and identify candidate genes that control this specificity, highlighting the potential of our method to pinpoint determinants of inter-individual variation in immunity. Our work sets the stage for a systematic dissection of the immune signaling at the host-microbiome interface and for targeted engineering of DCs for neoantigen vaccination.


Subject(s)
CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Dendritic Cells/immunology , Gene Editing , Genomics , Immunity, Innate/genetics , Bacteroides thetaiotaomicron/immunology , CRISPR-Associated Protein 9/metabolism , Cells, Cultured , Cytokines/genetics , Cytokines/metabolism , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Gene Expression Regulation , Humans , Immunity, Innate/drug effects , Lipopolysaccharides/pharmacology , Phenotype , Signal Transduction , Toll-Like Receptor 4/agonists , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism
16.
J Immunol ; 207(9): 2310-2324, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1497461

ABSTRACT

IFN-γ, a proinflammatory cytokine produced primarily by T cells and NK cells, activates macrophages and engages mechanisms to control pathogens. Although there is evidence of IFN-γ production by murine macrophages, IFN-γ production by normal human macrophages and their subsets remains unknown. Herein, we show that human M1 macrophages generated by IFN-γ and IL-12- and IL-18-stimulated monocyte-derived macrophages (M0) produce significant levels of IFN-γ. Further stimulation of IL-12/IL-18-primed macrophages or M1 macrophages with agonists for TLR-2, TLR-3, or TLR-4 significantly enhanced IFN-γ production in contrast to the similarly stimulated M0, M2a, M2b, and M2c macrophages. Similarly, M1 macrophages generated from COVID-19-infected patients' macrophages produced IFN-γ that was enhanced following LPS stimulation. The inhibition of M1 differentiation by Jak inhibitors reversed LPS-induced IFN-γ production, suggesting that differentiation with IFN-γ plays a key role in IFN-γ induction. We subsequently investigated the signaling pathway(s) responsible for TLR-4-induced IFN-γ production in M1 macrophages. Our results show that TLR-4-induced IFN-γ production is regulated by the ribosomal protein S6 kinase (p70S6K) through the activation of PI3K, the mammalian target of rapamycin complex 1/2 (mTORC1/2), and the JNK MAPK pathways. These results suggest that M1-derived IFN-γ may play a key role in inflammation that may be augmented following bacterial/viral infections. Moreover, blocking the mTORC1/2, PI3K, and JNK MAPKs in macrophages may be of potential translational significance in preventing macrophage-mediated inflammatory diseases.


Subject(s)
Interferon-gamma/biosynthesis , Macrophages/drug effects , Poly I-C/pharmacology , COVID-19/immunology , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/immunology , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/pharmacology , MAP Kinase Kinase Kinases/antagonists & inhibitors , MAP Kinase Kinase Kinases/immunology , Macrophages/immunology , Phosphatidylinositol 3-Kinases/immunology , Ribosomal Protein S6 Kinases, 70-kDa/antagonists & inhibitors , Ribosomal Protein S6 Kinases, 70-kDa/immunology , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/immunology , Toll-Like Receptor 4/agonists
17.
J Cell Physiol ; 237(2): 1521-1531, 2022 02.
Article in English | MEDLINE | ID: covidwho-1490820

ABSTRACT

Mechanical forces can modulate the immune response, mostly described as promoting the activation of immune cells, but the role and mechanism of pathological levels of mechanical stress in lymphocyte activation have not been focused on before. By an ex vivo experimental approach, we observed that mechanical stressing of murine spleen lymphocytes with 50 mmHg for 3 h induced the nuclear localization of NFAT1, increased C-Jun, and increased the expression of early activation marker CD69 in resting CD8+ cells. Interestingly, 50 mmHg mechanical stressing induced the nuclear localization of NFAT1; but conversely decreased C-Jun and inhibited the expression of CD69 in lymphocytes under lipopolysaccharide or phorbol 12-myristate 13-acetate/ionomycin stimulation. Additionally, we observed similar changes trends when comparing RNA-seq data of hypertensive and normotensive COVID-19 patients. Our results indicate a biphasic effect of mechanical stress on lymphocyte activation, which provides insight into the variety of immune responses in pathologies involving elevated mechanical stress.


Subject(s)
Lymphocyte Activation/immunology , Stress, Mechanical , Animals , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Biomarkers/metabolism , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , COVID-19/complications , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Comorbidity , Gene Expression Regulation/drug effects , Humans , Hypertension/complications , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Ion Channels/metabolism , Lectins, C-Type/metabolism , Lipopolysaccharides/pharmacology , Lymphocyte Activation/drug effects , Lymphocyte Activation/genetics , Male , Mice, Inbred C57BL , NFATC Transcription Factors/metabolism , Protein Transport/drug effects , Proto-Oncogene Proteins c-jun/metabolism , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacology
18.
Int J Mol Sci ; 22(20)2021 Oct 18.
Article in English | MEDLINE | ID: covidwho-1470894

ABSTRACT

Infection caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2) in many cases is accompanied by the release of a large amount of proinflammatory cytokines in an event known as "cytokine storm", which is associated with severe coronavirus disease 2019 (COVID-19) cases and high mortality. The excessive production of proinflammatory cytokines is linked, inter alia, to the enhanced activity of receptors capable of recognizing the conservative regions of pathogens and cell debris, namely TLRs, TREM-1 and TNFR1. Here we report that peptides derived from innate immunity protein Tag7 inhibit activation of TREM-1 and TNFR1 receptors during acute inflammation. Peptides from the N-terminal fragment of Tag7 bind only to TREM-1, while peptides from the C-terminal fragment interact solely with TNFR1. Selected peptides are capable of inhibiting the production of proinflammatory cytokines both in peripheral blood mononuclear cells (PBMCs) from healthy donors and in vivo in the mouse model of acute lung injury (ALI) by diffuse alveolar damage (DAD). Treatment with peptides significantly decreases the infiltration of mononuclear cells to lungs in animals with DAD. Our findings suggest that Tag7-derived peptides might be beneficial in terms of the therapy or prevention of acute lung injury, e.g., for treating COVID-19 patients with severe pulmonary lesions.


Subject(s)
Acute Lung Injury/pathology , Cytokines/chemistry , Peptides/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Triggering Receptor Expressed on Myeloid Cells-1/metabolism , Acute Lung Injury/metabolism , Animals , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Humans , Interferon-gamma/genetics , Interferon-gamma/metabolism , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/pharmacology , Lung/metabolism , Lung/pathology , Lymphocyte Activation/drug effects , Male , Mice , Mice, Inbred ICR , Peptides/chemistry , Peptides/pharmacology , Protein Binding , Receptors, Tumor Necrosis Factor, Type I/antagonists & inhibitors , Triggering Receptor Expressed on Myeloid Cells-1/antagonists & inhibitors
19.
J Immunol ; 207(9): 2310-2324, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1436143

ABSTRACT

IFN-γ, a proinflammatory cytokine produced primarily by T cells and NK cells, activates macrophages and engages mechanisms to control pathogens. Although there is evidence of IFN-γ production by murine macrophages, IFN-γ production by normal human macrophages and their subsets remains unknown. Herein, we show that human M1 macrophages generated by IFN-γ and IL-12- and IL-18-stimulated monocyte-derived macrophages (M0) produce significant levels of IFN-γ. Further stimulation of IL-12/IL-18-primed macrophages or M1 macrophages with agonists for TLR-2, TLR-3, or TLR-4 significantly enhanced IFN-γ production in contrast to the similarly stimulated M0, M2a, M2b, and M2c macrophages. Similarly, M1 macrophages generated from COVID-19-infected patients' macrophages produced IFN-γ that was enhanced following LPS stimulation. The inhibition of M1 differentiation by Jak inhibitors reversed LPS-induced IFN-γ production, suggesting that differentiation with IFN-γ plays a key role in IFN-γ induction. We subsequently investigated the signaling pathway(s) responsible for TLR-4-induced IFN-γ production in M1 macrophages. Our results show that TLR-4-induced IFN-γ production is regulated by the ribosomal protein S6 kinase (p70S6K) through the activation of PI3K, the mammalian target of rapamycin complex 1/2 (mTORC1/2), and the JNK MAPK pathways. These results suggest that M1-derived IFN-γ may play a key role in inflammation that may be augmented following bacterial/viral infections. Moreover, blocking the mTORC1/2, PI3K, and JNK MAPKs in macrophages may be of potential translational significance in preventing macrophage-mediated inflammatory diseases.


Subject(s)
Interferon-gamma/biosynthesis , Macrophages/drug effects , Poly I-C/pharmacology , COVID-19/immunology , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/immunology , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/pharmacology , MAP Kinase Kinase Kinases/antagonists & inhibitors , MAP Kinase Kinase Kinases/immunology , Macrophages/immunology , Phosphatidylinositol 3-Kinases/immunology , Ribosomal Protein S6 Kinases, 70-kDa/antagonists & inhibitors , Ribosomal Protein S6 Kinases, 70-kDa/immunology , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/immunology , Toll-Like Receptor 4/agonists
20.
Int J Biol Sci ; 17(13): 3305-3319, 2021.
Article in English | MEDLINE | ID: covidwho-1372133

ABSTRACT

An inflammatory cytokine storm is considered an important cause of death in severely and critically ill COVID-19 patients, however, the relationship between the SARS-CoV-2 spike (S) protein and the host's inflammatory cytokine storm is not clear. Here, the qPCR results indicated that S protein induced a significantly elevated expression of multiple inflammatory factor mRNAs in peripheral blood mononuclear cells (PBMCs), whereas RS-5645 ((4-(thiophen-3-yl)-1-(p-tolyl)-1H-pyrrol-3-yl)(3,4,5-trimethoxyphenyl)methanone) attenuated the expression of the most inflammatory factor mRNAs. RS-5645 also significantly reduced the cellular ratios of CD45+/IFNγ+, CD3+/IFNγ+, CD11b+/IFNγ+, and CD56+/IFNγ+ in human PBMCs. In addition, RS-5645 effectively inhibited the activation of inflammatory cells and reduced inflammatory damage to lung tissue in mice. Sequencing results of 16S rRNA v3+v4 in mouse alveolar lavage fluid showed that there were 494 OTUs overlapping between the alveolar lavage fluid of mice that underwent S protein+ LPS-combined intervention (M) and RS-5645-treated mice (R), while R manifested 64 unique OTUs and M exhibited 610 unique OTUs. In the alveoli of group R mice, the relative abundances of microorganisms belonging to Porphyromonas, Rothia, Streptococcus, and Neisseria increased significantly, while the relative abundances of microorganisms belonging to Psychrobacter, Shimia, and Sporosarcina were significantly diminished. The results of KEGG analysis indicated that the alveolar microbiota of mice in the R group can increase translation and reduce the activity of amino acid metabolism pathways. COG analysis results indicated that the abundance of proteins involved in ribosomal structure and biogenesis related to metabolism was augmented in the alveolar microbiota of the mice in the R group, while the abundance of proteins involved in secondary metabolite biosynthesis was significantly reduced. Therefore, our research results showed that RS-5645 attenuated pulmonary inflammatory cell infiltration and the inflammatory storm induced by the S protein and LPS by modulating the pulmonary microbiota.


Subject(s)
Anti-Inflammatory Agents/pharmacology , COVID-19/immunology , Cytokine Release Syndrome/prevention & control , Lipopolysaccharides/pharmacology , Lung/microbiology , Microbiota/drug effects , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/physiology , Animals , Antigens, CD/immunology , COVID-19/virology , Cytokine Release Syndrome/immunology , Disease Models, Animal , Humans , Interferon-gamma/immunology , Male , Mice , Mice, Inbred BALB C
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