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1.
PLoS One ; 18(6): e0286211, 2023.
Article in English | MEDLINE | ID: covidwho-20232587

ABSTRACT

BACKGROUND AND PURPOSE: Cytokine storm invoked during acute and chronic lung injury promotes alveolar damage and remodeling. The current study shows that degraded elastin-targeted nanoparticles releasing doxycycline (Doxy NPs) are potent in mitigating cytokines storm, migration of immune cells in the lungs, and inhibiting inflammasome pathways in the LPS mouse model. EXPERIMENTAL APPROACH: Cytokine storm and lung injury were induced using LPS and elastase in C57BL/6 mice (rodent model for emphysema). The mice were then treated with I.V. Doxy NPs, blank NPs, or Doxy a day before LPS administration. Cytokine levels, immune cell population, and MMP activity were analyzed in broncheo-alveolar lavage fluid (BALF) 4 hours after LPS administration. Additionally, gene expression of IL-6, IL-1beta, MCP-1, NLRP3, Caspase 1 and MMPs were investigated in alveolar cells on day 3 after LPS administration. KEY RESULTS: Doxycycline NPs but not Doxycycline significantly decreased IL-6, TNF-α, IL-23 and were significantly more effective in decreasing the percentage of immune cells in the BALF. This is the first in-vivo study to demonstrate that Doxycycline can effectively inhibit inflammasome pathways in the lungs. CONCLUSION AND IMPLICATIONS: IV administration of elastin antibody conjugated Doxycycline-loaded albumin NPs can effectively modulate the local immune environment in the lungs, which is not achieved by IV Doxycycline even at 100-fold higher dose. This novel method of drug delivery can effectively lead to the repurposing of traditional Doxycycline as a potential adjunct treatment for managing the cytokine storm in the lungs in COPD and viral infections.


Subject(s)
Lung Injury , Nanoparticles , Pneumonia , Mice , Animals , Lipopolysaccharides/pharmacology , Inflammasomes/metabolism , Interleukin-6/metabolism , Cytokine Release Syndrome , Elastin/metabolism , Mice, Inbred C57BL , Pneumonia/metabolism , Lung/metabolism , Cytokines/metabolism , Lung Injury/metabolism
2.
J Am Chem Soc ; 145(24): 13261-13272, 2023 06 21.
Article in English | MEDLINE | ID: covidwho-20240992

ABSTRACT

Activating antigen-presenting cells is essential to generate adaptive immunity, while the efficacy of conventional activation strategies remains unsatisfactory due to suboptimal antigen-specific priming. Here, in situ polymerization-mediated antigen presentation (IPAP) is described, in which antigen-loaded nanovaccines are spontaneously formed and efficiently anchored onto the surface of dendritic cells in vivo through co-deposition with dopamine. The resulting chemically bound nanovaccines can promote antigen presentation by elevating macropinocytosis-based cell uptake and reducing lysosome-related antigen degradation. IPAP is able to prolong the duration of antigen reservation in the injection site and enhance subsequent accumulation in the draining lymph nodes, thereby eliciting robust antigen-specific cellular and humoral immune responses. IPAP is also applicable for different antigens and capable of circumventing the disadvantages of complicated preparation and purification. By implementation with ovalbumin, IPAP induces a significant protective immunity against ovalbumin-overexpressing tumor cell challenge in a prophylactic murine model. The use of the SARS-CoV-2 Spike protein S1 subunit also remarkably increases the production of S1-specific immunoglobulin G in mice. IPAP offers a unique strategy for stimulating antigen-presenting cells to boost antigen-specific adaptive responses and proposes a facile yet versatile method for immunization against various diseases.


Subject(s)
Antigen Presentation , COVID-19 , Mice , Humans , Animals , Ovalbumin , Polymerization , Dendritic Cells , COVID-19/metabolism , SARS-CoV-2 , Antigens , Mice, Inbred C57BL
3.
Proc Natl Acad Sci U S A ; 120(22): e2300155120, 2023 05 30.
Article in English | MEDLINE | ID: covidwho-2323651

ABSTRACT

Obesity has been recognized as one of the most significant risk factors for the deterioration and mortality associated with COVID-19, but the significance of obesity itself differs among ethnicity. Multifactored analysis of our single institute-based retrospective cohort revealed that high visceral adipose tissue (VAT) burden, but not other obesity-associated markers, was related to accelerated inflammatory responses and the mortality of Japanese COVID-19 patients. To elucidate the mechanisms how VAT-dominant obesity induces severe inflammation after severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection, we infected two different strains of obese mice, C57BL/6JHamSlc-ob/ob (ob/ob), C57BLKS/J-db/db (db/db), genetically impaired in the leptin ligand and receptor, respectively, and control C57BL/6 mice with mouse-adapted SARS-CoV-2. Here, we revealed that VAT-dominant ob/ob mice were extremely more vulnerable to SARS-CoV-2 due to excessive inflammatory responses when compared to SAT-dominant db/db mice. In fact, SARS-CoV-2 genome and proteins were more abundant in the lungs of ob/ob mice, engulfed in macrophages, resulting in increased cytokine production including interleukin (IL)-6. Both an anti-IL-6 receptor antibody treatment and the prevention of obesity by leptin replenishment improved the survival of SARS-CoV-2-infected ob/ob mice by reducing the viral protein burden and excessive immune responses. Our results have proposed unique insights and clues on how obesity increases the risk of cytokine storm and death in patients with COVID-19. Moreover, earlier administration of antiinflammatory therapeutics including anti-IL-6R antibody to VAT-dominant patients might improve clinical outcome and stratification of the treatment for COVID-19, at least in Japanese patients.


Subject(s)
COVID-19 , Malus , Mice , Animals , Leptin/genetics , Cytokines , COVID-19/complications , Retrospective Studies , SARS-CoV-2 , Mice, Inbred C57BL , Obesity/complications , Obesity/genetics , Interleukin-6 , Mice, Obese
4.
Int J Mol Sci ; 24(9)2023 May 06.
Article in English | MEDLINE | ID: covidwho-2320574

ABSTRACT

Extracellular collagen remodeling is one of the central mechanisms responsible for the structural and compositional coherence of myocardium in patients undergoing myocardial infarction (MI). Activated primary cardiac fibroblasts following myocardial infarction are extensively investigated to establish anti-fibrotic therapies to improve left ventricular remodeling. To systematically assess vitamin C functions as a potential modulator involved in collagen fibrillogenesis in an in vitro model mimicking heart tissue healing after MI. Mouse primary cardiac fibroblasts were isolated from wild-type C57BL/6 mice and cultured under normal and profibrotic (hypoxic + transforming growth factor beta 1) conditions on freshly prepared coatings mimicking extracellular matrix (ECM) remodeling during healing after an MI. At 10 µg/mL, vitamin C reprogramed the respiratory mitochondrial metabolism, which is effectively associated with a more increased accumulation of intracellular reactive oxygen species (iROS) than the number of those generated by mitochondrial reactive oxygen species (mROS). The mRNA/protein expression of subtypes I, III collagen, and fibroblasts differentiations markers were upregulated over time, particularly in the presence of vitamin C. The collagen substrate potentiated the modulator role of vitamin C in reinforcing the structure of types I and III collagen synthesis by reducing collagen V expression in a timely manner, which is important in the initiation of fibrillogenesis. Altogether, our study evidenced the synergistic function of vitamin C at an optimum dose on maintaining the equilibrium functionality of radical scavenger and gene transcription, which are important in the initial phases after healing after an MI, while modulating the synthesis of de novo collagen fibrils, which is important in the final stage of tissue healing.


Subject(s)
Ascorbic Acid , Myocardial Infarction , Mice , Animals , Ascorbic Acid/pharmacology , Ascorbic Acid/metabolism , Reactive Oxygen Species/metabolism , Mice, Inbred C57BL , Myocardial Infarction/metabolism , Myocardium/metabolism , Collagen/metabolism , Fibroblasts/metabolism , Vitamins/metabolism , Ventricular Remodeling/physiology
5.
Front Immunol ; 14: 1030879, 2023.
Article in English | MEDLINE | ID: covidwho-2309368

ABSTRACT

Introduction: There is an unmet medical need for effective anti-inflammatory agents for the treatment of acute and post-acute lung inflammation caused by respiratory viruses. The semi-synthetic polysaccharide, Pentosan polysulfate sodium (PPS), an inhibitor of NF-kB activation, was investigated for its systemic and local anti-inflammatory effects in a mouse model of influenza virus A/PR8/1934 (PR8 strain) mediated infection. Methods: Immunocompetent C57BL/6J mice were infected intranasally with a sublethal dose of PR8 and treated subcutaneously with 3 or 6 mg/kg PPS or vehicle. Disease was monitored and tissues were collected at the acute (8 days post-infection; dpi) or post-acute (21 dpi) phase of disease to assess the effect of PPS on PR8-induced pathology. Results: In the acute phase of PR8 infection, PPS treatment was associated with a reduction in weight loss and improvement in oxygen saturation when compared to vehicle-treated mice. Associated with these clinical improvements, PPS treatment showed a significant retention in the numbers of protective SiglecF+ resident alveolar macrophages, despite uneventful changes in pulmonary leukocyte infiltrates assessed by flow cytometry. PPS treatment in PR8- infected mice showed significant reductions systemically but not locally of the inflammatory molecules, IL-6, IFN-g, TNF-a, IL-12p70 and CCL2. In the post-acute phase of infection, PPS demonstrated a reduction in the pulmonary fibrotic biomarkers, sICAM-1 and complement factor C5b9. Discussion: The systemic and local anti-inflammatory actions of PPS may regulate acute and post-acute pulmonary inflammation and tissue remodeling mediated by PR8 infection, which warrants further investigation.


Subject(s)
Influenzavirus A , Pneumonia , Mice , Animals , Pentosan Sulfuric Polyester/pharmacology , Pentosan Sulfuric Polyester/therapeutic use , Mice, Inbred C57BL , Pneumonia/drug therapy , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Disease Models, Animal
6.
Bone ; 172: 116762, 2023 07.
Article in English | MEDLINE | ID: covidwho-2300445

ABSTRACT

Social isolation is a potent form of psychosocial stress and is a growing public health concern, particularly among older adults. Even prior to the onset of the COVID-19 pandemic, which has significantly increased the prevalence of isolation and loneliness, researchers have been concerned about a rising "epidemic" of loneliness. Isolation is associated with an increased risk for many physical and mental health disorders and increased overall mortality risk. In addition to social isolation, older adults are also at greater risk for osteoporosis and related fractures. While researchers have investigated the negative effects of other forms of psychosocial stress on bone, including depression and PTSD, the effects of social isolation on bone have not been thoroughly investigated. The aim of this study was to test the hypothesis that social isolation would lead to bone loss in male and female C57BL/6J mice. 16-week-old mice were randomized into social isolation (1 mouse/cage) or grouped housing (4 mice/cage) for four weeks. Social isolation significantly decreased trabecular (BV/TV, BMD, Tb. N., Tb. Th.) and cortical bone (Ct.Th., Ct.Ar., Ct.Ar./Tt.Ar., pMOI) parameters in male, but not female mice. Isolated male mice had signs of reduced bone remodeling represented by reduced osteoblast numbers, osteoblast-related gene expression and osteoclast-related gene expression. However, isolated females had increased bone resorption-related gene expression, without any change in bone mass. Overall, our data suggest that social isolation has negative effects on bone in male, but not female mice, although females showed suggestive effects on bone resorption. These results provide critical insight into the effects of isolation on bone and have key clinical implications as we grapple with the long-term health impacts of the rise in social isolation related to the COVID-19 pandemic.


Subject(s)
Bone Resorption , COVID-19 , Female , Male , Mice , Humans , Animals , Mice, Inbred C57BL , Housing , Pandemics , Bone Density , Cortical Bone , Social Isolation
7.
Cells ; 12(7)2023 04 06.
Article in English | MEDLINE | ID: covidwho-2294986

ABSTRACT

The COVID-19 pandemic was triggered by the coronavirus SARS-CoV-2, whose peak occurred in the years 2020 and 2021. The main target of this virus is the lung, and the infection is associated with an accentuated inflammatory process involving mainly the innate arm of the immune system. Here, we described the induction of a pulmonary inflammatory process triggered by the intranasal (IN) instillation of UV-inactivated SARS-CoV-2 in C57BL/6 female mice, and then the evaluation of the ability of vitamin D (VitD) to control this process. The assays used to estimate the severity of lung involvement included the total and differential number of cells in the bronchoalveolar lavage fluid (BALF), histopathological analysis, quantification of T cell subsets, and inflammatory mediators by RT-PCR, cytokine quantification in lung homogenates, and flow cytometric analysis of cells recovered from lung parenchyma. The IN instillation of inactivated SARS-CoV-2 triggered a pulmonary inflammatory process, consisting of various cell types and mediators, resembling the typical inflammation found in transgenic mice infected with SARS-CoV-2. This inflammatory process was significantly decreased by the IN delivery of VitD, but not by its IP administration, suggesting that this hormone could have a therapeutic potential in COVID-19 if locally applied. To our knowledge, the local delivery of VitD to downmodulate lung inflammation in COVID-19 is an original proposition.


Subject(s)
COVID-19 , Pneumonia , Mice , Animals , Female , Humans , SARS-CoV-2 , Vitamin D/pharmacology , Pandemics , Mice, Inbred C57BL , Vitamins , Mice, Transgenic
8.
Behav Brain Res ; 448: 114441, 2023 06 25.
Article in English | MEDLINE | ID: covidwho-2292068

ABSTRACT

Opioid misuse has dramatically increased over the last few decades resulting in many people suffering from opioid use disorder (OUD). The prevalence of opioid overdose has been driven by the development of new synthetic opioids, increased availability of prescription opioids, and more recently, the COVID-19 pandemic. Coinciding with increases in exposure to opioids, the United States has also observed increases in multiple Narcan (naloxone) administrations as a life-saving measures for respiratory depression, and, thus, consequently, naloxone-precipitated withdrawal. Sleep dysregulation is a main symptom of OUD and opioid withdrawal syndrome, and therefore, should be a key facet of animal models of OUD. Here we examine the effect of precipitated and spontaneous morphine withdrawal on sleep behaviors in C57BL/6 J mice. We find that morphine administration and withdrawal dysregulate sleep, but not equally across morphine exposure paradigms. Furthermore, many environmental triggers promote relapse to drug-seeking/taking behavior, and the stress of disrupted sleep may fall into that category. We find that sleep deprivation dysregulates sleep in mice that had previous opioid withdrawal experience. Our data suggest that the 3-day precipitated withdrawal paradigm has the most profound effects on opioid-induced sleep dysregulation and further validates the construct of this model for opioid dependence and OUD.


Subject(s)
COVID-19 , Morphine Dependence , Opioid-Related Disorders , Substance Withdrawal Syndrome , Male , Female , Mice , Animals , Humans , Morphine/adverse effects , Analgesics, Opioid/pharmacology , Mice, Inbred C57BL , Narcotic Antagonists/pharmacology , Narcotic Antagonists/therapeutic use , Pandemics , Naloxone/pharmacology , Naloxone/therapeutic use , Narcotics/adverse effects , Opioid-Related Disorders/drug therapy , Sleep , Substance Withdrawal Syndrome/drug therapy , Morphine Dependence/drug therapy
9.
Virol J ; 20(1): 75, 2023 04 20.
Article in English | MEDLINE | ID: covidwho-2302137

ABSTRACT

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes non-symptomatic infection, mild influenza-like symptoms to pneumonia, severe acute respiratory distress syndrome, and even death, reflecting different clinical symptoms of viral infection. However, the mechanism of its pathogenicity remains unclear. Host-specific traits have a breakthrough significance for studying the pathogenicity of SARS-CoV-2. We previously reported SARS-CoV-2/BMA8, a mouse-adapted strain, was lethal to aged BALB/c mice but not to aged C57BL/6N mice. Here, we further investigate the differences in pathogenicity of BMA8 strain against wild-type aged C57BL/6N and BALB/c mice. METHODS: Whole blood and tissues were collected from mice before and after BMA8 strain infection. Viral replication and infectivity were assessed by detection of viral RNA copies and viral titers; the degree of inflammation in mice was tested by whole blood cell count, ELISA and RT-qPCR assays; the pathogenicity of SARS-CoV-2/BMA8 in mice was measured by Histopathology and Immunohistochemistry; and the immune level of mice was evaluated by flow cytometry to detect the number of CD8+ T cells. RESULTS: Our results suggest that SARS-CoV-2/BMA8 strain caused lower pathogenicity and inflammation level in C57BL/6N mice than in BALB/c mice. Interestingly, BALB/c mice whose MHC class I haplotype is H-2Kd showed more severe pathogenicity after infection with BMA8 strain, while blockade of H-2Kb in C57BL/6N mice was also able to cause this phenomenon. Furthermore, H-2Kb inhibition increased the expression of cytokines/chemokines and accelerated the decrease of CD8+ T cells caused by SARS-CoV-2/BMA8 infection. CONCLUSIONS: Taken together, our work shows that host MHC molecules play a crucial role in the pathogenicity differences of SARS-CoV-2/BMA8 infection. This provides a more profound insight into the pathogenesis of SARS-CoV-2, and contributes enlightenment and guidance for controlling the virus spread.


Subject(s)
COVID-19 , SARS-CoV-2 , Mice , Animals , CD8-Positive T-Lymphocytes , Virulence , COVID-19/pathology , Mice, Inbred C57BL , Mice, Inbred BALB C , Inflammation , Lung/pathology , Disease Models, Animal
10.
Phytomedicine ; 114: 154753, 2023 Jun.
Article in English | MEDLINE | ID: covidwho-2302077

ABSTRACT

BACKGROUND: Dehydroandrographolide (Deh) from Andrographis paniculata (Burm.f.) Wall has strong anti-inflammatory and antioxidant activities. PURPOSE: To explore the role of Deh in acute lung injury (ALI) of coronavirus disease 19 (COVID-19) and its inflammatory molecular mechanism. METHODS: Liposaccharide (LPS) was injected into a C57BL/6 mouse model of ALI, and LPS + adenosine triphosphate (ATP) was used to stimulate BMDMs in an in vitro model of ALI. RESULTS: In an in vivo and in vitro model of ALI, Deh considerably reduced inflammation and oxidative stress by inhibiting NLRP3-mediated pyroptosis and attenuated mitochondrial damage to suppress NLRP3-mediated pyroptosis through the suppression of ROS production by inhibiting the Akt/Nrf2 pathway. Deh inhibited the interaction between Akt at T308 and PDPK1 at S549 to promote Akt protein phosphorylation. Deh directly targeted PDPK1 protein and accelerated PDPK1 ubiquitination. 91-GLY, 111-LYS, 126-TYR, 162-ALA, 205-ASP and 223-ASP may be the reason for the interaction between PDPK1 and Deh. CONCLUSION: Deh from Andrographis paniculata (Burm.f.) Wall presented NLRP3-mediated pyroptosis in a model of ALI through ROS-induced mitochondrial damage through inhibition of the Akt/Nrf2 pathway by PDPK1 ubiquitination. Therefore, it can be concluded that Deh may be a potential therapeutic drug for the treatment of ALI in COVID-19 or other respiratory diseases.


Subject(s)
Acute Lung Injury , COVID-19 , Mice , Animals , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Andrographis paniculata , Proto-Oncogene Proteins c-akt/metabolism , Reactive Oxygen Species/metabolism , Medicine, Chinese Traditional , Pyroptosis , Lipopolysaccharides/pharmacology , NF-E2-Related Factor 2 , Mice, Inbred C57BL , Acute Lung Injury/chemically induced , Inflammasomes
11.
Emerg Microbes Infect ; 12(1): 2203782, 2023 Dec.
Article in English | MEDLINE | ID: covidwho-2296691

ABSTRACT

Multiple clinical and epidemiological studies have shown an interconnection between coronavirus disease 2019 (COVID-19) and diabetes, but experimental evidence is still lacking. Understanding the interplay between them is important because of the global health burden of COVID-19 and diabetes. We found that C57BL/6J mice were susceptible to the alpha strain of SARS-CoV-2. Moreover, diabetic C57BL/6J mice with leptin receptor gene deficiency (db/db mice) showed a higher viral load in the throat and lung and slower virus clearance in the throat after infection than C57BL/6J mice. Histological and multifactor analysis revealed more advanced pulmonary injury and serum inflammation in SARS-CoV-2 infected diabetic mice. Moreover, SARS-CoV-2 infected diabetic mice exhibited more severe insulin resistance and islet cell loss than uninfected diabetic mice. By RNA sequencing analysis, we found that diabetes may reduce the collagen level, suppress the immune response and aggravate inflammation in the lung after infection, which may account for the greater susceptibility of diabetic mice and their more severe lung damage after infection. In summary, we successfully established a SARS-CoV-2 infected diabetic mice model and demonstrated that diabetes and COVID-19 were risk factors for one another.


Subject(s)
COVID-19 , Diabetes Mellitus, Experimental , Mice , Animals , SARS-CoV-2 , Mice, Inbred C57BL , Inflammation
12.
Viruses ; 15(2)2023 01 18.
Article in English | MEDLINE | ID: covidwho-2272025

ABSTRACT

The COVID-19 pandemic remains a global health threat and novel antiviral strategies are urgently needed. SARS-CoV-2 employs the cellular serine protease TMPRSS2 for entry into lung cells, and TMPRSS2 inhibitors are being developed for COVID-19 therapy. However, the SARS-CoV-2 Omicron variant, which currently dominates the pandemic, prefers the endo/lysosomal cysteine protease cathepsin L over TMPRSS2 for cell entry, raising doubts as to whether TMPRSS2 inhibitors would be suitable for the treatment of patients infected with the Omicron variant. Nevertheless, the contribution of TMPRSS2 to the spread of SARS-CoV-2 in the infected host is largely unclear. In this study, we show that the loss of TMPRSS2 strongly reduced the replication of the Beta variant in the nose, trachea and lung of C57BL/6 mice, and protected the animals from weight loss and disease. The infection of mice with the Omicron variant did not cause disease, as expected, but again, TMPRSS2 was essential for efficient viral spread in the upper and lower respiratory tract. These results identify the key role of TMPRSS2 in SARS-CoV-2 Beta and Omicron infection, and highlight TMPRSS2 as an attractive target for antiviral intervention.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Humans , Mice , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Mice, Inbred C57BL , Pandemics , Serine Endopeptidases/genetics
13.
Am J Physiol Lung Cell Mol Physiol ; 324(5): L722-L736, 2023 05 01.
Article in English | MEDLINE | ID: covidwho-2271860

ABSTRACT

SARS-CoV-2 viremia is associated with increased acute lung injury (ALI) and mortality in children and adults. The mechanisms by which viral components in the circulation mediate ALI in COVID-19 remain unclear. We tested the hypothesis that the SARS-CoV-2 envelope (E) protein induces Toll-like receptor (TLR)-mediated ALI and lung remodeling in a model of neonatal COVID-19. Neonatal C57BL6 mice given intraperitoneal E protein injections revealed a dose-dependent increase in lung cytokines [interleukin 6 (Il6), tumor necrosis factor (Tnfα), and interleukin 1 beta (Il1ß)] and canonical proinflammatory TLR signaling. Systemic E protein induced endothelial immune activation, immune cell influx, and TGFß signaling and lung matrix remodeling inhibited alveolarization in the developing lung. E protein-mediated ALI and transforming growth factor beta (TGFß) signaling was repressed in Tlr2-/-, but not Tlr4-/- mice. A single dose of intraperitoneal E protein injection induced chronic alveolar remodeling as evidenced by a decrease in radial alveolar counts and increase in mean linear intercepts. Ciclesonide, a synthetic glucocorticoid, inhibited E protein-induced proinflammatory TLR signaling and ALI. In vitro, E protein-mediated inflammation and cell death were TLR2-dependent in human primary neonatal lung endothelial cells and were rescued by ciclesonide. This study provides insight into the pathogenesis of ALI and alveolar remodeling with SARS-CoV-2 viremia in children, whereas revealing the efficacy of steroids.NEW & NOTEWORTHY We reveal that the envelope protein of SARS-CoV-2 mediates acute lung injury (ALI) and alveolar remodeling through Toll-like receptor activation, which is rescued by the glucocorticoid, ciclesonide.


Subject(s)
Acute Lung Injury , COVID-19 , Animals , Child , Humans , Mice , Acute Lung Injury/chemically induced , COVID-19/complications , Endothelial Cells/metabolism , Glucocorticoids , Lipopolysaccharides/adverse effects , Mice, Inbred C57BL , SARS-CoV-2/metabolism , Toll-Like Receptor 2 , Toll-Like Receptor 4/metabolism , Toll-Like Receptors , Transforming Growth Factor beta , Viremia/complications , Viral Envelope/metabolism
14.
mBio ; 14(2): e0041623, 2023 04 25.
Article in English | MEDLINE | ID: covidwho-2278130

ABSTRACT

Recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant B.1.1.529 (Omicron) has rapidly become the dominant strain, with an unprecedented number of mutations within its spike gene. However, it remains unknown whether these variants have alterations in their entry efficiency, host tropism, and sensitivity to neutralizing antibodies and entry inhibitors. In this study, we found that Omicron spike has evolved to escape neutralization by three-dose inactivated-vaccine-elicited immunity but remains sensitive to an angiotensin-converting enzyme 2 (ACE2) decoy receptor. Moreover, Omicron spike could use human ACE2 with a slightly increased efficiency while gaining a significantly increased binding affinity for a mouse ACE2 ortholog, which exhibits limited binding with wild-type (WT) spike. Furthermore, Omicron could infect wild-type C57BL/6 mice and cause histopathological changes in the lungs. Collectively, our results reveal that evasion of neutralization by vaccine-elicited antibodies and enhanced human and mouse ACE2 receptor engagement may contribute to the expanded host range and rapid spread of the Omicron variant. IMPORTANCE The recently emerged SARS-CoV-2 Omicron variant with numerous mutations in the spike protein has rapidly become the dominant strain, thereby raising concerns about the effectiveness of vaccines. Here, we found that the Omicron variant exhibits a reduced sensitivity to serum neutralizing activity induced by a three-dose inactivated vaccine but remains sensitive to entry inhibitors or an ACE2-Ig decoy receptor. Compared with the ancestor strain isolated in early 2020, the spike protein of Omicron utilizes the human ACE2 receptor with enhanced efficiency while gaining the ability to utilize mouse ACE2 for cell entry. Moreover, Omicron could infect wild-type mice and cause pathological changes in the lungs. These results reveal that antibody evasion, enhanced human ACE2 utilization, and an expanded host range may contribute to its rapid spread.


Subject(s)
COVID-19 , Immune Evasion , Humans , Animals , Mice , Mice, Inbred C57BL , Angiotensin-Converting Enzyme 2/genetics , Host Specificity , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Antibodies, Neutralizing , Antibodies, Viral
15.
Sci Transl Med ; 15(686): eabn3464, 2023 03 08.
Article in English | MEDLINE | ID: covidwho-2277682

ABSTRACT

As mRNA vaccines have proved to be very successful in battling the coronavirus disease 2019 (COVID-19) pandemic, this new modality has attracted widespread interest for the development of potent vaccines against other infectious diseases and cancer. Cervical cancer caused by persistent human papillomavirus (HPV) infection is a major cause of cancer-related deaths in women, and the development of safe and effective therapeutic strategies is urgently needed. In the present study, we compared the performance of three different mRNA vaccine modalities to target tumors associated with HPV-16 infection in mice. We generated lipid nanoparticle (LNP)-encapsulated self-amplifying mRNA as well as unmodified and nucleoside-modified non-replicating mRNA vaccines encoding a chimeric protein derived from the fusion of the HPV-16 E7 oncoprotein and the herpes simplex virus type 1 glycoprotein D (gDE7). We demonstrated that single low-dose immunizations with any of the three gDE7 mRNA vaccines induced activation of E7-specific CD8+ T cells, generated memory T cell responses capable of preventing tumor relapses, and eradicated subcutaneous tumors at different growth stages. In addition, the gDE7 mRNA-LNP vaccines induced potent tumor protection in two different orthotopic mouse tumor models after administration of a single vaccine dose. Last, comparative studies demonstrated that all three gDE7 mRNA-LNP vaccines proved to be superior to gDE7 DNA and gDE7 recombinant protein vaccines. Collectively, we demonstrated the immunogenicity and therapeutic efficacy of three different mRNA vaccines in extensive comparative experiments. Our data support further evaluation of these mRNA vaccines in clinical trials.


Subject(s)
Cancer Vaccines , Neoplasms , Papillomavirus Infections , Papillomavirus Vaccines , Vaccines, DNA , Animals , Female , Mice , CD8-Positive T-Lymphocytes , Disease Models, Animal , Immunization , Mice, Inbred C57BL , Neoplasms/therapy , Papillomavirus E7 Proteins/genetics , Papillomavirus Infections/complications , Papillomavirus Infections/prevention & control , Papillomavirus Vaccines/genetics , Recombinant Proteins , RNA, Messenger/genetics
16.
Int J Mol Sci ; 24(5)2023 Mar 06.
Article in English | MEDLINE | ID: covidwho-2289135

ABSTRACT

In the course of the SARS-CoV-2 pandemic, vaccination safety and risk factors of SARS-CoV-2 mRNA-vaccines were under consideration after case reports of vaccine-related side effects, such as myocarditis, which were mostly described in young men. However, there is almost no data on the risk and safety of vaccination, especially in patients who are already diagnosed with acute/chronic (autoimmune) myocarditis from other causes, such as viral infections, or as a side effect of medication and treatment. Thus, the risk and safety of these vaccines, in combination with other therapies that could induce myocarditis (e.g., immune checkpoint inhibitor (ICI) therapy), are still poorly assessable. Therefore, vaccine safety, with respect to worsening myocardial inflammation and myocardial function, was studied in an animal model of experimentally induced autoimmune myocarditis. Furthermore, it is known that ICI treatment (e.g., antibodies (abs) against PD-1, PD-L1, and CTLA-4, or a combination of those) plays an important role in the treatment of oncological patients. However, it is also known that treatment with ICIs can induce severe, life-threatening myocarditis in some patients. Genetically different A/J (most susceptible strain) and C57BL/6 (resistant strain) mice, with diverse susceptibilities for induction of experimental autoimmune myocarditis (EAM) at various age and gender, were vaccinated twice with SARS-CoV-2 mRNA-vaccine. In an additional A/J group, an autoimmune myocarditis was induced. In regard to ICIs, we tested the safety of SARS-CoV-2 vaccination in PD-1-/- mice alone, and in combination with CTLA-4 abs. Our results showed no adverse effects related to inflammation and heart function after mRNA-vaccination, independent of age, gender, and in different mouse strains susceptible for induction of experimental myocarditis. Moreover, there was no worsening effect on inflammation and cardiac function when EAM in susceptible mice was induced. However, in the experiments with vaccination and ICI treatment, we observed, in some mice, low elevation of cardiac troponins in sera, and low scores of myocardial inflammation. In sum, mRNA-vaccines are safe in a model of experimentally induced autoimmune myocarditis, but patients undergoing ICI therapy should be closely monitored when vaccinated.


Subject(s)
COVID-19 , Drug-Related Side Effects and Adverse Reactions , Myocarditis , Male , Animals , Humans , Mice , Mice, Inbred C57BL , COVID-19 Vaccines , CTLA-4 Antigen , SARS-CoV-2 , Programmed Cell Death 1 Receptor , Inflammation , Antibodies , Models, Animal , RNA, Messenger , Vaccination
17.
Viruses ; 15(3)2023 03 16.
Article in English | MEDLINE | ID: covidwho-2286270

ABSTRACT

T-cell immunity plays an important role in the control of SARS-CoV-2 and has a great cross-protective effect on the variants. The Omicron BA.1 variant contains more than 30 mutations in the spike and severely evades humoral immunity. To understand how Omicron BA.1 spike mutations affect cellular immunity, the T-cell epitopes of SARS-CoV-2 wild-type and Omicron BA.1 spike in BALB/c (H-2d) and C57BL/6 mice (H-2b) were mapped through IFNγ ELISpot and intracellular cytokine staining assays. The epitopes were identified and verified in splenocytes from mice vaccinated with the adenovirus type 5 vector encoding the homologous spike, and the positive peptides involved in spike mutations were tested against wide-type and Omicron BA.1 vaccines. A total of eleven T-cell epitopes of wild-type and Omicron BA.1 spike were identified in BALB/c mice, and nine were identified in C57BL/6 mice, only two of which were CD4+ T-cell epitopes and most of which were CD8+ T-cell epitopes. The A67V and Del 69-70 mutations in Omicron BA.1 spike abolished one epitope in wild-type spike, and the T478K, E484A, Q493R, G496S and H655Y mutations resulted in three new epitopes in Omicron BA.1 spike, while the Y505H mutation did not affect the epitope. These data describe the difference of T-cell epitopes in SARS-CoV-2 wild-type and Omicron BA.1 spike in H-2b and H-2d mice, providing a better understanding of the effects of Omicron BA.1 spike mutations on cellular immunity.


Subject(s)
COVID-19 , Epitopes, T-Lymphocyte , Animals , Mice , Mice, Inbred C57BL , Epitopes, T-Lymphocyte/genetics , SARS-CoV-2/genetics , Mutation , Mice, Inbred BALB C
18.
J Hepatol ; 79(1): 150-166, 2023 Jul.
Article in English | MEDLINE | ID: covidwho-2261801

ABSTRACT

BACKGROUND & AIMS: Patients with chronic liver disease (CLD), including cirrhosis, are at increased risk of intractable viral infections and are hyporesponsive to vaccination. Hallmarks of CLD and cirrhosis include microbial translocation and elevated levels of type I interferon (IFN-I). We aimed to investigate the relevance of microbiota-induced IFN-I in the impaired adaptive immune responses observed in CLD. METHODS: We combined bile duct ligation (BDL) and carbon tetrachloride (CCl4) models of liver injury with vaccination or lymphocytic choriomeningitis virus infection in transgenic mice lacking IFN-I in myeloid cells (LysM-Cre IFNARflox/flox), IFNAR-induced IL-10 (MX1-Cre IL10flox/flox) or IL-10R in T cells (CD4-DN IL-10R). Key pathways were blocked in vivo with specific antibodies (anti-IFNAR and anti-IL10R). We assessed T-cell responses and antibody titers after HBV and SARS-CoV-2 vaccinations in patients with CLD and healthy individuals in a proof-of-concept clinical study. RESULTS: We demonstrate that BDL- and CCL4-induced prolonged liver injury leads to impaired T-cell responses to vaccination and viral infection in mice, subsequently leading to persistent infection. We observed a similarly defective T-cell response to vaccination in patients with cirrhosis. Innate sensing of translocated gut microbiota induced IFN-I signaling in hepatic myeloid cells that triggered excessive IL-10 production upon viral infection. IL-10R signaling in antigen-specific T cells rendered them dysfunctional. Antibiotic treatment and inhibition of IFNAR or IL-10Ra restored antiviral immunity without detectable immune pathology in mice. Notably, IL-10Ra blockade restored the functional phenotype of T cells from vaccinated patients with cirrhosis. CONCLUSION: Innate sensing of translocated microbiota induces IFN-/IL-10 expression, which drives the loss of systemic T-cell immunity during prolonged liver injury. IMPACT AND IMPLICATIONS: Chronic liver injury and cirrhosis are associated with enhanced susceptibility to viral infections and vaccine hyporesponsiveness. Using different preclinical animal models and patient samples, we identified that impaired T-cell immunity in BDL- and CCL4-induced prolonged liver injury is driven by sequential events involving microbial translocation, IFN signaling leading to myeloid cell-induced IL-10 expression, and IL-10 signaling in antigen-specific T cells. Given the absence of immune pathology after interference with IL-10R, our study highlights a potential novel target to reconstitute T-cell immunity in patients with CLD that can be explored in future clinical studies.


Subject(s)
COVID-19 , Interferon Type I , Mice , Animals , Interleukin-10 , SARS-CoV-2 , Mice, Transgenic , Liver Cirrhosis , Mice, Inbred C57BL
19.
Front Cell Infect Microbiol ; 13: 1134511, 2023.
Article in English | MEDLINE | ID: covidwho-2268706

ABSTRACT

Introduction: Inflammation play important roles in the initiation and progression of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), septic shock, clotting dysfunction, or even death associated with SARS-CoV-2 infection. However, the pathogenic mechanisms underlying SARS-CoV-2-induced hyperinflammation are still largely unknown. Methods: The animal model of septic shock and ALI was established after LPS intraperitoneal injection or intratracheal instillation. Bone marrow-derived macrophages (BMDMs) from WT and BPOZ-2 KO mouse strains were harvested from the femurs and tibias of mice. Immunohistology staining, ELISA assay, coimmunoprecipitation, and immunoblot analysis were used to detect the histopathological changes of lung tissues and the expression of inflammatory factors and protein interaction. Results and conclusions: We show a distinct mechanism by which the SARS-CoV-2 N (SARS-2-N) protein targets Bood POZ-containing gene type 2 (BPOZ-2), a scaffold protein for the E3 ubiquitin ligase Cullin 3 that we identified as a negative regulator of inflammatory responses, to promote NLRP3 inflammasome activation. We first demonstrated that BPOZ-2 knockout (BPOZ-2 KO) mice were more susceptible to lipopolysaccharide (LPS)-induced septic shock and ALI and showed increased serum IL-1ß levels. In addition, BMDMs isolated from BPOZ-2 KO mice showed increased IL-1ß production in response to NLRP3 stimuli. Mechanistically, BPOZ-2 interacted with NLRP3 and mediated its degradation by recruiting Cullin 3. In particular, the expression of BPOZ-2 was significantly reduced in lung tissues from mice infected with SARS-CoV-2 and in cells overexpressing SARS-2-N. Importantly, proinflammatory responses triggered by the SARS-2-N were significantly blocked by BPOZ-2 reintroduction. Thus, we concluded that BPOZ-2 is a negative regulator of the NLPR3 inflammasome that likely contributes to SARS-CoV-2-induced hyperinflammation.


Subject(s)
Acute Lung Injury , COVID-19 , NLR Family, Pyrin Domain-Containing 3 Protein , Nuclear Proteins , Shock, Septic , Animals , Mice , Acute Lung Injury/metabolism , Cullin Proteins , Inflammasomes/metabolism , Lipopolysaccharides/pharmacology , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , SARS-CoV-2/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism
20.
Virol J ; 20(1): 51, 2023 03 25.
Article in English | MEDLINE | ID: covidwho-2265323

ABSTRACT

BACKGROUND: Multiple sclerosis (MS) is characterized by neuroinflammation and demyelination orchestrated by activated neuroglial cells, CNS infiltrating leukocytes, and their reciprocal interactions through inflammatory signals. An inflammatory stimulus triggers inducible nitric oxide synthase (NOS2), a pro-inflammatory marker of microglia/macrophages (MG/Mφ) to catalyze sustained nitric oxide production. NOS2 during neuroinflammation, has been associated with MS disease pathology; however, studies dissecting its role in demyelination are limited. We studied the role of NOS2 in a recombinant ß-coronavirus-MHV-RSA59 induced neuroinflammation, an experimental animal model mimicking the pathological hallmarks of MS: neuroinflammatory demyelination and axonal degeneration. OBJECTIVE: Understanding the role of NOS2 in murine-ß-coronavirus-MHV-RSA59 demyelination. METHODS: Brain and spinal cords from mock and RSA59 infected 4-5-week-old MHV-free C57BL/6 mice (WT) and NOS2-/- mice were harvested at different disease phases post infection (p.i.) (day 5/6-acute, day 9/10-acute-adaptive and day 30-chronic phase) and compared for pathological outcomes. RESULTS: NOS2 was upregulated at the acute phase of RSA59-induced disease in WT mice and its deficiency resulted in severe disease and reduced survival at the acute-adaptive transition phase. Low survival in NOS2-/- mice was attributed to (i) high neuroinflammation resulting from increased accumulation of macrophages and neutrophils and (ii) Iba1 + phagocytic MG/Mφ mediated-early demyelination as observed at this phase. The phagocytic phenotype of CNS MG/Mφ was confirmed by significantly higher mRNA transcripts of phagocyte markers-CD206, TREM2, and Arg1 and double immunolabelling of Iba1 with MBP and PLP. Further, NOS2 deficiency led to exacerbated demyelination at the chronic phase as well. CONCLUSION: Taken together the results imply that the immune system failed to control the disease progression in the absence of NOS2. Thus, our observations highlight a protective role of NOS2 in murine-ß-coronavirus induced demyelination.


Subject(s)
Coronavirus Infections , Demyelinating Diseases , Murine hepatitis virus , Nitric Oxide Synthase Type II , Animals , Mice , Demyelinating Diseases/pathology , Demyelinating Diseases/virology , Membrane Glycoproteins , Mice, Inbred C57BL , Murine hepatitis virus/metabolism , Neuroinflammatory Diseases , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Receptors, Immunologic , Coronavirus Infections/pathology
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