SARS-CoV-2 infection induces thymic atrophy mediated by IFN-γ in hACE2 transgenic mice.

Pathogenic infections cause thymic atrophy, perturb thymic T-cell development, and alter immunological response. Previous studies reported dysregulated T-cell function and lymphopenia in coronavirus disease-19 (COVID-19). However, immunopathological changes in the thymus associated with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection have not been elucidated. Here, we report that SARS-CoV-2 infects thymocytes, and induces CD4+CD8+ (double positive; DP) T-cell apoptosis leading to thymic atrophy and loss of peripheral TCR repertoire in K18-hACE2 transgenic mice. Infected thymus led to increased CD44+CD25- T-cells, indicating an early arrest in the T-cell maturation pathway. Thymic atrophy was notably higher in male hACE2-Tg mice than in females and involved an upregulated de-novo synthesis pathway of thymic glucocorticoid. Further, IFN-γ was crucial for thymic atrophy, as anti-IFN-γ -antibody neutralization blunted thymic involution. Therapeutic use of Remdesivir also rescued thymic atrophy. While the Omicron variant and its sub-lineage BA.5 variant caused marginal thymic atrophy, the delta variant of SARS-CoV-2 exhibited severe thymic atrophy characterized by severely depleted DP T-cells. Recently characterized broadly SARS-CoV-2 neutralizing monoclonal antibody P4A2 was able to rescue thymic atrophy and restore the thymic maturation pathway of T-cells. Together, we report SARS-CoV-2-associated thymic atrophy resulting from impaired T-cell maturation pathway which may contribute to dyregulated T cell response during COVID-19.

VapC12 ribonuclease toxin modulates host immune response during Mycobacterium tuberculosis infection.

Mechanistic understanding of antibiotic persistence is a prerequisite in controlling the emergence of MDR cases in Tuberculosis (TB). We have reported that the cholesterol-induced activation of VapC12 ribonuclease is critical for disease persistence in TB. In this study, we observed that relative to the wild type, mice infected with ΔvapC12 induced a pro-inflammatory response, had a higher pathogen load, and responded better to the anti-TB treatment. In a high-dose infection model, all the mice infected with ΔvapC12 succumbed early to the disease. Finally, we reported that the above phenotype of ΔvapC12 was dependent on the presence of the TLR4 receptor. Overall, the data suggests that failure of a timely resolution of the early inflammation by the ΔvapC12 infected mice led to hyperinflammation, altered T-cell response and high bacterial load. In conclusion, our findings suggest the role of the VapC12 toxin in modulating the innate immune response of the host in ways that favor the long-term survival of the pathogen inside the host.

Fangchinoline inhibits SARS-CoV-2 and MERS-CoV entry.

The COVID-19 pandemic caused by SARS-CoV-2, lead to mild to severe respiratory illness and resulted in 6.9 million deaths worldwide. Although vaccines are effective in preventing COVID-19, they may not be sufficient to protect immunocompromised individuals from this respiratory illness. Moreover, novel emerging variants of SARS-CoV-2 pose a risk of new COVID-19 waves. Therefore, identification of effective antivirals is critical in controlling SARS and other coronaviruses, such as MERS-CoV. We show that Fangchinoline (Fcn), a bisbenzylisoquinoline alkaloid, inhibits replication of SARS-CoV, SARS-CoV-2, and MERS-CoV in a range of in vitro assays, by blocking entry. Therapeutic use of Fcn inhibited viral loads in the lungs, and suppressed associated airway inflammation in hACE2. Tg mice and Syrian hamster infected with SARS-CoV-2. Combination of Fcn with remdesivir (RDV) or an anti-leprosy drug, Clofazimine, exhibited synergistic antiviral activity. Compared to Fcn, its synthetic derivative, MK-04-003, more effectively inhibited SARS-CoV-2 and its variants B.1.617.2 and BA.5 in mice. Taken together these data demonstrate that Fcn is a pan beta coronavirus inhibitor, which possibly can be used to combat novel emerging coronavirus diseases.

Omicron sub-lineage BA.5 infection results in attenuated pathology in hACE2 transgenic mice.

A recently emerged sub-lineage of Omicron, BA.5, together with BA.4, caused a fifth wave of coronavirus disease (COVID-19) in South Africa and subsequently emerged as a predominant strain globally due to its high transmissibility. The lethality of BA.5 infection has not been studied in an acute hACE2 transgenic (hACE2.Tg) mouse model. Here, we investigated tissue-tropism and immuno-pathology induced by BA.5 infection in hACE2.Tg mice. Our data show that intranasal infection of BA.5 in hACE2.Tg mice resulted in attenuated pulmonary infection and pathology with diminished COVID-19-induced clinical and pathological manifestations. BA.5, similar to Omicron (B.1.1.529), infection led to attenuated production of inflammatory cytokines, anti-viral response and effector T cell response as compared to the ancestral strain of SARS-CoV-2, Wuhan-Hu-1. We show that mice recovered from B.1.1.529 infection showed robust protection against BA.5 infection associated with reduced lung viral load and pathology. Together, our data provide insights as to why BA.5 infection escapes previous SARS-CoV-2 exposure induced-T cell immunity but may result in milder immuno-pathology and alleviated chances of re-infectivity in Omicron-recovered individuals.

IL-9 aggravates SARS-CoV-2 infection and exacerbates associated airway inflammation.

SARS-CoV-2 infection is known for causing broncho-alveolar inflammation. Interleukin 9 (IL-9) induces airway inflammation and bronchial hyper responsiveness in respiratory viral illnesses and allergic inflammation, however, IL-9 has not been assigned a pathologic role in COVID-19. Here we show, in a K18-hACE2 transgenic (ACE2.Tg) mouse model, that IL-9 contributes to and exacerbates viral spread and airway inflammation caused by SARS-CoV-2 infection. ACE2.Tg mice with CD4+ T cell-specific deficiency of the transcription factor Forkhead Box Protein O1 (Foxo1) produce significantly less IL-9 upon SARS-CoV-2 infection than the wild type controls and they are resistant to the severe inflammatory disease that characterises the control mice. Exogenous IL-9 increases airway inflammation in Foxo1-deficient mice, while IL-9 blockade reduces and suppresses airway inflammation in SARS-CoV-2 infection, providing further evidence for a Foxo1-Il-9 mediated Th cell-specific pathway playing a role in COVID-19. Collectively, our study provides mechanistic insight into an important inflammatory pathway in SARS-CoV-2 infection, and thus represents proof of principle for the development of host-directed therapeutics to mitigate disease severity.

G4-binding drugs, chlorpromazine and prochlorperazine, repurposed against COVID-19 infection in hamsters.

The COVID-19 pandemic caused by SARS-CoV-2 has caused millions of infections and deaths worldwide. Limited treatment options and the threat from emerging variants underline the need for novel and widely accessible therapeutics. G-quadruplexes (G4s) are nucleic acid secondary structures known to affect many cellular processes including viral replication and transcription. We identified heretofore not reported G4s with remarkably low mutation frequency across >5 million SARS-CoV-2 genomes. The G4 structure was targeted using FDA-approved drugs that can bind G4s - Chlorpromazine (CPZ) and Prochlorperazine (PCZ). We found significant inhibition in lung pathology and lung viral load of SARS-CoV-2 challenged hamsters when treated with CPZ or PCZ that was comparable to the widely used antiviral drug Remdesivir. In support, in vitro G4 binding, inhibition of reverse transcription from RNA isolated from COVID-infected humans, and attenuated viral replication and infectivity in Vero cell cultures were clear in case of both CPZ and PCZ. Apart from the wide accessibility of CPZ/PCZ, targeting relatively invariant nucleic acid structures poses an attractive strategy against viruses like SARS-CoV-2, which spread fast and accumulate mutations quickly.

Pharmacological potential of Withania somnifera (L.) Dunal and Tinospora cordifolia (Willd.) Miers on the experimental models of COVID-19, T cell differentiation, and neutrophil functions.

Cytokine release syndrome (CRS) due to severe acute respiratory coronavirus-2 (SARS-CoV-2) infection leads to life-threatening pneumonia which has been associated with coronavirus disease (COVID-19) pathologies. Centuries-old Asian traditional medicines such as Withania somnifera (L.) Dunal (WS) and Tinospora cordifolia (Willd.) Miers (TC) possess potent immunomodulatory effects and were used by the AYUSH ministry, in India during the COVID-19 pandemic. In the present study, we investigated WS and TC's anti-viral and immunomodulatory efficacy at the human equivalent doses using suitable in vitro and in vivo models. While both WS and TC showed immuno-modulatory potential, WS showed robust protection against loss in body weight, viral load, and pulmonary pathology in the hamster model of SARS-CoV2. In vitro pretreatment of mice and human neutrophils with WS and TC had no adverse effect on PMA, calcium ionophore, and TRLM-induced ROS generation, phagocytosis, bactericidal activity, and NETs formation. Interestingly, WS significantly suppressed the pro-inflammatory cytokines-induced Th1, Th2, and Th17 differentiation. We also used hACE2 transgenic mice to further investigate the efficacy of WS against acute SARS-CoV2 infection. Prophylactic treatment of WS in the hACE2 mice model showed significant protection against body weight loss, inflammation, and the lung viral load. The results obtained indicate that WS promoted the immunosuppressive environment in the hamster and hACE2 transgenic mice models and limited the worsening of the disease by reducing inflammation, suggesting that WS might be useful against other acute viral infections. The present study thus provides pre-clinical efficacy data to demonstrate a robust protective effect of WS against COVID-19 through its broader immunomodulatory activity.

Intrinsic D614G and P681R/H mutations in SARS-CoV-2 VoCs Alpha, Delta, Omicron and viruses with D614G plus key signature mutations in spike protein alters fusogenicity and infectivity.

The SARS-CoV-2 virus has been rapidly evolving over the time and the genetic variation has led to the generation of Variants of Concerns (VoC), which have shown increased fitness. These VoC viruses contain the key mutations in the spike protein which have allowed better survival and evasion of host defense mechanisms. The D614G mutation in the spike domain is found in the majority of VoC; additionally, the P681R/H mutation at the S1/S2 furin cleavage site junction is also found to be highly conserved in major VoCs; Alpha, Delta, Omicron, and its' current variants. The impact of these genetic alterations of the SARS-CoV-2 VoCs on the host cell entry, transmissibility, and infectivity has not been clearly identified. In our study, Delta and D614G + P681R synthetic double mutant pseudoviruses showed a significant increase in the cell entry, cell-to-cell fusion and infectivity. In contrast, the Omicron and P681H synthetic single mutant pseudoviruses showed TMPRSS2 independent cell entry, less fusion and infectivity as compared to Delta and D614G + P681R double mutants. Addition of exogenous trypsin further enhanced fusion in Delta viruses as compared to Omicron. Furthermore, Delta viruses showed susceptibility to both E64d and Camostat mesylate inhibitors suggesting, that the Delta virus could exploit both endosomal and TMPRSS2 dependent entry pathways as compared to the Omicron virus. Taken together, these results indicate that the D614G and P681R/H mutations in the spike protein are pivotal which might be favoring the VoC replication in different host compartments, and thus allowing a balance of mutation vs selection for better long-term adaptation.

Prophylactic treatment of Glycyrrhiza glabra mitigates COVID-19 pathology through inhibition of pro-inflammatory cytokines in the hamster model and NETosis.

Severe coronavirus disease (COVID-19) is accompanied by acute respiratory distress syndrome and pulmonary pathology, and is presented mostly with an inflammatory cytokine release, a dysregulated immune response, a skewed neutrophil/lymphocyte ratio, and a hypercoagulable state. Though vaccinations have proved effective in reducing the COVID-19-related mortality, the limitation of the use of vaccine against immunocompromised individuals, those with comorbidity, and emerging variants remains a concern. In the current study, we investigate for the first time the efficacy of the Glycyrrhiza glabra (GG) extract, a potent immunomodulator, against SARS-CoV-2 infection in hamsters. Prophylactic treatment with GG showed protection against loss in body weight and a 35%-40% decrease in lung viral load along with reduced lung pathology in the hamster model. Remarkably, GG reduced the mRNA expression of pro-inflammatory cytokines and plasminogen activator inhibitor-1 (PAI-1). In vitro, GG acted as a potent immunomodulator by reducing Th2 and Th17 differentiation and IL-4 and IL-17A cytokine production. In addition, GG also showed robust potential to suppress ROS, mtROS, and NET generation in a concentration-dependent manner in both human polymorphonuclear neutrophils (PMNs) and murine bone marrow-derived neutrophils (BMDNs). Taken together, we provide evidence for the protective efficacy of GG against COVID-19 and its putative mechanistic insight through its immunomodulatory properties. Our study provides the proof of concept for GG efficacy against SARS-CoV-2 using a hamster model and opens the path for further studies aimed at identifying the active ingredients of GG and its efficacy in COVID-19 clinical cases.

Golden Syrian hamster as a model to study cardiovascular complications associated with SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in the Golden Syrian hamster causes lung pathology that resembles human coronavirus disease (COVID-19). However, extrapulmonary pathologies associated with SARS-CoV-2 infection and post-COVID sequelae remain to be understood. Here, we show, using a hamster model, that the early phase of SARS-CoV-2 infection leads to an acute inflammatory response and lung pathologies, while the late phase of infection causes cardiovascular complications (CVCs) characterized by ventricular wall thickening associated with increased ventricular mass/body mass ratio and interstitial coronary fibrosis. Molecular profiling further substantiated our findings of CVC as SARS-CoV-2-infected hamsters showed elevated levels of serum cardiac troponin I, cholesterol, low-density lipoprotein, and long-chain fatty acid triglycerides. Serum metabolomics profiling of SARS-CoV-2-infected hamsters identified N-acetylneuraminate, a functional metabolite found to be associated with CVC, as a metabolic marker was found to be common between SARS-CoV-2-infected hamsters and COVID-19 patients. Together, we propose hamsters as a suitable animal model to study post-COVID sequelae associated with CVC, which could be extended to therapeutic interventions.

Self- vs provider-referral differences for coronary artery calcium testing.

Study objectives: The objectives of this study were to identify independent predictors for moderate/accentuated coronary artery calcium (CAC) score and compare patients who self-referred for CAC Computed Tomography (CT) testing to those who were provider-referred. Design: Patients underwent CAC between January to July 2019. The analysis was divided into self-referred patients influenced by a CAC community campaign who identified themselves as having cardiovascular risk factors compared to provider-referred intermediate-risk patients who were asymptomatic. SAS version 9.4 (SAS Institute, Inc., Cary, NC) was used for all analyses. Setting: Seven southwest Ohio hospitals from a single network. Participants: 2124 adult patients who received CAC CT (163 self and 1961 provider-referred). Interventions: CAC CT. Main outcome measures: Demographics, risk factors, lab values, prescriptions, and referral status were used to compare CAC score differences between self- and provider-referred patients. Results: For 2124 patients, three predictors for moderate/accentuated CAC score remained significant after multiple logistic regression: CKD (OR 0.24, CI 0.008-0.68, p < 0.05), COPD (OR 0.39, CI 0.19-0.80, p < 0.05), and CAD (OR 0.46, CI 0.22-0.98, p < 0.05). There were four differences between referred groups: history of PVD (OR 0.21, CI 0.05-0.86, p < 0.05), higher triglyceride (OR 1.004, CI 1.00-1.01, p < 0.05), higher LDL levels (OR 0.991, CI 0.98-1.00, p < 0.05), and beta blocker prescription (OR 4.38, CI 1.49-12.85, p < 0.05) in self-referred patients. Conclusions: CAC CT testing is associated with independent risk predictors and can be used to clarify cardiovascular risk in self- and provider-referred patients with statistical similarity. Patients reliably self-refer for CAC CT when risk is present during a community initiative. Such initiatives may have a preventive benefit and lead to earlier pursuit and optimization of anti-lipid therapies.

High-salt diet mediates interplay between NK cells and gut microbiota to induce potent tumor immunity.

High-salt diet (HSD) modulates effector and regulatory T cell functions and promotes tissue inflammation in autoimmune diseases. However, effects of HSD and its association with gut microbiota in tumor immunity remain undefined. Here, we report that HSD induces natural killer (NK) cell­mediated tumor immunity by inhibiting PD-1 expression while enhancing IFNγ and serum hippurate. Salt enhanced tumor immunity when combined with a suboptimal dose of anti-PD1 antibody. While HSD-induced tumor immunity was blunted upon gut microbiota depletion, fecal microbiota transplantation (FMT) from HSD mice restored the tumor immunity associated with NK cell functions. HSD increased the abundance of Bifidobacterium and caused increased gut permeability leading to intratumor localization of Bifidobacterium, which enhanced NK cell functions and tumor regression. Intratumoral injections of Bifidobacterium activated NK cells, which inhibited tumor growth. These results indicate that HSD modulates gut microbiome that induces NK cell­dependent tumor immunity with a potential translational perspective.

Gefitinib Results in Robust Host-Directed Immunity Against Salmonella Infection Through Proteo-Metabolomic Reprogramming.

The global rise of antibiotic-resistant strains of Salmonella has necessitated the development of alternative therapeutic strategies. Recent studies have shown that targeting host factors may provide an alternative approach for the treatment of intracellular pathogens. Host-directed therapy (HDT) modulates host cellular factors that are essential to support the replication of the intracellular pathogens. In the current study, we identified Gefitinib as a potential host directed therapeutic drug against Salmonella. Further, using the proteome analysis of Salmonella-infected macrophages, we identified EGFR, a host factor, promoting intracellular survival of Salmonella via mTOR-HIF-1α axis. Blocking of EGFR, mTOR or HIF-1α inhibits the intracellular survival of Salmonella within the macrophages and in mice. Global proteo-metabolomics profiling indicated the upregulation of host factors predominantly associated with ATP turn over, glycolysis, urea cycle, which ultimately promote the activation of EGFR-HIF1α signaling upon infection. Importantly, inhibition of EGFR and HIF1α restored both proteomics and metabolomics changes caused by Salmonella infection. Taken together, this study identifies Gefitinib as a host directed drug that holds potential translational values against Salmonella infection and might be useful for the treatment of other intracellular infections.

Transcription factor Foxo1 is essential for IL-9 induction in T helper cells.

Interleukin 9 (IL-9)-producing helper T (Th9) cells have a crucial function in allergic inflammation, autoimmunity, immunity to extracellular pathogens and anti-tumor immune responses. In addition to Th9, Th2, Th17 and Foxp3+ regulatory T (Treg) cells produce IL-9. A transcription factor that is critical for IL-9 induction in Th2, Th9 and Th17 cells has not been identified. Here we show that the forkhead family transcription factor Foxo1 is required for IL-9 induction in Th9 and Th17 cells. We further show that inhibition of AKT enhances IL-9 induction in Th9 cells while it reciprocally regulates IL-9 and IL-17 in Th17 cells via Foxo1. Mechanistically, Foxo1 binds and transactivates IL-9 and IRF4 promoters in Th9, Th17 and iTreg cells. Furthermore, loss of Foxo1 attenuates IL-9 in mouse and human Th9 and Th17 cells, and ameliorates allergic inflammation in asthma. Our findings thus identify that Foxo1 is essential for IL-9 induction in Th9 and Th17 cells.The transcription factor Foxo1 can control regulatory T cell and Th1 function. Here the authors show that Foxo1 is also critical for IL-9 production by Th9 cells and other IL-9-producing cells.

Comparison of six-month outcomes and hospitalization rates in heart failure patients with and without preserved left ventricular ejection fraction and with and without intraventricular conduction defect.

Prolonged QRS duration (>120 ms), as a marker of ventricular dyssynchrony, is an independent predictor of mortality in systolic heart failure (HF). Little information exists about the characteristics of patients with preserved ejection fractions (EFs) and prolonged QRS (intraventricular conduction defects [IVCDs]). The electronic records of 334 consecutive patients hospitalized with acutely decompensated HF were reviewed. A significant number of patients hospitalized with decompensated HF had preserved EFs with IVCD. They had similar readmission and mortality rates compared with their systolic HF counterparts and higher rates compared with those with preserved EFs without IVCD. These findings and the resulting possible therapeutic interventions (resynchronization) need further analysis in a larger prospective cohort.