TNFα aggravates detrimental effects of SARS-CoV-2 infection in the liver.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus does not only lead to pulmonary infection but can also infect other organs such as the gut, the kidney, or the liver. Recent studies confirmed that severe cases of COVID-19 are often associated with liver damage and liver failure, as well as the systemic upregulation of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFα). However, the impact these immune mediators in the liver have on patient survival during SARS-CoV-2 infection is currently unknown. Here, by performing a post-mortem analysis of 45 patients that died from a SARS-CoV-2 infection, we find that an increased expression of TNFA in the liver is associated with elevated mortality. Using publicly available single-cell sequencing datasets, we determined that Kupffer cells and monocytes are the main sources of this TNFα production. Further analysis revealed that TNFα signaling led to the upregulation of pro-inflammatory genes that are associated with an unfavorable outcome. Moreover, high levels of TNFA in the liver were associated with lower levels of interferon alpha and interferon beta. Thus, TNFα signaling in the infected SARS-CoV-2 liver correlates with reduced interferon levels and overall survival time.

Neutrophil activation and neutrophil extracellular traps (NETs) in COVID-19 ARDS and immunothrombosis.

Acute respiratory distress syndrome (ARDS) is an acute inflammatory condition with a dramatic increase in incidence since the beginning of the coronavirus disease 19 (COVID-19) pandemic. Neutrophils play a vital role in the immunopathology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by triggering the formation of neutrophil extracellular traps (NETs), producing cytokines including interleukin-8 (CXCL8), and mediating the recruitment of other immune cells to regulate processes such as acute and chronic inflammation, which can lead to ARDS. CXCL8 is involved in the recruitment, activation, and degranulation of neutrophils, and therefore contributes to inflammation amplification and severity of disease. Furthermore, activation of neutrophils also supports a prothrombotic phenotype, which may explain the development of immunothrombosis observed in COVID-19 ARDS. This review aims to describe hyperinflammatory ARDS due to SARS-CoV-2 infection. In addition, we address the critical role of polymorphonuclear neutrophils, inflammatory cytokines, and the potential targeting of CXCL8 in treating the hyperinflammatory ARDS population.

Evaluation Role of CXCL-8 levels and SARS-CoV-2 Variants in Progression of Infection

Background: The SARS-CoV-2 virus underwent genetic alterations during viral genome replication, leading to the development of numerous variations including Alpha, Delta, Delta plus, Omicron, and other variants. The chemokine CXCL8 is known to directly restrict viral proteins by producing interferon as the antiviral efficacy. Subjects and methods: A total of 137 nasal swabs and blood samples were collected from 89 positively corona virus infection disease-19 (COVID-19) patients and 48 healthy individuals. RNA was extracted and real-time reverse transcriptase polymerase chain reaction (rRT-PCR) was performed to variants detection by using special kits. CXCL8 levels were determined in Enzyme linked immune-sorbent assay (ELISA) assay. Results: Significant difference (p < 0.001) in the median levels of CXCL8 in patient higher than healthy control groups. Highest median of CXCL8 levels was shown with severe infection 308.6 (IQR: 66.6 – 783.5). Receiver-operating characteristic (ROC) analysis revealed that CXCL8 level was a good biomarker for immune response (Area under the curve (AUC) was 0.707). CXCL8 showed a significant difference with variants of SARS-CoV-2 (p < 0.001), where Delta/Delta plus variant patients had the highest median of CXCL8 level than other SARS-CoV-2 variant infections. Conclusion: Patients infected with Delta/Delta plus and Omicron had higher level of CXCL-8. CXCL-8 had positive relation with cardiac disease, renal failure, leukemia with COVID-19 infection. © 2022, Ain Shams University Faculty of Medicine. All rights reserved.

Expression profiling of inflammation-related genes including IFI-16, NOTCH2, CXCL8, THBS1 in COVID-19 patients.

The present study aimed to scrutinize the expression profile of inflammatory-related genes (IFI-16, NOTCH2, CXCL8, and THBS1) from acute to post-acute stage of this infectious epidemic. The current cross-sectional study consisted of 53 acute-phase COVID-19 patients and 53 healthy individuals between February and March 2021. The extraction of total RNA was performed from PBMC specimens and also expression level of selected genes (IFI-16, NOTCH2, CXCL8, and THBS1) was evaluated by real-time PCR. Subsequently, levels of these factors were re-measured six weeks after the acute phase to determine if the levels of chosen genes returned to normal after the acute phase of COVID-19. Receiver operating characteristic (ROC) curve was plotted to test potential of genes as a diagnostic biomarker. The expression levels of inflammatory-related genes were significantly different between healthy and COVID-19 subjects. Besides, a significant higher CXCL8 level was found in the acute-phase COVID-19 compared to post-acute-phase infection which may be able to be considered as a potential biomarker for distinguishing between the acute phases from the post-acute-phase status. Deregulation of the inflammatory-related genes in COVID-19 patients, especially CXCL-8, can be serving as potent biomarkers to manage the COVID-19 infection.

Elevated Expression of RGS2 May Underlie Reduced Olfaction in COVID-19 Patients.

Anosmia is common in COVID-19 patients, lasting for weeks or months following recovery. The biological mechanism underlying olfactory deficiency in COVID-19 does not involve direct damage to nasal olfactory neurons, which do not express the proteins required for SARS-CoV-2 infection. A recent study suggested that anosmia results from downregulation of olfactory receptors. We hypothesized that anosmia in COVID-19 may also reflect SARS-CoV-2 infection-driven elevated expression of regulator of G protein signaling 2 (RGS2), a key regulator of odorant receptors, thereby silencing their signaling. To test our hypothesis, we analyzed gene expression of nasopharyngeal swabs from SARS-CoV-2 positive patients and non-infected controls (two published RNA-sequencing datasets, 580 individuals). Our analysis found upregulated RGS2 expression in SARS-CoV-2 positive patients (FC = 14.5, Padj = 1.69 × 10-5 and FC = 2.4; Padj = 0.001, per dataset). Additionally, RGS2 expression was strongly correlated with PTGS2, IL1B, CXCL8, NAMPT and other inflammation markers with substantial upregulation in early infection. These observations suggest that upregulated expression of RGS2 may underlie anosmia in COVID-19 patients. As a regulator of numerous G-protein coupled receptors, RGS2 may drive further neurological symptoms of COVID-19. Studies are required for clarifying the cellular mechanisms by which SARS-CoV-2 infection drives the upregulation of RGS2 and other genes implicated in inflammation. Insights on these pathway(s) may assist in understanding anosmia and additional neurological symptoms reported in COVID-19 patients.

The effect of reparixin on survival in patients at high risk for in-hospital mortality: a meta-analysis of randomized trials.

Introduction: A great number of anti-inflammatory drugs have been suggested in the treatment of SARS-CoV-2 infection. Reparixin, a non-competitive allosteric inhibitor of the CXCL8 (IL-8) receptors C-X-C chemokine receptor type 1 (CXCR1) and C-X-C chemokine receptor type 2 (CXCR2), has already been tried out as a treatment in different critical settings. Due to the contrasting existing literature, we decided to perform the present meta-analysis of randomized controlled trials (RCTs) to investigate the effect of the use of reparixin on survival in patients at high risk for in-hospital mortality. Methods: We created a search strategy to include any human RCTs performed with reparixin utilization in patients at high risk for in-hospital mortality, excluding oncological patients. Two trained, independent authors searched PubMed, EMBASE, and the Cochrane Central Register of Controlled Trials (CENTRAL) for appropriate studies. Furthermore, references of review articles and included RCTs were screened to identify more studies. No language restrictions were enforced. To assess the risk of bias of included trials, the Revised Cochrane risk-of-bias tool for randomized trials (RoB 2) was used. Results: Overall, six studies were included and involved 406 patients (220 received reparixin and 186 received the comparator). The all-cause mortality in the reparixin group was significantly lower than that in the control group [5/220 (2.3%) in the reparixin group vs. 12/186 (6.5%) in the control group, odds ratio = 0.33 (95% confidence interval 0.12 to 0.96), p-value for effect 0.04, p for heterogeneity 0.20, I2 = 36%]. In addition, no difference in the rate of pneumonia, sepsis, or non-serious infections was shown between the two groups. Conclusion: Our meta-analysis of randomized trials suggests that short-term inhibition of CXCL8 activity improved survival in patients at high risk for in-hospital mortality without increasing the risk of infection. Meta-analysis registration: PROSPERO, identifier CRD42021254467.

SARS-CoV-2 Envelope (E) Protein Binds and Activates TLR2 Pathway: A Novel Molecular Target for COVID-19 Interventions.

This paper presents a molecular characterization of the interaction between the SARS-CoV-2 envelope (E) protein and TLR2. We demonstrated that the E protein, both as a recombinant soluble protein and as a native membrane protein associated with SARS-CoV-2 viral particles, interacts physically with the TLR2 receptor in a specific and dose-dependent manner. Furthermore, we showed that the specific interaction with the TLR2 pathway activates the NF-κB transcription factor and stimulates the production of the CXCL8 inflammatory chemokine. In agreement with the importance of NF-κB in the TLR signaling pathway, we showed that the chemical inhibition of this transcription factor leads to significant inhibition of CXCL8 production, while the blockade of the P38 and ERK1/2 MAP kinases only results in partial CXCL8 inhibition. Overall, our findings propose the envelope (E) protein as a novel molecular target for COVID-19 interventions: either (i) by exploring the therapeutic effect of anti-E blocking/neutralizing antibodies in symptomatic COVID-19 patients, or (ii) as a promising non-spike SARS-CoV-2 antigen candidate for inclusion in the development of next-generation prophylactic vaccines against COVID-19 infection and disease.

SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome.

Viral infection involves complex set of events orchestrated by multiple viral proteins. To identify functions of SARS-CoV-2 proteins, we performed transcriptomic analyses of cells expressing individual viral proteins. Expression of Nsp14, a protein involved in viral RNA replication, provoked a dramatic remodeling of the transcriptome that strongly resembled that observed following SARS-CoV-2 infection. Moreover, Nsp14 expression altered the splicing of more than 1000 genes and resulted in a dramatic increase in the number of circRNAs, which are linked to innate immunity. These effects were independent of the Nsp14 exonuclease activity and required the N7-guanine-methyltransferase domain of the protein. Activation of the NFkB pathway and increased expression of CXCL8 occurred early upon Nsp14 expression. We identified IMPDH2, which catalyzes the rate-limiting step of guanine nucleotides biosynthesis, as a key mediator of these effects. Nsp14 expression caused an increase in GTP cellular levels, and the effect of Nsp14 was strongly decreased in the presence of IMPDH2 inhibitors. Together, our data demonstrate an unknown role for Nsp14 with implications for therapy.

Viruses are parasites, relying on the cells they infect to make more of themselves. In doing so they change how an infected cell turns its genes on and off, forcing it to build new virus particles and turning off the immune surveillance that would allow the body to intervene. This is how SARS-CoV-2, the virus that causes COVID, survives with a genome that carries instructions to make just 29 proteins. One of these proteins, known as Nsp14, is involved in both virus reproduction and immune escape. Previous work has shown that it interacts with IMPDH2, the cellular enzyme that controls the production of the building blocks of the genetic code. The impact of this interaction is not clear. To find out more, Zaffagni et al. introduced 26 of the SARS-CoV-2 proteins into human cells one at a time. Nsp14 had the most dramatic effect, dialing around 4,000 genes up or down and changing how the cell interprets over 1,000 genes. Despite being just one protein, it mimicked the genetic changes seen during real SARS-CoV-2 infection. Blocking IMPDH2 partially reversed the effects, which suggests that the interaction of Nsp14 with the enzyme might be responsible for the effects of SARS-CoV-2 on the genes of the cell. Understanding how viral proteins affect cells can explain what happens during infection. This could lead to the discovery of new treatments designed to counteract the effects of the virus. Further work could investigate whether interfering with Nsp14 helps cells to overcome infection.

MicroRNAs based regulation of cytokine regulating immune expressed genes and their transcription factors in COVID-19.

BACKGROUND: Coronavirus disease 2019 is characterized by the elevation of a broad spectrum of inflammatory mediators associated with poor disease outcomes. We aimed at an in-silico analysis of regulatory microRNA and their transcription factors (TF) for these inflammatory genes that may help to devise potential therapeutic strategies in the future. METHODS: The cytokine regulating immune-expressed genes (CRIEG) were sorted from literature and the GEO microarray dataset. Their co-differentially expressed miRNA and transcription factors were predicted from publicly available databases. Enrichment analysis was done through mienturnet, MiEAA, Gene Ontology, and pathways predicted by KEGG and Reactome pathways. Finally, the functional and regulatory features were analyzed and visualized through Cytoscape. RESULTS: Sixteen CRIEG were observed to have a significant protein-protein interaction network. The ontological analysis revealed significantly enriched pathways for biological processes, molecular functions, and cellular components. The search performed in the miRNA database yielded ten miRNAs that are significantly involved in regulating these genes and their transcription factors. CONCLUSION: An in-silico representation of a network involving miRNAs, CRIEGs, and TF, which take part in the inflammatory response in COVID-19, has been elucidated. Thus, these regulatory factors may have potentially critical roles in the inflammatory response in COVID-19 and may be explored further to develop targeted therapeutic strategies and mechanistic validation.

Immunoglobulin G Immune Complexes May Contribute to Neutrophil Activation in the Course of Severe Coronavirus Disease 2019.

Severe coronavirus disease 2019 (COVID-19) is associated with an overactive inflammatory response mediated by macrophages. Here, we analyzed the phenotype and function of neutrophils in patients with COVID-19. We found that neutrophils from patients with severe COVID-19 express high levels of CD11b and CD66b, spontaneously produce CXCL8 and CCL2, and show a strong association with platelets. Production of CXCL8 correlated with plasma concentrations of lactate dehydrogenase and D-dimer. Whole blood assays revealed that neutrophils from patients with severe COVID-19 show a clear association with immunoglobulin G (IgG) immune complexes. Moreover, we found that sera from patients with severe disease contain high levels of immune complexes and activate neutrophils through a mechanism partially dependent on FcγRII (CD32). Interestingly, when integrated in immune complexes, anti-severe acute respiratory syndrome coronavirus 2 IgG antibodies from patients with severe COVID-19 displayed a higher proinflammatory profile compared with antibodies from patients with mild disease. Our study suggests that IgG immune complexes might promote the acquisition of an inflammatory signature by neutrophils, worsening the course of COVID-19.

The cytokine storm in COVID-19: Further advances in our understanding the role of specific chemokines involved.

SARS-COV-2 infection represents the greatest pandemic of the world, counting daily increasing number of subjects positive to the virus and, sadly, increasing number of deaths. Current studies reported that the cytokine/chemokine network is crucial in the onset and maintenance of the "cytokine storm", the event occurring in those patients in whom the progression of COVID-19 will progress, in most cases, to a very severe and potentially threatening disease. Detecting a possible "immune signature" in patients, as assessed by chemokines status in patients with COVID-19, could be helpful for individual risk stratification for developing a more or less severe clinical course of the disease. The present review is specifically aimed at overviewing current evidences provided by in vitro and in vivo studies addressing the issue of which chemokines seems to be involved, at least at present, in COVID-19. Currently available experimental and clinical studies regarding those chemokines more deeply studied in COVID-19, with a specific focus on their role in the cytokine storm and ultimately with their ability to predict the clinical course of the disease, will be taken into account. Moreover, similarities and differences between chemokines and cytokines, which both contribute to the onset of the pro-inflammatory loop characterizing SARS-COV-2 infection, will be briefly discussed. Future studies will rapidly accumulate in the next months and their results will hopefully provide more insights as to the complex physiopathology of COVID-19-related cytokine storm. This will likely make the present review somehow "dated" in a short time, but still the present review provides an overview of the scenario of the current knowledge on this topic.

Re-analysis of Single Cell Transcriptome Reveals That the NR3C1-CXCL8-Neutrophil Axis Determines the Severity of COVID-19.

SARS-CoV-2 infection has recently been declared a pandemic. Some patients showing severe symptoms exhibit drastic inflammation and airway damage. In this study, we re-analyzed published scRNA-seq data of COVID-19 patient bronchoalveolar lavage fluid to further classify and compare immunological features according to the patient's disease severity. Patients with severe symptoms showed DNA damage and apoptotic features of epithelial cells. Our results suggested that epithelial damage was associated with neutrophil infiltration. Myeloid cells of severe patients showed higher expression of proinflammatory cytokines and chemokines such as CXCL8. As a result, neutrophils were abundant in lungs of patients from the severe group. Furthermore, recruited neutrophils highly expressed genes related to neutrophil extracellular traps. Neutrophil-mediated inflammation was regulated by glucocorticoid receptor expression and activity. Based on these results, we suggest that severe COVID-19 symptoms may be determined by differential expression of glucocorticoid receptors and neutrophils.

The cytokine storm in COVID-19: An overview of the involvement of the chemokine/chemokine-receptor system.

In 2019-2020 a new coronavirus named SARS-CoV-2 was identified as the causative agent of a several acute respiratory infection named COVID-19, which is causing a worldwide pandemic. There are still many unresolved questions regarding the pathogenesis of this disease and especially the reasons underlying the extremely different clinical course, ranging from asymptomatic forms to severe manifestations, including the Acute Respiratory Distress Syndrome (ARDS). SARS-CoV-2 showed phylogenetic similarities to both SARS-CoV and MERS-CoV viruses, and some of the clinical features are shared between COVID-19 and previously identified beta-coronavirus infections. Available evidence indicate that the so called "cytokine storm" an uncontrolled over-production of soluble markers of inflammation which, in turn, sustain an aberrant systemic inflammatory response, is a major responsible for the occurrence of ARDS. Chemokines are low molecular weight proteins with powerful chemoattractant activity which play a role in the immune cell recruitment during inflammation. This review will be aimed at providing an overview of the current knowledge on the involvement of the chemokine/chemokine-receptor system in the cytokine storm related to SARS-CoV-2 infection. Basic and clinical evidences obtained from previous SARS and MERS epidemics and available data from COVID-19 will be taken into account.