Peripheral blood mononuclear cell tissue factor (F3 gene) transcript levels and circulating extracellular vesicles are elevated in severe coronavirus 2019 (COVID-19) disease.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with excessive coagulation, thrombosis, and mortality. OBJECTIVE: To provide insight into mechanisms that contribute to excessive coagulation in coronavirus 2019 (COVID-19) disease. PATIENTS/METHODS: Blood from COVID-19 patients was investigated for coagulation-related gene expression and functional activities. RESULTS: Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells from severe COVID-19 patients revealed a 5.2-fold increase in tissue factor (TF [F3 gene]) transcript expression levels (P < .05), the trigger of extrinsic coagulation; a 7.7-fold increase in C1-inhibitor (SERPING1 gene; P < .01) transcript expression levels, an inhibitor of intrinsic coagulation; and a 4.4-fold increase in anticoagulant thrombomodulin (TM [THBD gene]) transcript expression levels (P < .001). Bulk RNA-seq analysis of sorted CD14+ monocytes on an independent cohort of COVID-19 patients confirmed these findings (P < .05). Indicative of excessive coagulation, 41% of COVID-19 patients' plasma samples contained high D-dimer levels (P < .0001); of these, 19% demonstrated extracellular vesicle TF activity (P = .109). COVID-19 patients' ex vivo plasma-based thrombin generation correlated positively with D-dimer levels (P < .01). Plasma procoagulant extracellular vesicles were elevated ∼9-fold in COVID-19 patients (P < .01). Public scRNA-seq data sets from bronchoalveolar lung fluid and our peripheral blood mononuclear cell scRNA-seq data show CD14+ monocytes/macrophages TF transcript expression levels are elevated in severe but not mild or moderate COVID-19 patients. CONCLUSIONS: Beyond local lung injury, SARS-CoV-2 infection increases systemic TF (F3) transcript levels and elevates circulating extracellular vesicles that likely contribute to disease-associated coagulation, thrombosis, and related mortality.

COVID-19 plasma exosomes promote proinflammatory immune responses in peripheral blood mononuclear cells.

Elevated serum cytokine production in COVID-19 patients is associated with disease progression and severity. However, the stimuli that initiate cytokine production in patients remain to be fully revealed. Virus-infected cells release virus-associated exosomes, extracellular vesicles of endocytic origin, into the blood to deliver viral cargoes able to regulate immune responses. Here, we report that plasma exosomes of COVID-19 patients contain SARS-CoV-2 double stranded RNA (dsRNA) and stimulate robust production of interleukin-6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α), and other inflammatory cytokines and chemokines by human peripheral mononuclear cells. Exosome depletion abolished these stimulated responses. COVID-19 plasma exosomes induced proinflammatory responses in CD4+ T cells, CD8+ T cells, and CD14+ monocytes but not significantly in regulatory T cells, Th17 T cells, or central memory T cells. COVID-19 plasma exosomes protect the SARS-CoV-2 dsRNA cargo from RNase and deliver the dsRNA into recipient cells. These exosomes significantly increase expression of endosomal toll-like receptor 3 (TLR3), TLR7, TLR8, and TLR9 in peripheral T cells and monocytes. A pharmacological inhibitor of TLR3 considerably reduced cytokine and chemokine production by CD4+ and CD8+ T cells but not by CD14+ monocytes, highlighting divergent signaling pathways of immune cells in response to COVID-19 plasma exosomes. Our results identify a novel model of intercellular crosstalk following SARS-CoV-2 infection that evoke immune responses positioned to contribute to elevated cytokine production associated with COVID-19 progression, severity, and long-haul symptoms.

Phenotypical and Functional Alteration of γδ T Lymphocytes in COVID-19 Patients: Reversal by Statins.

(1) Background: statins have been considered an attractive class of drugs in the pharmacological setting of COVID-19 due to their pleiotropic properties and their use correlates with decreased mortality in hospitalized COVID-19 patients. Furthermore, it is well known that statins, which block the mevalonate pathway, affect γδ T lymphocyte activation. As γδ T cells participate in the inflammatory process of COVID-19, we have investigated the therapeutical potential of statins as a tool to inhibit γδ T cell pro-inflammatory activities; (2) Methods: we harvested peripheral blood mononuclear cells (PBMCs) from COVID-19 patients with mild clinical manifestations, COVID-19 recovered patients, and healthy controls. We performed ex vivo flow cytometry analysis to study γδ T cell frequency, phenotype, and exhaustion status. PBMCs were treated with Atorvastatin followed by non-specific and specific stimulation, to evaluate the expression of pro-inflammatory cytokines; (3) Results: COVID-19 patients had a lower frequency of circulating Vδ2+ T lymphocytes but showed a pronounced pro-inflammatory profile, which was inhibited by in vitro treatment with statins; (4) Conclusions: the in vitro capacity of statins to inhibit Vδ2+ T lymphocytes in COVID-19 patients highlights a new potential biological function of these drugs and supports their therapeutical use in these patients.

Phenotypical and Functional Alteration of γδ T Lymphocytes in COVID-19 Patients: Reversal by Statins.

(1) Background: statins have been considered an attractive class of drugs in the pharmacological setting of COVID-19 due to their pleiotropic properties and their use correlates with decreased mortality in hospitalized COVID-19 patients. Furthermore, it is well known that statins, which block the mevalonate pathway, affect γδ T lymphocyte activation. As γδ T cells participate in the inflammatory process of COVID-19, we have investigated the therapeutical potential of statins as a tool to inhibit γδ T cell pro-inflammatory activities; (2) Methods: we harvested peripheral blood mononuclear cells (PBMCs) from COVID-19 patients with mild clinical manifestations, COVID-19 recovered patients, and healthy controls. We performed ex vivo flow cytometry analysis to study γδ T cell frequency, phenotype, and exhaustion status. PBMCs were treated with Atorvastatin followed by non-specific and specific stimulation, to evaluate the expression of pro-inflammatory cytokines; (3) Results: COVID-19 patients had a lower frequency of circulating Vδ2+ T lymphocytes but showed a pronounced pro-inflammatory profile, which was inhibited by in vitro treatment with statins; (4) Conclusions: the in vitro capacity of statins to inhibit Vδ2+ T lymphocytes in COVID-19 patients highlights a new potential biological function of these drugs and supports their therapeutical use in these patients.

Upregulation of hsa_circ_0004812 promotes COVID-19 cytokine storm via hsa-miR-1287-5p/IL6R, RIG-I axis.

BACKGROUND: SARS-CoV-2 is one of the most contagious viruses in the Coronaviridae (CoV) family, which has become a pandemic. The aim of this study is to understand more about the role of hsa_circ_0004812 in the SARS-CoV-2 related cytokine storm and its associated molecular mechanisms. MATERIALS AND METHODS: cDNA synthesis was performed after total RNA was extracted from the peripheral blood mononuclear cells (PBMC) of 46 patients with symptomatic COVID-19, 46 patients with asymptomatic COVID-19, and 46 healthy controls. The expression levels of hsa_circ_0004812, hsa-miR-1287-5p, IL6R, and RIG-I were determined using qRT-PCR, and the potential interaction between these molecules was confirmed by bioinformatics tools and correlation analysis. RESULTS: hsa_circ_0004812, IL6R, and RIG-I are expressed higher in the severe symptom group compared with the negative control group. Also, the relative expression of these genes in the asymptomatic group is lower than in the severe symptom group. The expression level of hsa-miR-1287-5p was positively correlated with symptoms in patients. The results of the bioinformatics analysis predicted the sponging effect of hsa_circ_0004812 as a competing endogenous RNA on hsa-miR-1287-5p. Moreover, there was a significant positive correlation between hsa_circ_0004812, RIG-I, and IL-6R expressions, and also a negative expression correlation between hsa_circ_0004812 and hsa-miR-1287-5p and between hsa-miR-1287-5p, RIG-I, and IL-6R. CONCLUSION: The results of this in-vitro and in silico study show that hsa_circ_0004812/hsa-miR-1287-5p/IL6R, RIG-I can play an important role in the outcome of COVID-19.

SOCS1 Haploinsufficiency Presenting as Severe Enthesitis, Bone Marrow Hypocellularity, and Refractory Thrombocytopenia in a Pediatric Patient with Subsequent Response to JAK Inhibition.

Haploinsufficiency of suppressor of cytokine signaling 1 (SOCS1) is a recently discovered autoinflammatory disorder with significant rheumatologic, immunologic, and hematologic manifestations. Here we report a case of SOCS1 haploinsufficiency in a 5-year-old child with profound arthralgias and immune-mediated thrombocytopenia unmasked by SARS-CoV-2 infection. Her clinical manifestations were accompanied by excessive B cell activity, eosinophilia, and elevated IgE levels. Uniquely, this is the first report of SOCS1 haploinsufficiency in the setting of a chromosomal deletion resulting in complete loss of a single SOCS1 gene with additional clinical findings of bone marrow hypocellularity and radiologic evidence of severe enthesitis. Immunologic profiling showed a prominent interferon signature in the patient's peripheral blood mononuclear cells, which were also hypersensitive to stimulation by type I and type II interferons. The patient showed excellent clinical and functional laboratory response to tofacitinib, a Janus kinase inhibitor that disrupts interferon signaling. Our case highlights the need to utilize a multidisciplinary diagnostic approach and consider a comprehensive genetic evaluation for inborn errors of immunity in patients with an atypical immune-mediated thrombocytopenia phenotype.

Targeting Doublecortin-Like Kinase 1 (DCLK1)-Regulated SARS-CoV-2 Pathogenesis in COVID-19.

Host factors play critical roles in SARS-CoV-2 infection-associated pathology and the severity of COVID-19. In this study, we systematically analyzed the roles of SARS-CoV-2-induced host factors, doublecortin-like kinase 1 (DCLK1), and S100A9 in viral pathogenesis. In autopsied subjects with COVID-19 and pre-existing chronic liver disease, we observed high levels of DCLK1 and S100A9 expression and immunosuppressive (DCLK1+S100A9+CD206+) M2-like macrophages and N2-like neutrophils in lungs and livers. DCLK1 and S100A9 expression were rarely observed in normal controls, COVID-19-negative subjects with chronic lung disease, or COVID-19 subjects without chronic liver disease. In hospitalized patients with COVID-19, we detected 2 to 3-fold increased levels of circulating DCLK1+S100A9+ mononuclear cells that correlated with disease severity. We validated the SARS-CoV-2-dependent generation of these double-positive immune cells in coculture. SARS-CoV-2-induced DCLK1 expression correlated with the activation of ß-catenin, a known regulator of the DCLK1 promoter. Gain and loss of function studies showed that DCLK1 kinase amplified live virus production and promoted cytokine, chemokine, and growth factor secretion by peripheral blood mononuclear cells. Inhibition of DCLK1 kinase blocked pro-inflammatory caspase-1/interleukin-1ß signaling in infected cells. Treatment of SARS-CoV-2-infected cells with inhibitors of DCLK1 kinase and S100A9 normalized cytokine/chemokine profiles and attenuated DCLK1 expression and ß-catenin activation. In conclusion, we report previously unidentified roles of DCLK1 in augmenting SARS-CoV-2 viremia, inflammatory cytokine expression, and dysregulation of immune cells involved in innate immunity. DCLK1 could be a potential therapeutic target for COVID-19, especially in patients with underlying comorbid diseases associated with DCLK1 expression. IMPORTANCE High mortality in COVID-19 is associated with underlying comorbidities such as chronic liver diseases. Successful treatment of severe/critical COVID-19 remains challenging. Herein, we report a targetable host factor, DCLK1, that amplifies SARS-CoV-2 production, cytokine secretion, and inflammatory pathways via activation of ß-catenin(p65)/DCLK1/S100A9/NF-κB signaling. Furthermore, we observed in the lung, liver, and blood an increased prevalence of immune cells coexpressing DCLK1 and S100A9, a myeloid-derived proinflammatory protein. These cells were associated with increased disease severity in COVID-19 patients. Finally, we used a novel small-molecule inhibitor of DCLK1 kinase (DCLK1-IN-1) and S100A9 inhibitor (tasquinimod) to decrease virus production in vitro and normalize hyperinflammatory responses known to contribute to disease severity in COVID-19.

Measurement of Mitochondrial Respiration in Cryopreserved Human Peripheral Blood Mononuclear Cells (PBMCs).

Inflammatory diseases caused by infectious agents such as the SARS-CoV-2 virus can lead to impaired reductive-oxidative (REDOX) balance and disrupted mitochondrial function. Peripheral blood mononuclear cells (PBMCs) provide a useful model for studying the effects of inflammatory diseases on mitochondrial function but can be limited by the need to store these cells by cryopreservation prior to assay. Here, we describe a method for improving and determining PBMC viability with normalization of values to number of living cells. The approach can be applied not only to PBMC samples derived from patients with diseases marked by an altered inflammatory response such as viral infections.

Soluble P2X7 Receptor Is Elevated in the Plasma of COVID-19 Patients and Correlates With Disease Severity.

Inflammation is a tightly coordinated response against bacterial and viral infections, triggered by the production of pro-inflammatory cytokines. SARS-CoV-2 infection induces COVID-19 disease, characterized by an inflammatory response mediated through the activation of the NLRP3 inflammasome, which results in the production of IL-1ß and IL-18 along with pyroptotic cell death. The NLRP3 inflammasome could be also activated by sterile danger signals such as extracellular ATP triggering the purinergic P2X7 receptor. Severe inflammation in the lungs of SARS-CoV-2-infected individuals is associated with pneumonia, hypoxia and acute respiratory distress syndrome, these being the causes of death associated with COVID-19. Both the P2X7 receptor and NLRP3 have been considered as potential pharmacological targets for treating inflammation in COVID-19. However, there is no experimental evidence of the involvement of the P2X7 receptor during COVID-19 disease. In the present study, we determined the concentration of different cytokines and the P2X7 receptor in the plasma of COVID-19 patients and found that along with the increase in IL-6, IL-18 and the IL-1 receptor antagonist in the plasma of COVID-19 patients, there was also an increase in the purinergic P2X7 receptor. The increase in COVID-19 severity and C-reactive protein concentration positively correlated with increased concentration of the P2X7 receptor in the plasma, but not with the IL-18 cytokine. The P2X7 receptor was found in the supernatant of human peripheral blood mononuclear cells after inflammasome activation. Therefore, our data suggest that determining the levels of the P2X7 receptor in the plasma could be a novel biomarker of COVID-19 severity.

Bioinformatics approach reveals the critical role of the NOD-like receptor signaling pathway in COVID-19-associated multiple sclerosis syndrome.

Multiple sclerosis (MS) is a kind of central nervous system (CNS) autoimmune disease, which mainly damages nerves, the brain, and the spinal cord. Recently, several clinical cases reported the relativity between Coronavirus Disease 2019 (COVID-19) and the development of MS, but the mechanism of how COVID-19 affects the occurrence of MS was still not clear. It is bioinformatics technology that we use to explore the potential association at the gene level. The genetic information related to the two diseases was collected from the DisGNET platform for functional protein network analysis and used STRING to identify the complete gene set. The protein-protein interaction (PPI) network was analyzed by STRING. Finally, in the GEO database, we selected peripheral blood mononuclear cell (PBMC) RNA sequencing data (GSE164805, GSE21942) from COVID-19 patients and MS patients to verify the potential cross mechanism between the two diseases. The similar gene set of immune or inflammation existed between the patients with COVID-19 and ones with MS, including L2RA, IFNG, IL1B, NLRP3, and TNF. Interaction network analysis among proteins revealed that IL1B, P2RX7, IFNB1, IFNB1, TNF, and CASP1 enhanced the network connectivity between the combined gene set of COVID-19 and MS associated with NOD-like receptor (NLR) signaling. The involvement of NLR signaling in both diseases was further confirmed by comparing peripheral blood monocyte samples from COVID-19 and MS patients. Activation of NLR signaling was found in both COVID-19 and MS. The PBMC samples analyses also indicated the involvement of the NLR signaling pathway. Taken together, our data analyses revealed that the NLR signaling pathway might play a critical role in the COVID-19-related MS.

RCA2: a scalable supervised clustering algorithm that reduces batch effects in scRNA-seq data.

The transcriptomic diversity of cell types in the human body can be analysed in unprecedented detail using single cell (SC) technologies. Unsupervised clustering of SC transcriptomes, which is the default technique for defining cell types, is prone to group cells by technical, rather than biological, variation. Compared to de-novo (unsupervised) clustering, we demonstrate using multiple benchmarks that supervised clustering, which uses reference transcriptomes as a guide, is robust to batch effects and data quality artifacts. Here, we present RCA2, the first algorithm to combine reference projection (batch effect robustness) with graph-based clustering (scalability). In addition, RCA2 provides a user-friendly framework incorporating multiple commonly used downstream analysis modules. RCA2 also provides new reference panels for human and mouse and supports generation of custom panels. Furthermore, RCA2 facilitates cell type-specific QC, which is essential for accurate clustering of data from heterogeneous tissues. We demonstrate the advantages of RCA2 on SC data from human bone marrow, healthy PBMCs and PBMCs from COVID-19 patients. Scalable supervised clustering methods such as RCA2 will facilitate unified analysis of cohort-scale SC datasets.

Young infants exhibit robust functional antibody responses and restrained IFN-γ production to SARS-CoV-2.

Severe COVID-19 appears rare in children. This is unexpected, especially in young infants, who are vulnerable to severe disease caused by other respiratory viruses. We evaluate convalescent immune responses in 4 infants under 3 months old with confirmed COVID-19 who presented with mild febrile illness, alongside their parents, and adult controls recovered from confirmed COVID-19. Although not statistically significant, compared to seropositive adults, infants have high serum levels of IgG and IgA to SARS-CoV-2 spike protein, with a corresponding functional ability to block SARS-CoV-2 cellular entry. Infants also exhibit robust saliva anti-spike IgG and IgA responses. Spike-specific IFN-γ production by infant peripheral blood mononuclear cells appears restrained, but the frequency of spike-specific IFN-γ- and/or TNF-α-producing T cells is comparable between infants and adults. On principal-component analysis, infant immune responses appear distinct from their parents. Robust functional antibody responses alongside restrained IFN-γ production may help protect infants from severe COVID-19.

Lysosome activation in peripheral blood mononuclear cells and prognostic significance of circulating LC3B in COVID-19.

Coronavirus disease 2019 (COVID-19) has spread rapidly worldwide, causing significant mortality. There is a mechanistic relationship between intracellular coronavirus replication and deregulated autophagosome-lysosome system. We performed transcriptome analysis of peripheral blood mononuclear cells (PBMCs) from COVID-19 patients and identified the aberrant upregulation of genes in the lysosome pathway. We further determined the capability of two circulating markers, namely microtubule-associated proteins 1A/1B light chain 3B (LC3B) and (p62/SQSTM1) p62, both of which depend on lysosome for degradation, in predicting the emergence of moderate-to-severe disease in COVID-19 patients requiring hospitalization for supplemental oxygen therapy. Logistic regression analyses showed that LC3B was associated with moderate-to-severe COVID-19, independent of age, sex and clinical risk score. A decrease in LC3B concentration <5.5 ng/ml increased the risk of oxygen and ventilatory requirement (adjusted odds ratio: 4.6; 95% CI: 1.1-22.0; P = 0.04). Serum concentrations of p62 in the moderate-to-severe group were significantly lower in patients aged 50 or below. In conclusion, lysosome function is deregulated in PBMCs isolated from COVID-19 patients, and the related biomarker LC3B may serve as a novel tool for stratifying patients with moderate-to-severe COVID-19 from those with asymptomatic or mild disease. COVID-19 patients with a decrease in LC3B concentration <5.5 ng/ml will require early hospital admission for supplemental oxygen therapy and other respiratory support.

Dysregulated transcriptional responses to SARS-CoV-2 in the periphery.

SARS-CoV-2 infection has been shown to trigger a wide spectrum of immune responses and clinical manifestations in human hosts. Here, we sought to elucidate novel aspects of the host response to SARS-CoV-2 infection through RNA sequencing of peripheral blood samples from 46 subjects with COVID-19 and directly comparing them to subjects with seasonal coronavirus, influenza, bacterial pneumonia, and healthy controls. Early SARS-CoV-2 infection triggers a powerful transcriptomic response in peripheral blood with conserved components that are heavily interferon-driven but also marked by indicators of early B-cell activation and antibody production. Interferon responses during SARS-CoV-2 infection demonstrate unique patterns of dysregulated expression compared to other infectious and healthy states. Heterogeneous activation of coagulation and fibrinolytic pathways are present in early COVID-19, as are IL1 and JAK/STAT signaling pathways, which persist into late disease. Classifiers based on differentially expressed genes accurately distinguished SARS-CoV-2 infection from other acute illnesses (auROC 0.95 [95% CI 0.92-0.98]). The transcriptome in peripheral blood reveals both diverse and conserved components of the immune response in COVID-19 and provides for potential biomarker-based approaches to diagnosis.

Single-cell transcriptomes of peripheral blood cells indicate and elucidate severity of COVID-19.

The blood and immune system of coronavirus disease 2019 (COVID-19) infected patients are dysfunctional, and numerous studies have been conducted to resolve their characteristics and pathogenic mechanisms. Nevertheless, the variations of immune responses along with disease severity have not been comprehensively documented. Here, we profiled the single-cell transcriptomes of 96,313 peripheral blood mononuclear cells (PBMCs) derived from 12 COVID-19 patients (including four moderate, four severe and four critical cases) and three healthy donors. We showed that proliferative CD8 effector T cells with declined immune functions and cytotoxicity accumulated in the critical stage. By contrast, the quantity of natural killer (NK) cells was significantly reduced, while they exhibited enhanced immune activities. Notably, a gradually attenuated responseto COVID-19 along with disease severity was observed in monocytes, in terms of cellular composition, transcriptional discrepancy and transcription factor regulatory network. Furthermore, we identified immune cell-type dependent cytokine signatures distinguishing the severity of COVID-19 patients. In addition, cell interactions between CD8 effector T/NK cells and monocytes mediated by inflammatory cytokines were enhanced in moderate and severe stages, but weakened in critical cases. Collectively, our work uncovers the cellular and molecular players underlying the disordered and heterogeneous immune responses associated with COVID-19 severity, which could provide valuable insights for the treatment of critical COVID-19 patients.

Higher expression of monocyte chemotactic protein 1 in mild COVID-19 patients might be correlated with inhibition of Type I IFN signaling.

BACKGROUND: Chemokine levels in severe coronavirus disease 2019 (COVID-19) patients have been shown to be markedly elevated. But the role of chemokines in mild COVID-19 has not yet been established. According to the epidemiological statistics, most of the COVID-19 cases in Shiyan City, China, have been mild. The purpose of this study was to evaluate the level of chemokines in mild COVID-19 patients and explore the correlation between chemokines and host immune response. METHODS: In this study, we used an enzyme-linked immunosorbent assay to detect serum levels of chemokines in COVID-19 patients in Shiyan City. Expression of chemokine receptors and of other signaling molecules was measured by real-time polymerase chain reaction. RESULTS: We first demonstrated that COVID-19 patients, both sever and mild cases, are characterized by higher level of chemokines. Specifically, monocyte chemotactic protein 1 (MCP-1) is expressed at higher levels both in severe and mild cases of COVID-19. The receptor of MCP-1, C-C chemokine receptor type 2, was expressed at higher levels in mild COVID-19 patients. Finally, we observed a significant negative correlation between expression levels of interferon (IFN) regulatory factor 3 (IRF3) and serum levels of MCP-1 in mild COVID-19 patients. CONCLUSION: Higher expression of MCP-1 in mild COVID-19 patients might be correlated with inhibition of IFN signaling. The finding adds to our understanding of the immunopathological mechanisms of severe acute respiratory syndrome coronavirus 2 infection and provides potential therapeutic targets and strategies.

Longitudinal transcriptome analyses show robust T cell immunity during recovery from COVID-19.

Understanding the processes of immune regulation in patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for improving treatment. Here, we performed longitudinal whole-transcriptome RNA sequencing on peripheral blood mononuclear cell (PBMC) samples from 18 patients with coronavirus disease 2019 (COVID-19) during their treatment, convalescence, and rehabilitation. After analyzing the regulatory networks of differentially expressed messenger RNAs (mRNAs), microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) between the different clinical stages, we found that humoral immunity and type I interferon response were significantly downregulated, while robust T-cell activation and differentiation at the whole transcriptome level constituted the main events that occurred during recovery from COVID-19. The formation of this T cell immune response might be driven by the activation of activating protein-1 (AP-1) related signaling pathway and was weakly affected by other clinical features. These findings uncovered the dynamic pattern of immune responses and indicated the key role of T cell immunity in the creation of immune protection against this disease.

Distinct inflammatory profiles distinguish COVID-19 from influenza with limited contributions from cytokine storm.

We pursued a study of immune responses in coronavirus disease 2019 (COVID-19) and influenza patients. Compared to patients with influenza, patients with COVID-19 exhibited largely equivalent lymphocyte counts, fewer monocytes, and lower surface human leukocyte antigen (HLA)-class II expression on selected monocyte populations. Furthermore, decreased HLA-DR on intermediate monocytes predicted severe COVID-19 disease. In contrast to prevailing assumptions, very few (7 of 168) patients with COVID-19 exhibited cytokine profiles indicative of cytokine storm syndrome. After controlling for multiple factors including age and sample time point, patients with COVID-19 exhibited lower cytokine levels than patients with influenza. Up-regulation of IL-6, G-CSF, IL-1RA, and MCP1 predicted death in patients with COVID-19 but were not statistically higher than patients with influenza. Single-cell transcriptional profiling revealed profound suppression of interferon signaling among patients with COVID-19. When considered across the spectrum of peripheral immune profiles, patients with COVID-19 are less inflamed than patients with influenza.

Mitochondrial metabolic manipulation by SARS-CoV-2 in peripheral blood mononuclear cells of patients with COVID-19.

The COVID-19 pandemic has been the primary global health issue since its outbreak in December 2019. Patients with metabolic syndrome suffer from severe complications and a higher mortality rate due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We recently proposed that SARS-CoV-2 can hijack host mitochondrial function and manipulate metabolic pathways for their own advantage. The aim of the current study was to investigate functional mitochondrial changes in live peripheral blood mononuclear cells (PBMCs) from patients with COVID-19 and to decipher the pathways of substrate utilization in these cells and corresponding changes in the inflammatory pathways. We demonstrate mitochondrial dysfunction, metabolic alterations with an increase in glycolysis, and high levels of mitokine in PBMCs from patients with COVID-19. Interestingly, we found that levels of fibroblast growth factor 21 mitokine correlate with COVID-19 disease severity and mortality. These data suggest that patients with COVID-19 have a compromised mitochondrial function and an energy deficit that is compensated by a metabolic switch to glycolysis. This metabolic manipulation by SARS-CoV-2 triggers an enhanced inflammatory response that contributes to the severity of symptoms in COVID-19. Targeting mitochondrial metabolic pathway(s) can help define novel strategies for COVID-19.

COVID-19-activated SREBP2 disturbs cholesterol biosynthesis and leads to cytokine storm.

Sterol regulatory element binding protein-2 (SREBP-2) is activated by cytokines or pathogen, such as virus or bacteria, but its association with diminished cholesterol levels in COVID-19 patients is unknown. Here, we evaluated SREBP-2 activation in peripheral blood mononuclear cells of COVID-19 patients and verified the function of SREBP-2 in COVID-19. Intriguingly, we report the first observation of SREBP-2 C-terminal fragment in COVID-19 patients' blood and propose SREBP-2 C-terminal fragment as an indicator for determining severity. We confirmed that SREBP-2-induced cholesterol biosynthesis was suppressed by Sestrin-1 and PCSK9 expression, while the SREBP-2-induced inflammatory responses was upregulated in COVID-19 ICU patients. Using an infectious disease mouse model, inhibitors of SREBP-2 and NF-κB suppressed cytokine storms caused by viral infection and prevented pulmonary damages. These results collectively suggest that SREBP-2 can serve as an indicator for severity diagnosis and therapeutic target for preventing cytokine storm and lung damage in severe COVID-19 patients.