Comprehensive Immune Profiling Reveals CD56+ Monocytes and CD31+ Endothelial Cells Are Increased in Severe COVID-19 Disease.

Immune response dysregulation plays a key role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis. In this study, we evaluated immune and endothelial blood cell profiles of patients with coronavirus disease 2019 (COVID-19) to determine critical differences between those with mild, moderate, or severe COVID-19 using spectral flow cytometry. We examined a suite of immune phenotypes, including monocytes, T cells, NK cells, B cells, endothelial cells, and neutrophils, alongside surface and intracellular markers of activation. Our results showed progressive lymphopenia and depletion of T cell subsets (CD3+, CD4+, and CD8+) in patients with severe disease and a significant increase in the CD56+CD14+Ki67+IFN-γ+ monocyte population in patients with moderate and severe COVID-19 that has not been previously described. Enhanced circulating endothelial cells (CD45-CD31+CD34+CD146+), circulating endothelial progenitors (CD45-CD31+CD34+/-CD146-), and neutrophils (CD11b+CD66b+) were coevaluated for COVID-19 severity. Spearman correlation analysis demonstrated the synergism among age, obesity, and hypertension with upregulated CD56+ monocytes, endothelial cells, and decreased T cells that lead to severe outcomes of SARS-CoV-2 infection. Circulating monocytes and endothelial cells may represent important cellular markers for monitoring postacute sequelae and impacts of SARS-CoV-2 infection during convalescence and for their role in immune host defense in high-risk adults after vaccination.

Hyperactive immature state and differential CXCR2 expression of neutrophils in severe COVID-19.

Neutrophils are vital in defence against pathogens, but excessive neutrophil activity can lead to tissue damage and promote acute respiratory distress syndrome. COVID-19 is associated with systemic expansion of immature neutrophils, but the functional consequences of this shift to immaturity are not understood. We used flow cytometry to investigate activity and phenotypic diversity of circulating neutrophils in acute and convalescent COVID-19 patients. First, we demonstrate hyperactivation of immature CD10- subpopulations in severe disease, with elevated markers of secondary granule release. Partially activated immature neutrophils were detectable 12 wk post-hospitalisation, indicating long term myeloid dysregulation in convalescent COVID-19 patients. Second, we demonstrate that neutrophils from moderately ill patients down-regulate the chemokine receptor CXCR2, whereas neutrophils from severely ill individuals fail to do so, suggesting an altered ability for organ trafficking and a potential mechanism for induction of disease tolerance. CD10- and CXCR2hi neutrophil subpopulations were enriched in severe disease and may represent prognostic biomarkers for the identification of individuals at high risk of progressing to severe COVID-19.

The Roles of Immune Cells in the Pathogenesis of Fibrosis.

Tissue injury and inflammatory response trigger the development of fibrosis in various diseases. It has been recognized that both innate and adaptive immune cells are important players with multifaceted functions in fibrogenesis. The activated immune cells produce various cytokines, modulate the differentiation and functions of myofibroblasts via diverse molecular mechanisms, and regulate fibrotic development. The immune cells exhibit differential functions during different stages of fibrotic diseases. In this review, we summarized recent advances in understanding the roles of immune cells in regulating fibrotic development and immune-based therapies in different disorders and discuss the underlying molecular mechanisms with a focus on mTOR and JAK-STAT signaling pathways.

CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation.

BACKGROUND: Coronavirus-induced disease 19 (COVID-19) infects more than three hundred and sixty million patients worldwide, and people with severe symptoms frequently die of acute respiratory distress syndrome (ARDS). Recent studies indicated that excessive neutrophil extracellular traps (NETs) contributed to immunothrombosis, thereby leading to extensive intravascular coagulopathy and multiple organ dysfunction. Thus, understanding the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced NET formation would be helpful to reduce thrombosis and prevent ARDS in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: We incubated SARS-CoV-2 with neutrophils in the presence or absence of platelets to observe NET formation. We further isolated extracellular vesicles from COVID-19 patients' sera (COVID-19-EVs) to examine their ability to induce NET formation. RESULTS: We demonstrated that antagonistic mAbs against anti-CLEC5A mAb and anti-TLR2 mAb can inhibit COVID-19-EVs-induced NET formation, and generated clec5a-/-/tlr2-/- mice to confirm the critical roles of CLEC5A and TLR2 in SARS-CoV-2-induced lung inflammation in vivo. We found that virus-free extracellular COVID-19 EVs induced robust NET formation via Syk-coupled C-type lectin member 5A (CLEC5A) and TLR2. Blockade of CLEC5A inhibited COVID-19 EVs-induced NETosis, and simultaneous blockade of CLEC5A and TLR2 further suppressed SARS-CoV-2-induced NETosis in vitro. Moreover, thromboinflammation was attenuated dramatically in clec5a-/-/tlr2-/- mice. CONCLUSIONS: This study demonstrates that SARS-CoV-2-activated platelets produce EVs to enhance thromboinflammation via CLEC5A and TLR2, and highlight the importance of CLEC5A and TLR2 as therapeutic targets to reduce the risk of ARDS in COVID-19 patients.

Complement Activation via the Lectin and Alternative Pathway in Patients With Severe COVID-19.

Complement plays an important role in the direct defense to pathogens, but can also activate immune cells and the release of pro-inflammatory cytokines. However, in critically ill patients with COVID-19 the immune system is inadequately activated leading to severe acute respiratory syndrome (SARS) and acute kidney injury, which is associated with higher mortality. Therefore, we characterized local complement deposition as a sign of activation in both lungs and kidneys from patients with severe COVID-19. Using immunohistochemistry we investigated deposition of complement factors C1q, MASP-2, factor D (CFD), C3c, C3d and C5b-9 as well as myeloperoxidase (MPO) positive neutrophils and SARS-CoV-2 virus particles in lungs and kidneys from 38 patients who died from COVID-19. In addition, tissue damage was analyzed using semi-quantitative scores followed by correlation with complement deposition. Autopsy material from non-COVID patients who died from cardiovascular causes, cerebral hemorrhage and pulmonary embolism served as control (n=8). Lung injury in samples from COVID-19 patients was significantly more pronounced compared to controls with formation of hyaline membranes, thrombi and edema. In addition, in the kidney tubular injury was higher in these patients and correlated with lung injury (r=0.361*). In autopsy samples SARS-CoV-2 spike protein was detected in 22% of the lungs of COVID-19 patients but was lacking in kidneys. Complement activation was significantly stronger in lung samples from patients with COVID-19 via the lectin and alternative pathway as indicated by deposition of MASP-2, CFD, C3d and C5b9. Deposits in the lung were predominantly detected along the alveolar septa, the hyaline membranes and in the alveolar lumina. In the kidney, complement was significantly more deposited in patients with COVID-19 in peritubular capillaries and tubular basement membranes. Renal COVID-19-induced complement activation occurred via the lectin pathway, while activation of the alternative pathway was similar in both groups. Furthermore, MPO-positive neutrophils were found in significantly higher numbers in lungs and kidneys of COVID-19 patients and correlated with local MASP-2 deposition. In conclusion, in patients who died from SARS-CoV-2 infection complement was activated in both lungs and kidneys indicating that complement might be involved in systemic worsening of the inflammatory response. Complement inhibition might thus be a promising treatment option to prevent deregulated activation and subsequent collateral tissue injury in COVID-19.

Innate immune deficiencies are associated with severity and poor prognosis in patients with COVID-19.

COVID-19 can cause acute respiratory distress syndrome, leading to death in many individuals. Evidence of a deleterious role of the innate immune system is accumulating, but the precise mechanisms involved remain unclear. In this study, we investigated the links between circulating innate phagocytes and severity in COVID-19 patients. We performed in-depth phenotyping of neutrophil and monocyte subpopulations and measured soluble activation markers in plasma. Additionally, anti-microbial functions (phagocytosis, oxidative burst, and NETosis) were evaluated on fresh cells from patients. Neutrophils and monocytes had a strikingly disturbed phenotype, and elevated concentrations of activation markers (calprotectin, myeloperoxidase, and neutrophil extracellular traps) were measured in plasma. Critical patients had increased CD13low immature neutrophils, LOX-1 + and CCR5 + immunosuppressive neutrophils, and HLA-DRlow downregulated monocytes. Markers of immature and immunosuppressive neutrophils were strongly associated with severity. Moreover, neutrophils and monocytes of critical patients had impaired antimicrobial functions, which correlated with organ dysfunction, severe infections, and mortality. Together, our results strongly argue in favor of a pivotal role of innate immunity in COVID-19 severe infections and pleads for targeted therapeutic options.

Brain motor and fear circuits regulate leukocytes during acute stress.

The nervous and immune systems are intricately linked1. Although psychological stress is known to modulate immune function, mechanistic pathways linking stress networks in the brain to peripheral leukocytes remain poorly understood2. Here we show that distinct brain regions shape leukocyte distribution and function throughout the body during acute stress in mice. Using optogenetics and chemogenetics, we demonstrate that motor circuits induce rapid neutrophil mobilization from the bone marrow to peripheral tissues through skeletal-muscle-derived neutrophil-attracting chemokines. Conversely, the paraventricular hypothalamus controls monocyte and lymphocyte egress from secondary lymphoid organs and blood to the bone marrow through direct, cell-intrinsic glucocorticoid signalling. These stress-induced, counter-directional, population-wide leukocyte shifts are associated with altered disease susceptibility. On the one hand, acute stress changes innate immunity by reprogramming neutrophils and directing their recruitment to sites of injury. On the other hand, corticotropin-releasing hormone neuron-mediated leukocyte shifts protect against the acquisition of autoimmunity, but impair immunity to SARS-CoV-2 and influenza infection. Collectively, these data show that distinct brain regions differentially and rapidly tailor the leukocyte landscape during psychological stress, therefore calibrating the ability of the immune system to respond to physical threats.

Infiltration of inflammatory macrophages and neutrophils and widespread pyroptosis in lung drive influenza lethality in nonhuman primates.

Severe influenza kills tens of thousands of individuals each year, yet the mechanisms driving lethality in humans are poorly understood. Here we used a unique translational model of lethal H5N1 influenza in cynomolgus macaques that utilizes inhalation of small-particle virus aerosols to define mechanisms driving lethal disease. RNA sequencing of lung tissue revealed an intense interferon response within two days of infection that resulted in widespread expression of interferon-stimulated genes, including inflammatory cytokines and chemokines. Macaques with lethal disease had rapid and profound loss of alveolar macrophages (AMs) and infiltration of activated CCR2+ CX3CR1+ interstitial macrophages (IMs) and neutrophils into lungs. Parallel changes of AMs and neutrophils in bronchoalveolar lavage (BAL) correlated with virus load when compared to macaques with mild influenza. Both AMs and IMs in lethal influenza were M1-type inflammatory macrophages which expressed neutrophil chemotactic factors, while neutrophils expressed genes associated with activation and generation of neutrophil extracellular traps (NETs). NETs were prominent in lung and were found in alveolar spaces as well as lung parenchyma. Genes associated with pyroptosis but not apoptosis were increased in lung, and activated inflammatory caspases, IL-1ß and cleaved gasdermin D (GSDMD) were present in bronchoalveolar lavage fluid and lung homogenates. Cleaved GSDMD was expressed by lung macrophages and alveolar epithelial cells which were present in large numbers in alveolar spaces, consistent with loss of epithelial integrity. Cleaved GSDMD colocalized with viral NP-expressing cells in alveoli, reflecting pyroptosis of infected cells. These novel findings reveal that a potent interferon and inflammatory cascade in lung associated with infiltration of inflammatory macrophages and neutrophils, elaboration of NETs and cell death by pyroptosis mediates lethal H5N1 influenza in nonhuman primates, and by extension humans. These innate pathways represent promising therapeutic targets to prevent severe influenza and potentially other primary viral pneumonias in humans.

Single-Cell Landscape of Lungs Reveals Key Role of Neutrophil-Mediated Immunopathology during Lethal SARS-CoV-2 Infection.

Due to the limitation of human studies with respect to individual difference or the accessibility of fresh tissue samples, how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in pathological complications in lung, the main site of infection, is still incompletely understood. Therefore, physiologically relevant animal models under realistic SARS-CoV-2 infection conditions would be helpful to our understanding of dysregulated inflammation response in lung in the context of targeted therapeutics. Here, we characterized the single-cell landscape in lung and spleen upon SARS-CoV-2 infection in an acute severe disease mouse model that replicates human symptoms, including severe lung pathology and lymphopenia. We showed a reduction of lymphocyte populations and an increase of neutrophils in lung and then demonstrated the key role of neutrophil-mediated lung immunopathology in both mice and humans. Under severe conditions, neutrophils recruited by a chemokine-driven positive feedback produced elevated "fatal signature" proinflammatory genes and pathways related to neutrophil activation or releasing of granular content. In addition, we identified a new Cd177high cluster that is undergoing respiratory burst and Stfahigh cluster cells that may dampen antigen presentation upon infection. We also revealed the devastating effect of overactivated neutrophil by showing the highly enriched neutrophil extracellular traps in lung and a dampened B-cell function in either lung or spleen that may be attributed to arginine consumption by neutrophil. The current study helped our understanding of SARS-CoV-2-induced pneumonia and warranted the concept of neutrophil-targeting therapeutics in COVID-19 treatment. IMPORTANCE We demonstrated the single-cell landscape in lung and spleen upon SARS-CoV-2 infection in an acute severe disease mouse model that replicated human symptoms, including severe lung pathology and lymphopenia. Our comprehensive study revealed the key role of neutrophil-mediated lung immunopathology in SARS-CoV-2-induced severe pneumonia, which not only helped our understanding of COVID-19 but also warranted the concept of neutrophil targeting therapeutics in COVID-19 treatment.

Comparative analysis of antibody- and lipid-based multiplexing methods for single-cell RNA-seq.

BACKGROUND: Multiplexing of samples in single-cell RNA-seq studies allows a significant reduction of the experimental costs, straightforward identification of doublets, increased cell throughput, and reduction of sample-specific batch effects. Recently published multiplexing techniques using oligo-conjugated antibodies or -lipids allow barcoding sample-specific cells, a process called "hashing." RESULTS: Here, we compare the hashing performance of TotalSeq-A and -C antibodies, custom synthesized lipids and MULTI-seq lipid hashes in four cell lines, both for single-cell RNA-seq and single-nucleus RNA-seq. We also compare TotalSeq-B antibodies with CellPlex reagents (10x Genomics) on human PBMCs and TotalSeq-B with different lipids on primary mouse tissues. Hashing efficiency was evaluated using the intrinsic genetic variation of the cell lines and mouse strains. Antibody hashing was further evaluated on clinical samples using PBMCs from healthy and SARS-CoV-2 infected patients, where we demonstrate a more affordable approach for large single-cell sequencing clinical studies, while simultaneously reducing batch effects. CONCLUSIONS: Benchmarking of different hashing strategies and computational pipelines indicates that correct demultiplexing can be achieved with both lipid- and antibody-hashed human cells and nuclei, with MULTISeqDemux as the preferred demultiplexing function and antibody-based hashing as the most efficient protocol on cells. On nuclei datasets, lipid hashing delivers the best results. Lipid hashing also outperforms antibodies on cells isolated from mouse brain. However, antibodies demonstrate better results on tissues like spleen or lung.

New Discovery of Myeloid-Derived Suppressor Cell's Tale on Viral Infection and COVID-19.

Myeloid-derived suppressor cells (MDSCs) are generated under biological stress such as cancer, inflammatory tissue damage, and viral infection. In recent years, with occurrence of global infectious diseases, new discovery on MDSCs functions has been significantly expanded during viral infection and COVID-19. For a successful viral infection, pathogens viruses develop immune evasion strategies to avoid immune recognition. Numerous viruses induce the differentiation and expansion of MDSCs in order to suppress host immune responses including natural killer cells, antigen presenting cells, and T-cells. Moreover, MDSCs play an important role in regulation of immunopathogenesis by balancing viral infection and tissue damage. In this review article, we describe the overview of immunomodulation and genetic regulation of MDSCs during viral infection in the animal model and human studies. In addition, we include up-to-date review of role of MDSCs in SARS-CoV-2 infection and COVID-19. Finally, we discuss potential therapeutics targeting MDSCs.

Neutrophils predominate the immune signature of cerebral thrombi in COVID-19 stroke patients.

Coronavirus disease 2019 (COVID-19) is associated with an increased risk of thrombotic events. Ischemic stroke in COVID-19 patients entails high severity and mortality rates. Here we aimed to analyze cerebral thrombi of COVID-19 patients with large vessel occlusion (LVO) acute ischemic stroke to expose molecular evidence for SARS-CoV-2 in the thrombus and to unravel any peculiar immune-thrombotic features. We conducted a systematic pathological analysis of cerebral thrombi retrieved by endovascular thrombectomy in patients with LVO stroke infected with COVID-19 (n = 7 patients) and non-covid LVO controls (n = 23). In thrombi of COVID-19 patients, the SARS-CoV-2 docking receptor ACE2 was mainly expressed in monocytes/macrophages and showed higher expression levels compared to controls. Using polymerase chain reaction and sequencing, we detected SARS-CoV-2 Clade20A, in the thrombus of one COVID-19 patient. Comparing thrombus composition of COVID-19 and control patients, we noted no overt differences in terms of red blood cells, fibrin, neutrophil extracellular traps (NETs), von Willebrand Factor (vWF), platelets and complement complex C5b-9. However, thrombi of COVID-19 patients showed increased neutrophil density (MPO+ cells) and a three-fold higher Neutrophil-to-Lymphocyte Ratio (tNLR). In the ROC analysis both neutrophils and tNLR had a good discriminative ability to differentiate thrombi of COVID-19 patients from controls. In summary, cerebral thrombi of COVID-19 patients can harbor SARS-CoV2 and are characterized by an increased neutrophil number and tNLR and higher ACE2 expression. These findings suggest neutrophils as the possible culprit in COVID-19-related thrombosis.

Distinguishing Incubation and Acute Disease Stages of Mild-to-Moderate COVID-19.

While numerous studies have already compared the immune responses against SARS-CoV-2 in severely and mild-to-moderately ill COVID-19 patients, longitudinal trajectories are still scarce. We therefore set out to analyze serial blood samples from mild-to-moderately ill patients in order to define the immune landscapes for differently progressed disease stages. Twenty-two COVID-19 patients were subjected to consecutive venipuncture within seven days after diagnosis or admittance to hospital. Flow cytometry was performed to analyze peripheral blood immune cell compositions and their activation as were plasma levels of cytokines and SARS-CoV-2 specific immunoglobulins. Healthy donors served as controls. Integrating the kinetics of plasmablasts and SARS-CoV-2 specific antibodies allowed for the definition of three disease stages of early COVID-19. The incubation phase was characterized by a sharp increase in pro-inflammatory monocytes and terminally differentiated cytotoxic T cells. The latter correlated significantly with elevated concentrations of IP-10. Early acute infection featured a peak in PD-1+ cytotoxic T cells, plasmablasts and increasing titers of virus specific antibodies. During late acute infection, immature neutrophils were enriched, whereas all other parameters returned to baseline. Our findings will help to define landmarks that are indispensable for the refinement of new anti-viral and anti-inflammatory therapeutics, and may also inform clinicians to optimize treatment and prevent fatal outcomes.

Adenovirus Co-Opts Neutrophilic Inflammation to Enhance Transduction of Epithelial Cells.

Human adenoviruses (HAdV) cause a variety of infections in human hosts, from self-limited upper respiratory tract infections in otherwise healthy people to fulminant pneumonia and death in immunocompromised patients. Many HAdV enter polarized epithelial cells by using the primary receptor, the Coxsackievirus and adenovirus receptor (CAR). Recently published data demonstrate that a potent neutrophil (PMN) chemoattractant, interleukin-8 (IL-8), stimulates airway epithelial cells to increase expression of the apical isoform of CAR (CAREx8), which results in increased epithelial HAdV type 5 (HAdV5) infection. However, the mechanism for PMN-enhanced epithelial HAdV5 transduction remains unclear. In this manuscript, the molecular mechanisms behind PMN mediated enhancement of epithelial HAdV5 transduction are characterized using an MDCK cell line that stably expresses human CAREx8 under a doxycycline inducible promoter (MDCK-CAREx8 cells). Contrary to our hypothesis, PMN exposure does not enhance HAdV5 entry by increasing CAREx8 expression nor through activation of non-specific epithelial endocytic pathways. Instead, PMN serine proteases are responsible for PMN-mediated enhancement of HAdV5 transduction in MDCK-CAREx8 cells. This is evidenced by reduced transduction upon inhibition of PMN serine proteases and increased transduction upon exposure to exogenous human neutrophil elastase (HNE). Furthermore, HNE exposure activates epithelial autophagic flux, which, even when triggered through other mechanisms, results in a similar enhancement of epithelial HAdV5 transduction. Inhibition of F-actin with cytochalasin D partially attenuates PMN mediated enhancement of HAdV transduction. Taken together, these findings suggest that HAdV5 can leverage innate immune responses to establish infections.

Formation of Calprotectin-Derived Peptides in the Airways of Children with Cystic Fibrosis.

Calprotectin is released by activated neutrophils along with myeloperoxidase (MPO) and proteases. It plays numerous roles in inflammation and infection, and is used as an inflammatory biomarker. However, calprotectin is readily oxidized by MPO-derived hypohalous acids to form covalent dimers of its S100A8 and S100A9 subunits. The dimers are susceptible to degradation by proteases. We show that detection of human calprotectin by ELISA declines markedly because of its oxidation by hypochlorous acid and subsequent degradation. Also, proteolysis liberates specific peptides from oxidized calprotectin that is present at inflammatory sites. We identified six calprotectin-derived peptides by mass spectrometry and detected them in the bronchoalveolar lavage fluid of children with cystic fibrosis (CF). We assessed the peptides as biomarkers of neutrophilic inflammation and infection. The content of the calprotectin peptide ILVI was related to calprotectin (r = 0.72, p = 0.01, n = 10). Four of the peptides were correlated with the concentration of MPO (r > 0.7, p ≤ 0.01, n = 21), while three were higher (p < 0.05) in neutrophil elastase-positive (n = 14) than -negative samples (n = 7). Also, five of the peptides were higher (p < 0.05) in the bronchoalveolar lavage fluid from children with CF with infections (n = 21) than from non-CF children without infections (n = 6). The specific peptides liberated from calprotectin will signal uncontrolled activity of proteases and MPO during inflammation. They may prove useful in tracking inflammation in respiratory diseases dominated by neutrophils, including coronavirus disease 2019.

Essential metals, vitamins and antioxidant enzyme activities in COVID-19 patients and their potential associations with the disease severity.

The role of micronutrient deficiency in the pathogenesis of COVID-19 has been reviewed in the literature; however, the data are limited and conflicting. This study investigated the association between the status of essential metals, vitamins, and antioxidant enzyme activities in COVID-19 patients and disease severity. We recruited 155 patients, who were grouped into four classes based on the Adults guideline for the Management of Coronavirus Disease 2019 at King Faisal Specialist & Research Centre (KFSH&RC): asymptomatic (N = 16), mild (N = 49), moderate (N = 68), and severe (N = 22). We measured serum levels of copper (Cu), zinc (Zn), selenium (Se), vitamin D3, vitamin A, vitamin E, total antioxidant capacity, and superoxide dismutase (SOD). Among the patients, 30%, 25%, 37%, and 68% were deficient in Se (< 70.08 µg/L), Zn (< 0.693 µg/mL), vitamin A (< 0.343 µg/mL), and vitamin D3 (< 20.05 µg/L), respectively, and SOD activity was low. Among the patients, 28% had elevated Cu levels (> 1.401 µg/mL, KFSH&RC upper reference limit). Multiple regression analysis revealed an 18% decrease in Se levels in patients with severe symptoms, which increased to 30% after adjusting the model for inflammatory markers. Regardless of inflammation, Se was independently associated with COVID-19 severity. In contrast, a 50% increase in Cu levels was associated with disease severity only after adjusting for C-reactive protein, reflecting its possible inflammatory and pro-oxidant role in COVID-19 pathogenesis. We noted an imbalance in the ratio between Cu and Zn, with ~ 83% of patients having a Cu/Zn ratio > 1, which is an indicator of inflammation. Cu-to-Zn ratio increased to 45% in patients with mild symptoms and 34%-36% in patients with moderate symptoms compared to asymptomatic patients. These relationships were only obtained when one of the laboratory parameters (lymphocyte or monocyte) or inflammatory markers (neutrophil-to-lymphocyte ratio) was included in the regression model. These findings suggest that Cu/Zn might further exacerbate inflammation in COVID-19 patients and might be synergistically associated with disease severity. A 23% decrease in vitamin A was seen in patients with severe symptoms, which disappeared after adjusting for inflammatory markers. This finding may highlight the potential role of inflammation in mediating the relationship between COVID-19 severity and vitamin A levels. Despite our patients' low status of Zn, vitamin D3, and antioxidant enzyme (SOD), there is no evidence of their role in COVID-19 progression. Our findings reinforce that deficiency or excess of certain micronutrients plays a role in the pathogenesis of COVID-19. More studies are required to support our results.

DEspRhigh neutrophils are associated with critical illness in COVID-19.

SARS-CoV-2 infection results in a spectrum of outcomes from no symptoms to widely varying degrees of illness to death. A better understanding of the immune response to SARS-CoV-2 infection and subsequent, often excessive, inflammation may inform treatment decisions and reveal opportunities for therapy. We studied immune cell subpopulations and their associations with clinical parameters in a cohort of 26 patients with COVID-19. Following informed consent, we collected blood samples from hospitalized patients with COVID-19 within 72 h of admission. Flow cytometry was used to analyze white blood cell subpopulations. Plasma levels of cytokines and chemokines were measured using ELISA. Neutrophils undergoing neutrophil extracellular traps (NET) formation were evaluated in blood smears. We examined the immunophenotype of patients with COVID-19 in comparison to that of SARS-CoV-2 negative controls. A novel subset of pro-inflammatory neutrophils expressing a high level of dual endothelin-1 and VEGF signal peptide-activated receptor (DEspR) at the cell surface was found to be associated with elevated circulating CCL23, increased NETosis, and critical-severity COVID-19 illness. The potential to target this subpopulation of neutrophils to reduce secondary tissue damage caused by SARS-CoV-2 infection warrants further investigation.

An integrative bioinformatics analysis for identifying hub genes associated with infection of lung samples in patients infected with SARS-CoV-2.

BACKGROUND: At the end of 2019, the world witnessed the emergence and ravages of a viral infection induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Also known as the coronavirus disease 2019 (COVID-19), it has been identified as a public health emergency of international concern (PHEIC) by the World Health Organization (WHO) because of its severity. METHODS: The gene data of 51 samples were extracted from the GSE150316 and GSE147507 data set and then processed by means of the programming language R, through which the differentially expressed genes (DEGs) that meet the standards were screened. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed on the selected DEGs to understand the functions and approaches of DEGs. The online tool STRING was employed to construct a protein-protein interaction (PPI) network of DEGs and, in turn, to identify hub genes. RESULTS: A total of 52 intersection genes were obtained through DEG identification. Through the GO analysis, we realized that the biological processes (BPs) that have the deepest impact on the human body after SARS-CoV-2 infection are various immune responses. By using STRING to construct a PPI network, 10 hub genes were identified, including IFIH1, DDX58, ISG15, EGR1, OASL, SAMD9, SAMD9L, XAF1, IFITM1, and TNFSF10. CONCLUSION: The results of this study will hopefully provide guidance for future studies on the pathophysiological mechanism of SARS-CoV-2 infection.

Prolonged activation of nasal immune cell populations and development of tissue-resident SARS-CoV-2-specific CD8+ T cell responses following COVID-19.

Systemic immune cell dynamics during coronavirus disease 2019 (COVID-19) are extensively documented, but these are less well studied in the (upper) respiratory tract, where severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates1-6. Here, we characterized nasal and systemic immune cells in individuals with COVID-19 who were hospitalized or convalescent and compared the immune cells to those seen in healthy donors. We observed increased nasal granulocytes, monocytes, CD11c+ natural killer (NK) cells and CD4+ T effector cells during acute COVID-19. The mucosal proinflammatory populations positively associated with peripheral blood human leukocyte antigen (HLA)-DRlow monocytes, CD38+PD1+CD4+ T effector (Teff) cells and plasmablasts. However, there was no general lymphopenia in nasal mucosa, unlike in peripheral blood. Moreover, nasal neutrophils negatively associated with oxygen saturation levels in blood. Following convalescence, nasal immune cells mostly normalized, except for CD127+ granulocytes and CD38+CD8+ tissue-resident memory T cells (TRM). SARS-CoV-2-specific CD8+ T cells persisted at least 2 months after viral clearance in the nasal mucosa, indicating that COVID-19 has both transient and long-term effects on upper respiratory tract immune responses.