Single-cell RNA-seq analysis identifies distinct myeloid cells in a case with encephalitis temporally associated with COVID-19 vaccination.

Recently accumulating evidence has highlighted the rare occurrence of COVID-19 vaccination-induced inflammation in the central nervous system. However, the precise information on immune dysregulation related to the COVID-19 vaccination-associated autoimmunity remains elusive. Here we report a case of encephalitis temporally associated with COVID-19 vaccination, where single-cell RNA sequencing (scRNA-seq) analysis was applied to elucidate the distinct immune signature in the peripheral immune system. Peripheral blood mononuclear cells (PBMCs) were analyzed using scRNA-seq to clarify the cellular components of the patients in the acute and remission phases of the disease. The data obtained were compared to those acquired from a healthy cohort. The scRNA-seq analysis identified a distinct myeloid cell population in PBMCs during the acute phase of encephalitis. This specific myeloid population was detected neither in the remission phase of the disease nor in the healthy cohort. Our findings illustrate induction of a unique myeloid subset in encephalitis temporally associated with COVID-19 vaccination. Further research into the dysregulated immune signature of COVID-19 vaccination-associated autoimmunity including the cerebrospinal fluid (CSF) cells of central nervous system (CNS) is warranted to clarify the pathogenic role of the myeloid subset observed in our study.

Microscale combinatorial stimulation of human myeloid cells reveals inflammatory priming by viral ligands.

Cells sense a wide variety of signals and respond by adopting complex transcriptional states. Most single-cell profiling is carried out today at cellular baseline, blind to cells' potential spectrum of functional responses. Exploring the space of cellular responses experimentally requires access to a large combinatorial perturbation space. Single-cell genomics coupled with multiplexing techniques provide a useful tool for characterizing cell states across several experimental conditions. However, current multiplexing strategies require programmatic handling of many samples in macroscale arrayed formats, precluding their application in large-scale combinatorial analysis. Here, we introduce StimDrop, a method that combines antibody-based cell barcoding with parallel droplet processing to automatically formulate cell population × stimulus combinations in a microfluidic device. We applied StimDrop to profile the effects of 512 sequential stimulation conditions on human dendritic cells. Our results demonstrate that priming with viral ligands potentiates hyperinflammatory responses to a second stimulus, and show transcriptional signatures consistent with this phenomenon in myeloid cells of patients with severe COVID-19.

Ablation of Adar1 in myeloid cells imprints a global antiviral state in the lung and heightens early immunity against SARS-CoV-2.

Under normal homeostatic conditions, self-double-stranded RNA (self-dsRNA) is modified by adenosine deaminase acting on RNA 1 (ADAR1) to prevent the induction of a type I interferon-mediated inflammatory cascade. Antigen-presenting cells (APCs) sense pathogen-associated molecular patterns, such as dsRNA, to activate the immune response. The impact of ADAR1 on the function of APCs and the consequences to immunity are poorly understood. Here, we show that ADAR1 deletion in CD11c+ APCs leads to (1) a skewed myeloid cell compartment enriched in inflammatory cDC2-like cells, (2) enhanced numbers of activated tissue resident memory T cells in the lung, and (3) the imprinting of a broad antiviral transcriptional signature across both immune and non-immune cells. The resulting changes can be partially reversed by blocking IFNAR1 signaling and promote early resistance against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our study provides insight into the consequences of self-dsRNA sensing in APCs on the immune system.

Single-cell immune profiling reveals long-term changes in myeloid cells and identifies a novel subset of CD9+ monocytes associated with COVID-19 hospitalization.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in severe immune dysfunction, hospitalization, and death. Many patients also develop long-COVID-19, experiencing symptoms months after infection. Although significant progress has been made in understanding the immune response to acute SARS-CoV-2 infection, gaps remain in our knowledge of how innate immunity influences disease kinetics and severity. We hypothesized that cytometry by time-of-flight analysis of PBMCs from healthy and infected subjects would identify novel cell surface markers and innate immune cell subsets associated with COVID-19 severity. In this pursuit, we identified monocyte and dendritic cell subsets that changed in frequency during acute SARS-CoV-2 infection and correlated with clinical parameters of disease severity. Subsets of nonclassical monocytes decreased in frequency in hospitalized subjects, yet increased in the most severe patients and positively correlated with clinical values associated with worse disease severity. CD9, CD163, PDL1, and PDL2 expression significantly increased in hospitalized subjects, and CD9 and 6-Sulfo LacNac emerged as the markers that best distinguished monocyte subsets amongst all subjects. CD9+ monocytes remained elevated, whereas nonclassical monocytes remained decreased, in the blood of hospitalized subjects at 3-4 months postinfection. Finally, we found that CD9+ monocytes functionally released more IL-8 and MCP-1 after LPS stimulation. This study identifies new monocyte subsets present in the blood of COVID-19 patients that correlate with disease severity, and links CD9+ monocytes to COVID-19 progression.

Myeloid cell interferon responses correlate with clearance of SARS-CoV-2.

Emergence of mutant SARS-CoV-2 strains associated with an increased risk of COVID-19-related death necessitates better understanding of the early viral dynamics, host responses and immunopathology. Single cell RNAseq (scRNAseq) allows for the study of individual cells, uncovering heterogeneous and variable responses to environment, infection and inflammation. While studies have reported immune profiling using scRNAseq in terminal human COVID-19 patients, performing longitudinal immune cell dynamics in humans is challenging. Macaques are a suitable model of SARS-CoV-2 infection. Our longitudinal scRNAseq of bronchoalveolar lavage (BAL) cell suspensions from young rhesus macaques infected with SARS-CoV-2 (n = 6) demonstrates dynamic changes in transcriptional landscape 3 days post- SARS-CoV-2-infection (3dpi; peak viremia), relative to 14-17dpi (recovery phase) and pre-infection (baseline) showing accumulation of distinct populations of both macrophages and T-lymphocytes expressing strong interferon-driven inflammatory gene signature at 3dpi. Type I interferon response is induced in the plasmacytoid dendritic cells with appearance of a distinct HLADR+CD68+CD163+SIGLEC1+ macrophage population exhibiting higher angiotensin-converting enzyme 2 (ACE2) expression. These macrophages are significantly enriched in the lungs of macaques at 3dpi and harbor SARS-CoV-2 while expressing a strong interferon-driven innate anti-viral gene signature. The accumulation of these responses correlated with decline in viremia and recovery.

Communication Pattern Changes Along With Declined IGF1 of Immune Cells in COVID-19 Patients During Disease Progression.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the coronavirus disease 2019 (COVID-19) pandemic, represents a global crisis. Most patients developed mild/moderate symptoms, and the status of immune system varied in acute and regulatory stages. The crosstalk between immune cells and the dynamic changes of immune cell contact is rarely described. Here, we analyzed the features of immune response of paired peripheral blood mononuclear cell (PBMC) samples from the same patients during acute and regulatory stages. Consistent with previous reports, both myeloid and T cells turned less inflammatory and less activated at recovery phase. Additionally, the communication patterns of myeloid-T cell and T-B cell are obviously changed. The crosstalk analysis reveals that typical inflammatory cytokines and several chemokines are tightly correlated with the recovery of COVID-19. Intriguingly, the signal transduction of metabolic factor insulin-like growth factor 1 (IGF1) is altered at recovery phase. Furthermore, we confirmed that the serum levels of IGF1 and several inflammatory cytokines are apparently dampened after the negative conversion of SARS-CoV-2 RNA. Thus, these results reveal several potential detection and therapeutic targets that might be used for COVID-19 recovery.

People critically ill with COVID-19 exhibit peripheral immune profiles predictive of mortality and reflective of SARS-CoV-2 lung viral burden.

Despite extensive analyses, there remains an urgent need to delineate immune cell states that contribute to mortality in people critically ill with COVID-19. Here, we present high-dimensional profiling of blood and respiratory samples from people with severe COVID-19 to examine the association between cell-linked molecular features and mortality outcomes. Peripheral transcriptional profiles by single-cell RNA sequencing (RNA-seq)-based deconvolution of immune states are associated with COVID-19 mortality. Further, persistently high levels of an interferon signaling module in monocytes over time lead to subsequent concerted upregulation of inflammatory cytokines. SARS-CoV-2-infected myeloid cells in the lower respiratory tract upregulate CXCL10, leading to a higher risk of death. Our analysis suggests a pivotal role for viral-infected myeloid cells and protracted interferon signaling in severe COVID-19.

Postmortem high-dimensional immune profiling of severe COVID-19 patients reveals distinct patterns of immunosuppression and immunoactivation.

A complete diagnostic autopsy is the gold-standard to gain insight into Coronavirus disease 2019 (COVID-19) pathogenesis. To delineate the in situ immune responses to SARS-CoV-2 viral infection, here we perform comprehensive high-dimensional transcriptional and spatial immune profiling in 22 COVID-19 decedents from Wuhan, China. We find TIM-3-mediated and PD-1-mediated immunosuppression as a hallmark of severe COVID-19, particularly in men, with PD-1+ cells being proximal rather than distal to TIM-3+ cells. Concurrently, lymphocytes are distal, while activated myeloid cells are proximal, to SARS-CoV-2 viral antigens, consistent with prevalent SARS-CoV-2 infection of myeloid cells in multiple organs. Finally, viral load positively correlates with specific immunosuppression and dendritic cell markers. In summary, our data show that SARS-CoV-2 viral infection induces lymphocyte suppression yet myeloid activation in severe COVID-19, so these two cell types likely have distinct functions in severe COVID-19 disease progression, and should be targeted differently for therapy.

Ethyl pyruvate, a versatile protector in inflammation and autoimmunity.

Ethyl pyruvate (EP) has potent influence on redox processes, cellular metabolism, and inflammation. It has been intensively studied in numerous animal models of systemic and organ-specific disorders whose pathogenesis involves a strong immune component. Here, basic chemical and biological properties of EP are discussed, with an emphasis on its redox and metabolic activity. Further, its influence on myeloid and T cells is considered, as well as on intracellular signaling beyond its effect on immune cells. Also, the effects of EP on animal models of chronic inflammatory and autoimmune disorders are presented. Finally, a possibility to apply EP as a treatment for such diseases in humans is discussed. Scientific papers cited in this review were identified using the PubMed search engine that relies on the MEDLINE database. The reference list covers the most important findings in the field in the past twenty years.

A virus-specific monocyte inflammatory phenotype is induced by SARS-CoV-2 at the immune-epithelial interface.

Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled, by in vitro coculture, the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV-2-specific inflammatory gene cluster distinct from that seen in influenza A or Ebola virus-infected cocultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV-2 proteins (Spike and some nonstructural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV-2 induces in epithelia.

The potential of COVID-19 patients' sera to cause antibody-dependent enhancement of infection and IL-6 production.

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), many vaccine trials have been initiated. An important goal of vaccination is the development of neutralizing antibody (Ab) against SARS-CoV-2. However, the possible induction of antibody-dependent enhancement (ADE) of infection, which is known for other coronaviruses and dengue virus infections, is a particular concern in vaccine development. Here, we demonstrated that human iPS cell-derived, immortalized, and ACE2- and TMPRSS2-expressing myeloid cell lines are useful as host cells for SARS-CoV-2 infection. The established cell lines were cloned and screened based on their function in terms of susceptibility to SARS-CoV-2-infection or IL-6 productivity. Using the resulting K-ML2 (AT) clone 35 for SARS-CoV-2-infection or its subclone 35-40 for IL-6 productivity, it was possible to evaluate the potential of sera from severe COVID-19 patients to cause ADE and to stimulate IL-6 production upon infection with SARS-CoV-2.

Understanding COVID-19: From Dysregulated Immunity to Vaccination Status Quo.

The development of vaccines against infectious diseases has helped us battle the greatest threat to public health. With the emergence of novel viruses, targeted immunotherapeutics ranging from informed vaccine development to personalized medicine may be the very thing that separates us between life and death. Late in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), made a remarkable entrance to human civilization, being one of many to cross the species barrier. This review discusses the important aspects of COVID-19, providing a brief overview of our current understanding of dysregulated immune responses developed using various experimental models, a brief outline of experimental models of COVID-19 and more importantly, the rapid development of vaccines against COVID-19.

Plasma Gradient of Soluble Urokinase-Type Plasminogen Activator Receptor Is Linked to Pathogenic Plasma Proteome and Immune Transcriptome and Stratifies Outcomes in Severe COVID-19.

Disease caused by SARS-CoV-2 coronavirus (COVID-19) led to significant morbidity and mortality worldwide. A systemic hyper-inflammation characterizes severe COVID-19 disease, often associated with acute respiratory distress syndrome (ARDS). Blood biomarkers capable of risk stratification are of great importance in effective triage and critical care of severe COVID-19 patients. Flow cytometry and next-generation sequencing were done on peripheral blood cells and urokinase-type plasminogen activator receptor (suPAR), and cytokines were measured from and mass spectrometry-based proteomics was done on plasma samples from an Indian cohort of COVID-19 patients. Publicly available single-cell RNA sequencing data were analyzed for validation of primary data. Statistical analyses were performed to validate risk stratification. We report here higher plasma abundance of suPAR, expressed by an abnormally expanded myeloid cell population, in severe COVID-19 patients with ARDS. The plasma suPAR level was found to be linked to a characteristic plasma proteome, associated with coagulation disorders and complement activation. Receiver operator characteristic curve analysis to predict mortality identified a cutoff value of suPAR at 1,996.809 pg/ml (odds ratio: 2.9286, 95% confidence interval 1.0427-8.2257). Lower-than-cutoff suPAR levels were associated with a differential expression of the immune transcriptome as well as favorable clinical outcomes, in terms of both survival benefit (hazard ratio: 0.3615, 95% confidence interval 0.1433-0.912) and faster disease remission in our patient cohort. Thus, we identified suPAR as a key pathogenic circulating molecule linking systemic hyperinflammation to the hypercoagulable state and stratifying clinical outcomes in severe COVID-19 patients with ARDS.

Myeloid dysregulation and therapeutic intervention in COVID-19.

The dysregulation of myeloid cell responses is increasingly demonstrated to be a major mechanism of pathogenesis for COVID-19. The pathological cellular and cytokine signatures associated with this disease point to a critical role of a hyperactivated innate immune response in driving pathology. Unique immunopathological features of COVID-19 include myeloid-cell dominant inflammation and cytokine release syndrome (CRS) alongside lymphopenia and acute respiratory distress syndrome (ARDS), all of which correlate with severe disease. Studies suggest a range of causes mediating myeloid hyperactivation, such as aberrant innate sensing, asynchronized immune cellular responses, as well as direct viral protein/host interactions. These include the recent identification of new myeloid cell receptors that bind SARS-CoV-2, which drive myeloid cell hyperinflammatory responses independently of lung epithelial cell infection via the canonical receptor, angiotensin-converting enzyme 2 (ACE2). The spectrum and nature of myeloid cell dysregulation in COVID-19 also differs from, at least to some extent, what is observed in other infectious diseases involving myeloid cell activation. While much of the therapeutic effort has focused on preventative measures with vaccines or neutralizing antibodies that block viral infection, recent clinical trials have also targeted myeloid cells and the associated cytokines as a means to resolve CRS and severe disease, with promising but thus far modest effects. In this review, we critically examine potential mechanisms driving myeloid cell dysregulation, leading to immunopathology and severe disease, and discuss potential therapeutic strategies targeting myeloid cells as a new paradigm for COVID-19 treatment.

Immune response pattern across the asymptomatic, symptomatic and convalescent periods of COVID-19.

We present an integrated analysis of urine and serum proteomics and clinical measurements in asymptomatic, mild/moderate, severe and convalescent cases of COVID-19. We identify the pattern of immune response during COVID-19 infection. The immune response is activated in asymptomatic infection, but is dysregulated in mild and severe COVID-19 patients. Our data suggest that the turning point depends on the function of myeloid cells and neutrophils. In addition, immune defects persist into the recovery stage, until 12 months after diagnosis. Moreover, disorders of cholesterol metabolism span the entire progression of the disease, starting from asymptomatic infection and lasting to recovery. Our data suggest that prolonged dysregulation of the immune response and cholesterol metabolism might be the pivotal causative agent of other potential sequelae. Our study provides a comprehensive understanding of COVID-19 immunopathogenesis, which is instructive for the development of early intervention strategies to ameliorate complex disease sequelae.

Myeloid cells in COVID-19 microenvironment.

Varying differentiation of myeloid cells is common in tumors, inflammation, autoimmune diseases, and metabolic diseases. The release of cytokines from myeloid cells is an important driving factor that leads to severe COVID-19 cases and subsequent death. This review briefly summarizes the results of single-cell sequencing of peripheral blood, lung tissue, and cerebrospinal fluid of COVID-19 patients and describes the differentiation trajectory of myeloid cells in patients. Moreover, we describe the function and mechanism of abnormal differentiation of myeloid cells to promote disease progression. Targeting myeloid cell-derived cytokines or checkpoints is essential in developing a combined therapeutic strategy for patients with severe COVID-19.

COVID19 Disease Map, a computational knowledge repository of virus-host interaction mechanisms.

We need to effectively combine the knowledge from surging literature with complex datasets to propose mechanistic models of SARS-CoV-2 infection, improving data interpretation and predicting key targets of intervention. Here, we describe a large-scale community effort to build an open access, interoperable and computable repository of COVID-19 molecular mechanisms. The COVID-19 Disease Map (C19DMap) is a graphical, interactive representation of disease-relevant molecular mechanisms linking many knowledge sources. Notably, it is a computational resource for graph-based analyses and disease modelling. To this end, we established a framework of tools, platforms and guidelines necessary for a multifaceted community of biocurators, domain experts, bioinformaticians and computational biologists. The diagrams of the C19DMap, curated from the literature, are integrated with relevant interaction and text mining databases. We demonstrate the application of network analysis and modelling approaches by concrete examples to highlight new testable hypotheses. This framework helps to find signatures of SARS-CoV-2 predisposition, treatment response or prioritisation of drug candidates. Such an approach may help deal with new waves of COVID-19 or similar pandemics in the long-term perspective.