Metabolomics meets systems immunology.

Metabolic processes play a critical role in immune regulation. Metabolomics is the systematic analysis of small molecules (metabolites) in organisms or biological samples, providing an opportunity to comprehensively study interactions between metabolism and immunity in physiology and disease. Integrating metabolomics into systems immunology allows the exploration of the interactions of multilayered features in the biological system and the molecular regulatory mechanism of these features. Here, we provide an overview on recent technological developments of metabolomic applications in immunological research. To begin, two widely used metabolomics approaches are compared: targeted and untargeted metabolomics. Then, we provide a comprehensive overview of the analysis workflow and the computational tools available, including sample preparation, raw spectra data preprocessing, data processing, statistical analysis, and interpretation. Third, we describe how to integrate metabolomics with other omics approaches in immunological studies using available tools. Finally, we discuss new developments in metabolomics and its prospects for immunology research. This review provides guidance to researchers using metabolomics and multiomics in immunity research, thus facilitating the application of systems immunology to disease research.

Weather variability and transmissibility of COVID-19: a time series analysis based on effective reproductive number.

COVID-19 is causing a significant burden on medical and healthcare resources globally due to high numbers of hospitalisations and deaths recorded as the pandemic continues. This research aims to assess the effects of climate factors (i.e., daily average temperature and average relative humidity) on effective reproductive number of COVID-19 outbreak in Wuhan, China during the early stage of the outbreak. Our research showed that effective reproductive number of COVID-19 will increase by 7.6% (95% Confidence Interval: 5.4% ~ 9.8%) per 1°C drop in mean temperature at prior moving average of 0-8 days lag in Wuhan, China. Our results indicate temperature was negatively associated with COVID-19 transmissibility during early stages of the outbreak in Wuhan, suggesting temperature is likely to effect COVID-19 transmission. These results suggest increased precautions should be taken in the colder seasons to reduce COVID-19 transmission in the future, based on past success in controlling the pandemic in Wuhan, China.

Single-cell transcriptomics identifies different immune signatures between macrophage activation-like syndrome and immune paralysis in sepsis (preprint)

Different immune phenotypes characterize sepsis patients, including hyperinflammation and/or immunosuppression, but the biological mechanisms driving this heterogeneity remain largely unknown. We used single-cell RNA sequencing to profile circulating leukocytes of healthy controls and sepsis patients classified as either hyperinflammatory (macrophage activation-like syndrome [MALS]), immune paralysis, or unclassified (when criteria for neither of these two immune subgroups were applicable). Pronounced differences were detected in the transcriptional signature of monocytes from sepsis patients, with clear distinction between MALS and immune paralysis patients. Unsupervised clustering analysis revealed the existence of MALS-specific monocyte clusters, as well as one sepsis-specific monocyte cluster that was linked to disease severity. In separate cohorts, urosepsis was characterized by heterogeneous MALS and immunosuppression monocyte signatures, while MALS-specific monocyte clusters showed overlapping transcriptional signatures with severe COVID-19. In conclusion, our findings shed light on the heterogeneous immune landscape underlying sepsis, and provide opportunities for patient stratification for future therapeutic development.

Altered and allele-specific open chromatin landscape reveals epigenetic and genetic regulators of innate immunity in COVID-19.

SARS-CoV-2 infection causes severe COVID-19 in some patients and mild in others. Dysfunctional innate immune responses have been identified to contribute to COVID-19 severity, but the key regulators are still unknown. Here, we present an integrative single-cell multi-omics analysis of peripheral blood mononuclear cells from hospitalized and convalescent COVID-19 patients. In classical monocytes, we identified genes that were potentially regulated by differential chromatin accessibility. Then, sub-clustering and motif-enrichment analyses reveals disease condition-specific regulation by transcription factors and their targets, including an interaction between C/EBPs and a long-noncoding RNA LUCAT1, which we validated through loss-of-function experiments. Finally, we investigated genetic risk variants that exhibit allele-specific open chromatin (AsoC) in COVID-19 patients and identified a SNP rs6800484-C, which is associated with lower expression of CCR2 and may contribute to higher viral loads and higher risk of COVID-19 hospitalization. Altogether, our study highlights the diverse genetic and epigenetic regulators that contribute to COVID-19.

Targeted proteomics identifies circulating biomarkers associated with active COVID-19 and post-COVID-19.

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic is caused by the highly infectious Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). There is an urgent need for biomarkers that will help in better stratification of patients and contribute to personalized treatments. We performed targeted proteomics using the Olink platform and systematically investigated protein concentrations in 350 hospitalized COVID-19 patients, 186 post-COVID-19 individuals, and 61 healthy individuals from 3 independent cohorts. Results revealed a signature of acute SARS-CoV-2 infection, which is represented by inflammatory biomarkers, chemokines and complement-related factors. Furthermore, the circulating proteome is still significantly affected in post-COVID-19 samples several weeks after infection. Post-COVID-19 individuals are characterized by upregulation of mediators of the tumor necrosis (TNF)-α signaling pathways and proteins related to transforming growth factor (TGF)-ß. In addition, the circulating proteome is able to differentiate between patients with different COVID-19 disease severities, and is associated with the time after infection. These results provide important insights into changes induced by SARS-CoV-2 infection at the proteomic level by integrating several cohorts to obtain a large disease spectrum, including variation in disease severity and time after infection. These findings could guide the development of host-directed therapy in COVID-19.

The Genetic Risk for COVID-19 Severity Is Associated With Defective Immune Responses.

Recent genome-wide association studies (GWASs) of COVID-19 patients of European ancestry have identified genetic loci significantly associated with disease severity. Here, we employed the detailed clinical, immunological and multi-omics dataset of the Human Functional Genomics Project (HFGP) to explore the physiological significance of the host genetic variants that influence susceptibility to severe COVID-19. A genomics investigation intersected with functional characterization of individuals with high genetic risk for severe COVID-19 susceptibility identified several major patterns: i. a large impact of genetically determined innate immune responses in COVID-19, with ii. increased susceptibility for severe disease in individuals with defective cytokine production; iii. genetic susceptibility related to ABO blood groups is probably mediated through the von Willebrand factor (VWF) and endothelial dysfunction. We further validated these identified associations at transcript and protein levels by using independent disease cohorts. These insights allow a physiological understanding of genetic susceptibility to severe COVID-19, and indicate pathways that could be targeted for prevention and therapy.

Protective effect of pneumococcal conjugate vaccination on the short-term association between low temperatures and childhood pneumonia hospitalizations: Interrupted time-series and case-crossover analyses in Matlab, Bangladesh.

Studies have shown that ambient extreme temperatures (heat and cold) were associated with an increased risk of childhood pneumonia, but the evidence is very limited in low-middle-income countries. It also remains unknown whether pneumococcal conjugate vaccine (PCV) could prevent temperature-related childhood pneumonia. This study collected data on ambient temperature and hospitalizations for childhood pneumonia in Matlab, Bangladesh from 2012 to 2016. Interrupted time series (ITS) analysis was employed to assess the impact of PCV (10-valent) intervention on childhood pneumonia hospitalizations. A time-stratified case-crossover analysis with a conditional logistic regression was performed to examine the association of childhood pneumonia hospitalizations with extreme temperatures and heatwaves before and after PCV10 intervention. Subgroup analyses were conducted to explore the modification effects of seasons, age, gender, and socioeconomic levels on temperature-related childhood pneumonia hospitalizations. We found that after PCV10 intervention, there was a sharp decrease in hospitalizations for childhood pneumonia (relative risk (RR): 0.59, 95% confidence interval (CI): 0.43-0.83). During the study period, heat effects on childhood pneumonia appeared immediately on the current day (odds ratio (OR): 1.28; 95% CI: 1.02-1.60, lag 0), while cold effects appeared 4 weeks later (OR: 1.53, 95% CI: 1.06-2.22, lag 28). Importantly, cold effects decreased significantly after PCV10 (p-value＜0.05), but heat and heatwave effects increased after PCV10 (p-value＜0.05). Particularly, children from families with a middle or low socioeconomic level, boys, and infants were more susceptible to heat-related pneumonia. This study suggests that PCV10 intervention in Bangladesh may help decrease cold-related not heat-related childhood pneumonia.

Multi-Omics Integration Reveals Only Minor Long-Term Molecular and Functional Sequelae in Immune Cells of Individuals Recovered From COVID-19.

The majority of COVID-19 patients experience mild to moderate disease course and recover within a few weeks. An increasing number of studies characterized the long-term changes in the specific anti-SARS-CoV-2 immune responses, but how COVID-19 shapes the innate and heterologous adaptive immune system after recovery is less well known. To comprehensively investigate the post-SARS-CoV-2 infection sequelae on the immune system, we performed a multi-omics study by integrating single-cell RNA-sequencing, single-cell ATAC-sequencing, genome-wide DNA methylation profiling, and functional validation experiments in 14 convalescent COVID-19 and 15 healthy individuals. We showed that immune responses generally recover without major sequelae after COVID-19. However, subtle differences persist at the transcriptomic level in monocytes, with downregulation of the interferon pathway, while DNA methylation also displays minor changes in convalescent COVID-19 individuals. However, these differences did not affect the cytokine production capacity of PBMCs upon different bacterial, viral, and fungal stimuli, although baseline release of IL-1Ra and IFN-γ was higher in convalescent individuals. In conclusion, we propose that despite minor differences in epigenetic and transcriptional programs, the immune system of convalescent COVID-19 patients largely recovers to the homeostatic level of healthy individuals.

Review and Thinking on Forensic Pathological Changes of Coronavirus-Infected Diseases.

Since the beginning of this century, three types of coronavirus have widely transmitted and caused severe diseases and deaths, which strongly indicates that severe infectious diseases caused by coronavirus infection are not accidental events. Coronavirus-infected diseases are mainly manifested by respiratory symptoms, with multiple organ dysfunctions. Precisely investigating the pathological process, characteristics and pathogenesis of coronavirus-infected diseases will be beneficial for us to understand clinical manifestations and provide targeted suggestions on prophylaxis and treatment. This paper briefly reviews the pathological findings of three known coronavirus-infected diseases, and attempts to construct the pathological spectrum of coronavirus-infected diseases, aiming to provide reference and thinking for autopsy, histopathological examination and animal infection model study of coronavirus-infected diseases.

Single-cell RNA sequencing reveals induction of distinct trained-immunity programs in human monocytes.

Trained immunity refers to the long-lasting memory traits of innate immunity. Recent studies have shown that trained immunity is orchestrated by sustained changes in epigenetic marks and metabolic pathways, leading to an altered transcriptional response to a second challenge. However, the potential heterogeneity of trained-immunity induction in innate immune cells has not been explored. In this study, we demonstrate cellular transcriptional programs in response to 4 different inducers of trained immunity in monocyte populations at single-cell resolution. Specifically, we identified 3 monocyte subpopulations upon the induction of trained immunity, and replicated these findings in an in vivo study. In addition, we found gene signatures consistent with these functional programs in patients with ulcerative colitis, sepsis, and COVID-19, suggesting the impact of trained-immunity programs in immune-mediated diseases.

Induction of trained immunity by influenza vaccination - impact on COVID-19.

Non-specific protective effects of certain vaccines have been reported, and long-term boosting of innate immunity, termed trained immunity, has been proposed as one of the mechanisms mediating these effects. Several epidemiological studies suggested cross-protection between influenza vaccination and COVID-19. In a large academic Dutch hospital, we found that SARS-CoV-2 infection was less common among employees who had received a previous influenza vaccination: relative risk reductions of 37% and 49% were observed following influenza vaccination during the first and second COVID-19 waves, respectively. The quadrivalent inactivated influenza vaccine induced a trained immunity program that boosted innate immune responses against various viral stimuli and fine-tuned the anti-SARS-CoV-2 response, which may result in better protection against COVID-19. Influenza vaccination led to transcriptional reprogramming of monocytes and reduced systemic inflammation. These epidemiological and immunological data argue for potential benefits of influenza vaccination against COVID-19, and future randomized trials are warranted to test this possibility.

Induction of trained immunity by influenza vaccination - impact on COVID-19 (preprint)

Non-specific protective effects of certain vaccines have been reported, and long-term boosting of innate immunity, termed trained immunity, has been proposed as one of the mechanisms mediating these effects. Several epidemiological studies suggested cross-protection between influenza vaccination and COVID-19. In a large academic Dutch hospital, we found that SARS-CoV-2 infection was less common among employees who had received a previous influenza vaccination: relative risk reductions of 37% and 49% were observed following influenza vaccination during the first and second COVID-19 waves, respectively. The quadrivalent inactivated influenza vaccine induced a trained immunity program that boosted innate immune responses against various viral stimuli and fine-tuned the anti-SARS-CoV-2 response, which may result in better protection against COVID-19. Influenza vaccination led to transcriptional reprogramming of monocytes and reduced systemic inflammation. These epidemiological and immunological data argue for potential benefits of influenza vaccination against COVID-19, and future randomized trials are warranted to test this possibility.

Comparison of clinical characteristics between patients with coronavirus disease 2019 (COVID-19) who retested RT-PCR positive versus negative: a retrospective study of data from Nanjing.

BACKGROUND: The epidemiological and clinical characteristics of patients with coronavirus disease 2019 (COVID-19) have been reported. However, the prevalence of retesting positive by RT-PCR for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated patient characteristics, remain unclear. METHODS: We included 90 confirmed cases of COVID-19 treated in the Nanjing Public Health Center from January 20, 2020 to February 16, 2020 in this retrospective study. All patients completed treatment for COVID-19 and were retested by RT-PCR for SARS-CoV-2 4-20 days after completion of therapy. The clinical characteristics between patients with who retested positive versus negative by RT-PCR were compared, and the factors predictive of positive retesting were analyzed. Positive retesting was modeled with the area under the receiver operating characteristic curve (AUC). RESULTS: The age range of the study population was 0.8-97 years, and all patients were cured or showed improvement. A total of 10 (11%) patients retested positive by RT-PCR 4-20 days after completion of therapy. As compared with patients who retested negative, those who retested positive had a lower percentage of pre-admission fever, a higher percentage of post-admission fever, a lower percentage of bilateral lung infection, higher white blood cell (WBC) count and creatine phosphokinase, and lower hypersensitive c-reactive protein (hs-CRP), interleukin-6 and erythrocyte sedimentation rates (all P<0.05). Logistic regression analysis of the above eight key variables showed that lower hs-CRP and higher WBC were independently associated with positive retesting by RT-PCR. A combination of hs-CRP and WBC were predictive of positive retesting, with an AUC of 0.859. CONCLUSIONS: Patients with COVID-19 who retested positive by RT-PCR for SARS-CoV-2 had mild symptoms and better blood testing results. A combination of hs-CRP and WBC may predict positive retesting by RT-PCR; however, the sensitivity and specificity should be studied further.

A functional genomics approach to understand host genetic regulation ofCOVID-19 severity (preprint)

A recent genome-wide association study of European ancestry has identified 3p21.31 and 9q34.2 (ABO gene) to be significantly associated with COVID-19 respiratory failure (1). Here, we employed the detailed clinical, immunological and multi-omics data of the Human Functional Genomics Projects (HFGP) to explore the physiological significance of the host genetic variants that influence susceptibility to severe COVID-19. A functional genomics investigation based on functional characterization of individuals with high genetic risk for severe COVID-19 susceptibility identified several major patterns: i. a large impact of genetically determined innate immune responses in COVID-19, with increased susceptibility for severe disease in individuals with defective monocyte-derived cytokine production; ii. genetic susceptibility related to ABO blood groups is probably mediated through the von Willebrand factor (VWF) and endothelial dysfunction; and iii. the increased susceptibility for severe COVID-19 in men is at least partially mediated by chromosome X-mediated genetic variation. These insights allow a physiological understanding of genetic susceptibility to severe COVID-19, and indicate pathways that could be targeted for prevention and therapy.

Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.

Suppressive myeloid cells are a hallmark of severe COVID-19 (preprint)

Severe Acute Respiratory Syndrome - Coronavirus-2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (COVID-19), a mild to moderate respiratory tract infection in the majority of patients. A subset of patients, however, progresses to severe disease and respiratory failure with acute respiratory distress syndrome (ARDS). Severe COVID-19 has been associated with increased neutrophil counts and dysregulated immune responses. The mechanisms of protective immunity in mild forms and the pathogenesis of dysregulated inflammation in severe courses of COVID-19 remain largely unclear. Here, we combined two single-cell RNA-sequencing technologies and single-cell proteomics in whole blood and peripheral blood mononuclear cells (PBMC) to determine changes in immune cell composition and activation in two independent dual-center patient cohorts (n=46+n=54 COVID-19 samples), each with mild and severe cases of COVID-19. We observed a specific increase of HLA-DRhiCD11chi inflammatory monocytes that displayed a strong interferon (IFN)-stimulated gene signature in patients with mild COVID-19, which was absent in severe disease. Instead, we found evidence of emergency myelopoiesis, marked by the occurrence of immunosuppressive pre-neutrophils and immature neutrophils and populations of dysfunctional and suppressive mature neutrophils, as well as suppressive HLA-DRto monocytes in severe COVID-19. Our study provides detailed insights into systemic immune response to SARS-CoV-2 infection and it reveals profound alterations in the peripheral myeloid cell compartment associated with severe courses of COVID-19.

Live poultry market closure and avian influenza A (H7N9) infection in cities of China, 2013-2017: an ecological study.

BACKGROUND: Previous studies have proven that the closure of live poultry markets (LPMs) was an effective intervention to reduce human risk of avian influenza A (H7N9) infection, but evidence is limited on the impact of scale and duration of LPMs closure on the transmission of H7N9. METHOD: Five cities (i.e., Shanghai, Suzhou, Shenzhen, Guangzhou and Hangzhou) with the largest number of H7N9 cases in mainland China from 2013 to 2017 were selected in this study. Data on laboratory-confirmed H7N9 human cases in those five cities were obtained from the Chinese National Influenza Centre. The detailed information of LPMs closure (i.e., area and duration) was obtained from the Ministry of Agriculture. We used a generalized linear model with a Poisson link to estimate the effect of LPMs closure, reported as relative risk reduction (RRR). We used classification and regression trees (CARTs) model to select and quantify the dominant factor of H7N9 infection. RESULTS: All five cities implemented the LPMs closure, and the risk of H7N9 infection decreased significantly after LPMs closure with RRR ranging from 0.80 to 0.93. Respectively, a long-term LPMs closure for 10-13 weeks elicited a sustained and highly significant risk reduction of H7N9 infection (RRR = 0.98). Short-time LPMs closure with 2 weeks in every epidemic did not reduce the risk of H7N9 infection (p > 0.05). Partially closed LPMs in some suburbs contributed only 35% for reduction rate (RRR = 0.35). Shenzhen implemented partial closure for first 3 epidemics (p > 0.05) and all closure in the latest 2 epidemic waves (RRR = 0.64). CONCLUSION: Our findings suggest that LPMs all closure in whole city can be a highly effective measure comparing with partial closure (i.e. only urban closure, suburb and rural remain open). Extend the duration of closure and consider permanently closing the LPMs will help improve the control effect. The effect of LPMs closure seems greater than that of meteorology on H7N9 transmission.