Single-cell transcriptomic analysis of hematopoietic progenitor cells from patients with systemic lupus erythematosus reveals interferon-inducible reprogramming in early progenitors.

Introduction: Immune cells that contribute to the pathogenesis of systemic lupus erythematosus (SLE) derive from adult hematopoietic stem and progenitor cells (HSPCs) within the bone marrow (BM). For this reason, we reasoned that fundamental abnormalities in SLE can be traced to a BM-derived HSPC inflammatory signature. Methods: BM samples from four SLE patients, six healthy controls, and two umbilical cord blood (CB) samples were used. CD34+ cells were isolated from BM and CB samples, and single-cell RNA-sequencing was performed. Results: A total of 426 cells and 24,473 genes were used in the analysis. Clustering analysis resulted in seven distinct clusters of cell types. Mutually exclusive markers, which were characteristic of each cell type, were identified. We identified three HSPC subpopulations, one of which consisted of proliferating cells (MKI67 expressing cells), one T-like, one B-like, and two myeloid-like progenitor subpopulations. Differential expression analysis revealed i) cell cycle-associated signatures, in healthy BM of HSPC clusters 3 and 4 when compared with CB, and ii) interferon (IFN) signatures in SLE BM of HSPC clusters 3 and 4 and myeloid-like progenitor cluster 5 when compared with healthy controls. The IFN signature in SLE appeared to be deregulated following TF regulatory network analysis and differential alternative splicing analysis between SLE and healthy controls in HSPC subpopulations. Discussion: This study revealed both quantitative-as evidenced by decreased numbers of non-proliferating early progenitors-and qualitative differences-characterized by an IFN signature in SLE, which is known to drive loss of function and depletion of HSPCs. Chronic IFN exposure affects early hematopoietic progenitors in SLE, which may account for the immune aberrancies and the cytopenias in SLE.

Targeted genotyping of COVID-19 patients reveals a signature of complement C3 and factor B coding SNPs associated with severe infection.

We have attempted to explore further the involvement of complement components in the host COVID-19 (Coronavirus disease-19) immune responses by targeted genotyping of COVID-19 adult patients and analysis for missense coding Single Nucleotide Polymorphisms (coding SNPs) of genes encoding Alternative pathway (AP) components. We have identified a small group of common coding SNPs in Survivors and Deceased individuals, present in either relatively similar frequencies (CFH and CFI SNPs) or with stark differences in their relative abundance (C3 and CFB SNPs). In addition, we have identified several sporadic, potentially protective, coding SNPs of C3, CFB, CFD, CFH, CFHR1 and CFI in Survivors. No coding SNPs were detected for CD46 and CD55. Our demographic analysis indicated that the C3 rs1047286 or rs2230199 coding SNPs were present in 60 % of all the Deceased patients (n = 25) (the rs2230199 in 67 % of all Deceased Males) and in 31 % of all the Survivors (n = 105, p = 0.012) (the rs2230199 in 25 % of all Survivor Males). When we analysed these two major study groups using the presence of the C3 rs1047286 or rs2230199 SNPs as potential biomarkers, we noticed the complete absence of the protective CFB rs12614 and rs641153 coding SNPs from Deceased Males compared to Females (p = 0.0023). We propose that in these individuals, C3 carrying the R102G and CFB lacking the R32W or the R32Q amino acid substitutions, may contribute to enhanced association dynamics of the C3bBb AP pre-convertase complex assembly, thus enabling the exploitation of the activation of the Complement Alternative pathway (AP) by SARS-CoV-2.

Genetic prediction of ICU hospitalization and mortality in COVID-19 patients using artificial neural networks.

There is an unmet need of models for early prediction of morbidity and mortality of Coronavirus disease-19 (COVID-19). We aimed to a) identify complement-related genetic variants associated with the clinical outcomes of ICU hospitalization and death, b) develop an artificial neural network (ANN) predicting these outcomes and c) validate whether complement-related variants are associated with an impaired complement phenotype. We prospectively recruited consecutive adult patients of Caucasian origin, hospitalized due to COVID-19. Through targeted next-generation sequencing, we identified variants in complement factor H/CFH, CFB, CFH-related, CFD, CD55, C3, C5, CFI, CD46, thrombomodulin/THBD, and A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS13). Among 381 variants in 133 patients, we identified 5 critical variants associated with severe COVID-19: rs2547438 (C3), rs2250656 (C3), rs1042580 (THBD), rs800292 (CFH) and rs414628 (CFHR1). Using age, gender and presence or absence of each variant, we developed an ANN predicting morbidity and mortality in 89.47% of the examined population. Furthermore, THBD and C3a levels were significantly increased in severe COVID-19 patients and those harbouring relevant variants. Thus, we reveal for the first time an ANN accurately predicting ICU hospitalization and death in COVID-19 patients, based on genetic variants in complement genes, age and gender. Importantly, we confirm that genetic dysregulation is associated with impaired complement phenotype.

Genetic justification of severe COVID-19 using a rigorous algorithm.

Recent studies suggest excessive complement activation in severe coronavirus disease-19 (COVID-19). The latter shares common characteristics with complement-mediated thrombotic microangiopathy (TMA). We hypothesized that genetic susceptibility would be evident in patients with severe COVID-19 (similar to TMA) and associated with disease severity. We analyzed genetic and clinical data from 97 patients hospitalized for COVID-19. Through targeted next-generation-sequencing we found an ADAMTS13 variant in 49 patients, along with two risk factor variants (C3, 21 patients; CFH,34 patients). 31 (32%) patients had a combination of these, which was independently associated with ICU hospitalization (p = 0.022). Analysis of almost infinite variant combinations showed that patients with rs1042580 in thrombomodulin and without rs800292 in complement factor H did not require ICU hospitalization. We also observed gender differences in ADAMTS13 and complement-related variants. In light of encouraging results by complement inhibitors, our study highlights a patient population that might benefit from early initiation of specific treatment.

The haemostatic profile in critically ill COVID-19 patients receiving therapeutic anticoagulant therapy: An observational study.

Hypercoagulability and thrombosis remain a challenge in severe coronavirus disease 2019 (COVID-19) infections. Our aim is to investigate the hemostatic profile of critically ill COVID-19 patients on therapeutic anticoagulant treatment.Forty one patients were enrolled into the study. We recruited 11 consecutive, COVID-19, patients who received therapeutic anticoagulant treatment on intensive care unit (ICU) admission. Disease severity indexes, biochemical, hematological and haemostatic parameters, endogenous thrombin potential (ETP), plasminogen activator inhibitor-1 (PAI-1) activity and extrinsically activated rotational thromboelastometry assay (EXTEM) were recorded on days 1, 3, 7. We also enrolled 9 ICU non-COVID-19, 21 non-ICU COVID-19 patients and 20 healthy blood donors as control populations.Critically ill COVID-19 patients demonstrated a more hypercoagulable and hypofibrinolytic profile related to those with COVID-19 mild illness, based on EXTEM amplitude at 10 min (A10), maximum clot firmness (MCF) and lysis index at 60 min (LI60) variables (p = 0.020, 0.046 and 0.001, respectively). Similarly, a more hypercoagulable state was detected in COVID-19 ICU patients related to non-COVID-19 ICU patients based on A10 and MCF parameters (p = 0.03 and 0.04, respectively). On the contrary, ETP and EXTEM (clotting time) CT values were similar between patients with severe and mild form of the COVID-19 infection, probably due to anticoagulant treatment given.Critically ill COVID-19 patients showed a hypercoagulable profile despite the therapeutic anticoagulant doses given. Due to the small sample size and the study design, the prognostic role of the hypercoagulability in this clinical setting remains unknown and further research is required in order to be assessed.

Transcriptome reprogramming and myeloid skewing in haematopoietic stem and progenitor cells in systemic lupus erythematosus.

OBJECTIVES: Haematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to both myeloid and lymphoid cell lineages. We reasoned that the aberrancies of immune cells in systemic lupus erythematosus (SLE) could be traced back to HSPCs. METHODS: A global gene expression map of bone marrow (BM)-derived HSPCs was completed by RNA sequencing followed by pathway and enrichment analysis. The cell cycle status and apoptosis status of HSPCs were assessed by flow cytometry, while DNA damage was assessed via immunofluorescence. RESULTS: Transcriptomic analysis of Lin-Sca-1+c-Kit+ haematopoietic progenitors from diseased lupus mice demonstrated a strong myeloid signature with expanded frequencies of common myeloid progenitors (CMPs)-but not of common lymphoid progenitors-reminiscent of a 'trained immunity' signature. CMP profiling revealed an intense transcriptome reprogramming with suppression of granulocytic regulators indicative of a differentiation arrest with downregulation trend of major regulators such as Cebpe, Cebpd and Csf3r, and disturbed myelopoiesis. Despite the differentiation arrest, frequencies of BM neutrophils were markedly increased in diseased mice, suggesting an alternative granulopoiesis pathway. In patients with SLE with severe disease, haematopoietic progenitor cells (CD34+) demonstrated enhanced proliferation, cell differentiation and transcriptional activation of cytokines and chemokines that drive differentiation towards myelopoiesis, thus mirroring the murine data. CONCLUSIONS: Aberrancies of immune cells in SLE can be traced back to the BM HSPCs. Priming of HSPCs and aberrant regulation of myelopoiesis may contribute to inflammation and risk of flare. TRIAL REGISTRATION NUMBER: 4948/19-07-2016.

Autoimmune hemolytic anemia and autoimmune thrombocytopenia at diagnosis and during follow-up of Hodgkin lymphoma.

Autoimmune hemolytic anemia and thrombocytopenia (AIHA/AITP) frequently complicate the course of non-Hodgkin lymphomas, especially low-grade, but they are very rarely observed in Hodgkin lymphoma (HL). Consequently the frequency and the profile of patients with HL-associated AIHA/AITP have not been well defined. Among 1029 patients with HL diagnosed between 1990 and 2010, two cases of AIHA (0.19%) and three of AITP (0.29%) were identified at the presentation of disease. These patients were significantly older, and more frequently had features of advanced disease and non-nodular sclerosing histology, compared to the majority of patients, who did not have autoimmune cytopenias at diagnosis. ABVD combination chemotherapy (doxorubicin, bleomycin, vinblastine, dacarbazine) provided effective control of HL and the autoimmune condition as well. During approximately 6600 person-years of follow-up for the remaining 1024 patients, seven (0.7%) patients developed autoimmune cytopenias (three AITP, three AIHA, one autoimmune pancytopenia) for a 10- and 15-year actuarial incidence of 0.95% and 1.40%, respectively. Their features did not differ compared to the general population of adult HL. In this large series of consecutive, unselected patients, those who presented with autoimmune cytopenias had a particular demographic and disease-related profile. In contrast, patients developing autoimmune cytopenias during follow-up did not appear to differ significantly from those who did not.

Clonal expansion of B-cells in human systemic lupus erythematosus: evidence from studies before and after therapeutic B-cell depletion.

Anti-B-cell therapy may be beneficial for patients with SLE and active proliferative glomerulonephritis. Using genomic DNA, we examined how rituximab-induced transient B-cell depletion affected the composition of immunoglobulin heavy-chain (Ig-VH/DH/JH) repertoire at the time-point of 33-35% B-cell reconstitution in these patients. Clusters of clonaly-related Ig-VH/DH/JH sequences were evident in all 7 patients with active SLE but in none of 4 healthy subjects studied for comparison. In addition to original somatic mutation(s) shared in the majority of clonaly-related sequences, ongoing mutation suggested expansion of single B-cell precursors and clonal evolution. Otherwise, lupus repertoire was similar to that of healthy subjects with the exception of increased somatic hypermutation. The regenerating repertoire was diverse and comparable to baseline, albeit with fewer somatic mutations but more clonal expansions. Since clonal expansion may lead to preferential survival/growth of potentially autoreactive B-cells further studies are warranted, especially as therapies aiming to reduce B-cell survival emerge.