Single cell transcriptomics of cerebrospinal fluid cells from patients with recent-onset narcolepsy.

Narcolepsy is a rare cause of hypersomnolence and may be associated or not with cataplexy, i.e. sudden muscle weakness. These forms are designated narcolepsy-type 1 (NT1) and -type 2 (NT2), respectively. Notable characteristics of narcolepsy are that most patients carry the HLA-DQB1*06:02 allele and NT1-patients have strongly decreased levels of hypocretin-1 (synonym orexin-A) in the cerebrospinal fluid (CSF). The pathogenesis of narcolepsy is still not completely understood but the strong HLA-bias and increased frequencies of CD4+ T cells reactive to hypocretin in the peripheral blood suggest autoimmune processes in the hypothalamus. Here we analyzed the transcriptomes of CSF-cells from twelve NT1 and two NT2 patients by single cell RNAseq (scRNAseq). As controls, we used CSF cells from patients with multiple sclerosis, radiologically isolated syndrome, and idiopathic intracranial hypertension. From 27,255 CSF cells, we identified 20 clusters of different cell types and found significant differences in three CD4+ T cell and one monocyte clusters between narcolepsy and multiple sclerosis patients. Over 1000 genes were differentially regulated between patients with NT1 and other diseases. Surprisingly, the most strongly upregulated genes in narcolepsy patients as compared to controls were coding for the genome-encoded MTRNR2L12 and MTRNR2L8 peptides, which are homologous to the mitochondria-encoded HUMANIN peptide that is known playing a role in other neurological diseases including Alzheimer's disease.

Cerebrospinal fluid proteomics in recent-onset Narcolepsy type 1 reveals activation of the complement system.

Introduction: Narcolepsy type 1 (NT1) is a rare, chronic and disabling neurological disease causing excessive daytime sleepiness and cataplexy. NT1 is characterized pathologically by an almost complete loss of neurons producing the orexin neuropeptides in the lateral hypothalamus. Genetic and environmental factors strongly suggest the involvement of the immune system in the loss of orexin neurons. The cerebrospinal fluid (CSF), secreted locally and surrounding the central nervous system (CNS), represents an accessible window into CNS pathological processes. Methods: To gain insight into the biological and molecular changes in NT1 patients, we performed a comparative proteomics analysis of the CSF from 21 recent-onset NT1 patients and from two control groups: group 1 with somatoform disorders, and group 2 patients with hypersomnia other than NT1, to control for any potential effect of sleep disturbances on CSF composition. To achieve an optimal proteomic coverage analysis, the twelve most abundant CSF proteins were depleted, and samples were analyzed by nano-flow liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) using the latest generation of hybrid Orbitrap mass spectrometer. Results and discussion: Our study allowed the identification and quantification of up to 1943 proteins, providing a remarkably deep analysis of the CSF proteome. Interestingly, gene set enrichment analysis indicated that the complement and coagulation systems were enriched and significantly activated in NT1 patients in both cohorts analyzed. Notably, the lectin and alternative complement pathway as well as the downstream lytic membrane attack complex were congruently increased in NT1. Our data suggest that the complement dysregulation in NT1 patients can contribute to immunopathology either by directly promoting tissue damage or as part of local inflammatory responses. We therefore reveal an altered composition of the CSF proteome in NT1 patients, which points to an ongoing inflammatory process contributed, at least in part, by the complement system.

Preexisting comorbidities shape the immune response associated with severe COVID-19.

BACKGROUND: Comorbidities are risk factors for development of severe coronavirus disease 2019 (COVID-19). However, the extent to which an underlying comorbidity influences the immune response to severe acute respiratory syndrome coronavirus 2 remains unknown. OBJECTIVE: Our aim was to investigate the complex interrelations of comorbidities, the immune response, and patient outcome in COVID-19. METHODS: We used high-throughput, high-dimensional, single-cell mapping of peripheral blood leukocytes and algorithm-guided analysis. RESULTS: We discovered characteristic immune signatures associated not only with severe COVID-19 but also with the underlying medical condition. Different factors of the metabolic syndrome (obesity, hypertension, and diabetes) affected distinct immune populations, thereby additively increasing the immunodysregulatory effect when present in a single patient. Patients with disorders affecting the lung or heart, together with factors of metabolic syndrome, were clustered together, whereas immune disorder and chronic kidney disease displayed a distinct immune profile in COVID-19. In particular, severe acute respiratory syndrome coronavirus 2-infected patients with preexisting chronic kidney disease were characterized by the highest number of altered immune signatures of both lymphoid and myeloid immune branches. This overall major immune dysregulation could be the underlying mechanism for the estimated odds ratio of 16.3 for development of severe COVID-19 in this burdened cohort. CONCLUSION: The combinatorial systematic analysis of the immune signatures, comorbidities, and outcomes of patients with COVID-19 has provided the mechanistic immunologic underpinnings of comorbidity-driven patient risk and uncovered comorbidity-driven immune signatures.

Distinct immunological signatures discriminate severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia.

Immune profiling of COVID-19 patients has identified numerous alterations in both innate and adaptive immunity. However, whether those changes are specific to SARS-CoV-2 or driven by a general inflammatory response shared across severely ill pneumonia patients remains unknown. Here, we compared the immune profile of severe COVID-19 with non-SARS-CoV-2 pneumonia ICU patients using longitudinal, high-dimensional single-cell spectral cytometry and algorithm-guided analysis. COVID-19 and non-SARS-CoV-2 pneumonia both showed increased emergency myelopoiesis and displayed features of adaptive immune paralysis. However, pathological immune signatures suggestive of T cell exhaustion were exclusive to COVID-19. The integration of single-cell profiling with a predicted binding capacity of SARS-CoV-2 peptides to the patients' HLA profile further linked the COVID-19 immunopathology to impaired virus recognition. Toward clinical translation, circulating NKT cell frequency was identified as a predictive biomarker for patient outcome. Our comparative immune map serves to delineate treatment strategies to interfere with the immunopathologic cascade exclusive to severe COVID-19.