No abstract

A key aspect of nearly all single cell experiments is the necessity to dissociate intact tissues into single cell suspensions for processing. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression changes during this process. Microglia, the brain’s resident macrophages, are a highly dynamic population that are extremely sensitive to their microenvironment and have been shown to dramatically alter their transcriptome upon stimulation. We demonstrate that use of enzymatic dissociation methods on mouse central nervous system (CNS) tissue induces an aberrant gene expression signature in microglia that can significantly confound downstream analysis. To minimize this issue, we developed a flexible protocol, that can be used with existing enzymatic protocols for fresh tissue, to eliminate artifactual gene expression while allowing for increased cell type diversity and yield. We demonstrate efficacy of this protocol in analysis of diverse CNS cell types and sorted myeloid populations while using enzymatic dissociation. Generation of new and reanalysis of previously published human brain single nucleus RNAseq (snRNA-seq) datasets reveal that a similar signature is also present in post-mortem tissue. Through novel snRNA-seq analysis of acutely-resected neurosurgical tissue we demonstrate that this signature can be induced in human tissue due to technical differences in sample processing. These results provide key insight into the potential confounds of enzymatic digestion and provide a solution to allow for enzymatic digestion for scRNA-seq while avoiding ex vivo transcriptional artifacts. Analysis of human tissue reveals potential for artifacts in current and future snRNA-seq datasets that will require deeper analysis and careful consideration to separate true biology from artifacts related to post-mortem processes.

The first objective of this study was to separate the contributions of the Cognitive Style Questionnaire (CSQ) and the Dysfunctional Attitude Scale (DAS) to maladaptive cognitive patterns in their relations to symptoms of depression and their potential developmental origins: emotional maltreatment, parents' typical feedback styles, and parents' dysfunctional attitudes regarding their offspring. Other objectives were to examine these relations in the context of symptoms of anxiety and to learn whether selected aspects of the Cognitive Vulnerability to Depression Project (CVD Project; Alloy, & Abramson, 1999) would generalize to a distinctive sample (n = 98). The same relations between the DAS and the CSQ and depression and developmental origins emerged as in the CVD Project, but relations with the DAS were somewhat more robust.We use the generic term ''depression'' to apply to both diagnoses and symptoms of depression. We specify diagnoses and/or symptoms as required by the context.

Microglia have emerged as key players in the pathogenesis of neurodegenerative conditions such as Alzheimer’s disease (AD). In response to CNS stimuli, these cells adopt distinct transcriptional and functional subtypes known as states. However, an understanding of the function of these states has been elusive, especially in human microglia, due to lack of tools to model and manipulate this cell-type. Here, we provide a platform for modeling human microglia transcriptional states in vitro. Using single-cell RNA sequencing, we found that exposure of human stem-cell differentiated microglia (iMGLs) to brain-related challenges generated extensive transcriptional diversity which mapped to gene signatures identified in human brain microglia. We identified two in vitro transcriptional clusters that were analogous to human and mouse disease-associated microglia (DAMs), a state enriched in neurodegenerative disease contexts. To facilitate scalable functional analyses, we established a lentiviral approach enabling broad and highly efficient genetic transduction of microglia in vitro. Using this new technology, we demonstrated that MITF (Melanocyte Inducing Transcription Factor), an AD-enriched transcription factor in microglia, drives both a disease-associated transcriptional signature and a highly phagocytic state. Finally, we confirmed these results across iMGLs differentiated from multiple iPSC lines demonstrating the broad utility of this platform. Together, these tools provide a comprehensive resource that enables the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

Microglia, the macrophages of the brain parenchyma, are key players in neurodegenerative diseases such as Alzheimer’s disease. These cells adopt distinct transcriptional subtypes known as states. Understanding state function, especially in human microglia, has been elusive owing to a lack of tools to model and manipulate these cells. Here, we developed a platform for modeling human microglia transcriptional states in vitro. We found that exposure of human stem-cell-differentiated microglia to synaptosomes, myelin debris, apoptotic neurons or synthetic amyloid-beta fibrils generated transcriptional diversity that mapped to gene signatures identified in human brain microglia, including disease-associated microglia, a state enriched in neurodegenerative diseases. Using a new lentiviral approach, we demonstrated that the transcription factor MITF drives a disease-associated transcriptional signature and a highly phagocytic state. Together, these tools enable the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

Background: The Omicron variant of SARS-CoV-2 is now overtaking the Delta variant in many countries. Results showing that sera from double vaccinated individuals have minimal neutralizing activity against Omicron may indicate that the higher rate of transmission is due to evasion from vaccine-induced immunity. However, there is little information about activation of recall responses to Omicron in vaccinated individuals. Methods: We measured inflammatory mediators, antibodies to the SARS-CoV-2 spike and nucleocapsid proteins, and spike peptide-induced release of interferon gamma in whole blood in 51 vaccinated individuals infected with Omicron, in 14 infected with Delta, and in 18 healthy controls. The median time points for the first and second samples were 7 and 14 days after symptom onset, respectively. Findings: Infection with Omicron or Delta led to a rapid and similar increase in antibodies to the SARS-CoV-2 spike and nucleocapsid proteins and spike peptide-induced interferon gamma in whole blood. Both the Omicron and the Delta infected patients had a mild and transient increase in inflammatory parameters. Interpretation: The results suggest that vaccine-induced immunological memory yields similar coverage for the Omicron and Delta variants.

Background. T-cell activation is associated with an adverse outcome in COVID-19, but whether T-cell activation and exhaustion relate to persistent respiratory dysfunction and death is unknown.Objectives. To investigate whether T-cell activation and exhaustion persist and are associated with prolonged respiratory dysfunction and death after hospitalization for COVID-19.Methods. Plasma and serum from two Norwegian cohorts of hospitalized patients with COVID-19 (n = 414) were analyzed for soluble (s) markers of T-cell activation (sCD25) and exhaustion (sTim-3) during hospitalization and follow-up.Results. Both markers were strongly associated with acute respiratory failure, but only sTim-3 was independently associated with 60-day mortality. Levels of sTim-3 remained elevated 3 and 12 months after hospitalization and were associated with pulmonary radiological pathology after 3 months. Conclusion. Our findings suggest prolonged T-cell exhaustion is an importantTuva B. Dahl and Jan C. Holter contributed equally to this study.

Background Immune dysregulation is a major factor in the development of severe Covid-19. The homeostatic chemokines CCL19 and CCL21 have been implicated as mediators of tissue inflammation, but data on their regulation in SARS-CoV-2 infection is limited. We thus investigated the levels of these chemokines in Covid-19 patients. Methods Serial blood samples were obtained from patients hospitalized with Covid-19 (n = 414). Circulating CCL19 and CCL21 levels during hospitalization and three-month follow-up were analyzed. In vitro assays and analysis of RNAseq data from public repositories were performed to further explore possible regulatory mechanisms. Results A consistent increase in circulating levels of CCL19 and CCL21 was observed, with high levels correlating with disease severity measures, including respiratory failure, need for intensive care, and 60-day all-cause mortality. High levels of CCL21 at admission were associated with persisting impairment of pulmonary function at the three-month follow-up. Conclusions Our findings highlight CCL19 and CCL21 as markers of immune dysregulation in Covid-19. This may reflect aberrant regulation triggered by tissue inflammation, as observed in other chronic inflammatory and autoimmune conditions. Determination of the source and regulation of these chemokines and their effects on lung tissue is warranted to further clarify their role in Covid-19.