Transcriptome profiling of PBMCs and formalin-fixed autopsy tissues from COVID-19 patients.

Here we present an optimized protocol for transcriptome profiling of COVID-19 patient samples, including peripheral blood mononuclear cells (PBMCs) and formalin-fixed paraffin-embedded tissue samples obtained from the lung, liver, heart, kidney, and spleen, with the matched controls. We describe RNA extraction and subsequent transcriptome analysis using NanoString technology of the patient samples. The protocol provides information about sample preparation, RNA extraction, and NanoString profiling and analysis. It can be also applied to differentiated Th17 and Treg subsets or formalin-fixed colon tissue samples. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).

Blockade of PGK1 and ALDOA enhances bilirubin control of Th17 cells in Crohn's disease.

Unconjugated bilirubin (UCB) confers Th17-cells immunosuppressive features by activating aryl-hydrocarbon-receptor, a modulator of toxin and adaptive immune responses. In Crohn's disease, Th17-cells fail to acquire regulatory properties in response to UCB, remaining at an inflammatory/pathogenic state. Here we show that UCB modulates Th17-cell metabolism by limiting glycolysis and through downregulation of glycolysis-related genes, namely phosphoglycerate-kinase-1 (PGK1) and aldolase-A (ALDOA). Th17-cells of Crohn's disease patients display heightened PGK1 and ALDOA and defective response to UCB. Silencing of PGK1 or ALDOA restores Th17-cell response to UCB, as reflected by increase in immunoregulatory markers like FOXP3, IL-10 and CD39. In vivo, PGK1 and ALDOA silencing enhances UCB salutary effects in trinitro-benzene-sulfonic-acid-induced colitis in NOD/scid/gamma humanized mice where control over disease activity and enhanced immunoregulatory phenotypes are achieved. PGK1 and/or ALDOA blockade might have therapeutic effects in Crohn's disease by favoring acquisition of regulatory properties by Th17-cells along with control over their pathogenic potential.

Dysfunctional Immune Regulation in Autoimmune Hepatitis: From Pathogenesis to Novel Therapies.

Autoimmune hepatitis (AIH) is a chronic inflammatory disorder characterized by hypergammaglobulinemia, presence of serum autoantibodies and histological features of interface hepatitis. AIH therapeutic management still relies on the administration of corticosteroids, azathioprine and other immunosuppressants like calcineurin inhibitors and mycophenolate mofetil. Withdrawal of immunosuppression often results in disease relapse, and, in some cases, therapy is ineffective or associated with serious side effects. Understanding the mechanisms underlying AIH pathogenesis is therefore of paramount importance to develop more effective and well tolerated agents capable of restoring immunotolerance to liver autoantigens. Imbalance between effector and regulatory cells permits liver damage perpetuation and progression in AIH. Impaired expression and regulation of CD39, an ectoenzyme key to immunotolerance maintenance, have been reported in Tregs and effector Th17-cells derived from AIH patients. Interference with these altered immunoregulatory pathways may open new therapeutic avenues that, in addition to limiting aberrant inflammatory responses, would also reconstitute immune homeostasis. In this review, we highlight the most recent findings in AIH immunopathogenesis and discuss how these could inform and direct the development of novel therapeutic tools.

Limited TCR repertoire and ENTPD1 dysregulation mark late-stage COVID-19.

T cell exhaustion and dysfunction are hallmarks of severe COVID-19. To gain insights into the pathways underlying these alterations, we performed a comprehensive transcriptome analysis of peripheral-blood-mononuclear-cells (PBMCs), spleen, lung, kidney, liver, and heart obtained at autopsy from COVID-19 patients and matched controls, using the nCounter CAR-T-Characterization panel. We found substantial gene alterations in COVID-19-impacted organs, especially the lung where altered TCR repertoires are noted. Reduced TCR repertoires are also observed in PBMCs of severe COVID-19 patients. ENTPD1/CD39, an ectoenzyme defining exhausted T-cells, is upregulated in the lung, liver, spleen, and PBMCs of severe COVID-19 patients where expression positively correlates with markers of vasculopathy. Heightened ENTPD1/CD39 is paralleled by elevations in STAT-3 and HIF-1α transcription factors; and by markedly reduced CD39-antisense-RNA, a long-noncoding-RNA negatively regulating ENTPD1/CD39 at the post-transcriptional level. Limited TCR repertoire and aberrant regulation of ENTPD1/CD39 could have permissive roles in COVID-19 progression and indicate potential therapeutic targets to reverse disease.