Prostaglandin E2 regulates Th17 cell differentiation and function through cyclic AMP and EP2/EP4 receptor signaling

Prostaglandins, particularly prostaglandin E2 (PGE2), play an important role during inflammation. This is exemplified by the clinical use of cyclooxygenase 2 inhibitors, which interfere with PGE2 synthesis, as effective antiinflammatory drugs. Here, we show that PGE2 directly promotes differentiation and proinflammatory functions of human and murine IL-17–producing T helper (Th17) cells. In human purified naive T cells, PGE2 acts via prostaglandin receptor EP2- and EP4-mediated signaling and cyclic AMP pathways to up-regulate IL-23 and IL-1 receptor expression. Furthermore, PGE2 synergizes with IL-1β and IL-23 to drive retinoic acid receptor–related orphan receptor (ROR)-γt, IL-17, IL-17F, CCL20, and CCR6 expression, which is consistent with the reported Th17 phenotype. While enhancing Th17 cytokine expression mainly through EP2, PGE2 differentially regulates interferon (IFN)-γ production and inhibits production of the antiinflammatory cytokine IL-10 in Th17 cells predominantly through EP4. Furthermore, PGE2 is required for IL-17 production in the presence of antigen-presenting cells. Hence, the combination of inflammatory cytokines and noncytokine immunomodulators, such as PGE2, during differentiation and activation determines the ultimate phenotype of Th17 cells. These findings, together with the altered IL-12/IL-23 balance induced by PGE2 in dendritic cells, further highlight the crucial role of the inflammatory microenvironment in Th17 cell development and regulation

Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue

Abstract Background Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Methods Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. Results Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. Conclusions We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers

Safety of procuring research tissue during a clinically indicated kidney biopsy from patients with lupus: data from the Accelerating Medicines Partnership RA/SLE Network

Objectives In lupus nephritis the pathological diagnosis from tissue retrieved during kidney biopsy drives treatment and management. Despite recent approval of new drugs, complete remission rates remain well under aspirational levels, necessitating identification of new therapeutic targets by greater dissection of the pathways to tissue inflammation and injury. This study assessed the safety of kidney biopsies in patients with SLE enrolled in the Accelerating Medicines Partnership, a consortium formed to molecularly deconstruct nephritis.Methods 475 patients with SLE across 15 clinical sites in the USA consented to obtain tissue for research purposes during a clinically indicated kidney biopsy. Adverse events (AEs) were documented for 30 days following the procedure and were determined to be related or unrelated by all site investigators. Serious AEs were defined according to the National Institutes of Health reporting guidelines.Results 34 patients (7.2%) experienced a procedure-related AE: 30 with haematoma, 2 with jets, 1 with pain and 1 with an arteriovenous fistula. Eighteen (3.8%) experienced a serious AE requiring hospitalisation; four patients (0.8%) required a blood transfusion related to the kidney biopsy. At one site where the number of cores retrieved during the biopsy was recorded, the mean was 3.4 for those who experienced a related AE (n=9) and 3.07 for those who did not experience any AE (n=140). All related AEs resolved.Conclusions Procurement of research tissue should be considered feasible, accompanied by a complication risk likely no greater than that incurred for standard clinical purposes. In the quest for targeted treatments personalised based on molecular findings, enhanced diagnostics beyond histology will likely be required

EAE clinical course is not affected by CD73 deficiency.

<p>A: Mean clinical scores following EAE induction in WT and CD73^-/- mice. B: Percentage of mice that had developed EAE clinical signs on indicated days after EAE induction. C: Day of EAE onset in WT and CD73^-/- mice that developed signs of EAE by day 16 post-immunization (mean +/- S.D.). Data pooled from four independent experiments.</p

CD73 does not influence Th17 differentiation <i>in vitro</i>.

<p>A, B: CD4⁺ T cells from WT or CD73^-/- mice were cultured under Th0 (top panels) or Th17 (lower panels) differentiating conditions for three days, then expression of IL-17 (A) and RORγt (B) were analyzed. C. Expression of FoxP3 in WT and CD73^-/- Th0 (top panel) and Tregs (lower panel) at day 3 of differentiation. Numbers indicate mean % +/- S.D. of gated cells (n = 2), representative of three independent experiments with similar results.</p

Th17 and Treg cells are similar in WT and CD73^-/- during EAE.

<p>Cytokine production and Treg frequency were analyzed by FACS in draining LN and CNS of WT and CD73^-/- mice at day 12 (onset), day 16 (peak) and day 21 (chronic/resolution) phases of EAE. A: Frequencies of IL-17, IFNγ and GM-CSF expressing T cells, analyzed in draining lymph nodes on indicated days after EAE induction. B: Numbers of IL-17, IFNγ and GM-CSF expressing T cells infiltrating the CNS at indicated time points after immunization. A-B show mean +/- SEM of pooled data, each point represents an individual mouse. C: Cells from draining lymph nodes at days 12 (n = 4-5/group) and 16 (n = 2-3/group) post-immunization were re-challenged <i>in vitro</i> with MOG(35–55) for three days in the presence/absence of IL-23 (20ng/mL), and IL-17 expression was measured by ELISA. D: Percentage of Tregs in the draining lymph nodes and CNS at indicated timepoints of EAE, FACS plots show representative staining of Foxp3 and CD73 in CNS on day 16 post-immunization, shown as mean +/- S.D. from 4–7 mice/group except day 16 WT has n = 2 mice. Data are representative of two-three independent experiments with similar results.</p