Recombinant Acid Ceramidase Reduces Inflammation and Infection in Cystic Fibrosis.

Rationale: In cystic fibrosis the major cause of morbidity and mortality is lung disease characterized by inflammation and infection. The influence of sphingolipid metabolism is poorly understood with a lack of studies using human airway model systems.Objectives: To investigate sphingolipid metabolism in cystic fibrosis and the effects of treatment with recombinant human acid ceramidase on inflammation and infection.Methods: Sphingolipids were measured using mass spectrometry in fully differentiated cultures of primary human airway epithelial cells and cocultures with Pseudomonas aeruginosa. In situ activity assays, Western blotting, and quantitative PCR were used to investigate function and expression of ceramidase and sphingomyelinase. Effects of treatment with recombinant human acid ceramidase on sphingolipid profile and inflammatory mediator production were assessed in cell cultures and murine models.Measurements and Main Results: Ceramide is increased in cystic fibrosis airway epithelium owing to differential function of enzymes regulating sphingolipid metabolism. Sphingosine, a metabolite of ceramide with antimicrobial properties, is not upregulated in response to P. aeruginosa by cystic fibrosis airway epithelia. Tumor necrosis factor receptor 1 is increased in cystic fibrosis epithelia and activates NF-κB signaling, generating inflammation. Treatment with recombinant human acid ceramidase, to decrease ceramide, reduced both inflammatory mediator production and susceptibility to infection.Conclusions: Sphingolipid metabolism is altered in airway epithelial cells cultured from people with cystic fibrosis. Treatment with recombinant acid ceramidase ameliorates the two pivotal features of cystic fibrosis lung disease, inflammation and infection, and thus represents a therapeutic approach worthy of further exploration.

Real-time measurement of cellular bioenergetics in fully differentiated human nasal epithelial cells grown at air-liquid-interface.

Shifts in cellular metabolic phenotypes have the potential to cause disease-driving processes in respiratory disease. The respiratory epithelium is particularly susceptible to metabolic shifts in disease, but our understanding of these processes is limited by the incompatibility of the technology required to measure metabolism in real-time with the cell culture platforms used to generate differentiated respiratory epithelial cell types. Thus, to date, our understanding of respiratory epithelial metabolism has been restricted to that of basal epithelial cells in submerged culture, or via indirect end point metabolomics readouts in lung tissue. Here we present a novel methodology using the widely available Seahorse Analyzer platform to monitor real-time changes in the cellular metabolism of fully differentiated primary human airway epithelial cells grown at air-liquid interface (ALI). We show increased glycolytic, but not mitochondrial, ATP production rates in response to physiologically relevant increases in glucose availability. We also show that pharmacological inhibition of lactate dehydrogenase is able to reduce glucose-induced shifts toward aerobic glycolysis. This method is timely given the recent advances in our understanding of new respiratory epithelial subtypes that can only be observed in vitro through culture at ALI and will open new avenues to measure real-time metabolic changes in healthy and diseased respiratory epithelium, and in turn the potential for the development of novel therapeutics targeting metabolic-driven disease phenotypes.

Human Regulatory T Cells Mediate Transcriptional Modulation of Dendritic Cell Function.

Regulatory T cells (Treg) attenuate dendritic cell (DC) maturation and stimulatory function. Current knowledge on the functional impact of semimature DC is limited to CD4+ T cell proliferation and cytokine production. Little is known about the molecular basis underpinning the functional effects of Treg-treated DC (Treg-DC). We present novel evidence that Treg-DC skewed CD4+ naive T cell polarization toward a regulatory phenotype and impaired CD8+ T cell allo-reactive responses, including their ability to induce target tissue damage in a unique in vitro human graft-versus-host disease skin explant model. Microarray analysis clustered Treg-DC as a discrete population from mature-DC and immature-DC, with 51 and 93 genes that were significantly over- or underexpressed, respectively, compared with mature-DC. Quantitative real-time PCR analysis revealed an intermediate expression level of CD38, CD83, CD80 and CD86 mRNA in Treg-DC, lower than mature-DC, higher than immature-DC. We also observed an attenuation of NF-κB pathway, an upstream regulator of the aforementioned genes, concomitant with reduced expression of two NF-κB-signaling related genes RELB and NFκBIZ, in the Treg-DC, together with an increased expression of Wnt5a, a negative regulator of DC differentiation. We further confirmed that the Treg-DC-mediated skewed CD4+ naive T cell polarization resulted from decreased IL-12 secretion by Treg-DC, which may be post-transcriptionally modulated by decreased expression of microRNA-155 in Treg-DC. To our knowledge, this is the first study demonstrating a transcriptional modulation of DC function by human Treg, partially via attenuation of the NF-κB signaling pathway and upregulation of Wnt5a, suggesting Treg may interfere with DC reprogramming during maturation, thereby modulating DC function.

Chemokine receptor CXCR3 agonist prevents human T-cell migration in a humanized model of arthritic inflammation.

The recruitment of T lymphocytes during diseases such as rheumatoid arthritis is regulated by stimulation of the chemokine receptors expressed by these cells. This study was designed to assess the potential of a CXCR3-specific small-molecule agonist to inhibit the migration of activated human T cells toward multiple chemokines. Further experiments defined the molecular mechanism for this anti-inflammatory activity. Analysis in vitro demonstrated agonist induced internalization of both CXCR3 and other chemokine receptors coexpressed by CXCR3(+) T cells. Unlike chemokine receptor-specific antagonists, the CXCR3 agonist inhibited migration of activated T cells toward the chemokine mixture in synovial fluid from patients with active rheumatoid arthritis. A humanized mouse air-pouch model showed that intravenous treatment with the CXCR3 agonist prevented inflammatory migration of activated human T cells toward this synovial fluid. A potential mechanism for this action was defined by demonstration that the CXCR3 agonist induces receptor cross-phosphorylation within CXCR3-CCR5 heterodimers on the surface of activated T cells. This study shows that generalized chemokine receptor desensitization can be induced by specific stimulation of a single chemokine receptor on the surface of activated human T cells. A humanized mouse model was used to demonstrate that this receptor desensitization inhibits the inflammatory response that is normally produced by the chemokines present in synovial fluid from patients with active rheumatoid arthritis.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Dendritic Cell Maturation and Function.

Mesenchymal stromal cells (MSCs) are potent regulators of immune responses largely through paracrine signaling. MSC secreted extracellular vesicles (MSC-EVs) are increasingly recognized as the key paracrine factors responsible for the biological and therapeutic function of MSCs. We report the first comprehensive study demonstrating the immunomodulatory effect of MSC-EVs on dendritic cell (DC) maturation and function. MSC-EVs were isolated from MSC conditioned media using differential ultracentrifugation. Human monocyte-derived DCs were generated in the absence or presence of MSC-EVs (20 ug/ml) then subjected to phenotypic and functional analysis in vitro. MSC-EV treatment impaired antigen uptake by immature DCs and halted DC maturation resulting in reduced expression of the maturation and activation markers CD83, CD38, and CD80, decreased secretion of pro-inflammatory cytokines IL-6 and IL-12p70 and increased production of anti-inflammatory cytokine TGF-ß. MSC-EV treated DCs also demonstrated a diminished CCR 7 expression after LPS stimulation, coupled with a significantly reduced ability to migrate toward the CCR7-ligand CCL21, although they were still able to stimulate allogeneic T cell proliferation in vitro. Through microRNA profiling we have identified 49 microRNAs, which were significantly enriched in MSC-EVs compared to their parent MSCs. MicroRNAs with known effect on DC maturation and functions, including miR-21-5p, miR-142-3p, miR-223-3p, and miR-126-3p, were detected within the top 10 most enriched miRNAs in MSC-EVs, with MiR-21-5p as the third highest expressed miRNA in MSC-EVs. In silico analysis revealed that miR-21-5p targets the CCR7 gene for degradation. To verify these observations, DCs were transfected with miR-21-5p mimics and analyzed for their ability to migrate toward the CCR7-ligand CCL21 in vitro. MiR-21-5p mimic transfected DCs showed a clear trend of reduced CCR7 expression and a significantly decreased migratory ability toward the CCL21. Our findings suggest that MSC-EVs are able to recapitulate MSC mediated DC modulation and MSC-EV enclosed microRNAs may represent a novel mechanism through which MSCs modulate DC functions. As MSCs are currently used in clinical trials to treat numerous diseases associated with immune dysregulation, such as graft-versus-host disease and inflammatory bowel disease, our data provide novel evidence to inform potential future application of MSC-EVs as a cell-free therapeutic agent.

Pathophysiology of GvHD and Other HSCT-Related Major Complications.

For over 60 years, hematopoietic stem cell transplantation has been the major curative therapy for several hematological and genetic disorders, but its efficacy is limited by the secondary disease called graft versus host disease (GvHD). Huge advances have been made in successful transplantation in order to improve patient quality of life, and yet, complete success is hard to achieve. This review assimilates recent updates on pathophysiology of GvHD, prophylaxis and treatment of GvHD-related complications, and advances in the potential treatment of GvHD.

Regulatory T cells inhibit CD8(+) T-cell tissue invasion in human skin graft-versus-host reactions.

BACKGROUND: Regulatory T cells (Tregs) effectively ameliorate graft-versus-host disease (GVHD). The mechanisms underlying Treg therapeutic effect on GVHD are not fully elucidated. This study investigates whether Treg prevention of GVH tissue damage is associated with blocking CD8 effector T-cell tissue invasion, a question not yet addressed in humans. METHOD: Tissue-infiltrating T cells and histopathology scores were detected using an in vitro human GVHD skin explant model, together with immunohistochemistry, cytometric bead array, functional adhesion and migration assays, flow cytometry, and quantitative real-time polymerase chain reaction. RESULTS: Treg intervention during priming significantly decreased effector T-cell infiltration into target tissue (P<0.01) resulting in a striking reduction in the histopathology score of tissue injury (P<0.0001). These results were coupled with reduced CXCR3 and cutaneous lymphocyte antigen expression by effector T cells, together with decreased CXCL10 and CXCL11 expression in target tissue. Treg intervention also impaired the functional interaction of CXCR3 and cutaneous lymphocyte antigen with their specific ligands (P<0.01) and suppressed the secretion of CXCL9, CXCL10, and interferon-γ (P<0.01, P<0.05, and P<0.001, respectively). Late addition of Tregs into the effector phase abolished their ability to suppress effector T-cell tissue invasion, resulting in a total loss of their ability to ameliorate GVH tissue damage. CONCLUSION: Preventing effector T-cell tissue invasion is a critical mechanistic event leading to Treg attenuation of GVH tissue damage. This therapeutic effect is associated with a failure of CD8 T cells to increase tissue homing receptors after allo-stimulation, together with a breakdown of interferon-γ-induced chemoattractant expression in the target tissue.