Anna Peired

Signals from the Sympathetic Nervous System Regulate Hematopoietic Stem Cell Egress from Bone Marrow

Hematopoietic stem and progenitor cells (HSPC), attracted by the chemokine CXCL12, reside in specific niches in the bone marrow (BM). HSPC migration out of the BM is a critical process that underlies modern clinical stem cell transplantation. Here we demonstrate that enforced HSPC egress from BM niches depends critically on the nervous system. UDP-galactose ceramide galactosyltransferase-deficient (Cgt(-/-)) mice exhibit aberrant nerve conduction and display virtually no HSPC egress from BM following granulocyte colony-stimulating factor (G-CSF) or fucoidan administration. Adrenergic tone, osteoblast function, and bone CXCL12 are dysregulated in Cgt(-/-) mice. Pharmacological or genetic ablation of adrenergic neurotransmission indicates that norepinephrine (NE) signaling controls G-CSF-induced osteoblast suppression, bone CXCL12 downregulation, and HSPC mobilization. Further, administration of a beta(2) adrenergic agonist enhances mobilization in both control and NE-deficient mice. Thus, these results indicate that the sympathetic nervous system regulates the attraction of stem cells to their niche.

Complete Identification of E-Selectin Ligands on Neutrophils Reveals Distinct Functions of PSGL-1, ESL-1, and CD44

The selectins and their ligands are required for leukocyte extravasation during inflammation. Several glycoproteins have been suggested to bind to E-selectin in vitro, but the complete identification of its physiological ligands has remained elusive. Here, we showed that E-selectin ligand-1 (ESL-1), P-selectin glycoprotein ligand-1 (PSGL-1), and CD44 encompassed all endothelial-selectin ligand activity on neutrophils by using gene- and RNA-targeted loss of function. PSGL-1 played a major role in the initial leukocyte capture, whereas ESL-1 was critical for converting initial tethers into steady slow rolling. CD44 controlled rolling velocity and mediated E-selectin-dependent redistribution of PSGL-1 and L-selectin to a major pole on slowly rolling leukocytes through p38 signaling. These results suggest distinct and dynamic contributions of these three glycoproteins in selectin-mediated neutrophil adhesion and signaling.

Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury

Selectins and their ligands mediate leukocyte rolling, allowing interactions with chemokines that lead to integrin activation and arrest. Here we show that E-selectin is crucial for generating a secondary wave of activating signals, transduced specifically by E-selectin ligand-1, that induces polarized, activated αMβ2 integrin clusters at the leading edge of crawling neutrophils, allowing capture of circulating erythrocytes or platelets. In a humanized mouse model of sickle cell disease, the capture of erythrocytes by αMβ2 microdomains leads to acute lethal vascular occlusions. In a model of transfusion-related acute lung injury, polarized neutrophils capture circulating platelets, resulting in the generation of oxidative species that produce vascular damage and lung injury. Inactivation of E-selectin or αMβ2 prevents tissue injury in both inflammatory models, suggesting broad implications of this paradigm in thromboinflammatory diseases. These results indicate that endothelial selectins can influence neutrophil behavior beyond its canonical rolling step through delayed, organ-damaging, polarized activation.

CD161 is a marker of all human IL-17-producing T-cell subsets and is induced by RORC

We have previously shown that human Th17 lymphocytes are characterized by the selective expression of IL‐23 receptor (IL‐23R), CCR6, CD161, and the transcription factor retinoic acid‐related orphan receptor C (RORC), and originate from a CD161+CD4+ naïve T‐cell precursor in response to the combined activity of IL‐1β and IL‐23. We show here that not only CD4+TCRαβ+, but also CD8+TCRαβ+, CD4−CD8− TCRαβ+, and CD4−CD8− TCRγδ+ circulating lymphocytes that produce IL‐17 express the distinctive marker CD161 on their surface. In addition, we demonstrate that CD161 expression identifies CD8+ and CD4−CD8− umbilical cord blood T cells that already express RORC and IL‐23R mRNA and that can be induced to differentiate into IL‐17‐producing cells in the presence of IL‐1β and IL‐23. Finally, we provide evidence that umbilical cord blood naïve CD4+CD161− T cells, upon lentivirus‐mediated transduction with RORC2 can acquire the ability to express IL‐23R, IL‐1RI, and CD161, as well as to produce IL‐17. Taken together, these data allow to conclude that T‐cell subsets able to produce IL‐17, as well as precursors of IL‐17‐producing T cells, exhibit surface expression of CD161, and that this feature is at least in part RORC2‐dependent.

Characterization of Renal Progenitors Committed Toward Tubular Lineage and Their Regenerative Potential in Renal Tubular Injury

Recent studies implicated the existence in adult human kidney of a population of renal progenitors with the potential to regenerate glomerular as well as tubular epithelial cells and characterized by coexpression of surface markers CD133 and CD24. Here, we demonstrate that CD133+CD24+ renal progenitors can be distinguished in distinct subpopulations from normal human kidneys based on the surface expression of vascular cell adhesion molecule 1, also known as CD106. CD133+CD24+CD106+ cells were localized at the urinary pole of Bowmans capsule, while a distinct population of scattered CD133+CD24+CD106− cells was localized in the proximal tubule as well as in the distal convoluted tubule. CD133+CD24+CD106+ cells exhibited a high proliferative rate and could differentiate toward the podocyte as well as the tubular lineage. By contrast, CD133+CD24+CD106− cells showed a lower proliferative capacity and displayed a committed phenotype toward the tubular lineage. Both CD133+CD24+CD106+ and CD133+CD24+CD106− cells showed higher resistance to injurious agents in comparison to all other differentiated cells of the kidney. Once injected in SCID mice affected by acute tubular injury, both of these populations displayed the capacity to engraft within the kidney, generate novel tubular cells, and improve renal function. These properties were not shared by other tubular cells of the adult kidney. Finally, CD133+CD24+CD106− cells proliferated upon tubular injury, becoming the predominating part of the regenerating epithelium in patients with acute or chronic tubular damage. These data suggest that CD133+CD24+CD106− cells represent tubular‐committed progenitors that display resistance to apoptotic stimuli and exert regenerative potential for injured tubular tissue. STEM CELLS2012;30:1714–1725

CD44 is a physiological E-selectin ligand on neutrophils

The selectin family of adhesion molecules and their glycoconjugated ligands are essential for blood polymorphonuclear neutrophil (PMN) extravasation into inflammatory and infectious sites. However, E-selectin ligands on PMNs are not well characterized. We show here that CD44 immunopurified from G-CSF–differentiated 32D cells or from peripheral blood PMNs binds specifically to E-selectin. In contrast, CD44 extracted from bone marrow stromal or brain endothelial cell lines does not interact with E-selectin, suggesting cell-specific posttranslational modifications of CD44. PMN-derived CD44 binding activity is mediated by sialylated, α(1,3) fucosylated, N-linked glycans. CD44 enables slow leukocyte rolling on E-selectin expressed on inflamed endothelium in vivo and cooperates with P-selectin glycoprotein ligand–1 to recruit neutrophils into thioglycollate-induced peritonitis and staphylococcal enterotoxin A–injected skin pouch. CD44 extracted from human PMNs also binds to E-selectin. Moreover, we demonstrate that CD44 is hypofucosylated in PMNs from a patient with leukocyte adhesion deficiency type II, suggesting that it contributes to the syndrome. These findings thus suggest broader roles for CD44 in the innate immune response and uncover a potential new target for diseases in which selectins play a prominent role.

Notch Activation Differentially Regulates Renal Progenitors Proliferation and Differentiation Toward the Podocyte Lineage in Glomerular Disorders

Glomerular diseases account for 90% of end‐stage kidney disease. Podocyte loss is a common determining factor for the progression toward glomerulosclerosis. Mature podocytes cannot proliferate, but recent evidence suggests that they can be replaced by renal progenitors localized within the Bowmans capsule. Here, we demonstrate that Notch activation in human renal progenitors stimulates entry into the S‐phase of the cell cycle and cell division, whereas its downregulation is required for differentiation toward the podocyte lineage. Indeed, a persistent activation of the Notch pathway induced podocytes to cross the G2/M checkpoint, resulting in cytoskeleton disruption and death by mitotic catastrophe. Notch expression was virtually absent in the glomeruli of healthy adult kidneys, while a strong upregulation was observed in renal progenitors and podocytes in patients affected by glomerular disorders. Accordingly, inhibition of the Notch pathway in mouse models of focal segmental glomerulosclerosis ameliorated proteinuria and reduced podocyte loss during the initial phases of glomerular injury, while inducing reduction of progenitor proliferation during the regenerative phases of glomerular injury with worsening of proteinuria and glomerulosclerosis. Taken altogether, these results suggest that the severity of glomerular disorders depends on the Notch‐regulated balance between podocyte death and regeneration provided by renal progenitors. STEM CELLS 2010; 28:1674–1685.

Proteinuria Impairs Podocyte Regeneration by Sequestering Retinoic Acid

In CKD, the risk of kidney failure and death depends on the severity of proteinuria, which correlates with the extent of podocyte loss and glomerular scarring. We investigated whether proteinuria contributes directly to progressive glomerulosclerosis through the suppression of podocyte regeneration and found that individual components of proteinuria exert distinct effects on renal progenitor survival and differentiation toward a podocyte lineage. In particular, albumin prevented podocyte differentiation from human renal progenitors in vitro by sequestering retinoic acid, thus impairing retinoic acid response element (RARE)-mediated transcription of podocyte-specific genes. In mice with Adriamycin nephropathy, a model of human FSGS, blocking endogenous retinoic acid synthesis increased proteinuria and exacerbated glomerulosclerosis. This effect was related to a reduction in podocyte number, as validated through genetic podocyte labeling in NPHS2.Cre;mT/mG transgenic mice. In RARE-lacZ transgenic mice, albuminuria reduced retinoic acid bioavailability and impaired RARE activation in renal progenitors, inhibiting their differentiation into podocytes. Treatment with retinoic acid restored RARE activity and induced the expression of podocyte markers in renal progenitors, decreasing proteinuria and increasing podocyte number, as demonstrated in serial biopsy specimens. These results suggest that albumin loss through the damaged filtration barrier impairs podocyte regeneration by sequestering retinoic acid and promotes the generation of FSGS lesions. Our findings may explain why reducing proteinuria delays CKD progression and provide a biologic rationale for the clinical use of pharmacologic modulators to induce regression of glomerular diseases.

Podocyte Regeneration Driven by Renal Progenitors Determines Glomerular Disease Remission and Can Be Pharmacologically Enhanced

Summary Podocyte loss is a general mechanism of glomerular dysfunction that initiates and drives the progression of chronic kidney disease, which affects 10% of the world population. Here, we evaluate whether the regenerative response to podocyte injury influences chronic kidney disease outcome. In models of focal segmental glomerulosclerosis performed in inducible transgenic mice where podocytes are tagged, remission or progression of disease was determined by the amount of regenerated podocytes. When the same model was established in inducible transgenic mice where renal progenitors are tagged, the disease remitted if renal progenitors successfully differentiated into podocytes, while it persisted if differentiation was ineffective, resulting in glomerulosclerosis. Treatment with BIO, a GSK3s inhibitor, significantly increased disease remission by enhancing renal progenitor sensitivity to the differentiation effect of endogenous retinoic acid. These results establish renal progenitors as critical determinants of glomerular disease outcome and a pharmacological enhancement of their differentiation as a possible therapeutic strategy.

The Antiviral Cytokines IFN-α and IFN-β Modulate Parietal Epithelial Cells and Promote Podocyte Loss: Implications for IFN Toxicity, Viral Glomerulonephritis, and Glomerular Regeneration

Interferon (IFN)-α and IFN-β are the central regulators of antiviral immunity but little is known about their roles in viral glomerulonephritis (eg, HIV nephropathy). We hypothesized that IFN-α and IFN-β would trigger local inflammation and podocyte loss. We found that both IFNs consistently activated human and mouse podocytes and parietal epithelial cells to express numerous IFN-stimulated genes. However, only IFN-β significantly induced podocyte death and increased the permeability of podocyte monolayers. In contrast, only IFN-α caused cell-cycle arrest and inhibited the migration of parietal epithelial cells. Both IFNs suppressed renal progenitor differentiation into mature podocytes. In Adriamycin nephropathy, injections with either IFN-α or IFN-β aggravated proteinuria, macrophage influx, and glomerulosclerosis. A detailed analysis showed that only IFN-β induced podocyte mitosis. This did not, however, lead to proliferation, but was associated with podocyte loss via podocyte detachment and/or mitotic podocyte death (mitotic catastrophe). We did not detect TUNEL-positive podocytes. Thus, IFN-α and IFN-β have both common and differential effects on podocytes and parietal epithelial cells, which together promote glomerulosclerosis by enhancing podocyte loss while suppressing podocyte regeneration from local progenitors.