Guglielmo Rosignoli

Annexin 1-deficient neutrophils exhibit enhanced transmigration in vivo and increased responsiveness in vitro

The role of the endogenous anti‐inflammatory mediator annexin 1 (AnxA1) in controlling polymorphonuclear leukocyte (PMN) trafficking and activation was addressed using the recently generated AnxA1 null mouse. In the zymosan peritonitis model, AnxA1 null mice displayed a higher degree (50–70%) of PMN recruitment compared with wild‐type littermate mice, and this was associated with reduced numbers of F4/80+ cells. Intravital microscopy analysis of the cremaster microcirculation inflamed by zymosan (6 h time‐point) indicated a greater extent of leukocyte emigration, but not rolling or adhesion, in AnxA1 null mice. Real‐time analysis of the cremaster microcirculation did not show spontaneous activation in the absence of AnxA1; however, superfusion with a direct‐acting PMN activator (1 nM platelet‐activating factor) revealed a subtle yet significant increase in leukocyte emigration, but not rolling or adhesion, in this genotype. Changes in the microcirculation were not secondary to alterations in hemodynamic parameters. The phenotype of the AnxA1 null PMN was investigated in two in vitro assays of cell activation (CD11b membrane expression and chemotaxis): the data obtained indicated a higher degree of cellular responses irrespective of the stimulus used. In conclusion, we have used a combination of inflammatory protocols and in vitro assays to address the specific counter‐regulatory role of endogenous AnxA1, demonstrating its inhibitory control on PMN activation and the consequent impact on the inflamed microcirculation.

Carbon Monoxide-Releasing Molecules Modulate Leukocyte-Endothelial Interactions under Flow

Carbon monoxide (CO) generated by the enzyme heme oxygenase during the breakdown of heme is known to mediate a number of biological effects. Here, we investigated whether CO liberated from a water-soluble CO-releasing molecule (CO-RM) is capable of modulating leukocyte-endothelial interactions. Tricarbonylchoro(glycinato)ruthenium (II) (CORM-3), a fast CO releaser, proved to be anti-inflammatory in two distinct models of acute inflammation in vivo. In both cases, a significant reduction in neutrophil extravasation was observed. Subsequent in vitro static experiments showed that CORM-3 produced a direct effect on neutrophil (polymorphonuclear neutrophil; PMN) adhesion molecule expression; dose-dependently inhibiting platelet-activating factor stimulated CD11b up-regulation and L-selectin shedding, whereas no effect was observed on up-regulation of human umbilical vein endothelial cell (HUVEC) adhesion molecules intercellular adhesion molecule-1 or E-selectin nor on interleukin-8 chemokine production. In addition, when PMN interaction with HUVECs was studied, an inhibitory effect of CORM-3 on cell capture and rolling was observed. The effect of CORM-3 on PMN CD11b expression was mimicked by the incubation of PMN with the selective large potassium channel opener 1,3-dihydro-1-(2-hydroxy-5-(trifluoromethyl)-phenyl)-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619), which suggests that CORM-3 actions in this instance are mediated, at least in part, via opening of this channel. In conclusion, we have reported that CORM-3 possesses acute anti-inflammatory effects in vivo and that these are probably the result of targeting PMN activation and rolling upon the endothelium.

Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation

Acute myeloid leukemia (AML) induces bone marrow (BM) failure in patients, predisposing them to life-threatening infections and bleeding. The mechanism by which AML mediates this complication is unknown but one widely accepted explanation is that AML depletes the BM of hematopoietic stem cells (HSCs) through displacement. We sought to investigate how AML affects hematopoiesis by quantifying residual normal hematopoietic subpopulations in the BM of immunodeficient mice transplanted with human AML cells with a range of genetic lesions. The numbers of normal mouse HSCs were preserved whereas normal progenitors and other downstream hematopoietic cells were reduced following transplantation of primary AMLs, findings consistent with a differentiation block at the HSC–progenitor transition, rather than displacement. Once removed from the leukemic environment, residual normal hematopoietic cells differentiated normally and outcompeted steady-state hematopoietic cells, indicating that this effect is reversible. We confirmed the clinical significance of this by ex vivo analysis of normal hematopoietic subpopulations from BM of 16 patients with AML. This analysis demonstrated that the numbers of normal CD34+CD38− stem-progenitor cells were similar in the BM of AML patients and controls, whereas normal CD34+CD38+ progenitors were reduced. Residual normal CD34+ cells from patients with AML were enriched in long-term culture, initiating cells and repopulating cells compared with controls. In conclusion the data do not support the idea that BM failure in AML is due to HSC depletion. Rather, AML inhibits production of downstream hematopoietic cells by impeding differentiation at the HSC–progenitor transition.

CFTR inhibition provokes an inflammatory response associated with an imbalance of the annexin A1 pathway.

Cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, is characterized by chronic bacterial infections and inflammation in the lung. Having previously shown that deletion of CFTR is associated with lower expression of the endogenous anti-inflammatory protein Annexin A1 (AnxA1), we investigated further this possible functional connection using a validated CFTR inhibitor. Treatment of mice with the CFTR inhibitor-172 (CFTR(172)) augmented the acute peritonitis promoted by zymosan, an effect associated with lower AnxA1 levels in peritoneal cells. Similar results were obtained with another, chemically distinct, CFTR inhibitor. The pro-inflammatory effect of CFTR(172) was lost in AnxA1(-/-), as well as CFTR(-/-) mice. Importantly, administration of hrAnxA1 and its peptido-mimetic to CFTR(-/-) animals or to animals treated with CFTR(172) corrected the exaggerated leukocyte migration seen in these animals. In vitro assays with human Polymorphonuclear leukocyte (PMN) demonstrated that CFTR(172) reduced cell-associated AnxA1 by promoting release of the protein in microparticles. We propose that the reduced impact of the counterregulatory properties of AnxA1 in CF cells contributes to the inflammatory phenotype characteristic of this disease. Thus, these findings provide an important insight into the mechanism underlying the inflammatory disease associated with CFTR inhibition while, at the same time, providing a novel pharmacological target for controlling the inflammatory phenotype of CF.

High Throughput Sequencing Analysis of the Immunoglobulin Heavy Chain Gene from Flow-Sorted B Cell Sub-Populations Define the Dynamics of Follicular Lymphoma Clonal Evolution

Understanding the dynamics of evolution of Follicular Lymphoma (FL) clones during disease progression is important for monitoring and targeting this tumor effectively. Genetic profiling of serial FL biopsies and examples of FL transmission following bone marrow transplant suggest that this disease may evolve by divergent evolution from a common ancestor cell. However where this ancestor cell resides and how it evolves is still unclear. The analysis of the pattern of somatic hypermutation of the immunoglobulin gene (Ig) is traditionally used for tracking the physiological clonal evolution of B cells within the germinal center and allows to discriminate those cells that have just entered the germinal center and display features of ancestor cells from those B cells that keep re-circulating across different lymphoid organs. Here we investigated the pattern of somatic hypermutation of the heavy chain of the immunoglobulin gene (IgH-VH) in 4 flow-sorted B cells subpopulations belonging to different stages of differentiation, from sequential lymph node biopsies of cases displaying diverse patterns of evolution, using the GS-FLX Titanium sequencing platform. We observed an unexpectedly high level of clonality, with hundreds of distinct tumor subclones in the different subpopulations from the same sample, the majority detected at a frequency <10−2. By using a lineage trees analysis we observed in all our FL and t-FL cases that the oligoclonal FL population was trapped in a narrow intermediate stage of maturation that maintains the capacity to undergo SHM, but was unable to further differentiate. The presence of such a complex architecture highlights challenges currently encountered in finding a cure for this disease.