Platelets from WAS patients show an increased susceptibility to ex vivo phagocytosis.

The thrombocytopenia of Wiskott-Aldrich syndrome (WAS) is thought to be due to both reduced platelet production and accelerated platelet consumption. We have previously demonstrated that platelets from WASP-deficient mice are consumed more rapidly in vivo than are WT platelets, and that opsonization accelerates their uptake by bone marrow- derived macrophages more than it does that of WT platelets. Here we asked whether platelets from WAS patients show similar features. We show that ex vivo phagocytosis by activated THP-1 cells of DIO-labeled platelets from a series of WAS or XLT patients is increased in comparison to that of normal control platelets. Using a numerical analysis method, we distinguish this effect from a concurrent effect on the amount of detectable fluorescent signal transferred to the macrophage per phagocytosed platelet. We show that the latter quantity is reduced by platelet WASP deficiency, as might be expected if the fluorescence transferred from these smaller platelets is more rapidly quenched. We are unable to detect a differential effect of opsonization with anti-CD61 antibody on the uptake of WASP(-) vs. WT platelets. However, the high probability of phagocytosis per adsorbed WASP(-) platelet could limit the sensitivity of the assay in this case. We also see no effect of sera from WAS patients on the uptake of normal control platelets, suggesting that in vivo opsonization is not the cause of increased uptake of WASP(-) platelets. Finally, we show little, if any, increase in the reticulated platelet fraction in WAS patients, suggesting that impaired production of reticulated platelets contributes to the thrombocytopenia. Our findings suggest that rapid in vivo platelet consumption contributes significantly to the thrombocytopenia of WAS. They also demonstrate the feasibility of routinely performing functional assays of phagocytosis of small numbers of platelets obtained at remote locations, a method which should be applicable to the study of other types of thrombocytopenia such as ITP.

p38 MAPK inhibition suppresses the TLR-hypersensitive phenotype in FANCC- and FANCA-deficient mononuclear phagocytes.

Fanconi anemia, complementation group C (FANCC)-deficient hematopoietic stem and progenitor cells are hypersensitive to a variety of inhibitory cytokines, one of which, TNFα, can induce BM failure and clonal evolution in Fancc-deficient mice. FANCC-deficient macrophages are also hypersensitive to TLR activation and produce TNFα in an unrestrained fashion. Reasoning that suppression of inhibitory cytokine production might enhance hematopoiesis, we screened small molecules using TLR agonist-stimulated FANCC- and Fanconi anemia, complementation group A (FANCA)-deficient macrophages containing an NF-κB/AP-1-responsive reporter gene (SEAP). Of the 75 small molecules screened, the p38 MAPK inhibitor BIRB 796 and dasatinib potently suppressed TLR8-dependent expression of the reporter gene. Fanconi anemia (FA) macrophages were hypersensitive to the TLR7/8 activator R848, overproducing SEAP and TNFα in response to all doses of the agonist. Low doses (50nM) of both agents inhibited p38 MAPK-dependent activation of MAPKAPK2 (MK2) and suppressed MK2-dependent TNFα production without substantially influencing TNFα gene transcription. Overproduction of TNFα by primary FA cells was likewise suppressed by these agents and involved inhibition of MK2 activation. Because MK2 is also known to influence production and/or sensitivity to 2 other suppressive factors (MIP-1α and IFNγ) to which FA hematopoietic progenitor cells are uniquely vulnerable, targeting of p38 MAPK in FA hematopoietic cells is a rational objective for preclinical evaluation.

A numerical analysis model for interpretation of flow cytometric studies of ex vivo phagocytosis.

The study of ex vivo phagocytosis via flow cytometry requires that one distinguish experimentally between uptake and adsorption of fluorescently labeled targets by phagocytes. Removal of the latter quantity from the analysis is the most common means of analyzing such data. Because the probability of phagocytosis is a function of the probability of adsorption, and because partially quenched fluorescence after uptake often overlaps with that of negative controls, this approach is suboptimal at best. Here, we describe a numerical analysis model which overcomes these limitations. We posit that the random adsorption of targets to macrophages, and subsequent phagocytosis, is a function of three parameters: the ratio of targets to macrophages (m), the mean fluorescence intensity imparted to the phagocyte by the internalized target (alpha), and the probability of phagocytosis per adsorbed target (p). The potential values of these parameters define a parameter space and their values at any point in parameter space can be used to predict the fraction of adsorption(+) and [adsorption(-), phagocytosis(+)] cells that might be observed experimentally. By systematically evaluating the points in parameter space for the latter two values and comparing them to experimental data, the model arrives at sets of parameter values that optimally predict such data. Using activated THP-1 cells as macrophages and platelets as targets, we validate the model by demonstrating that it can distinguish between the effects of experimental changes in m, alpha, and p. Finally, we use the model to demonstrate that platelets from a congenitally thrombocytopenic WAS patient show an increased probability of ex vivo phagocytosis. This finding correlates with other evidence that rapid in vivo platelet consumption contributes significantly to the thrombocytopenia of WAS. Our numerical analysis method represents a useful and innovative approach to multivariate analysis.

Fancd2-/- mice have hematopoietic defects that can be partially corrected by resveratrol.

Progressive bone marrow failure is a major cause of morbidity and mortality in human Fanconi Anemia patients. In an effort to develop a Fanconi Anemia murine model to study bone marrow failure, we found that Fancd2(-/-) mice have readily measurable hematopoietic defects. Fancd2 deficiency was associated with a significant decline in the size of the c-Kit(+)Sca-1(+)Lineage(-) (KSL) pool and reduced stem cell repopulation and spleen colony-forming capacity. Fancd2(-/-) KSL cells showed an abnormal cell cycle status and loss of quiescence. In addition, the supportive function of the marrow microenvironment was compromised in Fancd2(-/-) mice. Treatment with Sirt1-mimetic and the antioxidant drug, resveratrol, maintained Fancd2(-/-) KSL cells in quiescence, improved the marrow microenvironment, partially corrected the abnormal cell cycle status, and significantly improved the spleen colony-forming capacity of Fancd2(-/-) bone marrow cells. We conclude that Fancd2(-/-) mice have readily quantifiable hematopoietic defects, and that this model is well suited for pharmacologic screening studies.

Genetic disruption of both Fancc and Fancg in mice recapitulates the hematopoietic manifestations of Fanconi anemia.

Fanconi anemia (FA) is an inherited chromosomal instability syndrome characterized by bone marrow failure, myelodysplasia (MDS), and acute myeloid leukemia (AML). Eight FA proteins associate in a nuclear core complex to monoubiquitinate FANCD2/FANCI in response to DNA damage. Additional functions have been described for some of the core complex proteins; however, in vivo genetic proof has been lacking. Here we show that double-mutant Fancc(-/-);Fancg(-/-) mice develop spontaneous hematologic sequelae including bone marrow failure, AML, MDS and complex random chromosomal abnormalities that the single-mutant mice do not. This genetic model provides evidence for unique core complex protein function independent of their ability to monoubiquitinate FANCD2/FANCI. Importantly, this model closely recapitulates the phenotypes found in FA patients and may be useful as a preclinical platform to evaluate the molecular pathogenesis of spontaneous bone marrow failure, MDS and AML in FA.

TLR8-dependent TNF-(alpha) overexpression in Fanconi anemia group C cells.

Tumor necrosis factor alpha (TNF-alpha) production is abnormally high in Fanconi anemia (FA) cells and contributes to the hematopoietic defects seen in FA complementation group C-deficient (Fancc(-/-)) mice. Applying gene expression microarray and proteomic methods to studies on FANCC-deficient cells we found that genes encoding proteins directly involved in ubiquitinylation are overrepresented in the signature of FA bone marrow cells and that ubiquitinylation profiles of FA-C and complemented cells were substantially different. Finding that Toll-like receptor 8 (TLR8) was one of the proteins ubiquitinylated only in mutant cells, we confirmed that TLR8 (or a TLR8-associated protein) is ubiquitinylated in mutant FA-C cells and that TNF-alpha production in mutant cells depended upon TLR8 and the canonical downstream signaling intermediates interleukin 1 receptor-associated kinase (IRAK) and IkappaB kinase-alpha/beta. FANCC-deficient THP-1 cells and macrophages from Fancc(-/-) mice overexpressed TNF-alpha in response to TLR8 agonists but not other TLR agonists. Ectopically expressed FANCC point mutants were capable of fully complementing the mitomycin-C hypersensitivity phenotype of FA-C cells but did not suppress TNF-alpha overproduction. In conclusion, FANCC suppresses TNF-alpha production in mononuclear phagocytes by suppressing TLR8 activity and this particular function of FANCC is independent of its function in protecting the genome from cross-linking agents.