Alexander Gerbaulet

The receptor tyrosine kinase c-Kit controls IL-33 receptor signaling in mast cells

Members of the Toll/interleukin-1 receptor (TIR) family are of importance for host defense and inflammation. Here we report that the TIR-family member interleukin-33R (IL-33R) cross-activates the receptor tyrosine kinase c-Kit in human and murine mast cells. The IL-33R-induced activation of signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase B (PKB), and Jun NH(2)-terminal kinase 1 (JNK1) depends on c-Kit and is required to elicit optimal effector functions. Costimulation with the c-Kit ligand stem cell factor (SCF) is necessary for IL-33-induced cytokine production in primary mast cells. The structural basis for this cross-activation is the complex formation between c-Kit, IL-33R, and IL-1R accessory protein (IL-1RAcP). We found that c-Kit and IL-1RAcP interact constitutively and that IL-33R joins this complex upon ligand binding. Our findings support a model in which signals from seemingly disparate receptors are integrated for full cellular responses.

Mouse SAMHD1 Has Antiretroviral Activity and Suppresses a Spontaneous Cell-Intrinsic Antiviral Response

SUMMARY Aicardi-Goutières syndrome (AGS), a hereditary autoimmune disease, clinically and biochemically overlaps with systemic lupus erythematosus (SLE) and, like SLE, is characterized by spontaneous type I interferon (IFN) production. The finding that defects of intracellular nucleases cause AGS led to the concept that intracellular accumulation of nucleic acids triggers inappropriate production of type I IFN and autoimmunity. AGS can also be caused by defects of SAMHD1, a 3′ exonuclease and deoxy-nucleotide (dNTP) triphosphohydrolase. Human SAMHD1 is an HIV-1 restriction factor that hydrolyzes dNTPs and decreases their concentration below the levels required for retroviral reverse transcription. We show in gene-targeted mice that also mouse SAMHD1 reduces cellular dNTP concentrations and restricts retroviral replication in lymphocytes, macrophages, and dendritic cells. Importantly, the absence of SAMHD1 triggered IFN-β-dependent transcriptional upregulation of type I IFN-inducible genes in various cell types indicative of spontaneous IFN production. SAMHD1-deficient mice may be instrumental for elucidating the mechanisms that trigger pathogenic type I IFN responses in AGS and SLE.

Mast cell hyperplasia, B-cell malignancy, and intestinal inflammation in mice with conditional expression of a constitutively active kit

Signaling through the receptor tyrosine kinase kit controls proliferation and differentiation of hematopoietic precursor cells and mast cells. Somatic point mutations of the receptor that constitutively activate kit signaling are associated with mastocytosis and various hematopoietic malignancies. We generated a Cre/loxP-based bacterial artificial chromosome transgenic mouse model that allows conditional expression of a kit gene carrying the kitD814V mutation (the murine homolog of the most common mutation in human mastocytosis, kitD816V) driven by the kit promoter. Expression of the mutant kit in cells of adult mice, including hematopoietic precursors, caused severe mastocytosis with 100% penetrance at young age frequently associated with additional hematopoietic (mostly B lineage-derived) neoplasms and focal colitis. Restriction of transgene expression to mature mast cells resulted in a similar mast cell disease developing with slower kinetics. Embryonic expression led to a hyperproliferative dysregulation of the erythroid lineage with a high rate of perinatal lethality. In addition, most adult animals developed colitis associated with mucosal mast cell accumulation. Our findings demonstrate that the effects of constitutive kit signaling critically depend on the developmental stage and the state of differentiation of the cell hit by the gain-of-function mutation.

The bulk of the hematopoietic stem cell population is dispensable for murine steady-state and stress hematopoiesis.

Long-term repopulating (LT) hematopoietic stem cells (HSCs) are the most undifferentiated cells at the top of the hematopoietic hierarchy. The regulation of HSC pool size and its contribution to hematopoiesis are incompletely understood. We depleted hematopoietic stem and progenitor cells (HSPCs) in adult mice in situ and found that LT-HSCs recovered from initially very low levels (<1%) to below 10% of normal numbers but not more, whereas progenitor cells substantially recovered shortly after depletion. In spite of the persistent and massive reduction of LT-HSCs, steady-state hematopoiesis was unaffected and residual HSCs remained quiescent. Hematopoietic stress, although reported to recruit quiescent HSCs into cycle, was well tolerated by HSPC-depleted mice and did not induce expansion of the small LT-HSC compartment. Only upon 5-fluorouracil treatment was HSPC-depleted bone marrow compromised in reconstituting hematopoiesis, demonstrating that HSCs and early progenitors are crucial to compensate myeloablation. Hence, a contracted HSC compartment cannot recover in situ to its original size, and normal steady-state blood cell generation is sustained with <10% of normal LT-HSC numbers without increased contribution of the few residual cells.Long-term repopulating (LT) hematopoietic stem cells (HSCs) are the most undifferentiated cells at the top of the hematopoietic hierarchy. The regulation of HSC pool size and its contribution to hematopoiesis are incompletely understood. We depleted hematopoietic stem and progenitor cells (HSPCs) in adult mice in situ and found that LT-HSCs recovered from initially very low levels (<1%) to below 10% of normal numbers but not more, whereas progenitor cells substantially recovered shortly after depletion. In spite of the persistent and massive reduction of LT-HSCs, steady-state hematopoiesis was unaffected and residual HSCs remained quiescent. Hematopoietic stress, although reported to recruit quiescent HSCs into cycle, was well tolerated by HSPC-depleted mice and did not induce expansion of the small LT-HSC compartment. Only upon 5-fluorouracil treatment was HSPC-depleted bone marrow compromised in reconstituting hematopoiesis, demonstrating that HSCs and early progenitors are crucial to compensate myeloablation. Hence, a contracted HSC compartment cannot recover in situ to its original size, and normal steady-state blood cell generation is sustained with <10% of normal LT-HSC numbers without increased contribution of the few residual cells.

Loss of Trex1 in Dendritic Cells Is Sufficient To Trigger Systemic Autoimmunity

Defects of the intracellular enzyme 3′ repair exonuclease 1 (Trex1) cause the rare autoimmune condition Aicardi–Goutières syndrome and are associated with systemic lupus erythematosus. Trex1−/− mice develop type I IFN–driven autoimmunity, resulting from activation of the cytoplasmic DNA sensor cyclic GMP–AMP synthase by a nucleic acid substrate of Trex1 that remains unknown. To identify cell types responsible for initiation of autoimmunity, we generated conditional Trex1 knockout mice. Loss of Trex1 in dendritic cells was sufficient to cause IFN release and autoimmunity, whereas Trex1-deficient keratinocytes and microglia produced IFN but did not induce inflammation. In contrast, B cells, cardiomyocytes, neurons, and astrocytes did not show any detectable response to the inactivation of Trex1. Thus, individual cell types differentially respond to the loss of Trex1, and Trex1 expression in dendritic cells is essential to prevent breakdown of self-tolerance ensuing from aberrant detection of endogenous DNA.

Loss of Function of TET2 Cooperates with Constitutively Active KIT in Murine and Human Models of Mastocytosis

Systemic Mastocytosis (SM) is a clonal disease characterized by abnormal accumulation of mast cells in multiple organs. Clinical presentations of the disease vary widely from indolent to aggressive forms, and to the exceedingly rare mast cell leukemia. Current treatment of aggressive SM and mast cell leukemia is unsatisfactory. An imatinib-resistant activating mutation of the receptor tyrosine kinase KIT (KIT D816V) is most frequently present in transformed mast cells and is associated with all clinical forms of the disease. Thus the etiology of the variable clinical aggressiveness of abnormal mast cells in SM is unclear. TET2 appears to be mutated in primary human samples in aggressive types of SM, suggesting a possible role in disease modification. In this report, we demonstrate the cooperation between KIT D816V and loss of function of TET2 in mast cell transformation and demonstrate a more aggressive phenotype in a murine model of SM when both mutations are present in progenitor cells. We exploit these findings to validate a combination treatment strategy targeting the epigenetic deregulation caused by loss of TET2 and the constitutively active KIT receptor for the treatment of patients with aggressive SM.

Lack of Trex1 Causes Systemic Autoimmunity despite the Presence of Antiretroviral Drugs

Biallelic mutations of three prime repair exonuclease 1 (TREX1) cause the lupus-like disease Aicardi–Goutières syndrome in which accumulation of a yet unknown endogenous DNA substrate of TREX1 triggers a cyclic GMP–AMP synthase-dependent type I IFN response and systemic autoimmunity. Products of reverse transcription originating from endogenous retroelements have been suggested to be a major substrate for TREX1, and reverse transcriptase inhibitors (RTIs) were proposed as a therapeutic option in autoimmunity ensuing from defects of TREX1. In this study, we treated Trex1−/− mice with RTIs. The serum RTI levels reached were sufficient to block retrotransposition of endogenous retroelements. However, the treatment did not reduce the spontaneous type I IFN response and did not ameliorate lethal inflammation. Furthermore, long interspersed nuclear elements 1 retrotransposition was not enhanced in the absence of Trex1. Our data do not support the concept of retroelement-derived cDNA as key triggers of systemic autoimmunity in Trex1-deficient humans and mice and motivate the continuing search for the pathogenic IFN-inducing Trex1 substrate.

Mast Cell Apoptosis

Apoptosis is a physiological form of cell death. Cells undergoing apoptosis execute a genetically controlled program that leads to organized breakdown of cellular structures and ends in phagocytosis of their remains. In mast cells, several mechanisms regulating apoptosis have been identified including growth factors, tumor necrosis factor-alpha receptors, monomeric IgE, Toll-like receptors, and proteins of the bcl-2 family. Methods used to characterize apoptosis of mast cells are reviewed, with special attention to flow cytometric analysis of annexin V staining, analysis of deoxyribonucleic acid fragmentation by gel electrophoresis and end-labeling techniques, measurement of caspase activity by enzymatic assays, and characterization of pro- and anti-apoptotic proteins by immunoblotting.

Hematopoietic stem cells can differentiate into restricted myeloid progenitors before cell division in mice

Hematopoietic stem cells (HSCs) continuously replenish all blood cell types through a series of differentiation steps and repeated cell divisions that involve the generation of lineage-committed progenitors. However, whether cell division in HSCs precedes differentiation is unclear. To this end, we used an HSC cell-tracing approach and Ki67RFP knock-in mice, in a non-conditioned transplantation model, to assess divisional history, cell cycle progression, and differentiation of adult HSCs. Our results reveal that HSCs are able to differentiate into restricted progenitors, especially common myeloid, megakaryocyte-erythroid and pre-megakaryocyte progenitors, without undergoing cell division and even before entering the S phase of the cell cycle. Additionally, the phenotype of the undivided but differentiated progenitors correlated with the expression of lineage-specific genes and loss of multipotency. Thus HSC fate decisions can be uncoupled from physical cell division. These results facilitate a better understanding of the mechanisms that control fate decisions in hematopoietic cells.Dependence of hematopoietic stem cell (HSC) fate on the phase of the cell cycle has not been demonstrated in vivo. Here, the authors find that HSCs can differentiate into a downstream progenitor without physical division, even before progressing into the S phase of the cell cycle.

SCA-1 Expression Level Identifies Quiescent Hematopoietic Stem and Progenitor Cells

Summary Blood cell generation depends on continuous cellular output by the sequential hierarchy of hematopoietic stem cell (HSC) and progenitor populations that all contain quiescent and actively cycling cells. Hematopoietic stem and progenitor cells (HSPCs) express the surface molecule Stem cell antigen 1 (SCA-1/LY6A). Using histone 2B-red fluorescent fusion protein label retention and cell-cycle reporter mice, we demonstrate that high SCA-1 expression (SCA-1hi) identifies not only quiescent HSCs but quiescent cells on all hierarchical levels within the lineage−SCA-1+KIT+ (LSK) population. Each transplanted SCA-1hi HSPC population also displayed self-renewal potential superior to that of the respective SCA-1lo population. SCA-1 expression is inducible by type I interferon (IFN). We show, however, that quiescence and high self-renewal capacity of cells with brighter SCA-1 expression at steady state were independent of type I IFN signaling. We conclude that SCA-1 expression levels can be used to prospectively isolate functionally heterogeneous HSPC subpopulations.