CX3CR1 defines functionally distinct intestinal mononuclear phagocyte subsets which maintain their respective functions during homeostatic and inflammatory conditions

Intestinal mononuclear phagocytes (iMNP) are critically involved in mucosal immunity and tissue homeostasis. Two major non-overlapping populations of iMNP have been identified in mice. CD103(+) iMNP represent a migratory population capable of inducing tolerogenic responses, whereas CX3CR1(+) iMNP are resident cells with disease-promoting potential. CX3CR1(+) iMNP can further be subdivided based on differential expression of CX3CR1. Using CX3CR1(GFP/+) ×RAG2(-/-) mice, we demonstrate that CX3CR1(hi) and CX3CR1(lo) iMNP clearly differ with respect to their morphological and functional properties. Compared with CX3CR1(hi) iMNP, CX3CR1(lo) iMNP are polarised towards pro-inflammatory responses already under homeostatic conditions. During a CD4(+) T-cell-induced colitis, CX3CR1(lo) cells accumulate in the inflamed mucosa and upregulate the expression of pro-inflammatory cytokines and triggering receptor expressed on myeloid cells-1 (TREM-1). In contrast, CX3CR1(hi) iMNP retain their non-inflammatory profile even during intestinal inflammation. These findings identify two functionally distinct iMNP subsets based on differential expression of CX3CR1 and indicate an unanticipated stability of iMNP.

In vitro cytotoxicity of silver nanoparticles on osteoblasts and osteoclasts at antibacterial concentrations

Abstract Nanoparticulate silver coatings for orthopaedic implants promise to decrease postoperative infection rates. However, silver-induced cytotoxicity on bone cells has not been investigated in detail. This study investigated the cytotoxic effects of silver nano- and microparticles and Ag(+) on osteoblasts (OBs) and osteoclasts (OCs) and correlated their effects with the antibacterial efficacy on Staphylococcus epidermidis. Silver nanoparticles (50 nm) exhibited strong cytotoxic effects on OBs and OCs. Weak cytotoxic effects were observed for silver microparticles (3 μm). The cytotoxicity was primarily mediated by a size-dependent release of Ag(+). Antibacterial effects occurred at Ag(+) concentrations that were 2-4 times higher than those inducing cytotoxic effects. Such adverse effects on OB and OC survival may have deleterious effects on the biocompatibility of orthopaedic implants. Our study represents an important step toward the detailed investigation of orthopaedic implant with nanoparticulate silver coatings prior to their widespread clinical usage.

Bacterial meningitis impairs hippocampal neurogenesis

Bacterial meningitis causes persisting neurofunctional sequelae. Theoccurrence of apoptotic cell death in the hippocampal subgranular zone of the dentate gyrus characterizes the disease in patients and relates to deficits in learning and memory in corresponding experimental models. Here, we investigated why neurogenesis fails to regenerate the damage in the hippocampus associated with the persistence of neurofunctional deficits. In an infant rat model of bacterial meningitis, the capacity of hippocampal-derived cells to multiply and form neurospheres was significantly impaired comparedto that in uninfected littermates. In an in vitro model of differentiating hippocampal cells, challenges characteristic of bacterial meningitis (i.e. bacterial components, tumor necrosis factor [20 ng/mL], or growth factor deprivation) caused significantly more apoptosis in stem/progenitor cells and immature neurons than in mature neurons. These results demonstrate that bacterial meningitis injures hippocampal stem and progenitor cells, a finding that may explain the persistence of neurofunctional deficits after bacterial meningitis.

Reduced incidence of insect-bite hypersensitivity in Icelandic horses is associated with a down-regulation of interleukin-4 by interleukin-10 and transforming growth factor-beta1

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of insects of the genus Culicoides. IBH does not occur in Iceland due to the absence of Culicoides. However, Icelandic horses exported to mainland Europe as adults (1st generation) have a >/=50% incidence of developing IBH. In contrast, their progeny (2nd generation) has a <10% incidence of IBH. Here we show that peripheral blood mononuclear cells (PBMC) from Icelandic horses born in mainland Europe and belonging either to the IBH or healthy subgroup produce less interleukin (IL)-4 after polyclonal or allergen-specific stimulation when compared with counterparts from horses born in Iceland. We examined a role of IL-10 and transforming growth factor (TGF)-beta1 in down-regulation of IL-4 in healthy 2nd generation Icelandic horses. Supernatants of PBMC from 2nd generation healthy horses down-regulated the proportion of IL-4-producing cells and IL-4 production in stimulated cultures of PBMC from 1st generation IBH. This inhibition was mimicked by a combination of IL-10 and TGF-beta1 but not by the single cytokines. Cultures of stimulated PBMC of healthy 2nd generation horses produced a low level of IL-4, but IL-4 production was increased by anti-equine IL-10 and anti-human TGF-beta1. This shows for the first time that in horses, IL-10 and TGF-beta1 combined regulate IL-4 production in vitro. It is suggested that in this naturally occurring IgE-mediated allergy, IL-10 and TGF-beta1 have a role in the down-regulation of IL-4-induced allergen-specific Th2 cells, thereby reducing the incidence of IBH.

Topo IIIalpha and BLM act within the Fanconi anemia pathway in response to DNA-crosslinking agents

The Bloom protein (BLM) and Topoisomerase IIIalpha are found in association with proteins of the Fanconi anemia (FA) pathway, a disorder manifesting increased cellular sensitivity to DNA crosslinking agents. In order to determine if the association reflects a functional interaction for the maintenance of genome stability, we have analyzed the effects of siRNA-mediated depletion of the proteins in human cells. Depletion of Topoisomerase IIIalpha or BLM leads to increased radial formation, as is seen in FA. BLM and Topoisomerase IIIalpha are epistatic to the FA pathway for suppression of radial formation in response to DNA interstrand crosslinks since depletion of either of them in FA cells does not increase radial formation. Depletion of Topoisomerase IIIalpha or BLM also causes an increase in sister chromatid exchanges, as is seen in Bloom syndrome cells. Human Fanconi anemia cells, however, do not demonstrate increased sister chromatid exchanges, separating this response from radial formation. Primary cell lines from mice defective in both Blm and Fancd2 have the same interstrand crosslink-induced genome instability as cells from mice deficient in the Fancd2 protein alone. These observations demonstrate that the association of BLM and Topoisomerase IIIalpha with Fanconi proteins is a functional one, delineating a BLM-Topoisomerase IIIalpha-Fanconi pathway that is critical for suppression of chromosome radial formation.

Dendritic Cell-Based Immunotherapy for Myeloid Leukemias

Acute and chronic myeloid leukemia (AML, CML) are hematologic malignancies arising from oncogene-transformed hematopoietic stem/progenitor cells known as leukemia stem cells (LSCs). LSCs are selectively resistant to various forms of therapy including irradiation or cytotoxic drugs. The introduction of tyrosine kinase inhibitors has dramatically improved disease outcome in patients with CML. For AML, however, prognosis is still quite dismal. Standard treatments have been established more than 20 years ago with only limited advances ever since. Durable remission is achieved in less than 30% of patients. Minimal residual disease (MRD), reflected by the persistence of LSCs below the detection limit by conventional methods, causes a high rate of disease relapses. Therefore, the ultimate goal in the treatment of myeloid leukemia must be the eradication of LSCs. Active immunotherapy, aiming at the generation of leukemia-specific cytotoxic T cells (CTLs), may represent a powerful approach to target LSCs in the MRD situation. To fully activate CTLs, leukemia antigens have to be successfully captured, processed, and presented by mature dendritic cells (DCs). Myeloid progenitors are a prominent source of DCs under homeostatic conditions, and it is now well established that LSCs and leukemic blasts can give rise to "malignant" DCs. These leukemia-derived DCs can express leukemia antigens and may either induce anti-leukemic T cell responses or favor tolerance to the leukemia, depending on co-stimulatory or -inhibitory molecules and cytokines. This review will concentrate on the role of DCs in myeloid leukemia immunotherapy with a special focus on their generation, application, and function and how they could be improved in order to generate highly effective and specific anti-leukemic CTL responses. In addition, we discuss how DC-based immunotherapy may be successfully integrated into current treatment strategies to promote remission and potentially cure myeloid leukemias.

The effect of ultraviolet radiation on the pathogenesis of {\it Candida albicans} in mice

Ultraviolet (UV) radiation produces immunological alterations in both humans and animals that include a decrease in the delayed type hypersensitivity (DTH) response to complex antigens, and to the induction of the suppressor T cell pathway. Cell-mediated immunity of the type that is altered by UV radiation has been shown to be important in host resistance against microorganisms. My dissertation addresses questions concerning the effects of UV radiation on the pathogenesis of opportunistic fungal pathogens such as Candida albicans.^ The (DTH) response of C3H mice exposed to ultraviolet (UV) radiation before (afferent arm of DTH) or after (efferent arm of DTH) infection with Candida albicans was markedly and systemically suppressed. Although suppression of both the afferent and efferent phases of DTH were caused by similar wavebands within the ultraviolet region, the dose of UV radiation that suppressed the efferent arm of DTH was 10-fold higher than the dose that suppressed the afferent arm of the DTH reaction.^ The DTH response of C57BL/6 mice was also suppressed by UV radiation; however the suppression was accomplished by exposure to significantly lower doses UV radiation compared to C3H mice. In C57BL/6 mice, the dose of UV radiation that suppressed the afferent phase of DTH was 5-fold higher than the dose that suppressed the efferent phase.^ Exposure of C3H mice to UV radiation before sensitization induced splenic suppressor T cells that upon transfer to normal recipients, impaired the induction of DTH to Candida. In contrast, the suppression caused by UV irradiation of mice after sensitization was not transferable. Spleen cells from sensitized mice exhibited altered homing patterns in animals that were exposed to UV radiation shortly before receiving cells, suggesting that UV-induced suppression of the efferent arm of DTH could result from an alteration in the distribution of effector cells.^ UV radiation decreased the survival of Candida-infected mice; however, no correlation was found between suppression of the DTH response and the course of lethal infection. This suggested that DTH was not protective against lethal disease with this organism. UV radiation also changed the persistence of the organism in the internal organs. UV-irradiated, infected animals had increased numbers of Candida in their kidneys compared to non-irradiated mice. Sensitization prior to UV irradiation aided clearance of the organism from the kidneys of UV-irradiated mice.^ These data show that UV radiation suppresses cell-mediated immunity to Candida albicans in mice and increases mortality of Candida-infected mice. Moreover, the data suggest that an increase in environmental UV radiation could increase the severity of pathogenic infections. ^

Accessing novel developmental mechanisms in the mouse by gene trapping

Genetic analysis is a powerful method for analyzing the function of specific genes in development. I sought to identify novel genes in the mouse using a genetic analysis relying on the expression pattern and phenotype of mutated genes. To this end, I have conducted a gene trap screen using the vector $\rm SA\beta geo,$ a promoterless DNA construct that encodes a fusion protein with lacZ and neomycin resistance activities. Productive integration and expression of the $\beta$geo protein in embryonic stem (ES) cells requires integration into an active transcription unit. The endogenous regulatory elements direct reporter gene expression which reflects the expression of the endogenous gene. Of eight mouse lines generated from gene trap ES cell clones, four showed differential regulation of $\beta$geo activity during embryogenesis. These four were analyzed in more detail.^ Three of the lines RNA 1, RNA2 and RNA 3 had similar expression patterns, within subsets of cells in sites of embryonic hematopoiesis. Cloning of the trapped genes revealed that all three integrations had occurred within 45S rRNA precursor transcription units. These results imply that there exists in these cells some mechanism responsible for the efficient production of the $\beta$geo protein from an RNA polymerase I transcript that is not present in most of the cells in the embryo.^ The fourth line, GT-2, showed widespread, dynamic expression. Many of the sites of expression were important classic embryonic induction systems. Cloning of the sequences fused to the $5\sp\prime$ end of the $\beta$geo sequence revealed that the trapped gene contained significant sequence homology with a previously identified human sequence HumORF5. An open reading frame of this sequence is homologous to a group of eukaryotic proteins that are members of the RNA helicase superfamily I.^ Analysis of the gene trap lines suggests that potentially novel developmental mechanisms have been uncovered. In the case of RNA 1, 2 and 3, the differential production of ribosomal RNAs may be required for differentiation or function of the $\beta$geo positive hematopoietic cells. In the GT-2 line, a previously unsuspected temporal and spatial regulation of a putative RNA helicase implies a role for this activity during specific aspects of mouse development. ^

Mechanisms of apoptosis in chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by the accumulation of terminally differentiated, mature B cells that do not progress beyond the G1 stage of cell cycle, suggesting that these cells possess intrinsic defects in apoptosis. Treatment relies heavily on chemotherapy (primarily nucleoside analogs and glucocorticoids) that may initially be effective in patients, but ultimately give rise to refractory, untreatable disease. The purpose of this study was to determine whether key components of the apoptotic machinery were intact in CLL lymphocytes, especially in patients refractory to therapy. ^ Activation of proteases has been shown to be at the core of the apoptotic pathway and this work demonstrates that protease activation is required for glucocorticoid and nucleoside analog-induced apoptosis in CLL cells. Inhibitors of serine proteases as well as caspase inhibitors blocked induced DNA fragmentation, and a peptide inhibitor of the nuclear scaffold (NS) protease completely suppressed both induced and spontaneous apoptosis. However, the NS protease inhibitor actually promoted several pro-apoptotic events, such as caspase activation, exposure of surface phosphatidylserine, and loss of mitochondrial membrane potential. These results suggested that the NS protease may interact with the apoptotic program in CLL cells at two separate points. ^ In order to further investigate the role of the NS protease in CLL, patient isolates were treated with proteasome inhibitors because of previous results suggesting that the ISIS protease might be a β subunit of the proteasome. Proteasome inhibitors induced massive DNA fragmentation in every patient tested, even in those resistant to the effects of glucocorticoid and nucleoside analogs in vitro. Several other features of apoptosis were also promoted by the proteasome inhibitor, including mitochondrial alterations such as release of cytochrome c and drops in mitochondrial membrane potential. Proteasome inhibitor-induced apoptosis was associated with inhibition of NFκB, a proteasome-regulated transcription factor that has been implicated in the suppression of apoptosis in a number of systems. The NS protease inhibitor also caused a decrease in active NFκB, suggesting that the proapoptotic effects of this agent might be due to depletion of NFκB. ^ Given these findings, the role of NFκB, in conferring survival in CLL was investigated. Glucocorticoid hormone treatment was shown to cause decreases in the activity of the transcription factor, while phorbol dibutyrate, which blocks glucocorticoid-induced DNA fragmentation, was capable of upregulating NFκB. Compellingly, introduction of an undegradable form of the constitutive NFκB inhibitor, IκB, caused DNA fragmentation in several patient isolates, some of which were resistant to glucocorticoid in vitro. Transcription of anti-apoptotic proteins by NFκB was postulated to be responsible for its effects on survival, but Bcl-2 levels did not fluctuate with glucocorticoid or proteasome inhibitor treatment. ^ The in vitro values generated from these studies were organized into a database containing numbers for over 250 patients. Correlation of relevant clinical parameters revealed that levels of spontaneous apoptosis in vitro differ significantly between Rai stages. Importantly, in vitro resistance to nucleoside analogs or glucocorticoids predicted resistance to chemotherapy in vivo, and inability to achieve remission. ^

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro.

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.

Ca2+-dependent repair of pneumolysin pores: A new paradigm for host cellular defense against bacterial pore-forming toxins

Pneumolysin (PLY), a key virulence factor of Streptococcus pneumoniae, permeabilizes eukaryotic cells by forming large trans-membrane pores. PLY imposes a puzzling multitude of diverse, often mutually excluding actions on eukaryotic cells. Whereas cytotoxicity of PLY can be directly attributed to the pore-mediated effects, mechanisms that are responsible for the PLY-induced activation of host cells are poorly understood. We show that PLY pores can be repaired and thereby PLY-induced cell death can be prevented. Pore-induced Ca2+ entry from the extracellular milieu is of paramount importance for the initiation of plasmalemmal repair. Nevertheless, active Ca2+ sequestration that prevents excessive Ca2+ elevation during the execution phase of plasmalemmal repair is of no less importance. The efficacy of plasmalemmal repair does not only define the fate of targeted cells but also intensity, duration and repetitiveness of PLY-induced Ca2+ signals in cells that were able to survive after PLY attack. Intracellular Ca2+ dynamics evoked by the combined action of pore formation and their elimination mimic the pattern of receptor-mediated Ca2+ signaling, which is responsible for the activation of host immune responses. Therefore, we postulate that plasmalemmal repair of PLY pores might provoke cellular responses that are similar to those currently ascribed to the receptor-mediated PLY effects. Our data provide new insights into the understanding of the complexity of cellular non-immune defense responses to a major pneumococcal toxin that plays a critical role in the establishment and the progression of life-threatening diseases. Therapies boosting plasmalemmal repair of host cells and their metabolic fitness might prove beneficial for the treatment of pneumococcal infections.

MicroRNA miR-199a-5p regulates smooth muscle cell proliferation and morphology by targeting Wnt2 signaling pathway

MicroRNA miR-199a-5p impairs tight junction formation leading to increased urothelial permeability in bladder pain syndrome. Now using transcriptome analysis in urothelial TEU-2 cells we implicate it in the regulation of cell cycle, cytoskeleton remodeling, TGF and Wnt signaling pathways. MiR-199a-5p is highly expressed in the smooth muscle layer of the bladder and we altered its levels in bladder smooth muscle cells (SMC) to validate the pathway analysis. Inhibition of miR-199a-5p with antimiR increased SMC proliferation, reduced cell size and up-regulated miR-199a-5p targets, including Wnt2. Overexpression of Wnt2 protein or treating SMCs with recombinant Wnt2 closely mimicked the miR-199a-5p inhibition, whereas down-regulation of Wnt2 in antimiR-expressing SMCs with shRNA restored cell phenotype and proliferation rates. Overexpression of miR-199a-5p in the bladder SMCs significantly increased cell size and up-regulated SM22, SM alpha-actin and SM myosin heavy chain mRNA and protein levels. These changes, as well as increased expression of ACTG2, TGFB1I1, and CDKN1A were mediated by up-regulation of smooth muscle-specific transcriptional activator myocardin at mRNA and protein levels. Myocardin-related transcription factor (MRTF-A) downstream targets Id3 and MYL9 were also induced. Up-regulation of myocardin was accompanied by down-regulation of Wnt-dependent inhibitory Kruppel-like transcription factor 4 (KLF4) in miR-199a-5p overexpressing cells. In contrast, KLF4 was induced in antimiR-expressing cells following the activation of Wnt2 signaling, leading to repression of myocardin-dependent genes. MiR-199a-5p plays a critical role in the Wnt2-mediated regulation of proliferative and differentiation processes in the smooth muscle and may behave as a key modulator of smooth muscle hypertrophy, relevant for organ remodeling.

Alpha1a-adrenoceptor genetic variant induces cardiomyoblast-to-fibroblast-like cell transition via distinct signaling pathways.

The role of naturally occurring human α1a-Adrenergic Receptor (α1aAR) genetic variants associated with cardiovascular disorders is poorly understood. Here, we present the novel findings that expression of human α1aAR-247R (247R) genetic variant in cardiomyoblasts leads to transition of cardiomyoblasts into a fibroblast-like phenotype, evidenced by morphology and distinct de novo expression of characteristic genes. These fibroblast-like cells exhibit constitutive, high proliferative capacity and agonist-induced hypertrophy compared with cells prior to transition. We demonstrate that constitutive, synergistic activation of EGFR, Src and ERK kinases is the potential molecular mechanism of this transition. We also demonstrate that 247R triggers two distinct EGFR transactivation-dependent signaling pathways: 1) constitutive Gq-independent β-arrestin-1/Src/MMP/EGFR/ERK-dependent hyperproliferation and 2) agonist-induced Gq- and EGFR/STAT-dependent hypertrophy. Interestingly, in cardiomyoblasts agonist-independent hyperproliferation is MMP-dependent, but in fibroblast-like cells it is MMP-independent, suggesting that expression of α1aAR genetic variant in cardiomyocytes may trigger extracellular matrix remodeling. Thus, these novel findings demonstrate that EGFR transactivation by α1aAR-247R leads to hyperproliferation, hypertrophy and alterations in cardiomyoblasts, suggesting that these unique genetically-mediated alterations in signaling pathways and cellular function may lead to myocardial fibrosis. Such extracellular matrix remodeling may contribute to the genesis of arrhythmias in certain types of heart failure.

VEGFR2 Signaling Prevents Colorectal Cancer Cell Senescence to Promote Tumorigenesis in Mice With Colitis.

BACKGROUND & AIMS Senescence prevents cellular transformation. We investigated whether vascular endothelial growth factor (VEGF) signaling via its receptor, VEGFR2, regulates senescence and proliferation of tumor cells in mice with colitis-associated cancer (CAC). METHODS CAC was induced in VEGFR2(ΔIEC) mice, which do not express VEGFR2 in the intestinal epithelium, and VEGFR2(fl/fl) mice (controls) by administration of azoxymethane followed by dextran sodium sulfate. Tumor development and inflammation were determined by endoscopy. Colorectal tissues were collected for immunoblot, immunohistochemical, and quantitative polymerase chain reaction analyses. Findings from mouse tissues were confirmed in human HCT116 colorectal cancer cells. We analyzed colorectal tumor samples from patients before and after treatment with bevacizumab. RESULTS After colitis induction, VEGFR2(ΔIEC) mice developed significantly fewer tumors than control mice. A greater number of intestinal tumor cells from VEGFR2(ΔIEC) mice were in senescence than tumor cells from control mice. We found VEGFR2 to activate phosphatidylinositol-4,5-bisphosphate-3-kinase and AKT, resulting in inactivation of p21 in HCT116 cells. Inhibitors of VEGFR2 and AKT induced senescence in HCT116 cells. Tumor cell senescence promoted an anti-tumor immune response by CD8(+) T cells in mice. Patients whose tumor samples showed an increase in the proportion of senescent cells after treatment with bevacizumab had longer progression-free survival than patients in which the proportion of senescent tumor cells did not change before and after treatment. CONCLUSIONS Inhibition of VEGFR2 signaling leads to senescence of human and mouse colorectal cancer cells. VEGFR2 interacts with phosphatidylinositol-4,5-bisphosphate-3-kinase and AKT to inactivate p21. Colorectal tumor senescence and p21 level correlate with patient survival during treatment with bevacizumab.

IL-33 signaling contributes to the pathogenesis of myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) are characterized by the clonal expansion of one or more myeloid cell lineage. In most cases, proliferation of the malignant clone is ascribed to defined genetic alterations. MPNs are also associated with aberrant expression and activity of multiple cytokines; however, the mechanisms by which these cytokines contribute to disease pathogenesis are poorly understood. Here, we reveal a non-redundant role for steady-state IL-33 in supporting dysregulated myelopoiesis in a murine model of MPN. Genetic ablation of the IL-33 signaling pathway was sufficient and necessary to restore normal hematopoiesis and abrogate MPN-like disease in animals lacking the inositol phosphatase SHIP. Stromal cell-derived IL-33 stimulated the secretion of cytokines and growth factors by myeloid and non-hematopoietic cells of the BM, resulting in myeloproliferation in SHIP-deficient animals. Additionally, in the transgenic JAK2V617F model, the onset of MPN was delayed in animals lacking IL-33 in radio-resistant cells. In human BM, we detected increased numbers of IL-33-expressing cells, specifically in biopsies from MPN patients. Exogenous IL-33 promoted cytokine production and colony formation by primary CD34+ MPN stem/progenitor cells from patients. Moreover, IL-33 improved the survival of JAK2V617F-positive cell lines. Together, these data indicate a central role for IL-33 signaling in the pathogenesis of MPNs.