Frann Antignano

Comprehensive microRNA expression profiling of the hematopoietic hierarchy

The hematopoietic system produces a large number of highly specialized cell types that are derived through a hierarchical differentiation process from a common stem cell population. miRNAs are critical players in orchestrating this differentiation. Here, we report the development and application of a high-throughput microfluidic real-time quantitative PCR (RT-qPCR) approach for generating global miRNA profiles for 27 phenotypically distinct cell populations isolated from normal adult mouse hematopoietic tissues. A total of 80,000 RT-qPCR assays were used to map the landscape of miRNA expression across the hematopoietic hierarchy, including rare progenitor and stem cell populations. We show that miRNA profiles allow for the direct inference of cell lineage relations and functional similarity. Our analysis reveals a close relatedness of the miRNA expression patterns in multipotent progenitors and stem cells, followed by a major reprogramming upon restriction of differentiation potential to a single lineage. The analysis of miRNA expression in single hematopoietic cells further demonstrates that miRNA expression is very tightly regulated within highly purified populations, underscoring the potential of single-cell miRNA profiling for assessing compartment heterogeneity.

Group 2 innate lymphoid cells facilitate sensitization to local, but not systemic, TH2-inducing allergen exposures

BACKGROUND Allergic inflammation involves the sensitization of naive CD4(+) T cells to allergens, resulting in a TH2-skewed inflammatory response. Although antigen presentation by dendritic cells to T cells in the lymph node is crucial for TH2 cell development, the innate signals that initiate adaptive type 2 inflammation and the role of group 2 innate lymphoid cells (ILC2s) are poorly understood. OBJECTIVE We sought to investigate the influence of ILC2s and the route of priming on the development of an adaptive type 2 immune response to lung allergens. METHODS Wild-type and ILC2-deficient mice were exposed intranasally or systemically to the TH2-inducing antigens house dust mite or ovalbumin in a model of allergic airway inflammation or the TH17-inducing bacterial antigen Saccharopolyspora rectivirgula in a model of hypersensitivity pneumonitis. The formation of an adaptive immune response was evaluated based on serum antibody titers and production of T cell-derived cytokines (IL-4, IL-5, IL-13 and IL-17A). RESULTS We find that lung ILC2s play a critical role in priming the adaptive type 2 immune response to inhaled allergens, including the recruitment of eosinophils, TH2 cytokine production and serum IgE levels. Surprisingly, systemic priming with ovalbumin, with or without adjuvants, circumvents the requirement for ILC2s in inducing TH2-driven lung inflammation. ILC2s were also found to be dispensable for the sensitization to TH1- or TH17-inducing antigens. CONCLUSION These data highlight a critical role for ILC2s in the development of adaptive type 2 responses to local, but not systemic, antigen exposure.

SHIP Represses the Generation of IL-3-Induced M2 Macrophages by Inhibiting IL-4 Production from Basophils

There is a great deal of interest in determining what regulates the generation of classically activated (M1) vs alternatively activated (M2) macrophages (Mφs) because of the opposing effects that these two Mφ subsets have on tumor progression. We show herein that IL-3 and, to a lesser extent, GM-CSF skew murine Mφ progenitors toward an M2 phenotype, especially in the absence of SHIP. Specifically, the addition of these cytokines, with or without M-CSF, to adherence- or lineage-depleted (Lin−) SHIP−/− bone marrow (BM) cells induces high levels of the M2 markers, arginase I, and Ym1 in the resulting mature Mφs. These in vitro-derived mature Mφs also display other M2 characteristics, including an inability to enhance anti-CD3-stimulated splenic T cell secretion of IFN-γ and low IL-12 and high IL-10 production in response to LPS. Not surprisingly, given that IL-3 and GM-CSF utilize STAT5 to trigger many downstream signaling pathways, this M2 phenotype is suppressed when STAT5−/− BM cells are used. Unexpectedly, however, this M2 phenotype is also suppressed when STAT6−/− BM cells are used, suggesting that IL-4- or IL-13-induced signaling might be involved. Consistent with this, we found that IL-3 and GM-CSF stimulate the production of IL-4, especially from SHIP−/− Lin− BM cells, and that neutralizing anti-IL-4 Abs block IL-3-induced M2 skewing. Moreover, we found that basophil progenitors within the Lin− BM are responsible for this IL-3- and GM-CSF-induced IL-4 production, and that SHIP represses M2 skewing not by preventing skewing within Mφs themselves but by inhibiting IL-4 production from basophils.

Control of the Hippo Pathway by Set7-Dependent Methylation of Yap

Methylation of nonhistone proteins is emerging as a regulatory mechanism to control protein function. Set7 (Setd7) is a SET-domain-containing lysine methyltransferase that methylates and alters function of a variety of proteins in vitro, but the in vivo relevance has not been established. We found that Set7 is a modifier of the Hippo pathway. Mice that lack Set7 have a larger progenitor compartment in the intestine, coinciding with increased expression of Yes-associated protein (Yap) target genes. Mechanistically, monomethylation of lysine 494 of Yap is critical for cytoplasmic retention. These results identify a methylation-dependent checkpoint in the Hippo pathway.

Bone Marrow-Derived Mast Cells Accumulate in the Central Nervous System During Inflammation but Are Dispensable for Experimental Autoimmune Encephalomyelitis Pathogenesis

Reports showing that W/Wv mice are protected from experimental autoimmune encephalomyelitis (EAE, a murine model of multiple sclerosis), have implicated mast cells as an essential component in disease susceptibility, but the role of mast cell trafficking has not been addressed. In this study, we have used both mast cell transplantation and genetic mutations (Cd34−/−, W/Wv, Wsh/Wsh) to investigate the role of mast cell trafficking in EAE in detail. We show, for the first time, that bone marrow-derived mast cells are actively recruited to the CNS during EAE. Unexpectedly, however, we found that EAE develops unabated in two independent genetic backgrounds in the complete absence of mast cells or bone marrow-derived mast cell reconstitution. We conclude that although mast cells do accumulate in the brain and CNS during demyelinating disease via peripheral mast cell trafficking, they are completely dispensable for development of disease.

SHIP Is Required for Dendritic Cell Maturation

Although several groups have investigated the role of SHIP in macrophage (Mϕ) development and function, SHIP’s contribution to the generation, maturation, and innate immune activation of dendritic cells (DCs) is poorly understood. We show herein that SHIP negatively regulates the generation of DCs from bone marrow precursors in vitro and in vivo, as illustrated by the enhanced expansion of DCs from SHIP−/− GM-CSF cultures, as well as increased numbers of DCs in the spleens of SHIP-deficient mice. Interestingly, however, these SHIP−/− DCs display a relatively immature phenotype and secrete substantially lower levels of IL-12 after TLR ligand stimulation than wild type DCs. This, in turn, leads to a dramatically reduced stimulation of Ag-specific T cell proliferation and Th1 cell responses in vitro and in vivo. This immature phenotype of SHIP−/− DCs could be reversed with the PI3K inhibitors LY294002 and wortmannin, suggesting that SHIP promotes DC maturation by reducing the levels of the PI3K second messenger phosphatidylinositol-3,4,5-trisphosphate. These results are consistent with SHIP being a negative regulator of GM-CSF–derived DC generation but a positive regulator of GM-CSF–derived DC maturation and function.

Methyltransferase G9A regulates T cell differentiation during murine intestinal inflammation

Inflammatory bowel disease (IBD) pathogenesis is associated with dysregulated CD4⁺ Th cell responses, with intestinal homeostasis depending on the balance between IL-17-producing Th17 and Foxp3⁺ Tregs. Differentiation of naive T cells into Th17 and Treg subsets is associated with specific gene expression profiles; however, the contribution of epigenetic mechanisms to controlling Th17 and Treg differentiation remains unclear. Using a murine T cell transfer model of colitis, we found that T cell-intrinsic expression of the histone lysine methyltransferase G9A was required for development of pathogenic T cells and intestinal inflammation. G9A-mediated dimethylation of histone H3 lysine 9 (H3K9me2) restricted Th17 and Treg differentiation in vitro and in vivo. H3K9me2 was found at high levels in naive Th cells and was lost following Th cell activation. Loss of G9A in naive T cells was associated with increased chromatin accessibility and heightened sensitivity to TGF-β1. Pharmacological inhibition of G9A methyltransferase activity in WT T cells promoted Th17 and Treg differentiation. Our data indicate that G9A-dependent H3K9me2 is a homeostatic epigenetic checkpoint that regulates Th17 and Treg responses by limiting chromatin accessibility and TGF-β1 responsiveness, suggesting G9A as a therapeutic target for treating intestinal inflammation.

MyD88-Dependent SHIP1 Regulates Proinflammatory Signaling Pathways in Dendritic Cells after Monophosphoryl Lipid A Stimulation of TLR4

We previously showed that monophosphoryl lipid A (MLA) activates TLR4 in dendritic cells (DCs) in a Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF)–biased manner: MLA produced from Salmonella minnesota Re595 induced signaling events and expression of gene products that were primarily TRIF dependent, whereas MyD88-dependent signaling was impaired. Moreover, when tested in TRIF-intact/MyD88-deficient DCs, synthetic MLA of the Escherichia coli chemotype (sMLA) showed the same activity as its diphosphoryl, inflammatory counterpart (synthetic diphosphoryl lipid A), indicating that TRIF-mediated signaling is fully induced by sMLA. Unexpectedly, we found that the transcript level of one proinflammatory cytokine was increased in sMLA-treated cells by MyD88 deficiency to the higher level induced by synthetic diphosphoryl lipid A, which suggested MyD88 may paradoxically help restrain proinflammatory signaling by TRIF-biased sMLA. In this article, we demonstrate that sMLA induces MyD88 recruitment to TLR4 and activates the anti-inflammatory lipid phosphatase SHIP1 in an MyD88-dependent manner. At the same time, MyD88-dependent signaling activity at the level of IL-1R–associated kinase 1 is markedly reduced. Increased SHIP1 activity is associated with reductions in sMLA-induced IκB kinase α/β and IFN regulatory factor 3 activation and with restrained expression of their downstream targets, endothelin-1 and IFN-β, respectively. Results of this study identify a pattern that is desirable in the context of vaccine adjuvant design: TRIF-biased sMLA can stimulate partial MyD88 activity, with MyD88-dependent SHIP1 helping to reduce proinflammatory signaling in DCs.

SHIP Represses Th2 Skewing by Inhibiting IL-4 Production from Basophils

We report that SHIP−/− mice, compared to SHIP+/+ mice, are Th2 skewed with elevated serum IgE and twice as many splenic CD4+ Th2 cells that, when stimulated with anti-CD3, produce more IL-4 and less IFN-γ. Exploring the reason for this Th2 skewing, we found that freshly isolated SHIP−/− splenic and bone marrow basophils are present in elevated numbers and secrete far more IL-4 in response to IL-3 or to FcεRI stimulation than do WT basophils. These SHIP−/− basophils markedly skew wild-type macrophage colony stimulating factor–derived macrophages toward an M2 phenotype, stimulate OT-II CD4+ Th cells to differentiate into Th2 cells, and trigger SHIP+/+ B cells to become IgE-producing cells. All these effects are completely abrogated with neutralizing anti–IL-4 Ab. Exploring the cell signaling pathways responsible for hyperproduction of IL-4 by SHIP−/− basophils, we found that IL-3–induced activation of the PI3K pathway is significantly enhanced and that PI3K inhibitors, especially a p110α inhibitor, dramatically suppresses IL-4 production from these cells. In vivo studies, in which basophils were depleted from mast cell-deficient SHIP+/+ and SHIP−/− mice, confirmed the central role that basophils play in the Th2 skewing of naive SHIP-deficient mice. Taken together, these studies demonstrate that SHIP is a potent negative regulator of IL-4 production from basophils and thus may be a novel therapeutic target for Th1- and Th2-related diseases.

SETD7 Controls Intestinal Regeneration and Tumorigenesis by Regulating Wnt/β-Catenin and Hippo/YAP Signaling

Intestinal tumorigenesis is a result of mutations in signaling pathways that control cellular proliferation, differentiation, and survival. Mutations in the Wnt/β-catenin pathway are associated with the majority of intestinal cancers, while dysregulation of the Hippo/Yes-Associated Protein (YAP) pathway is an emerging regulator of intestinal tumorigenesis. In addition, these closely related pathways play a central role during intestinal regeneration. We have previously shown that methylation of the Hippo transducer YAP by the lysine methyltransferase SETD7 controls its subcellular localization and function. We now show that SETD7 is required for Wnt-driven intestinal tumorigenesis and regeneration. Mechanistically, SETD7 is part of a complex containing YAP, AXIN1, and β-catenin, and SETD7-dependent methylation of YAP facilitates Wnt-induced nuclear accumulation of β-catenin. Collectively, these results define a methyltransferase-dependent regulatory mechanism that links the Wnt/β-catenin and Hippo/YAP pathways during intestinal regeneration and tumorigenesis.