TH2 heterogeneity: Does function follow form? Prussin, Calman; Yin, Yuzhi; Upadhyaya, Bhaskar
Journal of allergy and clinical immunology,
12/2010, Letnik:
126, Številka:
6
Journal Article
Recenzirano
Odprti dostop
TH2 immune responses are required for the 2 fundamental pathological processes characteristic of allergic disease: IgE-mediated hypersensitivity and eosinophilic inflammation. The 3 established TH2 ...cytokines, IL-4, IL-5, and IL-13, each play a nonredundant role in allergic disease pathology. The recent explosion of TH subpopulations combined with the wide availability of polychromatic cytokine staining has facilitated the discovery of TH2 lineage heterogeneity. In this article we review TH2 heterogeneity and ask the following question: At what point do these subpopulations graduate from in vitro curiosities to immunologically robust therapeutic targets? We propose criteria to establish a T-cell subset as a biologically relevant entity and address the evidence to support these TH2 subpopulations having a unique function or specific contribution to allergic pathology or host defense.
Mast cell hyperactivity and accumulation in tissues are associated with allergy and other mast cell-related disorders. However, the molecular pathways regulating mast cell survival in homeostasis and ...disease are not completely understood. As glioma-associated oncogene (GLI) proteins are involved in both tissue homeostasis and in the hematopoietic system by regulating cell fate decisions, we sought to investigate the role for GLI proteins in the control of proliferation and survival of human mast cells. GLI1 transcripts were present in primary human mast cells and mast cell lines harboring or not activating mutations in the tyrosine kinase receptor KIT (HMC-1.1 and HMC-1.2, and LAD2 cells, respectively), while GLI2 transcripts were only present in HMC-1.1 and HMC-1.2 cells, suggesting a role for oncogenic KIT signaling in the regulation of GLI2. Reduction in GLI activity by small molecule inhibitors, or by shRNA-mediated knockdown of GLI1 or GLI2, led to increases in apoptotic cell death in both cultured human and murine mast cells, and reduced the number of peritoneal mast cells in mice. Although GLI proteins are typically activated
the hedgehog pathway, steady-state activation of GLI in mast cells occurred primarily
non-canonical pathways. Apoptosis induced by GLI silencing was associated with a downregulation in the expression of KIT and of genes that influence p53 stability and function including USP48, which promotes p53 degradation; and iASPP, which inhibits p53-induced transcription, thus leading to the induction of p53-regulated apoptotic genes. Furthermore, we found that GLI silencing inhibited the proliferation of neoplastic mast cell lines, an effect that was more pronounced in rapidly growing cells. Our findings support the conclusion that GLI1/2 transcription factors are critical regulators of mast cell survival and that their inhibition leads to a significant reduction in the number of mast cells
and
, even in cells with constitutively active KIT variants. This knowledge can potentially be applicable to reducing mast cell burden in mast cell-related diseases.
Each of the three Th2 cytokine genes, IL-4, IL-5, and IL-13, has different functions. We hypothesized that Th2 heterogeneity could yield Th2 subpopulations with different cytokine expression and ...effector functions. Using multiple approaches, we demonstrate that human Th2 cells are composed of two major subpopulations: a minority IL-5(+) (IL-5(+), IL-4(+), IL-13(+)) and majority IL-5(-) Th2 (IL-5(-), IL-4(+), IL-13(+)) population. IL-5(+) Th2 cells comprised only 20% of all Th2 cells. Serial rounds of in vitro differentiation initially yielded IL-5(-) Th2, but required multiple rounds of differentiation to generate IL-5(+) Th2 cells. IL-5(+) Th2 cells expressed less CD27 and greater programmed cell death-1 than IL-5(-) Th2 cells, consistent with their being more highly differentiated, Ag-exposed memory cells. IL-5(+) Th2 cells expressed greater IL-4, IL-13, and GATA-3 relative to IL-5(-) Th2 cells. GATA-3 and H3K4me(3) binding to the IL5 promoter (IL5p) was greater in IL-5(+) relative to IL-5(-) Th2 cells, whereas there was no difference in their binding to the IL4p and IL13p. Conversely, H3K27me(3) binding to the IL5p was greater in IL-5(-) Th2 cells. These findings demonstrate Th2 lineage heterogeneity, in which the IL5 gene is regulated in a hierarchical manner relative to other Th2 genes. IL-5(+) Th2 cells are phenotypically distinct and have epigenetic changes consistent with greater IL5p accessibility. Recurrent antigenic exposure preferentially drives the differentiation of IL-5(+) Th2 cells. These results demonstrate that IL-5(+) and IL-5(-) Th2 cells, respectively, represent more and less highly differentiated Th2 cell subpopulations. Such Th2 subpopulations may differentially contribute to Th2-driven pathology.
Laboratory of allergic diseases 2 (LAD2) human mast cells were developed over 15 years ago and have been distributed worldwide for studying mast cell proliferation, receptor expression, mediator ...release/inhibition, and signaling. LAD2 cells were derived from CD34+ cells following marrow aspiration of a patient with aggressive mastocytosis with no identified mutations in
. Another aspiration gave rise to a second cell line which has recently been re-established (LADR). We queried whether LADR had unique properties for the preclinical study of human mast cell biology.
LADR and LAD2 cells were cultured under identical conditions. Experiments examined proliferation, beta-hexosaminidase (β-hex) release, surface receptor and granular protease expression, infectivity with HIV, and gene expression.
LADR cells were larger and more granulated as seen with Wright-Giemsa staining and flow cytometry, with cell numbers doubling in 4 weeks, in contrast to LAD2 cells, which doubled every 2 weeks. Both LADR and LAD2 cells released granular contents following aggregation of FcεRI. LADR cells showed log-fold increases in FcεRI/CD117 and expressed CD13, CD33, CD34, CD63, CD117, CD123, CD133, CD184, CD193, and CD195, while LAD2 cells expressed CD33, CD34, CD63, CD117, CD133, CD193 but not CD13, CD123, CD184, or CD195. LADR tryptase expression was one-log-fold increased. LADR cell and LAD2 cell chymase expression were similar. Both cell lines could be infected with T-tropic, M-tropic, and dual tropic HIV. Following monomeric human IgE stimulation, LADR cells showed greater surface receptor and mRNA expression for CD184 and CD195. Expression arrays revealed differences in gene upregulation, especially for the suppressor of cytokine signaling (SOCS) family of genes with their role in JAK2/STAT3 signaling and cellular myelo
ytomatosis oncogene (c-MYC) in cell growth and regulation.
LADR cells are thus unique in that they exhibit a slower proliferation rate, are more advanced in development, have increased FcεRI/CD117 and tryptase expression, have a different profile of gene expression, and show earlier infectivity with HIV-BAL, LAV, and TYBE when compared to LAD2 cells. This new cell line is thus a valuable addition to the few FcεRI+ human mast cell lines previously described and available for scientific inquiry.
The diversity of autologous cells being used and investigated for cancer therapy continues to increase. Mast cells (MCs) are tissue cells that contain a unique set of anti-cancer mediators and are ...found in and around tumors. We sought to exploit the anti-tumor mediators in MC granules to selectively target them to tumor cells using tumor specific immunoglobin E (IgE) and controllably trigger release of anti-tumor mediators upon tumor cell engagement. We used a human HER2/
-specific IgE to arm human MCs through the high affinity IgE receptor (FcεRI). The ability of MCs to bind to and induce apoptosis of HER2/
-positive cancer cells
and
was assessed. The interactions between MCs and cancer cells were investigated in real time using confocal microscopy. The mechanism of action using cytotoxic MCs was examined using gene array profiling. Genetically manipulating autologous MC to assess the effects of MC-specific mediators have on apoptosis of tumor cells was developed using siRNA. We found that HER2/
tumor-specific IgE-sensitized MCs bound, penetrated, and killed HER2/
-positive tumor masses
. Tunneling nanotubes formed between MCs and tumor cells are described that parallel tumor cell apoptosis. In solid tumor, human breast cancer (BC) xenograft mouse models, infusion of HER2/
IgE-sensitized human MCs co-localized to BC cells, decreased tumor burden, and prolonged overall survival without indications of toxicity. Gene microarray of tumor cells suggests a dependence on TNF and TGFβ signaling pathways leading to apoptosis. Knocking down MC-released tryptase did not affect apoptosis of cancer cells. These studies suggest MCs can be polarized from Type I hypersensitivity-mediating cells to cytotoxic cells that selectively target tumor cells and specifically triggered to release anti-tumor mediators. A strategy to investigate which MC mediators are responsible for the observed tumor killing is described so that rational decisions can be made in the future when selecting which mediators to target for deletion or those that could further polarize them to cytotoxic MC by adding other known anti-tumor agents. Using autologous human MC may provide further options for cancer therapeutics that offers a unique anti-cancer mechanism of action using tumor targeted IgE's.
To the Editor: The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that integrates metabolic and activation signals to regulate cell proliferation and differentiation. mTOR plays ...a major role in T-cell proliferation and differentiation.1-3 The transplant drug rapamycin is the canonical inhibitor of mTOR and in the 5 to 10 nM concentration range used clinically, rapamycin largely exerts its effect via inhibition of mTOR complex 1 (mTORC1).4 Recently, several groups have characterized an IL-5+ pathogenic effector TH2 (peTh2) subpopulation with enhanced effector function.4 Notably, peTH2 cells demonstrate greater proinflammatory function in murine models of asthma and atopic dermatitis (AD) and are increased in human eosinophilic gastrointestinal diseases (EGID) and AD.5-9 Because of their pathogenic role in eosinophilic inflammation, we sought to examine mTOR function within the human peTH2 subpopulation in samples from subjects with eosinophilic asthma or EGID (see Table E1 in this article's Online Repository at www.jacionline.org). To investigate the mTOR pathway in human TH1 and TH2 cell responses, we used dye dilution proliferation cultures coupled with intracellular cytokine staining (see this article's Methods section in the Online Repository at www.jacionline.org) to examine the dominant TH1- or TH2-cell proliferative responses associated with respiratory syncytial virus or house dust mite (HDM) antigen, respectively (see Fig E1 in this article's Online Repository at www.jacionline.org). ...a dose response using a Boolean gating strategy was performed to examine the differential effect of rapamycin on IL-5+ TH2 (IL-5+, IL-13+) versus IL-5− TH2 (IL-5−, IL-13+) versus TH1 (IFN-γ+) cells. No inhibition was noted when...
Stem cell antigen (Sca)-1/Ly6A, a glycerophosphatidylinositol-linked surface protein, was found to be associated with murine stem cell-and progenitor cell-enriched populations, and also has been ...linked to the capacity of tumor-initiating cells. Despite these interesting associations, this protein's functional role in these processes remains largely unknown. To identify the mechanism underlying the protein's possible role in mammary tumorigenesis, Sca-1 expression was examined in Sca-1+/EGFP mice during carcinogenesis. Mammary tumor cells derived from these mice readily engrafted in syngeneic mice, and tumor growth was markedly inhibited on down-regulation of Sca-1 expression. The latter effect was associated with significantly elevated expression of the TGF-β ligand growth differentiation factor-10 (GDF10), which was found to selectively activate TGF-β receptor (TβRI/II)-dependent Smad3 phosphorylation. Overexpression of GDF10 attenuated tumor formation; conversely, silencing of GDF10 expression reversed these effects. Sca-1 attenuated GDF10-dependent TGF-β signaling by disrupting the heterodimerization of TßRI and TßRIl receptors. These findings suggest a new functional role for Sca-1 in maintaining tumorigenicity, in part by acting as a potent suppressor of TGF-β signaling.
Results Rapamycin inhibited antigen specific proliferation of Th2 cells more than Th1 cells (p< 0.001). ...IL-5+ Th2 cell responses demonstrated the greatest inhibition.
Mastocytosis is a disorder resulting from an abnormal mast cell (MC) accumulation in tissues that is often associated with the D816V mutation in KIT, the tyrosine kinase receptor for stem cell ...factor. Therapies available to treat aggressive presentations of mastocytosis are limited, thus exploration of novel pharmacological targets that reduce MC burden is desirable. Since increased generation of the lipid mediator sphingosine-1-phosphate (S1P) by sphingosine kinase (SPHK) has been linked to oncogenesis, we studied the involvement of the two SPHK isoforms (SPHK1 and SPHK2) in the regulation of neoplastic human MC growth. While SPHK2 inhibition prevented entry into the cell cycle in normal and neoplastic human MCs with minimal effect on cell survival, SPHK1 inhibition caused cell cycle arrest in G2/M and apoptosis, particularly in D816V-KIT MCs. This was mediated
activation of the DNA damage response (DDR) cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated M-phase inducer phosphatase 3 depletion, and p53 activation. Combination treatment of SPHK inhibitors with KIT inhibitors showed greater growth inhibition of D816V-KIT MCs than either inhibitor alone. Furthermore, inhibition of SPHK isoforms reduced the number of malignant bone marrow MCs from patients with mastocytosis and the growth of D816V-KIT MCs in a xenograft mouse model. Our results reveal a role for SPHK isoforms in the regulation of growth and survival in normal and neoplastic MCs and suggest a regulatory function for SPHK1 in the DDR in MCs with KIT mutations. The findings also suggest that targeting the SPHK/S1P axis may provide an alternative to tyrosine kinase inhibitors, alone or in combination, for the treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation.
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