Src Homology 2–Containing Inositol 5′-Phosphatase 1 Negatively Regulates IFN-γ Production by Natural Killer Cells Stimulated with Antibody-Coated Tumor Cells and Interleukin-12

The signal transduction pathways that lead activated natural killer (NK) cells to produce cytokines, releases cytotoxic granules, or do both, are not clearly dissected. For example, phosphoinositide 3-kinases (PI3Ks) are key players in the execution of both functions, but the relative contribution of each isoform is unknown. We show here that the catalytic isoform p110␦, not p110␥, was required for interferon-␥ (IFN-␥), tumor necrosis factor-␣ (TNF-␣), and granulocyte macrophage colony-stimulating factor (GM-CSF) secretion, whereas neither was necessary for cytotoxicity. Yet, when both p110␦ and p110␥ isoforms were inactivated by a combination of genetic and biochemical approaches, cytotoxicity was decreased. NK-cell numbers were also affected by the lack of p110␦ but not p110␥ and more severely so in mice lacking both subunits. These results provide genetic evidence that p110␦ is the dominant PI3K isoform for cytokine secretion by NK cells and suggest that PI3Ks cooperate during NK-cell development and cytotoxicity. ( IntroductionCellular cytotoxicity and production of inflammatory cytokines are the 2 main functions of natural killer (NK) cells. Although distinct cell subpopulations can specialize in one function rather than the other, 1-3 most NK cells develop the potential to do both. Multiple signal transduction pathways can activate NK cells, including some that are also involved in B and T lymphocyte stimulations. 4 Some of the pathways are independent of Syk-family kinases and revolve around phosphoinositide 3-kinases (PI3Ks). 5,6 PI3Ks are divided into 3 subclasses on the basis of their structure. Class I PI3Ks, which are the most studied in mammalian cell signaling, are further subdivided in 2 groups. Class IA consists of 5 isoforms of regulatory subunits (p85␣, p50␣, p55␣, p85␤, and p55␥) and 3 isoforms of catalytic subunits (p110␣, p110␤, and p110␦), which can interact in various combinations and are mostly activated downstream of tyrosine kinase-associated receptors for growth factors, antigens, cytokines, and costimulatory molecules. 7 Class IB includes only one regulatory isoform (p101) and one catalytic isoform (p110␥), and they are mostly activated downstream of G-coupled protein receptors. 8 P110␥ and p110␦ are expressed primarily in leukocytes, whereas p110␣ and p110␤ are expressed in all cells. The recruitment and activation of class I PI3Ks take place through adaptor molecules containing PI3K binding motif (YXXM), which interacts with SH2 domains of the regulatory subunits. Activated class I PI3Ks convert phosphatidylinositol-4,5-bisphospate (PIP 2 ) to phosphatidylinositol-3,4,5-trisphospate (PIP 3 ). 9 PIP 3 acts as a binding site for various intracellular molecules containing pleckstrin-homology (PH) domains that mediate crucial downstream effector functions. For example, AKT/PKB, 10 Vav, 11 BTK (Bruton agammaglobulinemia tyrosine kinase), ITK (IL-2-inducible T-cell kinase), 12 and PLC␥ (phospholipase C-␥) contain PH domains. Class II and III PI3Ks regulate vesicle trafficking, 13 but lit...

Section: Discussionmentioning

confidence: 54%

Section: Introductionmentioning

confidence: 97%

The p110delta catalytic isoform of PI3K is a key player in NK-cell development and cytokine secretion

Kim

Saudemont

Webb

et al. 2007

“…Because SHIP1 normally targets the PI-3K lipid product phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P 3 ), 29 its suppression in miR-155 tg NK cells, in fact, leads to enhanced ERK activation. 10,30,41 One aspect of miR-mediated gene regulation is that each specific miR is predicted to target hundreds of genes. 42 As miR-155 has multiple targets, it is very likely that the mechanism and observed effects of miR-155 overexpression on NK-cell development, homeostasis, and function are not yet completely understood.…”

Section: Discussionmentioning

confidence: 99%

Overexpression of miR-155 causes expansion, arrest in terminal differentiation and functional activation of mouse natural killer cells

Trotta

Chen

Costinean³

et al. 2013

Self Cite

• miR-155 tg mice have increased NK-cell number, enhanced NK-cell survival, excess immature CD11b low CD27 high NK cells, and an activated phenotype.• miR-155 tg NK cells exhibit enhanced expansion, interferon-g production, AKT and ERK activation, and killing of lymphoma in vivo.It is known that microRNAs (miRs) are involved in lymphocyte development, homeostasis, activation, and occasionally malignant transformation. In this study, a miR-155 transgene (tg) was driven to be overexpressed off of the lck promoter in order to assess its effects on natural killer (NK) cell biology in vivo. miR-155 tg mice have an increase in NK-cell number with an excess of the CD11b low CD27 high NK subset, indicative of a halt in terminal NK-cell differentiation that proved to be intrinsic to the cell itself. The increase in NK cells results, in part, from improved survival in medium alone and enhanced expansion with endogenous or exogenous interleukin 15. Phenotypic and functional data from miR-155 tg NK cells showed constitutive activation and enhanced target cell conjugation, resulting in more potent antitumor activity in vitro and improved survival of lymphoma-bearing mice in vivo when compared with wild type NK cells. The enhanced NK-cell survival, expansion, activation, and tumor control that result from overexpression of miR-155 in NK cells could be explained, in part, via diminished expression of the inositol phosphatase SHIP1 and increased activation of ERK and AKT kinases. Thus, the regulation of miR-155 is important for NKcell development, homeostasis, and activation. (Blood. 2013;121(16):3126-3134)

“…Regulation of SHIP1 expression in resting and IL-12 and/or IL-18 activated primary human NK cells SHIP1 5Ј inositol phosphatase is a negative regulator of IFN-␥ in both human and mouse NK cells, 13,16 and a primary target of miR-155. 28,32,33 To address whether there is an inverse correlation between SHIP1 expression and miR-155 expression in NK cells, we first analyzed SHIP1 mRNA and protein expression in primary NK cells stimulated with IL-12 and/or IL-18.…”

Section: The Role Of Mir-155 In the Regulation Of Ifn-␥ Production Bymentioning

confidence: 99%

“…13 SHIP1, by dampening the PI3K pathway, is able to negatively regulate IFN-␥ production by monokines and CD16 stimulation in both human and mouse NK cells. 13,16 MicroRNAs (miRs) are a highly conserved class of small, noncoding RNAs with important regulatory functions in proliferation, differentiation, signal transduction, immune responses, and carcinogenesis. 17 miRs regulate gene expression posttranscriptionally by forming imperfect base pairs with sequences in the 3Ј untranslated region (UTR) of genes.…”

Section: Introductionmentioning

confidence: 99%

miR-155 regulates IFN-γ production in natural killer cells

Trotta¹,

Chen²,

Ciarlariello³

et al. 2012

Self Cite

184

165

IntroductionHuman natural killer (NK) cells are CD56 ϩ CD3 Ϫ large granular lymphocytes of the innate immune system. 1,2 NK cells participate in early responses against infection or malignant transformation. In addition to their potent cytolytic activity, NK cells have an important immunoregulatory function in that they produce cytokines and chemokines when activated. In particular, NK cells produce IFN-␥, a critical cytokine for the clearance of infectious pathogens and tumor surveillance, 3 in response to a wide variety of stimuli, including both soluble factors and cellular interactions. 4,5 Dendritic cells and monocytes stimulated with bacterial cell wall components release monokines such as IL-12 and IL-18, which synergistically induce rapid and robust production of IFN-␥ by NK cells. 6 NK cells also express the low-affinity receptor for the Fc fragment of immunoglobulin (Ig)G (Fc␥RIIIA, CD16), which is the activating receptor required for triggering antibody dependent cellular cytotoxicity (ADCC) as well as the induction of IFN-␥. 7 IL-12 monokine stimulation in combination with CD16 activation induces a synergistic induction of IFN-␥ in NK cells, but to a lesser extent than does IL-12 and IL-18 costimulation. 8 This observation has recently been shown to have implications in the antibody therapy of breast cancer patients. In fact, the antitumor actions of the anti-HER2 monoclonal antibody trastuzumab are enhanced by IL-12 treatment in vivo, and this effect is dependent on NK cell production of IFN-␥. 9 The regulation of NK cell IFN-␥ production involves positive and negative mediators, such as kinases and phosphatases, as well as transcription factors. 10-14 SHIP1 is a hematopoietic cell specific 5Ј inositol phosphatase. 15 We have previously shown that SHIP1 is expressed differentially in CD56 bright and CD56 dim NK cell subsets, and is negatively modulated by the costimulation of IL-12 and IL-18. 13 SHIP1, by dampening the PI3K pathway, is able to negatively regulate IFN-␥ production by monokines and CD16 stimulation in both human and mouse NK cells. 13,16 MicroRNAs (miRs) are a highly conserved class of small, noncoding RNAs with important regulatory functions in proliferation, differentiation, signal transduction, immune responses, and carcinogenesis. 17 miRs regulate gene expression posttranscriptionally by forming imperfect base pairs with sequences in the 3Ј untranslated region (UTR) of genes. In turn, this prevents protein accumulation by repressing translation or by inducing mRNA degradation. 18 Recently, a general role of miRs in regulation of NK cell activation, survival, and function has been shown using conditional deletion of Dicer or Dgcr8. 19 A specific role of miR-150 in regulating development and maturation of mouse NK cells has also been reported. 20 Further, it has been shown that miR-181 promotes human NK cell development by regulating Notch signaling. 21 26 Of interest, miR-155 is overexpressed in NK-cell lymphoma/leukemia and this correlates with low levels of SHIP1 expression and u...