Regulation of Reactive Oxygen Species and the Antioxidant Protein DJ-1 in Mastocytosis

Neoplastic accumulation of mast cells in systemic mastocytosis (SM) associates with activating mutations in the receptor tyrosine kinase KIT. Constitutive activation of tyrosine kinase oncogenes has been linked to imbalances in oxidant/antioxidant mechanisms in other myeloproliferative disorders. However, the impact of KIT mutations on the redox status in SM and the potential therapeutic implications are not well understood. Here, we examined the regulation of reactive oxygen species (ROS) and of the antioxidant protein DJ-1 (PARK-7), which increases with cancer progression and acts to lessen oxidative damage to malignant cells, in relationship with SM severity. ROS levels were increased in both indolent (ISM) and aggressive variants of the disease (ASM). However, while DJ-1 levels were reduced in ISM with lower mast cell burden, they rose in ISM with higher mast cell burden and were significantly elevated in patients with ASM. Studies on mast cell lines revealed that activating KIT mutations induced constant ROS production and consequent DJ-1 oxidation and degradation that could explain the reduced levels of DJ-1 in the ISM population, while IL-6, a cytokine that increases with disease severity, caused a counteracting transcriptional induction of DJ-1 which would protect malignant mast cells from oxidative damage. A mouse model of mastocytosis recapitulated the biphasic changes in DJ-1 and the escalating IL-6, ROS and DJ-1 levels as mast cells accumulate, findings which were reversed with anti-IL-6 receptor blocking antibody. Our findings provide evidence of increased ROS and a biphasic regulation of the antioxidant DJ-1 in variants of SM and implicate IL-6 in DJ-1 induction and expansion of mast cells with KIT mutations. We propose consideration of IL-6 blockade as a potential adjunctive therapy in the treatment of patients with advanced mastocytosis, as it would reduce DJ-1 levels making mutation-positive mast cells vulnerable to oxidative damage

S1P4 Regulates Passive Systemic Anaphylaxis in Mice but Is Dispensable for Canonical IgE-Mediated Responses in Mast Cells

Mast cells are key players in the development of inflammatory allergic reactions. Cross-linking of the high-affinity receptor for IgE (FcεRI) on mast cells leads to the generation and secretion of the sphingolipid mediator, sphingosine-1-phosphate (S1P) which is able, in turn, to transactivate its receptors on mast cells. Previous reports have identified the expression of two of the five receptors for S1P on mast cells, S1P1 and S1P2, with functions in FcεRI-mediated chemotaxis and degranulation, respectively. Here, we show that cultured mouse mast cells also express abundant message for S1P4. Genetic deletion of S1pr4 did not affect the differentiation of bone marrow progenitors into mast cells or the proliferation of mast cells in culture. A comprehensive characterization of IgE-mediated responses in S1P4-deficient bone marrow-derived and peritoneal mouse mast cells indicated that this receptor is dispensable for mast cell degranulation, cytokine/chemokine production and FcεRI-mediated chemotaxis in vitro. However, interleukin-33 (IL-33)-mediated enhancement of IgE-induced degranulation was reduced in S1P4-deficient peritoneal mast cells, revealing a potential negative regulatory role for S1P4 in an IL-33-rich environment. Surprisingly, genetic deletion of S1pr4 resulted in exacerbation of passive systemic anaphylaxis to IgE/anti-IgE in mice, a phenotype likely related to mast cell-extrinsic influences, such as the high circulating levels of IgE in these mice which increases FcεRI expression and consequently the extent of the response to FcεRI engagement. Thus, we provide evidence that S1P4 modulates anaphylaxis in an unexpected manner that does not involve regulation of mast cell responsiveness to IgE stimulation

IL-6 increases extracellular ROS levels in HMC-1 cells.

<p>(A) Changes in intracellular (left) and extracellular ROS (right) levels induced by IL-6 (50 ng/ml) in HMC-1.2 cells compared to LAD2 cells. LAD2 were pre-stimulated with SCF for 48 h (100 ng/ml) prior to IL-6 stimulation. (B) Changes in intracellular (left) and extracellular ROS (right) levels induced by 50 ng/ml IL-6 in P815 cells compared to normal BMMC. ROS was measured using a fluorescence assay. All experiments were repeated at least 3 times and data represents mean±SEM. <i>*P<0</i>.<i>05</i> and <i>**P<0</i>.<i>01</i>.</p

Proposed mechanisms for the alterations in ROS and DJ-1 homeostasis in SM.

<p>ISM patients with lower mast cell burden, tryptase values and IL-6 levels show a high ratio of ROS to DJ-1 (>22) as compared to healthy controls (<4) due to their reduced levels of DJ-1. This is driven by constitutive KIT activity in <i>KIT</i> mutated MCs which induces ROS production and causes oxidation and proteosomal degradation of oxidized DJ-1 (left panel). In ISM patients with higher mast cell burden and with advanced disease, the increasing levels of DJ-1 offset the ROS to DJ-1 ratio toward normal values (<8; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162831#pone.0162831.g001" target="_blank">Fig 1C</a>). This is driven by elevated levels of IL-6 in these patients, which causes DJ-1 transcription and compensates for the enhanced DJ-1 turnover. Reduction of oxidative imbalances may allow for MC expansion.</p

Constitutive KIT activation in cells with gain-of function KIT mutations causes increased ROS levels and decreased DJ-1.

<p>(A) Comparison of the intracellular levels of ROS (left panel) and (B) oxidized DJ-1 (Cys 106) and total DJ-1 in human mast cells carrying normal <i>KIT</i> (LAD2) or mutated <i>KIT</i> (HMC-1.1 and HMC-1.2). DJ-1 expression was silenced in HMC-1.2 using specific sh-RNA sequences to target DJ-1 (DJ-1 KD) or sh-RNA non-target control sequences (sh-RNA Con) (B, right panels). ROS levels in DJ-1 KD and sh-RNA Con cells were measured side by side, but for simplicity, ROS levels in sh-RNA Con are not shown in A since the levels were identical to untreated HMC-1.2 cells (sh-RNA Con: 4.24 μM±0.075 μM). Levels of ROS (C) and DJ-1 (D) in murine MCBS1 mast cells transduced with WT-human <i>KIT</i>, D816V-human <i>KIT</i> or empty vector as indicated. Presence of KIT and phosphorylated KIT in cells with D816V are shown by western blot (D, bottom panels). The values under the blots indicate average fold increases in the band intensities of DJ-1 or oxidized DJ-1 (corrected to their respective β-actin loading control) as compared to non-stimulated cells (n = 3); SDs were less than 10% of the mean</p

Adoptive transfer of mastocytoma P815 murine cells into DBA/2 mice causes serum alterations in DJ-1 and ROS that are reversed by anti-IL-6R antibody treatment.

<p>(A-C) Serum levels of IL-6 (A), ROS (B), and DJ-1 (C) in mice injected with P815 cells and given daily injections of 200 μg/mouse of anti-IL-6R antibody, isotype control antibody or PBS on days 10 through 16 as indicate. (D) Representative histologic images of dorsal skin and spleen samples stained with alcian blue/safranin to detect mast cell in naïve and P185-injected mice untreated or treated with either an isotype control or anti-IL-6R antibody. Below, average number of mast cells per field (5 fields/mouse, n = 4–5) (Axioskop2 plus microscope, total magnification 400X). (E) Representative plots of APC-KIT positive cells (P815 cells lack FcεRI in culture) and APC-KIT and PE-FcεRI double positive cells from blood sorted by FACS in the same experiment. Shown below are the average percentages of mast cells (KIT and FcεRI double positive) in circulation from the various groups of mice. The percentages of KIT positive cells in circulation were as follows: naïve: 0.36±0.07; P815-PBS: 0.91±0.03; P815-Isotype Ab: 0.58±0.03; P815-Anti-IL-6R Ab: 0.21±0.09. (F) Body weights of mice before adoptive transfer of P815 cells and at day 16 after transfer in all the indicated groups. Values represent mean ± SEM of a representative experiment using 4–5 mice per condition. *<i>P</i><0.05 and **<i>P</i><0.01. The experiment was repeated twice with similar results.</p