Kiyomi Mizugishi

IgE-dependent Activation of Sphingosine Kinases 1 and 2 and Secretion of Sphingosine 1-Phosphate Requires Fyn Kinase and Contributes to Mast Cell Responses

Engagement of the high affinity receptor for IgE (FcϵRI) on mast cells results in the production and secretion of sphingosine 1-phosphate (S1P), a lipid metabolite present in the lungs of allergen-challenged asthmatics. Herein we report that two isoforms of sphingosine kinase (SphK1 and SphK2) are expressed and activated upon FcϵRI engagement of bone marrow-derived mast cells (BMMC). Fyn kinase is required for FcϵRI coupling to SphK1 and -2 and for subsequent S1P production. Normal activation of SphK1 and -2 was restored by expression of wild type Fyn but only partly with a kinase-defective Fyn, indicating that induction of SphK1 and SphK2 depended on both catalytic and noncatalytic properties of Fyn. Downstream of Fyn, the requirements for SphK1 activation differed from that of SphK2. Whereas SphK1 was considerably dependent on the adapter Grb2-associated binder 2 and phosphatidylinositol 3-OH kinase, SphK2 showed minimal dependence on these molecules. Fyn-deficient BMMC were defective in chemotaxis and, as previously reported, in degranulation. These functional responses were partly reconstituted by the addition of exogenous S1P to FcϵRI-stimulated cells. Taken together with our previous study, which demonstrated delayed SphK activation in Lyn-deficient BMMC, we propose a cooperative role between Fyn and Lyn kinases in the activation of SphKs, which contributes to mast cell responses.

Maternal disturbance in activated sphingolipid metabolism causes pregnancy loss in mice

Uterine decidualization, a process that occurs in response to embryo implantation, is critical for embryonic survival and thus is a key event for successful pregnancy. Here we show that the sphingolipid metabolic pathway is highly activated in the deciduum during pregnancy and disturbance of the pathway by disruption of sphingosine kinase (Sphk) genes causes defective decidualization with severely compromised uterine blood vessels, leading to early pregnancy loss. Sphk-deficient female mice (Sphk1(-/-)Sphk2(+/-)) exhibited both an enormous accumulation of dihydrosphingosine and sphingosine and a reduction in phosphatidylethanolamine levels in pregnant uteri. These mice also revealed increased cell death in decidual cells, decreased cell proliferation in undifferentiated stromal cells, and massive breakage of decidual blood vessels, leading to uterine hemorrhage and early embryonic lethality. Thus, sphingolipid metabolism regulates proper uterine decidualization and blood vessel stability. Our findings also suggest that disturbance in sphingolipid metabolism may be considered as a cause of pregnancy loss in humans.

Uric acid induces NADPH oxidase-independent neutrophil extracellular trap formation.

Neutrophil extracellular traps (NETs) are composed of extracellular DNA fibers with antimicrobial peptides that capture and kill microbes. NETs play a critical role in innate host defense and in autoimmune and inflammatory diseases. While the mechanism of NET formation remains unclear, reactive oxygen species (ROS) produced via activation of NADPH oxidase (Nox) are known to be an important requirement. In this study, we investigated the effect of uric acid (UA) on NET formation. UA, a well-known ROS scavenger, was found to suppress Nox-dependent ROS release in a dose-dependent manner. Low concentrations of UA significantly inhibited Nox-dependent NET formation. However, high concentrations of UA unexpectedly induced, rather than inhibited, NET formation. NETs were directly induced by UA alone in a Nox-independent manner, as revealed by experiments using control neutrophils treated with ROS inhibitors or neutrophils of patients with chronic granulomatous disease who have a congenital defect in ROS production. Furthermore, we found that UA-induced NET formation was partially mediated by NF-κB activation. Our study is the first to demonstrate the novel function of UA in NET formation and may provide insight into the management of patients with hyperuricemia.

Serum Neutrophil Extracellular Trap Levels Predict Thrombotic Microangiopathy after Allogeneic Stem Cell Transplantation

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a devastating complication of hematopoietic stem cell transplantation. TA-TMA likely represents the final stage of vascular endothelial injury; however, its pathophysiology is largely unknown, making clinical management difficult. Recently, the association of neutrophil extracellular traps (NETs) with the development of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome has been reported. Thus, we explored whether NETs are also relevant to the occurrence of TA-TMA. We retrospectively analyzed post-transplant trends of serum NET levels in 90 patients, 11 of whom developed TA-TMA. Relative to baseline (before the conditioning regimen), elevated serum NET levels either at 4 weeks after transplantation or as early as the day of transplantation were associated with significantly increased risk of TA-TMA. In contrast, thrombomodulin, a potential marker for TA-TMA, was not helpful to predict the occurrence of TA-TMA in our study. In addition, we directly detected glomerular deposition of NETs in 2 TA-TMA patients. Increased NET levels are a significant risk factor for TA-TMA, suggesting that NET level is a useful biomarker for TA-TMA.

Ozone production by amino acids contributes to killing of bacteria

Reactive oxygen species produced by phagocytosing neutrophils are essential for innate host defense against invading microbes. Previous observations revealed that antibody-catalyzed ozone formation by human neutrophils contributed to the killing of bacteria. In this study, we discovered that 4 amino acids themselves were able to catalyze the production of an oxidant with the chemical signature of ozone from singlet oxygen in the water-oxidation pathway, at comparable level to antibodies. The resultant oxidant with the chemical signature of ozone exhibited significant bactericidal activity in our distinct cell-free system and in human neutrophils. The results also suggest that an oxidant with the chemical signature of ozone produced by neutrophils might potentiate a host defense system, when the host is challenged by high doses of infectious agents. Our findings provide biological insights into the killing of bacteria by neutrophils.

Phagocytosis by human monocytes is required for the secretion of presepsin

BACKGROUND Presepsin, a soluble CD14 subtype, is increasingly recognized as a useful biomarker for sepsis. However, little is known about the biological characteristics of presepsin in humans. Furthermore, there are no studies evaluating clinical validity of measuring the presepsin levels in patients after allogeneic hematopoietic cell transplantation, irrespective of the high frequency of sepsis. METHODS For in vitro assays, neutrophils and monocytes were isolated from the peripheral blood of healthy controls and treated with bacteria or inflammatory stimuli. Presepsin levels in the culture supernatants were measured by enzyme linked immunosorbent assay (ELISA). For a cohort study of patients undergoing allogeneic hematopoietic cell transplantation, serum samples were subjected to ELISA for presepsin, and the relationship of presepsin levels with the incidence of transplantation-related complications was statistically analyzed. RESULTS We found that monocytes were the main source of presepsin in humans. Presepsin secretion by human monocytes was triggered by bacterial phagocytosis or sterile phagocytic stimulus, such as monosodium urate crystals, rather than soluble inflammatory stimuli. Elastase, a serine protease in human monocytes, mediated CD14 cleavage to produce presepsin. The cohort study demonstrated that high presepsin values were significantly associated with an increased incidence of hemophagocytic syndrome, as well as bacteremia. Moreover, patients with higher presepsin values revealed inferior overall survival, suggesting that presepsin can also be a prognostic marker for transplantation. CONCLUSIONS In this study, we clarified the biological features of presepsin in humans. Our study may be useful for increasing the clinical application of presepsin as a biomarker.

Plasmacytoid Dendritic Cell Activation and IFN-α Production Are Prominent Features of Murine Autoimmune Pancreatitis and Human IgG4-Related Autoimmune Pancreatitis

The abnormal immune response accompanying IgG4-related autoimmune pancreatitis (AIP) is presently unclear. In this study, we examined the role of plasmacytoid dendritic cell (pDC) activation and IFN-α production in this disease as well as in a murine model of AIP (MRL/Mp mice treated with polyinosinic-polycytidylic acid). We found that the development of AIP in treated MRL/Mp mice occurred in parallel with pancreatic accumulation of pDCs producing IFN-α, and with pDC depletion and IFN-α-blocking studies, we showed that such accumulation was necessary for AIP induction. In addition, we found that the pancreas of treated MRL/Mp mice contained neutrophil extracellular traps (NETs) shown previously to stimulate pDCs to produce IFN-α. Consistent with these findings, we found that patients with IgG4-related AIP also exhibited pancreatic tissue localization of IFN-α–expressing pDCs and had significantly higher serum IFN-α levels than healthy controls. In addition, the inflamed pancreas of these patients but not controls also contained NETs that were shown to be capable of pDC activation. More importantly, patient pDCs cultured in the presence of NETs produced greatly increased levels of IFN-α and induced control B cells to produce IgG4 (but not IgG1) as compared with control pDCs. These data suggest that pDC activation and production of IFN-α is a major cause of murine AIP; in addition, the increased pDC production of IFN-α and its relation to IgG4 production observed in IgG4-related AIP suggest that this mechanism also plays a role in the human disease.

Risk factors for hypogammaglobulinemia after allo-SCT

Allo-SCT is an established therapy primarily used to eradicate various types of hematological malignancy. However, defects in cellular and humoral immunity persist for months (even years) after allo-SCT. Successful reconstitution of the immune system is important for decreasing the complications of allo-SCT, such as infections and GVHD. Thus, identification of the risk factors for hypogammaglobulinemia will help in improving the outcomes of patients undergoing allo-SCT. However, to the best of our knowledge, only one large-scale study has explored the risk factors for hypogammaglobulinemia in adults. The authors of that study found that acute GVHD (aGVHD), younger patients, lack of treatment with anti-thymocyte globulin (ATG), female donor to male recipient and treatment with CsA and MTX as prophylaxis against aGVHD were associated significantly with an increased risk for hypogammaglobulinemia after allo-SCT. However, that study did not involve newly developed procedures such as cord blood transplantation (CBT) or analyses of mycophenolate mofetil (MMF) usage as prophylaxis for GVHD. In a recent study of 185 pediatric patients undergoing allo-SCT, the risk factors identified for hypogammaglobulinemia were younger age, lower pretransplant IgG level, malignant disease, use of unrelated donor stem cell source and development of aGVHD. We retrospectively investigated the risk factors for hypogammaglobulinemia in adult patients who underwent allo-SCT within our department between January 2001 and June 2012. This study was approved by the ethics committee of Kyoto University (E1714). These subjects survived ⩾28 days, and their Ig levels were measured before conditioning regimens and at least twice after allo-SCT. We assessed 299 cases of allo-SCT in 278 patients. Patient characteristics and allo-SCT procedures are summarized in Table 1. Patients with multiple myeloma were excluded. Patients were divided into ‘high-risk’ and ‘standard-risk’ groups according to transplantation risk. Disease status at allo-SCT was divided into ‘controlled’ and ‘progressive’ disease. With respect to conditioning regimens, the definitions of myeloablative conditioning and reduced-intensity conditioning (RIC) were consistent with those of the RIC regimen workshop. The prophylaxis of aGVHD comprised a calcineurin inhibitor (CsA or FK506) plus short-term MTX, MMF (30mg/kg/day, orally from day 0 until day 30), or both. ATG was not used in any case. Grade 2–4 aGVHD and extensive chronic GVHD (cGVHD) were treated with corticosteroids. Serum IgG levels were measured before conditioning regimens, at 2 weeks, 1, 3, 6 and 9 months and 1 and 3 years after allo-SCT. Once relapse was diagnosed, subsequent data on IgG levels were excluded from the study. IgG levels were additionally measured as clinically indicated. Hypogammaglobulinemia was defined as serum IgG level o400mg/dL, and patients whose IgG level was o400 mg/ dL at least once were included in the hypogammaglobulinemia group. Patients with hypogammaglobulinemia or other conditions (for example, severe infection) received i.v. Ig replacement therapy according to the decision of the attending physician. Serum IgG levels after allo-SCT were compared with those before conditioning regimens using unpaired t tests. Univariate analyses of the cumulative incidence of hypogammaglobulinemia were undertaken using Kaplan–Meier methods considering relapse and TRM as a censor. Patients with GVHD only after the diagnosis of hypogammaglobulinemia were included in the non-GVHD group in this analysis. Factors with Po0.1 and occurrence of GVHD were subjected to multivariate analyses using Cox proportional hazard models. Statistical analyses were performed using R ver 2.13.0 (R Foundation for Statistical Computing, Vienna, Austria). The alpha level of all tests or the P-value was set at 0.05. Hypogammaglobulinemia was observed in 63 patients at 5–2077 (median, 89) days after allo-SCT. The cumulative incidence of hypogammaglobulinemia 1 or 3 years after allo-SCT was 24.1% or 27.1% (95% confidence interval, 18.2%–29.5% or 20.7%–33.1%), respectively. More than 88% of the patients in the hypogammaglobulinemia group fulfilled the criteria for hypogammaglobulinemia within 1 year after allo-SCT. Serum IgG levels decreased significantly as early as 2 weeks after allo-SCT compared with preconditioning levels (mean ± s.e., 963.1 ± 28.0 mg/dL vs 810.5 ± 24.9 mg/dL, Po0.01). IgG levels were lowest at 6 months (757.6 ± 39.9 mg/dL, Po0.01) and recovered to the preconditioning level 1 year after allo-SCT (958.8 ± 52.4 mg/dL, P= 0.99). Next, we explored the risk factors for hypogammaglobulinemia after allo-SCT. Univariate analyses revealed a significantly higher incidence of hypogammaglobulinemia in patients with lymphoid malignancies (particularly with rituximab treatment), high-risk SCT, progressive disease, history of allo-SCT, HLA mismatch, MMF usage, low pre-SCT IgG levels (o800mg/dL) and grade 2–4 aGVHD (Table 2). Nonsignificant risk factors were sex, age, donor sources, conditioning regimens, type of calcineurin inhibitor, MTX usage and cGVHD. Multivariate analyses showed that lymphoid

Risk factors for late-onset neutropenia after rituximab treatment of B-cell lymphoma

Abstract Objectives Late-onset neutropenia after rituximab (RTX) therapy (R-LON) has been widely reported, but clinical studies on a large number of cases are limited. In this study, we aimed to investigate the incidence and risk factors of R-LON. Patients and methods In this study, we retrospectively analyzed data of 213 enrolled B-cell lymphoma patients (male 114; female 99) treated with RTX at a single institution. R-LON was defined as otherwise unexplained grade III–IV neutropenia after RTX. The median age of the patients was 62 years, and 129 of them were initially diagnosed at advanced stages (stage III–IV). Results R-LON occurred in 19 patients within a median of 121 (range, 49–474) days after the last RTX administration. The 1-year cumulative incidence was 9.0%. On univariate analysis, older age (>60 years), advanced stage, and purine analog or methotrexate administration were significant or borderline significant risk factors for R-LON, whereas sex, disease type, bone marrow invasion, combination with cytotoxic chemotherapeutic drugs, intensified therapy (compared with R-CHOP), prior autologous transplantation, and repeated RTX administration were not. On multivariate analysis, older age (hazard ratio (HR), 2.95) and advanced stage (HR, 3.56) were significant risk factors. Treatment with granulocyte colony-stimulating factor was feasible in grade IV R-LON patients with high risk of infection. Discussion and conclusion Careful follow-up is therefore necessary after B-cell lymphoma treatment, especially in high-risk patients with advanced disease or of older age.

Enhanced generation of reactive oxygen species by interferon-γ may have contributed to successful treatment of invasive pulmonary aspergillosis in a patient with chronic granulomatous disease

Invasive pulmonary aspergillosis (IPA) is a life-threatening complication of chronic granulomatous disease (CGD), a rare inherited disorder of phagocytes that is characterized by a defect in the production of reactive oxygen species (ROS) caused by mutations in NADPH oxidase 2. Here, we report a case of successful treatment of IPA complicated with CGD by the administration of interferon-γ (IFN-γ) in combination with voriconazole. The patient carried a splice site mutation in the CYBB gene, and the neutrophils could produce a certain amount of ROS. In this case, augmentation of ROS generation in the patient’s neutrophils was observed after in vivo IFN-γ treatment, which may be attributable to the induction of a normal CYBB gene in the myeloid progenitor cells. This treatment, in combination with voriconazole, may have contributed to the reversal of IPA in this patient. These results suggest that the in vivo use of IFN-γ may augment ROS generation in CGD neutrophils, thus leading to the successful treatment of severe IPA.