Lina Kozhaya

CCR5 is a receptor for Staphylococcus aureus leukotoxin ED

Pore-forming toxins are critical virulence factors for many bacterial pathogens and are central to Staphylococcus aureus-mediated killing of host cells. S. aureus encodes pore-forming bi-component leukotoxins that are toxic towards neutrophils, but also specifically target other immune cells. Despite decades since the first description of staphylococcal leukocidal activity, the host factors responsible for the selectivity of leukotoxins towards different immune cells remain unknown. Here we identify the human immunodeficiency virus (HIV) co-receptor CCR5 as a cellular determinant required for cytotoxic targeting of subsets of myeloid cells and T lymphocytes by the S. aureus leukotoxin ED (LukED). We further demonstrate that LukED-dependent cell killing is blocked by CCR5 receptor antagonists, including the HIV drug maraviroc. Remarkably, CCR5-deficient mice are largely resistant to lethal S. aureus infection, highlighting the importance of CCR5 targeting in S. aureus pathogenesis. Thus, depletion of CCR5+ leukocytes by LukED suggests a new immune evasion mechanism of S. aureus that can be therapeutically targeted.

The CLN9 Protein, a Regulator of Dihydroceramide Synthase

A new variant of a group of pediatric neurodegenerative diseases known as neuronal ceroid lipofuscinosis (NCL) or Batten disease has been identified. It is termed CLN9-deficient. CLN9-deficient fibroblasts have a distinctive phenotype of rapid growth and increased apoptosis and diminished levels of ceramide, dihydroceramide, and sphingomyelin. Transfection with CLN8 but not other NCL genes corrected growth and apoptosis in CLN9-deficient cells, although the entire CLN8 sequence was normal. CLN8 is one of the TRAM-Lag1-CLN8 proteins containing a Lag1 motif. The latter imparts (dihydro)ceramide synthase activity to yeast cells. Transfection with the yeast gene Lag1 Sc and the human homolog LASS1 increased ceramide levels and partially corrected growth and apoptosis in CLN9-deficient cells. LASS2,-4,,-5, and -6 also corrected growth and apoptosis. Dihydroceramide levels and dihydroceramide synthase activity were markedly diminished in CLN9-deficient cells. Sequencing of LASS1, LASS2, LASS4, LASS5, and LASS6 genes was normal, and expression levels were increased or normal in CLN9-deficient cells by reverse transcription-PCR. N-(4-Hydroxyphenyl)retinamide (4-HPR), a dihydroceramide synthase activator, corrected growth and apoptosis and increased dihydroceramide synthase activity. Ceramide levels dropped further, and there was no increase in de novo ceramide synthesis, probably due to the effects of 4-HPR as activator of dihydroceramide synthase and inhibitor of dihydroceramide desaturase. Fumonisin B1, a dihydroceramide synthase inhibitor, exaggerated the CLN9-deficient phenotype of accelerated growth, decreased ceramide and increased apoptosis. This was neutralized by 4-HPR. We conclude that the CLN9 protein may be a regulator of dihydroceramide synthase and that 4-HPR could be developed as a treatment for CLN9-deficient patients.

Cytokine signals through PI-3 kinase pathway modulate Th17 cytokine production by CCR6+ human memory T cells

PI-3K–mediated repression of FOXO1 and KLF2 promotes proinflammatory cytokine expression by lineage-committed human CCR6+ Th17/Th22 memory cells.

De novo N-palmitoylsphingosine synthesis is the major biochemical mechanism of ceramide accumulation following p53 up-regulation.

The tumor suppressor protein p53 and the putative lipid tumor suppressor ceramide play pivotal roles in inducing cell cycle arrest or in driving the cell towards apoptosis. Previously we had shown that, in a p53-dependent model of cell death, ceramide accumulated in a p53-dependent manner [Dbaibo GS, Pushkareva MY, Rachid RA, Alter N, Smyth MJ, Obeid LM, Hannun YA. J Clin Invest 1998;102:329-339]. In the current study, we investigated the biochemical pathways by which ceramide accumulated following p53 up-regulation. In both Molt-4 LXSN leukemia cells exposed to gamma-irradiation and in EB-1 colon cancer cells treated with ZnCl(2), p53 up-regulation led to de novo ceramide synthesis with predominance of N-palmitoylsphingosine (C16-ceramide) synthesis. The activation of the de novo pathway was not associated with increased activity of the key enzyme serine palmitoyltransferase (SPT) but rather with the increased activity of ceramide synthase. Furthermore, transcriptional up-regulation of the palmitoyl-specific Lass5 ceramide synthase gene was observed in Molt-4 but not in EB-1 cells. The SPT inhibitor ISP-1 or the ceramide synthase inhibitor fumonisin B1 led to substantial inhibition of ceramide accumulation in response to p53 up-regulation. Other biochemical pathways of ceramide generation such as sphingomyelinase activation were examined and found unlikely to contribute to p53-dependent ceramide formation. These studies indicate that p53 specifically drives de novo ceramide synthesis by activation of a ceramide synthase that favors the synthesis of N-palmitoylsphingosine.

Ceramide Inhibits IL-2 Production by Preventing Protein Kinase C-Dependent NF-κB Activation: Possible Role in Protein Kinase Cθ Regulation

The role of the sphingolipid ceramide in modulating the immune response has been controversial, in part because of conflicting data regarding its ability to regulate the transcription factor NF-κB. To help clarify this role, we investigated the effects of ceramide on IL-2, a central NF-κB target. We found that ceramide inhibited protein kinase C (PKC)-mediated activation of NF-κB. Ceramide was found to significantly reduce the kinase activity of PKCθ as well as PKCα, the critical PKC isozymes involved in TCR-induced NF-κB activation. This was followed by strong inhibition of IL-2 production in both Jurkat T leukemia and primary T cells. Exogenous sphingomyelinase, which generates ceramide at the cell membrane, also inhibited IL-2 production. As expected, the repression of NF-κB activation by ceramide led to the reduction of transcription of the IL-2 gene in a dose-dependent manner. Inhibition of IL-2 production by ceramide was partially overcome when NF-κB nuclear translocation was reconstituted with activation of a PKC-independent pathway by TNF-α or when PKCθ was overexpressed. Importantly, neither the conversion of ceramide to complex glycosphingolipids, which are known to have immunosuppressive effects, nor its hydrolysis to sphingosine, a known inhibitor of PKC, was necessary for its inhibitory activity. These results indicate that ceramide plays a negative regulatory role in the activation of NF-κB and its targets as a result of inhibition of PKC.

Ceramide and glutathione define two independently regulated pathways of cell death initiated by p53 in Molt-4 leukaemia cells

The tumour suppressor p53 induces cell death by launching several pathways that are either dependent on or independent of gene transcription. Accumulation of the sphingolipid ceramide and reactive oxygen species are among these pathways. Crossregulation of these two pathways is possible owing to the demonstrated inhibition of neutral sphingomyelinase by glutathione, the predominant cellular antioxidant, and has been observed in some cytokine-dependent cell-death models. In a model of irradiation-induced cell death of Molt-4 leukaemia cells, it was found that ceramide accumulation and glutathione depletion were dependent on p53 up-regulation. The loss of p53 owing to expression of the papilloma virus E6 protein inhibited both pathways after irradiation. However, in this model, these two pathways appeared to be independently regulated on the basis of the following observations: (1) glutathione supplementation or depletion did not alter irradiation-induced ceramide accumulation, (2) exogenous ceramide treatment did not induce glutathione depletion, (3) glutathione depletion was dependent on new protein synthesis, whereas ceramide accumulation was independent of it and (4) caspase activation was required for ceramide accumulation but not for glutathione depletion. Furthermore, caspase 9 activation, which is dependent on the release of mitochondrial cytochrome c, was not required for ceramide accumulation. This suggested that a caspase, other than caspase 9, was necessary for ceramide accumulation. Interestingly, Bcl-2 expression inhibited these pathways, indicating a possible role for mitochondria in regulating both pathways. These findings indicate that these two pathways exhibit cross-regulation in cytokine-dependent, but not in p53-dependent, cell-death models.

Human T-cell lymphotropic virus type I-transformed T-cells have a partial defect in ceramide synthesis in response to N-(4-hydroxyphenyl)retinamide

Treatment with the synthetic retinoid HPR [N-(4-hydroxyphenyl)-retinamide] causes growth arrest and apoptosis in HTLV-I (human T-cell lymphotropic virus type-I)-positive and HTLV-I-negative malignant T-cells. It was observed that HPR-mediated growth inhibition was associated with ceramide accumulation only in HTLV-I-negative cells. The aim of the present study was to investigate the mechanism by which HPR differentially regulates ceramide metabolism in HTLV-I-negative and HTLV-I-positive malignant T-cells. Clinically achievable concentrations of HPR caused early dose-dependent increases in ceramide levels only in HTLV-I-negative cells and preceded HPR-induced growth suppression. HPR induced de novo synthesis of ceramide in HTLV-I-negative, but not in HTLV-I-positive, cells. Blocking ceramide glucosylation in HTLV-I-positive cells, which leads to accumulation of endogenous ceramide, rendered these cells more sensitive to HPR. Exogenous cell-permeant ceramides that function partially by generating endogenous ceramide induced growth suppression in all tested malignant lymphocytes, were consistently found to be less effective in HTLV-I-positive cells confirming their defect in de novo ceramide synthesis. Owing to its multipotent activities, the HTLV-I-encoded Tax protein was suspected to inhibit ceramide synthesis. Tax-transfected Molt-4 and HELA cells were less sensitive to HPR and C6-ceramide mediated growth inhibition respectively and produced lower levels of endogenous ceramide. Together, these results indicate that HTLV-I-positive cells are defective in de novo synthesis of ceramide and that therapeutic modalities that bypass this defect are more likely to be successful.

Time of Initiating Enzyme Replacement Therapy Affects Immune Abnormalities and Disease Severity in Patients with Gaucher Disease

Gaucher disease (GD) patients often present with abnormalities in immune response that may be the result of alterations in cellular and/or humoral immunity. However, how the treatment and clinical features of patients impact the perturbation of their immunological status remains unclear. To address this, we assessed the immune profile of 26 GD patients who were part of an enzyme replacement therapy (ERT) study. Patients were evaluated clinically for onset of GD symptoms, duration of therapy and validated outcome measures for ERT. According to DS3 disease severity scoring system criteria, they were assigned to have mild, moderate or severe GD. Flow cytometry based immunophenotyping was performed to analyze subsets of T, B, NK, NKT and dendritic cells. GD patients showed multiple types of immune abnormalities associated to T and B lymphocytes with respect to their subpopulations as well as memory and activation markers. Skewing of CD4 and CD8 T cell numbers resulting in lower CD4/CD8 ratio and an increase in overall T cell activation were observed. A decrease in the overall B cells and an increase in NK and NKT cells were noted in the GD patients compared to controls. These immune alterations do not correlate with GD clinical type or level of biomarkers. However, subjects with persistent immune alterations, especially in B cells and DCs correlate with longer delay in initiation of ERT (ΔTX). Thus, while ERT may reverse some of these immune abnormalities, the immune cell alterations become persistent if therapy is further delayed. These findings have important implications in understanding the immune disruptions before and after treatment of GD patients.

Ceramide inhibits PKCθ by regulating its phosphorylation and translocation to lipid rafts in Jurkat cells

Protein kinase C theta (PKCθ) is a novel, calcium-independent member of the PKC family of kinases that was identified as a central player in T cell signaling and proliferation. Upon T cell activation by antigen-presenting cells, PKCθ gets phosphorylated and activated prior to its translocation to the immunological synapse where it couples with downstream effectors. PKCθ may be regulated by ceramide, a crucial sphingolipid that is known to promote differentiation, growth arrest, and apoptosis. To further investigate the mechanism, we stimulated human Jurkat T cells with either PMA or anti-CD3/anti-CD28 antibodies following induction of ceramide accumulation by adding exogenous ceramide, bacterial sphingomyelinase, or Fas ligation. Our results suggest that ceramide regulates the PKCθ pathway through preventing its critical threonine 538 (Thr538) phosphorylation and subsequent activation, thereby inhibiting the kinase’s translocation to lipid rafts. Moreover, this inhibition is not likely to be a generic effect of ceramide on membrane reorganization. Other lipids, namely dihydroceramide, palmitate, and sphingosine, did not produce similar effects on PKCθ. Addition of the phosphatase inhibitors okadaic acid and calyculin A reversed the inhibition exerted by ceramide, and this suggests involvement of a ceramide-activated protein phosphatase. Such previously undescribed mechanism of regulation of PKCθ raises the possibility that ceramide, or one of its derivatives, and may prove valuable in novel therapeutic approaches for disorders involving autoimmunity or excessive inflammation—where PKCθ plays a critical role.

Genomic Profiling of T-Cell Neoplasms Reveals Frequent JAK1 and JAK3 Mutations With Clonal Evasion From Targeted Therapies

Purpose The promise of precision oncology is that identification of genomic alterations will direct the rational use of molecularly targeted therapy. This approach is particularly applicable to neoplasms that are resistant to standard cytotoxic chemotherapy, like T-cell leukemias and lymphomas. In this study, we tested the feasibility of targeted next-generation sequencing in profiles of diverse T-cell neoplasms and focused on the therapeutic utility of targeting activated JAK1 and JAK3 in an index case. Patients and Methods Using Foundation One and Foundation One Heme assays, we performed genomic profiling on 91 consecutive T-cell neoplasms for alterations in 405 genes. The samples were sequenced to high uniform coverage with an Illumina HiSeq and averaged a coverage depth of greater than 500× for DNA and more than 8M total pairs for RNA. An index case of T-cell prolymphocytic leukemia (T-PLL), which was analyzed by targeted next-generation sequencing, is presented. T-PLL cells were analyzed by RNA-seq, in vitro drug testing, mass cytometry, and phospho-flow. Results One third of the samples had genomic aberrations in the JAK-STAT pathway, most often composed of JAK1 and JAK3 gain-of-function mutations. We present an index case of a patient with T-PLL with a clonal JAK1 V658F mutation that responded to ruxolitinib therapy. After relapse developed, an expanded clone that harbored mutant JAK3 M511I and downregulation of the phosphatase, CD45, was identified. We demonstrate that the JAK missense mutations were activating, caused pathway hyperactivation, and conferred cytokine hypersensitivity. Conclusion These results underscore the utility of profiling occurrences of resistance to standard regimens and support JAK enzymes as rational therapeutic targets for T-cell leukemias and lymphomas.