Monocyte- and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice.

Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was >100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and proinflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate ß-glucans, whereas inflammation in transgenic and wild-type mice was mild and transient. Taken together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation.

Downregulation of BCL2 by AT-101 enhances the antileukaemic effect of lenalidomide both by an immune dependant and independent manner.

Over-expression of anti-apoptotic BCL2 has been reported in chronic lymphocytic leukaemia (CLL), but targeting BCL2 alone did not yield appreciable clinical results. However, it was demonstrated that BCL2 inhibitors enhanced the clinical efficacy of chemo and immunotherapeutics. Lenalidomide, an immunomodulator, is clinically effective in CLL and can enhance the anti-CLL effects of CD20 targeting monoclonal antibody, rituximab. Here, we investigated the mechanism of immune-directed killing of lenalidomide in CLL and evaluated if concurrent targeting of CD20 and BCL2 can enhance this effect. In vitro treatment with lenalidomide enhanced the antibody-mediated cellular cytotoxicity (ADCC) directed by rituximab in autologous leukaemic cells. Furthermore, peripheral blood mononuclear cells obtained from patients after treatment with lenalidomide and rituximab showed increased ADCC in vitro versus control (pre-treatment sample). This effect was further enhanced with pre-treatment of tumour cells with AT-101 (a BH3 mimetic that functions as BCL2 antagonist). Our data suggest that AT-101 in combination with lenalidomide can potentially be an effective therapeutic regimen for CLL.

Histone deacetylase regulation of immune gene expression in tumor cells.

Epigenetic modifications of chromatin, such as histone acetylation, are involved in repression of tumor antigens and multiple immune genes that are thought to facilitate tumor escape. The status of acetylation in a cell is determined by the balance of the activities of histone acetyltransferases and histone deacetylases. Inhibitors of histone deacetylase (HDACi) can enhance the expression of immunologically important molecules in tumor cells and HDACi treated tumor cells are able to induce immune responses in vitro and in vivo. Systemic HDACi are in clinical trails in cancer and also being used in several autoimmune disease models. To date, 18 HDACs have been reported in human cells and more than thirty HDACi identified, although only a few immune targets of these inhibitors have been identified. Here, we discuss the molecular pathways employed by HDACi and their potential role in inducing immune responses against tumors. We review data suggesting that selection of target specific HDACi and combinations with other agents and modalities, including those that activate stress pathways, may further enhance the efficacy of epigenetic therapies.

An epigenetic vaccine model active in the prevention and treatment of melanoma.

BACKGROUND: Numerous immune genes are epigenetically silenced in tumor cells and agents such as histone deacetylase inhibitors (HDACi), which reverse these effects, could potentially be used to develop therapeutic vaccines. The conversion of cancer cells to antigen presenting cells (APCs) by HDACi treatment could potentially provide an additional pathway, together with cross-presentation of tumor antigens by host APCs, to establish tumor immunity. METHODS: HDACi-treated B16 melanoma cells were used in a murine vaccine model, lymphocyte subset depletion, ELISpot and Cytotoxicity assays were employed to evaluate immunity. Antigen presentation assays, vaccination with isolated apoptotic preparations and tumorigenesis in MHC-deficient mice and radiation chimeras were performed to elucidate the mechanisms of vaccine-induced immunity. RESULTS: HDACi treatment enhanced the expression of MHC class II, CD40 and B7-1/2 on B16 cells and vaccination with HDACi-treated melanoma cells elicited tumor specific immunity in both prevention and treatment models. Cytotoxic and IFN-gamma-producing cells were identified in splenocytes and CD4+, CD8+ T cells and NK cells were all involved in the induction of immunity. Apoptotic cells derived from HDACi treatments, but not H2O2, significantly enhanced the effectiveness of the vaccine. HDACi-treated B16 cells become APCs in vitro and studies in chimeras defective in cross presentation demonstrate direct presentation in vivo and short-term but not memory responses and long-term immunity. CONCLUSION: The efficacy of this vaccine derives mainly from cross-presentation which is enhanced by HDACi-induced apoptosis. Additionally, epigenetic activation of immune genes may contribute to direct antigen presentation by tumor cells. Epigenetically altered cancer cells should be further explored as a vaccine strategy.

Neem leaf extract induces cell death by apoptosis and autophagy in B-chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia and is currently incurable. To expand the therapeutic armamentarium, we investigated neem leaf extract (NLE) after a patient with CLL demonstrated disease regression upon taking oral NLE. NLE-mediated apoptosis was examined in peripheral blood mononuclear cells (PBMCs) from 41 patients with CLL. NLE induced a dose-dependent reduction in CLL cell viability with significant apoptosis observed at 0.06% (w/v) by 24 h. Annexin-V staining and poly(ADP-ribose) polymerase 1 (PARP-1) and caspase 3 cleavage were observed after NLE treatment. However, a pan-caspase inhibitor only partially blocked NLE-mediated cell death. NLE also caused loss of mitochondrial outer membrane permeability and nuclear translocation of apoptosis-inducing factor. Furthermore, NLE treatment resulted in LC3-I cleavage. Biochemical analyses revealed that NLE also inhibits Bcl-2 and p53 proteins. In summary, NLE exhibits anti-leukemic properties in patient primary CLL cells and demonstrates clinical efficacy, warranting further investigation as a potential therapy for CLL.

Myeloid-derived suppressor cells modulate immune responses independently of NADPH oxidase in the ovarian tumor microenvironment in mice.

The phagocyte NADPH oxidase generates superoxide anion and downstream reactive oxidant intermediates in response to infectious threat, and is a critical mediator of antimicrobial host defense and inflammatory responses. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that are recruited by cancer cells, accumulate locally and systemically in advanced cancer, and can abrogate anti-tumor immunity. Prior studies have implicated the phagocyte NADPH oxidase as being an important component promoting MDSC accumulation and immunosuppression in cancer. We therefore used engineered NADPH oxidase-deficient (p47 (phox-/-)) mice to delineate the role of this enzyme complex in MDSC accumulation and function in a syngeneic mouse model of epithelial ovarian cancer. We found that the presence of NADPH oxidase did not affect tumor progression. The accumulation of MDSCs locally and systemically was similar in tumor-bearing wild-type (WT) and p47 (phox-/-) mice. Although MDSCs from tumor-bearing WT mice had functional NADPH oxidase, the suppressive effect of MDSCs on ex vivo stimulated T cell proliferation was NADPH oxidase-independent. In contrast to other tumor-bearing mouse models, our results show that MDSC accumulation and immunosuppression in syngeneic epithelial ovarian cancer is NADPH oxidase-independent. We speculate that factors inherent to the tumor, tumor microenvironment, or both determine the specific requirement for NADPH oxidase in MDSC accumulation and function.

Interferon regulatory factor-8 is important for histone deacetylase inhibitor-mediated antitumor activity.

The notion that epigenetic alterations in neoplasia are reversible has provided the rationale to identify epigenetic modifiers for their ability to induce or enhance tumor cell death. Histone deacetylase inhibitors (HDACi) represent one such class of anti-neoplastic agents. Despite great interest for clinical use, little is known regarding the molecular targets important for response to HDACi-based cancer therapy. We had previously shown that interferon regulatory factor (IRF)-8, originally discovered as a leukemia suppressor gene by regulating apoptosis, also regulates Fas-mediated killing in non-hematologic tumor models. Furthermore, we and others have shown that epigenetic mechanisms are involved in repression of IRF-8 in tumors. Therefore, in our preclinical tumor model, we tested the hypothesis that IRF-8 expression is important for response to HDACi-based antitumor activity. In the majority of experiments, we selected the pan-HDACi, Trichostatin A (TSA), because it was previously shown to restore Fas sensitivity to tumor cells. Overall, we found that: 1) TSA alone and more so in combination with IFN-γ enhanced both IRF-8 expression and Fas-mediated death of tumor cells in vitro; 2) TSA treatment enhanced IRF-8 promoter activity via a STAT1-dependent pathway; and 3) IRF-8 was required for this death response, as tumor cells rendered IRF-8 incompetent were significantly less susceptible to Fas-mediated killing in vitro and to HDACi-mediated antitumor activity in vivo. Thus, IRF-8 status may underlie a novel molecular basis for response to HDACi-based antitumor treatment.

Regulation of innate immunity by NADPH oxidase.

NADPH oxidase is a critical regulator of both antimicrobial host defense and inflammation. Activated in nature by microbes and microbial-derived products, the phagocyte NADPH oxidase is rapidly assembled, and generates reactive oxidant intermediates (ROIs) in response to infectious threat. Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by recurrent and severe bacterial and fungal infections, and pathology related to excessive inflammation. Studies in CGD patients and CGD mouse models indicate that NADPH oxidase plays a key role in modulating inflammation and injury that is distinct from its antimicrobial function. The mechanisms by which NADPH oxidase mediates killing of pathogens and regulation of inflammation have broad relevance to our understanding of normal physiological immune responses and pathological states, such as acute lung injury and bacterial or fungal infections.

Histone deacetylase inhibitors induce TAP, LMP, Tapasin genes and MHC class I antigen presentation by melanoma cells.

Histone deacetylase inhibitors (HDACi), including trichostatin A (TSA) and valproic acid, can alter the acetylation of histones in chromatin and enhance gene transcription. Previously we demonstrated that HDACi-treated tumor cells are capable of presenting antigen via the MHC class II pathway. In this study, we show that treatment with HDACi enhances the expression of molecules (TAP1, TAP2, LMP2, LMP7, Tapasin and MHC class I) involved in antigen processing and presentation via the MHC class I pathway in melanoma cells. HDACi treatment of B16F10 cells also enhanced cell surface expression of class I and costimulatory molecules CD40 and CD86. Enhanced transcription of these genes is associated with a significant increase in direct presentation of whole protein antigen and MHC class I-restricted peptides by TSA-treated B16F10 cells. Our data indicate that epigenetic modification can convert a tumor cell to an antigen presenting cell capable of activating IFN-gamma secreting T cells via the class I pathway. These findings suggest that the abnormalities, observed in some tumors in the expression of MHC class I antigen processing and presentation molecules, may result from epigenetic repression.

Epigenetic regulation of immune escape genes in cancer.

According to the concept of immune surveillance, the appearance of a tumor indicates that it has earlier evaded host defenses and subsequently must have escaped immunity to evolve into a full-blown cancer. Tumor escape mechanisms have focused mainly on mutations of immune and apoptotic pathway genes. However, data obtained over the past few years suggest that epigenetic silencing in cancer may be as frequent a cause of gene inactivation as are mutations. Here, we discuss the evidence that tumor immune evasion is mediated by non-mutational epigenetic events involving chromatin and that epigenetics collaborates with mutations in determining tumor progression. Since epigenetic changes are potentially reversible, the relative contribution of mutations and epigenetics, to the gene defects in any given tumor, may be a factor in determining the efficacy of treatments. We review new developments in basic chromatin mechanisms and in this context describe the rationale for the current use of epigenetic agents in cancer therapy and for a novel epigenetically generated tumor vaccine model. We emphasize that epigenetic cancer treatments are currently a 'blunt-sword' and suggest future directions for designing chromatin-based programs of potential value in the diagnosis and treatment of cancer.

Histone acetylation regulates the cell type specific CIITA promoters, MHC class II expression and antigen presentation in tumor cells.

The regulation of MHC class II expression by the class II transactivator (CIITA) is complex and differs in various cell types depending on the relative activity of three CIITA promoters. Here we show that, in plasma cell tumors, the deacetylase inhibitor trichostatin A (TSA) elicits PIII-CIITA but does not activate the IFN-gamma-inducible PIV-CIITA promoter. In trophoblast cells, all CIITA promoter types are constitutively silent and not induced by IFN-gamma or TSA treatment. TSA induction of PI-CIITA was restricted to macrophage and dendritic cell lines. In the Colon 26 tumor IFN-gamma induced endogenous PIV-CIITA but not PIII-CIITA while TSA activated class II in the apparent absence of CIITA. Reporter assays in Colon 26 showed that TSA induced PIII-CIITA but not PIV-CIITA. Transfection of a dominant negative CIITA plasmid in Colon 26 inhibited induction of class II by IFN-gamma but not TSA. Thus, the potential for both CIITA-dependent and -independent pathways of MHC induction exists within a single cell. Further evidence of CIITA-independent class II expression elicited by TSA was obtained using knockout mice with defects in CIITA, STAT-1alpha and IRF-1 expression. TSA treatment can also activate class II expression in mutant cell lines with deficiencies in signaling molecules, transcription factors and the BRG-1 cofactor that are required for IFN-gamma-induced CIITA expression. Importantly, after epigenetic activation by the deacetylase inhibitor, MHC class II is transported and displayed on the cell surface of a plasma cell tumor and it is converted to an efficient antigen presenting cell for protein and class II-peptide presentation.

An epigenetically altered tumor cell vaccine.

Functional inactivation of genes critical to immunity may occur by mutation and/or by repression, the latter being potentially reversible with agents that modify chromatin. This study was constructed to determine whether reversal of gene silencing, by altering the acetylation status of chromatin, might lead to an effective tumor vaccine. We show that the expression of selected genes important to tumor immunity, including MHC class II, CD40, and B7-1/2 are altered by treating tumor cells in vitro with a histone deacetylase inhibitor, trichostatin A (TSA). Tumor cells treated in vitro with TSA showed delayed onset and rate of tumor growth in 70% of the J558 plasmacytoma and 100% of the B16 melanoma injected animals. Long-term tumor specific immunity was elicited to rechallenge with wild-type cells in approximately 30% in both tumor models. Splenic T cells from immune mice lysed untreated tumor cells, and SCID mice did not manifest immunity, suggesting that T cells may be involved in immunity. We hypothesize that repression of immune genes is involved in the evasion of immunity by tumors and suggest that epigenetically altered cancer cells should be further explored as a strategy for the induction of tumor immunity.

Exosomes from plasmacytoma cells as a tumor vaccine.

Exosomes are membrane-bound vesicles derived from multivesicular bodies that are externalized by cells through fusion with the plasma membrane. Exosomes have been implicated in cell-to-cell signaling, and those derived from immunologic cells may be involved in both direct and cross-presentation of antigens to T cells. The research presented here evaluated their efficacy as a prophylactic cancer vaccine in a mouse plasmacytoma model. Plasmacytoma cells were shown to release exosomes in vitro, and vaccination with a single dose (5 microg) of exosome protein protected 80% of mice against challenge with wild-type tumors. Protection could be linked to the immune system since vaccinated mice generated specific cytotoxic T lymphocytes, the effects were not seen in SCID mice, and immunity was tumor-specific. Several proteins involved in immunity, including two potential tumor antigens (P1A and intracisternal A particle protein) as well as Hsp70, were demonstrated to be present in exosomes. The authors conclude that exosomes can induce tumor-specific immunity and prevent tumor development and are a potential strategy for future therapeutic tumor vaccination.